Extracellular signal-regulated kinases 1/2 control claudin-2 expression in Madin-Darby canine kidney strain I and II cells

The tight junction of the epithelial cell determines the characteristics of paracellular permeability across epithelium. Recent work points toward the claudin family of tight junction proteins as leading candidates for the molecular components that regulate paracellular permeability properties in epithelial tissues. Madin-Darby canine kidney (MDCK) strain I and II cells are models for the study of tight junctions and based on transepithelial electrical resistance (TER) contain "tight" and "leaky" tight junctions, respectively. Overexpression studies suggest that tight junction leakiness in these two strains of MDCK cells is conferred by expression of the tight junction protein claudin-2. Extracellular signal-regulated kinase (ERK) 1/2 activation by hepatocyte growth factor treatment of MDCK strain II cells inhibited claudin-2 expression and transiently increased TER. This process was blocked by the ERK 1/2 inhibitor U0126. Transfection of constitutively active mitogen-activated protein kinase/extracellular signal-regulated kinase kinase into MDCK strain II cells also inhibited claudin-2 expression and increased TER. MDCK strain I cells have higher levels of active ERK 1/2 than do MDCK strain II cells. U0126 treatment of MDCK strain I cells decreased active ERK 1/2 levels, induced expression of claudin-2 protein, and decreased TER by approximately 20-fold. U0126 treatment also induced claudin-2 expression and decreased TER in a high resistance mouse cortical collecting duct cell line (94D). These data show for the first time that the ERK 1/2 signaling pathway negatively controls claudin-2 expression in mammalian renal epithelial cells and provide evidence for regulation of tight junction paracellular transport by alterations in claudin composition within tight junction complexes.     

Proinflammatory cytokines tumor necrosis factor-α and interferon-γ modulate epithelial barrier function in Madin-Darby canine kidney cells through mitogen activated protein kinase signaling

Background  

The tight junction is a dynamic structure that is regulated by a number of cellular signaling processes. Occludin, claudin-1, claudin-2 and claudin-3 are integral membrane proteins found in the tight junction of MDCK cells. These proteins are restricted to this region of the membrane by a complex array of intracellular proteins which are tethered to the cytoskeleton. Alteration of these tight junction protein complexes during pathological events leads to impaired epithelial barrier function that perturbs water and electrolyte homeostasis. We examined MDCK cell barrier function in response to challenge by the proinflammatory cytokines tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ).     

Claudin-8 Expression in Renal Epithelial Cells Augments the Paracellular Barrier by Replacing Endogenous Claudin-2

Claudins are transmembrane proteins of the tight junction that determine and regulate paracellular ion permeability. We previously reported that claudin-8 reduces paracellular cation permeability when expressed in low-resistance Madin-Darby canine kidney (MDCK) II cells. Here, we address how the interaction of heterologously expressed claudin-8 with endogenous claudin isoforms impacts epithelial barrier properties. In MDCK II cells, barrier improvement by claudin-8 is accompanied by a reduction of endogenous claudin-2 protein at the tight junction. Here, we show that this is not because of relocalization of claudin-2 into the cytosolic pool but primarily due to a decrease in gene expression. Claudin-8 also affects the trafficking of claudin-2, which was displaced specifically from the junctions at which claudin-8 was inserted. To test whether replacement of cation-permeable claudin-2 mediates the effect of claudin-8 on the electrophysiological phenotype of the host cell line, we expressed claudin-8 in high-resistance MDCK I cells, which lack endogenous claudin-2. Unlike in MDCK II cells, induction of claudin-8 in MDCK I cells (which did not affect levels of endogenous claudins) did not alter paracellular ion permeability. Furthermore, when endogenous claudin-2 in MDCK II cells was downregulated by epidermal growth factor to create a cell model with low transepithelial resistance and low levels of claudin-2, the permeability effects of claudin-8 were also abolished. Our findings demonstrate that claudin overexpression studies measure the combined effect of alterations in both endogenous and exogenous claudins, thus explaining the dependence of the phenotype on the host cell line.     

