Endoplasmic Reticulum Stress and Autophagy

*Mohammad Fazlul Kabir, Hyung-Ryong Kim and Han-Jung Chae*

#### **Abstract**

In eukaryotic cells, the aggregation of the endoplasmic reticulum (ER)-mediated unfolded or misfolded proteins leads to disruption of the ER homeostasis, which can trigger ER stress. To restore the ER homeostasis, the ER stress activates the intracellular signaling cascade from the ER to the nucleus, referred to as the unfolded protein response (UPR). Autophagy primitively portrayed as an evolutionarily conserved process is involved in cellular homeostasis by facilitating the lysosomal degradation pathway for the recycling and elimination of intracellular defective macromolecules and organelles. Autophagy is tightly regulated by the protective mechanism of UPR. The UPR and autophagy are interlinked, which indicates that the ER stress can not only induce autophagy but also suppress it. Here, we discuss the molecular mechanism of ER stress and autophagy and their induction and inhibition signaling network.

**Keywords:** ER stress, autophagy, calcium, lysosome

#### **1. Endoplasmic reticulum**

The endoplasmic reticulum (ER) is a central membrane-bound organelle constructed from a dynamic network of tubules involved in cellular processes such as protein synthesis, gluconeogenesis, lipid synthesis and processing, and calcium storage and release in the cell and contributes to the generation of autophagosomes and peroxisomes [1]. The extension of ER morphology depends on the cell's activity and lineage; it is organized into subcompartments of different shapes, such as cisternae and tubules. ER appears as two main interconnected compartments, namely, the smooth endoplasmic reticulum (SER) and the rough endoplasmic reticulum (RER), which are abundant in different proportions in different cell lineages [2]. RER is less tubular than the SER, which forms an interrelated network of subdomains of ER; the RER is illuminated with ribosomes on their membranes, which are absent in the SER. RER has appeared in all cells and its density is higher, similar to that of the Golgi apparatus and nucleus because in all cells the nascent polypeptide is cotranslationally inserted into their membranes from the ER membrane. However, SER is present in only certain cell types, such as the liver cells, steroid-synthesizing cells, neurons, and muscle cells. SER is involved in the generation of steroid hormones within the adrenal cortex and endocrine glands and acts as a center for detoxification and protein transportation [3, 4]. A remarkable number of proteins are Ca2+ dependent and need a completely oxidizing environment [5]. In the lumen, the

abundant molecular chaperones bind to the proteins and prevent them from aggregation, which makes the ER an ideal and unique milieu for proper protein folding. In fact, the ER quality control checkpoints allow the existence of only the precisely folded proteins. In addition, the ER facilitates the formation of three-dimensional structures by cotranslational and posttranslational modifications of the proteins [6].

#### **2. ER stress**

The ER is a subcellular organelle predominantly known as a protein-folding checkpoint, which has an important role to ensure the proper folding and maturation of newly secreted proteins and transmembrane proteins. Several pathological and physiological conditions, such as perturbation in the cellular ATP level, calcium fluctuation, hypoxia, viral infection, inflammatory cytokines, nutrient deprivation, and environmental toxins, result in the loss of ER homeostasis and a reduction in the protein-folding potential of ER, eventually leading to the accumulation and aggregation of unfolded proteins in the ER lumen, acknowledged as the ER stress [7]. In experimental settings, the ER stress and protein misfolding or aggregation is instigated by treating cells with ER stress-inducing toxic chemicals. Versatile mechanisms of the UPR, under these nonphysiological conditions, are unable to maintain the homeostasis in the ER and the cells finally undergo apoptosis [8].

#### **3. The unfolded protein response (UPR)**

The UPR can be viewed as a process that is involved in the sensing of the ER stress and transduces this signal to the regulation of downstream transcription factors that are involved in stress reduction or the induction of proapoptotic programs [9]. The ER stress enacts the UPR as an adaptive response for maintaining protein homeostasis [10, 11]. The UPR is initiated by three ER transmembrane proteins: the inositolrequiring 1α (IRE1α), PKR-like ER kinase (PERK), and activating transcription factor 6α (ATF6α). Under normal conditions, the ER chaperone, luminal domain binding immunoglobulin protein (BiP), binds to these proteins and keeps them inactive; but, when ER stress occurs, the BiP dissociates from these three proteins, UPR arms are activated [12]. This activates the UPR, which has three noteworthy functions: (a) adaptive feedback, which encompasses decreasing the ER workload, in anticipation of further augmentation of unfolded proteins, by the upregulation of molecular chaperones and protein-processing enzymes that maximize the folding efficiency, and an accompanying increase in the ER-associated protein degradation (ERAD) and the upregulation of the autophagy components to aid in the removal of misfolded proteins; (b) feedback control, which includes prevention of the hyperactivation of UPR, when the ER homeostasis is retrieved; (c) cell fate regulation, by the coordination of apoptotic and antiapoptotic signals, in the form of a switch between life and death of ER-stressed cells [12, 13]. The gene targets of the UPR change depending on the type of tissue and the nature of the physiological trigger that induces the ER stress. In distinctive hereditary backgrounds like the mouse and human cells, the different gene expression patterns triggered by the ER stress have been reported [9, 14].

#### **4. The UPR signaling pathway**

The UPR and misfolded or unfolded proteins as a prominent characteristic of mammalian cell ER stress were first reported in the 1980s by Kozutsumi et al. [10].

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*nucleus where they actuate the expression of target genes.*

**Figure 1.**

*Endoplasmic Reticulum Stress and Autophagy DOI: http://dx.doi.org/10.5772/intechopen.81381*

ATF6α is translocated to the Golgi apparatus [16].

**4.1 IRE1α**

The UPR signaling pathway consists of three main branches involving the proteins IRE1α, PERK, and ATF6α. These proteins present in the ER resident transmembrane are major signaling elements. These three signaling sensors are all confederate with the ER chaperones, such as GRP78 (glucose-regulated protein 78 kDa, the main ER chaperone that is also named as BiP), which regulate their activation by fastening or discharge mechanism [15]. Primarily, IRE1α, PERK, and ATF6α are activated by the interaction with GRP78. GRP78 is well established to bind to the hydrophobic domains of proteins with its C-terminal binding domain, to prevent misfolding and unfolded protein aggregation (**Figure 1**). GRP78 accelerates the oligomerization and autophosphorylation of IRE1α and PERK activates them, while

IRE1α is the most conserved ER stress signaling branch, and its activation mechanism has been studied thoroughly. IRE1α is a bifunctional type 1 transmembrane protein kinase containing three domains: an N-terminal luminal domain, a cytosolic endoribonuclease (RNase) domain, and a cytosolic serine/threonine kinase domain [17]. In response to the accumulation of unfolded proteins under the ER stress condition, IRE1α dimerizes and transautophosphorylates leading to activation of

*Schematic overview of UPR signaling. The three sensors of UPR, namely, IRE1α, PERK, and ATF6α, are activated when the misfolded protein aggregates recruit GRP78 or Bip, by dissociating them from the sensors. Activated IRE1α dimerization and phosphorylation induces XBP1 mRNA splicing to generate active XBP1s, which increase the expression of UPR functional gene. UPR also activates another cellular pathway by interacting with Jun N-terminal kinase (JNK), via recruit TRAF2 and ASK1. PERK phosphorylates the downstream translation initiation factor eIF2α, leading to the attenuation of overall protein translation and the activation of ATF4, which activates the expression of CHOP. Under ER stress conditions, the ATF6α is transported to the Golgi apparatus and its cytosolic domain is cleaved by S1P and S2P proteases, which triggers the transcription of the ER chaperones. XBP1, ATF4, and ATF6α transcription factors are translocated to the* 

*Endoplasmic Reticulum Stress and Autophagy DOI: http://dx.doi.org/10.5772/intechopen.81381*

The UPR signaling pathway consists of three main branches involving the proteins IRE1α, PERK, and ATF6α. These proteins present in the ER resident transmembrane are major signaling elements. These three signaling sensors are all confederate with the ER chaperones, such as GRP78 (glucose-regulated protein 78 kDa, the main ER chaperone that is also named as BiP), which regulate their activation by fastening or discharge mechanism [15]. Primarily, IRE1α, PERK, and ATF6α are activated by the interaction with GRP78. GRP78 is well established to bind to the hydrophobic domains of proteins with its C-terminal binding domain, to prevent misfolding and unfolded protein aggregation (**Figure 1**). GRP78 accelerates the oligomerization and autophosphorylation of IRE1α and PERK activates them, while ATF6α is translocated to the Golgi apparatus [16].

#### **4.1 IRE1α**

*Endoplasmic Reticulum*

**2. ER stress**

**3. The unfolded protein response (UPR)**

abundant molecular chaperones bind to the proteins and prevent them from aggregation, which makes the ER an ideal and unique milieu for proper protein folding. In fact, the ER quality control checkpoints allow the existence of only the precisely folded proteins. In addition, the ER facilitates the formation of three-dimensional structures by cotranslational and posttranslational modifications of the proteins [6].

The ER is a subcellular organelle predominantly known as a protein-folding checkpoint, which has an important role to ensure the proper folding and maturation of newly secreted proteins and transmembrane proteins. Several pathological and physiological conditions, such as perturbation in the cellular ATP level, calcium fluctuation, hypoxia, viral infection, inflammatory cytokines, nutrient deprivation, and environmental toxins, result in the loss of ER homeostasis and a reduction in the protein-folding potential of ER, eventually leading to the accumulation and aggregation of unfolded proteins in the ER lumen, acknowledged as the ER stress [7]. In experimental settings, the ER stress and protein misfolding or aggregation is instigated by treating cells with ER stress-inducing toxic chemicals. Versatile mechanisms of the UPR, under these nonphysiological conditions, are unable to maintain the homeostasis in the ER and the cells finally undergo apoptosis [8].

The UPR can be viewed as a process that is involved in the sensing of the ER stress and transduces this signal to the regulation of downstream transcription factors that are involved in stress reduction or the induction of proapoptotic programs [9]. The ER stress enacts the UPR as an adaptive response for maintaining protein homeostasis [10, 11]. The UPR is initiated by three ER transmembrane proteins: the inositolrequiring 1α (IRE1α), PKR-like ER kinase (PERK), and activating transcription factor 6α (ATF6α). Under normal conditions, the ER chaperone, luminal domain binding immunoglobulin protein (BiP), binds to these proteins and keeps them inactive; but, when ER stress occurs, the BiP dissociates from these three proteins, UPR arms are activated [12]. This activates the UPR, which has three noteworthy functions: (a) adaptive feedback, which encompasses decreasing the ER workload, in anticipation of further augmentation of unfolded proteins, by the upregulation of molecular chaperones and protein-processing enzymes that maximize the folding efficiency, and an accompanying increase in the ER-associated protein degradation (ERAD) and the upregulation of the autophagy components to aid in the removal of misfolded proteins; (b) feedback control, which includes prevention of the hyperactivation of UPR, when the ER homeostasis is retrieved; (c) cell fate regulation, by the coordination of apoptotic and antiapoptotic signals, in the form of a switch between life and death of ER-stressed cells [12, 13]. The gene targets of the UPR change depending on the type of tissue and the nature of the physiological trigger that induces the ER stress. In distinctive hereditary backgrounds like the mouse and human cells, the different gene

expression patterns triggered by the ER stress have been reported [9, 14].

The UPR and misfolded or unfolded proteins as a prominent characteristic of mammalian cell ER stress were first reported in the 1980s by Kozutsumi et al. [10].

**4. The UPR signaling pathway**

**50**

IRE1α is the most conserved ER stress signaling branch, and its activation mechanism has been studied thoroughly. IRE1α is a bifunctional type 1 transmembrane protein kinase containing three domains: an N-terminal luminal domain, a cytosolic endoribonuclease (RNase) domain, and a cytosolic serine/threonine kinase domain [17]. In response to the accumulation of unfolded proteins under the ER stress condition, IRE1α dimerizes and transautophosphorylates leading to activation of

#### **Figure 1.**

*Schematic overview of UPR signaling. The three sensors of UPR, namely, IRE1α, PERK, and ATF6α, are activated when the misfolded protein aggregates recruit GRP78 or Bip, by dissociating them from the sensors. Activated IRE1α dimerization and phosphorylation induces XBP1 mRNA splicing to generate active XBP1s, which increase the expression of UPR functional gene. UPR also activates another cellular pathway by interacting with Jun N-terminal kinase (JNK), via recruit TRAF2 and ASK1. PERK phosphorylates the downstream translation initiation factor eIF2α, leading to the attenuation of overall protein translation and the activation of ATF4, which activates the expression of CHOP. Under ER stress conditions, the ATF6α is transported to the Golgi apparatus and its cytosolic domain is cleaved by S1P and S2P proteases, which triggers the transcription of the ER chaperones. XBP1, ATF4, and ATF6α transcription factors are translocated to the nucleus where they actuate the expression of target genes.*

the cytosolic region RNase domain, resulting in the conformational change that activates the excision of the 26-nucleotide intron from the mRNA encoding the transcription factor XBP1 (**Figure 1**) [18–21]. This splicing event results in a frame shift in the mRNA and leads to the expression of an active and stable form of the transcription factor XBP1. The XBP1 is then translocated to the nucleus where it upregulates target genes that are involved in prosurvival events, such as quality control, maintaining ER homeostasis (via the ER chaperones GRP78, ERDj4, HEDJ, and PDI-P5) and ERAD (ER-associated degradation) [22, 23]. In the ER and Golgi compartment, XBP1 also increases the secretion rate of proteins. In addition, the RNase domain of IRE1α can rapidly cleave a group of mRNAs and microRNAs, degradation through a process known as IRE1α-dependent decay (RIDD) [24, 25]. IRE1α activation is associated with the reduction of levels of a myriad of cytosolic RNAs, ribosomal RNAs, and microRNAs that have significant roles in inflammation, glucose metabolism, and apoptosis. Furthermore, active IRE1α not only promotes UPR but also mediates other pathways, including the mitogen-activated protein (MAPK) kinase pathway, where the activated IRE1α interacts with the adaptor protein tumor necrosis factor receptor-associated protein (TRAF-2) to form the complex IRE1α-TRAF2. This complex interacts with the apoptosis signal-regulated kinase 1 (ASK1) to form the IRE1α-TRAF2-ASK1 complex, which interacts with the ER stress-triggered c-Jun N terminal kinase (JNK) and results in the production of reactive oxygen species and activation of the autophagy and inflammatory pathways that involve the nuclear factor-κB [26–28].

#### **4.2 PERK**

PERK is a type 1 transmembrane kinase that is structurally and functionally related to IRE1α and is activated by transautophosphorylation and dimerization [29]. Under the ER stress conditions, PERK phosphorylates the downstream substrate eukaryotic translation initiator factor-2α (eIF2α) at serine 51, which leads to the inhibition of protein synthesis within the ER lumen (**Figure 1**) [30–32]. This blockade reduces the continuous accumulation of unfolded proteins in the ER, thus reducing the ER stress. In addition, it allows the selective translation of the mRNA encoding the transcription factor ATF4, which has a ribosome entry site in its 5′ nontranslated region, enabling its cap-independent translation [33, 34]. ATF4 is translocated to the nucleus where it upregulates the expression of the ER chaperone proteins (GRP78 and GRP94), the genes involved in macroautophagy, amino acid biosynthesis, protein secretion, antioxidant response, and the proapoptotic transcription factor CHOP [34, 35]. In addition to its role in UPR, eIF2α phosphorylation assumes the role of a confluent marker of a particular stress pathway known as the integrated stress response," which is led by the unambiguous kinase that triggered during nutrient deficiency, viral infection, inflammation, and heme deficiency [35].

#### **4.3 ATF6α**

A third sensor of the ER stress, ATF6α, is an ER-targeted type 2 transmembrane protein that includes a basic leucine zipper transcription factor domain (**Figure 1**) [36, 37]. Under upregulation of UPR, ATF6α is translocated to the Golgi apparatus for cleavage by the endopeptidases S1P and S2P, thereby releasing the activated form of ATF6α. In response to the ER stress condition and GRP78, GRP94 agglomeration, similar to that of IRE1α and PERK activation, the redox state is involved in the activation of ATF6α [38].

The activation of IRE1α, PERK, and ATF6α has several effects, such as reduced translation, enhanced ER protein-folding capacity, and clearance of misfolded ER

**53**

*Endoplasmic Reticulum Stress and Autophagy DOI: http://dx.doi.org/10.5772/intechopen.81381*

**5. Autophagy**

proteins. The UPR stress sensors interact with and activate several transcription

Autophagy, derived from the Greek words "auto," meaning "self," and "phagy," meaning "to eat," is a lysosomal pathway for cell survival used by eukaryotes, in which the cells digest and recycle their own cytoplasmic contents [39]. In the past three decades, several studies, especially in yeast, have revealed the molecular mechanisms involved in autophagy. Cells attune the number of components or vitiate parts of the organelle to maintain the optimum activity by assisting the minimal basal level of autophagy [40, 41]. In response, the basal autophagy can be activated to play a crucial role in cellular starvation and other cellular stresses, by lysosomal degradation and the exclusion of perennial and misfolded proteins, pernicious cellular substances, and pernicious organelles and infecting pathogens [42, 43]. In addition, autophagy can involve the rearrangement of the cellular membrane to concede parts of the cytoplasm being transported to the compartment, and it also acts as an energy source for the biosynthesis of new macromolecules produced by recycling metabolites of lysosomal proteolysis [44, 45]. Autophagy can maintain the energy homeostasis not only in particular organelle but also in the entire cell, through the increase of metabolic activity [45]. Moreover, autophagy plays critical roles in physiological processes such as cell growth, cell cycle, differentiation, tumor suppression, and programmed cell death including apoptosis and cellular senescence. In these ways, autophagy plays crucial roles throughout the life cycle of the cells [46, 47].

In mammal cells, there are three types of autophagy that have been documented; they are distinguished according to their physiological function and mechanism of cargo sequestration at the known destination lysosomes. These subtypes include macroautophagy, microautophagy, and chaperone-mediated autophagy [43, 48]. Macroautophagy is a major type of autophagy, and it has been the most studied compared to microautophagy and chaperone-mediated autophagy. It uses cytosolic double-membrane sequestering vesicles formed from phagophores, known as the autophagosomes, which transport cytosolic content to the lysosome [43, 49]. In microautophagy, the lysosome itself is a component of the cytoplasm where it engulfs cytoplasmic protein and small components of the lysosomal membrane. Macroautophagy and microautophagy both carry out the nonselective degradation of proteins, lipids, and organelles [50, 51]. In contrast, the chaperone-mediated autophagy does not involve the membrane rearrangement; instead, the protease of the lysosomal matrix acts on the substrate unfolded protein by directly translocating across the lysosomal membrane, which is dependent on LAMP2A (lysosomalassociated membrane protein 2A) and the lysosomal molecular chaperon HSPA8/

The mechanism of autophagy is a complex process that can be categorized into multiple steps. It involves the formation of double-membrane vesicles containing cellular and external malformed proteins. Long-lived proteins can be induced autophagy, which are ensued by cargo recognition and packaging, an extension of the phagophore membrane, and closure to form the complete autophagosome. Fusion of the autophagosome with the lysosome occurs, which leads to the derogation of the autophagosomal contents, and the breakdown products are finally

HSC73/lys-HSC70 (heat shock cognate 70) [52, 53].

**6. Molecular mechanism of autophagy**

factors, which indicate the functional role of the UPR in proteostasis.

proteins. The UPR stress sensors interact with and activate several transcription factors, which indicate the functional role of the UPR in proteostasis.

#### **5. Autophagy**

*Endoplasmic Reticulum*

**4.2 PERK**

**4.3 ATF6α**

the activation of ATF6α [38].

the cytosolic region RNase domain, resulting in the conformational change that activates the excision of the 26-nucleotide intron from the mRNA encoding the transcription factor XBP1 (**Figure 1**) [18–21]. This splicing event results in a frame shift in the mRNA and leads to the expression of an active and stable form of the transcription factor XBP1. The XBP1 is then translocated to the nucleus where it upregulates target genes that are involved in prosurvival events, such as quality control, maintaining ER homeostasis (via the ER chaperones GRP78, ERDj4, HEDJ, and PDI-P5) and ERAD (ER-associated degradation) [22, 23]. In the ER and Golgi compartment, XBP1 also increases the secretion rate of proteins. In addition, the RNase domain of IRE1α can rapidly cleave a group of mRNAs and microRNAs, degradation through a process known as IRE1α-dependent decay (RIDD) [24, 25]. IRE1α activation is associated with the reduction of levels of a myriad of cytosolic RNAs, ribosomal RNAs, and microRNAs that have significant roles in inflammation, glucose metabolism, and apoptosis. Furthermore, active IRE1α not only promotes UPR but also mediates other pathways, including the mitogen-activated protein (MAPK) kinase pathway, where the activated IRE1α interacts with the adaptor protein tumor necrosis factor receptor-associated protein (TRAF-2) to form the complex IRE1α-TRAF2. This complex interacts with the apoptosis signal-regulated kinase 1 (ASK1) to form the IRE1α-TRAF2-ASK1 complex, which interacts with the ER stress-triggered c-Jun N terminal kinase (JNK) and results in the production of reactive oxygen species and activation of the autophagy and

inflammatory pathways that involve the nuclear factor-κB [26–28].

PERK is a type 1 transmembrane kinase that is structurally and functionally related to IRE1α and is activated by transautophosphorylation and dimerization [29]. Under the ER stress conditions, PERK phosphorylates the downstream substrate eukaryotic translation initiator factor-2α (eIF2α) at serine 51, which leads to the inhibition of protein synthesis within the ER lumen (**Figure 1**) [30–32]. This blockade reduces the continuous accumulation of unfolded proteins in the ER, thus reducing the ER stress. In addition, it allows the selective translation of the mRNA encoding the transcription factor ATF4, which has a ribosome entry site in its 5′ nontranslated region, enabling its cap-independent translation [33, 34]. ATF4 is translocated to the nucleus where it upregulates the expression of the ER chaperone proteins (GRP78 and GRP94), the genes involved in macroautophagy, amino acid biosynthesis, protein secretion, antioxidant response, and the proapoptotic transcription factor CHOP [34, 35]. In addition to its role in UPR, eIF2α phosphorylation assumes the role of a confluent marker of a particular stress pathway known as the integrated stress response," which is led by the unambiguous kinase that triggered during nutrient deficiency, viral infection, inflammation, and heme deficiency [35].

A third sensor of the ER stress, ATF6α, is an ER-targeted type 2 transmembrane protein that includes a basic leucine zipper transcription factor domain (**Figure 1**) [36, 37]. Under upregulation of UPR, ATF6α is translocated to the Golgi apparatus for cleavage by the endopeptidases S1P and S2P, thereby releasing the activated form of ATF6α. In response to the ER stress condition and GRP78, GRP94 agglomeration, similar to that of IRE1α and PERK activation, the redox state is involved in

The activation of IRE1α, PERK, and ATF6α has several effects, such as reduced translation, enhanced ER protein-folding capacity, and clearance of misfolded ER

**52**

Autophagy, derived from the Greek words "auto," meaning "self," and "phagy," meaning "to eat," is a lysosomal pathway for cell survival used by eukaryotes, in which the cells digest and recycle their own cytoplasmic contents [39]. In the past three decades, several studies, especially in yeast, have revealed the molecular mechanisms involved in autophagy. Cells attune the number of components or vitiate parts of the organelle to maintain the optimum activity by assisting the minimal basal level of autophagy [40, 41]. In response, the basal autophagy can be activated to play a crucial role in cellular starvation and other cellular stresses, by lysosomal degradation and the exclusion of perennial and misfolded proteins, pernicious cellular substances, and pernicious organelles and infecting pathogens [42, 43]. In addition, autophagy can involve the rearrangement of the cellular membrane to concede parts of the cytoplasm being transported to the compartment, and it also acts as an energy source for the biosynthesis of new macromolecules produced by recycling metabolites of lysosomal proteolysis [44, 45]. Autophagy can maintain the energy homeostasis not only in particular organelle but also in the entire cell, through the increase of metabolic activity [45]. Moreover, autophagy plays critical roles in physiological processes such as cell growth, cell cycle, differentiation, tumor suppression, and programmed cell death including apoptosis and cellular senescence. In these ways, autophagy plays crucial roles throughout the life cycle of the cells [46, 47].

