**Necrosis**

**Chapter 18**

## **HMGB1 in Cell Death**

### Daolin Tang and Rui Kang

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61208

#### **Abstract**

High mobility group box 1 (HMGB1) is named for its electrophoretic mobility on pol‐ yacrylamide gels when it was first identified in calf thymus in 1973. HMGB1 plays a critical role in the stress response not only inside the cell as a DNA chaperone and cell death regulator, but also outside the cell as the prototypic damage-associated molecu‐ lar pattern molecule. The physiological and pathological role of HMGB1 in health and disease has been widely studied for years. In this chapter, we will focus on the release and function of HMGB1 in cell death types such as apoptosis, autophagy, and ne‐ crosis.

**Keywords:** hmgb1, autophagy, necrosis, apoptosis

### **1. Introduction**

Cell death is the cell's process of losing its biological ability to carry out all the essential life processes. The Nomenclature Committee on Cell Death proposes several cell death classifica‐ tion criteria. According to morphological appearance, cell death is divided into apoptosis (type I), autophagy (type II), and necrosis (type III) [1, 2]. According to enzymological qualities, cell death is divided into several subtypes depending on the involvement or noninvolvement of nuclei or distinct protease classes such as caspases, calpains, cathepsins, and transglutami‐ nases. According to immunological characteristics, cell death is divided into immunogenic or tolerogenic cell death [3]. For example, apoptosis is generally considered nonimmunogenic cell death, whereas necrosis is considered immunogenic cell death. In addition, cell death can be classified into regulated or accidental cell death based on functional aspects [4]. Accidental cell death is caused by unexpected and accidental cell damage (e.g., ischemic and trauma), whereas regulated cell death is mediated by an expected program in response to different stimuli. The list of regulated cell death subtypes is rapidly increasing and includes anoikis, autophagic cell death, apoptosis, cornification, entosis, ferroptosis, mitotic catastrophe,

© 2015 The Author(s). Licensee InTech. 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.

necroptosis, netosis, parthanatos, and pyroptosis [4]. Cell death is essential for a plethora of physiological processes, and its deregulation is implicated in several human diseases such as infections, neurodegeneration, cancer, autoimmunity, and ischemic disease [5-7]. During the past few decades, a number of important concepts regarding the regulation of cell death and its roles in human health and disease have arisen. Understanding the molecular mechanisms and signaling pathways of cell death is crucial for identifying new diagnostic and therapeutic targets.

Compared to pathogen-associated molecular pattern molecules (PAMPs), which are generated from the components of foreign pathogens such as bacteria and viruses, damage-associated molecular pattern molecules (DAMPs) are endogenous or self-molecules that are secreted, released, or undergo surface exposure by dead, dying, or injured cells [8-12]. Both PAMPs and DAMPs are mainly recognized by pattern recognition receptors such as receptor for advanced glycation end products (RAGE) and toll-like receptors (TLRs) to mediate the inflammatory, immunity, and metabolism response. The release and activity of DAMPs during cell death can determine whether cell death is immunogenic or tolerogenic [13]. Thus, DAMPs are suitable emergent targets for cell-death-associated immune therapy.

High mobility group box 1 (HMGB1) is named for its electrophoretic mobility on polyacryla‐ mide gels when it was first identified in calf thymus in 1973 [14]. As an extremely conserved protein, HMGB1 originated before the divergence of the protostomes and deuterostomes, approximately 525 million years ago [15]. HMGB1 shares 100% amino acid sequence identity between mice and rats, and a 99% homology between rodents and humans [16-18]. The homolog of mammalian HMGB1 has been reported for several species such as *Nhp6A/B* in yeast and *HMG-D* and *DSP1* in *Drosophila* [19-21]. HMGB1 plays a critical role in the stress response not only inside the cell as a DNA chaperone and cell death regulator, but also outside the cell as the prototypic DAMP. The physiological and pathological role of HMGB1 in health and disease has been widely studied for years [22]. In this chapter, we will focus on the release and function of HMGB1 in cell death types such as apoptosis, autophagy, and necrosis.

### **2. HMGB1 structure and function**

### **2.1. Structure**

Human HMGB1 consists of 215 amino acid residues and has two L-shaped DNA-binding domains (HMG A box [9-79aa], HMG B box [95-163aa]) and a shorter C-terminal tail (186-215aa) [23]. Both A- and B-box domains are necessary for efficient DNA bending and flexure. HMGB1 binds to DNA without apparent sequence specificity. HMGB1 normally locates in the nucleus due to two nuclear-localization signals (NLS): NLS1 (28-44aa) and NLS2 (179-185aa) [24]. In contrast, HMGB1 contains nuclear-emigration signals in DNA-binding domains, which contributes to extranuclear HMGB1 during stress in a nuclear exportin chromosome-region maintenance 1-dependent manner. In addition to DNA, HMGB1 can bind a number of proteins involved in multiple biologic processes. For example, HMGB1 binds to RAGE, TLR4, and p53 by residues 150-183, 89-108, and 7-74, which mediates cell migration [25], cytokine production [26], and gene transcription [27], respectively. The recombinant B box protein exhibits proinflammatory activity, whereas the recombinant A box protein displays anti-inflammatory activity [28], although the potential mechanism remains unknown. The C terminus is composed of 30 acidic amino acid residues and is able to regulate DNA binding/bending by intramolecular interaction with the A- and B- box [29, 30] or by intermo‐ lecular interaction with histones (e.g., H1 and H3) [31, 32]. Additionally, residues 201-205 in the C-terminal acidic tail region contribute to the antibacterial activity of recombinant HMGB1 [33]. Hence, the structural basis of HMGB1 determines its biological function.

#### **2.2. Intracellular HMGB1**

#### *2.2.1. Nuclear HMGB1*

necroptosis, netosis, parthanatos, and pyroptosis [4]. Cell death is essential for a plethora of physiological processes, and its deregulation is implicated in several human diseases such as infections, neurodegeneration, cancer, autoimmunity, and ischemic disease [5-7]. During the past few decades, a number of important concepts regarding the regulation of cell death and its roles in human health and disease have arisen. Understanding the molecular mechanisms and signaling pathways of cell death is crucial for identifying new diagnostic and therapeutic

Compared to pathogen-associated molecular pattern molecules (PAMPs), which are generated from the components of foreign pathogens such as bacteria and viruses, damage-associated molecular pattern molecules (DAMPs) are endogenous or self-molecules that are secreted, released, or undergo surface exposure by dead, dying, or injured cells [8-12]. Both PAMPs and DAMPs are mainly recognized by pattern recognition receptors such as receptor for advanced glycation end products (RAGE) and toll-like receptors (TLRs) to mediate the inflammatory, immunity, and metabolism response. The release and activity of DAMPs during cell death can determine whether cell death is immunogenic or tolerogenic [13]. Thus, DAMPs are suitable

High mobility group box 1 (HMGB1) is named for its electrophoretic mobility on polyacryla‐ mide gels when it was first identified in calf thymus in 1973 [14]. As an extremely conserved protein, HMGB1 originated before the divergence of the protostomes and deuterostomes, approximately 525 million years ago [15]. HMGB1 shares 100% amino acid sequence identity between mice and rats, and a 99% homology between rodents and humans [16-18]. The homolog of mammalian HMGB1 has been reported for several species such as *Nhp6A/B* in yeast and *HMG-D* and *DSP1* in *Drosophila* [19-21]. HMGB1 plays a critical role in the stress response not only inside the cell as a DNA chaperone and cell death regulator, but also outside the cell as the prototypic DAMP. The physiological and pathological role of HMGB1 in health and disease has been widely studied for years [22]. In this chapter, we will focus on the release and function of HMGB1 in cell death types such as apoptosis, autophagy, and necrosis.

Human HMGB1 consists of 215 amino acid residues and has two L-shaped DNA-binding domains (HMG A box [9-79aa], HMG B box [95-163aa]) and a shorter C-terminal tail (186-215aa) [23]. Both A- and B-box domains are necessary for efficient DNA bending and flexure. HMGB1 binds to DNA without apparent sequence specificity. HMGB1 normally locates in the nucleus due to two nuclear-localization signals (NLS): NLS1 (28-44aa) and NLS2 (179-185aa) [24]. In contrast, HMGB1 contains nuclear-emigration signals in DNA-binding domains, which contributes to extranuclear HMGB1 during stress in a nuclear exportin chromosome-region maintenance 1-dependent manner. In addition to DNA, HMGB1 can bind a number of proteins involved in multiple biologic processes. For example, HMGB1 binds to RAGE, TLR4, and p53 by residues 150-183, 89-108, and 7-74, which mediates cell migration

emergent targets for cell-death-associated immune therapy.

**2. HMGB1 structure and function**

**2.1. Structure**

targets.