Multiple domains of occludin are involved in the regulation of paracellular permeability

Tight junctions form selective paracellular diffusion barriers that regulate the diffusion of solutes across epithelia and constitute intramembrane diffusion barriers that prevent the intermixing of apical and basolateral lipids in the extracytoplasmic leaflet of the plasma membrane. In MDCK cells, previous expression experiments demonstrated that occludin, a tight junction protein with four transmembrane domains, is critically involved in both of these tight junction functions and that its COOH-terminal cytoplasmic domain is of functional importance. By expressing mutant and chimeric occludin that exert a dominant negative effect on selective paracellular diffusion, we now demonstrate that the extracytoplasmic domains and at least one of the transmembrane domains are also critically involved in selective paracellular permeability. Multiple domains of occludin are thus important for the regulation of paracellular permeability. Expression of chimeras containing at least one transmembrane domain of occludin also resulted in an enhanced intracellular accumulation of claudin-4, another transmembrane protein of tight junctions, suggesting that the two proteins may cooperate in the regulation of paracellular permeability.     

Claudin-8 interacts with multi-PDZ domain protein 1 (MUPP1) and reduces paracellular conductance in epithelial cells.

The claudin family is a set of integral membrane proteins found at cell-cell interactions in tight junctions. To identify proteins that interact with claudin-8, we used the yeast two-hybrid system to search for binding partners. Using the C-terminal 37 amino acids of claudin-8 as bait, we screened a human kidney cDNA library and identified multi-PDZ domain protein 1 (MUPP1) as a claudin-8 binding protein. MUPP1 contains 13 PDZ domains and binds to claudin-8 though its PDZ9 domain. When MDCK cells were transfected with epitope-tagged claudin-8 or MUPP1, both molecules were concentrated at cell-cell junctions. The interaction of claudin-8 and MUPP1 in vivo was confirmed by co-immunolocalization and co-immunoprecipitation in MDCK cells. Expression of claudin-8-myc increased transepithelial electrical resistance (TER) and reduced paracellular flux using FITC-dextran as a tracer. Over-expression of FLAG-MUPP1 in MDCK cells also reduced the epithelial paracelhular conductance. Our results indicate that claudin-8 and MUPP1 interact in tight junctions of epithelial cells and are involved in the tight junction barrier function.     

Conversion of zonulae occludentes from tight to leaky strand type by introducing claudin-2 into Madin-Darby canine kidney I cells

There are two strains of MDCK cells, MDCK I and II. MDCK I cells show much higher transepithelial electric resistance (TER) than MDCK II cells, although they bear similar numbers of tight junction (TJ) strands. We examined the expression pattern of claudins, the major components of TJ strands, in these cells: claudin-1 and -4 were expressed both in MDCK I and II cells, whereas the expression of claudin-2 was restricted to MDCK II cells. The dog claudin-2 cDNA was then introduced into MDCK I cells to mimic the claudin expression pattern of MDCK II cells. Interestingly, the TER values of MDCK I clones stably expressing claudin-2 (dCL2-MDCK I) fell to the levels of MDCK II cells (>20-fold decrease). In contrast, when dog claudin-3 was introduced into MDCK I cells, no change was detected in their TER. Similar results were obtained in mouse epithelial cells, Eph4. Morphometric analyses identified no significant differences in the density of TJs or in the number of TJ strands between dCL2-MDCK I and control MDCK I cells. These findings indicated that the addition of claudin-2 markedly decreased the tightness of individual claudin-1/4-based TJ strands, leading to the speculation that the combination and mixing ratios of claudin species determine the barrier properties of individual TJ strands.     