In mammal cells, there are three types of autophagy that have been documented; they are distinguished according to their physiological function and mechanism of cargo sequestration at the known destination lysosomes. These subtypes include macroautophagy, microautophagy, and chaperone-mediated autophagy [43, 48]. Macroautophagy is a major type of autophagy, and it has been the most studied compared to microautophagy and chaperone-mediated autophagy. It uses cytosolic double-membrane sequestering vesicles formed from phagophores, known as the autophagosomes, which transport cytosolic content to the lysosome [43, 49]. In microautophagy, the lysosome itself is a component of the cytoplasm where it engulfs cytoplasmic protein and small components of the lysosomal membrane. Macroautophagy and microautophagy both carry out the nonselective degradation of proteins, lipids, and organelles [50, 51]. In contrast, the chaperone-mediated autophagy does not involve the membrane rearrangement; instead, the protease of the lysosomal matrix acts on the substrate unfolded protein by directly translocating across the lysosomal membrane, which is dependent on LAMP2A (lysosomalassociated membrane protein 2A) and the lysosomal molecular chaperon HSPA8/ HSC73/lys-HSC70 (heat shock cognate 70) [52, 53].

#### **6. Molecular mechanism of autophagy**

The mechanism of autophagy is a complex process that can be categorized into multiple steps. It involves the formation of double-membrane vesicles containing cellular and external malformed proteins. Long-lived proteins can be induced autophagy, which are ensued by cargo recognition and packaging, an extension of the phagophore membrane, and closure to form the complete autophagosome. Fusion of the autophagosome with the lysosome occurs, which leads to the derogation of the autophagosomal contents, and the breakdown products are finally

eliminated [54–56]. The initiation of autophagy can be observed by TEM (transmission electron microscopy) during the expansion of phagophore and autophagosome. The induction of autophagy, vesicle nucleation, and formation of autophagosomes are regulated by the proteins named as autophagy-related genes (ATGs) [50]. They are highly conserved genes and were originally discovered in yeasts. Mammalian orthologs of the ATGs have also been discovered [57]. Autophagy induction is controlled at the molecular level by the multiprotein complex of unc-51-like autophagy-activating kinase 1 (ULK1, the mammalian homolog of yeast Atg1), ATG13, ATG101a, and RB1 inducible coiled coil 1 (RB1CC1, also known as FIP200) [58, 59]. This complex is regulated by the mammalian target of kanamycin complex 1 (mTORC1), which remains inhibited by mTORC by the phosphorylation of ULK1/2 and ATG13, which suppresses the phosphorylation activity of ULK1/2- ATG13-FIP200 complex [59–61]. Under starvation and other stress conditions, the inhibition of mTORC1 dissociates it from the ULK complex followed by the dephosphorylating of specific residues within the ULK1/2 and ATG13 (phosphorylated by mTORC1) complex, which in turn promotes the induction of the phagophores [61]. Formation of phagophores includes a class III phosphatidylinositol 3-kinase complex (PtdIns3K) consisting of Beclin-1 (ATG6 in yeast), VPS34 (class III PI3K), VPS15 (also known as p150 in mammals), PIK3R4/p150, ATG14, UV radiation resistanceassociated gene (UVRAG), and nuclear receptor binding factor 2 (NRBF2) [62–64]. In addition, the nonapoptotic proteins, such as the B-cell lymphoma-2 (BCL2) and BCL2L1/BCL-XL, hold Beclin-1 directly interacting with Beclin-1(BECN-1s) BH3 domain and negatively regulating autophagy inducing the PtdIns3K. The c-Jun protein kinase (JNK1) and death-associated protein kinase (DAPK) phosphorylate BCL2 and are positive regulators involved in the induction of autophagy [65, 66].

The elongation or obstruction of phagophore depends on two diverse ubiquitinlike protein conjugation reactions [67, 68]. The first pathway involves the covalent conjugation reaction of ATG12 to ATG5, with the assist of the E1-like enzyme ATG7 and the E2-like enzyme ATG10. This conjugate ATG12-ATG5 complex interacts with ATG16L in a no covalent reaction to form the multiprotein complex ATG12-ATG5- ATG16L, which performs the E3 ligase reaction of the cytosolic MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3), LC3-I to the membrane-bound lipidated form, LC3-II [50, 69–71]. The second pathway includes the ubiquitin-like system, which plays a role in the conjugation to phosphatidylethanolamine (PE) lipid and glycine residue of the yeast ATG8 (LC3 in the mammal), and is processed by the cysteine protease ATG4 and then ATG8 is conjugated to PE by E1-like enzyme ATG7 and E2-like enzyme ATG3. Based on that , the ATG4 can act as delipidation or deconjugation enzyme which is involved in the recycling of membrane bound LC3-II on the external layer to the internal layer of the autophagosome [50, 67, 72]. Accordingly, the lipidated form of LC3-II is a stable marker protein associated with the biochemical and microscopic detection of cellular autophagy [73]. In mammals, six orthologs of ATG8 and four of ATG4 exist, among which the LC3, GATE-16 (Golgi-associated ATPase enhancer of 16kDa), and GABARAP (G-amino butyric acid type A receptor-associated protein) have been the most studied [74]. The lipidation of ATG8/LC3 expedites the interaction with the autophagosome membrane, which leads to the autophagosome maturation steps, such as the extension and shrinkage of the membranes and cargo induction to autophagosome [75]. Once the autophagosome has surrounded the substrate of autophagy, it may merge with the late lysosome or endosome to create the autolysosome [76]. The cellular and molecular machinery that important for the fusion is activated by the small GTPase, RAB7A/RAB7 member of RAS oncogene family, which is necessary for autophagosome maturation [77]; and the RAB7 effector pleckstrin homology and RUN domain containing M1 (PLEKHM1) [78]; other soluble

**55**

nucleation [93, 94].

*Endoplasmic Reticulum Stress and Autophagy DOI: http://dx.doi.org/10.5772/intechopen.81381*

pump known as the V-type ATPase) [85].

**7. ER stress and autophagy**

N-ethylmaleimide-sensitive factor proteins trigger set of SNARE protein including syntaxin-17 (STX17), SNAP29,and VAMP8 [79, 80]; the PI3P-binding protein tectonin beta-propeller repeat containing 1 (TECPR1) [81]; inositol polyphosphate-5-phosphatase-E (INPP5E) [82]; ectopic P-granules autophagy protein 5 homolog (EPG5) [83]; as well as the homotypic fusion and vacuole protein sorting (HOPS) complexes ATG14 [78]; LAMP2B (but not LAMP2A) as well as the phosphorylated and lipidated LC3 which are also involved in the formation of autolysosomes [84]; finally, the autophagosomal-sequestered cargo undergoes degradation upon the acidification of the lysosomal lumen (by the activity of an ATP-dependent proton

Several studies have demonstrated that the ER stress and autophagy are mechanistically interconnected, in which the UPR, the key ER stress pathway, stimulates the autophagy. The three canonical divisions of the UPR intervened by the three ER membrane-associated proteins, IRE1α (inositol-requiring enzyme 1), PERK (PKRlike eIF2α also known as EIF2AK3), and ATF6α (activating transcription factor-6), regulate the autophagy in distinctive manners during the ER stress. The relationship between autophagosome and the ER stress was first described in 2006 [86, 87].

IRE1α-mediated MAPK8 (mitogen-activated protein kinases 8) phosphorylation

is the major regulatory step in this pathway [88]. MAPK8 is considered stressassociated protein kinase," which is involved in numerous manners in stress-induced autophagy and apoptosis, which depend on MAPK8 activation [89]. In particular, the activation of IRE1α leads to MAPK8 phosphorylation, which induces autophagy. JNK (c-Jun N-terminal kinase) interacts with the MAPK8 family, which triggers the downstream mediators of autophagy, both directly and indirectly [90]. Directly, JNK can stimulate cell apoptosis in cancer cells by inducing Atg5 and p53. Indirectly, JNK inhibits the association of Bcl-2 with Beclin-1 and upregulates Beclin-1 expression by c-Jun phosphorylation. Beclin-1 is the autophagy-related gene and is the downstream regulator of MAPK8 and is activated by the direct phosphorylation of Bcl-2, which then obstructs the interaction between Beclin-1 and Bcl-2 and activation of the phosphoinositide-3-kinase (PI3K) complex and induces autophagy in the cancer cell (**Figure 2**) [90, 91]. Additionally, SP600125, a pharmacological inhibitor of JNK, also blocks the Beclin-1 expression and autophagy [92]. Wei Y et al [91] elucidated the starvation-induced autophagy by JNK1, via phosphorylation of ER-specific Bcl-2, at multiresidues T69, S70, and S87A, followed by Beclin-1 disruption from ER-localized Bcl-2 and the induction of autophagy [91]. Similarly, Beclin-1 expression is regulated by the JNK1 pathway, which plays a crucial role at the transcription level, following the ceramide-induced autophagy in mammalian CNE2 and Hep3B cancer cell lines [92]. SP600125 inhibited the autophagosome formation and ceramide-induced upregulation of Beclin-1, and similar phenomenon was observed using the small interfering RNA targeting JNK mRNA. Moreover, immunoprecipitation of chromatin and luciferase reporter analysis demonstrated that c-Jun, a target of JNK1, was activated and directly interacted with the Beclin-1 promoter in ceramide-treated cancer cells. In this respect, the IRE1α/JNK1/c-Jun pathway is the key mechanism for the induction of autophagy. The IRE1α/JNK1-induced autophagy pathways interact with the ATG protein and Beclin-1, which play a key role in vesicle

In addition, the IRE1α-XBP1s axis has been involved in the induction of autophagy [95]. Initially, the spliced XBP1 indirectly regulates the Bcl-2 expression to induce autophagy (**Figure 2**) [66, 96]. Along with this, the autophagy induction

*Endoplasmic Reticulum Stress and Autophagy DOI: http://dx.doi.org/10.5772/intechopen.81381*

*Endoplasmic Reticulum*

eliminated [54–56]. The initiation of autophagy can be observed by TEM (transmission electron microscopy) during the expansion of phagophore and autophagosome. The induction of autophagy, vesicle nucleation, and formation of autophagosomes are regulated by the proteins named as autophagy-related genes (ATGs) [50]. They are highly conserved genes and were originally discovered in yeasts. Mammalian orthologs of the ATGs have also been discovered [57]. Autophagy induction is controlled at the molecular level by the multiprotein complex of unc-51-like autophagy-activating kinase 1 (ULK1, the mammalian homolog of yeast Atg1), ATG13, ATG101a, and RB1 inducible coiled coil 1 (RB1CC1, also known as FIP200) [58, 59]. This complex is regulated by the mammalian target of kanamycin complex 1 (mTORC1), which remains inhibited by mTORC by the phosphorylation of ULK1/2 and ATG13, which suppresses the phosphorylation activity of ULK1/2- ATG13-FIP200 complex [59–61]. Under starvation and other stress conditions, the inhibition of mTORC1 dissociates it from the ULK complex followed by the dephosphorylating of specific residues within the ULK1/2 and ATG13 (phosphorylated by mTORC1) complex, which in turn promotes the induction of the phagophores [61]. Formation of phagophores includes a class III phosphatidylinositol 3-kinase complex (PtdIns3K) consisting of Beclin-1 (ATG6 in yeast), VPS34 (class III PI3K), VPS15 (also known as p150 in mammals), PIK3R4/p150, ATG14, UV radiation resistanceassociated gene (UVRAG), and nuclear receptor binding factor 2 (NRBF2) [62–64]. In addition, the nonapoptotic proteins, such as the B-cell lymphoma-2 (BCL2) and BCL2L1/BCL-XL, hold Beclin-1 directly interacting with Beclin-1(BECN-1s) BH3 domain and negatively regulating autophagy inducing the PtdIns3K. The c-Jun protein kinase (JNK1) and death-associated protein kinase (DAPK) phosphorylate BCL2 and are positive regulators involved in the induction of autophagy [65, 66]. The elongation or obstruction of phagophore depends on two diverse ubiquitinlike protein conjugation reactions [67, 68]. The first pathway involves the covalent conjugation reaction of ATG12 to ATG5, with the assist of the E1-like enzyme ATG7 and the E2-like enzyme ATG10. This conjugate ATG12-ATG5 complex interacts with ATG16L in a no covalent reaction to form the multiprotein complex ATG12-ATG5- ATG16L, which performs the E3 ligase reaction of the cytosolic MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3), LC3-I to the membrane-bound lipidated form, LC3-II [50, 69–71]. The second pathway includes the ubiquitin-like system, which plays a role in the conjugation to phosphatidylethanolamine (PE) lipid and glycine residue of the yeast ATG8 (LC3 in the mammal), and is processed by the cysteine protease ATG4 and then ATG8 is conjugated to PE by E1-like enzyme ATG7 and E2-like enzyme ATG3. Based on that , the ATG4 can act as delipidation or deconjugation enzyme which is involved in the recycling of membrane bound LC3-II on the external layer to the internal layer of the autophagosome [50, 67, 72]. Accordingly, the lipidated form of LC3-II is a stable marker protein associated with the biochemical and microscopic detection of cellular autophagy [73]. In mammals, six orthologs of ATG8 and four of ATG4 exist, among which the LC3, GATE-16 (Golgi-associated ATPase enhancer of 16kDa), and GABARAP (G-amino butyric acid type A receptor-associated protein) have been the most studied [74]. The lipidation of ATG8/LC3 expedites the interaction with the autophagosome membrane, which leads to the autophagosome maturation steps, such as the extension and shrinkage of the membranes and cargo induction to autophagosome [75]. Once the autophagosome has surrounded the substrate of autophagy, it may merge with the late lysosome or endosome to create the autolysosome [76]. The cellular and molecular machinery that important for the fusion is activated by the small GTPase, RAB7A/RAB7 member of RAS oncogene family, which is necessary for autophagosome maturation [77]; and the RAB7 effector pleckstrin homology and RUN domain containing M1 (PLEKHM1) [78]; other soluble

**54**

N-ethylmaleimide-sensitive factor proteins trigger set of SNARE protein including syntaxin-17 (STX17), SNAP29,and VAMP8 [79, 80]; the PI3P-binding protein tectonin beta-propeller repeat containing 1 (TECPR1) [81]; inositol polyphosphate-5-phosphatase-E (INPP5E) [82]; ectopic P-granules autophagy protein 5 homolog (EPG5) [83]; as well as the homotypic fusion and vacuole protein sorting (HOPS) complexes ATG14 [78]; LAMP2B (but not LAMP2A) as well as the phosphorylated and lipidated LC3 which are also involved in the formation of autolysosomes [84]; finally, the autophagosomal-sequestered cargo undergoes degradation upon the acidification of the lysosomal lumen (by the activity of an ATP-dependent proton pump known as the V-type ATPase) [85].

#### **7. ER stress and autophagy**

Several studies have demonstrated that the ER stress and autophagy are mechanistically interconnected, in which the UPR, the key ER stress pathway, stimulates the autophagy. The three canonical divisions of the UPR intervened by the three ER membrane-associated proteins, IRE1α (inositol-requiring enzyme 1), PERK (PKRlike eIF2α also known as EIF2AK3), and ATF6α (activating transcription factor-6), regulate the autophagy in distinctive manners during the ER stress. The relationship between autophagosome and the ER stress was first described in 2006 [86, 87].

IRE1α-mediated MAPK8 (mitogen-activated protein kinases 8) phosphorylation is the major regulatory step in this pathway [88]. MAPK8 is considered stressassociated protein kinase," which is involved in numerous manners in stress-induced autophagy and apoptosis, which depend on MAPK8 activation [89]. In particular, the activation of IRE1α leads to MAPK8 phosphorylation, which induces autophagy. JNK (c-Jun N-terminal kinase) interacts with the MAPK8 family, which triggers the downstream mediators of autophagy, both directly and indirectly [90]. Directly, JNK can stimulate cell apoptosis in cancer cells by inducing Atg5 and p53. Indirectly, JNK inhibits the association of Bcl-2 with Beclin-1 and upregulates Beclin-1 expression by c-Jun phosphorylation. Beclin-1 is the autophagy-related gene and is the downstream regulator of MAPK8 and is activated by the direct phosphorylation of Bcl-2, which then obstructs the interaction between Beclin-1 and Bcl-2 and activation of the phosphoinositide-3-kinase (PI3K) complex and induces autophagy in the cancer cell (**Figure 2**) [90, 91]. Additionally, SP600125, a pharmacological inhibitor of JNK, also blocks the Beclin-1 expression and autophagy [92]. Wei Y et al [91] elucidated the starvation-induced autophagy by JNK1, via phosphorylation of ER-specific Bcl-2, at multiresidues T69, S70, and S87A, followed by Beclin-1 disruption from ER-localized Bcl-2 and the induction of autophagy [91]. Similarly, Beclin-1 expression is regulated by the JNK1 pathway, which plays a crucial role at the transcription level, following the ceramide-induced autophagy in mammalian CNE2 and Hep3B cancer cell lines [92]. SP600125 inhibited the autophagosome formation and ceramide-induced upregulation of Beclin-1, and similar phenomenon was observed using the small interfering RNA targeting JNK mRNA. Moreover, immunoprecipitation of chromatin and luciferase reporter analysis demonstrated that c-Jun, a target of JNK1, was activated and directly interacted with the Beclin-1 promoter in ceramide-treated cancer cells. In this respect, the IRE1α/JNK1/c-Jun pathway is the key mechanism for the induction of autophagy. The IRE1α/JNK1-induced autophagy pathways interact with the ATG protein and Beclin-1, which play a key role in vesicle nucleation [93, 94].

In addition, the IRE1α-XBP1s axis has been involved in the induction of autophagy [95]. Initially, the spliced XBP1 indirectly regulates the Bcl-2 expression to induce autophagy (**Figure 2**) [66, 96]. Along with this, the autophagy induction

is also observed in endothelial cells that overexpress XBP1s, which enhances the transformation of LC3-I to LC3-II and increases the Beclin-1 expression [95]. Furthermore, XBP1s binds directly to the −537 and −755 region of the Beclin-1 gene promoter in the nucleus and enhances an autophagy induction via the transcriptional upregulated expression of Beclin-1 gene [97]. The deficiency in XBP1s leads to increased expression of Forkhead box O1, a transcriptional factor that elevates the induction of autophagy in neurons [98].

The major events in autophagy, such as the induction of phagophore and maturation, are coordinated by the LC3-II and the ATG12-ATG5 conjugate [99]. To maintain the autophagy flux, the upregulation of the transcription of the congruent autophagy genes is important [100]. Under the ER stress conditions, the PERK branch of UPR aids in the regulation of the autophagy-related genes. The association of PERK in ER stress-mediated induction of autophagy was first reported by Kouroku et al. [101]. In particular, they demonstrated that the aggregated polyglutamine (72Q ) protein in the cytosol decreases the activity of proteasomes and leads to autophagy induction through the activation of the PERK branch of the UPR [102]. Under the hypoxic response, PERK mediates the transcriptional activation of LC3 and Atg5 proteins, through the action of the transcription factors ATF4, CHOP, and DDIT3 induction (**Figure 2**) [101, 103]. PERK may also reduce IkBα translation, as well as NF-kB activation, which promotes the induction of

#### **Figure 2.**

*Overview of the mechanism of UPR-mediated autophagy. The ER stress can activate autophagy through Ca2+, IRE1α, PERK, and the ATF6α signaling pathway. Ca2+ from ER lumen can be released through the IP3R channel, which phosphorylates CaMKKβ and activates AMPK, which in turn inactivates ULK1 complex through the inhibition of mTOR; Ca2+ activates DAPK which phosphorylates Beclin1 and Bcl2 lead to autophagy induction. The IRE1α arm of UPR activation of JNK1 mediates phosphorylation of Bcl2, which causes Beclin-1 dissociation and induction of autophagy. In addition, spliced XBP1 also enhances the formation of LC3-I and LC3-II, which triggers the Beclin-1 via decrease of FoxO1 activity. Another arm of UPR activated PERK induce autophagy via expression of ATG12, DDIT3, ATG12, ATG16L by ATF4 transcription factor similarly CHOP activate TRIB3 which suppress the activity of Akt/mTOR pathway induced autophagy. ATF6α arm of UPR can also induce autophagy by inhibiting phosphorylation at Akt and mTOR pathway.*

**57**

*Endoplasmic Reticulum Stress and Autophagy DOI: http://dx.doi.org/10.5772/intechopen.81381*

of Bcl-2 expression [106].

induction of autophagy [113].

autophagy [104]. PERK phosphorylates the downstream regulator eukaryotic initiation factor 2a (eIF2α), at the residue serine 51, and also increases the ATG12 mRNA and protein levels [105]. PERK-mediated ATF4 activation is required for expression of the autophagy genes, including MAP1LC3B, BECN1, ATG3, ATG12, and ATG16L1, while interaction of ATF4 and DDIT3 causes the upregulation of the transcription of SQSTM1/p62, BR1, and ATG7 [100]. In addition, ATF4 directly binds to cyclic AMP response component binding site of the promoter of microtubule-associated protein 1 light chain 3β (LC3β), a vital component of autophagosomal membranes, which alleviates the induction of autophagy. In addition, DDIT3 can activate the formation of autophagosome through downregulation

CHOP is another potent transcription factor, which is involved in the induction of autophagy [107, 108]. It has been elucidated that the expression levels of ATG5 and BH3 domain proteins are elevated by upregulation of the CHOP expression. Besides, the Bcl-2 expression level is downregulated, which assists in the release of Beclin-1 from Bcl-2. Moreover, the PERK-CHOP pathway instigates tribbles-related protein 3 (TRIB3), which inhibits the activation of the protein kinase B (Akt) [103, 109]. TRIB3-mediated inhibition of Akt regulates the phosphorylation of TSC2 (tuberous sclerosis complex 2) by the serine/threonine kinase, Ras homolog enriched in brain (Rheb), and the inhibition of mTORC1, which dephosphorylates ATG13 and the ULK1/2 complex and results in the induction of autophagosome formation [110]. The ATF6α branch of the UPR is the least understood branch in relation to ER stress and autophagy. Nonetheless, the ATF6α transcription regulator is involved in the initiation of autophagy by the elevated expression of HSPA5 (heat shock70kDa protein 5) (**Figure 2**) and followed by the inhibition of expression and activation of protein kinase B of AKT1/AKT [111]. In addition, the ATF6α interacts with CEBPB (CCAAT/enhancer binding protein) to form a transcriptional heterodimer complex and binds to the CRE/ATF components of DAPK1 (death-associated protein kinase 1) to induce DAPK1 expression. ATF6α knockdown with specific shRNA and ATF6α−/<sup>−</sup> cells leads to reduced expression of DAPK1, followed by the inhibition of formation of autophagosomes [112]. Beclin-1 phosphorylation leads to decreased Bcl-2 expression and initiates the formation of a complex between the autophagosome initiator Beclin-1 and PIK3C3. Simultaneously, the ATF6α-mediated upregulation of CHOP, XBP1, and GRP78 expression is also initiated, resulting in the

**8. ER stress induces autophagy via the PI3K/AKT/mTORC pathway**

The serine/threonine kinase of mTORC is the main regulator of ER stress [114]. It forms two complexes, the mTORC1 and mTORC2, both of which are triggered by extracellular and intracellular stimuli, under favorable conditions for growth [114, 115]. Accordingly, mTORC1 is a critical regulator of the UPR-mediated autophagy and nutrient signaling [116]. mTORC1 is involved in the regulation of the major signaling pathway. Interaction of growth factors with insulin triggers the PI3K complex, which accelerates the plasma membrane adaptation of the lipid phosphatidylinositol-3-phosphate (PtdIns(3)P) to generate PtdIns(3,4,5)P2 and PtdIns(3,4,5)P3. These increase the membrane recruitment of pleckstrin homology domain proteins such as the serine/threonine kinase PDK1 (phosphoinositidedependent kinase 1) and its substrate Akt protein kinase B to activate Akt in the plasma membrane [117]. The PI3K is elicited as a vesicular protein trafficking mediator, which binds to PtdIns(3)P, resulting in its translocation to intracellular membranes such as endosomal and lysosomal membranes. PI3K is a member of Vps34 family, which plays an important role in the formation of autophagosomes,

#### *Endoplasmic Reticulum Stress and Autophagy DOI: http://dx.doi.org/10.5772/intechopen.81381*

*Endoplasmic Reticulum*

the induction of autophagy in neurons [98].

is also observed in endothelial cells that overexpress XBP1s, which enhances the transformation of LC3-I to LC3-II and increases the Beclin-1 expression [95]. Furthermore, XBP1s binds directly to the −537 and −755 region of the Beclin-1 gene promoter in the nucleus and enhances an autophagy induction via the transcriptional upregulated expression of Beclin-1 gene [97]. The deficiency in XBP1s leads to increased expression of Forkhead box O1, a transcriptional factor that elevates

The major events in autophagy, such as the induction of phagophore and maturation, are coordinated by the LC3-II and the ATG12-ATG5 conjugate [99]. To maintain the autophagy flux, the upregulation of the transcription of the congruent autophagy genes is important [100]. Under the ER stress conditions, the PERK branch of UPR aids in the regulation of the autophagy-related genes. The association of PERK in ER stress-mediated induction of autophagy was first reported by Kouroku et al. [101]. In particular, they demonstrated that the aggregated polyglutamine (72Q ) protein in the cytosol decreases the activity of proteasomes and leads to autophagy induction through the activation of the PERK branch of the UPR [102]. Under the hypoxic response, PERK mediates the transcriptional activation of LC3 and Atg5 proteins, through the action of the transcription factors ATF4, CHOP, and DDIT3 induction (**Figure 2**) [101, 103]. PERK may also reduce IkBα translation, as well as NF-kB activation, which promotes the induction of

*Overview of the mechanism of UPR-mediated autophagy. The ER stress can activate autophagy through Ca2+, IRE1α, PERK, and the ATF6α signaling pathway. Ca2+ from ER lumen can be released through the IP3R channel, which phosphorylates CaMKKβ and activates AMPK, which in turn inactivates ULK1 complex through the inhibition of mTOR; Ca2+ activates DAPK which phosphorylates Beclin1 and Bcl2 lead to autophagy induction. The IRE1α arm of UPR activation of JNK1 mediates phosphorylation of Bcl2, which causes Beclin-1 dissociation and induction of autophagy. In addition, spliced XBP1 also enhances the formation of LC3-I and LC3-II, which triggers the Beclin-1 via decrease of FoxO1 activity. Another arm of UPR activated PERK induce autophagy via expression of ATG12, DDIT3, ATG12, ATG16L by ATF4 transcription factor similarly CHOP activate TRIB3 which suppress the activity of Akt/mTOR pathway induced autophagy. ATF6α arm of UPR can also induce autophagy by inhibiting phosphorylation at Akt and* 

**56**

*mTOR pathway.*

**Figure 2.**

autophagy [104]. PERK phosphorylates the downstream regulator eukaryotic initiation factor 2a (eIF2α), at the residue serine 51, and also increases the ATG12 mRNA and protein levels [105]. PERK-mediated ATF4 activation is required for expression of the autophagy genes, including MAP1LC3B, BECN1, ATG3, ATG12, and ATG16L1, while interaction of ATF4 and DDIT3 causes the upregulation of the transcription of SQSTM1/p62, BR1, and ATG7 [100]. In addition, ATF4 directly binds to cyclic AMP response component binding site of the promoter of microtubule-associated protein 1 light chain 3β (LC3β), a vital component of autophagosomal membranes, which alleviates the induction of autophagy. In addition, DDIT3 can activate the formation of autophagosome through downregulation of Bcl-2 expression [106].

