Occludin: a novel integral membrane protein localizing at tight junctions

Recently, we found that ZO-1, a tight junction-associated protein, was concentrated in the so called isolated adherens junction fraction from the liver (Itoh, M., A. Nagafuchi, S. Yonemura, T. Kitani-Yasuda, Sa. Tsukita, and Sh. Tsukita. 1993. J. Cell Biol. 121:491-502). Using this fraction derived from chick liver as an antigen, we obtained three monoclonal antibodies specific for a approximately 65-kD protein in rats. This antigen was not extractable from plasma membranes without detergent, suggesting that it is an integral membrane protein. Immunofluorescence and immunoelectron microscopy with these mAbs showed that this approximately 65-kD membrane protein was exclusively localized at tight junctions of both epithelial and endothelial cells: at the electron microscopic level, the labels were detected directly over the points of membrane contact in tight junctions. To further clarify the nature and structure of this membrane protein, we cloned and sequenced its cDNA. We found that the cDNA encoded a 504-amino acid polypeptide with 55.9 kDa. A search of the data base identified no proteins with significant homology to this membrane protein. A most striking feature of its primary structure was revealed by a hydrophilicity plot: four putative membrane-spanning segments were included in the NH2-terminal half. This hydrophilicity plot was very similar to that of connexin, an integral membrane protein in gap junctions. These findings revealed that an integral membrane protein localizing at tight junctions is now identified, which we designated as "occludin."     

The 220-kD protein colocalizing with cadherins in non-epithelial cells is identical to ZO-1, a tight junction-associated protein in epithelial cells: cDNA cloning and immunoelectron microscopy

We previously identified a 220-kD constitutive protein of the plasma membrane undercoat which colocalizes at the immunofluorescence microscopic level with cadherins and occurs not only in epithelial M., S. Yonemura, A. Nagafuchi, Sa. Tsukita, and Sh. Tsukita. 1991. J. Cell Biol. 115:1449-1462). To clarify the nature and possible functions of this protein, we cloned its full-length cDNA and sequenced it. Unexpectedly, we found mouse 220-kD protein to be highly homologous to rat protein ZO-1, only a part of which had been already sequenced. This relationship was confirmed by immunoblotting with anti-ZO-1 antibody. As protein ZO-1 was originally identified as a component exclusively underlying tight junctions in epithelial cells, where cadherins are not believed to be localized, we analyzed the distribution of cadherins and the 220-kD protein by ultrathin cryosection immunoelectron microscopy. We found that in non-epithelial cells lacking tight junctions cadherins and the 220-kD protein colocalize, whereas in epithelial cells (e.g., intestinal epithelial cells) bearing well-developed tight junctions cadherins and the 220-kD protein are clearly segregated into adherens and tight junctions, respectively. Interestingly, in epithelial cells such as hepatocytes, which tight junctions are not so well developed, the 220-kD protein is detected not only in the tight junction zone but also at adherens junctions. Furthermore, we show in mouse L cells transfected with cDNAs encoding N-, P-, E-cadherins that cadherins interact directly or indirectly with the 220-kD protein. Possible functions of the 220-kD protein (ZO-1) are discussed with special reference to the molecular mechanism for adherens and tight junction formation.     

Shoichiro Tsukita: a life exploring the molecular architecture of the tight junction

On December 11, 2005, Shoichiro Tsukita died at the young age of 52, after 14 months of treatment for cancer. Early in his career, Tsukita succeeded in isolating and purifying the adherens junction with his wife Sachiko, an accomplishment that he followed up with an impressive series of discoveries of cell adhesion and cytoskeletal molecules, including what may have been his greatest contribution to the field, the identification of occludin and the claudin family of molecules, which were watershed discoveries in the study of the molecular nature of tight junctions.     

