Keywords

Junctional complexes; Tight junctions; Claudins; Occludins; Nectins

Introduction

There multiple types of epithelial cells depending on the organ and the function they perform. They are squamous, cuboidal or glandular and columnar epithelium and they can be single or multiple layered. The cells in epithelial tissues are tightly packed together with very little intercellular and extracellular space. The epithelial cells are connected to one another and to the basement membrane by junctional complexes. They are tight junctions (TJs), adherens junctions (AJs), gap junctions, desmosomes and hemidesmosomes. AJs are mediated by E-cadherin. Desmosomes are mediated by desmoglein and desmocollin that also belong to cadherin family. GJs constitute of tube like cylinder formed by transmembrane proteins called connexins and the hemidesmosomes consist of integrins that help the cells adhere to the basement membrane. In this review, we focus on the TJ and AJ proteins and their role in tumor suppression and the various signaling events at these junctions [1-3].

Tight Junctions

TJs are present the apical most region at the cell-cell junctions, followed by AJs. TJs consist of four types of transmembrane proteins: Claudins, Occludins, Nectins and Junctional Adhesion Molecules. TJs help maintain cell polarity, perform barrier function, hold the cells together, and regulate the movement of ions, and other molecules. They perform these functions either by homotypic or heterotypic interactions, by binding to peripheral proteins with their cytoplasmic tail that link them to the actin cytoskeleton [4-6].

Claudins

Claudins (CLDN) are tetra span proteins expressed at the tight junctions. However, unlike occludins they are expressed in cells that do not form TJs but are highly expressed in cells that form TJs. There 27 claudins 1 to 24, and 3 CLDNs from splice variants of CLDN10, 18 and 19. In general they are about 20 to 34 KD in size. Like occludins their N and C- termini extend into the cytoplasm and c-terminus is longer to be able to bind to different scaffolding proteins. Claudins act as para-cellular barrier in the absence of occludin. Like occludins the two claudin extracellular loops interact with the extracellular loops of claudin on adjacent cells. This interaction and cell-cell adhesion is Ca+2 independent. Claudins bind to ZO-1, ZO-2, ZO-3, PAR3 and PAR6 at their cytoplasmic tail. Mutations in different types of claudins are indicative of disease and some CLDNs are abnormally expressed in cancers while some are downregulated. CLDN1 is known as a tumor suppressor for breast cancer. It is also known as senescence-associated epithelial membrane protein1. Claudins play a role in regulation of paracellular permeability of specific ions to maintain physiological homeostasis. CLDN3 and 4 are upregulated in several cancers and play a role in motility, invasion and anoikis resistance. Basal type breast cancers are referred to a claudin-low type which is a very aggressive subtype [7-9].

Junctional Adhesion Molecules

JAMs are about 40KD in size. There are six types of JAMs that are so far identified. They are JAM-A, JAM-B, JAM-C, CAR (coxsackie and adenovirus receptor), ESAM (endothelial cell-selective adhesion molecule) and JAM4. JAMs are expressed on leukocytes, platelets, epithelial and endothelial cells. In the epithelial cells they are found to be localized at tight junctions. JAM-c is highly expressed during embryogenesis, lymphatic vessels, lymph nodes, endothelial venules and vascular structures in the kidney [10-12]. JAMs have membrane distal V-type Ig-domain and membrane-proximal C2-type Ig-domain in the N-terminus and a PDZ domain in the c-terminu. Both v-type Ig domain and C2- type Ig domain have conserved disulphide bridges. They undergo homotypic and heterotypic interactions. JAMs interact with occludins and this interaction is important for TJs assembly. The JAMs interact with leukocytes β2, α4β1, β1, LFA1 and Mac- 1 through their extracellular domains. The cytoplasmic tail of JAMs anchor or bind to various tight junction proteins like zonula occludens (ZO-1), PAR-3 (cell polarity protein), AF-6 and MUPP1 that link JAMs to the actin cytoskeleton. Therefore, JAMs play a role in regulating immune cells and endothelial cell association in the immune surveillance and also maintain cell polarity by tight junction formation in endothelial and epithelial cells [13,14].

Cell Signaling Events at TJs

Bves is a transmembrane cell-cell adhesion protein expressed at TJs that is known to play a role in TJ formation and function. Bves has been shown to interact with ZO-1. Patricia et al. used human corneal epithelial cells (HCE) to study how Bves regulates TJ associated signaling. ZO-1 binds to GEF-H1 via cingulin and directly to ZONAB; and GEF-H1 activates RhoA at TJs. Patricia et al. found that Bves regulates TJ formation and function by sequestering ZO-1 at the TJs. In the presence of Bves, GEF H1 and ZONAB were localized to the membrane but its loss caused an increase in free ZO-1; cytosolic GEF-H1 and nuclear ZONAB. This resulted in high RhoA by GEF-H1 and thus increases in phosphorylated myosin light chain (MLC). MLC is a target of Rock which is a Rho kinase that is down stream of RhoA. P-MLC causes cell contraction and important for motility [15-17].