CHOP is another potent transcription factor, which is involved in the induction of autophagy [107, 108]. It has been elucidated that the expression levels of ATG5 and BH3 domain proteins are elevated by upregulation of the CHOP expression. Besides, the Bcl-2 expression level is downregulated, which assists in the release of Beclin-1 from Bcl-2. Moreover, the PERK-CHOP pathway instigates tribbles-related protein 3 (TRIB3), which inhibits the activation of the protein kinase B (Akt) [103, 109]. TRIB3-mediated inhibition of Akt regulates the phosphorylation of TSC2 (tuberous sclerosis complex 2) by the serine/threonine kinase, Ras homolog enriched in brain (Rheb), and the inhibition of mTORC1, which dephosphorylates ATG13 and the ULK1/2 complex and results in the induction of autophagosome formation [110].

The ATF6α branch of the UPR is the least understood branch in relation to ER stress and autophagy. Nonetheless, the ATF6α transcription regulator is involved in the initiation of autophagy by the elevated expression of HSPA5 (heat shock70kDa protein 5) (**Figure 2**) and followed by the inhibition of expression and activation of protein kinase B of AKT1/AKT [111]. In addition, the ATF6α interacts with CEBPB (CCAAT/enhancer binding protein) to form a transcriptional heterodimer complex and binds to the CRE/ATF components of DAPK1 (death-associated protein kinase 1) to induce DAPK1 expression. ATF6α knockdown with specific shRNA and ATF6α−/<sup>−</sup> cells leads to reduced expression of DAPK1, followed by the inhibition of formation of autophagosomes [112]. Beclin-1 phosphorylation leads to decreased Bcl-2 expression and initiates the formation of a complex between the autophagosome initiator Beclin-1 and PIK3C3. Simultaneously, the ATF6α-mediated upregulation of CHOP, XBP1, and GRP78 expression is also initiated, resulting in the induction of autophagy [113].

#### **8. ER stress induces autophagy via the PI3K/AKT/mTORC pathway**

The serine/threonine kinase of mTORC is the main regulator of ER stress [114]. It forms two complexes, the mTORC1 and mTORC2, both of which are triggered by extracellular and intracellular stimuli, under favorable conditions for growth [114, 115]. Accordingly, mTORC1 is a critical regulator of the UPR-mediated autophagy and nutrient signaling [116]. mTORC1 is involved in the regulation of the major signaling pathway. Interaction of growth factors with insulin triggers the PI3K complex, which accelerates the plasma membrane adaptation of the lipid phosphatidylinositol-3-phosphate (PtdIns(3)P) to generate PtdIns(3,4,5)P2 and PtdIns(3,4,5)P3. These increase the membrane recruitment of pleckstrin homology domain proteins such as the serine/threonine kinase PDK1 (phosphoinositidedependent kinase 1) and its substrate Akt protein kinase B to activate Akt in the plasma membrane [117]. The PI3K is elicited as a vesicular protein trafficking mediator, which binds to PtdIns(3)P, resulting in its translocation to intracellular membranes such as endosomal and lysosomal membranes. PI3K is a member of Vps34 family, which plays an important role in the formation of autophagosomes,

by directly interacting with Beclin-1 [118]. Similarly, PtdIns(3)P and PtdIns(3,4,5) P3 initiate autophagy by phosphorylation of the phosphatidylinositol to activate PtdIns(3,4,5)P3 and contributes to the autophagic vacuole sequestration [119].

Akt is a serine/threonine kinase, which is an upstream regulator of mTORC. Several hormone growth factors and the phosphorylation of the oncogene PI3K-Akt-mTORC can stimulate mTORC and the ribosomal protein S6 kinase (RPS6KB1) and inhibit the expression and phosphorylation of TSC1 (tuberous sclerosis 1) and TSC2, which under ER stress conditions inhibits mTORC [90]. Similarly, the inhibition of TSC triggers mTORC activity, which suppresses the initiation of ER stress-mediated autophagy. Furthermore, the knockdown of TSC1/2 can regulate the activation of mTORC, which is elevated under ER stress conditions. This indicates that TSC is essential for the canonical ER stress feedback [120, 121]. Thus, TSC1/2 is a crucial coordinator of several signals, including mTORC and the well-known PI3K-Akt pathway, for the induction of autophagy.

The opposite branch of this pathway is downregulated by mTORC release, and ULK1 initiates the autophagosome formation [122]. Accordingly, ER stress can inhibit the expression of Akt and suppress the mTORC regulation, which can induce autophagy. ATF6α increases the expression of ER chaperone HSPA5 (heat shock 70 kDa protein 5), which can block the phosphorylation of Akt activity, in turn activating the induction of autophagy in placental choriocarcinoma cell [90].

TRIB3 (tribbles homolog 3) is an ER stress-associated protein, which can interact with Akt and downregulate the expression of Akt-mTORC [123, 124]. The defective ATF4-DDIT3 complex in malignant gliomas can activate TRIB3 under ER stress condition, which indicates that TRIB3 activation is ATF4-DDIT3 dependent. Δ9-Tetrahydrocannabinol (THC), the main active compound of marijuana, triggers the TRIB3-dependent autophagy pathway of ER stress, by the suppression of the Akt/mTORC1 pathway. The overactivation of TRIB3 can reduce the transcriptional activity of ATF4 and DDIT3. This indicates that the ER stress-mediated induction of autophagy via the PI3K/AKT/mTOR pathway plays a key role in cell survival [123].

#### **9. ER stress induces autophagy via the AMPK/TSC/mTORC1 pathway**

The AMP-activated kinase (AMPK) is a key cellular energy sensor that regulates the transcription of the autophagy genes through the regulation of many downstream kinases [125]. AMPK is a cellular energy sensor that detects increased level of intracellular ATP/AMP concentration ratio [126]. Under several metabolic stress conditions, AMPK is phosphorylated by a serine/threonine kinase and activates genes including liver kinase B1 (LKB1, which is activated upon energy depletion), calcium/calmodulin kinase (CaMKKβ, which is activated by cytosolic Ca2+), and TGFβ-activated kinase-1 (TAK-1, which is involved in IKK activation) [126]. AMPK induces autophagy through the inactivation of mTORC1 via the phosphorylation of the tuberous sclerosis complex 2 (TSC2) and the regulation of the associated protein RAPTOR, after the dissociation and activation of ULK1 [127]. In addition, AMPK-induced autophagy not only inhibits mTORC1 but also directly phosphorylates ULK1 and Beclin-1. AMPK has a major role in preventing the ER stressinduced autophagy-mediated cytotoxicity. In addition, albumin-treated cellular toxicity leads to the activation of AMPK. Similarly, silenced RPS6KA3 (ribosomal S6 kinase 90 kDa polypeptide 3) decreased expression of AMPK induce autophagy which aggregates ER stress mammalian breast cancer model [128, 129]. Involvement of PERK-AMPK mediated and inactivation mTORC initiate autophagy has also demonstrated detachment of extracellular matrix in human epithelial cell. Moreover, AMPK inhibits synthesis protein by inactivation of mTORC and

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*Endoplasmic Reticulum Stress and Autophagy DOI: http://dx.doi.org/10.5772/intechopen.81381*

induction of autophagy by starvation.

**10. Ca2+ in ER stress regulates autophagy**

phosphorylating EIF4EBP1/4E-BP1 and RPS6KB/p70S6K [130]. Moreover, the phosphorylation of eIF2α [101] and the activation of IKK [131] are indispensable for

The ER plays a major role in maintaining the intracellular Ca2+ store that can compile Ca2+ concentrations of 10–100 mM, while in the cytoplasm and remaining cell concentration, the range is 100–300 nm [132]. The multifunctional organelle ER maintains Ca2+ homeostasis, which is necessary for proper functioning including protein folding, lipid and protein biosynthesis, and posttranslational modification and regulation of gene expression [133]. The majority of ER-associated proteins participate in maintaining ER Ca2+ homeostasis. For maintaining ER Ca2+ homeostasis, most of the ER-associated proteins, such as calreticulin, GRP94 or GRP78, histidine-rich Ca2+-binding protein, and protein disulfide isomerase (PDI), uphold to Ca2+ buffer in the lumen of ER [134]. Ca2+-binding protein mainly GRP78 is involved in sensing unfolded protein accumulation in the ER and interacts with three other UPRs of ER transmembrane proteins, ATF6α, IRE1α, and PERK [135]. As noted, loss of Ca2+ homeostasis in the ER followed to initiate ER stress [136]. In addition, ER lumenal Ca2+ can reduce because of ER stress. Upon incitement of plasma membrane ER influx and discharge formation of Ca2+ signal, whereas ER reservoir influx and release depend on replenishment of Ca2+. Activity of Ca2+ across the membrane of ER is expedited by three kinds of protein receptor: Ca2+ release channels—RYR (ryanodine receptor) and ITPR/IP3R (inositol 1, 4, 5-trisphosphate receptor); in the ER, cytosolic Ca2+ enters through a Ca2+ pump called ATP2A/SERCA (sarco/endoplasmic reticulum Ca2+) [137]. There is multitudinous Ca2+ movement through the membrane of ER that assures appropriate functioning of numerous kinases and proteases. It is already well established that cytosolic Ca2+ signal regulates protein intricate in several stages of autophagosome formation [138]. In addition, a number of Ca2+ dependent pathways involved in autophagy induction have been studied. Indeed, cytosolic Ca2+ initiation of autophagy it is ambiguous in many conditions. The numerous Ca2+ origin has already involved merely various downstream effectors containing protein kinase C, Ca2+/calmodulin-dependent kinase β (CaMKKβ or CaMKK2), ERK, and Vps34 (a calmodulin protein) [139, 140]. It is already proven that CaMKKβ or CaMKK2 has perceived the majority experimental support, whereas Ca2+ refinement of Vps34 and ERK is unsupportable. Activation of Vps34 by Ca2+ or calmodulin is insinuated although the activity of Vps34 in cellulo was not affected by cytosolic Ca2+ or calmodulin antagonist [139]. CaMKKβ is an inrease the activity of AMPK, thereby inhibition of mTORC1 leads to activate autophagy [141]. Høyer-Hansen et al. demonstrated that in MCF-7 breast cancer cells the mobilize of cytosolic Ca2+ from ER by stimulate IP3R generating agonist, such as thasigargin, ionomycin and vitamin D analogue activate CaMKKβ which is initiate autophagy by downregulating of mTORC1 and activation AMPK dependent pathway [142]. In addition, deficient autophagy in T lymphocyte has an extension of ER compartment due to more Ca2+ in the ER. Depletion of Ca2+ in the ER leads to extension of Ca2+ reservoir, which could be the purpose behind unfit to store diminished. This invasion of Ca2+ can be recovered by SERCA/ATPase pump blocking with thapsigargin, which means autophagy can maintain Ca2+ mobilization across the ER [143]. In total, the connection between autophagy and Ca2+ mobilization intimates that they can have impact on each other. Moreover, the elevation of cytosolic Ca2+ endogenously induction of autophagy by precipitation of Ca2+ phosphate without modifying the condition of ER. In consequence, ER Ca2+ plays a key role for induced *Endoplasmic Reticulum*

by directly interacting with Beclin-1 [118]. Similarly, PtdIns(3)P and PtdIns(3,4,5) P3 initiate autophagy by phosphorylation of the phosphatidylinositol to activate PtdIns(3,4,5)P3 and contributes to the autophagic vacuole sequestration [119]. Akt is a serine/threonine kinase, which is an upstream regulator of

mTORC. Several hormone growth factors and the phosphorylation of the oncogene PI3K-Akt-mTORC can stimulate mTORC and the ribosomal protein S6 kinase (RPS6KB1) and inhibit the expression and phosphorylation of TSC1 (tuberous sclerosis 1) and TSC2, which under ER stress conditions inhibits mTORC [90]. Similarly, the inhibition of TSC triggers mTORC activity, which suppresses the initiation of ER stress-mediated autophagy. Furthermore, the knockdown of TSC1/2 can regulate the activation of mTORC, which is elevated under ER stress conditions. This indicates that TSC is essential for the canonical ER stress feedback [120, 121]. Thus, TSC1/2 is a crucial coordinator of several signals, including mTORC and the

The opposite branch of this pathway is downregulated by mTORC release, and ULK1 initiates the autophagosome formation [122]. Accordingly, ER stress can inhibit the expression of Akt and suppress the mTORC regulation, which can induce autophagy. ATF6α increases the expression of ER chaperone HSPA5 (heat shock 70 kDa protein 5), which can block the phosphorylation of Akt activity, in turn activating the induction of autophagy in placental choriocarcinoma cell [90]. TRIB3 (tribbles homolog 3) is an ER stress-associated protein, which can interact with Akt and downregulate the expression of Akt-mTORC [123, 124]. The defective ATF4-DDIT3 complex in malignant gliomas can activate TRIB3 under ER stress condition, which indicates that TRIB3 activation is ATF4-DDIT3 dependent. Δ9-Tetrahydrocannabinol (THC), the main active compound of marijuana, triggers the TRIB3-dependent autophagy pathway of ER stress, by the suppression of the Akt/mTORC1 pathway. The overactivation of TRIB3 can reduce the transcriptional activity of ATF4 and DDIT3. This indicates that the ER stress-mediated induction of autophagy via the PI3K/AKT/mTOR pathway plays a key role in cell survival [123].

**9. ER stress induces autophagy via the AMPK/TSC/mTORC1 pathway**

the transcription of the autophagy genes through the regulation of many downstream kinases [125]. AMPK is a cellular energy sensor that detects increased level of intracellular ATP/AMP concentration ratio [126]. Under several metabolic stress conditions, AMPK is phosphorylated by a serine/threonine kinase and activates genes including liver kinase B1 (LKB1, which is activated upon energy depletion), calcium/calmodulin kinase (CaMKKβ, which is activated by cytosolic Ca2+), and TGFβ-activated kinase-1 (TAK-1, which is involved in IKK activation) [126]. AMPK induces autophagy through the inactivation of mTORC1 via the phosphorylation of the tuberous sclerosis complex 2 (TSC2) and the regulation of the associated protein RAPTOR, after the dissociation and activation of ULK1 [127]. In addition, AMPK-induced autophagy not only inhibits mTORC1 but also directly phosphorylates ULK1 and Beclin-1. AMPK has a major role in preventing the ER stressinduced autophagy-mediated cytotoxicity. In addition, albumin-treated cellular toxicity leads to the activation of AMPK. Similarly, silenced RPS6KA3 (ribosomal S6 kinase 90 kDa polypeptide 3) decreased expression of AMPK induce autophagy

which aggregates ER stress mammalian breast cancer model [128, 129].

Involvement of PERK-AMPK mediated and inactivation mTORC initiate autophagy has also demonstrated detachment of extracellular matrix in human epithelial cell. Moreover, AMPK inhibits synthesis protein by inactivation of mTORC and

The AMP-activated kinase (AMPK) is a key cellular energy sensor that regulates

well-known PI3K-Akt pathway, for the induction of autophagy.

**58**

phosphorylating EIF4EBP1/4E-BP1 and RPS6KB/p70S6K [130]. Moreover, the phosphorylation of eIF2α [101] and the activation of IKK [131] are indispensable for induction of autophagy by starvation.

### **10. Ca2+ in ER stress regulates autophagy**

The ER plays a major role in maintaining the intracellular Ca2+ store that can compile Ca2+ concentrations of 10–100 mM, while in the cytoplasm and remaining cell concentration, the range is 100–300 nm [132]. The multifunctional organelle ER maintains Ca2+ homeostasis, which is necessary for proper functioning including protein folding, lipid and protein biosynthesis, and posttranslational modification and regulation of gene expression [133]. The majority of ER-associated proteins participate in maintaining ER Ca2+ homeostasis. For maintaining ER Ca2+ homeostasis, most of the ER-associated proteins, such as calreticulin, GRP94 or GRP78, histidine-rich Ca2+-binding protein, and protein disulfide isomerase (PDI), uphold to Ca2+ buffer in the lumen of ER [134]. Ca2+-binding protein mainly GRP78 is involved in sensing unfolded protein accumulation in the ER and interacts with three other UPRs of ER transmembrane proteins, ATF6α, IRE1α, and PERK [135]. As noted, loss of Ca2+ homeostasis in the ER followed to initiate ER stress [136]. In addition, ER lumenal Ca2+ can reduce because of ER stress. Upon incitement of plasma membrane ER influx and discharge formation of Ca2+ signal, whereas ER reservoir influx and release depend on replenishment of Ca2+. Activity of Ca2+ across the membrane of ER is expedited by three kinds of protein receptor: Ca2+ release channels—RYR (ryanodine receptor) and ITPR/IP3R (inositol 1, 4, 5-trisphosphate receptor); in the ER, cytosolic Ca2+ enters through a Ca2+ pump called ATP2A/SERCA (sarco/endoplasmic reticulum Ca2+) [137].

There is multitudinous Ca2+ movement through the membrane of ER that assures appropriate functioning of numerous kinases and proteases. It is already well established that cytosolic Ca2+ signal regulates protein intricate in several stages of autophagosome formation [138]. In addition, a number of Ca2+ dependent pathways involved in autophagy induction have been studied. Indeed, cytosolic Ca2+ initiation of autophagy it is ambiguous in many conditions. The numerous Ca2+ origin has already involved merely various downstream effectors containing protein kinase C, Ca2+/calmodulin-dependent kinase β (CaMKKβ or CaMKK2), ERK, and Vps34 (a calmodulin protein) [139, 140]. It is already proven that CaMKKβ or CaMKK2 has perceived the majority experimental support, whereas Ca2+ refinement of Vps34 and ERK is unsupportable. Activation of Vps34 by Ca2+ or calmodulin is insinuated although the activity of Vps34 in cellulo was not affected by cytosolic Ca2+ or calmodulin antagonist [139]. CaMKKβ is an inrease the activity of AMPK, thereby inhibition of mTORC1 leads to activate autophagy [141]. Høyer-Hansen et al. demonstrated that in MCF-7 breast cancer cells the mobilize of cytosolic Ca2+ from ER by stimulate IP3R generating agonist, such as thasigargin, ionomycin and vitamin D analogue activate CaMKKβ which is initiate autophagy by downregulating of mTORC1 and activation AMPK dependent pathway [142]. In addition, deficient autophagy in T lymphocyte has an extension of ER compartment due to more Ca2+ in the ER. Depletion of Ca2+ in the ER leads to extension of Ca2+ reservoir, which could be the purpose behind unfit to store diminished. This invasion of Ca2+ can be recovered by SERCA/ATPase pump blocking with thapsigargin, which means autophagy can maintain Ca2+ mobilization across the ER [143]. In total, the connection between autophagy and Ca2+ mobilization intimates that they can have impact on each other. Moreover, the elevation of cytosolic Ca2+ endogenously induction of autophagy by precipitation of Ca2+ phosphate without modifying the condition of ER. In consequence, ER Ca2+ plays a key role for induced autophagy by the UPR, while other sources of Ca2+ can induce autophagy but not interaction with the UPR [144, 145].

IP3R receptor is another important cellular pathways which is involved in regulating Ca2+ and induced autophagy. This pathway is mTORC-dependent autophagy and ER stress through upon activation of UPR [146]. IP3R is a second messenger which is known for regulating cell survival signaling although its negative role initiating autophagy is also emerging from several experimental studies that suggest the pharmacological and genetic inhibition of IP3R induction of autophagyindependent Ca2+ flux [147]. The role of ER Ca2+ depletion (SERCA/ATPase antagonist thapsigargin) and luminal ER Ca2+-stimulating compound IP3R antagonist xetospongin B, both of contradictory role, can activate autophagy. Inversely, inhibition of IP3Rs can activate autophagy signal that might be mechanically different from ER stress-attenuated autophagy. Apart from IP3Rs, RYRs have also induced autophagy. In hippocampal neuronal stem cells treated of insulin lead to increase expression of RYR3 isoform which instigate cell death through elevate induction of autophagy [148]. Accordingly, endogenous expression of RYRs in skeletal muscle cells and HEK cells segregates rat hippocampal neurons inhibit the autophagy flux particularly at the autophagosome-lysosome fusion. Inhibition of RYRs increased autophagy flux by mTORC independent pathway [149].

Under ER stress condition, Ca2+-mediated autophagy is induced by known tumor inhibitor DAPK1. Activated DAPK1 mediated direct phosphorylation on BH3 domain of Beclin-1 elevated from Bcl2L1, which promotes autophagy [113]. Accordingly, under hypoxic condition, decrease synthesis of protein through PERKeIF2α-ATF4 and AMPK-mTORC1 pathway. Similarly, autophagy can be induced upon hypoxic condition, whereas Ca2+ influx by initiation of hypoxia and triggered CaMKKβ or CaMKK2 promotes WIPI1 and autophagosome formation [150, 151].

Many evidences suggest that cytosolic Ca2+ can initiate autophagy although many reports demonstrate that chelating Ca2+ suppresses autophagy. BAPTA-AM (1,2-bis (O-aminophenoxy) ethane-N, N, N′, N′-tetraacetic acid tetra (acetoxymethyl) ester), a cell permeable Ca2+ buffering agent, can also suppress autophagy initiation following ER stress induced by inhibition of proteasome [152]. In many studies, stimulation of exogenous cytosolic Ca2+ signal and the BAPTA-AM effect on autophagy can be rational inhibition activate the influx of Ca2+. In addition, BAPTA-AM effect on cell did not alter the production of IP3Rs by Vps34 but mutated the aggregation of the IP3Rs protein receptor WIPI-1 to the formation of phagophore. Likewise, BAPTA-AM was observed to suppress lysosome fusion [153]. Furthermore, BAPTA-AM inhibits initiating autophagy by experimentally increasing influx Ca2+ signal but blocks formation of autophagosome. In the meantime, autophagy inhibition by BAPTA-AM continuously remarks that there are some consequences using Ca2+ chelating agents which also defect lysosomal function followed by inhibiting degradation of autophagosome [154]. In addition, hydrolysis of the acetoxymethyl ester modification of Ca2+-dependent intracellular signaling process directly involved autophagy [154]. Nevertheless, BAPTA is Ca2+ chelator and limitaion is when Ca2+ enters the cell and it can be replete by the influx of Ca2+. In a similar way, mobilization of intracellular Ca2+ led to defects in plasma membrane, resulting in the expanded interplay between lysosome and SNAREs, which are more important for membrane fusion, and thereby increase of Ca2+ could alleviate autophagosome-lysosome fusion, which induces autophagy [155].