Induction of tight junctions in human connexin 32 (hCx32)-transfected mouse hepatocytes: connexin 32 interacts with occludin

Small gap junction plaques are associated with tight junction strands in some cell types including hepatocytes and it is thought that they may be closely related to tight junctions and the establishment of cell polarity. In order to examine roles of gap junctions in regulating expression and structure of tight junctions, we transfected human Cx32 cDNA into immortalized mouse hepatocytes (CHST8 cells) which lack endogenous Cx32 and Cx26. Immunocytochemistry revealed that endogenous integral tight junction protein occludin was strongly localized and was colocalized with Cx32 at cell borders in transfectants, whereas neither was detected in parental cells. In Northern blots, mRNAs encoding occludin and the other integral tight junction proteins, claudin-1 and -2, were induced in the transfectants compared to parental cells. In Western blots, occludin protein was increased in the transfectants compared to parental cells, and binding of occludin to Cx32 protein was demonstrated by immunoprecipitation. In freeze fracture of the transfectants, tight junction strands were more numerous and complex compared to parental cells, and small gap junction plaques appeared within induced tight junction strands. Nevertheless, no change in barrier function of tight junctions was observed. These results indicate that in hepatocytes, gap junction, and tight junction expression are closely coordinated, and that Cx32 may play a role in regulating occludin expression.     

Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical- basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein

Tight junctions, the most apical of the intercellular junctions that connect individual cells in a epithelial sheet, are thought to form a seal that restricts paracellular and intramembrane diffusion. To analyze the functioning of tight junctions, we generated stable MDCK strain 2 cell lines expressing either full-length or COOH-terminally truncated chicken occludin, the only known transmembrane component of tight junctions. Confocal immunofluorescence and immunoelectron microscopy demonstrated that mutant occludin was incorporated into tight junctions but, in contrast to full-length chicken occludin, exhibited a discontinuous junctional staining pattern and also disrupted the continuous junctional ring formed by endogenous occludin. This rearrangement of occludin was not paralleled by apparent changes in the junctional morphology as seen by thin section electron microscopy nor apparent discontinuities of the junctional strands observed by freeze-fracture. Nevertheless, expression of both wild-type and mutant occludin induced increased transepithelial electrical resistance (TER). In contrast to TER, particularly the expression of COOH-terminally truncated occludin led to a severalfold increase in paracellular flux of small molecular weight tracers. Since the selectivity for size or different types of cations was unchanged, expression of wild-type and mutant occludin appears to have activated an existing mechanism that allows selective paracellular flux in the presence of electrically sealed tight junctions. Occludin is also involved in the formation of the apical/basolateral intramembrane diffusion barrier, since expression of the COOH-terminally truncated occludin was found to render MDCK cells incapable of maintaining a fluorescent lipid in a specifically labeled cell surface domain.     

Loss of Occludin and Functional Tight Junctions, but Not ZO-1, during Neural Tube Closure—Remodeling of the Neuroepithelium Prior to Neurogenesis

Neuroepithelial cells can generate nonepithelial cells, the neurons. Here we have investigated, for chick and mouse embryos, the epithelial character of neuroepithelial cells in the context of neurogenesis by examining the presence of molecular components of tight junctions during the transition from the neural plate to the neural tube. Immunoreactivity for occludin, a transmembrane protein specific to tight junctions, was detected at the apical end of the lateral membrane of neuroepithelial cells throughout the chick neural plate. During neural tube closure, occludin disappeared from all neuroepithelial cells. Correspondingly, the addition of horseradish peroxidase to the apical side of the neuroepithelium by injection into the amniotic cavity of mouse embryos revealed the presence of functional tight junctions in the neural plate (Embryonic Day 8), but not the neural tube (Embryonic Day 9). In contrast to occludin, expression of ZO-1, a peripheral membrane protein of tight junctions, increased from the neural plate to the neural tube stage, also being confined to the apical end of the lateral neuroepithelial cell membrane. This localization coincided with that of N-cadherin, whose expression increased concomitantly with the disappearance of occludin. We propose that the loss of tight junctions from neuroepithelial cells reflects an overall decrease in their epithelial nature, which precedes the generation of neurons.     

Na-K-ATPase regulates tight junction permeability through occludin phosphorylation in pancreatic epithelial cells

Tight junctions are crucial for maintaining the polarity and vectorial transport functions of epithelial cells. We and others have shown that Na-K-ATPase plays a key role in the organization and permeability of tight junctions in mammalian cells and analogous septate junctions in Drosophila. However, the mechanism by which Na-K-ATPase modulates tight junctions is not known. In this study, using a well-differentiated human pancreatic epithelial cell line HPAF-II, we demonstrate that Na-K-ATPase is present at the apical junctions and forms a complex with protein phosphatase-2A, a protein known to be present at tight junctions. Inhibition of Na-K-ATPase ion transport function reduced protein phosphatase-2A activity, hyperphosphorylated occludin, induced rearrangement of tight junction strands, and increased permeability of tight junctions to ionic and nonionic solutes. These data suggest that Na-K-ATPase is required for controlling the tight junction gate function.     

