Claudin-based Tight Junctions Research Articles

Tight junction formation by a claudin mutant lacking the COOH-terminal PDZ domain-binding motif.

Claudin-based tight junctions (TJs) are formed at the most apical part of cell-cell contacts in epithelial cells. Previous studies suggest that scaffolding proteins ZO-1 and ZO-2 (ZO proteins) determine the location of TJs by interacting with claudins, but this idea is not conclusive. To address the role of the ZO proteins binding to claudins at TJs, a COOH-terminal PDZ domain binding motif-deleted claudin-3 mutant, which lacks the ZO protein binding, was stably expressed in claudin-deficient MDCK cells. The COOH-terminus-deleted claudin-3 was localized at the apicolateral region similar to full-length claudin-3. Consistently, freeze-fracture electron microscopy revealed that the COOH-terminus-deleted claudin-3-expressing cells reconstituted belts of TJs at the most apical region of the lateral membrane and restored functional epithelial barriers. These results suggest that the interaction of claudins with ZO proteins is not a prerequisite for TJ formation at the most apical part of cell-cell contacts.

Intestinal deletion of Claudin-7 enhances paracellular organic solute flux and initiates colonic inflammation in mice

ObjectiveTo design novel anti-inflammation treatments, it is important to recognise two distinct steps of inflammation: initiation and acceleration. In IBDs, intestinal inflammation is reported to be accelerated by dysfunction in...

Transcriptional Control of Tight Junction Proteins via a Protein Kinase C Signal Pathway in Human Telomerase Reverse Transcriptase-Transfected Human Pancreatic Duct Epithelial Cells

In human pancreatic cancer, integral membrane proteins of tight junction claudins are abnormally regulated, making these proteins promising molecular diagnostic and therapeutic targets. However, the regulation of claudin-based tight junctions remains unknown not only in the pancreatic cancer cells but also in normal human pancreatic duct epithelial (HPDE) cells. To investigate the regulation of tight junction molecules including claudins in normal HPDE cells, we introduced the human telomerase reverse transcriptase (hTERT) gene into HPDE cells in primary culture. The hTERT-transfected HPDE (hTERT-HPDE) cells were positive for the pancreatic duct epithelial markers such as CK7, CK19, and carbonic anhydrase isozyme 2 and expressed epithelial tight junction molecules claudin-1, -4, -7 and, -18, occludin, JAM-A, ZO-1, ZO-2, and tricellulin. By treatment with fetal bovine serum or 12-O-tetradecanoylphorbol 13-acetate (TPA), the tight junction molecules were up-regulated at the transcriptional level via a protein kinase C (PKC) signal pathway. A PKC-alpha inhibitor, Gö6976, prevented up-regulation of claudin-4 by TPA. Furthermore, a PKC-delta inhibitor, rottlerin, prevented up-regulation of claudin-7, occludin, ZO-1, and ZO-2 by TPA. By GeneChip analysis, up-regulation of the transcription factor ELF3 was observed in both fetal bovine serum- and TPA-treated cells. Treatment with small interfering RNAs of ELF3 prevented up-regulation of claudin-7 by TPA. These data suggest that tight junctions of normal HPDE cells were at least in part regulated via a PKC signal pathway by transcriptional control.

Tight junction-based epithelial microenvironment and cell proliferation

Belt-like tight junctions (TJs), referred to as zonula occludens, have long been regarded as a specialized differentiation of epithelial cell membranes. They are required for cell adhesion and paracellular barrier functions, and are now thought to be partly involved in fence functions and in cell polarization. Recently, the molecular bases of TJs have gradually been unveiled. TJs are constructed by TJ strands, whose basic frameworks are composed of integral membrane proteins with four transmembrane domains, designated claudins. The claudin family is supposedly composed of at least 24 members in mice and humans. Other types of integral membrane proteins with four transmembrane domains, namely occludin and tricellulin, as well as the single transmembrane proteins, JAMs (junctional adhesion molecules) and CAR (coxsackie and adenovirus receptor), are associated with TJ strands, and the high-level organization of TJ strands is likely to be established by membrane-anchored scaffolding proteins, such as ZO-1/2. Recent functional analyses of claudins in cell cultures and in mice have suggested that claudin-based TJs may have pivotal functions in the regulation of the epithelial microenvironment, which is critical for various biological functions such as control of cell proliferation. These represent the dawn of 'Barriology' (defined by Shoichiro Tsukita as the science of barriers in multicellular organisms). Taken together with recent reports regarding changes in claudin expression levels, understanding the regulation of the TJ-based microenvironment system will provide new insights into the regulation of polarization in the respect of epithelial microenvironment system and new viewpoints for developing anticancer strategies.