Alternatively, many compounds that inhibit Ca2+ signaling led to an ascent of cytosolic Ca2+ that blocks initiation of autophagy. Particularly, voltage-operated Ca2+ channel antagonist and the IP3R signal can induce autophagy by suppressing activity of Ca2+-sensitive protease called as calpain [156]. Calpain is activated by elevation of cytosolic Ca2+. Inhibition of calpain by pharmacological calpestatin

**61**

*Endoplasmic Reticulum Stress and Autophagy DOI: http://dx.doi.org/10.5772/intechopen.81381*

cytosolic Ca2+ signal activated by mTORC1 [157].

**11. ER stress mediates autophagy in pathological condition**

The UPR pathway is not always a reason for autophagy induction. When ER stress is divergent in some contagious situation, defective regulation of autophagy occurs. Notably, in some pathological conditions such as neurodegenerative, cardiovascular, and liver diseases, ER stress negatively regulates autophagy. Alzheimer disease (AD) is one of the most common neurodegenerative diseases, which is mainly caused by the accumulation of extracellular amyloid-β (Aβ), senile plaques, and neurofibrillary tangles protein. Aβ is originating from the cleavage of the amyloid precursor protein (APP) by two aspartic enzymes β-secretase (BACE1) and γ-secretase. This γ-secretase is a membrane-associated complex consisting of a presenilin-1/2 (PS1/PS2) in the ER [158]. UPR and autophagy play a key role in maintaining normal neuron against aggregation of Aβ and PS1 mutation that affect the form of AD. Many reports suggest that mutation in PS1 and accumulation of intracellular Aβ activate ER stress in neurons [159]. However, mutation of Aβ leads to upregulation of the HSPA5 (heat shock 70 kDa protein 5) expression in the neuron, which is the main neuroprotective role despite the ER stress-associated cell death and sustaining Ca2+ stability [160]. Interestingly, mutation of ps1 and Aβ suppresses the main arms of UPR, including IRE1α, PERK, and ATF6α [161]. Activation of ER stress is an early sequence of the AD, which initiates autophagy by phosphorylation of PERK-positive neuron via accumulation of MAP1LC3B induced autophagy in cardinal direction for abasement of Aβ and APP [162]. Defective regulation of autophagic function leads to AD progression; Pickford et al. report that downregulation of Beclin-1 was observed in the middle frontal lobe in the brain cortex of AD patients similar to the observation in the mouse model of AD [163]. Similarly, in Parkinson disease model, synaptic protein α-synuclein (α-syn) decreases accumulation of the expression of Beclin-1 gene that suppresses the induction of autophagy [164]. In addition, Huntington's diseases (HD) is also neuropathological disease condition, whereas ER stress impaired the regulation of autophagy. Knockdown of IRE1α-XBP1 increases autophagy in HD model which initiates pathological condition [165, 166]. Similarly, in HD-upregulated expression, USP14 is the deubiquitinating enzyme with His and Cys domains that increase autophagic discharge of mutant HTT protein (huntingtin protein) through nonphosphorylated IRE1α. Phosphorylated IRE1α has not much affinity to interact with USP14, thus increasing accumulation of mutant HTT by suppressing autophagy regulation [167]. Therefore, activation of UPR will not be regulated properly as a result of negative induction of autophagy, which fails to eradicate the accumulation of contagious protein and then consequently leads to neurodegenerative diseases. UPR and autophagy are also interconnected for inflammation of bowel in the epithelial cell. In cultured intestinal epithelial cell initiate PERK-eIF2α dependent

and calpeptin or knockdown of calpain enhances autophagy flux without turbulence mTORC1 [156]. In addition, in neuronal disease cells, abnormal Ca2+ signal obstructs the clearance accumulation of nascent protein through inhibition of autophagy induction. Nonetheless, these studies demonstrate that calpain can suppress autophagy induction although other experimental studies suggest that the activation of calpain is essential for autophagy induction [156]. Cytosolic Ca2+ can activate mTORC1, which led to inhibition of autophagy induction. For instance, knockdown of TRPML1 (transient receptor potential cation channel, mucolipin subfamily, member 1) lysosomal Ca2+ channel inhibits mTORC1 activity. However, knockdown of TRPML1 channel reversed by thapsigargin, lead to downstream

*Endoplasmic Reticulum Stress and Autophagy DOI: http://dx.doi.org/10.5772/intechopen.81381*

*Endoplasmic Reticulum*

interaction with the UPR [144, 145].

autophagy by the UPR, while other sources of Ca2+ can induce autophagy but not

the pharmacological and genetic inhibition of IP3R induction of autophagyindependent Ca2+ flux [147]. The role of ER Ca2+ depletion (SERCA/ATPase antagonist thapsigargin) and luminal ER Ca2+-stimulating compound IP3R antagonist xetospongin B, both of contradictory role, can activate autophagy. Inversely, inhibition of IP3Rs can activate autophagy signal that might be mechanically different from ER stress-attenuated autophagy. Apart from IP3Rs, RYRs have also induced autophagy. In hippocampal neuronal stem cells treated of insulin lead to increase expression of RYR3 isoform which instigate cell death through elevate induction of autophagy [148]. Accordingly, endogenous expression of RYRs in skeletal muscle cells and HEK cells segregates rat hippocampal neurons inhibit the autophagy flux particularly at the autophagosome-lysosome fusion. Inhibition of RYRs increased

Under ER stress condition, Ca2+-mediated autophagy is induced by known tumor inhibitor DAPK1. Activated DAPK1 mediated direct phosphorylation on BH3 domain of Beclin-1 elevated from Bcl2L1, which promotes autophagy [113]. Accordingly, under hypoxic condition, decrease synthesis of protein through PERKeIF2α-ATF4 and AMPK-mTORC1 pathway. Similarly, autophagy can be induced upon hypoxic condition, whereas Ca2+ influx by initiation of hypoxia and triggered CaMKKβ or CaMKK2 promotes WIPI1 and autophagosome formation [150, 151]. Many evidences suggest that cytosolic Ca2+ can initiate autophagy although many reports demonstrate that chelating Ca2+ suppresses autophagy. BAPTA-AM (1,2-bis (O-aminophenoxy) ethane-N, N, N′, N′-tetraacetic acid tetra (acetoxymethyl) ester), a cell permeable Ca2+ buffering agent, can also suppress autophagy initiation following ER stress induced by inhibition of proteasome [152]. In many studies, stimulation of exogenous cytosolic Ca2+ signal and the BAPTA-AM effect on autophagy can be rational inhibition activate the influx of Ca2+. In addition, BAPTA-AM effect on cell did not alter the production of IP3Rs by Vps34 but mutated the aggregation of the IP3Rs protein receptor WIPI-1 to the formation of phagophore. Likewise, BAPTA-AM was observed to suppress lysosome fusion [153]. Furthermore, BAPTA-AM inhibits initiating autophagy by experimentally increasing influx Ca2+ signal but blocks formation of autophagosome. In the meantime, autophagy inhibition by BAPTA-AM continuously remarks that there are some consequences using Ca2+ chelating agents which also defect lysosomal function followed by inhibiting degradation of autophagosome [154]. In addition, hydrolysis of the acetoxymethyl ester modification of Ca2+-dependent intracellular signaling process directly involved autophagy [154]. Nevertheless, BAPTA is Ca2+ chelator and limitaion is when Ca2+ enters the cell and it can be replete by the influx of Ca2+. In a similar way, mobilization of intracellular Ca2+ led to defects in plasma membrane, resulting in the expanded interplay between lysosome and SNAREs, which are more important for membrane fusion, and thereby increase of Ca2+ could

alleviate autophagosome-lysosome fusion, which induces autophagy [155].

Alternatively, many compounds that inhibit Ca2+ signaling led to an ascent of cytosolic Ca2+ that blocks initiation of autophagy. Particularly, voltage-operated Ca2+ channel antagonist and the IP3R signal can induce autophagy by suppressing activity of Ca2+-sensitive protease called as calpain [156]. Calpain is activated by elevation of cytosolic Ca2+. Inhibition of calpain by pharmacological calpestatin

autophagy flux by mTORC independent pathway [149].

IP3R receptor is another important cellular pathways which is involved in regulating Ca2+ and induced autophagy. This pathway is mTORC-dependent autophagy and ER stress through upon activation of UPR [146]. IP3R is a second messenger which is known for regulating cell survival signaling although its negative role initiating autophagy is also emerging from several experimental studies that suggest

**60**

and calpeptin or knockdown of calpain enhances autophagy flux without turbulence mTORC1 [156]. In addition, in neuronal disease cells, abnormal Ca2+ signal obstructs the clearance accumulation of nascent protein through inhibition of autophagy induction. Nonetheless, these studies demonstrate that calpain can suppress autophagy induction although other experimental studies suggest that the activation of calpain is essential for autophagy induction [156]. Cytosolic Ca2+ can activate mTORC1, which led to inhibition of autophagy induction. For instance, knockdown of TRPML1 (transient receptor potential cation channel, mucolipin subfamily, member 1) lysosomal Ca2+ channel inhibits mTORC1 activity. However, knockdown of TRPML1 channel reversed by thapsigargin, lead to downstream cytosolic Ca2+ signal activated by mTORC1 [157].

#### **11. ER stress mediates autophagy in pathological condition**

The UPR pathway is not always a reason for autophagy induction. When ER stress is divergent in some contagious situation, defective regulation of autophagy occurs. Notably, in some pathological conditions such as neurodegenerative, cardiovascular, and liver diseases, ER stress negatively regulates autophagy. Alzheimer disease (AD) is one of the most common neurodegenerative diseases, which is mainly caused by the accumulation of extracellular amyloid-β (Aβ), senile plaques, and neurofibrillary tangles protein. Aβ is originating from the cleavage of the amyloid precursor protein (APP) by two aspartic enzymes β-secretase (BACE1) and γ-secretase. This γ-secretase is a membrane-associated complex consisting of a presenilin-1/2 (PS1/PS2) in the ER [158]. UPR and autophagy play a key role in maintaining normal neuron against aggregation of Aβ and PS1 mutation that affect the form of AD. Many reports suggest that mutation in PS1 and accumulation of intracellular Aβ activate ER stress in neurons [159]. However, mutation of Aβ leads to upregulation of the HSPA5 (heat shock 70 kDa protein 5) expression in the neuron, which is the main neuroprotective role despite the ER stress-associated cell death and sustaining Ca2+ stability [160]. Interestingly, mutation of ps1 and Aβ suppresses the main arms of UPR, including IRE1α, PERK, and ATF6α [161]. Activation of ER stress is an early sequence of the AD, which initiates autophagy by phosphorylation of PERK-positive neuron via accumulation of MAP1LC3B induced autophagy in cardinal direction for abasement of Aβ and APP [162]. Defective regulation of autophagic function leads to AD progression; Pickford et al. report that downregulation of Beclin-1 was observed in the middle frontal lobe in the brain cortex of AD patients similar to the observation in the mouse model of AD [163]. Similarly, in Parkinson disease model, synaptic protein α-synuclein (α-syn) decreases accumulation of the expression of Beclin-1 gene that suppresses the induction of autophagy [164]. In addition, Huntington's diseases (HD) is also neuropathological disease condition, whereas ER stress impaired the regulation of autophagy. Knockdown of IRE1α-XBP1 increases autophagy in HD model which initiates pathological condition [165, 166]. Similarly, in HD-upregulated expression, USP14 is the deubiquitinating enzyme with His and Cys domains that increase autophagic discharge of mutant HTT protein (huntingtin protein) through nonphosphorylated IRE1α. Phosphorylated IRE1α has not much affinity to interact with USP14, thus increasing accumulation of mutant HTT by suppressing autophagy regulation [167]. Therefore, activation of UPR will not be regulated properly as a result of negative induction of autophagy, which fails to eradicate the accumulation of contagious protein and then consequently leads to neurodegenerative diseases.

UPR and autophagy are also interconnected for inflammation of bowel in the epithelial cell. In cultured intestinal epithelial cell initiate PERK-eIF2α dependent pathway autophagy because of loss IRE1α activity which intimate that UPR signal maintaining normal mechanism also conserve balance need to possible rebuttal mechanism [168]. In addition, XBP1 conditional knock in intestinal epithelial cell lead to induced autophagy in small intestinal paneth cell, essential for the formation of antimicrobial agents followed by inflammation in small intestine, which is more exacerbated when codeletion of ATG gene like ATG7 or ATG16L1. Double knockout mice XBP1<sup>−</sup>/<sup>−</sup>, ATG7<sup>−</sup>/<sup>−</sup> and XBP1<sup>−</sup>/<sup>−</sup>, ATG16L<sup>−</sup>/<sup>−</sup> demonstrate that Crohn diseases stimulate nuclear factor kappa B (NF-kB) in IRE1α-dependent manner. Moreover, In ATG16L conditional knockout mice enhance GRP78 expression along with phosphorylation of eIF2a and activation of JNK, terminating the expression of IRE1a and increased the XBP1 spicing in intestinal glands, these circumstances increase the inflammation state, which changes the interaction between ER stress and autophagy that increases cell death, which is negative retroaction of ER stressinduced autophagy [168]. Notably, inactivation of XBP1 can induce autophagy but this UPR also can downregulate the induction of autophagy. Nevertheless, defective regulation of XBP1 integrates FoxO1 (Forkhead box O1), a transcription factor that sequentially provokes expression of many genes that positively induce autophagy [98]. The unspliced XBP1 (uXBP1) under glutamine starvation condition regulated FoxO1 depravation by interacting FoxO1 for the 20s proteasome. Similarly, this interaction between uXBP1 and FoxO1 based on phosphorylation of uXBP1 by the extracellular signal-regulated kinase 1/2 (ERK1/2), as well as spliced XBP1 (XBP1s) in overexpression which also interacted, evolved degradation of FoxO1 [169]. Accordingly, recently, the FoxO1 and XBP1 interaction in auditory cells regulates autophagy [170]. Prominently, the consistent mechanism has been proved under severe ER stress in which the UPR loses its activity, whereas it can be considered that another regulatory mechanism FoxO1 maintains the autophagy induction.

#### **12. Conclusion**

During the last decade, research has been conducted to determine the mechanism by which ER stress and autophagy maintain intracellular homeostasis. Here, we described the UPR and autophagy in detail with respect to their molecular mechanism and interaction between ER stress and autophagy. However, the detailed mechanism of ER stress and autophagy is yet to be fully understood. In the last few years, research has shown that the ER stress response can not only initiate autophagy but can also negatively regulate autophagy to maintain cell survival. Elucidation of the interactions between the UPR and autophagy will help in the development of novel treatments for several diseases.

#### **Acknowledgements**

The study was supported by Korean National Research Foundation (2017R1E1A1A01073796 and 2017M3A9G707219). We acknowledge Mr. Raghu Patil Junjappa and Mr. Ziaur Rahman (Department of Pharmacology, Medical School, Chonbuk National University) for their contribution in preparing the first draft.

**63**

**Author details**

Daegu, South Korea

Mohammad Fazlul Kabir1

provided the original work is properly cited.

\*Address all correspondence to: hjchae@jbnu.ac.kr

*Endoplasmic Reticulum Stress and Autophagy DOI: http://dx.doi.org/10.5772/intechopen.81381*

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

, Hyung-Ryong Kim2

Development, Chonbuk National University, Jeonju, South Korea

1 Department of Pharmacology, School of Medicine, Institute of New Drug

2 Graduate School, Daegu Gyeongbuk Institute of Science and Technology,

and Han-Jung Chae1

\*

#### **Conflict of interest**

The authors declare that there is no conflict of interest.

*Endoplasmic Reticulum Stress and Autophagy DOI: http://dx.doi.org/10.5772/intechopen.81381*

*Endoplasmic Reticulum*

pathway autophagy because of loss IRE1α activity which intimate that UPR signal maintaining normal mechanism also conserve balance need to possible rebuttal mechanism [168]. In addition, XBP1 conditional knock in intestinal epithelial cell lead to induced autophagy in small intestinal paneth cell, essential for the formation of antimicrobial agents followed by inflammation in small intestine, which is more exacerbated when codeletion of ATG gene like ATG7 or ATG16L1. Double knockout mice XBP1<sup>−</sup>/<sup>−</sup>, ATG7<sup>−</sup>/<sup>−</sup> and XBP1<sup>−</sup>/<sup>−</sup>, ATG16L<sup>−</sup>/<sup>−</sup> demonstrate that Crohn diseases stimulate nuclear factor kappa B (NF-kB) in IRE1α-dependent manner. Moreover, In ATG16L conditional knockout mice enhance GRP78 expression along with phosphorylation of eIF2a and activation of JNK, terminating the expression of IRE1a and increased the XBP1 spicing in intestinal glands, these circumstances increase the inflammation state, which changes the interaction between ER stress and autophagy that increases cell death, which is negative retroaction of ER stressinduced autophagy [168]. Notably, inactivation of XBP1 can induce autophagy but this UPR also can downregulate the induction of autophagy. Nevertheless, defective regulation of XBP1 integrates FoxO1 (Forkhead box O1), a transcription factor that sequentially provokes expression of many genes that positively induce autophagy [98]. The unspliced XBP1 (uXBP1) under glutamine starvation condition regulated FoxO1 depravation by interacting FoxO1 for the 20s proteasome. Similarly, this interaction between uXBP1 and FoxO1 based on phosphorylation of uXBP1 by the extracellular signal-regulated kinase 1/2 (ERK1/2), as well as spliced XBP1 (XBP1s) in overexpression which also interacted, evolved degradation of FoxO1 [169]. Accordingly, recently, the FoxO1 and XBP1 interaction in auditory cells regulates autophagy [170]. Prominently, the consistent mechanism has been proved under severe ER stress in which the UPR loses its activity, whereas it can be considered that another regulatory mechanism FoxO1 maintains the autophagy

During the last decade, research has been conducted to determine the mechanism by which ER stress and autophagy maintain intracellular homeostasis. Here, we described the UPR and autophagy in detail with respect to their molecular mechanism and interaction between ER stress and autophagy. However, the detailed mechanism of ER stress and autophagy is yet to be fully understood. In the last few years, research has shown that the ER stress response can not only initiate autophagy but can also negatively regulate autophagy to maintain cell survival. Elucidation of the interactions between the UPR and autophagy will help in the

The study was supported by Korean National Research Foundation

The authors declare that there is no conflict of interest.

(2017R1E1A1A01073796 and 2017M3A9G707219). We acknowledge Mr. Raghu Patil Junjappa and Mr. Ziaur Rahman (Department of Pharmacology, Medical School, Chonbuk National University) for their contribution in preparing the first draft.

development of novel treatments for several diseases.

**62**

induction.

**12. Conclusion**

**Acknowledgements**

**Conflict of interest**

### **Author details**

Mohammad Fazlul Kabir1 , Hyung-Ryong Kim2 and Han-Jung Chae1 \*

1 Department of Pharmacology, School of Medicine, Institute of New Drug Development, Chonbuk National University, Jeonju, South Korea

2 Graduate School, Daegu Gyeongbuk Institute of Science and Technology, Daegu, South Korea

\*Address all correspondence to: hjchae@jbnu.ac.kr

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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**73**

follicular atresia.

**Chapter 5**

Atresia

**Abstract**

paraptosis, necroptosis

**1. Introduction**

*and Gerardo H. Vázquez-Nin*

Endoplasmic Reticulum Stress

during Mammalian Follicular

*Nayeli Torres-Ramírez, Rosario Ortiz-Hernández,* 

*M. Luisa Escobar-Sánchez, Olga M. Echeverría-Martínez* 

Follicles are ovarian structures that contain a single germ cell. During the mammalian reproductive lifetime, ovarian follicles mature through the process of follicular development, with the aim of selecting oocytes for ovulation. As part of this process, several follicles are eliminated by means of follicular atresia, a mechanism that mainly involves apoptosis. Nevertheless, it has been shown that there are other routes of programmed cell death in the ovary including autophagy, paraptosis, and necroptosis. Surprisingly, the endoplasmic reticulum is involved in these different programmed cell death pathways. Moreover, there are several evidences for the pathways triggered by intra- and extracellular signals in endoplasmic reticulum-induced cell death. Thus, it is important to analyze the participation of endoplasmic reticulum in follicular atresia.

**Keywords:** ovary, follicular atresia, endoplasmic reticulum, apoptosis, autophagy,

The endoplasmic reticulum plays several important roles in normal cellular physiology. Some functions include protein synthesis, folding, and distribution to the Golgi apparatus. Alterations in protein synthesis inside the endoplasmic reticulum have been related to the trigger of different programmed cell death routes such as necroptosis, apoptosis, autophagy, and paraptosis, with apoptosis being the most studied process. The mammalian ovary is an excellent model to study the mechanisms of programmed cell death because 99% of the follicles, the functional units of the ovary, undergo degeneration through follicular atresia, which maintains intraovarian homeostasis. Follicular atresia involves the physiological elimination of most germinal cells (oocytes) before they are ovulated, both in fetal and reproductive lives. The presence of different programmed cell death pathways in follicular atresia have recently been shown, and these can be directly related to endoplasmic reticulum signaling. In this chapter we describe evidences of the linkage between endoplasmic reticulum alterations and programmed cell death, with special emphasis on

#### **Chapter 5**

*Endoplasmic Reticulum*

2013;**2013**(2):122-125

eLife. 2016;**5**:1-13

2004;**23**(3):483-488

2013;**12**(2):292-302

endosome-lysosome heterotypic fusion and in the reformation of lysosomes from hybrid organelles. The Journal of Cell Biology. 2000;**149**(5):1053-1062

disease. Cell Death and Differentiation.

[163] Pickford F et al. The autophagyrelated protein beclin 1 shows reduced

expression in early Alzheimer disease and regulates amyloid beta accumulation in mice. The Journal of Clinical Investigation.

[164] Spencer B et al. Beclin 1 gene transfer activates autophagy and ameliorates the neurodegenerative pathology in alpha-synuclein models of Parkinson's and Lewy body

diseases. The Journal of Neuroscience.

[165] Lee H et al. IRE1 plays an essential role in ER stress-mediated aggregation of mutant huntingtin via the inhibition of autophagy flux. Human Molecular

2011;**18**(6):1071-1081

2011;**18**(6):1071-1081

2009;**29**(43):13578-13588

Genetics. 2012;**21**(1):101-114

2014;**23**(22):5928-5939

2013;**21**(1):9-13

[166] Hyrskyluoto A et al. Ubiquitinspecific protease-14 reduces cellular aggregates and protects against mutant huntingtin-induced cell degeneration: Involvement of the proteasome and ER stress-activated kinase

IRE1alpha. Human Molecular Genetics.

[167] Jarome TJ et al. The ubiquitinspecific protease 14 (USP14) is a critical regulator of long-term memory formation. Learning & Memory.

[168] Adolph TE et al. Paneth cells as a site of origin for

autophagy by promoting the

2013;**503**(7475):272-276

Reports. 2017;**7**(1):4442

intestinal inflammation. Nature.

[169] Zhao Y et al. XBP-1u suppresses

degradation of FoxO1 in cancer cells. Cell Research. 2013;**23**(4):491-507

[170] Kishino A et al. XBP1-FoxO1 interaction regulates ER stress-induced autophagy in auditory cells. Scientific

[154] Bootman MD et al. Loading fluorescent Ca2+ indicators into living cells. Cold Spring Harbor Protocols.

target. Journal of Molecular Cell Biology. 2013;**5**(4):214-226

[156] Williams A et al. Novel targets for Huntington's disease in an mTORindependent autophagy pathway. Nature Chemical Biology. 2008;**4**(5):295-305

[157] Li RJ et al. Regulation of mTORC1 by lysosomal calcium and calmodulin.

[158] Haass C. Take five--BACE and the gamma-secretase quartet conduct Alzheimer's amyloid beta-peptide generation. The EMBO Journal.