Restoration of tight junction structure and barrier function by down-regulation of the mitogen-activated protein kinase pathway in ras-transformed Madin-Darby canine kidney cells

In the Madin-Darby canine kidney epithelial cell line, the proteins occludin and ZO-1 are structural components of the tight junctions that seal the paracellular spaces between the cells and contribute to the epithelial barrier function. In Ras-transformed Madin-Darby canine kidney cells, occludin, claudin-1, and ZO-1 were absent from cell-cell contacts but were present in the cytoplasm, and the adherens junction protein E-cadherin was weakly expressed. After treatment of the Ras-transformed cells with the mitogen-activated protein kinase kinase (MEK1) inhibitor PD98059, which blocks the activation of mitogen-activated protein kinase (MAPK), occludin, claudin-1, and ZO-1 were recruited to the cell membrane, tight junctions were assembled, and E-cadherin protein expression was induced. Although it is generally believed that E-cadherin-mediated cell-cell adhesion is required for tight junction assembly, the recruitment of occludin to the cell-cell contact area and the restoration of epithelial cell morphology preceded the appearance of E-cadherin at cell-cell contacts. Both electron microscopy and a fourfold increase in the transepithelial electrical resistance indicated the formation of functional tight junctions after MEK1 inhibition. Moreover, inhibition of MAPK activity stabilized occludin and ZO-1 by differentially increasing their half-lives. We also found that during the process of tight junction assembly after MEK1 inhibition, tyrosine phosphorylation of occludin and ZO-1, but not claudin-1, increased significantly. Our study demonstrates that down-regulation of the MAPK signaling pathway causes the restoration of epithelial cell morphology and the assembly of tight junctions in Ras-transformed epithelial cells and that tyrosine phosphorylation of occludin and ZO-1 may play a role in some aspects of tight junction formation.     

Suppression of the transformed phenotype of breast cancer by tropomyosin-1

Gap junctional intercellular communication (GJIC) is thought to play a crucial role in cell differentiation. Small gap junction plaques are frequently associated with tight junction strands in hepatocytes, suggesting that gap junctions may be closely related to the role of tight junctions in the establishment of cell polarity. To examine the exact role of gap junctions in regulating tight junctions, we transfected connexin 32 (Cx32), Cx26, or Cx43 cDNAs into immortalized mouse hepatocytes derived from Cx32-deficient mice and examined the expression and function of the endogenous tight junction molecules. In transient wild-type Cx32 transfectants, immunocytochemistry revealed that endogenous occludin was in part localized at cell borders, where it was colocalized with Cx32, whereas neither was detected in parental cells. In Cx32 null hepatocytes transfected with Cx32 truncated at position 220 (R220stop), wild-type Cx26, or wild-type Cx43 cDNAs, occludin was not detected at cell borders. In stable wild-type Cx32 transfectants, occludin, claudin-1, and ZO-1 mRNAs and proteins were significantly increased compared to parental cells and all of the proteins were colocalized with Cx32 at cell borders. Treatment with a GJIC blocker, 18 beta-glycyrrhetinic acid, resulted in decreases of occludin and claudin-1 at cell borders in the stable transfectants. The induction of tight junction proteins in the stable transfectants was accompanied by an increase in both fence and barrier functions of tight junctions. Furthermore, in the stable transfectants, circumferencial actin filaments were also increased without a change of actin protein. These results indicate that Cx32 formation and/or Cx32-mediated intercellular communication may participate in the formation of functional tight junctions and actin organization.     