FGF Signaling Preserves the Integrity of Endothelial Adherens Junctions

Fibroblast growth factor (FGF)-activated signaling regulates an array of cellular processes ranging from embryonic development to tissue repair. A recent paper by Murakami et al. identifies a potentially important role for FGF signaling in maintenance of endothelial barrier homeostasis through the regulation of adherens junctions.

The host adherens junction molecule nectin-1 is downregulated in Chlamydia trachomatis-infected genital epithelial cells

Nectin-1, a member of the immunoglobulin superfamily, is a Ca(2+)-independent cell adhesion protein implicated in the organization of E-cadherin-based adherens junctions (AJs) and claudin-based tight junctions (TJs) in epithelial cells. Nectin-1 also regulates cell-cell adhesion and cell polarization in a Cdc42- and Rac-dependent manner. Western blot analyses demonstrated that accumulation of host nectin-1 is decreased by 85 % at 48 hours post-infection (h.p.i.) in Chlamydia trachomatis serovar E-infected HeLa cells. Time-course experiments demonstrated that this decrease was sustained to 60 h.p.i. Nectin-1 downregulation in C. trachomatis-infected cells was prevented by both chloramphenicol exposure and prior inactivation of the chlamydiae with UV light, demonstrating that active C. trachomatis replication was required. Penicillin G-exposure studies demonstrated that nectin-1 accumulation was also altered during persistent infection. Finally, RT-PCR analyses indicated that chlamydial infection did not alter accumulation of any nectin-1 transcripts, demonstrating that nectin-1 accumulation is reduced at a post-transcriptional level. Intesrestingly, N-cadherin-dependent cell-cell junctions can be disrupted by C. trachomatis infection, as reported by Prozialeck et al. (2002). Because interaction of nectin molecules on adjacent cells is essential for AJ formation, these data suggest that C. trachomatis may disrupt AJs, at least in part, by diminishing nectin-1 accumulation. Notably, release of chlamydiae-infected epithelial cells has been observed both in vitro from polarized monolayers and in vivo from tissues, suggesting that chlamydia-modulated downregulation of adhesion molecules and the subsequent disruption of host cell adherence may be involved in chlamydial dissemination or pathogenesis.

Requirement of nectin, but not cadherin, for formation of claudin-based tight junctions in annexin II-knockdown MDCK cells

Adherens junctions (AJs) and tight junctions (TJs) comprise a junctional complex which plays key roles not only in cell adhesion and polarization but also in regulation of cell movement and proliferation in epithelial cells. E-Cadherin and nectin are major cell-cell adhesion molecules (CAMs) at AJs, whereas claudin is a major CAM at TJs. We have shown that the cadherin-based cell-cell adhesion is not formed in MDCK cells in which annexin II, a Ca(2+)- and phospholipid-binding protein, is knocked down. Here, we found that TJs and the nectin-based cell-cell adhesions were formed in annexin II-knockdown cells. The formation of TJs in annexin II-knockdown MDCK cells required the nectin-based cell-cell adhesion and afadin, a nectin- and actin-filament-binding protein. In addition, it required the activation of Cdc42 and Rac small G proteins and subsequent reorganization of the IQGAP1-dependent actin cytoskeleton which were induced by the nectin-based cell-cell adhesion. These results indicate that the nectin-based cell-cell adhesion and afadin, but not the cadherin-based cell-cell adhesion, are necessary for the formation of TJs and that the signaling by nectin and the subsequent reorganization of the actin cytoskeleton are also necessary for the formation of TJs under certain conditions.