398 Cell Death - Autophagy, Apoptosis and Necrosis

HMGB1 translocates between the cytoplasm and the nucleus, but normally stays in the nucleus in most cells and tissues. Nuclear HMGB1 is the structural protein of chromatin and orchestrates a number of nuclear events by its DNA chaperone activity as follows: (1) Nucleosome stability and sliding. As basic unit of chromatin, nucleosome contains a short length of DNA wrapped around a core of histone proteins. HMGB1 and histone H1 can bind to linker DNA between successive nucleosomes in the chromatin fiber [34]. H1 stabilizes nucleosome with less mobility, whereas HMGB1 relaxes nucleosome and makes chromatin more accessible at the distorted site [35, 36]. (2) Nucleosome number and genome chromati‐ nization. Loss of HMGB1 in mammalian and yeast cells leads to 20-30% less histones and nucleosomes and more RNA transcripts [37]. (3) Nuclear catastrophe and nucleosome release. Conditional knockout of HMGB1 in the pancreas causes nuclear oxidative injury and proinflammatory nucleosome release, which mediates sterile inflammation [38]. (4) DNA bending and binding. HMGB1 binds to DNA with structure-specificity, but not sequencespecificity [39]. After binding DNA, the major function of HMGB1 is to bend and change DNA conformation by unwinding [40], looping [41], or compacting DNA [42]. This DNA chaperone activity of HMGB1 is implicated in the regulation of gene transcription [43], DNA repair [44], DNA replication [45], V(D)J recombination [46], gene delivery [47], and gene transfer [48]. (5) Telomere homeostasis. Loss of HMGB1 in yeast and mammalian cells inhibits telomerase activity, decreases telomere length, and increases DNA damage and chromoso‐ mal instability [49].

#### *2.2.2. Cytosolic HMGB1*

Several cell types (e.g., fibroblasts [50], thymocytes [51]), and tissue types (e.g., liver, kidney, heart, and lung) [52] have normal cytosolic HMGB1 expression. The ratio of nuclear to cytoplasmic HMGB1 is about 30:1 [52]. Importantly, the translation of HMGB1 from the nucleus to the cytosol, including mitochondria and lysosomes, are observed in response to various stressors (e.g., cytokines, chemokines, heat, hypoxia, oxidative stress, and oncogenes). Although the function of cytosolic HMGB1 still remains poorly studied, HMGB1 may act as a positive regulator of mitochondrial quality in an autophagy-dependent and autophagyindependent manner [53, 54], which will be discussed later in the "Autophagy" section. In addition to autophagy, cytosolic HMGB1 is involved in the regulation of unconventional secretory pathways based on mass spectrometry-mediated binding partner analysis [55]. In one study, several HMGB1-binding partners in nuclear and cytosol fraction were identified in several cancer cells [55]. Among them, nine of the cytosolic HMGB1-binding proteins were related to protein translocation and secretion. In particular, immunoprecipitation analysis further confirmed four cytosolic HMGB1-binding proteins, including annexin A2, myosin IC isoform a, myosin-9, and Ras-related protein Rab10 [55]. These proteins are directly implicated in the process of unconventional protein secretion. Further studies are needed to define the function of cytosolic HMGB1 in unconventional protein secretion. In addition to nuclear and cytosolic HMGB1, intracellular HMGB1 presents on cell surface membranes and regulates neurite outgrowth [56], platelet activation [57, 58], cell differentiation [59], erythroid matura‐ tion [60], adhesion [61], and innate immunity [62].

#### **2.3. Extracellular HMGB1**

HMGB1 is released in two different ways. On the one hand, HMGB1 can be actively secreted by normal cells, especially immune and endothelial cells [63, 64]. On the other hand, HMGB1 can be passively released by dead, dying, or injured cells in response to autophagic cell death [65], apoptosis [66, 67], necrosis [68], necroptosis [69, 70], netosis [71], and pyroptosis [72]. Oxidative stress refers to elevated intracellular levels of reactive oxygen species (ROS) that play a central role in the regulation of HMGB1 secretion and release, although the actual mechanism of action remains ambiguous [73]. Once released, HMGB1 acts as a cytokine, chemokine, and growth factor that is implicated in multiple biological processes including inflammation, immunity, migration, invasion, metabolism, proliferation, differentiation, antimicrobial defense, angiogenesis, tissue regeneration, death, autophagy, senescence, and efferocytosis. Extracellular HMGB1 plays important roles in the pathogenesis of human disease and is a potential therapy target in infection and sterile inflammation [74-76]. Several factors can affect HMGB1 activity in different experimental settings. For example, RAGE [77] and TLRs [78, 79] are positive receptors in macrophages and fibroblasts, whereas CD24 [80] and T cell immunoglobulin mucin 3 [81, 82] are negative receptors of HMGB1-mediated signaling in macrophages and dendritic cells (DCs). In addition to receptors, HMGB1 can be directly taken up and mediate the inflammatory and metabolism response [83, 84]. Ultra-pure HMGB1 (free from contaminating bacterial proteins and nucleic acids) exhibits very low immune activity in macrophages. In contrast, extracellular HMGB1 is in fact a "sticky" protein and a synergistic immune effect is observed between HMGB1 and PAMPs (e.g., lipopolysac‐ charide), DAMPs (e.g., DNA), and other molecules (e.g., cytokines, chemokine, and IgG) in multiple cells [85]. Thus, serum and plasma components (e.g., immunoglobulins, phospholi‐ pids, thrombomodulin, and proteoglycans) can interfere with HMGB1 detection by enzymelinked immunosorbent assay [86]. Another important factor affecting HMGB1 activity is its redox status [87]. HMGB1 contains three conserved redox-sensitive cysteine residues: C23, C45, and C106. Reduced all-thiol-HMGB1 only exhibits chemokine activity, whereas disulfide-HMGB1 displays only cytokine activity, and oxidized HMGB1 has neither in immune cells [88]. In addition, reduced HMGB1 induces autophagy, whereas oxidized HMGB1 triggers apoptosis in cancer cells [89]. This redox status of HMGB1 also affects the affinity between HMGB1 and its receptors [26]. A recent study demonstrates that HMGB1 is specifically cleaved by caspase-1 but not other caspases during inflammasome activation [90]. Collectively, the release and activity of HMGB1 is context-dependent.

### **3. HMGB1 regulates cell death**

secretory pathways based on mass spectrometry-mediated binding partner analysis [55]. In one study, several HMGB1-binding partners in nuclear and cytosol fraction were identified in several cancer cells [55]. Among them, nine of the cytosolic HMGB1-binding proteins were related to protein translocation and secretion. In particular, immunoprecipitation analysis further confirmed four cytosolic HMGB1-binding proteins, including annexin A2, myosin IC isoform a, myosin-9, and Ras-related protein Rab10 [55]. These proteins are directly implicated in the process of unconventional protein secretion. Further studies are needed to define the function of cytosolic HMGB1 in unconventional protein secretion. In addition to nuclear and cytosolic HMGB1, intracellular HMGB1 presents on cell surface membranes and regulates neurite outgrowth [56], platelet activation [57, 58], cell differentiation [59], erythroid matura‐

HMGB1 is released in two different ways. On the one hand, HMGB1 can be actively secreted by normal cells, especially immune and endothelial cells [63, 64]. On the other hand, HMGB1 can be passively released by dead, dying, or injured cells in response to autophagic cell death [65], apoptosis [66, 67], necrosis [68], necroptosis [69, 70], netosis [71], and pyroptosis [72]. Oxidative stress refers to elevated intracellular levels of reactive oxygen species (ROS) that play a central role in the regulation of HMGB1 secretion and release, although the actual mechanism of action remains ambiguous [73]. Once released, HMGB1 acts as a cytokine, chemokine, and growth factor that is implicated in multiple biological processes including inflammation, immunity, migration, invasion, metabolism, proliferation, differentiation, antimicrobial defense, angiogenesis, tissue regeneration, death, autophagy, senescence, and efferocytosis. Extracellular HMGB1 plays important roles in the pathogenesis of human disease and is a potential therapy target in infection and sterile inflammation [74-76]. Several factors can affect HMGB1 activity in different experimental settings. For example, RAGE [77] and TLRs [78, 79] are positive receptors in macrophages and fibroblasts, whereas CD24 [80] and T cell immunoglobulin mucin 3 [81, 82] are negative receptors of HMGB1-mediated signaling in macrophages and dendritic cells (DCs). In addition to receptors, HMGB1 can be directly taken up and mediate the inflammatory and metabolism response [83, 84]. Ultra-pure HMGB1 (free from contaminating bacterial proteins and nucleic acids) exhibits very low immune activity in macrophages. In contrast, extracellular HMGB1 is in fact a "sticky" protein and a synergistic immune effect is observed between HMGB1 and PAMPs (e.g., lipopolysac‐ charide), DAMPs (e.g., DNA), and other molecules (e.g., cytokines, chemokine, and IgG) in multiple cells [85]. Thus, serum and plasma components (e.g., immunoglobulins, phospholi‐ pids, thrombomodulin, and proteoglycans) can interfere with HMGB1 detection by enzymelinked immunosorbent assay [86]. Another important factor affecting HMGB1 activity is its redox status [87]. HMGB1 contains three conserved redox-sensitive cysteine residues: C23, C45, and C106. Reduced all-thiol-HMGB1 only exhibits chemokine activity, whereas disulfide-HMGB1 displays only cytokine activity, and oxidized HMGB1 has neither in immune cells [88]. In addition, reduced HMGB1 induces autophagy, whereas oxidized HMGB1 triggers apoptosis in cancer cells [89]. This redox status of HMGB1 also affects the affinity between HMGB1 and its receptors [26]. A recent study demonstrates that HMGB1 is specifically cleaved

tion [60], adhesion [61], and innate immunity [62].