[159] Alberdi E et al. Ca(2+)-dependent endoplasmic reticulum stress correlates with astrogliosis in oligomeric amyloid beta-treated astrocytes and in a model of Alzheimer's disease. Aging Cell.

[160] Yu Z et al. The endoplasmic reticulum stress-responsive protein GRP78 protects neurons against

[161] Katayama T et al. Induction of neuronal death by ER stress in

[162] Nijholt DA et al. Endoplasmic reticulum stress activates autophagy but not the proteasome in neuronal cells: Implications for Alzheimer's

excitotoxicity and apoptosis: Suppression of oxidative stress and stabilization of calcium homeostasis. Experimental Neurology. 1999;**155**(2):302-314

Alzheimer's disease. Journal of Chemical Neuroanatomy. 2004;**28**(1-2):67-78

[155] Appelqvist H et al. The lysosome: From waste bag to potential therapeutic

**72**

## Endoplasmic Reticulum Stress during Mammalian Follicular Atresia

*Nayeli Torres-Ramírez, Rosario Ortiz-Hernández, M. Luisa Escobar-Sánchez, Olga M. Echeverría-Martínez and Gerardo H. Vázquez-Nin*

#### **Abstract**

Follicles are ovarian structures that contain a single germ cell. During the mammalian reproductive lifetime, ovarian follicles mature through the process of follicular development, with the aim of selecting oocytes for ovulation. As part of this process, several follicles are eliminated by means of follicular atresia, a mechanism that mainly involves apoptosis. Nevertheless, it has been shown that there are other routes of programmed cell death in the ovary including autophagy, paraptosis, and necroptosis. Surprisingly, the endoplasmic reticulum is involved in these different programmed cell death pathways. Moreover, there are several evidences for the pathways triggered by intra- and extracellular signals in endoplasmic reticulum-induced cell death. Thus, it is important to analyze the participation of endoplasmic reticulum in follicular atresia.

**Keywords:** ovary, follicular atresia, endoplasmic reticulum, apoptosis, autophagy, paraptosis, necroptosis

#### **1. Introduction**

The endoplasmic reticulum plays several important roles in normal cellular physiology. Some functions include protein synthesis, folding, and distribution to the Golgi apparatus. Alterations in protein synthesis inside the endoplasmic reticulum have been related to the trigger of different programmed cell death routes such as necroptosis, apoptosis, autophagy, and paraptosis, with apoptosis being the most studied process.

The mammalian ovary is an excellent model to study the mechanisms of programmed cell death because 99% of the follicles, the functional units of the ovary, undergo degeneration through follicular atresia, which maintains intraovarian homeostasis. Follicular atresia involves the physiological elimination of most germinal cells (oocytes) before they are ovulated, both in fetal and reproductive lives.

The presence of different programmed cell death pathways in follicular atresia have recently been shown, and these can be directly related to endoplasmic reticulum signaling. In this chapter we describe evidences of the linkage between endoplasmic reticulum alterations and programmed cell death, with special emphasis on follicular atresia.

### **2. Follicular development and atresia**

The mammalian ovary is a paired organ that is responsible for generating competent oocytes for successful fertilization and early embryonic development. To do this, these germinal cells need to mature within transient functional complexes called follicles. Follicles form for an oocyte surrounded by somatic cells. During reproductive life, follicles are continuously recruited into the pool of growing follicles and change their size, morphology, and physiology, leading to different stage classifications including primordial, primary, secondary, and antral (**Figure 1**).

At birth, the ovaries contain a fixed number of nongrowing primordial follicles, characterized by an oocyte enclosed by flattened pre-granulosa cells. In primary follicles, the oocyte is surrounded by a monolayer of cubical granulosa cells. Secondary follicles are formed by two or more layers of granulosa cells. Antral follicles accumulate fluid and develop an antral cavity. The accumulation of fluid is useful for transporting nutrients and waste products.

Follicular growth is a continuous process that is under strict control by hormones, growth factors, cytokines, and environmental factors. Folliclestimulating hormone (FSH), luteinizing hormone (LH), insulin-like growth factor (IGF)-I, and estradiol are the principal regulators of follicular growth. FSH, a gonadotropin secreted by the pituitary gland, together with estradiol and IGF-I, is responsible for stimulating follicular growth and maturation. Moreover, FSH, LH, and estradiol enhance IGF-I secretion [1]. Additionally, FSH stimulates granulosa cells to develop LH receptor sites. The main function of LH is stimulating ovulation.

Several follicles grow and undergo ovulation, releasing an oocyte that is available for fertilization, but the principal destiny of ovarian follicles is follicular atresia, which is a physiological process that eliminates more than 99% of the follicles. Follicular atresia can occur in all stages of follicular development and ensures that only healthy follicles that contain optimal quality oocytes will be ovulated. Follicular degeneration occurs by programmed cell death (PCD). Apoptosis is the main route of follicular atresia, but may not be the only process involved (**Figure 2**). Other forms of PCD such as autophagy and paraptosis may also participate in this process [2–4].

#### **Figure 1.**

*Ovary of mouse. Follicles are in different stages of growth. Primordial (P), primary (head arrow), secondary (asterisk), and antral (A) follicles.*

**75**

**Figure 2.**

*Endoplasmic Reticulum Stress during Mammalian Follicular Atresia*

**3. The endoplasmic reticulum and cell death**

*(asterisk). Bars (a–c) 500 nm, and (d) 2 μm.*

The endoplasmic reticulum (ER) is the organelle that is responsible for the folding and maturation of both transmembrane proteins and proteins that follow the route of secretion. Protein folding is facilitated by chaperones and oxidoreductases including binding immunoglobulin protein/glucose-regulated protein 78-kDa (BiP/GRP78), calnexin, calreticulin, and protein disulfide isomerase (PDI). An increase of cellular translational activity is possible under both normal and altered conditions, causing an overload of accumulating misfolding or unfolded proteins inside the ER. During ER stress, damaged proteins need to be degraded, but there is a limited number of proteases in the ER, and thus misfolded proteins are ejected from the ER and returned to the cytoplasm to be ubiquitinated and degraded by the 26S proteasome. These events are collectively referred to as ER-associated degradation (ERAD) [5]. Also, ER stress triggers the unfolded protein response (UPR), which is orchestrated by three ER-resident UPR sensors, inositol-requiring kinase 1 (IRE1), protein kinase R-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6) [6, 7].

*Transmission electron microscope images of granulosa cells in different programmed cell death pathways. (a) healthy granulosa cell, (b) apoptotic body with highly condensed chromatin (cc), (c) autophagic cell with autophagic vesicles (head arrow), and (d) paraptotic granulosa cell with endoplasmic reticulum swelling* 

The UPR establishes an adaptive program aimed at re-establishing ER homeostasis by increasing the folding capacity of the cell, reducing protein synthesis, and enhancing the clearance of abnormally folded proteins and damaged organelles.

*DOI: http://dx.doi.org/10.5772/intechopen.82687*

*Endoplasmic Reticulum Stress during Mammalian Follicular Atresia DOI: http://dx.doi.org/10.5772/intechopen.82687*

#### **Figure 2.**

*Endoplasmic Reticulum*

antral (**Figure 1**).

ing ovulation.

process [2–4].

**2. Follicular development and atresia**

useful for transporting nutrients and waste products.

The mammalian ovary is a paired organ that is responsible for generating competent oocytes for successful fertilization and early embryonic development. To do this, these germinal cells need to mature within transient functional complexes called follicles. Follicles form for an oocyte surrounded by somatic cells. During reproductive life, follicles are continuously recruited into the pool of growing follicles and change their size, morphology, and physiology, leading to different stage classifications including primordial, primary, secondary, and

At birth, the ovaries contain a fixed number of nongrowing primordial follicles, characterized by an oocyte enclosed by flattened pre-granulosa cells. In primary follicles, the oocyte is surrounded by a monolayer of cubical granulosa cells. Secondary follicles are formed by two or more layers of granulosa cells. Antral follicles accumulate fluid and develop an antral cavity. The accumulation of fluid is

Follicular growth is a continuous process that is under strict control by hormones, growth factors, cytokines, and environmental factors. Folliclestimulating hormone (FSH), luteinizing hormone (LH), insulin-like growth factor (IGF)-I, and estradiol are the principal regulators of follicular growth. FSH, a gonadotropin secreted by the pituitary gland, together with estradiol and IGF-I, is responsible for stimulating follicular growth and maturation. Moreover, FSH, LH, and estradiol enhance IGF-I secretion [1]. Additionally, FSH stimulates granulosa cells to develop LH receptor sites. The main function of LH is stimulat-

Several follicles grow and undergo ovulation, releasing an oocyte that is available

for fertilization, but the principal destiny of ovarian follicles is follicular atresia, which is a physiological process that eliminates more than 99% of the follicles. Follicular atresia can occur in all stages of follicular development and ensures that only healthy follicles that contain optimal quality oocytes will be ovulated. Follicular degeneration occurs by programmed cell death (PCD). Apoptosis is the main route of follicular atresia, but may not be the only process involved (**Figure 2**). Other forms of PCD such as autophagy and paraptosis may also participate in this

*Ovary of mouse. Follicles are in different stages of growth. Primordial (P), primary (head arrow), secondary* 

**74**

**Figure 1.**

*(asterisk), and antral (A) follicles.*

*Transmission electron microscope images of granulosa cells in different programmed cell death pathways. (a) healthy granulosa cell, (b) apoptotic body with highly condensed chromatin (cc), (c) autophagic cell with autophagic vesicles (head arrow), and (d) paraptotic granulosa cell with endoplasmic reticulum swelling (asterisk). Bars (a–c) 500 nm, and (d) 2 μm.*

#### **3. The endoplasmic reticulum and cell death**

The endoplasmic reticulum (ER) is the organelle that is responsible for the folding and maturation of both transmembrane proteins and proteins that follow the route of secretion. Protein folding is facilitated by chaperones and oxidoreductases including binding immunoglobulin protein/glucose-regulated protein 78-kDa (BiP/GRP78), calnexin, calreticulin, and protein disulfide isomerase (PDI). An increase of cellular translational activity is possible under both normal and altered conditions, causing an overload of accumulating misfolding or unfolded proteins inside the ER. During ER stress, damaged proteins need to be degraded, but there is a limited number of proteases in the ER, and thus misfolded proteins are ejected from the ER and returned to the cytoplasm to be ubiquitinated and degraded by the 26S proteasome. These events are collectively referred to as ER-associated degradation (ERAD) [5]. Also, ER stress triggers the unfolded protein response (UPR), which is orchestrated by three ER-resident UPR sensors, inositol-requiring kinase 1 (IRE1), protein kinase R-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6) [6, 7].

The UPR establishes an adaptive program aimed at re-establishing ER homeostasis by increasing the folding capacity of the cell, reducing protein synthesis, and enhancing the clearance of abnormally folded proteins and damaged organelles.

The proteins PERK and IRE1α and β are important players during UPR because they undergo oligomerization and autophosphorylation due to their interactions with peptides and unfolded proteins [8, 9]. Additionally, IRE1 promotes the unconventional splicing of X-box binding protein 1 (XBP-1) mRNA and an unspecific decrease of mRNAs better known as regulated IRE1-dependent decay (RIDD) [10, 11]. Afterward, the protein XBP-1 is translocated to the nucleus to activate the transcription of chaperones and ERAD factors [12]. RIDD suppresses protein inflow by degrading the mRNA of proteins with signal peptides or proteins with transmembrane domains, and in this manner RIDD permits proteins that are incorrectly folded inside the ER to be folded correctly [10]. PERK phosphorylates eukaryotic translation initiation factor (eIF2a), which then accumulates on the cytosolic side and leads to the downregulation of translation and enhances the translation of Grp78 and the transcription factor ATF4 [13, 14]. It has been shown that during early mouse embryonic development, Grp78 suppresses ER stress and pro-apoptotic pathways via ER signaling [15]. ATF6 is regulated by proteolysis in the Golgi apparatus, allowing the N-terminal fragments to be translocated into the nucleus where they function as a transcription factor [16, 17]. The processing of both ATF6- and IRE1α-mediated splicing of XBP1 mRNA is required for the full activation of the UPR [18].

UPR works like a protection mechanism. For example, in pancreatic beta cell line INS-1E, glucosamine and high glucose induce UPR activation and generate a feedback loop at the level of insulin transcription [19]. However, chronic or irreversible UPR can trigger cell death pathways, mainly apoptosis, but ER stress can induce other programmed cell death mechanisms including autophagy, necroptosis, and paraptosis.

#### **3.1 The ER and follicular atresia**

Morphological ER disturbances during follicular atresia have been observed for a long time. Henderson et al. [20] observed a higher surface area of endoplasmic reticulum in granulosa cells cultured from atretic follicles. Moreover, researchers have used electron microscopy to observe the dilation and disintegration of RER cisterns and the swelling of mitochondria [21].

These morphological disturbances in ovaries are associated with ER stress and UPR activation under both physiological and pathological conditions [22]. UPR is present during follicular growth and maturation and follicular atresia and in the corpus luteum. ER stress during follicular growth and maturation has been evidenced by means of the expression of XPB1 and heat shock 70 kDa protein 5 (HSPA5) accompanied by the activation of IRE1 and PERK [23]. The ER stress level and cellular response depend on the signal and its intensity. It has been shown that a lipid-rich intrafollicular environment induces ER stress and impaired oocyte nuclear maturation [24]. Likewise, in the ovary a moderate activation of ER stress depends upon PERK and p38 signaling [25], evidencing a UPR response in the cells of this organ.

#### **4. Apoptosis**

Apoptosis, the term proposed by Kerr et al. [26], describes an intrinsic suicide mechanism that involves cell shrinkage and the loss of cell contacts, chromatin condensation, and cleavage [27]. This process is better known as programmed cell death type 1 (PCD type 1). The biochemical activation of apoptosis can be directed through extrinsic and intrinsic pathways. The extrinsic pathway is initiated by the activation of cell surface death receptors to their ligands, like the Fas Ligand and TNF. After binding, apoptotic signals are transmitted through dead effector domains

**77**

factors [33].

role (**Figure 2b**).

**4.1 The role of the ER in apoptosis**

stress-induced apoptosis [44].

mitochondrial apoptotic pathway [47].

*Endoplasmic Reticulum Stress during Mammalian Follicular Atresia*

activate the executor caspases (caspase-3, caspase-6, and caspase-7).

and caspase recruitment domains. The intrinsic pathway is governed by a variety of cellular stresses including DNA damage, endoplasmic reticulum stress, and nutrient deprivation, which culminates in mitochondrial outer membrane permeabilization (MOMP), resulting in the release of mitochondrial proteins including cytochrome c and Smac/DIABLO. Apoptosis pathways converge on a common machinery of cell destruction that is activated by caspases, a family of cysteine proteases that cleave after an aspartate residue [28, 29]. The caspases implicated in apoptosis are divided into initiators and executioners, where initiator caspases (caspase-8 and caspase-9)

The Bcl-2 family, which are central regulators of MOMP, are a large class of both pro- and anti-apoptotic proteins. The Bcl-2 family is divided into three subfamilies: multidomain anti-apoptotic such as BCL-2, BCL-XL (BCL2L1), MCL-1, BCL-W (BCL2L2), and A1 (BCL2A1), multidomain pro-apoptotic such as BAX and BAK, and pro-apoptotic BH3-only molecules that include BID, BIM, PUMA (p53 upregulated modulator of apoptosis), and NOXA [30]. BH3-only proteins antagonize anti-apoptotic BCL-2 proteins to release and activate Bak/Bax [31]. Bax and Bak induce external membrane mitochondrial permeabilization and cytochrome c release [32]. Nevertheless, some death stimuli can trigger caspase-independent cell death pathways where other organelles such as the endoplasmic reticulum and the mitochondria have an important function in the release and activation of death

In atretic follicles, this PCD was thoroughly described by Tilly et al. [34] and can be conducted through the intrinsic or the extrinsic pathway [35]. In ovaries, apoptosis can be triggered by deprivation of various signal molecules, survival factors, growth factors (IGF and EGF), and gonadotropins (FSH and LH). Apoptosis can occur in both oocytes and somatic cells. Cell elimination has been observed in follicles in different stages of development, from fetal to adult organisms [3, 36–38]. Although different routes of PCD can occur during follicular atresia, apoptosis plays a major

Apoptosis is triggered by chronic or irreversible ER stress and UPR and occurs through either the extrinsic or intrinsic pathway. Further, apoptosis can be carried out by two pathways, a classical Bax-/Bak-dependent apoptotic response that can be inhibited by ERK1/2 signaling and an alternative ERK1-/2- and Bax-/ Bak-independent pathway [39]. No single component is entirely necessary, but the interaction of many different mechanisms results in apoptosis during ER stress [40]. Under ER stress Bax and Bak interact with the cytosolic region of IRE1α,

The activity of the BH3-only protein Bim is induced through different pathways. The first one involves protein phosphatase 2A-mediated dephosphorylation, which prevents its ubiquitination and the proteasomal degradation of Bim. A second pathway is direct transcriptional induction that is C/EBP homologous protein (CHOP)-C/EBPalpha-mediated, and a third comprises a repression of miRNAs led by PERK [42, 43]. On the other hand, PUMA, p53, and NOXA contribute to ER

It has been reported that CHOP (a transcription factor of pro-apoptotic proteins such as Bim) increases during ER stress [45]. ATF4 and CHOP increase a generalized protein synthesis, provoking ATP depletion, oxidative stress, and cell death [46]. Also, IRE1α degrades the miRNA that represses caspase-2 mRNA translation, which causes an increase in the protein levels of this initiator protease of the

which is required for the modulation of IRE1α signaling [41].

*DOI: http://dx.doi.org/10.5772/intechopen.82687*

#### *Endoplasmic Reticulum Stress during Mammalian Follicular Atresia DOI: http://dx.doi.org/10.5772/intechopen.82687*

*Endoplasmic Reticulum*

and paraptosis.

**3.1 The ER and follicular atresia**

cisterns and the swelling of mitochondria [21].

The proteins PERK and IRE1α and β are important players during UPR because they undergo oligomerization and autophosphorylation due to their interactions with peptides and unfolded proteins [8, 9]. Additionally, IRE1 promotes the unconventional splicing of X-box binding protein 1 (XBP-1) mRNA and an unspecific decrease of mRNAs better known as regulated IRE1-dependent decay (RIDD) [10, 11]. Afterward, the protein XBP-1 is translocated to the nucleus to activate the transcription of chaperones and ERAD factors [12]. RIDD suppresses protein inflow by degrading the mRNA of proteins with signal peptides or proteins with transmembrane domains, and in this manner RIDD permits proteins that are incorrectly folded inside the ER to be folded correctly [10]. PERK phosphorylates eukaryotic translation initiation factor (eIF2a), which then accumulates on the cytosolic side and leads to the downregulation of translation and enhances the translation of Grp78 and the transcription factor ATF4 [13, 14]. It has been shown that during early mouse embryonic development, Grp78 suppresses ER stress and pro-apoptotic pathways via ER signaling [15]. ATF6 is regulated by proteolysis in the Golgi apparatus, allowing the N-terminal fragments to be translocated into the nucleus where they function as a transcription factor [16, 17]. The processing of both ATF6- and IRE1α-mediated

splicing of XBP1 mRNA is required for the full activation of the UPR [18].

UPR works like a protection mechanism. For example, in pancreatic beta cell line INS-1E, glucosamine and high glucose induce UPR activation and generate a feedback loop at the level of insulin transcription [19]. However, chronic or irreversible UPR can trigger cell death pathways, mainly apoptosis, but ER stress can induce other programmed cell death mechanisms including autophagy, necroptosis,

Morphological ER disturbances during follicular atresia have been observed for a long time. Henderson et al. [20] observed a higher surface area of endoplasmic reticulum in granulosa cells cultured from atretic follicles. Moreover, researchers have used electron microscopy to observe the dilation and disintegration of RER

These morphological disturbances in ovaries are associated with ER stress and UPR activation under both physiological and pathological conditions [22]. UPR is present during follicular growth and maturation and follicular atresia and in the corpus luteum. ER stress during follicular growth and maturation has been evidenced

Apoptosis, the term proposed by Kerr et al. [26], describes an intrinsic suicide mechanism that involves cell shrinkage and the loss of cell contacts, chromatin condensation, and cleavage [27]. This process is better known as programmed cell death type 1 (PCD type 1). The biochemical activation of apoptosis can be directed through extrinsic and intrinsic pathways. The extrinsic pathway is initiated by the activation of cell surface death receptors to their ligands, like the Fas Ligand and TNF. After binding, apoptotic signals are transmitted through dead effector domains

by means of the expression of XPB1 and heat shock 70 kDa protein 5 (HSPA5) accompanied by the activation of IRE1 and PERK [23]. The ER stress level and cellular response depend on the signal and its intensity. It has been shown that a lipid-rich intrafollicular environment induces ER stress and impaired oocyte nuclear maturation [24]. Likewise, in the ovary a moderate activation of ER stress depends upon PERK and p38 signaling [25], evidencing a UPR response in the cells of this organ.

**76**

**4. Apoptosis**

and caspase recruitment domains. The intrinsic pathway is governed by a variety of cellular stresses including DNA damage, endoplasmic reticulum stress, and nutrient deprivation, which culminates in mitochondrial outer membrane permeabilization (MOMP), resulting in the release of mitochondrial proteins including cytochrome c and Smac/DIABLO. Apoptosis pathways converge on a common machinery of cell destruction that is activated by caspases, a family of cysteine proteases that cleave after an aspartate residue [28, 29]. The caspases implicated in apoptosis are divided into initiators and executioners, where initiator caspases (caspase-8 and caspase-9) activate the executor caspases (caspase-3, caspase-6, and caspase-7).

The Bcl-2 family, which are central regulators of MOMP, are a large class of both pro- and anti-apoptotic proteins. The Bcl-2 family is divided into three subfamilies: multidomain anti-apoptotic such as BCL-2, BCL-XL (BCL2L1), MCL-1, BCL-W (BCL2L2), and A1 (BCL2A1), multidomain pro-apoptotic such as BAX and BAK, and pro-apoptotic BH3-only molecules that include BID, BIM, PUMA (p53 upregulated modulator of apoptosis), and NOXA [30]. BH3-only proteins antagonize anti-apoptotic BCL-2 proteins to release and activate Bak/Bax [31]. Bax and Bak induce external membrane mitochondrial permeabilization and cytochrome c release [32]. Nevertheless, some death stimuli can trigger caspase-independent cell death pathways where other organelles such as the endoplasmic reticulum and the mitochondria have an important function in the release and activation of death factors [33].

In atretic follicles, this PCD was thoroughly described by Tilly et al. [34] and can be conducted through the intrinsic or the extrinsic pathway [35]. In ovaries, apoptosis can be triggered by deprivation of various signal molecules, survival factors, growth factors (IGF and EGF), and gonadotropins (FSH and LH). Apoptosis can occur in both oocytes and somatic cells. Cell elimination has been observed in follicles in different stages of development, from fetal to adult organisms [3, 36–38]. Although different routes of PCD can occur during follicular atresia, apoptosis plays a major role (**Figure 2b**).

#### **4.1 The role of the ER in apoptosis**

Apoptosis is triggered by chronic or irreversible ER stress and UPR and occurs through either the extrinsic or intrinsic pathway. Further, apoptosis can be carried out by two pathways, a classical Bax-/Bak-dependent apoptotic response that can be inhibited by ERK1/2 signaling and an alternative ERK1-/2- and Bax-/ Bak-independent pathway [39]. No single component is entirely necessary, but the interaction of many different mechanisms results in apoptosis during ER stress [40]. Under ER stress Bax and Bak interact with the cytosolic region of IRE1α, which is required for the modulation of IRE1α signaling [41].

The activity of the BH3-only protein Bim is induced through different pathways. The first one involves protein phosphatase 2A-mediated dephosphorylation, which prevents its ubiquitination and the proteasomal degradation of Bim. A second pathway is direct transcriptional induction that is C/EBP homologous protein (CHOP)-C/EBPalpha-mediated, and a third comprises a repression of miRNAs led by PERK [42, 43]. On the other hand, PUMA, p53, and NOXA contribute to ER stress-induced apoptosis [44].

It has been reported that CHOP (a transcription factor of pro-apoptotic proteins such as Bim) increases during ER stress [45]. ATF4 and CHOP increase a generalized protein synthesis, provoking ATP depletion, oxidative stress, and cell death [46]. Also, IRE1α degrades the miRNA that represses caspase-2 mRNA translation, which causes an increase in the protein levels of this initiator protease of the mitochondrial apoptotic pathway [47].

