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.     

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.     

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.     

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.     

Juxtacrine activation of EGFR regulates claudin expression and increases transepithelial resistance

Heparin-binding (HB)-EGF, a ligand for EGF receptors, is synthesized as a membrane-anchored precursor that is potentially capable of juxtacrine activation of EGF receptors. However, the physiological importance of such juxtacrine signaling remains poorly described, due to frequent inability to distinguish effects mediated by membrane-anchored HB-EGF vs. mature "secreted HB-EGF." In our studies, using stable expression of a noncleavable, membrane-anchored rat HB-EGF isoform (MDCK(rat5aa) cells) in Madin-Darby canine kidney (MDCK) II cells, we observed a significant increase in transepithelial resistance (TER). Similar significant increases in TER were observed on stable expression of an analogous, noncleavable, membrane-anchored human HB-EGF construct (MDCK(human5aa) cells). The presence of noncleavable, membrane-anchored HB-EGF led to alterations in the expression of selected claudin family members, including a marked decrease in claudin-2 in MDCK(rat5aa) cells compared with the control MDCK cells. Reexpression of claudin-2 in MDCK(rat5aa) cells largely prevented the increases in TER. Ion substitution studies indicated decreased paracellular ionic permeability of Na(+) in MDCK(rat5aa) cells, further indicating that the altered claudin-2 expression mediated the increased TER seen in these cells. In a Ca(2+)-switch model, increased phosphorylation of EGF receptor and Akt was observed in MDCK(rat5aa) cells compared with the control MDCK cells, and inhibition of these pathways inhibited TER changes specifically in MDCK(rat5aa) cells. Therefore, we hypothesize that juxtacrine activation of EGFR by membrane-anchored HB-EGF may play an important role in the regulation of tight junction proteins and TER.     

Developmental regulation of claudin localization by fetal alveolar epithelial cells

Tight junction proteins in the claudin family regulate epithelial barrier function. We examined claudin expression by human fetal lung (HFL) alveolar epithelial cells cultured in medium containing dexamethasone, 8-bromo-cAMP, and isobutylmethylxanthanine (DCI), which promotes alveolar epithelial cell differentiation to a type II phenotype. At the protein level, HFL cells expressed claudin-1, claudin-3, claudin-4, claudin-5, claudin-7, and claudin-18, where levels of expression varied with culture conditions. DCI-treated differentiated HFL cells cultured on permeable supports formed tight transepithelial barriers, with transepithelial resistance (TER) >1,700 ohm/cm(2). In contrast, HFL cells cultured in control medium without DCI did not form tight barriers (TER <250 ohm/cm(2)). Consistent with this difference in barrier function, claudins expressed by HFL cells cultured in DCI medium were tightly localized to the plasma membrane; however, claudins expressed by HFL cells cultured in control medium accumulated in an intracellular compartment and showed discontinuities in claudin plasma membrane localization. In contrast to claudins, localization of other tight junction proteins, zonula occludens (ZO)-1, ZO-2, and occludin, was not sensitive to HFL cell phenotype. Intracellular claudins expressed by undifferentiated HFL cells were localized to a compartment containing early endosome antigen-1, and treatment of HFL cells with the endocytosis inhibitor monodansylcadaverine increased barrier function. This suggests that during differentiation to a type II cell phenotype, fetal alveolar epithelial cells use differential claudin expression and localization to the plasma membrane to help regulate tight junction permeability.     

Opposite regulation of transepithelial electrical resistance and paracellular permeability by Rho in Madin-Darby canine kidney cells.

Small GTPase Rho has been thought to be important for the formation and the maintenance of tight junction in epithelial cells, but the role of Rho in the regulation of barrier function of tight junction is not well understood. We here examined whether Rho was involved in the barrier function of tight junction in Madin-Darby canine kidney (MDCK) cells. The activation of prostaglandin EP3beta receptor, coupled to a Rho activation pathway, induced the increase in transepithelial electrical resistance (TER) but the increase in paracellular flux of mannitol in the preformed monolayer of the MDCK cells expressing the EP3beta receptor. This effect of the EP3 receptor was mimicked by the expression of constitutively active RhoA but not by active Rac1 in MDCK cells, using an isopropyl-beta-D-thiogalactoside-inducible expression system. On the other hand, the activation of EP3beta receptor suppressed the elevation of TER and the decrease in paracellular mannitol flux during Ca(2+) switch-induced tight junction formation, whereas the expression of active RhoA or Rac1 did not apparently affect the TER development in the Ca(2+) switch. These results demonstrate that the EP3 receptor and active RhoA regulate permeabilities of ionic and nonionic molecules in opposite directions in the preformed monolayer, and the EP3 receptor suppresses the elevation of TER during the tight junction formation.     