**2.3. Extracellular HMGB1**

400 Cell Death - Autophagy, Apoptosis and Necrosis

#### **3.1. Mechanism of HMGB1-mediated autophagy regulation**

Autophagy, including macroautophagy, microautophagy, and chaperone-mediated autoph‐ agy, is a highly conserved degradation process in organisms from yeasts to plants and animals [91]. The well-studied form of autophagy is macroautophagy (hereafter referred to as autoph‐ agy). As a complex dynamic process, autophagy is composed of the formation and maturation of three major membrane structures: the phagophore, autophagosome, and autolysosome [92]. Briefly, the phagophore originates from multiple membrane resources and engulfs the cytosolic materials, which leads to the formation of a closed autophagosome with a double membrane. Of note, microtubule-associated protein light chain 3 (LC3)-II is a widely used autophagosome marker [93]. Finally, autophagosomes fuse with lysosomes to form autolyso‐ somes, which results in degradation of the engulfed material, including LC3-II, by lysosomal enzymes into elementary pieces that can be used for protein synthesis and energy production. Thus, autophagy is a programmed cell survival pathway in response to intracellular and extracellular stress [94]. However, excessive or impaired autophagy can cause cell death, indicating a dual role of autophagy in cell survival and cell death. In particular, autosis is an Na+ , K+ -ATPase-dependent form of cell death triggered by autophagy-inducing peptides, starvation, and hypoxia–ischemia [95]. The process of autophagy is controlled by multiple posttranslational modifications of the autophagy-related gene (Atg) family and shares regulators derived from other cell death pathways [96].

HMGB1 promotes autophagy in a location- and modification-dependent manner. Nuclear HMGB1 regulates heat shock protein β-1 (HSPB1) expression at a transactional level [54]. The protein expression of HSPB1, but not other heat shock proteins, is significantly inhibited in HMGB1 knockout or knockdown cells. Both HMGB1 and HSPB1 regulate mitochondrial selective autophagy, namely mitophagy, following mitochondrial injury [54]. Like other ATGs, it was recently suggested that HMGB1-independent autophagy exists in the regula‐ tion of mitochondrial quality, including the mitochondrial DNA damage response [53]. Cytosolic HMGB1 is a Beclin-1 binding protein [97]. HMGB1 C23S and C45S mutants lose their ability to bind Beclin-1 and therefore cannot promote autophagy [97]. The binding of HMGB1 with Beclin-1 is positively regulated by unc-51-like kinase 1 [98] mitogen-activated kinase-like protein [99], and nucleus accumbens-1 [100]. In contrast, p53 [101], SNCA/αsynuclein [102], lysosomal thiol reductase [103], miR34A [104], and miR22 [105] negatively regulate HMGB1-mediated autophagy by disrupting HMGB1-Beclin-1 complex formation. Moreover, activation of poly [ADP-ribose] polymerase 1 (PARP1) is required for tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced ADP-ribosylation of HMGB1 and subsequent HMGB1-Beclin-1 complex formation in cancer cells [106]. Extracellular reduced HMGB1, but not oxidized HMGB1, significantly induced autophagy in cancer cells in a RAGE-dependent manner [89]. This process may sustain anaerobic energy production during tumor growth and development [107]. Collectively, these findings suggest an HMGB1-dependent autophagic pathway at multiple levels in response to stress. Howev‐ er, HMGB1-independent autophagy may exist in several organs, although the underlying mechanism of its action remains obscure [108].

### **3.2. Mechanism of HMGB1-mediated apoptosis regulation**

Apoptosis is the process of programmed cell death and includes classical "extrinsic" and "intrinsic" pathways and nonclassical T and natural killer cell-mediated cytolytic pathways. The extrinsic pathway is primarily mediated by the binding of a ligand to a transmembrane death receptor (DR). DRs are members of the TNF receptor gene superfamily, including FasR, TNFR1, lymphotoxin receptor, DR3, and DR4/DR5 [109]. In addition to DRs, dependence receptors mediate apoptosis by monitoring the absence of certain trophic factors or the presence of anti-trophic factors [110]. The intrinsic pathway for apoptosis involves activation of a mitochondrial pathway including altering mitochondrial permeability and subsequent release of mitochondrial proteins such as cytochrome c and second mitochondrial-derived activator of caspases [111]. The process of apoptosis is tightly regulated by the Bcl-2, caspase, and nuclease families [112-114]. Caspases are a family of endoproteases linking inflammation and cell death. Initiator caspases (e.g., caspases-8 and -9) activate executioner caspases (e.g., caspases-3, -6, and -7) that mediate the cleavage of key structural proteins such as PARP1. However, caspase-independent apoptosis may exist by translocation of apoptosis-inducing factor [115, 116] and endonuclease G [117] from the mitochondria to the nucleus, or activation of Omi/HTRA2 (a mitochondrial serine protease) [118]. Remarkably, several caspases (e.g., caspase-1, -4, -5, and -12 in humans; caspase-1, -11, and -12 in mice) are critical mediators of innate immune responses partly by activation of inflammasome, but not activation of the apoptosis pathway.

Intracellular HMGB1 is generally an anti-apoptotic protein in response to several apoptotic stimuli such as ultraviolet radiation, CD95, TRAIL, caspase-8, and Bax [119]. Knockdown of HMGB1 increases drug sensitivity in cancer cells [120]. Mechanically, HMGB1 plays tran‐ scriptional-dependent (e.g., regulation of Bcl-2 family protein expression) and transcriptionalindependent roles (e.g., regulation of autophagy and p53 location) in the regulation of apoptosis. For example, inhibition of HMGB1-mediated autophagy can increase caspase activity [121]. In addition to caspases, several non-caspase proteases such as calpain (Ca2+ dependent proteases) may play a role in the execution of apoptosis. Interestingly, HMGB1 deletion can enhance calpain activity and trigger cleavage of Beclin-1 and ATG5 [122]. Thus, HMGB1 is an important regulator of the cross talk between apoptosis and autophagy. *In vivo*, conditional knockout of HMGB1 in pancreas, liver, intestinal epithelial and myeloid cells enhances sterile inflammation and infection partly through inhibition of autophagy and induction of apoptosis [38, 122-124]. In some cases, overexpression of HMGB1 renders cells sensitive to apoptosis in response to chemotherapy agents [125]. In addition, extracellular oxidized HMGB1 can induce caspase-dependent apoptosis in cancer cells [89]. These findings suggest that HMGB1 plays dual roles in the regulation of apoptosis.

### **3.3. Mechanism of HMGB1-mediated necrosis regulation**

production during tumor growth and development [107]. Collectively, these findings suggest an HMGB1-dependent autophagic pathway at multiple levels in response to stress. Howev‐ er, HMGB1-independent autophagy may exist in several organs, although the underlying

Apoptosis is the process of programmed cell death and includes classical "extrinsic" and "intrinsic" pathways and nonclassical T and natural killer cell-mediated cytolytic pathways. The extrinsic pathway is primarily mediated by the binding of a ligand to a transmembrane death receptor (DR). DRs are members of the TNF receptor gene superfamily, including FasR, TNFR1, lymphotoxin receptor, DR3, and DR4/DR5 [109]. In addition to DRs, dependence receptors mediate apoptosis by monitoring the absence of certain trophic factors or the presence of anti-trophic factors [110]. The intrinsic pathway for apoptosis involves activation of a mitochondrial pathway including altering mitochondrial permeability and subsequent release of mitochondrial proteins such as cytochrome c and second mitochondrial-derived activator of caspases [111]. The process of apoptosis is tightly regulated by the Bcl-2, caspase, and nuclease families [112-114]. Caspases are a family of endoproteases linking inflammation and cell death. Initiator caspases (e.g., caspases-8 and -9) activate executioner caspases (e.g., caspases-3, -6, and -7) that mediate the cleavage of key structural proteins such as PARP1. However, caspase-independent apoptosis may exist by translocation of apoptosis-inducing factor [115, 116] and endonuclease G [117] from the mitochondria to the nucleus, or activation of Omi/HTRA2 (a mitochondrial serine protease) [118]. Remarkably, several caspases (e.g., caspase-1, -4, -5, and -12 in humans; caspase-1, -11, and -12 in mice) are critical mediators of innate immune responses partly by activation of inflammasome, but not activation of the

Intracellular HMGB1 is generally an anti-apoptotic protein in response to several apoptotic stimuli such as ultraviolet radiation, CD95, TRAIL, caspase-8, and Bax [119]. Knockdown of HMGB1 increases drug sensitivity in cancer cells [120]. Mechanically, HMGB1 plays tran‐ scriptional-dependent (e.g., regulation of Bcl-2 family protein expression) and transcriptionalindependent roles (e.g., regulation of autophagy and p53 location) in the regulation of apoptosis. For example, inhibition of HMGB1-mediated autophagy can increase caspase activity [121]. In addition to caspases, several non-caspase proteases such as calpain (Ca2+ dependent proteases) may play a role in the execution of apoptosis. Interestingly, HMGB1 deletion can enhance calpain activity and trigger cleavage of Beclin-1 and ATG5 [122]. Thus, HMGB1 is an important regulator of the cross talk between apoptosis and autophagy. *In vivo*, conditional knockout of HMGB1 in pancreas, liver, intestinal epithelial and myeloid cells enhances sterile inflammation and infection partly through inhibition of autophagy and induction of apoptosis [38, 122-124]. In some cases, overexpression of HMGB1 renders cells sensitive to apoptosis in response to chemotherapy agents [125]. In addition, extracellular oxidized HMGB1 can induce caspase-dependent apoptosis in cancer cells [89]. These findings

suggest that HMGB1 plays dual roles in the regulation of apoptosis.

mechanism of its action remains obscure [108].