Rab27 effector Slp2-a transports the apical signaling molecule podocalyxin to the apical surface of MDCK II cells and regulates claudin-2 expression

Most cells in tissues are polarized and usually have two distinct plasma membrane domains-an apical membrane and a basolateral membrane, which are the result of polarized trafficking of proteins and lipids. However, the mechanism underlying the cell polarization is not fully understood. In this study, we investigated the involvement of synaptotagmin-like protein 2-a (Slp2-a), an effector molecule for the small GTPase Rab27, in polarized trafficking by using Madin-Darby canine kidney II cells as a model of polarized cells. The results show that the level of Slp2-a expression in MDCK II cells increases greatly as the cells become polarized and that its expression is specifically localized at the apical membrane. The results also reveal that Slp2-a is required for targeting of the signaling molecule podocalyxin to the apical membrane in a Rab27A-dependent manner. In addition, ezrin, a downstream target of podocalyxin, and ERK1/2 are activated in Slp2-a-knockdown cells, and their activation results in a dramatic reduction in the amount of the tight junction protein claudin-2. Because both Slp2-a and claudin-2 are highly expressed in mouse renal proximal tubules, Slp2-a is likely to regulate claudin-2 expression through trafficking of podocalyxin to the apical surface in mouse renal tubule epithelial cells.     

The Cytoplasmic Tails of Claudins Can Influence Tight Junction Barrier Properties through Effects on Protein Stability

The tight junction seal formed between epithelial cells varies among tissues in both tightness and ionic charge selectivity. We recently demonstrated that the extracellular domains of the claudin family of proteins are determinants of both characteristics, but in that study other unidentified domains in the claudins clearly contributed to their physiological potency. To investigate the importance of the cytoplasmic carboxyl-terminal domains in determining the degree to which a claudin can influence barrier properties, we constructed chimeras by exchanging the tails of claudin-2 and -4 and expressing them in MDCK II cells. Although swapping these domains had little effect on claudin localization, we found that the tail of claudin-2 could stabilize claudin-4, with a concomitant increase in both protein level and physiologic influence. This difference in stability was not an artifact of their chimeric structure, since metabolic radio-labeling experiments revealed that the half-life of endogenous claudin-2 is more than three times longer than claudin-4 (>12 h and ∼4 h respectively). Further, half-life was not affected by removing the carboxyl-terminal three amino acids, which form a PDZ-binding motif. The finding that cytoplasmic tails of claudins strongly influence stability reveals a potential mechanism by which cells can establish their tight junction protein composition and thus function.This revised version was published online in August 2005 with a corrected cover date.     

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.     

The PIKfyve inhibitor YM201636 blocks the continuous recycling of the tight junction proteins claudin-1 and claudin-2 in MDCK cells

Tight junctions mediate the intercellular diffusion barrier found in epithelial tissues but they are not static complexes; instead there is rapid movement of individual proteins within the junctions. In addition some tight junction proteins are continuously being endocytosed and recycled back to the plasma membrane. Understanding the dynamic behaviour of tight junctions is important as they are altered in a range of pathological conditions including cancer and inflammatory bowel disease. In this study we investigate the effect of treating epithelial cells with a small molecule inhibitor (YM201636) of the lipid kinase PIKfyve, a protein which is involved in endocytic trafficking. We show that MDCK cells treated with YM201636 accumulate the tight junction protein claudin-1 intracellularly. In contrast YM201636 did not alter the localization of other junction proteins including ZO-1, occludin and E-cadherin. A biochemical trafficking assay was used to show that YM201636 inhibited the endocytic recycling of claudin-1, providing an explanation for the intracellular accumulation. Claudin-2 was also found to constantly recycle in confluent MDCK cells and treatment with YM201636 blocked this recycling and caused accumulation of intracellular claudin-2. However, claudin-4 showed negligible endocytosis and no detectable intracellular accumulation occurred following treatment with YM201636, suggesting that not all claudins show the same rate of endocytic trafficking. Finally, we show that, consistent with the defects in claudin trafficking, incubation with YM201636 delayed formation of the epithelial permeability barrier. Therefore, YM201636 treatment blocks the continuous recycling of claudin-1/claudin-2 and delays epithelial barrier formation.     

Expression and function of tight junction associated molecules in human breast tumor cells is not affected by the Ras-MEK1 pathway.