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.     

TGF-β1 mediates sirolimus and cyclosporine A-induced alteration of barrier function in renal epithelial cells via a noncanonical ERK1/2 signaling pathway

The immunosuppressant drugs cyclosporine A (CsA) and sirolimus (SRL) used in combination demonstrated beneficial effects in organ transplantation, but this combination can also result in increased adverse effects. We previously showed that not only CsA treatment but also its combination with SRL decreased paracellular permeability in renal proximal tubular cells by modification of the tight junction proteins, claudins, through ERK1/2 signaling pathway. In this present study, evidence is presented that not only CsA but also the combination of CsA/SRL may have adverse effects on the barrier function of renal proximal cells, at least in part, through the expression of the cytokine transforming growth factor (TGF)-β(1). CsA treatment upregulated TGF-β(1) gene expression and this upregulation was enhanced when CsA and SRL were applied together. Addition of TGF-β(1) (5 ng/ml) altered the barrier function with increased transepithelial electrical resistance (TER) and claudin-1 expression. Use of a TGF-β(1)-blocking antibody or blockage of TGF-β(1) receptor kinase activity with SD208 prevented the CsA- and CsA/SRL-induced increase in TER. No evidence was found in the present studies to indicate that CsA or CsA/SRL treatment activated the TGF-β(1) Smad canonical signaling pathway, whereas addition of TGF-β(1) (5 ng/ml) did activate the Smad pathway. Addition of the ERK1/2 signaling inhibitor U0126 was able to prevent the TGF-β(1)-mediated increase in TER and claudin expression. It is most likely that the CsA- and CsA/SRL-induced increases in TGF-β(1) expression may not be sufficient to trigger the Smad pathway but however may trigger other TGF-β(1) receptor-mediated signaling including the ERK1/2 signaling pathway.     

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.     

Comparative characterization of mouse rectum CMT93-I and -II cells by expression of claudin isoforms and tight junction morphology and function

Recent studies suggest that the morphological and physiological properties of tight junctions (TJs) are determined by the combination and mixing ratios of claudin isoforms. In this study, we tried to characterize mouse cell lines by expression of claudin isoforms to use for studying epithelial TJs by overexpression or suppression of claudin(s) in the cells and found that claudin-2 was expressed in a few mouse rectum carcinoma cells, CMT93 cells. We have isolated CMT93-I and -II cells from CMT93 cells by immunohistochemical screening for the presence or absence of claudin-2 expression. Immunofluorescence and RT-PCR analyses showed that expression of claudin-4, -6, -7 and -12 was detected in both cell lines, but claudin-2 was only expressed in CMT93-II cells. There were no differences in paracellular permeability between CMT93-I and -II cells examined by 4 kDa FITC-dextran and fluorescein sodium, or in the number of TJ strands examined by freeze-fracture electron microscopy. However, the transepithelial electrical resistance (TER) of CMT93-I cells was approximately 6.5 times higher than that of CMT93-II cells, suggesting that expression of claudin-2 may be related to decreased TER. Comparative examinations of CMT93-I and -II cells provide a clue how the combination and mixing ratios of claudin isoforms regulate the paracellular permeability.     

Extracellular Mg regulates the tight junctional localization of claudin-16 mediated by ERK-dependent phosphorylation

Claudin-16 is involved in the paracellular reabsorption of Mg²⁺ in the thick ascending limb of Henle. Little is known about the mechanism regulating the tight junctional localization of claudin-16. Here, we examined the effect of Mg²⁺ deprivation on the distribution and function of claudin-16 using Madin-Darby canine kidney (MDCK) cells expressing FLAG-tagged claudin-16. Mg²⁺ deprivation inhibited the localization of claudin-16 at tight junctions, but did not affect the localization of other claudins. Re-addition of Mg²⁺ induced the tight junctional localization of claudin-16, which was inhibited by U0126, a MEK inhibitor. Transepithelial permeability to Mg²⁺ was also inhibited by U0126. The phosphorylation of ERK was reduced by Mg²⁺ deprivation, and recovered by re-addition of Mg²⁺. These results suggest that the MEK/ERK-dependent phosphorylation of claudin-16 affects the tight junctional localization and function of claudin-16. Mg²⁺ deprivation decreased the phosphothreonine levels of claudin-16. The phosphothreonine levels of T225A and T233A claudin-16 were decreased in the presence of Mg²⁺ and these mutants were widely distributed in the plasma membrane. Furthermore, TER and transepithelial Mg²⁺ permeability were decreased in the mutants. We suggest that the tight junctional localization of claudin-16 requires a physiological Mg²⁺ concentration and the phosphorylation of threonine residues via a MEK/ERK-dependent pathway.     