402 Cell Death - Autophagy, Apoptosis and Necrosis

apoptosis pathway.

**3.2. Mechanism of HMGB1-mediated apoptosis regulation**

Necrosis includes accidental and regulated necrosis [2]. Partially, the term "necroptosis" has recently been used to describe regulated necrosis when cells lack the capacity to activate caspase [126]. Necroptosis is mediated by a signaling complex called necrosome, containing receptor-interacting protein (RIP)1, RIP3, and mixed-lineage kinase domain-like (MLKL) [127, 128], and can be inhibited by small molecule inhibitors necrostatin 1 and necrosulfonamide [129, 130] [126]. The fundamental causes of necrosis include calcium overload, ROS generation, cellular energy depletion, and membrane lipid injury [131]. PARP is a protein family involved in a number of cellular processes such as DNA repair and programmed cell death. Induced overactivation of PARP1 can lead to adenosine triphosphate (ATP) depletion and subsequent necrosis [132]. The process of necrosis ends with the leaking out of enzymes from lysosomes to digest cell components that are associated with HMGB1 release. *In vivo*, loss of HMGB1 in the pancreas increases L-arginine-induced apoptosis and necrosis due to oxidative injury [38]. However, the role of HMGB1 in necroptosis remains undefined.

### **4. HMGB1 release in cell death**

#### **4.1. Mechanism of HMGB1 release in autophagy**

Autophagic cell death is not only a morphologic notion such as cell death associated with autophagosomes and autolysosomes, but also a functional description that excessive autoph‐ agy can cause cell death. Induction of autophagy facilitates both active secretion and passive release of HMGB1. For example, the release of HMGB1 is significantly increased in response to epidermal growth factor (EGF) receptor-targeted diphtheria toxin (DT-EGF)-induced autophagic cell death [65]. In contrast, suppression of ATG5, ATG7, or ATG12 expression by RNA interference (RNAi) inhibits autophagy and subsequent HMGB1 release after treatment with DT-EGF in cancer cells [65]. In addition, ATG5-dependent autophagy promotes HMGB1 secretion in fibroblasts and macrophages after treatment with Hank's balanced salt solution and lipopolysaccharide [97, 133]. Antioxidant (e.g., N-acetyl-L-cysteine) inhibits the cytosolic translocation and release of HMGB1 in starvation-induced autophagy [97]. In contrast, ROS and knockdown of superoxide dismutases (SOD)-1 and SOD2 by RNAi promotes cytosolic HMGB1 expression and extracellular release [134]. These findings suggest that oxidative stress is involved in autophagy-mediated HMGB1 release.

#### **4.2. Mechanism of HMGB1 release in apoptosis**

An early study indicated that HMGB1 is released only by necrotic cells, but not apoptotic cells [68]. However, recent studies demonstrated that activation of caspases and deoxyribonuclease (DNase) in apoptosis regulates HMGB1 release and activity in apoptosis. Caspase-3 and caspase-7 are important executioner caspases in apoptosis through amplified initiation signals from caspase-8 and caspase-9. Activation of caspase-3 and -7 induces mitochondrial complex 1 protein p75 NDUFS1 cleavage, which results in mitochondrial ROS production and subse‐ quent HMGB1 release during apoptosis in DCs [135]. Interestingly, the activity of released HMGB1 in apoptosis is impaired, which promotes immunological resistance due to its oxidized form [135]. In addition to caspase-3 and -7, caspase-1 is responsible for HMGB1 cleavage and release in the response to pyroptosis in immune cells [72, 136, 137]. This cas‐ pase-1-mediated HMGB1 fragment can rescue apoptosis-induced immune tolerance in a RAGE-dependent manner [137]. Thus, different caspases can determine HMGB1 release and action in apoptosis and pyroptosis.

DNase is responsible for DNA fragmentation during cell death. Activation of DNA endonu‐ clease (DNase-gamma) contributes to the degradation of DNA into nucleosomal units in apoptosis, whereas activation of DNase I and II facilitates degradation of DNA in necrosis [138]. The release of HMGB1 in apoptosis is triggered by DNase-gamma-mediated nucleoso‐ mal DNA fragmentation [139, 140]. Thus, inhibition of DNase gamma activity by small molecular compound DR396 can significantly diminish HMGB1 release in response to apoptotic stimuli [139, 140].

### **4.3. Mechanism of HMGB1 release in necrosis**

The nuclear enzyme PARP1, which catalyzes the synthesis of the biopolymer poly(ADPribose), exhibits an essential role in the DNA damage response and genomic stability. How‐ ever, overactivation of PARP1 may deplete the stores of cellular NAD+, which results in ATP depletion and subsequent necrosis [141]. In fact, HMGB1 release in necrosis is regulated by PARP1. Genetic and pharmacologic inhibition of PARP1 inhibits alkylating DNA damage agent-mediated necrosis as well as HMGB1 release [142]. In addition to necrosis, activation of PARP1 also contributes to HMGB1 translocation and release in autophagy and inflammation [106, 143]. Interestingly, loss of HMGB1 in tissue and cells accelerates DNA damage that results in PARP1 overactivation [144]. These findings suggest interplay between HMGB1 and PARP1 in response to cell death.

The RIP3-mediated signaling pathway is responsible for HMGB1 release in necroptosis. Upregulation of RIP3 expression *in vitro* triggers necroptosis, whereas suppression of RIP3 expression by RNAi *in vitro* or *in vivo* significantly inhibits inflammatory stimuli-induced necroptosis. RIP3-deficient mice exhibit resistance to sepsis and donor kidney inflammatory injury. This anti-inflammatory function of RIP3 is due partly to inhibition of HMGB1 and release of other DAMPs [145]. Additionally, RIP3-mediated necroptosis also contributes to dsRNA/poly (I:C)-induced HMGB1 release [146]. This process promotes retinal degeneration and triggers an inflammatory response in the mouse retina [146]. In addition to RIP3, inter‐ feron-β promoter stimulator 1 (an adaptor molecule for RIG-I-like receptors) may be critical for poly (I:C)-induced HMGB1 release in necroptosis in DCs.

Cysteine cathepsins are lysosomal proteases with housekeeping functions that also initiate a specific cell death pathway termed lysosomal cell death. This type of cell death includes morphological features of necrosis and apoptosis [147]. Cathepsin B, a critical lysosomal cysteine protease, mediates HMGB1 release following *L. pneumophila*-induced lysosomal cell death [148]. Mechanically, cathepsin B can translocate from the lysosome to the nucleus, where it interacts with HMGB1 and inhibits its cytosolic translocation. In addition to lysosomal cell death, cathepsin B is also important for HMGB1 release during inflammasome activation [149, 150]. In contrast, cathepsin D may facilitate HMGB1 release in necroptosis in DCs. The function of other cathepsins in the regulation of HMGB1 release remains unknown.

### **5. Concluding remarks**

quent HMGB1 release during apoptosis in DCs [135]. Interestingly, the activity of released HMGB1 in apoptosis is impaired, which promotes immunological resistance due to its oxidized form [135]. In addition to caspase-3 and -7, caspase-1 is responsible for HMGB1 cleavage and release in the response to pyroptosis in immune cells [72, 136, 137]. This cas‐ pase-1-mediated HMGB1 fragment can rescue apoptosis-induced immune tolerance in a RAGE-dependent manner [137]. Thus, different caspases can determine HMGB1 release and

DNase is responsible for DNA fragmentation during cell death. Activation of DNA endonu‐ clease (DNase-gamma) contributes to the degradation of DNA into nucleosomal units in apoptosis, whereas activation of DNase I and II facilitates degradation of DNA in necrosis [138]. The release of HMGB1 in apoptosis is triggered by DNase-gamma-mediated nucleoso‐ mal DNA fragmentation [139, 140]. Thus, inhibition of DNase gamma activity by small molecular compound DR396 can significantly diminish HMGB1 release in response to

The nuclear enzyme PARP1, which catalyzes the synthesis of the biopolymer poly(ADPribose), exhibits an essential role in the DNA damage response and genomic stability. How‐ ever, overactivation of PARP1 may deplete the stores of cellular NAD+, which results in ATP depletion and subsequent necrosis [141]. In fact, HMGB1 release in necrosis is regulated by PARP1. Genetic and pharmacologic inhibition of PARP1 inhibits alkylating DNA damage agent-mediated necrosis as well as HMGB1 release [142]. In addition to necrosis, activation of PARP1 also contributes to HMGB1 translocation and release in autophagy and inflammation [106, 143]. Interestingly, loss of HMGB1 in tissue and cells accelerates DNA damage that results in PARP1 overactivation [144]. These findings suggest interplay between HMGB1 and PARP1

The RIP3-mediated signaling pathway is responsible for HMGB1 release in necroptosis. Upregulation of RIP3 expression *in vitro* triggers necroptosis, whereas suppression of RIP3 expression by RNAi *in vitro* or *in vivo* significantly inhibits inflammatory stimuli-induced necroptosis. RIP3-deficient mice exhibit resistance to sepsis and donor kidney inflammatory injury. This anti-inflammatory function of RIP3 is due partly to inhibition of HMGB1 and release of other DAMPs [145]. Additionally, RIP3-mediated necroptosis also contributes to dsRNA/poly (I:C)-induced HMGB1 release [146]. This process promotes retinal degeneration and triggers an inflammatory response in the mouse retina [146]. In addition to RIP3, inter‐ feron-β promoter stimulator 1 (an adaptor molecule for RIG-I-like receptors) may be critical

Cysteine cathepsins are lysosomal proteases with housekeeping functions that also initiate a specific cell death pathway termed lysosomal cell death. This type of cell death includes morphological features of necrosis and apoptosis [147]. Cathepsin B, a critical lysosomal cysteine protease, mediates HMGB1 release following *L. pneumophila*-induced lysosomal cell death [148]. Mechanically, cathepsin B can translocate from the lysosome to the nucleus, where it interacts with HMGB1 and inhibits its cytosolic translocation. In addition to lysosomal cell

action in apoptosis and pyroptosis.

