**4.2 Proteolytic degradation of occludin**

Studies have found that degradation of tight junction proteins play an important regulatory feature in pathological and physiological tissue remodeling [43]. Basic studies demonstrated that the two fragments of cleaved Occludin released into circulation and the levels of blood occludin correlate well with the extent of blood brain barrier in cerebral ischemic model of rats [44]. In addition, occludin serve as a potential biomarker to predict the severity of acute ischemic stroke, hemorrhagic transformation, and patient prognosis [45]. These results suggested that the degradation of Occludin may be involved in the occurrence and development of many diseases.

## **4.3 MMPs-dependent degradation of occludin**

Matrix metalloproteinases (MMPs) are secreted by astrocytes, endothelial cells, pericytes and peripheral circulating cells and are capable to degrade extracellular matrix (ECM) proteins as well as non-ECM proteins, including cytokines, chemokines, membrane receptors, and antimicrobial peptides [46, 47]. Studied showed that MMPs are related to the development of cancer infiltration and metastasis, inflammatory response, and angiogenesis. Within the endothelial layer, MMPs can degrade intercellular junction molecules (such as cadherin, occludin, and claudins) and intracellular structural proteins (e.g., actins), enhancing the permeability of endothelial barrier [48].

Currently, a number of data have showed that occludin was mainly proteolytically cleaved *via* MMPs to inactive fragments, leading to endothelial barrier disruption. (1) Feng Chen et al. demonstrated MMP9 induced the degradation of occludin and suppressed the synthesis and expression of Occludin in brain endothelial cells and in brains of mice with experimental acute liver failure (ALF), which can cause severe vasogenic brain edema [49]. (2) Related studies showed that LPS/hypoxia induced brain blood barrier (BBB) leakage by MMP2/MMP9 contributed to the degradation of occludin in brain microvascular endothelial cells [34]. (3) TGF-β can promote the production of MMP9 in brain microvascular endothelial cells and retinal endothelial cells, accelerate the degradation of Occludin, and lead to increased vascular endothelial permeability [50]. (4) Several studies demonstrated that MMP2/9 leads to occludin fragmentation in brain microvessels from rat model of cerebral ischemic injury, with resultant brain leakage and brain edema [51–53]. (5) At the same time, Yang et al. firstly described the temporal dynamics of occludin degradation by MMPs in rodent models of cerebral ischemic injury, suggesting that MMP-2 cleaved occludin during the early phase of the ischemia (3 h), while MMP-9 caused further occludin degradation and more long-term (24-h) alterations to BBB integrity. In addition, MMP9 can promote the degradation of occludin through HIF-1α and AQP-4, ultimately triggering BBB disruption and brain edema [54]. (6) Simultaneous data show that MMP2/9 mediated occludin hydrolysis can be used as a marker of blood-brain barrier and blood-retinal barrier in type 2 diabetes and diabetic retinopathy [55, 56]. (7) Caron et al. have suggested that elevated ProMMP-2/9 and MMP9 correlate with increased levels of occludin degradation in rodent kidney endothelium in ischemic injury [57]. (8) The degradation of tight junction proteins (occludin, claudins) through MMP9 secreted by glioma cells is an important mechanism in the BBB breakdown mediated by TGF-β [58]. (9) In acute leukemia, MMP9 secreted by leukemic cells degraded occludin, which constituted an extreme mechanism of the BBB breakdown that contributes to the invasion of the central nervous system [59]. Overall, occludin contains

extracellular MMP cleavage sites and are a substrate of MMPs. In endothelial cells, the degradation of Occludin mediated by MMPs leads to vascular leakage.