#### **4.2 The role of the ER in apoptosis during follicular atresia**

ER stress and UPR during follicular atresia are not fully understood; however, there are several evidences of these processes in the ovary. For example, cisplatin, a widely used chemotherapeutic agent, can induce ER stress, which promotes apoptosis and autophagy in granulosa cells, causing excessive follicle loss and endocrine disorders [48].

In goat ovaries, ER stress is involved in follicular atresia through ATF6 and PERK/eIF2α/ATF4 signaling. Furthermore CHOP, caspase-12, and Grp78 proteins are upregulated in apoptotic granulosa cells during follicular atresia [49, 50]. ATF6 is a protein that is extensively distributed in the granulosa cells of ovarian follicles and oocytes in adult mice, and the amount of ATF6 increases in the presence of FSH and LH. ATF6 regulates apoptosis, the cell cycle, steroid hormone synthesis, and other modulators of folliculogenesis in granulosa cells, which may impact the development, ovulation, and atresia of ovarian follicles [51].

The presence of apoptosis-inducing factor (AIF) has been identified in granulosa cells. This protein mediates caspase-independent apoptosis and causes chromatin condensation and DNA fragmentation. AIF expression increases during follicular atresia, and AIF depletion protects ER stress-mediated goat granulosa cell apoptosis [52].

Reactive oxygen species (ROS) generation and oxidative stress can be upstream or downstream UPR targets. That is, UPR is interconnected with different enzymatic mechanisms of ROS generation, and they may depend on Ca2+ levels, ROS themselves, and PDI, which associates with NADPH oxidase and regulates its function [53]. ROS are pro-apoptotic factors in antral follicles. During oxidative stress, JNK activates FoxO1, which increases PUMA and induces apoptosis in granulosa cells [54]. Furthermore, pentosidine, a biomarker for advanced glycation end products, is accumulated in apoptotic human oocytes and increases with age [55].

UPR and ER stresses also have important roles in the regulation of corpus luteum (CL) regression. The overexpression of p-JNK, CHOP, caspase-12, and active caspase-3 during CL regression points to ER stress-dependent apoptosis [56, 57].

#### **5. Autophagy**

Autophagy is a catabolic pathway of cell constituents that contributes to cell survival in response to stress. Autophagy does not cause a loss of cell chemical components because the cell reutilizes them. There are three major types of autophagy, microautophagy, chaperon-mediated autophagy, and macroautophagy.

In microautophagy, vesicles bud into the lysosomal lumen by direct invagination of the boundary membrane, resulting in degradation of both cytoplasmic components and the lysosomal membrane by lysosomal hydrolases. This process involves sequential stages of vacuole invagination and vesicle scission [58].

Chaperon-mediated autophagy is the selective transport of proteins into lysosomes. The first step is protein recognition and lysosomal targeting. Protein recognition takes place in the cytosol through the binding of hsc70 to a KFERQ-like motif present in all chaperon-mediated autophagy substrates [59]. In the second step, proteins bind to receptors at the lysosomal membrane, Lamp2A, or a similar protein receptor for subsequent translocation and lysosomal degradation [60]. Receptors are subcompartmentalized in lipid microdomains to engage the processes of degradation, multimerization, and membrane retrieval [61].

**79**

autophagy.

homeostasis [70].

*Endoplasmic Reticulum Stress during Mammalian Follicular Atresia*

ysosome where cargo is degraded by acid hydrolases [62].

fusion, where sequestered cargos are digested.

**5.1 The role of the ER in autophagy**

Macroautophagy, also referred to as autophagy, involves the engulfment of cytoplasmic portions in a nonselective manner, as well as the degradation of specific proteins, organelles, and invading bacteria by a selective autophagy. Autophagy begins with the formation of an isolation membrane, the phagophore, which is a disk-like structure where the Atg machinery assembles. An isolation membrane grows to generate a double-membrane autophagosome, followed by elongation to form a mature autophagosome that captures cytosolic cargo. The fusion of mature autophagosomes with endosomes or lysosomes results in a single-membrane autol-

Autophagy (Atg)-related proteins are the core machinery for autophagosome biogenesis and consist of several functional units: the ULK1-Atg13-FIP200-Atg101 protein kinase complex; the PI3K class III complex containing the core proteins VPS34, VPS15, and beclin 1; the PI3P-binding WIPI/Atg18-Atg2 complex; Atg9A; and the ubiquitin-like Atg5/Atg12 and Atg8/LC3 conjugation systems [63].

Autophagosome maturation involves the clearance of PI3P by Ymr1, a PI3P phosphatase, triggering the dissociation of the Atg machinery. Mature autophagosomes are transported to lysosomes through the microtubule cytoskeleton. The FYVE and coiled-coil domain containing 1 (FYCO1) protein binds to LC3, PI3P, and the small GTPase Rab7 and acts as an adaptor between autophagosomes and microtubules [64, 65]. Finally, the autolysosome is generated by autophagosome and lysosome

Autophagy and ER stress can be physiological processes in organisms. For example, they regulate endometrial function by modulating the mTOR pathway [66]. Also, autophagy contributes to the recovery of cell homeostasis after ER stress. During ER stress, damaged proteins are degraded by ERAD. However, some misfolded proteins are resistant, so autophagy is a final cell protection strategy deployed against ER-accumulated cytotoxic aggregates that cannot be removed by ERAD [67]. Additionally, ubiquitin is a common signal for both the ubiquitin-proteasome system and autophagy. In the mouse neuroblastoma cell line neuro-2a treated with tunicamycin, an ER stress inductor, the proteins involved in proteasomal degradation were downregulated, while proteins involved in ubiquitination were upregulated. Moreover, tunicamycin triggered autophagy, suggesting that it may serve as a compensatory effect to proteasomal degradation [68]. Also, ER-resident chaperones and enzymes that reduce the overload of misfolded proteins need to be removed by

The structure or phagophore assembly site (PAS) localizes proximal to the ER. Autophagosome formation and transport to the vacuole are stimulated in an Atg protein-dependent manner. ER stress can induce an autophagic response because it increases Atg1 kinase activity and reflects both the nutritional status and autophagic state of the cell [69]. ER exit sites are essential for autophagy and are proximal to the PAS. Sec62, a constituent of the translocon complex that regulates protein import into the mammalian ER, intervenes during recovery from ER stress to selectively deliver ER components to the autolysosomal system for clearance and therefore is a critical molecular component in the maintenance and recovery of ER

The eIF2α/ATF4 pathway directs an autophagy gene transcriptional program in response to amino acid starvation or ER stress. The eIF2α kinase and the transcriptional factors ATF4 and CHOP are required to increase the transcription of a set of genes implicated in the formation, elongation, and function of the autophagosome,

*DOI: http://dx.doi.org/10.5772/intechopen.82687*

#### *Endoplasmic Reticulum Stress during Mammalian Follicular Atresia DOI: http://dx.doi.org/10.5772/intechopen.82687*

*Endoplasmic Reticulum*

disorders [48].

apoptosis [52].

increases with age [55].

**5. Autophagy**

macroautophagy.

**4.2 The role of the ER in apoptosis during follicular atresia**

development, ovulation, and atresia of ovarian follicles [51].

ER stress and UPR during follicular atresia are not fully understood; however, there are several evidences of these processes in the ovary. For example, cisplatin, a widely used chemotherapeutic agent, can induce ER stress, which promotes apoptosis and autophagy in granulosa cells, causing excessive follicle loss and endocrine

In goat ovaries, ER stress is involved in follicular atresia through ATF6 and PERK/eIF2α/ATF4 signaling. Furthermore CHOP, caspase-12, and Grp78 proteins are upregulated in apoptotic granulosa cells during follicular atresia [49, 50]. ATF6 is a protein that is extensively distributed in the granulosa cells of ovarian follicles and oocytes in adult mice, and the amount of ATF6 increases in the presence of FSH and LH. ATF6 regulates apoptosis, the cell cycle, steroid hormone synthesis, and other modulators of folliculogenesis in granulosa cells, which may impact the

The presence of apoptosis-inducing factor (AIF) has been identified in granulosa cells. This protein mediates caspase-independent apoptosis and causes chromatin condensation and DNA fragmentation. AIF expression increases during follicular atresia, and AIF depletion protects ER stress-mediated goat granulosa cell

Reactive oxygen species (ROS) generation and oxidative stress can be upstream or downstream UPR targets. That is, UPR is interconnected with different enzymatic mechanisms of ROS generation, and they may depend on Ca2+ levels, ROS themselves, and PDI, which associates with NADPH oxidase and regulates its function [53]. ROS are pro-apoptotic factors in antral follicles. During oxidative stress, JNK activates FoxO1, which increases PUMA and induces apoptosis in granulosa cells [54]. Furthermore, pentosidine, a biomarker for advanced glycation end products, is accumulated in apoptotic human oocytes and

UPR and ER stresses also have important roles in the regulation of corpus luteum

(CL) regression. The overexpression of p-JNK, CHOP, caspase-12, and active caspase-3 during CL regression points to ER stress-dependent apoptosis [56, 57].

Autophagy is a catabolic pathway of cell constituents that contributes to cell survival in response to stress. Autophagy does not cause a loss of cell chemical components because the cell reutilizes them. There are three major types of autophagy, microautophagy, chaperon-mediated autophagy, and

Chaperon-mediated autophagy is the selective transport of proteins into lysosomes. The first step is protein recognition and lysosomal targeting. Protein recognition takes place in the cytosol through the binding of hsc70 to a KFERQ-like motif present in all chaperon-mediated autophagy substrates [59]. In the second step, proteins bind to receptors at the lysosomal membrane, Lamp2A, or a similar protein receptor for subsequent translocation and lysosomal degradation [60]. Receptors are subcompartmentalized in lipid microdomains to engage the processes

sequential stages of vacuole invagination and vesicle scission [58].

of degradation, multimerization, and membrane retrieval [61].

In microautophagy, vesicles bud into the lysosomal lumen by direct invagination of the boundary membrane, resulting in degradation of both cytoplasmic components and the lysosomal membrane by lysosomal hydrolases. This process involves

**78**

Macroautophagy, also referred to as autophagy, involves the engulfment of cytoplasmic portions in a nonselective manner, as well as the degradation of specific proteins, organelles, and invading bacteria by a selective autophagy. Autophagy begins with the formation of an isolation membrane, the phagophore, which is a disk-like structure where the Atg machinery assembles. An isolation membrane grows to generate a double-membrane autophagosome, followed by elongation to form a mature autophagosome that captures cytosolic cargo. The fusion of mature autophagosomes with endosomes or lysosomes results in a single-membrane autolysosome where cargo is degraded by acid hydrolases [62].

Autophagy (Atg)-related proteins are the core machinery for autophagosome biogenesis and consist of several functional units: the ULK1-Atg13-FIP200-Atg101 protein kinase complex; the PI3K class III complex containing the core proteins VPS34, VPS15, and beclin 1; the PI3P-binding WIPI/Atg18-Atg2 complex; Atg9A; and the ubiquitin-like Atg5/Atg12 and Atg8/LC3 conjugation systems [63].

Autophagosome maturation involves the clearance of PI3P by Ymr1, a PI3P phosphatase, triggering the dissociation of the Atg machinery. Mature autophagosomes are transported to lysosomes through the microtubule cytoskeleton. The FYVE and coiled-coil domain containing 1 (FYCO1) protein binds to LC3, PI3P, and the small GTPase Rab7 and acts as an adaptor between autophagosomes and microtubules [64, 65]. Finally, the autolysosome is generated by autophagosome and lysosome fusion, where sequestered cargos are digested.

#### **5.1 The role of the ER in autophagy**

Autophagy and ER stress can be physiological processes in organisms. For example, they regulate endometrial function by modulating the mTOR pathway [66]. Also, autophagy contributes to the recovery of cell homeostasis after ER stress. During ER stress, damaged proteins are degraded by ERAD. However, some misfolded proteins are resistant, so autophagy is a final cell protection strategy deployed against ER-accumulated cytotoxic aggregates that cannot be removed by ERAD [67]. Additionally, ubiquitin is a common signal for both the ubiquitin-proteasome system and autophagy. In the mouse neuroblastoma cell line neuro-2a treated with tunicamycin, an ER stress inductor, the proteins involved in proteasomal degradation were downregulated, while proteins involved in ubiquitination were upregulated. Moreover, tunicamycin triggered autophagy, suggesting that it may serve as a compensatory effect to proteasomal degradation [68]. Also, ER-resident chaperones and enzymes that reduce the overload of misfolded proteins need to be removed by autophagy.

The structure or phagophore assembly site (PAS) localizes proximal to the ER. Autophagosome formation and transport to the vacuole are stimulated in an Atg protein-dependent manner. ER stress can induce an autophagic response because it increases Atg1 kinase activity and reflects both the nutritional status and autophagic state of the cell [69]. ER exit sites are essential for autophagy and are proximal to the PAS. Sec62, a constituent of the translocon complex that regulates protein import into the mammalian ER, intervenes during recovery from ER stress to selectively deliver ER components to the autolysosomal system for clearance and therefore is a critical molecular component in the maintenance and recovery of ER homeostasis [70].

The eIF2α/ATF4 pathway directs an autophagy gene transcriptional program in response to amino acid starvation or ER stress. The eIF2α kinase and the transcriptional factors ATF4 and CHOP are required to increase the transcription of a set of genes implicated in the formation, elongation, and function of the autophagosome, including Atgs and beclin 1, increasing the capacity to maintain autophagy in stressed cells. These autophagy genes exhibit different dependencies on ATF4 and CHOP, which means that they have a differential transcriptional response according to the stress intensity [71]. In human heart failure, the overexpression of the ER stress markers Grp78, PERK, CHOP, and ATF3 correlates with the expression of autophagy genes [72].

IRE1, a UPR sensor, has two isoforms, IRE1α and IRE1β, which both have RNase and kinase activities. However, in *Arabidopsis thaliana*, RNase activity of IRE1β, but not its protein kinase activity, is required for ER stress-mediated autophagy [73]. In *Dictyostelium*, the response to ER stress involves the combined activation of an IRE1α-dependent gene expression program and the autophagy pathway [74]. In mammalian cells, the spliced form of XBP 1 upregulates Nedd4-2, an E3 ubiquitin ligase involved in targeting proteins for subsequent degradation, in response to ER stress. It is also important for the induction of an appropriate autophagic response [75].

Different cancer cell models have allowed a better understanding of the mechanisms involved in autophagy triggered by ER stress. In cervical tumor cells, ER stress and UPR induced by X-ray exposition led to the activation of the NF-κB signaling pathway, autophagy, and apoptosis [76]. NF-κB is important for the proliferation, invasion, and metastasis of cervical cancer cells. Furthermore, in a model of breast cancer, autophagy and apoptosis were triggered through ER stress, UPR, and a high expression of CHOP and JNK [77].

Moreover, ERK and JNK activation is associated with cross talk between autophagy and another PCD. In L929 fibrosarcoma cells, ERK and JNK can link a signal from caspase-8 inhibition to autophagy, which in turn induce ROS production and PARP activation, leading to ATP depletion and necroptosis [78].

Ca2+ exchange between the ER and mitochondria is mediated through domains called mitochondria-associated membranes (MAMs). The interruption of Ca2+ flux between these organelles generates metabolic stress where AMPK present in MAMs triggers autophagy via beclin-1 phosphorylation [79, 80]. Autophagy activation might prevent proper interorganelle communication that would maintain mitochondrial function and cellular homeostasis [79].

In ER stress, some miRNAs promote the survival of the cells, while others promote cell death. In HeLa cells under RE stress, miR-346 positively regulates the expression of glycogen synthase kinase 3 beta (GSK3B) which reduces the interaction of beclin-1 and BCL2 to induce autophagy, ROS reduction, and cell death [81].

#### **5.2 The role of the ER in autophagy during follicular atresia**

Autophagy is mainly induced in granulosa cells (**Figure 2c**) during folliculogenesis and shows a high correlation with apoptosis, and furthermore, both routes of PCD could play active roles in oocyte depletion [82]. According to Meng et al. [83], antral follicular degeneration is initiated by granulosa cell apoptosis, while preantral follicular atresia occurs mainly via enhanced granulosa cell autophagy. Surprisingly, apoptosis and autophagy can be present in the same cell at the same time, just as cells can show caspase-3 active, DNA fragmentation, and immunodetection of LC3 and Lamp 1 [2, 3].

The signals that establish autophagy or apoptosis as the route of cell death are not fully understood. Consistent with Zhang et al. [84], atresia initiation is associated with a cross talk of different PCDs including apoptosis and autophagy, a dramatic shift of steroidogenic enzymes, deficient glutathione metabolism, and vascular degeneration. In a rat model, FSH, a survival factor, decreased autophagy through LC3-II inhibition and Akt-mTOR pathway activation [85]. Shen et al. [86]

**81**

*Endoplasmic Reticulum Stress during Mammalian Follicular Atresia*

assessed the mechanism involved in autophagy inhibition by the Akt-mTOR pathway in granulosa cells exposed to FSH and oxidative stress because mTOR, a negative regulator of autophagy, inhibits FOXO1, which promotes the expression of several autophagy genes. They found that FSH induced granulosa cell survival via FOXO1 inhibition by the PI3K-Akt-mTOR pathway [86]. Nevertheless, in mouse granulosa cells, FSH was related to follicle development and atresia because FSH

Despite the studies on the role of the ER in autophagy, its specific participation

Necroptosis is a subtype of regulated necrosis and shares the same morphological changes, including organelle swelling and membrane rupture. Necroptosis is a caspase-independent cell death, and its execution involves the active disintegration of mitochondrial, lysosomal, and plasma membranes. This PCD is triggered by various stimuli, such as TNF, Fas ligand, and TRAIL and depends on the serine/ threonine kinase activity of RIP1. Additionally, a set of 432 genes regulates necroptosis and cellular sensitivity to this PCD by a signaling network that mediates innate immunity [88]. Moreover, Bmf, a BH3-only protein, is required for death receptor-

Moreover, environmental toxicants like cadmium can activate necroptosis. Intermediate levels of cadmium are associated with lost plasma membrane integrity, a decrease of ATP levels, and mitochondrial membrane potential and cell swelling,

The core pathway of necroptosis relies on the assembly of an amyloid-like structure termed the necrosome. The necrosome is a multiprotein complex formed by receptor-interacting protein kinase 3 (RIPK3), RIPK1, and mixed lineage kinase domain-like (MLKL). Oligomerization and intramolecular autophosphorylation of RIPK3 lead to the recruitment and phosphorylation of MLKL. RIPK3 and MLKL continuously shuttle between the nucleus and the cytoplasm, whereas RIPK1 is constitutively present in both compartments [90]. Nuclear RIPK1 becomes ubiquitinated, and then nuclear MLKL becomes phosphorylated and oligomerized [90]. MLKL mediates plasma membrane rupture. MLKL forms cation channels that are preferentially

and K+

The role of the ER in necroptosis has been evidenced using necrostatin-1, an inhibitor of necroptosis, which has a protective effect on the endoplasmic reticulum and mitochondria and alleviates ER stress after spinal cord injury [92]. Furthermore, Grp78 promotes an inflammatory response through the upregulation of necroptosis and subsequent activation of NF-κB and AP-1 pathways [93]. The depletion of reticulocalbin 1, an ER-resident Ca2+-binding protein, induces Grp78, activates PERK, and phosphorylates eIF2α. Moreover, the activation of CaMKII and the inactivation of Akt are important for necroptosis in response to reticulocalbin 1 depletion [94]. The function of MLKL and RIPK in necroptosis has been widely studied. The signal transducer and activator of transcription 3 (STAT3) was demonstrated to be downstream of calpain and regulates RIPK3 expression and MLKL phosphorylation and induces ER stress and mitochondrial calcium dysregulation [95]. Moreover, in cardiomyocytes upregulated RIPK1 and RIPK3 evoke ER stress, accompanied by an

depolarization and cell death exhibit a positive correlation to channel activity.

[91]. MLKL-induced membrane

which are features associated with necroptotic cell death [89].

*DOI: http://dx.doi.org/10.5772/intechopen.82687*

induces autophagy signaling via HIF-1α [87].

in follicular atresia is still unknown.

**6. Necroptosis**

induced necroptosis [88].

permeable to Mg2+ in the presence of Na+

**6.1 The role of ER in necroptosis**

*Endoplasmic Reticulum Stress during Mammalian Follicular Atresia DOI: http://dx.doi.org/10.5772/intechopen.82687*

assessed the mechanism involved in autophagy inhibition by the Akt-mTOR pathway in granulosa cells exposed to FSH and oxidative stress because mTOR, a negative regulator of autophagy, inhibits FOXO1, which promotes the expression of several autophagy genes. They found that FSH induced granulosa cell survival via FOXO1 inhibition by the PI3K-Akt-mTOR pathway [86]. Nevertheless, in mouse granulosa cells, FSH was related to follicle development and atresia because FSH induces autophagy signaling via HIF-1α [87].

Despite the studies on the role of the ER in autophagy, its specific participation in follicular atresia is still unknown.

#### **6. Necroptosis**

*Endoplasmic Reticulum*

autophagy genes [72].

appropriate autophagic response [75].

UPR, and a high expression of CHOP and JNK [77].

chondrial function and cellular homeostasis [79].

detection of LC3 and Lamp 1 [2, 3].

PARP activation, leading to ATP depletion and necroptosis [78].

**5.2 The role of the ER in autophagy during follicular atresia**

including Atgs and beclin 1, increasing the capacity to maintain autophagy in stressed cells. These autophagy genes exhibit different dependencies on ATF4 and CHOP, which means that they have a differential transcriptional response according to the stress intensity [71]. In human heart failure, the overexpression of the ER stress markers Grp78, PERK, CHOP, and ATF3 correlates with the expression of

IRE1, a UPR sensor, has two isoforms, IRE1α and IRE1β, which both have RNase and kinase activities. However, in *Arabidopsis thaliana*, RNase activity of IRE1β, but not its protein kinase activity, is required for ER stress-mediated autophagy [73]. In *Dictyostelium*, the response to ER stress involves the combined activation of an IRE1α-dependent gene expression program and the autophagy pathway [74]. In mammalian cells, the spliced form of XBP 1 upregulates Nedd4-2, an E3 ubiquitin ligase involved in targeting proteins for subsequent degradation, in response to ER stress. It is also important for the induction of an

Different cancer cell models have allowed a better understanding of the mechanisms involved in autophagy triggered by ER stress. In cervical tumor cells, ER stress and UPR induced by X-ray exposition led to the activation of the NF-κB signaling pathway, autophagy, and apoptosis [76]. NF-κB is important for the proliferation, invasion, and metastasis of cervical cancer cells. Furthermore, in a model of breast cancer, autophagy and apoptosis were triggered through ER stress,

Moreover, ERK and JNK activation is associated with cross talk between autophagy and another PCD. In L929 fibrosarcoma cells, ERK and JNK can link a signal from caspase-8 inhibition to autophagy, which in turn induce ROS production and

Ca2+ exchange between the ER and mitochondria is mediated through domains called mitochondria-associated membranes (MAMs). The interruption of Ca2+ flux between these organelles generates metabolic stress where AMPK present in MAMs triggers autophagy via beclin-1 phosphorylation [79, 80]. Autophagy activation might prevent proper interorganelle communication that would maintain mito-

In ER stress, some miRNAs promote the survival of the cells, while others promote cell death. In HeLa cells under RE stress, miR-346 positively regulates the expression of glycogen synthase kinase 3 beta (GSK3B) which reduces the interaction of beclin-1 and BCL2 to induce autophagy, ROS reduction, and cell death [81].

Autophagy is mainly induced in granulosa cells (**Figure 2c**) during folliculogenesis and shows a high correlation with apoptosis, and furthermore, both routes of PCD could play active roles in oocyte depletion [82]. According to Meng et al. [83], antral follicular degeneration is initiated by granulosa cell apoptosis, while preantral follicular atresia occurs mainly via enhanced granulosa cell autophagy. Surprisingly, apoptosis and autophagy can be present in the same cell at the same time, just as cells can show caspase-3 active, DNA fragmentation, and immuno-

The signals that establish autophagy or apoptosis as the route of cell death are not fully understood. Consistent with Zhang et al. [84], atresia initiation is associated with a cross talk of different PCDs including apoptosis and autophagy, a dramatic shift of steroidogenic enzymes, deficient glutathione metabolism, and vascular degeneration. In a rat model, FSH, a survival factor, decreased autophagy through LC3-II inhibition and Akt-mTOR pathway activation [85]. Shen et al. [86]

**80**

Necroptosis is a subtype of regulated necrosis and shares the same morphological changes, including organelle swelling and membrane rupture. Necroptosis is a caspase-independent cell death, and its execution involves the active disintegration of mitochondrial, lysosomal, and plasma membranes. This PCD is triggered by various stimuli, such as TNF, Fas ligand, and TRAIL and depends on the serine/ threonine kinase activity of RIP1. Additionally, a set of 432 genes regulates necroptosis and cellular sensitivity to this PCD by a signaling network that mediates innate immunity [88]. Moreover, Bmf, a BH3-only protein, is required for death receptorinduced necroptosis [88].