404 Cell Death - Autophagy, Apoptosis and Necrosis

apoptotic stimuli [139, 140].

in response to cell death.

**4.3. Mechanism of HMGB1 release in necrosis**

for poly (I:C)-induced HMGB1 release in necroptosis in DCs.

HMGB1 is a member of family containing the evolutionarily conserved HMG box domains. The function of HMGB1 depends on its cellular location. Besides its functions in the nucleus and cytosol, HMGB1 plays a critical role in extracellular signaling associated with multiple biological processes. Both intracellular and extracellular HMGB1 are involved in the regulation of types of cell death such as apoptosis, necrosis, and autophagy. Intracellular HMGB1 regulates cell death in both transactional-dependent or transactional-independent manners. In many cases, HMGB1 is a negative regulator of apoptosis and necrosis, but a positive regulator of autophagy. In addition, the release and activity of HMGB1 in cell death is contextdependent, which may cause immunogenic cell death or tolerogenic cell death. Future studies are needed to define the upstream and downstream signaling of HMGB1 in the regulation of cell death; clarify the interplay and cooperative role of HMGB1 and other DAMPs in the celldeath-associated microenvironment; and develop new therapeutic strategies for targeting HMGB1 in cell-death-associated disorders.

### **Acknowledgements**

We apologize to the researchers who were not referenced due to space limitations. We thank Christine Heiner (Department of Surgery, University of Pittsburgh) for her critical reading of the manuscript. This work was supported by the USA National Institutes of Health (R01CA160417 and R01GM115366 to D.T.) and a 2013 Pancreatic Cancer Action Network-AACR Career Development Award (Grant Number 13-20-25-TANG). Work performed in support of findings reviewed in this manuscript was aided by core support of the University of Pittsburgh Cancer Institute (P30CA047904).

### **Author details**

Daolin Tang\* and Rui Kang\*

\*Address all correspondence to: tangd2@upmc.edu; kangr@upmc.edu

Department of Surgery, University of Pittsburgh Cancer Institute, University of Pittsburgh, USA

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## **Necrosis as Programmed Cell Death**

Ma. Luisa Escobar, Olga M. Echeverría and Gerardo H. Vázquez-Nin

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61483

#### **Abstract**

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418 Cell Death - Autophagy, Apoptosis and Necrosis

The process of cell death is the mechanism through which organisms eliminate useless cells. Hence, it is a normal process that maintains homeostasis. Cell removal can be effec‐ tuated by several pathways that involve complex and regulated molecular events specific to each type of cell death. Diverse studies have evidenced different types of cell death: apoptosis, autophagy, and necrosis. This chapter presents a brief review of the apoptotic and autophagic cell death processes but focuses attention primarily on necrosis because it has previously been considered an accidental and uncontrolled form of cell death. More recent evidence, however, has shown that, under certain circumstances, necrosis is con‐ ducted by a controlled program called necroptosis, which is now included as a program‐ med cell death process.

**Keywords:** Apoptosis, autophagy, cell death, necrosis, necroptosis

### **1. Introduction**

The tissular environment includes a series of signals that maintain the rates of cell proliferation and cell death so as to conserve structural integrity and functionality. Alterations in either one of these processes can cause certain pathologies, such as cancer. The cell death process is an ongoing event during the development of tissues and organs, one that is present right from embryonic development in the form of programmed cell death, which occurs under physio‐ logical conditions as a process that requires the active participation of highly regulated mechanisms. Traditionally, apoptosis was synonymous with programmed cell death; howev‐ er, different routes of cell death, such as autophagy and, more recently, necroptosis, are now included as forms of programmed cell death. Morphologically, each one of these cell death processes has features that make it possible to distinguish among them. The different molec‐ ular mechanisms involved in the cell death pathways are responsible for the morphological changes that occur in the affected cells. However, each pathway has specific characteristics;

© 2015 The Author(s). Licensee InTech. 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.

for instance, cellular shrinkage is a phenomenon that occurs in apoptosis [1], but is not present in other types of cell death, such as autophagy or necrosis. On the other hand, the extensive presence of vesicles evidences autophagy but does not appear to the same extent in the other types of cell elimination [2]. Necrosis, meanwhile, presents generalized swelling of membra‐ nous organelles that leads to cell rupture [3].

### **2. Brief description of two types of programmed cell death: apoptosis and autophagy**

Apoptosis, or type I programmed cell death, is the most widely studied of the forms of cell death. Its morphological characteristics can be identified under light microscopy, and include cell shrinkage, compacting of the chromatin, blebbing of the cytoplasmic membrane, and, finally, the formation of apoptotic bodies [1] (Figure 1). Biochemically, apoptosis is character‐ ized by the participation of proteases called caspases, orderly internucleosomal DNA frag‐ mentation, phosphatidylserine externalization, changes in mitochondrial membrane permeability, and the participation of members of the Bcl-2 protein family.

**Figure 1.** Schematic representation of the programmed cell death process type I (apoptosis) and type II (autophagy). Apoptosis is characterized by a cellular contraction, chromatin compaction, membranous blebs, and the formation of apoptotic bodies. Autophagy is characterized by the presence of a large number of autophagosomes with cytoplasmic content. Both types of cell death do not generate an inflammatory response since the cytoplasmic membrane is con‐ served until the cellular debris are eliminated by neighborhood or by specialized ones.

Caspases are cysteinyl-aspartate-specific proteases that are synthetized in an inactive form as zymogens called pro-caspases (Figure 2). It is this inactive form that allows the controlled execution of the cell death process. Caspases were first identified in the nematode *Ceanorhab‐ ditis elegans* [4], but homologous forms are present in mammals [5].

for instance, cellular shrinkage is a phenomenon that occurs in apoptosis [1], but is not present in other types of cell death, such as autophagy or necrosis. On the other hand, the extensive presence of vesicles evidences autophagy but does not appear to the same extent in the other types of cell elimination [2]. Necrosis, meanwhile, presents generalized swelling of membra‐

**2. Brief description of two types of programmed cell death: apoptosis and**

Apoptosis, or type I programmed cell death, is the most widely studied of the forms of cell death. Its morphological characteristics can be identified under light microscopy, and include cell shrinkage, compacting of the chromatin, blebbing of the cytoplasmic membrane, and, finally, the formation of apoptotic bodies [1] (Figure 1). Biochemically, apoptosis is character‐ ized by the participation of proteases called caspases, orderly internucleosomal DNA frag‐ mentation, phosphatidylserine externalization, changes in mitochondrial membrane

**Figure 1.** Schematic representation of the programmed cell death process type I (apoptosis) and type II (autophagy). Apoptosis is characterized by a cellular contraction, chromatin compaction, membranous blebs, and the formation of apoptotic bodies. Autophagy is characterized by the presence of a large number of autophagosomes with cytoplasmic content. Both types of cell death do not generate an inflammatory response since the cytoplasmic membrane is con‐

served until the cellular debris are eliminated by neighborhood or by specialized ones.

permeability, and the participation of members of the Bcl-2 protein family.

nous organelles that leads to cell rupture [3].

420 Cell Death - Autophagy, Apoptosis and Necrosis

**autophagy**

The hallmarks of apoptosis, such as DNA fragmentation and compacted chromatin, result from caspase activity. During apoptosis, DNA is fragmented into nucleosome size (200 bp) [6, 7]. The factor responsible for DNA fragmentation during apoptosis is a specific DNase (CAD, caspase-activated DNase) that is activated by active caspase-3 [8]. Active caspase-3, in turn, is involved in morphological cell changes during apoptosis, where it cleaves rho-associated kinase-1 (ROCK-1) in order to activate it and this, finally, affects the cytoskeletal arrangement causing the apoptotic shrinkage morphology [9].

**Figure 2.** Caspases involved in the apoptotic process are synthetized in an inactive form as zymogens, are constituted by a pro-domain, a large subunit, and a small subunit. The zymogens are activated forming tetramers.

Apoptotic cell death is highly regulated by members of the B-cell lymphoma 2 (Bcl-2) family [10]. Bcl-2 family members have been classified as anti-apoptotic and pro-apoptotic proteins according to their Bcl-2 homology (BH) and domain organization (Figure 3). The presence of domains BH1, BH2, BH3, and BH4 corresponds to the group that inhibits apoptosis. The proapoptotic group, in contrast, is divided in two groups: those with domains BH1, BH2, and BH3, and those with only the BH3 domains (defined as BH3 only; see the review in [11]). This family of proteins performs its functions at the intracellular level inside the mitochondria, a key element in apoptosis.