#### **4.4 MMP-independent proteolysis of occludin**

At present, a large number of data focus on MMPs-dependent occludin degradation; however, there are also some studies showing the existence of MMPs-independent occludin degradation. (1) Qian et al. found that tryptase can act on mouse brain microvascular endothelial cells to promote the production of MMP9/2, degrade the tight junction proteins occludin and Claudin5, and lead to the destruction of the blood-brain barrier [60]. (2) Wan et al. and Runs et al. verified both serine and cysteine peptidases cleavage the occludin with elevation of epithelial permeability, which reveals a pathological mechanism for allergen delivery across lung and nasal epithelial barriers in asthma and allergic rhinitis sufferers [35]. (3) Caspase-mediated cleavage of the occludin C-terminal promotes apoptosis in MDCKs [61]. The studies about the MMP-independent proteolysis of occludin occurred in the epithelial cells; therefore, more research is needed to further define the MMP-independent proteolysis of occludin in endothelial cells.

#### **4.5 Occludin phosphorylation**

Protein phosphorylation is a ubiquitous type of post-translational modification, whereby protein kinase catalyzes the phosphorylation reactions by transferring the phosphate group of ATP to the substrate protein amino acid residues, typically serine, threonine, and tyrosine, or bind GTP under the action of signal transduction. It was widely demonstrated that protein phosphorylation is the most basic and the most common key mechanism for regulating and controlling protein biological activity and function [62]. Notably, the phosphorylation status of occludin regulating endothelial barrier protection has been received extensive attention.

More than 40 phosphorylation residues are in human occludin; however, only nine sites are confirmed in cell levels by different kinases on certain stimuli, including Y398, T400, Y402, T403, T404, S408, T424, T438, and S490 [46]. All confirmed phosphorylation residues lie in the occludin C terminal. As early as 1997, Sakakibara et al. firstly observed increased phospho-serine [pSer] and phospho-threonine [pThr] occludin selectively localized to intact epithelial TJs as a detergent-insoluble form [63]. Subsequently, Kale and Elias et al. confirmed occludin phosphorylation on key serine, threonine, and tyrosine residues plays a crucial role in the assembly and maintenance of TJs in Caco-2 and MDCK cells [64, 65]. Dörfel and colleagues in 2013 studied that CK2-mediated phosphorylation [T400A/T404A/S408A] of occludin in MDCK-C11 cells bind with ZO1/2 interaction and protect the epithelial barrier [66]. The regulation of occludin phosphorylation in endothelium has also received extensive attention, with many studies focusing on how the phosphorylation status of occludin regulates the vessel barrier.

Different phosphorylation sites of occludin exerts specific functions in endothelial cells: (1) Antonetti and his colleagues investigate the role of tight junction protein occludin phosphorylation at S490 in modulating barrier properties and its impact on visual function. They found that endothelial-specific expression of the S490A form of occludin completely prevented diabetes-induced permeability to label dextran and inhibit leukostasis. Importantly, vascular-specific expression of the occludin mutant completely blocked the diabetes-induced decrease in visual acuity

*The Role of Occludin in Vascular Endothelial Protection DOI: http://dx.doi.org/10.5772/intechopen.107479*

and contrast sensitivity in the retinas of streptozotocin-induced diabetic mice [67]. (2) Treatment with glutamate increased tyrosine phosphorylation and decreased threonine phosphorylation of occluding in brain microvascular endothelial cells. It affects the redistribution of occludin. These may lead to opening of the blood-brain barrier (BBB) and induce further brain damage [68]. (3) The phosphorylation of occludin and claudin-5 by RhoK at specific sites disrupted the integrity of BBB. Antibodies against specific phosphorylation sites of occludin could be useful reagents for monitoring BBB dysfunction *in vivo* [69]. (4) Other recent studies confirm the importance of threonine phosphorylation with occludin C-terminal for mediating its ability to localize to the tight junction [70–72]. According to the above studies, it is demonstrated that the specific phosphorylation sites of occludin regulate the different function of endothelial cells. Other studies need to further focus on other phosphorylation residues of occludin in correlating with endothelial function.