Identification of a novel protein, LYRIC, localized to tight junctions of polarized epithelial cells

Tight junctions (TJ) are multiprotein complexes that function to regulate paracellular transport of molecules through epithelial and endothelial cell layers. Many new tight junction-associated proteins have been identified in the past few years, and their functional roles and interactions have just begun to be elucidated. In this paper, we describe a novel protein LYsine-RIch CEACAM1 co-isolated (LYRIC) that is widely expressed and highly conserved between species. LYRIC has no conserved domains that would indicate function and does not appear to be a member of a larger protein family. Data from analysis of rat and human tissue sections and cell lines show that LYRIC colocalizes with tight junction proteins ZO-1 and occludin in polarized epithelial cells, suggesting that LYRIC is part of the tight junction complex. LYRIC dissociates from ZO-1 when junctional complexes are disrupted, and as tight junctions reform, ZO-1 relocalizes before LYRIC. These results suggest that LYRIC is most likely not a structural component required for TJ formation, but rather is recruited during the maturation of the tight junction complex.     

Biphasic effects of 17-beta-estradiol on expression of occludin and transendothelial resistance and paracellular permeability in human vascular endothelial cells

Tight junctions govern the paracellular permeability of endothelial and epithelial cells. Aberrations of tight junction function are an early and key event during the vascular spread of cancer and inflammation. This study sought to determine the role of estrogen in the regulation of tight junctions and expression of molecules making tight junctions in endothelial cells. Human endothelial cell, HECV, which express ER-beta but not ER-alpha was used. 17-beta-estradiol induced a concentration- and time-dependent biphasic effect on tight junction. At 10(-9) and 10(-6) M, it decreased the level of occludin and increased in paracellular permeability of HECV cells, but at 10(-12) M it decreased in paracellular permeability and increased the level of occludin. The transendothelial electrical resistance (TER), however, was reduced by 17-beta-estradiol at lower concentrations (as low as 10(-12) M). Furthermore, the time-dependent biphasic effect was observed over a period of 4 days, with the first reduction of TER seen within 15 min and the second drop occurring 48 h after 17-beta-estradiol treatment. It was further revealed that protein and mRNA levels of occludin, but not claudin-1 and -5, and ZO-1, were reduced by 17-beta-estradiol, in line with changes of TER. This study shows that 17-beta-estradiol can induce concentration- and time-related biphasic effects on tight junction functions expression of occludin in endothelial cells and that this perturbation of tight junction functions may have implications in the etiology of mastalgia and the vascular spread of breast cancer.     

Assembly of tight junction is regulated by the antagonism of conventional and novel protein kinase C isoforms

Apparently conflicting observations indicated that protein kinase C both may block and support the assembly of tight junctions. We therefore tested the hypothesis that different isoenzymes antagonistically affect tight junction proteins and function. Thus, by using specific inhibitors we investigated the involvement of conventional and novel protein kinase C of kidney tubule cells in tight junction assembly. In low Ca2+ medium, the application of pan-protein kinase C inhibitor GF-109203X blocked the formation of tight junctions induced by protein kinase C agonist diacyglycerol. G?6976, inhibitor of conventional protein kinase C, promoted the formation of tight junctions and occludin phosphorylation in cells cultivated in low Ca2+ medium and attenuated the disruption of tight junction complex induced by the switch to low Ca2+ medium. In addition, G?6976 accelerated the occludin phosphorylation and the formation of tight junction barrier during assembly of tight junctions induced by Ca2+ re-addition. This phosphorylation was accompanied by accelerated occludin incorporation into newly forming tight junctions and by reducing the paracellular permeability. In contrast, inhibitor of novel protein kinase C rottlerin blocked the occludin phosphorylation and the formation of tight junction barrier, both caused by re-addition of normal Ca2+ medium. It is concluded that the conventional protein kinase C alpha participates in tight junction disassembly while the novel protein kinase C epsilon plays a role in tight junction formation of kidney epithelial cells. The discovered antagonism contributes to a better understanding of the regulation of the structure and function of tight junctions and hence to that of the epithelial barrier.     

Molecular characterization and tissue distribution of ZO-2, a tight junction protein homologous to ZO-1 and the Drosophila discs-large tumor suppressor protein