Apoptosis can be initiated by two well-described routes: the extrinsic and intrinsic pathways (Figure 4). Extrinsic activation is conducted through the participation of death ligands (such as the tumor necrosis factor – TNF – superfamily, and TNF-related apoptosis-induced ligands, or TRAIL) with their cognate cell surface death receptors (such as TNF receptor 1, Fas, TRAIL receptor 1, or TRAIL receptor 2) (reviewed in [12]). Once the ligand recognizes and bonds to

**Figure 3.** B-cell lymphoma 2 (Bcl-2) family proteins. A) General schematization of the structure of Bcl-2 proteins. B) The anti-apoptotic members – they possess all the four BH domains. C) The pro-apoptotic members which in turn are divided into two groups: multidomain and BH3 only.

its receptor, a series of intracellular complexes are formed to activate the initiator caspases (such as -8 and -10), which then activate the executioner caspases (such as -3, -6, and -7). In their activated form, these executioner caspases cleave multiple intracellular targets.

The intrinsic apoptotic pathway, in contrast, can be activated by various stimuli, including DNA damage, growth factor starvation, and oxidative stress [13]. During exposure of cells to these stimuli, the mitochondria are affected, since several members of the Bcl-2 family are activated and promote mitochondria outer membrane permeabilization (MOMP). The permeated external mitochondria membrane allows the release of cytochrome c (cyt c), which is associated with the Apaf-1 protein. The cyt c and Apaf-1 union then bonds to the initiator caspase-9 to form the complex that constitutes the apoptosome, which has the ability to activate the initiator caspases that perform their functions by cleaving specific cellular substrates.

The second process of cell death, autophagy, is a genetically programmed and evolutionarily conserved process that produces the degradation of obsolete organelles and proteins. It is activated by such extracellular stimuli as nutrient starvation, hypoxia, high temperature, and

**Figure 4.** Routes of activation of apoptosis. The extrinsic route is mediated by external signals – a ligand – that activate to the membrane receptor. The ligand–receptor interaction induces the assembly of the death-inducing signaling com‐ plex (DISC) to promote the activation of caspase-8, which in turn is able to activate to the executor caspase -3, -6, or -7, conducting to the morphological changes of the apoptosis. The intrinsic route is directed by the mitochondrial outer membrane permeabilization, which allows the release of pro-apoptotic elements as cytochrome-C. Cytochrome-C in‐ duces the apoptosis protease-activating factor 1 (Apaf-1) to promote the activation of caspase-9 to assemble the apop‐ tosome. The apoptosome is capable of activating to the executor caspases.

altered intracellular conditions, including the accumulation of damaged or superfluous organelles (reviewed in [2]).

In eukaryotic organisms, three types of autophagy have been described: microautophagy, macroautophagy (commonly called simply autophagy), and chaperone-mediated autophagy (Figure 5). Microautophagy involves the engulfing of cytoplasmic components directly at the level of the lysosome by means of an invagination process, while macroautophagy entails the formation of double-membrane vesicles that contain cellular components, which fuse with lysosomes to form an autophagolysosome. It is inside the autophagolysosome that the intravesicular components are degraded and, if possible, recycled by the cell (reviewed in [2 and 14]). Chaperone-mediated autophagy, finally, entails the participation of chaperones in recognizing the proteins designated for elimination by the lysosomes [14].

its receptor, a series of intracellular complexes are formed to activate the initiator caspases (such as -8 and -10), which then activate the executioner caspases (such as -3, -6, and -7). In

**Figure 3.** B-cell lymphoma 2 (Bcl-2) family proteins. A) General schematization of the structure of Bcl-2 proteins. B) The anti-apoptotic members – they possess all the four BH domains. C) The pro-apoptotic members which in turn are

The intrinsic apoptotic pathway, in contrast, can be activated by various stimuli, including DNA damage, growth factor starvation, and oxidative stress [13]. During exposure of cells to these stimuli, the mitochondria are affected, since several members of the Bcl-2 family are activated and promote mitochondria outer membrane permeabilization (MOMP). The permeated external mitochondria membrane allows the release of cytochrome c (cyt c), which is associated with the Apaf-1 protein. The cyt c and Apaf-1 union then bonds to the initiator caspase-9 to form the complex that constitutes the apoptosome, which has the ability to activate the initiator caspases that perform their functions by cleaving specific cellular substrates.

The second process of cell death, autophagy, is a genetically programmed and evolutionarily conserved process that produces the degradation of obsolete organelles and proteins. It is activated by such extracellular stimuli as nutrient starvation, hypoxia, high temperature, and

their activated form, these executioner caspases cleave multiple intracellular targets.

divided into two groups: multidomain and BH3 only.

422 Cell Death - Autophagy, Apoptosis and Necrosis

Autophagy is directed by *Atg* (AuTophaGy-related) genes, which are required to activate the signaling complex that triggers the formation of autophagosomes [15]. *Atg* genes were discovered in yeast, but many have orthologues in higher eukaryotes (Figure 6). Autophago‐ some formation entails the participation of the cytoplasmic protein LC3 (Atg8), which undergoes lipidation by phosphatidylethanolamine, and is then recruited to the nascent autophagosome membrane (Figure 7). Accumulation of lipidated LC3 protein (known as LC3- II) is used as a marker of autophagy [16].

**Figure 5.** Schematic representations of the different mechanisms of autophagy. Macroautophagy – autophagy- implies the formation of a double-membrane vesicle, which engulfs cytoplasmic content that will be conducted to the lyso‐ some to be degraded. Microautophagy is characterized by direct engulfing of cytoplasmic components by the lyso‐ some. This process involves the remodeling of the membrane of the organelle by forming a lysosomal membrane invagination. During chaperone-mediated autophagy, the proteins to be degraded are targeted for an Hsp70, which in turn transport the target cargo to the lysosome.


**Figure 6.** Atg protein family includes more than 30 members that participate in the different events that constitute the autophagic process.

Autophagic cell death, or type II programmed cell death, is characterized by a massive engulfing of the cytoplasm by autophagic vesicles. This intense autophagic activity differs substantially from autophagy that occurs continuously at basal levels. Ultrastructural studies in Drosophila have revealed the accumulation of autophagic vacuoles in most larval tissues.

**Figure 7.** The formation of autophagic vacuoles involves the participation of different Atg proteins since the initial phases until the sequestration of cytoplasmic content. LC3 protein is lipidated by the phosphatidylethanolamine.

This type of programmed cell death begins with the degradation of cytoplasmic organelles by autophagy, though the cytoskeletal elements are conserved until the late stages of the process (reviewed in [17]).

### **3. Morphological characteristics of the necrosis process**

**Figure 6.** Atg protein family includes more than 30 members that participate in the different events that constitute the

**Figure 5.** Schematic representations of the different mechanisms of autophagy. Macroautophagy – autophagy- implies the formation of a double-membrane vesicle, which engulfs cytoplasmic content that will be conducted to the lyso‐ some to be degraded. Microautophagy is characterized by direct engulfing of cytoplasmic components by the lyso‐ some. This process involves the remodeling of the membrane of the organelle by forming a lysosomal membrane invagination. During chaperone-mediated autophagy, the proteins to be degraded are targeted for an Hsp70, which in

Autophagic cell death, or type II programmed cell death, is characterized by a massive engulfing of the cytoplasm by autophagic vesicles. This intense autophagic activity differs substantially from autophagy that occurs continuously at basal levels. Ultrastructural studies in Drosophila have revealed the accumulation of autophagic vacuoles in most larval tissues.

autophagic process.

turn transport the target cargo to the lysosome.

424 Cell Death - Autophagy, Apoptosis and Necrosis

Cell death caused by necrosis is considered an accidental, unprogrammed event that occurs under total ATP depletion [3], and that results from such external stimuli as extreme physical– chemical stress, heat, osmotic shock, mechanical stress, freezing, thawing, and high concen‐ trations of hydrogen peroxide.

Necrotic cell death is characterized morphologically by generalized swelling of cell mem‐ branes, often accompanied by chromatin condensation and an irregular DNA degradation pattern [18]. The cytoplasmic membranes and membranous organelles dilate, and the in‐ creased cellular swelling causes the breakdown of the plasma membrane, which releases the cytoplasmic contents into the extracellular space (Figure 8). The release of the intracellular contents leads to massive cellular damage that affects neighboring cells, which explains why necrosis triggers inflammatory and autoimmune reactions. The necrosis process takes place in the absence of phagocytosis, and its final phase is characterized by the loss of the integrity of the cellular membrane. The release of the contents of necrotic cells includes molecules which act as signals that promote inflammation.

The most significant difference between programmed cell death (*i.e.*, apoptosis and autophagy) and necrosis is plasma membrane leakage and the consequent induction of inflammation in the affected tissue caused by the release of intracellular components [19].

**Figure 8.** Morphological characteristics of necrosis involve the membranous swelling of the organeles, DNA degrada‐ tion, and finally the release of the cytoplasmic content that affects the neighbor cells, provoking an inflammatory response.