Constitutive activation of Ras or Ras-mediated signaling pathways is one of the initial steps during tumorigenesis that promotes neoplastic transformation. Recently it was reported that in Ha-Ras overexpressing MDCK cells the tight junction proteins claudin-1, occludin and ZO-1 were absent at cell-cell contact sites but present in the cytoplasm. Inhibition of MEK1 activity recruited all three proteins to the cell membrane leading to a restoration of the tight junction barrier function in MDCK cells. In order to evaluate the relevance of the MEK1 pathway in tight junction regulation in breast cancer cells, we investigated the effect ofMEK1 inhibition on expression of claudin-1, occludin and ZO-1 in natively claudin-1 expressing T47-D cells (low Ras activity), claudin-1 negative MCF-7 cells (elevated Ras activity) as well as two retroviral claudin-1 transduced MCF-7 daughter cell lines with prominent membrane and cytoplasmic claudin-1 dominant homing, respectively. Although we effectively blocked phosphorylation of MAPKs ERK-1 and ERK-2 using the selective MEK1 inhibitor PD98059, no quantitative changes of mRNA or protein levels of claudin-1, occludin and ZO-1 could be detected in all cell lines investigated. Furthermore, immnfluorescence analysis of claudin-1 revealed that inhibition of the MAPK pathway did not alter th e subcellular cytoplasmic distribution of claudin-1 to be more membrane specific. Finally, the diffusion barrier properties of tight junctions as analyzed by transepithelial resistance (TER) or paracellular flux analysis of 3 and 40 kDa dextran of tight junctions were not altered in the claudin-1 positive T47-D and the MCF-7 cell lines. Our findings indicate that the proposed involvement of the Ras-MEK-ERK pathway is likely not involved in the dysregulated tight junction formation in breast tumor cells and indicates that elevated activity of Ras might not be of general importance for the disruption of tight junction structures in breast tumors.     

Rab14 regulation of claudin-2 trafficking modulates epithelial permeability and lumen morphogenesis

Regulation of epithelial barrier function requires targeted insertion of tight junction proteins that have distinct selectively permeable characteristics. The insertion of newly synthesized proteins and recycling of internalized tight junction components control both polarity and junction function. Here we show that the small GTPase Rab14 regulates tight junction structure. In Madin–Darby canine kidney (MDCK) II cells, Rab14 colocalizes with junctional proteins, and knockdown of Rab14 results in increased transepithelial resistance. In cells without Rab14, there are small changes in the trafficking of claudin-1 and occludin. In addition, there is substantial depletion of the leaky claudin, claudin-2, but not other tight junction components. The loss of claudin-2 is complemented by inhibition of lysosomal function, suggesting that Rab14 sorts claudin-2 out of the lysosome-directed pathway. MDCK I cells lack claudin-2 endogenously, and knockdown of Rab14 in these cells does not result in a change in transepithelial resistance, suggesting that the effect is specific to claudin-2 trafficking. Furthermore, leaky claudins have been shown to be required for epithelial morphogenesis, and knockdown of Rab14 results in failure to form normal single-lumen cysts in three-dimensional culture. These results implicate Rab14 in specialized trafficking of claudin-2 from the recycling endosome.     

Knockdown of tight junction protein claudin-2 prevents bile canalicular formation in WIF-B9 cells

The polarization of hepatocytes involves formation of functionally distinct sinusoidal (basolateral) and bile canalicular (apical) plasma membrane domains that are separated by tight junctions. Although various molecular mechanisms and signaling cascades including polarity complex proteins may contribute to bile canalicular formation in hepatocytes, the role of tight junction proteins in bile canalicular formation remains unclear. To investigate the role of the integral tight junction protein claudin-2 in bile canalicular formation, we depleted claudin-2 expression by siRNA in the polarized hepatic cell line WIF-B9 after treatment with or without phenobarbital. When WIF-B9 cells were treated with phenobarbital, claudin-2 expression and tight junction strands were markedly increased together with induction of canalicular formation with a biliary secretion function. Knockdown of claudin-2 prevented bile canalicular formation after treatment with or without phenobarbital. Furthermore, knockdown of claudin-2 caused a change from a hepatic polarized phenotype to a simple polarized phenotype, together with upregulation of pLKB1, pMAPK, pAkt and pp38 MAPK, but not pMLC, PTEN or cdc42, and an increase of intracellular vacuoles, which were present before bile canalicular formation. These results suggest that claudin-2 may affect not only the bile canalicular seal but also bile canalicular formation.     