ZO-1 is a 210-225-kD peripheral membrane protein associated with cytoplasmic surfaces of the zonula occludens or tight junction. A 160- kD polypeptide, designated ZO-2, was found to coimmunoprecipitate with ZO-1 from MDCK cell extracts prepared under conditions which preserve protein associations (Gumbiner, B., T. Lowenkopf, and D. Apatira. 1991. Proc. Natl. Acad. Sci. USA. 88: 3460-3464). We have isolated ZO-2 from MDCK cell monolayers by bulk coimmunoprecipitation with ZO-1 followed by electroelution from preparative SDS-PAGE gel slices. Amino acid sequence information obtained from a ZO-2 tryptic fragment was used to isolate a partial cDNA clone from an MDCK library. The deduced amino acid sequence revealed that canine ZO-2 contains a region that is very similar to sequences in human and mouse ZO-1. This region includes both a 90-amino acid repeat domain of unknown function and guanylate kinase- like domains which are shared among members of the family of proteins that includes ZO-1, erythrocyte p55, the product of the lethal(1)discs- large-1 (dlg) gene of Drosophila, and a synapse-associated protein from rat brain, PSD-95/SAP90. The dlg gene product has been shown to act as a tumor suppressor in the imaginal disc of the Drosophila larva, although the functions of other family members have not yet been defined. A polyclonal antiserum was raised against a unique region of ZO-2 and found to exclusively label the cytoplasmic surfaces of tight junctions in MDCK plasma membrane preparations, indicating that ZO-2 is a tight junction-associated protein. Immunohistochemical staining of frozen sections of whole tissue demonstrated that ZO-2 localized to the region of the tight junction in a number of epithelia, including liver, intestine, kidney, testis, and arterial endothelium, suggesting that this protein is a ubiquitous component of the tight junction. Double- label immunofluorescence microscopy performed on cryosections of heart, a nonepithelial tissue, revealed the presence of ZO-1 but no ZO-2 staining at the fascia adherens, a specialized junction of cardiac myocytes which has previously been shown to contain ZO-1 (Itoh, M., S. Yonemura, A. Nagafuchi, S. Tsukita, and Sh. Tsukita. 1991. J. Cell Biol. 115:1449-1462). Thus it appears that ZO-2 is not a component of the fascia adherens, and that unlike ZO-1, this protein is restricted to the epithelial tight junction.     

ERK is involved in EGF-mediated protection of tight junctions, but not adherens junctions, in acetaldehyde-treated Caco-2 cell monolayers

The role of mitogen-activated protein kinases (MAPK) in the mechanism of EGF-mediated prevention of acetaldehyde-induced tight junction disruption was evaluated in Caco-2 cell monolayers. Pretreatment of cell monolayers with EGF attenuated acetaldehyde-induced decrease in resistance and increase in inulin permeability and redistribution of occludin, zona occludens-1 (ZO-1), E-cadherin, and β-catenin from the intercellular junctions. EGF rapidly increased the levels of phospho-ERK1/2, phospho-p38 MAPK, and phospho-JNK1. Pretreatment of cell monolayers with U-0126 (inhibitor of ERK activation), but not SB-202190 and SP-600125 (p38 MAPK and JNK inhibitors), significantly attenuated EGF-mediated prevention of acetaldehyde-induced changes in resistance, inulin permeability, and redistribution of occludin and ZO-1. U-0126, but not SB-202190 and SP-600125, also attenuated EGF-mediated prevention of acetaldehyde effect on the midregion F-actin ring. However, EGF-mediated preservation of junctional distribution of E-cadherin and β-catenin was unaffected by all three inhibitors. Expression of wild-type or constitutively active MEK1 attenuated acetaldehyde-induced redistribution of occludin and ZO-1, whereas dominant-negative MEK1 prevented EGF-mediated preservation of occludin and ZO-1 in acetaldehyde-treated cells. MEK1 expression did not alter E-cadherin distribution in acetaldehyde-treated cells in the presence or absence of EGF. Furthermore, EGF attenuated acetaldehyde-induced tyrosine-phosphorylation of occludin, ZO-1, claudin-3, and E-cadherin. U-0126, but not SB-202190 and SP-600125, prevented EGF effect on tyrosine-phosphorylation of occludin and ZO-1, but not claudin-3, E-cadherin, or β-catenin. These results indicate that EGF-mediated protection of tight junctions from acetaldehyde requires the activity of ERK1/2, but not p38 MAPK or JNK1/2, and that EGF-mediated protection of adherens junctions is independent of MAPK activities.     