Protamine-induced epithelial barrier disruption involves rearrangement of cytoskeleton and decreased tight junction-associated protein expression in cultured MDCK strains

Natural and synthetic polycationic proteins, such as protamine, have been used to reproduce the tissue injury and changes in epithelial permeability caused by positively charged substances released by polymorphonuclear cells during inflammation. Protamine has diverse and often conflicting effects on epithelial permeability. The effects of this polycation on the distribution and expression of tight junction (TJ)-associated proteins have not yet been investigated. In this work, we examined the influence of protamine on paracellular barrier function and TJ structure using two strains of the epithelial Madin-Darby canine kidney (MDCK) cell line that differed in their TJ properties ("tight" TJ-strain I and "leaky" TJ-strain II). Protamine induced concentration-, time- and strain-dependent alterations in transepithelial electrical resistance (Rt) only when applied to apical or apical+basolateral monolayer surfaces, indicating a polarity of action. In MDCK II cells, protamine (50 microg/ml) caused a significant increase in Rt that returned to control values after 2 h. However, the treatment of this MDCK strain with a higher concentration of protamine (250 microg/ml) significantly decreased the Rt after 30 min. In contrast, treated MDCK I monolayers showed a significant decrease in Rt after apical treatment with protamine at both concentrations. The protamine-induced decrease in Rt was paralleled by an increase in the phenol red basal-to-apical flux in both MDCK strains, suggesting disruption of the paracellular barrier. Marked changes in cytoskeletal F-actin distribution/polymerization and a significant reduction in the junctional expression of the tight junctional proteins occludin and claudin-1 but subtle alterations in ZO-1 were observed following protamine-elicited paracellular barrier disruption. In conclusion, protamine induces alterations in the epithelial barrier function of MDCK monolayers that may involve the cytoskeleton and TJ-associated proteins. The various actions of protamine on epithelial function may reflect different degrees of interaction of protamine with the plasma membrane and different intracellular processes triggered by this polycation.     

Cultured Alveolar Epithelial Cells From Septic Rats Mimic In Vivo Septic Lung

Sepsis results in the formation of pulmonary edema by increasing in epithelial permeability. Therefore we hypothesized that alveolar epithelial cells isolated from septic animals develop tight junctions with different protein composition and reduced barrier function relative to alveolar epithelial cells from healthy animals. Male rats (200–300g) were sacrificed 24 hours after cecal ligation and double puncture (2CLP) or sham surgery. Alveolar epithelial cells were isolated and plated on fibronectin-coated flexible membranes or permeable, non-flexible transwell substrates. After a 5 day culture period, cells were either lysed for western analysis of tight junction protein expressin (claudin 3, 4, 5, 7, 8, and 18, occludin, ZO-1, and JAM-A) and MAPk (JNK, ERK, an p38) signaling activation, or barrier function was examined by measuring transepithelial resistance (TER) or the flux of two molecular tracers (5 and 20 Å). Inhibitors of JNK (SP600125, 20 µM) and ERK (U0126, 10 µM) were used to determine the role of these pathways in sepsis induced epithelial barrier dysfunction. Expression of claudin 4, claudin 18, and occludin was significantly lower, and activation of JNK and ERK signaling pathways was significantly increased in 2CLP monolayers, relative to sham monolayers. Transepithelial resistance of the 2CLP monolayers was reduced significantly compared to sham (769 and 1234 ohm-cm², respectively), however no significant difference in the flux of either tracer was observed. Inhibition of ERK, not JNK, significantly increased TER and expression of claudin 4 in 2CLP monolayers, and prevented significant differences in claudin 18 expression between 2CLP and sham monolayers. We conclude that alveolar epithelial cells isolated from septic animals form confluent monolayers with impaired barrier function compared to healthy monolayers, and inhibition of ERK signaling partially reverses differences between these monolayers. This model provides a unique preparation for probing the mechanisms by which sepsis alters alveolar epithelium.     