Moreover, environmental toxicants like cadmium can activate necroptosis. Intermediate levels of cadmium are associated with lost plasma membrane integrity, a decrease of ATP levels, and mitochondrial membrane potential and cell swelling, which are features associated with necroptotic cell death [89].

The core pathway of necroptosis relies on the assembly of an amyloid-like structure termed the necrosome. The necrosome is a multiprotein complex formed by receptor-interacting protein kinase 3 (RIPK3), RIPK1, and mixed lineage kinase domain-like (MLKL). Oligomerization and intramolecular autophosphorylation of RIPK3 lead to the recruitment and phosphorylation of MLKL. RIPK3 and MLKL continuously shuttle between the nucleus and the cytoplasm, whereas RIPK1 is constitutively present in both compartments [90]. Nuclear RIPK1 becomes ubiquitinated, and then nuclear MLKL becomes phosphorylated and oligomerized [90]. MLKL mediates plasma membrane rupture. MLKL forms cation channels that are preferentially permeable to Mg2+ in the presence of Na+ and K+ [91]. MLKL-induced membrane depolarization and cell death exhibit a positive correlation to channel activity.

#### **6.1 The role of ER in necroptosis**

The role of the ER in necroptosis has been evidenced using necrostatin-1, an inhibitor of necroptosis, which has a protective effect on the endoplasmic reticulum and mitochondria and alleviates ER stress after spinal cord injury [92]. Furthermore, Grp78 promotes an inflammatory response through the upregulation of necroptosis and subsequent activation of NF-κB and AP-1 pathways [93]. The depletion of reticulocalbin 1, an ER-resident Ca2+-binding protein, induces Grp78, activates PERK, and phosphorylates eIF2α. Moreover, the activation of CaMKII and the inactivation of Akt are important for necroptosis in response to reticulocalbin 1 depletion [94].

The function of MLKL and RIPK in necroptosis has been widely studied. The signal transducer and activator of transcription 3 (STAT3) was demonstrated to be downstream of calpain and regulates RIPK3 expression and MLKL phosphorylation and induces ER stress and mitochondrial calcium dysregulation [95]. Moreover, in cardiomyocytes upregulated RIPK1 and RIPK3 evoke ER stress, accompanied by an

increase in intracellular Ca2+ levels and xanthine oxidase expression, which raised cellular ROS that mediated the mitochondrial permeability transition pore opening and necroptosis [96, 97]. In addition, the activation of JNK1/2 is regulated by RIPK3 [96].

Moreover, there are proteins that can participate in necroptosis and other types of PCD such as AIF and MLKL. Apoptosis-inducing factor (AIF), a protein normally located within the intermembrane space of mitochondria, is linked to apoptosis and necrosis. However, it has been shown that mitochondrial depolarization induced by ER stress promotes AIF release and nuclear condensation, which is consistent with necroptotic cell death [98–100]. MLKL, a member of the necrosome, also participates in chelerythrine (CHE)-promoted apoptosis through nuclear MLKL translocation and a special band of MLKL, which is promoted by a mutual regulation between the MLKL and PERK-eIF2α pathways in response to ROS formation [101].

#### **6.2 The role of the RE in necroptosis during follicular atresia**

Necroptosis has been widely researched, but there is still much to investigate, including the mechanism that mediates its execution. Nevertheless, necroptosis studies have been carried out under pathological conditions, and thus it is important to use physiological models like follicular atresia.

Necroptosis contributes to follicular atresia and luteolysis [102]. The factors involved in granulosa cell necroptosis can be regulated by acetylcholinesterase (AChE), cytokines, starvation, and oxidative stress via TNFα [103]. Also, an ovarian AChE variant, the read-through isoform AChE-R, has a nonenzymatic function that stimulates RIPK1-/MLKL-dependent necroptosis [103]. Therefore, although the participation of the ER in necroptosis and the contribution of this PCD in follicular atresia have been shown, the interrelation between ER stress-induced necroptosis and follicular atresia is completely unknown.

#### **7. Paraptosis**

Sperandio et al. [104] introduced the term paraptosis to describe a route of caspase-independent PCD that has morphological, biochemical, and transcriptional features that are different from apoptosis [104]. Endoplasmic reticulum swelling, mitochondrial swelling, and resistance to apoptosis inhibitors without nuclear shrinkage or pyknosis characterize paraptosis. Although paraptosis is a caspase-independent cell death, participation of caspase-9 has been shown under experimental conditions [104].

Paraptosis can be triggered by different stimuli including insulin-like growth factor I receptor (IGFIR), JAY/TROY, and ROS. IGF-I is a regulator of multiple cell signaling pathways including PI3K-Akt1-RPS6 and ERK1/2 MAPK that are critical for cell proliferation, migration, and survival [105]. IGFIR-induced paraptosis is mediated by caspase-9, and at least two signal transduction pathways participate in the execution of paraptosis, the MAPK and JNK pathways [104, 106].

TAJ/TROY, a member of the tumor necrosis factor receptor superfamily, induces morphological features of paraptosis accompanied by phosphatidylserine externalization, the loss of the mitochondrial transmembrane potential, and independent caspase activation [105]. Moreover, programmed cell death 5 (PDCD5), an apoptosis-promoting protein, enhances TAJ-/TROY-induced paraptotic cell death [107].

ROS production can trigger paraptosis through PINK and mitophagy activation [108, 109]. Covalent modifications of free sulfhydryl groups on proteins cause protein misfolding and the accumulation of misfolded proteins, leading to ER stress, CHOP activation, and paraptosis [110, 111]. In malignant hepatoma cells with

**83**

*Endoplasmic Reticulum Stress during Mammalian Follicular Atresia*

Bcl-xL-mediated apoptotic defects, the disruption of thiol homeostasis and treatment with doxorubicin and pyrrolidine dithiocarbamate induced paraptotic cell death [112]. The full signal transduction pathway and identification of specific markers for paraptosis are still unclear. Nevertheless, phosphatidylethanolamine-binding protein (PEBP-1), a suppressor of the MAPK pathway, has been identified, and prohibitin, a mitochondrial protein, is a mediator of paraptosis [113]. Furthermore, the redistribution of α- and β-tubulin and tropomyosin has been observed in the early stages of paraptosis. Other characteristics of the paraptotic pathway involve alterations mainly in signal transduction proteins, mitochondrial proteins, and some metabolic proteins [113].

Cancer cells are the best model to study paraptosis because there can be apoptosis and/or autophagy resistance. In melanoma cells, the sustained activation of the IRE1α and ATF6 pathways driven by the MEK/ERK pathway avoids ER stress-

Different compounds for cancer treatment have shown paraptosis induction. For example, HeLa, A549, and PC-3 cells treated with celastrol induced vacuoles derived from the dilation of ER, a feature of apoptotic cell death; moreover, this was accompanied by autophagy and apoptosis. Furthermore, the ER swelling triggered by celastrol induced ER stress markers including Grp78, PERK, IRE, and CHOP and alterations to proteasome function that resulted in the accumulation of ubiquitinated protein [115, 116]. Moreover, paraptosis can be accelerated by pre-treatment with the proteasome inhibitor MG132 [117]. On the other hand, cyclosporine A treatment of cervical cancerous SiHa cells showed ER stress and UPR preceded by massive cytoplasmic vacuole formation that culminated in a paraptosis-like cell death [118]. Moreover, murine hepatoma 1c1c7 cells and the human non-small cell lung cancer A549 cell line exposed to a combination of photodamage and benzopor-

For the pathways involved in paraptosis, ER vacuoles can be dependent on the PI3K/Akt signaling pathway [120]. Moreover, in BC3H1 myoblast cell lines exposed to yessotoxin, paraptosis was accompanied by cytoskeletal alterations and the activation of JNK/SAPK1 [121]. However, in acute lymphoblastic leukemia cells, everolimus, a mTOR inhibitor, showed that JNK signaling was not required for paraptotic cell death [122]. Paraptosis in epithelial ovarian cancer (EOC) cells treated with morusin was characterized by VDAC-mediated Ca2+ influx into mitochondria, and subsequent mitochondrial Ca2+ overload contributes to mitochondrial swelling and dysfunction, leading to the accumulation of ER stress markers, the generation of ROS, and the loss of mitochondrial membrane potential (Δψm) in EOC cells [123].

Knowledge of the role of paraptosis during follicular atresia is still limited. In *Bombyx mori*, apoptosis, autophagy, and paraptosis occur in the ovarian nurse cell cluster during late vitellogenesis, whereas middle vitellogenesis is exclusively characterized by the presence of paraptosis, preceding both apoptosis and autophagy [124]. In mammals, paraptosis was evidenced by ER swelling (**Figure 2d**) and CHOP immunodetection in granulosa cells during follicular atresia in adult Wistar rats [4]. The mechanisms involved in paraptosis during follicular atresia are still unknown. The paraptotic inductor IGFR might be related because it is implicated in follicular growth and selection [104, 125]. Moreover, IGF2R and the binding protein genes IGFBP5 and IGFBP6 are overexpressed in atretic follicles [126]. However,

phyrin derivative result in ER swelling and paraptotic cell death [119].

**7.2 The role of the RE in paraptosis during follicular atresia**

more studies on paraptosis during follicular atresia are necessary.

*DOI: http://dx.doi.org/10.5772/intechopen.82687*

**7.1 The role of RE in paraptosis**

induced apoptosis [114].

#### *Endoplasmic Reticulum Stress during Mammalian Follicular Atresia DOI: http://dx.doi.org/10.5772/intechopen.82687*

Bcl-xL-mediated apoptotic defects, the disruption of thiol homeostasis and treatment with doxorubicin and pyrrolidine dithiocarbamate induced paraptotic cell death [112].

The full signal transduction pathway and identification of specific markers for paraptosis are still unclear. Nevertheless, phosphatidylethanolamine-binding protein (PEBP-1), a suppressor of the MAPK pathway, has been identified, and prohibitin, a mitochondrial protein, is a mediator of paraptosis [113]. Furthermore, the redistribution of α- and β-tubulin and tropomyosin has been observed in the early stages of paraptosis. Other characteristics of the paraptotic pathway involve alterations mainly in signal transduction proteins, mitochondrial proteins, and some metabolic proteins [113].

#### **7.1 The role of RE in paraptosis**

*Endoplasmic Reticulum*

increase in intracellular Ca2+ levels and xanthine oxidase expression, which raised cellular ROS that mediated the mitochondrial permeability transition pore opening and necroptosis [96, 97]. In addition, the activation of JNK1/2 is regulated by RIPK3 [96]. Moreover, there are proteins that can participate in necroptosis and other types of PCD such as AIF and MLKL. Apoptosis-inducing factor (AIF), a protein normally located within the intermembrane space of mitochondria, is linked to apoptosis and necrosis. However, it has been shown that mitochondrial depolarization induced by ER stress promotes AIF release and nuclear condensation, which is consistent with necroptotic cell death [98–100]. MLKL, a member of the necrosome, also participates in chelerythrine (CHE)-promoted apoptosis through nuclear MLKL translocation and a special band of MLKL, which is promoted by a mutual regulation between

the MLKL and PERK-eIF2α pathways in response to ROS formation [101].

Necroptosis has been widely researched, but there is still much to investigate, including the mechanism that mediates its execution. Nevertheless, necroptosis studies have been carried out under pathological conditions, and thus it is impor-

Necroptosis contributes to follicular atresia and luteolysis [102]. The factors involved in granulosa cell necroptosis can be regulated by acetylcholinesterase (AChE), cytokines, starvation, and oxidative stress via TNFα [103]. Also, an ovarian AChE variant, the read-through isoform AChE-R, has a nonenzymatic function that stimulates RIPK1-/MLKL-dependent necroptosis [103]. Therefore, although the participation of the ER in necroptosis and the contribution of this PCD in follicular atresia have been shown, the interrelation between ER stress-induced

Sperandio et al. [104] introduced the term paraptosis to describe a route of caspase-independent PCD that has morphological, biochemical, and transcriptional features that are different from apoptosis [104]. Endoplasmic reticulum swelling, mitochondrial swelling, and resistance to apoptosis inhibitors without nuclear shrinkage or pyknosis characterize paraptosis. Although paraptosis is a caspase-independent cell death, participation of caspase-9 has been shown under

Paraptosis can be triggered by different stimuli including insulin-like growth factor I receptor (IGFIR), JAY/TROY, and ROS. IGF-I is a regulator of multiple cell signaling pathways including PI3K-Akt1-RPS6 and ERK1/2 MAPK that are critical for cell proliferation, migration, and survival [105]. IGFIR-induced paraptosis is mediated by caspase-9, and at least two signal transduction pathways participate in

TAJ/TROY, a member of the tumor necrosis factor receptor superfamily, induces morphological features of paraptosis accompanied by phosphatidylserine externalization, the loss of the mitochondrial transmembrane potential, and independent caspase activation [105]. Moreover, programmed cell death 5 (PDCD5), an apoptosis-promoting protein, enhances TAJ-/TROY-induced paraptotic cell death [107]. ROS production can trigger paraptosis through PINK and mitophagy activation [108, 109]. Covalent modifications of free sulfhydryl groups on proteins cause protein misfolding and the accumulation of misfolded proteins, leading to ER stress, CHOP activation, and paraptosis [110, 111]. In malignant hepatoma cells with

the execution of paraptosis, the MAPK and JNK pathways [104, 106].

**6.2 The role of the RE in necroptosis during follicular atresia**

tant to use physiological models like follicular atresia.

necroptosis and follicular atresia is completely unknown.

**82**

**7. Paraptosis**

experimental conditions [104].

Cancer cells are the best model to study paraptosis because there can be apoptosis and/or autophagy resistance. In melanoma cells, the sustained activation of the IRE1α and ATF6 pathways driven by the MEK/ERK pathway avoids ER stressinduced apoptosis [114].

Different compounds for cancer treatment have shown paraptosis induction. For example, HeLa, A549, and PC-3 cells treated with celastrol induced vacuoles derived from the dilation of ER, a feature of apoptotic cell death; moreover, this was accompanied by autophagy and apoptosis. Furthermore, the ER swelling triggered by celastrol induced ER stress markers including Grp78, PERK, IRE, and CHOP and alterations to proteasome function that resulted in the accumulation of ubiquitinated protein [115, 116]. Moreover, paraptosis can be accelerated by pre-treatment with the proteasome inhibitor MG132 [117]. On the other hand, cyclosporine A treatment of cervical cancerous SiHa cells showed ER stress and UPR preceded by massive cytoplasmic vacuole formation that culminated in a paraptosis-like cell death [118]. Moreover, murine hepatoma 1c1c7 cells and the human non-small cell lung cancer A549 cell line exposed to a combination of photodamage and benzoporphyrin derivative result in ER swelling and paraptotic cell death [119].

For the pathways involved in paraptosis, ER vacuoles can be dependent on the PI3K/Akt signaling pathway [120]. Moreover, in BC3H1 myoblast cell lines exposed to yessotoxin, paraptosis was accompanied by cytoskeletal alterations and the activation of JNK/SAPK1 [121]. However, in acute lymphoblastic leukemia cells, everolimus, a mTOR inhibitor, showed that JNK signaling was not required for paraptotic cell death [122]. Paraptosis in epithelial ovarian cancer (EOC) cells treated with morusin was characterized by VDAC-mediated Ca2+ influx into mitochondria, and subsequent mitochondrial Ca2+ overload contributes to mitochondrial swelling and dysfunction, leading to the accumulation of ER stress markers, the generation of ROS, and the loss of mitochondrial membrane potential (Δψm) in EOC cells [123].

#### **7.2 The role of the RE in paraptosis during follicular atresia**

Knowledge of the role of paraptosis during follicular atresia is still limited. In *Bombyx mori*, apoptosis, autophagy, and paraptosis occur in the ovarian nurse cell cluster during late vitellogenesis, whereas middle vitellogenesis is exclusively characterized by the presence of paraptosis, preceding both apoptosis and autophagy [124]. In mammals, paraptosis was evidenced by ER swelling (**Figure 2d**) and CHOP immunodetection in granulosa cells during follicular atresia in adult Wistar rats [4].

The mechanisms involved in paraptosis during follicular atresia are still unknown. The paraptotic inductor IGFR might be related because it is implicated in follicular growth and selection [104, 125]. Moreover, IGF2R and the binding protein genes IGFBP5 and IGFBP6 are overexpressed in atretic follicles [126]. However, more studies on paraptosis during follicular atresia are necessary.

### **8. Conclusions**

Endoplasmic reticulum stress is a strong signal that triggers different programmed cell death pathways. Interestingly, programmed cell death via endoplasmic reticulum stress is not exclusive to pathological or experimental conditions but is present in physiological processes like follicular atresia. However, the specific mechanisms and signals for choosing a particular cell death pathway are still unknown. In this way, research on the pathways and mechanisms involved in programmed cell death activated by endoplasmic reticulum stress are fundamental, particularly for follicular atresia, as this process ensures the ovulation of competent oocytes for fertilization.

### **Acknowledgements**

This work was supported by PAPIIT IN225117 and PAPIIT IN227919.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Nayeli Torres-Ramírez, Rosario Ortiz-Hernández, M. Luisa Escobar-Sánchez, Olga M. Echeverría-Martínez and Gerardo H. Vázquez-Nin\* Laboratory of Electron Microscopy, Department of Cellular Biology, Faculty of Sciences, National Autonomous University of Mexico, Mexico City, Mexico

\*Address all correspondence to: vazqueznin@ciencias.unam.mx

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**85**

*Endoplasmic Reticulum Stress during Mammalian Follicular Atresia*

conformational switch activates mammalian IRE1. eLife. 2017;**6**:pii: e30700. DOI: 10.7554/eLife.30700

[10] Calfon M, Zeng H, Urano F, Till JH, Hubbard SR, Harding HP, et al. IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA. Nature.

2002;**415**(6867):92-96. DOI:

[11] Hollien J, Lin JH, Li H, Stevens N, Walter P, Weissman JS. Regulated Ire1 dependent decay of messenger RNAs in mammalian cells. The Journal of Cell Biology. 2009;**186**(3):323-331. DOI:

[12] Lee AH, Iwakoshi NN, Glimcher LH. XBP-1 regulates a subset of endoplasmic reticulum resident

chaperone genes in the unfolded protein response. Molecular and Cellular Biology. 2003;**23**(21):7448-7459

10.1038/415092a

10.1083/jcb.200903014

[13] Bertolotti A, Zhang Y,

10.1038/35014014

Hendershot LM, Harding HP, Ron D. Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response. Nature Cell Biology. 2000;**2**(6):326-332. DOI:

[14] Harding HP, Zhang Y, Bertolotti A, Zeng H, Ron D. Perk is essential for translational regulation and cell survival during the unfolded protein response. Molecular Cell. 2000;**5**(5):897-904

[15] Luo S, Mao C, Lee B, Lee AS. GRP78/BiP is required for cell proliferation and protecting the inner

[9] Su Q, Wang S, Gao HQ, Kazemi S, Harding HP, Ron D, et al. Modulation of the eukaryotic initiation factor 2 alpha-subunit kinase PERK by tyrosine phosphorylation. The Journal of Biological Chemistry. 2008;**283**(1):469- 475. DOI: 10.1074/jbc.M704612200

*DOI: http://dx.doi.org/10.5772/intechopen.82687*

Gonadotropins and estradiol stimulate immunoreactive insulin-like growth factor-I production by porcine

granulosa cells in vitro. Endocrinology. 1987;**120**(1):198-207. DOI: 10.1210/

[2] Escobar Sánchez ML, Echeverría Martínez OM, Vázquez-Nin GH. Immunohistochemical and

ultrastructural visualization of different routes of oocyte elimination in adult rats. European Journal of Histochemistry. 2012;**56**(2):e17. DOI: 10.4081/ejh.2012.17

[3] Escobar ML, Echeverría OM, Casasa AS, García G, Aguilar SJ, Vázquez-Nin GH. Involvement of pro-apoptotic and pro-autophagic proteins in granulosa cell death. Cell Biology. 2013;**1**(1):9-17.

DOI: 10.11648/j.cb.20130101.12

DOI: 10.1111/dgd.12322

[5] Brodsky JL. Cleaning up:

10.1016/j.cell.2012.11.012

1993;**73**(6):1197-1206

10.1126/science.1209038

[6] Cox JS, Shamu CE, Walter P. Transcriptional induction of genes encoding endoplasmic

ER-associated degradation to the rescue. Cell. 2012;**151**(6):1163-1167. DOI:

reticulum resident proteins requires a transmembrane protein kinase. Cell.

[7] Walter P, Ron D. The unfolded protein response: From stress pathway to homeostatic regulation. Science. 2011;**334**(6059):1081-1086. DOI:

[8] Karagöz GE, Acosta-Alvear D, Nguyen HT, Lee CP, Chu F, Walter P. An unfolded protein-induced

[4] Torres-Ramírez N, Escobar ML, Vázquez-Nin GH, Ortiz R, Echeverría OM. Paraptosis-like cell death in Wistar rat granulosa cells. Development, Growth & Differentiation. 2016;**58**(8):651-663.

[1] Hsu CJ, Hammond JM.

endo-120-1-198

**References**

*Endoplasmic Reticulum Stress during Mammalian Follicular Atresia DOI: http://dx.doi.org/10.5772/intechopen.82687*

#### **References**

*Endoplasmic Reticulum*

**8. Conclusions**

**Acknowledgements**

**Conflict of interest**

The authors declare no conflict of interest.

**84**

**Author details**

provided the original work is properly cited.

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Nayeli Torres-Ramírez, Rosario Ortiz-Hernández, M. Luisa Escobar-Sánchez,

Endoplasmic reticulum stress is a strong signal that triggers different programmed cell death pathways. Interestingly, programmed cell death via endoplasmic reticulum stress is not exclusive to pathological or experimental conditions but is present in physiological processes like follicular atresia. However, the specific mechanisms and signals for choosing a particular cell death pathway are still unknown. In this way, research on the pathways and mechanisms involved in programmed cell death activated by endoplasmic reticulum stress are fundamental, particularly for follicular atresia, as this process ensures the ovulation of competent oocytes for fertilization.

This work was supported by PAPIIT IN225117 and PAPIIT IN227919.