Cingulin regulates claudin-2 expression and cell proliferation through the small GTPase RhoA

In mouse embryoid bodies, mutation of the tight junction protein cingulin results in changes in gene expression. Here, we studied the function of cingulin using a gene silencing approach in Madin-Darby canine kidney (MDCK) cells. Cingulin-depleted cells show higher protein and mRNA levels of claudin-2 and ZO-3, increased RhoA activity, activation of G1/S phase transition, and increased cell density. The effects of cingulin depletion on claudin-2 expression, cell proliferation, and density are reversed by coexpression of either a dominant-negative form of RhoA (RhoAN19) or the Rho-inhibiting enzyme C3 transferase. However, the increase in ZO-3 protein and mRNA levels is not reversed by inhibition of either RhoA, p38, extracellular signal-regulated kinase (ERK), or c-Jun NH2-terminal kinase (JNK), suggesting that cingulin modulates ZO-3 expression by a different mechanism. JNK is implicated in the regulation of claudin-2 levels independently of cingulin depletion and RhoA activity, indicating distinct roles of RhoA- and JNK-dependent pathways in the control of claudin-2 expression. Finally, cingulin depletion does not significantly alter the barrier function of monolayers and the overall molecular organization of tight junctions. These results provide novel insights about the mechanisms of cingulin function and the signaling pathways controlling claudin-2 expression in MDCK cells.     

CLMP, a novel member of the CTX family and a new component of epithelial tight junctions

The CTX family is a growing group of type I transmembrane proteins within the immunoglobulin superfamily (IgSF). They localize to junctional complexes between endothelial and epithelial cells and seem to participate in cell-cell adhesion and transmigration of leukocytes. Here, we report the identification of a new member of the CTX family. This protein, which was designated CLMP (coxsackie- and adenovirus receptor-like membrane protein), is composed of 373 amino acids including an extracellular part containing a V- and a C2-type domain, a transmembrane region and a cytoplasmic tail. CLMP mRNA was detected in a variety of both human and mouse tissues and cell lines. The protein migrated with an Mr of around 48 on SDS-PAGE and was predominantly expressed in epithelial cells within different tissues. In cultured epithelial cells, CLMP was detected in areas of cell-cell contacts. When exogenously expressed in polarized MDCK cells, CLMP was restricted to the subapical area of the lateral cell surface, where it co-localized with the tight junction markers ZO-1 and occludin. Also endogenous CLMP showed association with tight junctions, as analyzed in polarized human CACO-2 cells. This suggested a role for CLMP in cell-cell adhesion and indeed, overexpressed CLMP induced aggregation of non-polarized CHO cells. Furthermore, CLMP-expressing MDCK cells showed significantly increased transepithelial resistance, indicating a role for CLMP in junctional barrier function. Thus, we conclude that CLMP is a novel cell-cell adhesion molecule and a new component of epithelial tight junctions. We also suggest, based on phylogenetic studies, that CLMP, CAR, ESAM, and BT-IgSF form a new group of proteins within the CTX family.     

Study of claudin function by RNA interference

Claudins are tight junction proteins that play a key selectivity role in the paracellular conductance of ions. Numerous studies of claudin function have been carried out using the overexpression strategy to add new claudin channels to an existing paracellular protein background. Here, we report the systematic knockdown of endogenous claudin gene expression in Madin-Darby canine kidney (MDCK) cells and in LLC-PK1 cells using small interfering RNA against claudins 1-4 and 7. In MDCK cells (showing cation selectivity), claudins 2, 4, and 7 are powerful effectors of paracellular Na+ permeation. Removal of claudin-2 depressed the permeation of Na+ and resulted in the loss of cation selectivity. Loss of claudin-4 or -7 expression elevated the permeation of Na+ and enhanced the proclivity of the tight junction for cations. On the other hand, LLC-PK1 cells express little endogenous claudin-2 and show anion selectivity. In LLC-PK1 cells, claudin-4 and -7 are powerful effectors of paracellular Cl- permeation. Knockdown of claudin-4 or -7 expression depressed the permeation of Cl- and caused the tight junction to lose the anion selectivity. In conclusion, claudin-2 functions as a paracellular channel to Na+ to increase the cation selectivity of the tight junction; claudin-4 and -7 function either as paracellular barriers to Na+ or as paracellular channels to Cl-, depending upon the cellular background, to decrease the cation selectivity of the tight junction.     