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.     

Clostridium perfringens enterotoxin fragment removes specific claudins from tight junction strands: Evidence for direct involvement of claudins in tight junction barrier.

Claudins, comprising a multigene family, constitute tight junction (TJ) strands. Clostridium perfringens enterotoxin (CPE), a single approximately 35-kD polypeptide, was reported to specifically bind to claudin-3/RVP1 and claudin-4/CPE-R at its COOH-terminal half. We examined the effects of the COOH-terminal half fragment of CPE (C-CPE) on TJs in L transfectants expressing claudin-1 to -4 (C1L to C4L, respectively), and in MDCK I cells expressing claudin-1 and -4. C-CPE bound to claudin-3 and -4 with high affinity, but not to claudin-1 or -2. In the presence of C-CPE, reconstituted TJ strands in C3L cells gradually disintegrated and disappeared from their cell surface. In MDCK I cells incubated with C-CPE, claudin-4 was selectively removed from TJs with its concomitant degradation. At 4 h after incubation with C-CPE, TJ strands were disintegrated, and the number of TJ strands and the complexity of their network were markedly decreased. In good agreement with the time course of these morphological changes, the TJ barrier (TER and paracellular flux) of MDCK I cells was downregulated by C-CPE in a dose-dependent manner. These findings provided evidence for the direct involvement of claudins in the barrier functions of TJs.     

Protein Kinase C Activation Has Distinct Effects on the Localization, Phosphorylation and Detergent Solubility of the Claudin Protein Family in Tight and Leaky Epithelial Cells

We have previously shown that protein kinase C (PKC) activation has distinct effects on the structure and barrier properties of cultured epithelial cells (HT29 and MDCK I). Since the claudin family of tight junction (TJ)-associated proteins is considered to be crucial for the function of mature TJ, we assessed their expression patterns and cellular destination, detergent solubility and phosphorylation upon PKC stimulation for 2 or 18 h with phorbol myristate acetate (PMA). In HT29 cells, claudins 1, 3, 4 and 5 and possibly claudin 2 were redistributed to apical cell–cell contacts after PKC activation and the amounts of claudins 1, 3 and 5, but not of claudin 2, were increased in cell lysates. By contrast, in MDCK I cells, PMA treatment resulted in redistribution of claudins 1, 3, 4 and 5 from the TJ and in reorganization of the proteins into more insoluble complexes. Claudins 1 and 4 were phosphorylated in both MDCK I and HT29 cells, but PKC-induced changes in claudin phosphorylation state were detected only in MDCK I cells. A major difference between HT29 and MDCK I cells, which have low and high basal transepithelial electrical resistance, respectively, was the absence of claudin 2 in the latter. Our findings show that PKC activation targets in characteristic ways the expression patterns, destination, detergent solubility and phosphorylation state of claudins in epithelial cells with different capacities to form an epithelial barrier.     

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.     

Interferon-gamma signals via an ERK1/2-ARF6 pathway to promote bacterial internalization by gut epithelia

The barrier function of the epithelium lining the intestine is essential for health by preventing the free passage of colonic bacteria into the mucosa. Epithelia treated with interferon (IFN)-γ display increased bacteria transcytosis. Much is known of how IFNγ affects the tight junction and paracellular permeability, yet its role in modifying transcellular traffic of commensal bacteria remains poorly understood. Using immunoblotting, ELISA and immunolocalization, IFNγ was found to activate extracellular regulated kinase (ERK)1/2 in the human colon-like T84 epithelial cell line. Pharmacological inhibition of MEK/ERK1/2 signalling with U0126 significantly inhibited IFNγ-induced increases in the transcytosis of non-invasive Escherichia coli (strain HB101). IFNγ treatment enhanced epithelial internalization of E. coli, some of which subsequently escaped the enterocyte. Molecular analyses revealed that ERK1/2 inhibition prevented activation of the ADP-ribosylation factor (ARF)-6, a protein associated with endocytosis, and that siRNA knock-down of ARF6 expression reduced IFNγ-induced E. coli internalization into T84 cells. None of these interventions affected the drop in transepithelial resistance caused by IFNγ. Thus, increased transcellular passage may be a major component of IFNγ-induced increases in epithelial permeability, and ERK1/2 and ARF6 are presented as important molecules in IFNγ-evoked transcytosis of bacteria across gut epithelia.