Involvement of the c-Src-Crk-C3G-Rap1 Signaling in the Nectin-induced Activation of Cdc42 and Formation of Adherens Junctions

Nectins, Ca(2+)-independent immunoglobulin-like cell-cell adhesion molecules, induce the activation of Cdc42 and Rac small G proteins, enhancing the formation of cadherin-based adherens junctions (AJs) and claudin-based tight junctions. Nectins recruit and activate c-Src at the nectin-based cell-cell contact sites. c-Src then activates Cdc42 through FRG, a Cdc42-GDP/GTP exchange factor. We showed here that Rap1 small G protein was involved in the nectin-induced activation of Cdc42 and formation of AJs. Rap1 was recruited to the nectin-based cell-cell contact sites and locally activated through the c-Src-Crk-C3G signaling there. The activation of either c-Src or Rap1 alone was insufficient for and the activation of both molecules was essential for the activation of FRG. The activation of Rap1 was not necessary for the c-Src-mediated phosphorylation or recruitment of FRG. The inhibition of the Crk, C3G, or Rap1 signaling reduced the formation of AJs. These results indicate that Rap1 is activated by nectins through the c-Src-Crk-C3G signaling and involved in the nectin-induced, c-Src- and FRG-mediated activation of Cdc42 and formation of AJs.

Dysfunction of keratinocyte adhesion.

Dysfunction of keratinocyte adhesion.

Expression patterns of claudin family of tight junction membrane proteins in developing mouse submandibular gland.

Here, we investigated the expression of the claudin family of tight junction transmembrane proteins in the developing mouse submandibular gland. Data obtained by reverse transcriptase-polymerase chain reaction, Western blot, and immunofluorescence microscopy showed the expression and localization of claudin-3 to -8, -10, and -11 at epithelial tight junctions. Examination of the glands taken from embryonic day (E) 14, E16, and newborn mice revealed differential expression patterns of these claudins in the developing epithelium. Claudin-3, -5, and -7 were expressed in all of the luminal epithelial cells of the ducts at all of the developmental stages examined and in those of terminal tubules at E16 and later. Claudin-4 was expressed mainly in the ducts at all the developmental stages. The expression of claudin-6 and -8 was also restricted to the ducts at E14 and E16; but after birth, the former was undetectable, whereas the latter was expressed in both the ducts and terminal tubules. Claudin-10 and -11 were detectable mainly in the terminal tubules at E16 and later. In addition to being found in the epithelium, claudin-5 was also expressed in certain mesenchymal cells, probably endothelial cells. These results will provide a valuable resource for further investigation of tubulogenesis and physiological regulation of claudin-based tight junctions.

Involvement of Nectin-activated Cdc42 Small G Protein in Organization of Adherens and Tight Junctions in Madin-Darby Canine Kidney Cells

Nectins, Ca2+-independent immunoglobulin-like cell-cell adhesion molecules, trans-interact and form cell-cell adhesion, which increases the velocities of the formation of the E-cadherin-based adherens junctions (AJs) and the claudin-based tight junctions (TJs) in Madin-Darby canine kidney (MDCK) cells. The trans-interactions of nectins furthermore induce activation of Cdc42 and Rac small G proteins, but the roles of these small G proteins activated in this way remain unknown. We examined here the role and the mode of action of Cdc42 in the organization of AJs and TJs in MDCK cells. We first made the NWASP-Cdc42 and Rac interactive binding (CRIB) domain, an inhibitor of activated Cdc42, fused to the Ki-Ras CAAX motif (NWASP-CRIB-CAAX; where A is aliphatic amino acid), which was targeted to the cell-cell adhesion sites. We then found that overexpression of NWASP-CRIB-CAAX reduced the velocities of the formation of AJs and TJs. Conversely, overexpression of a constitutively active mutant of Cdc42 (V12Cdc42) increased their velocities, and the inhibitory effect of NWASP-CRIB-CAAX was suppressed by co-expression with V12Cdc42. The inhibitory effect of NWASP-CRIB-CAAX on the formation of AJs and TJs was suppressed by co-expression of nectin-1 of which trans-interaction activated endogenous Cdc42. Moreover, the formation of the claudin-based TJs required a greater amount of activated Cdc42 than that of the E-cadherin-based AJs. These results indicate that the Cdc42 activated by the trans-interactions of nectins is involved in the organization of AJs and TJs in different mechanisms in MDCK cells.

Nectin and afadin: novel organizers of intercellular junctions.

