Claudin-2 is expressed in human lung adenocarcinoma tissue and cell lines, although it is absent in normal lung tissue. However, the role of claudin-2 in cell proliferation and the regulatory mechanism of intracellular distribution remain undefined. Proliferation of human adenocarcinoma A549 cells was decreased by claudin-2 knockdown together with a decrease in the percentage of S phase cells. This knockdown decreased the expression levels of ZONAB and cell cycle regulators. Claudin-2 was distributed in the nucleus in human adenocarcinoma tissues and proliferating A549 cells. The nuclear distribution of ZONAB and percentage of S phase cells were higher in cells exogenously expressing claudin-2 with a nuclear localization signal than in cells expressing claudin-2 with a nuclear export signal. Nuclear claudin-2 formed a complex with ZO-1, ZONAB, and cyclin D1. Nuclear distribution of S208A mutant, a dephosphorylated form of claudin-2, was higher than that of wild type. We suggest that nuclear distribution of claudin-2 is up-regulated by dephosphorylation and claudin-2 serves to retain ZONAB and cyclin D1 in the nucleus, resulting in the enhancement of cell proliferation in lung adenocarcinoma cells.

Although paracellin-1 (PCLN-1) is known to have a crucial role in the control of Mg2+ reabsorption in the kidney, the molecular pathways involved in the regulation of PCLN-1 have not been clarified. We used FLAG-tagged PCLN-1 to investigate these pathways further, and found that PCLN-1 is phosphorylated at Ser217 by protein kinase A (PKA) under physiological conditions in Madin-Darby canine kidney (MDCK) cells. PCLN-1 expression decreased Na+ permeability, resulting in a decrease in the transepithelial electrical resistance (TER). By contrast, PCLN-1 enhanced transepithelial Mg2+ transport. PKA inhibitors, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89) and myristoylated protein kinase A inhibitor 14-22 amide PKI, and an adenylate cyclase inhibitor, 2′,5′-dideoxy adenosine (DDA), reduced the phosphoserine level of PCLN-1. The inhibitory effect of DDA was rescued by 8-bromoadenosine-3′,5′-cyclic monophosphate (8-Br-cAMP). PKA and adenylate cyclase inhibitors decreased transepithelial Mg2+ transport and TER. Dephosphorylated PCLN-1 moved from detergent-insoluble to soluble fractions and was dissociated from ZO-1. A fusion protein of PCLN-1 with glutathione-S-transferase revealed that Ser217 was phosphorylated by PKA. Phosphorylated PCLN-1 was localized in the tight junction (TJ) along with ZO-1, whereas dephosphorylated PCLN-1 and the S217A mutant were translocated into the lysosome. The degradation of dephosphorylated PCLN-1 and S217A mutant was inhibited by chloroquine, a specific lysosome inhibitor. Thus, the PKA-dependent phosphorylation of Ser217 in PCLN-1 is essential for its localization in the TJ and transepithelial Mg2+ transport.

In human adenocarcinoma, claudin-2 expression is higher than that in normal lung tissue, but the regulatory mechanism of its expression has not been clarified. In human adenocarcinoma A549 cells, claudin-2 level time-dependently increased under the control conditions. In contrast, claudin-1 expression remained constant for 24h. The concentration of epidermal growth factor (EGF) in medium time-dependently increased, which was inhibited by matrix metalloproteinase (MMP) inhibitor II, an inhibitor of MMP-1, 3, 7, and 9. MMP inhibitor II decreased claudin-2 and phosphorylated ERK1/2 (p-ERK1/2) levels, which were recovered by EGF. Both claudin-2 and p-ERK1/2 levels were decreased by EGF neutralizing antibody, EGF receptor (EGFR) siRNA, AG1478, an inhibitor of EGFR, U0126, an inhibitor of MEK, and the exogenous expression of dominant negative-MEK. These results suggest that EGF is secreted from A549 cells by MMP and increases claudin-2 expression mediated via the activation of an EGFR/MEK/ERK pathway. The inhibition of the signaling pathway decreased phosphorylated c-Fos and nuclear c-Fos levels. The introduction of c-Fos siRNA decreased claudin-2 level without affecting claudin-1. The promoter activity of human claudin-2 was decreased by AG1478 and U0126. Furthermore, the activity was decreased by the deletion or mutation of the AP-1 binding site of claudin-2 promoter. Chromatin immunoprecipitation and avidin-biotin conjugated DNA assays showed that c-Fos binds to the AP-1 binding site. We suggest that a secreted EGF up-regulates the transcriptional activity of claudin-2 mediated by the activation of an EGFR/MEK/ERK/c-Fos pathway in A549 cells.

The relation of atrophic gastritis, other gastric lesions and lifestyle factors to stomach cancer risk was prospectively studied among 3,914 subjects who underwent gastroscopic examination and responded to a questionnaire survey at the Aichi Cancer Center Hospital. During 4.4 years of follow‐up on average, 45 incident cases of stomach cancer were identified at least three months after the initial examination. If the baseline endoscopic findings indicated the presence of atrophic gastritis, the risk of developing stomach cancer was increased 5.73‐fold, compared with no indication at the baseline. The risk further increased with advancing degree of atrophy and increasing extension of atrophy on the lesser curvature. These trends in the relative risks were statistically significant (P= 0.027 and P= 0.041, respectively). The risk of developing stomach cancer was statistically significantly increased among subjects with gastric polyps, but not among those with gastric ulcer. Stomach cancer cases tended to consume more cigarettes, alcohol, rice, pickles and salted fish gut/cod roe and less fruits and vegetables and to have more family histories of stomach cancer than noncases, although these differences were not statistically significant. The results of the present study provide additional evidence on the relation between atrophic gastritis and stomach cancer and suggest a need for intensive follow‐up of patients with atrophic gastritis and gastric polyps.