### **4. Biochemical events during necrotic cell death**

Despite these findings, however, necrosis is still considered an accidental process in which several factors exert effects on the cells to begin the elimination event. Cellular stress factors like low oxygen (hypoxia), cytokines, ischemia (restricted blood supply), heat, irradiation, pathogens, and toxin exposure can all lead to necrosis. These stimuli provoke several changes at the cellular level. While reactive oxygen species are produced by the mitochondria as a normal process, under pathological conditions, reactive oxygen molecules increase and induce damage in the biomolecules, which leads the cells toward necrosis. During necrosis, the levels of both reactive oxygen species and intracellular calcium increase (reviewed in [20]). It is important to consider that the internal cell environment is highly regulated, so certain stimuli are able to alter cell membrane permeability and thus produce an imbalance among different ions, such as potassium, sodium, and calcium. Calcium is regulated by the endoplasmic reticulum, and a loss of calcium homeostasis can lead to several intracellular alterations. In contrast, increased calcium levels can affect diverse mitochondrial functions and result in alterations of the production of reactive oxygen species. When high calcium levels are sustained over time, they disrupt mitochondrial inner membrane integrity and cause a loss of the ability to generate ATP [21] and, eventually, necrotic cell death (Figure 9). In addition to their effects inside the mitochondria, altered cytosolic calcium levels can activate different types of proteases, including calpains. Calpains are intracellular cysteine proteases present in inactive form, that may be activated by increased cytosolic calcium [22, 23]. Once activated, they can disrupt the lysosomal membrane with the resulting release of cathepsines B and L [24]. This group of reactions causes destabilization of the final membrane system. Together, these alterations cause the cell to lose its membranes such that the cellular contents are released into the extracellular space.

The molecular hallmark of necrosis is drastic ATP depletion, which is believed to be the underlying cause of cell death. There is a metabolic disruption accompanied by energy depletion and loss of ATP that leads to cellular edema, while the mitochondria become round and swollen, the endoplasmic reticulum dilates, the lysosomes are disrupted, and the forma‐ tion of plasma membrane protrusions called blebs is apparent [25].

**Figure 9.** Biochemical cellular changes during necrosis cell death. The levels of both reactive oxygen species (ROS) as well as intracellular calcium (Ca2+) are increased. Sustained high calcium levels alter cell permeability of the mem‐ branes, leading to the dysfunction and ruptureof membranes. During necrosis, the ATP depletion is conducted.

**4. Biochemical events during necrotic cell death**

426 Cell Death - Autophagy, Apoptosis and Necrosis

into the extracellular space.

Despite these findings, however, necrosis is still considered an accidental process in which several factors exert effects on the cells to begin the elimination event. Cellular stress factors like low oxygen (hypoxia), cytokines, ischemia (restricted blood supply), heat, irradiation, pathogens, and toxin exposure can all lead to necrosis. These stimuli provoke several changes at the cellular level. While reactive oxygen species are produced by the mitochondria as a normal process, under pathological conditions, reactive oxygen molecules increase and induce damage in the biomolecules, which leads the cells toward necrosis. During necrosis, the levels of both reactive oxygen species and intracellular calcium increase (reviewed in [20]). It is important to consider that the internal cell environment is highly regulated, so certain stimuli are able to alter cell membrane permeability and thus produce an imbalance among different ions, such as potassium, sodium, and calcium. Calcium is regulated by the endoplasmic reticulum, and a loss of calcium homeostasis can lead to several intracellular alterations. In contrast, increased calcium levels can affect diverse mitochondrial functions and result in alterations of the production of reactive oxygen species. When high calcium levels are sustained over time, they disrupt mitochondrial inner membrane integrity and cause a loss of the ability to generate ATP [21] and, eventually, necrotic cell death (Figure 9). In addition to their effects inside the mitochondria, altered cytosolic calcium levels can activate different types of proteases, including calpains. Calpains are intracellular cysteine proteases present in inactive form, that may be activated by increased cytosolic calcium [22, 23]. Once activated, they can disrupt the lysosomal membrane with the resulting release of cathepsines B and L [24]. This group of reactions causes destabilization of the final membrane system. Together, these alterations cause the cell to lose its membranes such that the cellular contents are released

**Figure 8.** Morphological characteristics of necrosis involve the membranous swelling of the organeles, DNA degrada‐ tion, and finally the release of the cytoplasmic content that affects the neighbor cells, provoking an inflammatory response.

The molecular hallmark of necrosis is drastic ATP depletion, which is believed to be the underlying cause of cell death. There is a metabolic disruption accompanied by energy depletion and loss of ATP that leads to cellular edema, while the mitochondria become round and swollen, the endoplasmic reticulum dilates, the lysosomes are disrupted, and the forma‐

tion of plasma membrane protrusions called blebs is apparent [25].

Necrosis cell death occurs due to a failure in plasma membrane permeability that disrupts the plasma membrane and releases cellular components. This cell death process is associated with the indiscriminate extracellular release of soluble intracellular constituents through the permeabilized plasma membrane.

### **5. The emergence of necrosis as programmed cell death (necroptosis)**

The way in which necrosis occurs allows us to determine that it is not merely accidental. Several examples of the presence of necrosis in different phases of an organism's development suggest that this process may be regulated during the embryonic stage. Necrosis is present during the longitudinal growth of bones in young animals [26], and is also found in intestinal epithelial cells in adults [27]. On the other hand, during caspase inhibition, necrosis may be activated as an alternative route of cell elimination, suggesting that it is not simply an accidental process, but that, under certain conditions could function as a programmed event called necroptosis.

The necrosis process has long been conceived as an accidental, passive event; however, recent detailed observations were able to identify dying cells by the expression of different proteins and the intracellular disposition of several proteins that may be active during the event. As mentioned above, programmed cell death, or apoptosis, can be activated by two routes, one of which entails the participation of receptors present in the cytoplasmic membrane. These receptors include TNFα, FasL, and TRAIL that, once activated, cause recruitment of a protein complex (death-inducing signaling complex, DISC) mediated by the adaptor protein FADD (Fas-associated protein with death domain) to activate the initiator caspase-8 [28]. This activation of the caspase system triggers execution of the apoptotic process. Diverse findings indicate that the receptors involved in apoptotic cell death may also participate in the occur‐ rence of a different type of cell death under distinct conditions of molecular resource availa‐ bility. For cells that do not express caspase-8, it was predicted that they could not respond to an apoptotic stimuli directed by FasL induction. However, those results were surprising because they included the multimerization of FADD in the absence of caspase activation. The morphology of apoptosis was not present, but ultrastructural analyses of those dying cells revealed necrotic morphological changes [29]. All these observations suggest that the receptor regulators of apoptosis were involved not only in that process, but also in the activation of a different signaling pathway that allows the formation of protein complexes which lead the cell toward a death process with necrotic features. These developments led to the emergence of a new concept of programmed cell death called necroptosis, whose morphological characteris‐ tics are similar to those of accidental necrosis, although the molecular events that occur indicate that it is a coordinated process. Necroptosis has been found under special conditions, where pro-apoptotic enzymes were absent or limited. In experimental embryo models, interdigital membrane regression in mouse embryos was effectuated by necrosis triggered by either caspase inhibition or drugs [30]. Necroptosis is thus a form of programmed cell death that has been demonstrated under experimental conditions, when apoptosis is inhibited.

### **6. Biochemical aspects of necroptosis**

Several studies have succeeded in discerning the molecular events that occur during necrop‐ tosis, and it is those events that differentiate between necrosis (an accidental process) and necroptosis (a programmed process). Necroptosis has been observed in several pathological cell death events, such as ischemic brain injury, myocardial infarction, exotoxicity, and chemotherapy-induced cell death [31].

Necroptosis is morphologically characterized by several cytoplasmic changes. In fact, it is sometimes possible to distinguish the different degrees of advance of this process as the organelles swell, the cell membrane fragments, and cytoplasmic and nuclear disintegration become evident. During necroptosis, the nuclei remain intact and there is no massive caspase activation, chromatin condensation, spillage of cell contents, phagocytosis by macropinocy‐ tosis, lysosomal leakage, or oxidative bursts [32]. The term necroptosis has been introduced to identify a process of cell death with morphological characteristics distinct from those of apoptosis. Because there was no caspase activation during this process, it is called "caspaseindependent".

Necroptosis is a programmed event that ends with the delivery of the cytoplasmic contents into the extracellular space. Membrane destabilization is a consequence of different intracel‐ lular mechanisms that generate osmotic changes by damaging the ion balance. When DNA damage occurred due to reactive oxygen species, the PARP protein was activated and began the reparation process; however, this process consumed abundant ATP and that reduction initiated a sequence of events that led to a deficient cellular efflux of calcium. The decreased ATP levels affected the activity of Na+-K+ ATPase, which requires a large amount of ATP in order to function correctly. This decreased Na+-K+ ATPase activity reduced calcium release and, as a result, increased intracellular calcium levels, leading to membrane destruction (reviewed in [33]). The breakdown of the cellular membrane, in turn, released several signals that activated the immune system. These soluble signals were proteins with pro-inflammatory properties that stimulate the recruitment of neutrophils to the site of cell death [34].

because they included the multimerization of FADD in the absence of caspase activation. The morphology of apoptosis was not present, but ultrastructural analyses of those dying cells revealed necrotic morphological changes [29]. All these observations suggest that the receptor regulators of apoptosis were involved not only in that process, but also in the activation of a different signaling pathway that allows the formation of protein complexes which lead the cell toward a death process with necrotic features. These developments led to the emergence of a new concept of programmed cell death called necroptosis, whose morphological characteris‐ tics are similar to those of accidental necrosis, although the molecular events that occur indicate that it is a coordinated process. Necroptosis has been found under special conditions, where pro-apoptotic enzymes were absent or limited. In experimental embryo models, interdigital membrane regression in mouse embryos was effectuated by necrosis triggered by either caspase inhibition or drugs [30]. Necroptosis is thus a form of programmed cell death that has

been demonstrated under experimental conditions, when apoptosis is inhibited.