Direct interaction of two polarity complexes implicated in epithelial tight junction assembly

Tight junctions help establish polarity in mammalian epithelia by forming a physical barrier that separates apical and basolateral membranes. Two evolutionarily conserved multi-protein complexes, Crumbs (Crb)-PALS1 (Stardust)-PATJ (DiscsLost) and Cdc42-Par6-Par3-atypical protein kinase C (aPKC), have been implicated in the assembly of tight junctions and in polarization of Drosophila melanogaster epithelia. Here we identify a biochemical and functional link between these two complexes that is mediated by Par6 and PALS1 (proteins associated with Lin7). The interaction between Par6 and PALS1 is direct, requires the amino terminus of PALS1 and the PDZ domain of Par6, and is regulated by Cdc42-GTP. The transmembrane protein Crb can recruit wild-type Par6, but not Par6 with a mutated PDZ domain, to the cell surface. Expression of dominant-negative PALS1-associated tight junction protein (PATJ) in MDCK cells results in mis-localization of PALS1, members of the Par3-Par6-aPKC complex and the tight junction marker, ZO-1. Similarly, overexpression of Par6 in MDCK cells inhibits localization of PALS1 to the tight junction. Our data highlight a previously unrecognized link between protein complexes that are essential for epithelial polarity and formation of tight junctions.     

The tight junction protein complex undergoes rapid and continuous molecular remodeling at steady state

The tight junction defines epithelial organization. Structurally, the tight junction is comprised of transmembrane and membrane-associated proteins that are thought to assemble into stable complexes to determine function. In this study, we measure tight junction protein dynamics in live confluent Madin-Darby canine kidney monolayers using fluorescence recovery after photobleaching and related methods. Mathematical modeling shows that the majority of claudin-1 (76 +/- 5%) is stably localized at the tight junction. In contrast, the majority of occludin (71 +/- 3%) diffuses rapidly within the tight junction with a diffusion constant of 0.011 microm(2)s(-1). Zonula occludens-1 molecules are also highly dynamic in this region, but, rather than diffusing within the plane of the membrane, 69 +/- 5% exchange between membrane and intracellular pools in an energy-dependent manner. These data demonstrate that the tight junction undergoes constant remodeling and suggest that this dynamic behavior may contribute to tight junction assembly and regulation.     

Claudin-3 acts as a sealing component of the tight junction for ions of either charge and uncharged solutes

The paracellular barrier of epithelia and endothelia is established by several tight junction proteins including claudin-3. Although claudin-3 is present in many epithelia including skin, lung, kidney, and intestine and in endothelia, its function is unresolved as yet. We therefore characterized claudin-3 by stable transfection of MDCK II kidney tubule cells with human claudin-3 cDNA. Two clone systems were analyzed, exhibiting high or low claudin-2 expression, respectively. Expression of other claudins was unchanged. Ultrastructurally, tight junction strands were changed toward uninterrupted and rounded meshwork loops. Functionally, the paracellular resistance of claudin-3-transfected monolayers was strongly elevated, causing an increase in transepithelial resistance compared to vector controls. Permeabilities for mono- and divalent cations and for anions were decreased. In the high-claudin-2 system, claudin-3 reduced claudin-2-induced cation selectivity, while in the low-claudin-2 system no charge preference was observed, the latter thus reflecting the "intrinsic" action of claudin-3. Furthermore, the passage of the paracellular tracers fluorescein (332 Da) and FD-4 (4 kDa) was decreased, whereas the permeability to water was not affected. We demonstrate that claudin-3 alters the tight junction meshwork and seals the paracellular pathway against the passage of small ions of either charge and uncharged solutes. Thus, in a kidney model epithelium, claudin-3 acts as a general barrier-forming protein.