The cadherin superfamily plays key roles in intercellular adhesion. An emerging intercellular adhesion system, consisting of nectin and afadin, also has roles in organization of a variety of intercellular junctions either in cooperation with, or independently of, cadherin. Nectin is a Ca(2+)-independent immunoglobulin-like intercellular adhesion molecule, and afadin is a nectin- and actin-filament-binding protein that connects nectin to the actin cytoskeleton. This novel intercellular adhesion system has roles in the organization of E-cadherin-based adherens junctions and claudin-based tight junctions in epithelial cells. The adhesion system is furthermore involved in the formation of synapses in neurons and the organization of heterotypic junctions between Sertoli cells and spermatids in the testis.

Ectodomain shedding of nectin-1α by SF/HGF and TPA in MDCK cells

Nectin is a Ca 2+-independent immunoglobulin-like cell–cell adhesion molecule implicated in the organization of the junctional complex comprised of E-cadherin-based adherens junctions and claudin-based tight junctions in epithelial cells. Scatter factor (SF)/hepatocyte growth factor (HGF) and 12- O-tetradecanoylphorbol-13-acetate (TPA), a tumor-promoting phorbol ester, induce cell spreading, followed by cell–cell dissociation and cell scattering, in Madin–Darby canine kidney (MDCK) cells. We found here that SF/HGF and TPA induced proteolytic cleavage of nectin-1α in the ectodomain, resulting in generation of the 80-kDa extracellular fragment and the 33-kDa fragment composed of the transmembrane and cytoplasmic domains, in MDCK cells. This shedding of nectin-1α was inhibited by metalloprotease inhibitors. These results indicate that SF/HGF and TPA induce the ectodomain shedding of nectin-1α presumably by a metalloprotease, and have raised the possibility that this shedding is involved in the SF/HGF- and TPA-induced cell–cell dissociation.

Role of nectin in organization of tight junctions in epithelial cells.

In polarized epithelial cells, cell-cell adhesion forms specialized membrane structures comprised of claudin-based tight junctions (TJs) and of E-cadherin-based adherens junctions (AJs). These structures are aligned from the apical to the basal side of the lateral membrane, but the mechanism of this organization remains unknown. Nectin is a Ca2+ independent immunoglobulin-like cell-cell adhesion molecule which localizes at AJs. Nectin is associated with E-cadherin through their respective cytoplasmic tail-binding proteins, afadin and catenins, and involved in the formation of AJs in cooperation with E-cadherin. We show here that nectin is also involved in the formation of TJs. During the formation of the junctional complex consisting of AJs and TJs in Madin-Darby canine kidney (MDCK) cells, claudin and occludin accumulated at the apical sites of the nectin-based cell-cell adhesion sites. This accumulation of claudin and occludin was inhibited by inhibitors acting on the trans interaction of nectin. The barrier function of TJs was also impaired by the nectin inhibitors. It has been shown that a phorbol ester promotes the formation of a TJ-like structure in an E-cadherin-independent manner. This phorbol ester-induced formation of the TJ-like structure was also inhibited by the nectin inhibitors. These results suggest a role of the nectin-afadin system in the organization of TJs as well as AJs in epithelial cells.

Claudin-based tight junctions are crucial for the mammalian epidermal barrier: a lesson from claudin-1-deficient mice.

The tight junction (TJ) and its adhesion molecules, claudins, are responsible for the barrier function of simple epithelia, but TJs have not been thought to play an important role in the barrier function of mammalian stratified epithelia, including the epidermis. Here we generated claudin-1–deficient mice and found that the animals died within 1 d of birth with wrinkled skin. Dehydration assay and transepidermal water loss measurements revealed that in these mice the epidermal barrier was severely affected, although the layered organization of keratinocytes appeared to be normal. These unexpected findings prompted us to reexamine TJs in the epidermis of wild-type mice. Close inspection by immunofluorescence microscopy with an antioccludin monoclonal antibody, a TJ-specific marker, identified continuous TJs in the stratum granulosum, where claudin-1 and -4 were concentrated. The occurrence of TJs was also confirmed by ultrathin section EM. In claudin-1–deficient mice, claudin-1 appeared to have simply been removed from these TJs, leaving occludin-positive (and also claudin-4–positive) TJs. Interestingly, in the wild-type epidermis these occludin-positive TJs efficiently prevented the diffusion of subcutaneously injected tracer (∼600 D) toward the skin surface, whereas in the claudin-1–deficient epidermis the tracer appeared to pass through these TJs. These findings provide the first evidence that continuous claudin-based TJs occur in the epidermis and that these TJs are crucial for the barrier function of the mammalian skin.