Contribution of claudin-5 to barrier properties in tight junctions of epithelial cells

Claudin-5 is a transmembrane protein reported to be primarily present in tight junctions of endothelia. Unexpectedly, we found expression of claudin-5 in HT-29/B6 cells, an epithelial cell line derived from human colon. Confocal microscopy showed colocalization of claudin-5 with occludin, indicating its presence in the tight junctions. By contrast, claudin-5 was absent in the human colonic cell line Caco-2 and in Madin-Darby canine kidney cells (MDCK sub-clones C7 and C11), an epithelial cell line derived from the collecting duct. To determine the contribution of claudin-5 to tight junctional permeability in cells of human origin, stable transfection of Caco-2 with FLAG-claudin-5 cDNA was performed. In addition, clone MDCK-C7 was transfected. Synthesis of the exogenous FLAG-claudin-5 was verified by Western blot analysis and confocal fluorescent imaging by employing FLAG-specific antibody. FLAG-claudin-5 was detected in transfected cells in colocalization with occludin, whereas cells transfected with the vector alone did not exhibit specific signals. Resistance measurements and mannitol fluxes after stable transfection with claudin-5 cDNA revealed a marked increase of barrier function in cells of low genuine transepithelial resistance (Caco-2). By contrast, no changes of barrier properties were detected in cells with a high transepithelial resistance (MDCK-C7) after stable transfection with claudin-5 cDNA. We conclude that claudin-5 is present in epithelial cells of colonic origin and that it contributes to some extent to the paracellular seal. Claudin-5 may thus be classified as a tight-junctional protein capable of contributing to the "sealing" of the tight junction.     

Occludin 蛋白与细胞间紧密连接关系及其临床意义

细胞间紧密连接(tight junctions)广泛存在于上皮细胞及内皮细胞之间,其作用是保持细胞间结构的完整性,确保其功能的正常发挥,紧密连接上有很多种蛋白,occludin蛋白是其中主要蛋白之一,occludin蛋白的结构发生变化会导致紧密连接结构及功能的改变,而紧密连接结构与功能的紊乱是很多临床疾病共同的病理生理学特点,如肿瘤、中风及炎症性肺疾病。Occludin蛋白的结构及功能的改变受很多机制的调控,本文主要对occludin蛋白的结构、功能、调控机制及其与紧密连接之间的关系进行叙述。     

Protein kinase Cζ phosphorylates occludin and promotes assembly of epithelial tight junctions

Protein kinases play an important role in the regulation of epithelial tight junctions. In the present study, we investigated the role of PKCζ (protein kinase Cζ) in tight junction regulation in Caco-2 and MDCK (Madin-Darby canine kidney) cell monolayers. Inhibition of PKCζ by a specific PKCζ pseudosubstrate peptide results in redistribution of occludin and ZO-1 (zona occludens 1) from the intercellular junctions and disruption of barrier function without affecting cell viability. Reduced expression of PKCζ by antisense oligonucleotide or shRNA (short hairpin RNA) also results in compromised tight junction integrity. Inhibition or knockdown of PKCζ delays calcium-induced assembly of tight junctions. Tight junction disruption by PKCζ pseudosubstrate is associated with the dephosphorylation of occludin and ZO-1 on serine and threonine residues. PKCζ directly binds to the C-terminal domain of occludin and phosphorylates it on threonine residues. Thr403, Thr404, Thr424 and Thr438 in the occludin C-terminal domain are the predominant sites of PKCζ-dependent phosphorylation. A T424A or T438A mutation in full-length occludin delays its assembly into the tight junctions. Inhibition of PKCζ also induces redistribution of occludin and ZO-1 from the tight junctions and dissociates these proteins from the detergent-insoluble fractions in mouse ileum. The present study demonstrates that PKCζ phosphorylates occludin on specific threonine residues and promotes assembly of epithelial tight junctions.     

JAM4, a junctional cell adhesion molecule interacting with a tight junction protein,MAGI-1

MAGI-1 is a membrane-associated guanylate kinase protein at tight junctions in epithelial cells. It interacts with various molecules and functions as a scaffold protein at cell junctions. We report here a novel MAGI-1-binding protein that we named junctional adhesion molecule 4 (JAM4). JAM4 belongs to an immunoglobulin protein family. JAM4 was colocalized with ZO-1 in kidney glomeruli and in intestinal epithelial cells. Biochemical in vitro studies revealed that JAM4 bound to MAGI-1 but not to ZO-1, whereas JAM1 did not bind to MAGI-1. JAM4 and MAGI-1 interacted with each other and formed clusters in COS-7 cells when coexpressed. JAM4 mediated calcium-independent homophilic adhesion and was accumulated at cell-cell contacts when expressed in L cells. MAGI-1, ZO-1, and occludin were recruited to JAM4-based cell contacts. JAM4 also reduced the permeability of CHO cell monolayers. MAGI-1 strengthened JAM4-mediated cell adhesion in L cells and sealing effects in CHO cells. These findings suggest that JAM4 together with MAGI-1 provides an adhesion machinery at tight junctions, which may regulate the permeability of kidney glomerulus and small intestinal epithelial cells.     