It is known that ZONAB is a transcriptional factor that regulates cell proliferation. To identify the genes that are induced by ZONAB activity Tony et al. overexpressed ZONAB or the SH3 domain of ZO-1 to inhibit ZONAB activity in MCF10A cells. They found that ZONAB binds to SH3 domain of ZO-1 but not to SH3 domains of Abl, Src, Crk, Grb2, ZO-2 or ZO-3. MCF10A cells showed increased proliferation in ZONAB overexpressed cells but not in cells that express both ZO-1 and ZONAB. With increase in ZONZAB, there was an increase in transcription factors like c-jun and c-myc and a significant increase in mRNA levels of proliferating cell nuclear antigen (PCNA) and cyclin D1. Chromatin immunoprecipitation assays show that both PCNA and cyclin D1 promoters have ZONAB binding sequences. The similar results for ZONAB activity are seen in MDCK cells.

The above are few of much evidence showing that TJs not only perform barrier function and maintain cell polarity of epithelial cells but also play a role in cell signaling. TJs regulate the transcriptional activity of multiple genes and as a result regulate cell proliferation which is important or tumor suppression.

Occludins

It is a 504 aa long polypeptide with about 60 to 65 KD. It has four transmembrane domains, with both N-terminus and C-terminus in the cytoplasm. The hydrophicity plot shows that its primary structure is similar to connexin, a gap junctional protein. The extracellular loops interact with the extracellular loops of other occludin on the neighboring cell in a Ca+2 independent way. This allows occludins to seal the para-cellular space [18,19]. Its cytoplasmic tail interacts with zonula occludens-1 (ZO-1) and this interaction is important for occludin to maintain its extracellular cell-cell adhesion [20-45]. Occludin is not expressed in cells that don’t form TJs but only in epithelial and endothelial cells that do form TJs. Occludin molecules are important for TJ assembly which is regulated by its phosphorylation. Smales et al. have showed that CK2 phosphorylates c-terminus of occludin in crude brain cell extracts [46]. Yu et al. showed that KD of occludin caused increased permeability to large cations.

Adherens Junctions

AJs are mediated by E-cadherin. E-cadherin is expressed on the lateral sides at cell-cell contacts special beneath the TJs. The human E-cadherin gene (CDH1) is located on chromosome 16q22.1. E-cadherin was first identified by Hyafil et al. They called it as uvomorulin (UM) because anti-uvomorulin antibodies were able to bind to E-cadherin. Electron microscopic images show that E-cadherin is localized to the adherens junctions of intestinal epithelium. Nagafuchi et al. first cloned E-cadherin cDNA from mouse and showed that it is a calcium cell-cell adhesion molecule. In addition to mediating cell-cell adhesion, E-cadherin is also important for maintaining apico-basal polarity and it is a type I transmembrane glycoprotein which is 728 amino acids long. It has a single transmembrane domain, cytoplasmic tail and five extracellular repeats. The extracellular domain binds calcium and E-cadherin forms a homodimer with E-cadherin on adjacent cells via histidine-alanine-valine (HAV) motif in its aminoterminus. Even though the extracellular domain mediates cellcell adhesion, the intracellular cytoplasmic domain is required for the same function. The cytoplasmic tail of E-cadherin connects the adherens junction to actin cytoskeleton via many adaptor proteins like α, β, γ catenin’s and p120 [47-49]. Therefore E-cadherin along with the adaptor molecules is important for adherens junction integrity and also many signaling pathways.

Cell Signaling Events at TJs

Beta catenin is a transcription factor that is sequestered at the AJs and it is an important molecule in Wnt signaling. On the other hand, loss of alpha catenin causes hyper activation of MAPK signaling and p120 catenin regulates NF-kβ and Rho pathways [50-53]. The loss of E-cadherin activates specific downstream signaling pathways that allow epithelial cells to become mesenchymal like via epithelial to mesenchymal transition (EMT). The loss or downregulation of E-cadherin can occur due to several reasons. They are somatic mutations, chromosomal deletions, promoter silencing by DNA hypermethylation or by transcription factors like Snail, Slug and Twist and proteolytic cleavage [54]. Chen et al. showed that under normal circumstances beta catenin is required for E-cadherin transport to the cell membrane, which otherwise causes proteosomal degradation of E-cadherin. On the otherhand, cytosolic beta-catenin is phosphorylated at the serine residues of its N-terminus by GSK-3β, axin and adenomatous polyposis coli (APC) complex. This results in beta-catenin ubiquitination and proteosomal degradation. But wnt signaling allows beta-catenin to accumulate in the cytosol and translocate to the nucleus to activate TCF/LEF transcription factors, c-myc and cyclin D1 that effects cell adhesion, tumor development, increase in cell migration and invasion. Phosphorylation of serine/threonine kinase on E-cadherin or beta-catenin stabilizes their complex but if beta-catenin gets phosphorylated at its tyrosine kinase site by intracellular signaling it disrupts E-cadherin complex and results in EMT [55]. Onder et al. showed that loss of E-cadherin cause’s increase in cytosolic beta-catenin, mesenchymal markers like N-cadherin, fibronectin and vimentin but a decrease in epithelial markers like cytokeratins. To assess if beta-catenin is the main downstream signaling molecule causing EMT upon E-cadherin loss, Onder et al. made double KD of E-cad and beta-catenin in HMLE cells. They found that there is a significant increase in mesenchymal markers like N-cad, vimentin and fibronectin but the overexpression of beta-catenin alone was not enough to induce EMT. So they compared the gene expression profiles of shEcad and DNEcad that still had E-cadherin cytoplasmic tail. They found that DN E-cad downregulated epithelial markers but no induction of EMT. Interestingly, they found that there is an increase in Twist and Zeb-1 that are actually TFs known to repress E-cadherin expression. Twist was upregulated in ShEcad but not in DNEcad. There was an increase in invasion and anoikis resistance with Twist induction in ShEcad cells. When Twist was inhibited there was a decrease in invasion and anoikis resistance. Therefore E-cadherin loss promotes EMT through induction of several downstream signaling pathways.

