唾液腺腺房細胞におけるmatrix metalloproteinase-9抑制と導管細胞におけるaquaporin-5誘導機構 : シェーグレン症候群唾液腺水分泌機能の再生

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Our previous in vitro study suggested that the suppression by cepharanthin of tumor necrosis factor (TNF)-α-induced matrix metalloproteinase (MMP)-9 could prevent the destruction of the acinar structure in Sjogren's syndrome (SS) salivary glands. In this study, we demonstrated that the in vivo administration of cepharanthin resulted in the prevention of severe damage to acinar tissues in the murine model of human SS. Cepharanthin was intraperitoneally administered five times a week at a dose of 0, 10, or 100μg/mouse to 4 to 10 week-old thymectomized female NFS/sld mice. Mononuclear cell infiltrates and the destruction of acinar tissue in the salivary and lacrimal glands were extensively observed in the control mice ; however, in the mice treated with cepharanthin for 6 weeks, both a significant decrease in the mononuclear cell infiltrates and an improvement of the severe damage to the acinar tissues were evident in the salivary and lacrimal glands. Immunohistochemical analysis revealed that phosphorylated IκB-α and MMP-9 were more strongly stained in the acinar cells of the control mice than in those of the cepharanthin-treated mice. In addition, although the lack of staining for type IV collagen was partially observed in the acinar tissues of the control mice, the continuity of type IV collagen was detected in the acinar tissues of the cepharanthin-treated mice. According to the results of a TUNEL analysis, the destruction of acinar tissues was attributed to the induction of apoptosis, suggesting that cepharanthin inhibits apoptosis by suppressing phosphorylation of IκB-α, followed by the prevention of MMP-9 activation. Our findings suggest that cepharanthin may be a promising agent for use in preventing the destruction of acinar tissues in murine SS. Moreover, to investigate the possibility that salivary gland cells, especially ductal cells, surviving in the salivary gland tissues of SS could acquire the functional expression of membrane water channel aquaporin-5 (AQP5), because ductal cells, but not acinar cells, preferentially survive and/or proliferate in SS salivary glands. Thus, in this study, we demonstrate that an immortalized normal human salivary gland ductal cell (NS-SV-DC) line, lacking the expression of AQP5, acquires AQP5 gene expression in response to treatment with 5-aza-2'-deoxycytidine (5-Aza-CdR), a DNA demethylating agent. Confocal microscopic analysis revealed the localization of AQP5 expression mainly at the apical and lateral sides of the plasma membrane. The expressed AQP5 protein was functionally active because AQP5 expression resulted in a significant increase in the osmotically directed net fluid rate across monolayers of NS-SV-DC cells. By the analysis of bisulfite sequencing of CpG islands in the AQP5 promoter, hypermethylation within the consensus Sp1-binding sites was commonly observed in parental cell clones, whereas demethylation at the CGs, one in the second consensus Sp1 element and the other outside of the third consensus Sp1 element in the AQP5 promoter, was detected in NS-SV-DC cells after treatment with 5-Aza-CdR. By analyzing the luciferase activity of transfected AQP5 promoter vectors, it became evident that demethylation at the CGs cooperatively functions between these two sites to induce AQP5 expression. Our data, therefore, suggest that treatment of ductal cells with 5-Aza-CdR could result in the expression of the AQP5 gene, thereby leading to increased fluid secretion from ductal cells in SS salivary glands.

唾液腺腺房細胞におけるmatrix metalloproteinase-9抑制と導管細胞におけるaquaporin-5誘導機構 : シェーグレン症候群唾液腺水分泌機能の再生

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