Laboratory of Electron Microscopy, Department of Cellular Biology, Faculty of Sciences, National Autonomous University of Mexico, Mexico City, Mexico

Olga M. Echeverría-Martínez and Gerardo H. Vázquez-Nin\*

\*Address all correspondence to: vazqueznin@ciencias.unam.mx

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[2] Escobar Sánchez ML, Echeverría Martínez OM, Vázquez-Nin GH. Immunohistochemical and ultrastructural visualization of different routes of oocyte elimination in adult rats. European Journal of Histochemistry. 2012;**56**(2):e17. DOI: 10.4081/ejh.2012.17

[3] Escobar ML, Echeverría OM, Casasa AS, García G, Aguilar SJ, Vázquez-Nin GH. Involvement of pro-apoptotic and pro-autophagic proteins in granulosa cell death. Cell Biology. 2013;**1**(1):9-17. DOI: 10.11648/j.cb.20130101.12

[4] Torres-Ramírez N, Escobar ML, Vázquez-Nin GH, Ortiz R, Echeverría OM. Paraptosis-like cell death in Wistar rat granulosa cells. Development, Growth & Differentiation. 2016;**58**(8):651-663. DOI: 10.1111/dgd.12322

[5] Brodsky JL. Cleaning up: ER-associated degradation to the rescue. Cell. 2012;**151**(6):1163-1167. DOI: 10.1016/j.cell.2012.11.012

[6] Cox JS, Shamu CE, Walter P. Transcriptional induction of genes encoding endoplasmic reticulum resident proteins requires a transmembrane protein kinase. Cell. 1993;**73**(6):1197-1206

[7] Walter P, Ron D. The unfolded protein response: From stress pathway to homeostatic regulation. Science. 2011;**334**(6059):1081-1086. DOI: 10.1126/science.1209038

[8] Karagöz GE, Acosta-Alvear D, Nguyen HT, Lee CP, Chu F, Walter P. An unfolded protein-induced

conformational switch activates mammalian IRE1. eLife. 2017;**6**:pii: e30700. DOI: 10.7554/eLife.30700

[9] Su Q, Wang S, Gao HQ, Kazemi S, Harding HP, Ron D, et al. Modulation of the eukaryotic initiation factor 2 alpha-subunit kinase PERK by tyrosine phosphorylation. The Journal of Biological Chemistry. 2008;**283**(1):469- 475. DOI: 10.1074/jbc.M704612200

[10] Calfon M, Zeng H, Urano F, Till JH, Hubbard SR, Harding HP, et al. IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA. Nature. 2002;**415**(6867):92-96. DOI: 10.1038/415092a

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[12] Lee AH, Iwakoshi NN, Glimcher LH. XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response. Molecular and Cellular Biology. 2003;**23**(21):7448-7459

[13] Bertolotti A, Zhang Y, Hendershot LM, Harding HP, Ron D. Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response. Nature Cell Biology. 2000;**2**(6):326-332. DOI: 10.1038/35014014

[14] Harding HP, Zhang Y, Bertolotti A, Zeng H, Ron D. Perk is essential for translational regulation and cell survival during the unfolded protein response. Molecular Cell. 2000;**5**(5):897-904

[15] Luo S, Mao C, Lee B, Lee AS. GRP78/BiP is required for cell proliferation and protecting the inner cell mass from apoptosis during early mouse embryonic development. Molecular and Cellular Biology. 2006;**26**(15):5688-5697. DOI: 10.1128/ MCB.00779-06

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[21] Kovács J, Forgó V, Péczely P. The fine structure of the follicular cells in growing and atretic ovarian follicles of the domestic goose. Cell and Tissue Research. 1992;**267**(3):561-569

[22] Huang N, Yu Y, Qiao J. Dual role for the unfolded protein response in the ovary: Adaption and apoptosis. Protein & Cell. 2017;**8**(1):14-24. DOI: 10.1007/ s13238-016-0312-3

[23] Harada M, Nose E, Takahashi N, Hirota Y, Hirata T, Yoshino O, et al. Evidence of the activation of unfolded protein response in granulosa and cumulus cells during follicular growth and maturation. Gynecological Endocrinology. 2015;**31**(10):783-787. DOI: 10.3109/09513590.2015.1062862

[24] Yang X, Wu LL, Chura LR, Liang X, Lane M, Norman RJ, et al. Exposure to lipid-rich follicular fluid is associated with endoplasmic reticulum stress and impaired oocyte maturation in cumulus-oocyte complexes. Fertility and Sterility. 2012;**97**(6):1438-1443. DOI: 10.1016/j.fertnstert.2012.02.034

[25] Lumley EC, Osborn AR, Scott JE, Scholl AG, Mercado V, McMahan YT, et al. Moderate endoplasmic reticulum stress activates a PERK and p38-dependent apoptosis. Cell Stress & Chaperones. 2017;**22**(1):43-54. DOI: 10.1007/s12192-016-0740-2

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[42] Gupta S, Read DE, Deepti A, Cawley K, Gupta A, Oommen D, et al. Perkdependent repression of miR-106b-25 cluster is required for ER stress-induced apoptosis. Cell Death & Disease. 2012;**3**:e333. DOI: 10.1038/cddis.2012.74

[43] Puthalakath H, O'Reilly LA, Gunn P, Lee L, Kelly PN, Huntington ND, et al. ER stress triggers apoptosis by activating BH3-only protein Bim. Cell. 2007;**129**(7):1337-1349. DOI: 10.1016/j.

[44] Li J, Lee B, Lee AS. Endoplasmic reticulum stress-induced apoptosis: Multiple pathways and activation of p53-up-regulated modulator of apoptosis (PUMA) and NOXA by p53. The Journal of Biological Chemistry. 2006;**281**(11):7260-7270. DOI: 10.1074/

journal.pone.0184907

10.1126/science.1123480

cell.2007.04.027

jbc.M509868200

*DOI: http://dx.doi.org/10.5772/intechopen.82687*

in mitochondrial apoptosis. Current Opinion in Physiology. 2018;**3**:71-81. DOI: 10.1016/j.cophys.2018.03.005

[31] Holinger EP, Chittenden T, Lutz RJ. Bak BH3 peptides antagonize Bcl-xL function and induce apoptosis through cytochrome c-independent activation of caspases. The Journal of Biological Chemistry. 1999;**274**(19):13298-13304

[32] Narita M, Shimizu S, Ito T, Chittenden T, Lutz RJ, Matsuda H, et al. Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria. Proceedings of the National Academy of Sciences of the United States of America. 1998;**95**(25):14681-14686

[33] Bröker LE, Kruyt FA, Giaccone G. Cell death independent of caspases: A review. Clinical Cancer Research. 2005;**11**(9):3155-3162. DOI: 10.1158/1078-0432.CCR-04-2223

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DOI: 10.1210/endo-129-5-2799

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[36] Coucouvanis EC, Sherwood SW, Carswell-Crumpton C, Spack EG, Jones PP. Evidence that the mechanism of prenatal germ cell death in the mouse is apoptosis. Experimental Cell Research. 1993;**209**(2):238-247. DOI: 10.1006/

[37] Escobar ML, Echeverría OM, Sánchez-Sánchez L, Méndez C,

10.1007/s10495-009-0448-1

Pedernera E, Vázquez-Nin GH. Analysis of different cell death processes of prepubertal rat oocytes in vitro. Apoptosis. 2010;**15**(4):511-526. DOI:

Endocrinology. 1991;**129**(5):2799-2801.

*Endoplasmic Reticulum Stress during Mammalian Follicular Atresia DOI: http://dx.doi.org/10.5772/intechopen.82687*

in mitochondrial apoptosis. Current Opinion in Physiology. 2018;**3**:71-81. DOI: 10.1016/j.cophys.2018.03.005

*Endoplasmic Reticulum*

MCB.00779-06

cell mass from apoptosis during early mouse embryonic development. Molecular and Cellular Biology. 2006;**26**(15):5688-5697. DOI: 10.1128/ [22] Huang N, Yu Y, Qiao J. Dual role for the unfolded protein response in the ovary: Adaption and apoptosis. Protein & Cell. 2017;**8**(1):14-24. DOI: 10.1007/

[23] Harada M, Nose E, Takahashi N, Hirota Y, Hirata T, Yoshino O, et al. Evidence of the activation of unfolded protein response in granulosa and cumulus cells during follicular growth and maturation. Gynecological Endocrinology. 2015;**31**(10):783-787. DOI: 10.3109/09513590.2015.1062862

[24] Yang X, Wu LL, Chura LR, Liang X, Lane M, Norman RJ, et al. Exposure to lipid-rich follicular fluid is associated with endoplasmic reticulum stress and impaired oocyte maturation in cumulus-oocyte complexes. Fertility and Sterility. 2012;**97**(6):1438-1443. DOI: 10.1016/j.fertnstert.2012.02.034

[25] Lumley EC, Osborn AR, Scott JE, Scholl AG, Mercado V, McMahan YT, et al. Moderate endoplasmic reticulum stress activates a PERK and p38-dependent apoptosis. Cell Stress & Chaperones. 2017;**22**(1):43-54. DOI:

10.1007/s12192-016-0740-2

Cell. 1996;**85**(6):781-784

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s13238-016-0312-3

[16] Schindler AJ, Schekman R. In vitro reconstitution of ER-stress induced ATF6 transport in COPII vesicles. Proceedings of the National Academy of Sciences of the United States of America. 2009;**106**(42):17775-17780. DOI: 10.1073/pnas.0910342106

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pathway flux dedifferentiates INS-1E cells and murine islets by an extracellular signal-regulated kinase (ERK)1/2-mediated signal transmission pathway. Diabetologia. 2012;**55**(1):141- 153. DOI: 10.1007/s00125-011-2315-1

[20] Henderson KM, McNatty KP, Smith P, Gibb M, O'Keeffe LE, Lun S, et al. Influence of follicular health on the steroidogenic and morphological characteristics of bovine granulosa cells in vitro. Journal of Reproduction and

Fertility. 1987;**79**(1):185-193

Research. 1992;**267**(3):561-569

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2001;**107**(7):881-891

**86**

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[105] Jeong W, Song G, Bazer FW, Kim J. Insulin-like growth factor I induces

proliferation and migration of porcine trophectoderm cells through multiple cell signaling pathways, including protooncogenic protein kinase 1 and mitogen-activated protein kinase. Molecular and Cellular Endocrinology. 2014;**384**(1-2):175-184. DOI: 10.1016/j. mce.2014.01.023

[106] Sperandio S, Poksay K, de Belle I, Lafuente MJ, Liu B, Nasir J, et al. Paraptosis: Mediation by MAP kinases and inhibition by AIP-1/Alix. Cell Death and Differentiation. 2004;**11**(10):1066- 1075. DOI: 10.1038/sj.cdd.4401465

[107] Wang Y, Li X, Wang L, Ding P, Zhang Y, Han W, et al. An alternative form of paraptosis-like cell death triggered by TAJ/TROY and enhanced by PDCD5 overexpression. Journal of Cell Science. 2004;**117**(Pt 8):1525-1532. DOI: 10.1242/jcs.00994

[108] Han H, Chou CC, Li R, Liu J, Zhang L, Zhu W, et al. Chalcomoracin is a potent anticancer agent acting through triggering oxidative stress via a mitophagy- and paraptosisdependent mechanism. Scientific Reports. 2018;**8**(1):9566. DOI: 10.1038/ s41598-018-27724-3

[109] Petrillo S, Chiabrando D, Genova T, Fiorito V, Ingoglia G, Vinchi F, et al. Heme accumulation in endothelial cells impairs angiogenesis by triggering paraptosis. Cell Death and Differentiation. 2018;**25**(3):573-588. DOI: 10.1038/s41418-017-0001-7

[110] Hager S, Korbula K, Bielec B, Grusch M, Pirker C, Schosserer M, et al. The thiosemicarbazone Me2NNMe2 induces paraptosis by disrupting the ER thiol redox homeostasis based on protein disulfide isomerase inhibition. Cell Death & Disease. 2018;**9**(11):1052. DOI: 10.1038/s41419-018-1102-z

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BRAFV600E in FRO anaplastic thyroid carcinoma cells. Anticancer Research.

2014;**34**(9):4857-4868

[118] Ram BM, Ramakrishna G. Endoplasmic reticulum vacuolation and unfolded protein response leading to paraptosis like cell death in cyclosporine a treated cancer cervix cells is mediated by cyclophilin B inhibition. Biochimica et Biophysica Acta. 2014;**1843**(11):2497-2512. DOI:

10.1016/j.bbamcr.2014.06.020

php.12805

[119] Kessel D, Reiners JJ Jr. Effects of combined Lysosomal and mitochondrial Photodamage in a non-small-cell Lung Cancer cell line: The role of paraptosis. Photochemistry and Photobiology. 2017;**93**(6):1502-1508. DOI: 10.1111/

[120] Monel B, Compton AA, Bruel T, Amraoui S, Burlaud-Gaillard J, Roy N, et al. Zika virus induces massive cytoplasmic vacuolization and paraptosis-like death in infected cells. The EMBO Journal. 2017;**36**(12):1653- 1668. DOI: 10.15252/embj.201695597

[121] Korsnes MS, Espenes A, Hetland DL, Hermansen LC. Paraptosis-like cell death induced by yessotoxin. Toxicology In Vitro. 2011;**25**(8):1764-1770. DOI:

[122] Baraz R, Cisterne A, Saunders PO, Hewson J, Thien M, Weiss J, et al. mTOR inhibition by everolimus in childhood acute lymphoblastic

independent cell death. PLoS One. 2014;**9**(7):e102494. DOI: 10.1371/

[123] Xue J, Li R, Zhao X, Ma C, Lv X, Liu L, et al. Morusin induces paraptosis-like cell death through mitochondrial calcium overload and dysfunction in epithelial ovarian cancer. Chemico-Biological Interactions. 2018;**283**:59-74. DOI:

10.1016/j.tiv.2011.09.005

leukemia induces caspase-

journal.pone.0102494

10.1016/j.cbi.2018.02.003

*DOI: http://dx.doi.org/10.5772/intechopen.82687*

inducing endoplasmic reticulum stress via disruption of thiol proteostasis. Oncotarget. 2017;**8**(63):106740-106752.

[112] Park SS, Lee DM, Lim JH, Lee D, Park SJ, Kim HM, et al. Pyrrolidine dithiocarbamate reverses Bcl-xLmediated apoptotic resistance to doxorubicin by inducing paraptosis. Carcinogenesis. 2018;**39**(3):458-470.

[113] Sperandio S, Poksay KS, Schilling B, Crippen D, Gibson BW, Bredesen DE. Identification of new modulators and protein alterations in nonapoptotic programmed cell death. Journal of Cellular Biochemistry. 2010;**111**(6):1401-1412. DOI: 10.1002/

DOI: 10.18632/oncotarget.22537

DOI: 10.1093/carcin/bgy003

[114] Tay KH, Luan Q, Croft A, Jiang CC, Jin L, Zhang XD, et al. Sustained IRE1 and ATF6 signaling is important for survival of melanoma cells undergoing ER stress. Cellular Signalling. 2014;**26**(2):287-294. DOI:

10.1016/j.cellsig.2013.11.008

[115] Nedungadi D, Binoy A, Pandurangan N, Pal S, Nair BG, Mishra N. 6-Shogaol induces caspaseindependent paraptosis in cancer cells via proteasomal inhibition. Experimental Cell Research.

yexcr.2018.02.018

2018;**364**(2):243-251. DOI: 10.1016/j.

[116] Wang WB, Feng LX, Yue QX, Wu WY, Guan SH, Jiang BH, et al. Paraptosis accompanied by autophagy and apoptosis was induced by celastrol, a natural compound with influence on proteasome, ER stress and Hsp 90. Journal of Cellular Physiology. 2012;**227**(5):2196-2206. DOI: 10.1002/

[117] Kim SH, Shin HY, Kim YS, Kang JG, Kim CS, Ihm SH, et al. Tunicamycin induces paraptosis potentiated by inhibition of

jcb.22870

*Endoplasmic Reticulum Stress during Mammalian Follicular Atresia DOI: http://dx.doi.org/10.5772/intechopen.82687*

inducing endoplasmic reticulum stress via disruption of thiol proteostasis. Oncotarget. 2017;**8**(63):106740-106752. DOI: 10.18632/oncotarget.22537

*Endoplasmic Reticulum*

pone.0063038

[99] Leon LJ, Pasupuleti N, Gorin F, Carraway KL 3rd. A cell-permeant amiloride derivative induces

proliferation and migration of porcine trophectoderm cells through multiple cell signaling pathways, including protooncogenic protein kinase 1 and mitogen-activated protein kinase. Molecular and Cellular Endocrinology. 2014;**384**(1-2):175-184. DOI: 10.1016/j.

[106] Sperandio S, Poksay K, de Belle I, Lafuente MJ, Liu B, Nasir J, et al. Paraptosis: Mediation by MAP kinases and inhibition by AIP-1/Alix. Cell Death and Differentiation. 2004;**11**(10):1066- 1075. DOI: 10.1038/sj.cdd.4401465

[107] Wang Y, Li X, Wang L, Ding P, Zhang Y, Han W, et al. An alternative form of paraptosis-like cell death triggered by TAJ/TROY and enhanced by PDCD5 overexpression. Journal of Cell Science. 2004;**117**(Pt 8):1525-1532.

[108] Han H, Chou CC, Li R, Liu J, Zhang L, Zhu W, et al. Chalcomoracin is a potent anticancer agent acting through triggering oxidative stress via a mitophagy- and paraptosisdependent mechanism. Scientific Reports. 2018;**8**(1):9566. DOI: 10.1038/

[109] Petrillo S, Chiabrando D, Genova T, Fiorito V, Ingoglia G, Vinchi F, et al. Heme accumulation in endothelial cells impairs angiogenesis by

triggering paraptosis. Cell Death and Differentiation. 2018;**25**(3):573-588. DOI: 10.1038/s41418-017-0001-7

[110] Hager S, Korbula K, Bielec B, Grusch M, Pirker C, Schosserer M, et al. The thiosemicarbazone Me2NNMe2 induces paraptosis by disrupting the ER thiol redox homeostasis based on protein disulfide isomerase inhibition. Cell Death & Disease. 2018;**9**(11):1052. DOI: 10.1038/s41419-018-1102-z

[111] Kim IY, Kwon M, Choi MK, Lee D, Lee DM, Seo MJ, et al. Ophiobolin a kills human glioblastoma cells by

DOI: 10.1242/jcs.00994

s41598-018-27724-3

mce.2014.01.023

caspase-independent, AIF-mediated programmed necrotic death of breast cancer cells. PLoS One.

2013;**8**(4):e63038. DOI: 10.1371/journal.

[100] Pasupuleti N, Leon L, Carraway KL 3rd, Gorin F. 5-Benzylglycinyl-amiloride kills proliferating and nonproliferating malignant glioma cells through caspaseindependent necroptosis mediated by apoptosis-inducing factor. The Journal of Pharmacology and Experimental Therapeutics. 2013;**344**(3):600-615.

[101] Cao WX, Li T, Tang ZH, Zhang LL, Wang ZY, Guo X, et al. MLKL mediates apoptosis via a mutual regulation with PERK/eIF2α pathway in response to reactive oxygen species generation. Apoptosis. 2018;**23**(9-10):521-531. DOI:

DOI: 10.1124/jpet.112.200519

10.1007/s10495-018-1475-6

10.1038/cddis.2015.51

DOI: 10.1002/jcp.27562

pnas.97.26.14376

[102] Blohberger J, Kunz L, Einwang D, Berg U, Berg D, Ojeda SR, et al. Readthrough acetylcholinesterase (AChE-R) and regulated necrosis: Pharmacological targets for the regulation of ovarian functions? Cell Death & Disease. 2015;**6**:e1685. DOI:

[103] Chaudhary GR, Yadav PK, Yadav AK, Tiwari M, Gupta A, Sharma A, et al. Necrosis and necroptosis in germ cell depletion from mammalian ovary. Journal of Cellular Physiology. 2018.

[104] Sperandio S, de Belle I, Bredesen DE. An alternative, nonapoptotic form of programmed cell death. Proceedings of the National Academy of Sciences of the United States of America.

2000;**97**(26):14376-14381. DOI: 10.1073/

[105] Jeong W, Song G, Bazer FW, Kim J. Insulin-like growth factor I induces

**92**

[112] Park SS, Lee DM, Lim JH, Lee D, Park SJ, Kim HM, et al. Pyrrolidine dithiocarbamate reverses Bcl-xLmediated apoptotic resistance to doxorubicin by inducing paraptosis. Carcinogenesis. 2018;**39**(3):458-470. DOI: 10.1093/carcin/bgy003

[113] Sperandio S, Poksay KS, Schilling B, Crippen D, Gibson BW, Bredesen DE. Identification of new modulators and protein alterations in nonapoptotic programmed cell death. Journal of Cellular Biochemistry. 2010;**111**(6):1401-1412. DOI: 10.1002/ jcb.22870

[114] Tay KH, Luan Q, Croft A, Jiang CC, Jin L, Zhang XD, et al. Sustained IRE1 and ATF6 signaling is important for survival of melanoma cells undergoing ER stress. Cellular Signalling. 2014;**26**(2):287-294. DOI: 10.1016/j.cellsig.2013.11.008

[115] Nedungadi D, Binoy A, Pandurangan N, Pal S, Nair BG, Mishra N. 6-Shogaol induces caspaseindependent paraptosis in cancer cells via proteasomal inhibition. Experimental Cell Research. 2018;**364**(2):243-251. DOI: 10.1016/j. yexcr.2018.02.018

[116] Wang WB, Feng LX, Yue QX, Wu WY, Guan SH, Jiang BH, et al. Paraptosis accompanied by autophagy and apoptosis was induced by celastrol, a natural compound with influence on proteasome, ER stress and Hsp 90. Journal of Cellular Physiology. 2012;**227**(5):2196-2206. DOI: 10.1002/ jcp.22956

[117] Kim SH, Shin HY, Kim YS, Kang JG, Kim CS, Ihm SH, et al. Tunicamycin induces paraptosis potentiated by inhibition of

BRAFV600E in FRO anaplastic thyroid carcinoma cells. Anticancer Research. 2014;**34**(9):4857-4868

[118] Ram BM, Ramakrishna G. Endoplasmic reticulum vacuolation and unfolded protein response leading to paraptosis like cell death in cyclosporine a treated cancer cervix cells is mediated by cyclophilin B inhibition. Biochimica et Biophysica Acta. 2014;**1843**(11):2497-2512. DOI: 10.1016/j.bbamcr.2014.06.020

[119] Kessel D, Reiners JJ Jr. Effects of combined Lysosomal and mitochondrial Photodamage in a non-small-cell Lung Cancer cell line: The role of paraptosis. Photochemistry and Photobiology. 2017;**93**(6):1502-1508. DOI: 10.1111/ php.12805

[120] Monel B, Compton AA, Bruel T, Amraoui S, Burlaud-Gaillard J, Roy N, et al. Zika virus induces massive cytoplasmic vacuolization and paraptosis-like death in infected cells. The EMBO Journal. 2017;**36**(12):1653- 1668. DOI: 10.15252/embj.201695597

[121] Korsnes MS, Espenes A, Hetland DL, Hermansen LC. Paraptosis-like cell death induced by yessotoxin. Toxicology In Vitro. 2011;**25**(8):1764-1770. DOI: 10.1016/j.tiv.2011.09.005

[122] Baraz R, Cisterne A, Saunders PO, Hewson J, Thien M, Weiss J, et al. mTOR inhibition by everolimus in childhood acute lymphoblastic leukemia induces caspaseindependent cell death. PLoS One. 2014;**9**(7):e102494. DOI: 10.1371/ journal.pone.0102494

[123] Xue J, Li R, Zhao X, Ma C, Lv X, Liu L, et al. Morusin induces paraptosis-like cell death through mitochondrial calcium overload and dysfunction in epithelial ovarian cancer. Chemico-Biological Interactions. 2018;**283**:59-74. DOI: 10.1016/j.cbi.2018.02.003

[124] Mpakou VE, Nezis IP, Stravopodis DJ, Margaritis LH, Papassideri IS. Different modes of programmed cell death during oogenesis of the silkmoth Bombyx mori. Autophagy. 2008;**4**(1):97-100

[125] Beg MA, Ginther OJ. Follicle selection in cattle and horses: Role of intrafollicular factors. Reproduction. 2006;**132**:365-377. DOI: 10.1530/ rep.1.01233

[126] Hatzirodos N, Hummitzsch K, Irving-Rodgers HF, Harland ML, Morris SE, Rodgers RJ. Transcriptome profiling of granulosa cells from bovine ovarian follicles during atresia. BMC Genomics. 2014;**15**:40. DOI: 10.1186/1471-2164-15-40

*Endoplasmic Reticulum*

2008;**4**(1):97-100

rep.1.01233

[124] Mpakou VE, Nezis IP, Stravopodis DJ, Margaritis LH, Papassideri IS. Different modes of programmed cell death during oogenesis of the silkmoth Bombyx mori. Autophagy.

[125] Beg MA, Ginther OJ. Follicle selection in cattle and horses: Role of intrafollicular factors. Reproduction. 2006;**132**:365-377. DOI: 10.1530/

[126] Hatzirodos N, Hummitzsch K, Irving-Rodgers HF, Harland ML, Morris SE, Rodgers RJ. Transcriptome profiling of granulosa cells from bovine ovarian follicles during atresia. BMC Genomics. 2014;**15**:40. DOI:

10.1186/1471-2164-15-40

**94**

### *Edited by Angel Català*

The purpose of this book is to concentrate on recent developments on endoplasmic reticulum. The articles collected in this book are contributions by invited researchers with a long-standing experience in different research areas. We hope that the material presented here is understandable to a broad audience, not only scientists but also people with general background in many different biological sciences. This volume offers you up-to-date, expert reviews of the fast-moving field of endoplasmic reticulum. The book is divided in two sections: 1. Introduction and 2. Endoplasmic Reticulum Properties and Functions.

Published in London, UK © 2019 IntechOpen © wir0man / iStock

Endoplasmic Reticulum

IntechOpen Book Series

Physiology, Volume 2

Endoplasmic Reticulum

*Edited by Angel Català*