Several studies have succeeded in discerning the molecular events that occur during necrop‐ tosis, and it is those events that differentiate between necrosis (an accidental process) and necroptosis (a programmed process). Necroptosis has been observed in several pathological cell death events, such as ischemic brain injury, myocardial infarction, exotoxicity, and

Necroptosis is morphologically characterized by several cytoplasmic changes. In fact, it is sometimes possible to distinguish the different degrees of advance of this process as the organelles swell, the cell membrane fragments, and cytoplasmic and nuclear disintegration become evident. During necroptosis, the nuclei remain intact and there is no massive caspase activation, chromatin condensation, spillage of cell contents, phagocytosis by macropinocy‐ tosis, lysosomal leakage, or oxidative bursts [32]. The term necroptosis has been introduced to identify a process of cell death with morphological characteristics distinct from those of apoptosis. Because there was no caspase activation during this process, it is called "caspase-

Necroptosis is a programmed event that ends with the delivery of the cytoplasmic contents into the extracellular space. Membrane destabilization is a consequence of different intracel‐ lular mechanisms that generate osmotic changes by damaging the ion balance. When DNA damage occurred due to reactive oxygen species, the PARP protein was activated and began the reparation process; however, this process consumed abundant ATP and that reduction initiated a sequence of events that led to a deficient cellular efflux of calcium. The decreased ATP levels affected the activity of Na+-K+ ATPase, which requires a large amount of ATP in order to function correctly. This decreased Na+-K+ ATPase activity reduced calcium release and, as a result, increased intracellular calcium levels, leading to membrane destruction (reviewed in [33]). The breakdown of the cellular membrane, in turn, released several signals

**6. Biochemical aspects of necroptosis**

428 Cell Death - Autophagy, Apoptosis and Necrosis

chemotherapy-induced cell death [31].

independent".

ThemechanismproposedfortheonsetofnecroptosisinvolvesparticipationoftheTNF-R(tumor necrosis factor-receptor), Fas, and TRAIL receptors, all of which belong to the tumor necrosis factor/nervegrowthfactorreceptor superfamilyandare involvedinapoptotic celldeath(Figure 10). Activation of TNF receptors by their ligands triggers different responses that involve prosurvival or pro-cell-death processes. Activated TNFR1 induces recruitment of TRADD, TRAF2/2, RIPK1, IAPs, and LUBAC to form a pro-survival complex that activates NF-kap‐ paB, JNK, and p38 MAPKs (reviewed in [35]). However, once this complex becomes establish‐ ed it is able to recruit FADD and procaspase-8, which produces a complex that could initiate either apoptosis or necroptosis. Under conditions of low levels of procaspase-8, a different complex is formed, – one that includes the receptor which interacts with protein 1 (RIP1 – a serine/threonine kinase activator -) and leads to the onset of necroptosis cell death. Biochemi‐ cally, necroptosis is defined as a form of cell death thatis dependent onRIP1, which is the target protein in necrotic cell death induced by the TNFα, TRAIL, and CD95 receptors [36].

**Figure 10.** Biochemical aspects of necroptosis. TNFR recruits TRADD; this recruitment allows the formation of differ‐ ent complexes related to the RIPK1 protein or to the pro-caspase-8. The TRADD-FADD-pro-caspase-8 allows caspase-8 activation, which in turn activates the executer caspases, promoting the apoptosis process. Under conditions where caspase-8 is inhibited, the formation of the complex TRADD-FADD-RIPK1 initiates necroptosis.

The RIP3 is implicated in necroptosis during inflammatory responses to virus infections [37], and during cellular necrosis in response to the TNF-alpha family of death-inducing cytokines [38]. RIP3 mediates necroptosis induced by Smac mimetic and TNFα [38].

Activation of RIP can be directed not only by TNFR, but also by other death receptors, such as Fas. Activation of ligands associated with apoptotic cell death, such as Fas, in conditions that are unfavorable for apoptosis, that is, when caspases are absent or inhibited, allows RIP activation, which leads to death by necrosis [36, 39].

The biochemical process of necroptosis is a new and active field, so not all the routes of activation of this event have been determined. Diverse studies concur that kinase RIP is involved, as we have mentioned. Another protein, PARP-1 (poly (ADP-ribose) polymerase-1), has been shown to be involved in necrotic cell death by means of DNA-damaging agents [40], since PARP-1 is an abundant repair nuclear protein. PARP-1 is activated via TRAIL-induced necroptosis that induces ATP depletion [41]. DNA strand breaks promote the activation of PARP-1 (poly(ADP-ribose) polymerase-1) for DNA repair; PARP-1 binds to DNA strand breaks using NAD+ as substrate, generating a negatively charged PARP-1, which in turn is dissociated from DNA ends, allowing the DNA repair process [42, 43]. Intensive PARP-1 activation can generate increased NAD+ depletion and, as a result, an energy failure that leads to necrosis. In neuronal cells under severe oxidative stress, PARP-1 activation resulted in NAD + and ATP depletion that caused cell death [44].

### **7. Concluding remarks**

Cell death is a normal event that controls tissue homeostasis. Today, we know that cells can be eliminated by means of different pathways that involve programmed or accidental mechanisms. Apoptotic cell death has been considered the major factor in physiological cell death, but recent evidence demonstrates that other routes of cell elimination – such as autophagy – also play important roles in maintaining homeostasis. A third route of cell death is necrosis, which was long considered an accidental form, characterized by general membrane swelling and ATP depletion. More recently, however, a new concept has been introduced: necroptosis. Necroptosis has been proposed as a kind of programmed cell death that is distinct from necrosis and apoptosis; one in which several signals involved in apoptosis participate significantly to initiate the process. It is important to note that necroptosis is an event that can be activated and regulated by such receptors as TNF or Fas – both of which are involved in the extrinsic route of apoptosis activation – when the pro-apoptotic signals are not available or are inhibited.

Activation of death receptors triggers a signaling cascade that includes activation of kinase RIP1, which in turn generates diverse intracellular reactions that lead the cell toward energy failure and conclude with the loss of intracellular homeostasis and the rupture of the mem‐ branes that, finally, generates an immunological response.

### **Acknowledgements**

The RIP3 is implicated in necroptosis during inflammatory responses to virus infections [37], and during cellular necrosis in response to the TNF-alpha family of death-inducing cytokines

Activation of RIP can be directed not only by TNFR, but also by other death receptors, such as Fas. Activation of ligands associated with apoptotic cell death, such as Fas, in conditions that are unfavorable for apoptosis, that is, when caspases are absent or inhibited, allows RIP

The biochemical process of necroptosis is a new and active field, so not all the routes of activation of this event have been determined. Diverse studies concur that kinase RIP is involved, as we have mentioned. Another protein, PARP-1 (poly (ADP-ribose) polymerase-1), has been shown to be involved in necrotic cell death by means of DNA-damaging agents [40], since PARP-1 is an abundant repair nuclear protein. PARP-1 is activated via TRAIL-induced necroptosis that induces ATP depletion [41]. DNA strand breaks promote the activation of PARP-1 (poly(ADP-ribose) polymerase-1) for DNA repair; PARP-1 binds to DNA strand breaks using NAD+ as substrate, generating a negatively charged PARP-1, which in turn is dissociated from DNA ends, allowing the DNA repair process [42, 43]. Intensive PARP-1 activation can generate increased NAD+ depletion and, as a result, an energy failure that leads to necrosis. In neuronal cells under severe oxidative stress, PARP-1 activation resulted in NAD

Cell death is a normal event that controls tissue homeostasis. Today, we know that cells can be eliminated by means of different pathways that involve programmed or accidental mechanisms. Apoptotic cell death has been considered the major factor in physiological cell death, but recent evidence demonstrates that other routes of cell elimination – such as autophagy – also play important roles in maintaining homeostasis. A third route of cell death is necrosis, which was long considered an accidental form, characterized by general membrane swelling and ATP depletion. More recently, however, a new concept has been introduced: necroptosis. Necroptosis has been proposed as a kind of programmed cell death that is distinct from necrosis and apoptosis; one in which several signals involved in apoptosis participate significantly to initiate the process. It is important to note that necroptosis is an event that can be activated and regulated by such receptors as TNF or Fas – both of which are involved in the extrinsic route of apoptosis activation – when the pro-apoptotic signals are not available

Activation of death receptors triggers a signaling cascade that includes activation of kinase RIP1, which in turn generates diverse intracellular reactions that lead the cell toward energy failure and conclude with the loss of intracellular homeostasis and the rupture of the mem‐

branes that, finally, generates an immunological response.

[38]. RIP3 mediates necroptosis induced by Smac mimetic and TNFα [38].

activation, which leads to death by necrosis [36, 39].

430 Cell Death - Autophagy, Apoptosis and Necrosis

+ and ATP depletion that caused cell death [44].

**7. Concluding remarks**

or are inhibited.

We thank CONACyT for grant 180526. The authors kindly thank Paul C. Kersey Johnson for reviewing the English word usage and grammar.

### **Author details**

Ma. Luisa Escobar, Olga M. Echeverría and Gerardo H. Vázquez-Nin\*

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

Laboratorio de Microscopía Electrónica, Depto. de Biología Celular, Facultad de Ciencias, Universidad Nacional Autónoma de México (UNAM), México

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