Nectins

Like JAMs, nectins are also immunoglobulin like cell-cell adhesion molecules. There are 4 members in nectin family; they have 3 immunoglobulin loops in their extracellular domain, one transmembrane domain and a cytoplasmic tail. They play a role in TJ formation by recruiting cell-cell adhesion molecules like JAMs, claudins and occludins. Nectin is connected to actin cytoskeleton via afidin. Nectin has been shown to recruit E-cadherin –β catenin complex and ZO-1 to nectin based cell-cell adhesion sites via afidin [55-57].

Discussion and Conclusion

E-cadherin binds to RhoA via p120 catenin and this inhibits Smad3 phosphorylation by TGFβ via RhoA. But loss of E-cadherin causes upregulation of TGFβ and its downstream targets. In human retinal pigment epithelial cells that have undergone EMT, there is an increase in TGFβ that upregulates anti-apoptotic protein survivin and thus inhibits apoptosis. Therefore loss of E-cadherin expression is enough to induce EMT in mammary epithelium.

Once the cancer cells undergo EMT they enter blood stream, survive there, extravasate and form colonies at a distant sites, called micro and macro metastasis. Usually the detached cells undergo apoptosis and it is called anoikis. But cancer cells gain resistance to anoikis by co-opting themselves with blood platelets to promote metastasis. Overexpression of anti-apoptotic proteins like BCL2, BCL-Xl and XIAP can make cancer cells resistant to anoikis and become more metastatic. Onder et al. showed that loss of E-cadherin caused a decrease in apoptotic marker Annexin V, suggesting that E-cadherin loss enhances anoikis resistance and increased invasiveness. Cancer cells that undergo EMT are more resistant to chemo and radiation therapies, because these cells gain stem like properties [56,57]. Piyush et al. showed that conventional chemotherapy helps select for these cancer stem cells. Therefore, EMT requires loss of cell-cell adhesion molecule E-cadherin that helps cancer cell to become more migratory, invasive and resistant to anoikis.

Walia et al. found that loss of CLCA2, downregulates E-cadherin and induces EMT in HMEC. We found that CLCA2 is expressed at basolateral membranes in epithelial cells and it interacts with EVA1. EVA1 is a transmembrane protein similar to JAMs structurally except it has only one Ig-V-like domain with disulphide bridges in the N-terminus and no PDZ domain in the c-terminus unlike JAMs [58]. However, EVA1 is also expressed on lymphocytes, epithelial and endothelial cells. EVA1 expression caused CHO cells to aggregate in suspension suggesting that it undergoes homotypic interaction. It has been also shown to regulate immune surveillance at blood-CSF barrier. However, we found that EVA1 is expressed at the AJs along with CLCA2 and E-cadherin. Interestingly, its loss leads to cell morphology change and thus cells undergo EMT. Both CLCA2 and EVA1 are tumor suppressor genes expressed at lateral junctions along with E-cadherin [59]. CLCA2 and EVA1 form a complex with ZO-1 and CLCA2 sequesters beta-catenin by itself, therefore inhibits EMT via multiple mechanisms.

On the other hand, TJs sequester transcription factors like ZONAB at cell membrane and inhibit cell proliferation. TJs are not shown to play a role in EMT. However, breast cancers with loss of claudins at TJs are characterized as the most aggressive and recurrent subtypes. Therefore TJs also might play a role in EMT. Junichi et al. showed that ZO-1 is required not only for TJ formation but also belt-like adherens junction formation during epithelial polarization. Anastasios et al. showed that ZO-1 knockdown caused an increase in cell proliferation as well as induced EMT in retinal pigment epithelial cells [60]. There is enough evidence that the proteins present at cell-cell junctions that can inhibit EMT in cancer cells are possible tumor suppressors.

Conflict of Interest

No conflict of interest.

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