Enhancement of cell-cell contact by claudin-4 in renal epithelial Madin-Darby canine kidney cells.

Claudin-4 regulates ion permeability via a paracellular pathway in renal epithelial cells, but its other physiological functions have not been examined. We found that hyperosmotic stress increases claudin-4 expression in Madin-Darby canine kidney cells. Here, we examined whether claudin-4 affects cell motility, cell association, and the intracellular distribution of endogenous junctional proteins. Doxycycline-inducible expression of claudin-4 did not change endogenous levels of claudin-1, claudin-2, claudin-3, occludin, E-cadherin, and ZO-1. Claudin-4 overexpression increased cell association and decreased cell migration without affecting cell proliferation. Doxycycline did not change cell junctional protein levels, cell association or cell migration in mock-transfected cells. The insolubility of claudin-1 and -3 in Triton X-100 was increased by claudin-4 overexpression, but that of claudin-2, occludin, ZO-1, and E-cadherin was unchanged. Immunocytochemistry showed that claudin-4 overexpression increases the accumulation of claudin-1 and -3 in tight junctions (TJs). Furthermore, claudin-4 overexpression increased the association of claudin-4 with claudin-1 and -3. These results suggest that claudin-4 accumulates claudin-1 and -3 in TJs to enhance cell-cell contact in renal tubular epithelial cells.

Epidermal growth factor increases clathrin-dependent endocytosis and degradation of claudin-2 protein in MDCK II cells.

Epidermal growth factor (EGF) decreases the mRNA and protein levels of claudin-2 (CLDN2) in Madin-Darby canine kidney (MDCK) II cells. Here we examined whether EGF affects the stability and intracellular distribution of CLDN2 protein. EGF decreased surface and total levels of CLDN2, which was inhibited by U0126, a MEK inhibitor. CLDN2 was co-localized at the tight junctions (TJs) with ZO-1, a scaffolding protein. The fluorescence signal for CLDN2 disappeared on treatment with EGF, which was inhibited by U0126. EGF accelerated the decrease in CLDN2 in the presence of cycloheximide, a translation inhibitor, indicating that EGF reduces the stability of the protein. Chloroquine, a lysosomal protease inhibitor, blocked the EGF-induced decrease in CLDN2 protein and caused the co-localization of CLDN2 with Lamp-1, a marker of lysosome. Monodancylcadaverine, an inhibitor of endocytosis, and clathrin siRNA blocked the EGF-induced decrease in CLDN2 and the translocation of CLDN2 from the TJs to the lysosome. EGF increased the association of CLDN2 with clathrin and adaptin α which was inhibited by U0126. These results suggest that EGF accelerates clathrin-dependent endocytosis and lysosomal degradation of CLDN2 protein mediated by the activation of a MEK/ERK pathway.

Decrease in claudin-2 expression enhances cell migration in renal epithelial Madin-Darby canine kidney cells.

Migration of renal epithelial cells increases after renal tubular damage, but its mechanism has not been clarified in detail. Hyperosmotic stress increased a cellular injury concomitant with a decrease in mRNA and protein expression of claudin-2 in renal tubular epithelial Madin-Darby canine kidney cells. We hypothesized that claudin-2 is involved in the regulation of cell migration. To knockdown claudin-2 expression, we made the cells expressing doxycycline-inducible claudin-2 shRNA vector. Claudin-2 knockdown affected neither the endogenous expression levels of claudin-1, -3, -4, and -7 nor the Triton X-100 solubility of these claudins. Transepithelial electrical resistance was increased by claudin-2 knockdown without affecting permeability to FITC-dextran (4,000 Da). BrdU incorporation assay and cell counting revealed that cell proliferation and viability are unaffected by claudin-2 knockdown. In the wound-healing assay, the recovery rate of wound area was increased by claudin-2 knockdown. The mRNA expression and activity of matrix metalloproteinase-9 (MMP-9) were increased by claudin-2 knockdown. A selective MMP-9 inhibitor suppressed cell migration in the claudin-2 knockdown cells. Hyperosmotic stress increased the expression and activity of MMP-9, which were inhibited by claudin-2 overexpression. These results suggest that the decrease in claudin-2 expression enhances cell migration mediated by the increase in the expression and activity of MMP-9.

Epidermal growth factor increases claudin-4 expression mediated by Sp1 elevation in MDCK cells.

Epidermal growth factor (EGF) increases claudin-4 expression in Madin-Darby canine kidney (MDCK) cells. Here we examined what regulatory mechanisms are involved in the EGF-induced claudin-4 elevation. EGF transiently increased claudin-4 mRNA at 3h and persistently increased its protein for 24h without affecting claudin-1 expression. EGF increased p-ERK1/2 levels, which were inhibited by U0126, a MEK inhibitor. The exogenous expression of constitutively activated MEK increased claudin-4 expression. These results indicate that the activation of ERK1/2 is involved in the EGF-induced claudin-4 elevation. EGF increased Sp1 expression within 1h, which was inhibited by U0126. In immunocytochemistry, Sp1 was distributed in nucleus in control and the EGF-treated cells. The EGF-induced claudin-4 elevation was inhibited by mithramycin, a Sp1 inhibitor, and Sp1 small interfering RNA. We suggest that EGF activates a MEK/ERK pathway and increases Sp1 expression, resulting in an elevation of claudin-4 expression.

Claudin-2 knockdown decreases matrix metalloproteinase-9 activity and cell migration via suppression of nuclear Sp1 in A549 cells.

AIMS: Claudin expression is altered in lung cancer, but the pathophysiological role of claudin is not well understood. We examined the effect of claudin-2 expression on cell migration using human adenocarcinoma A549 cells. MAIN METHODS: The mRNA level was measured by real time polymerase chain reaction. To knockdown claudin-2 expression, we made the cells expressing doxycycline-inducible claudin-2 shRNA vector. The protein level was examined by Western blotting. Cell migration was measured by wound-healing assay. The enzymatic activity of MMP-9 was assessed by gelatin zymography. KEY FINDINGS: In A549 cells, claudin-2 expression was higher than in normal lung tissue. Claudin-2 knockdown did not affect the expression of other junctional proteins including claudin-1, occludin and E-cadherin. Claudin-2 knockdown decreased cell migration concomitant with a decrease in the mRNA level and enzymatic activity of MMP-9. The expression level of Sp1 in the nuclei was decreased by claudin-2 knockdown. In contrast, the expression levels of c-Fos, c-Jun and NF-kB p65 in the nuclei were not changed by claudin-2 knockdown. The knockdown of Sp1 expression by siRNA decreased cell migration, and the mRNA expression, enzymatic activity, and promoter activity of MMP-9. SIGNIFICANCE: Claudin-2 may increase the mRNA level and enzymatic activity of MMP-9 mediated by the elevation of nuclear distribution of Sp1, resulting in the up-regulation of A549 cell migration.