### **4.6 Occludin ubiquitination**

Ubiquitination, also known as ubiquitylation, refers to the process in which the ubiquitin (a small 76-residue regulatory protein widely expressed in eukaryotes) molecules classify the proteins in cells under the action of a series of special enzymes, choose the target protein molecules from them, and specifically modify the target proteins. These special enzymes include ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), ubiquitin-protein ligase (E3), and degrading enzymes [73]. Ubiquitination plays an important role in protein localization, metabolism, function, regulation, and degradation [73, 74]. At the same time, ubiquitination takes part within the regulation of nearly all life activities, including cell cycle, proliferation, apoptosis, differentiation, metastasis, gene expression, transcriptional regulation, signal transmission, damage repair, inflammation, and immunity [73, 74]. In recent years, studies about the functional role of occludin ubiquitination in diseases have begun to emerge in a burst. And the modification way has become an important regulatory mechanism in epithelial and endothelial function.

Hannelore et al. identified a novel interaction between occludin N-terminal and the E3 ubiquitin-protein ligase Itch, a member of the HECT domain-containing ubiquitin-protein ligases by co-immunoprecipitation *in vivo* and *in vitro* [75]. In addition, the team provides evidence that Itch is specifically involved in the ubiquitination of occludin *in vivo*, and that the degradation of occludin is sensitive to proteasome inhibition. The team firstly confirmed that occludin can be ubiquitinated. Liu and Lee et al. reported that occludin degradation was associated with Itch and UBC-4 (an ubiquitin-conjugating enzyme), resulting in occludin ubiquitination to disrupt tight junctions in blood and testosterone barrier [76]. A slightly later study reported that a conserved C-terminal PY motif of occludin association with Nedd4-2 was involved in the paracellular permeability of mpk-CCD[c14] cells (a collecting duct epithelial cell line) by coimmunoprecipitation. These authors also showed that small interfering RNA [siRNA]-mediated knockdown of Nedd4-2 increased occludin expression and reduced the epithelial permeability, with Nedd4-2 overexpression having the opposite effects [77]. In conclusion, ubiquitinated occludin is taken part in the maintenance of cell barrier.

Currently, the study about the regulation of occludin ubiquitination in vascular endothelial function focuses on the following aspects. (1) Murakami et al. demonstrated that Ser-490 phosphorylation of occludin is an essential prerequisite for its ubiquitination in BRECs. The team showed that a C-terminal occludin-ubiquitin

chimera was internalized, bypassing the requirement for phosphorylation. Thus, VEGF, through PKCβ-mediated phosphorylation, promotes Itch-mediated ubiquitylation of occludin, which is required for its internalization and degradation, thereby enhancing retinal endothelial permeability [78]. (2) It is well known that blood-spinal cord barrier (BSCB) breakdown is a hallmark of amyotrophic lateral sclerosis (ALS). Results found that mutant SOD1 induced occludin phosphorylation, which promoted the subsequent occludin ubiquitination mediated by the E3 ligase ITCH. Moreover, ubiquitinated occludin interacted with Eps15 to initiate its internalization, then trafficked to Rab5-positive vesicles, and be degraded by proteasomes, resulting in a reduction in cell surface localization and total abundance [79]. (3) Feng et al. showed that the γ-secretase blocker DAPT reduced the permeability of the BBB by decreasing the ubiquitination and degradation of occludin during permanent brain ischemia [80]. Notwithstanding the information already generated about the role of occludin ubiquitination in endothelial cells, several avenues for future investigation still remain. The identification of new ubiquitin enzymes, characterization of tissue and cell-specific occludin ubiquitination, and deciphering the functional rapport between different modification events (e.g., phosphorylation, ubiquitination, proteolysis), will likely typify future studies in this field. This will ultimately yield a fuller understanding of how ubiquitination modifications to occludin affect TJ characteristics and will help to unlock the therapeutic potential of the TJ by identifying new cellular targets for intervention in diseases characterized by barrier dysregulation.