Contrasting effects of ERK on tight junction integrity in differentiated and under-differentiated Caco-2 cell monolayers

ERK (extracellular-signal-regulated kinase) activation leads to disruption of tight junctions in some epithelial monolayers, whereas it prevents disruption of tight junctions in other epithelia. The factors responsible for such contrasting influences of ERK on tight junction integrity are unknown. The present study investigated the effect of the state of cell differentiation on ERK-mediated regulation of tight junctions in Caco-2 cell monolayers. EGF (epidermal growth factor) potentiated H2O2-induced tight junction disruption in under-differentiated cell monolayers, which was attenuated by the MEK [MAPK (mitogen-activated protein kinase)/ERK kinase] inhibitor U0126. In contrast, EGF prevented H2O2-induced disruption of tight junctions in differentiated cell monolayers, which was also attenuated by U0126. Knockdown of ERK1/2 enhanced tight junction integrity and accelerated assembly of tight junctions in under-differentiated cell monolayers, whereas it had the opposite effect in differentiated cell monolayers. Regulated expression of wild-type and constitutively active MEK1 disrupted tight junctions, and the expression of dominant-negative MEK1 enhanced tight junction integrity in under-differentiated cells, whereas contrasting responses were recorded in differentiated cells. EGF prevented both H2O2-induced association of PP2A (protein phosphatase 2A), and loss of association of PKCζ (protein kinase Cζ), with occludin by an ERK-dependent mechanism in differentiated cell monolayers, but not in under-differentiated cell monolayers. Active ERK was distributed in the intracellular compartment in under-differentiated cell monolayers, whereas it was localized mainly in the perijunctional region in differentiated cell monolayers. Thus ERK may exhibit its contrasting influences on tight junction integrity in under-differentiated and differentiated epithelial cells by virtue of differences in its subcellular distribution and ability to regulate the association of PKCζ and PP2A with tight junction proteins.     

Xenopus laevis occludin. Identification of in vitro phosphorylation sites by protein kinase CK2 and association with cingulin.

Occludin is a protein component of the membrane domain of tight junctions, and has been shown to be phosphorylated in vivo in cultured cells and Xenopus laevis embryos. However, nothing is known about the identity of specific occludin kinase(s) and occludin phosphorylation site(s). Furthermore, nothing is known about the interaction of occludin with cingulin, a cytoplasmic plaque component of tight junctions. Here we report the isolation and sequencing of a complete X. laevis occludin cDNA, and experiments aimed at mapping X. laevis occludin in vitro phosphorylation site(s) and characterizing occludin interaction with cingulin. The sequence of Xenopus occludin is homologous to that of occludins from other species, with identities ranging from 41% to 58%. Bacterially expressed domain E of Xenopus occludin (amino acids 247-493) was a good substrate for protein kinase CK2 (stoichiometry 10.8%, Km 8.4 microM) but not for CK1 kinase, protein kinase A, cdc2 kinase, MAP kinase or syk kinase. Residues Thr375 and Ser379 were identified as potential CK2 phosphorylation sites in this region based on sequence analysis. Mutation of Ser379 to aspartic acid or alanine reduced phosphorylation by CK2 by approximately 50%, and double mutation of Ser379 into aspartic acid and Thr375 into aspartic acid essentially abolished phosphorylation. Glutathione S-transferase (GST) pull-down experiments using extracts of Xenopus A6 epithelial cells showed that constructs of GST fused to wild-type and mutant forms of the C-terminal region of X. laevis occludin associate with several polypeptides, and immunoblot analysis showed that one of these polypeptides is cingulin. GST pull-down experiments using in vitro translated, full-length Xenopus cingulin indicated that cingulin interacts directly with the C-terminal region of occludin.