ClC-2於人類腎上腺皮質細胞H295R對於醛固酮生成以及小鼠塞特利氏細胞TM4屏障完整性之影響

Abstract

氯離子通道蛋白2 (Chloride channel protein 2，ClC-2) 是由人類CLCN2基因所編碼的蛋白。ClC-2廣泛表現於哺乳類組織的細胞膜上，對於調控細胞離子的平衡、細胞與細胞之間物質的運輸具有重要的角色。近年來發現CLCN2基因功能獲得型突變與第二型家族性高醛固酮症有關。醛固酮是由腎上腺皮質絲球帶所分泌，主要調控電解質的平衡。本篇研究即利用人類腎上腺皮質癌細胞株H295R，探討ClC-2對於醛固酮生成相關基因之影響，包括CYP11B2 (醛固酮合成酶) 及其他上游基因StAR、CYP11A1、HSD3B2及CYP21A2。另外，在ClC-2基因剔除小鼠中發現睪丸萎縮、塞特利氏細胞 (Sertoli cell) 異常及精子無法產生而導致雄性不孕。臨床上也發現CLCN2基因突變男性不孕症的案例。塞特利氏細胞之間有細胞連結會形成血液睪丸屏障，對於精子的生成是相當重要；因此我們也利用小鼠塞特利氏細胞株TM4，探討ClC-2對於屏障完整性之影響。 我們在H295R細胞使用三種處理，包括ClC-2 knockdown、ClC-2抑制劑GaTx2及ClC-2促進劑lubiprostone，觀察其對於醛固酮生成相關基因表現之效應。結果顯示，ClC-2 knockdown會降低CYP11B2、StAR及CYP11A1 mRNA的量；但是GaTx2處理，對這些基因的表現則都沒有影響。相反地，給予lubiprostone能促進CYP11B2、StAR及CYP21A2 mRNA的量，亦能提升StAR蛋白質的量，但不影響CYP11A1及HSD3B2的表現。上述結果說明ClC-2對醛固酮生成具有重要性。 在TM4細胞我們發現lubiprostone處理能顯著提升細胞跨膜電阻 (TER)，說明其可能增進細胞屏障的完整性；GaTx2處理對TER則沒有效應。分析TM4細胞連結蛋白質的表現量，ClC-2 knockdown、GaTx2及lubiprostone處理都沒有顯著效應。因此，lubiprostone在睪丸屏障的功能有待更多的研究。

Chloride channel protein 2 (ClC-2) is encoded by the human CLCN2 gene. ClC-2 is broadly expressed in the plasma membranes of epithelial cells from many mammalian tissues. ClC-2 plays an important role in regulating ion homeostasis and cell to cell communication. Recently, it has been found that patients with gain-of-function mutation of CLCN2 gene are associated with familial hyperaldosteronism type II. Aldosterone is synthesized in the zona glomerulosa of the adrenal cortex, which regulates electrolyte homeostasis. In this study, we used human adrenocortical carcinoma cells H295R to investigate the effect of ClC-2 on expression of genes involved in aldosterone synthesis, including CYP11B2 (aldosterone synthase) and the upstream genes StAR, CYP11A1, HSD3B2 and CYP21A2. In addition, ClC-2 knockout mice suffered from testicular degeneration, abnormality of Sertoli cells and defective spermatogenesis that lead to male infertility. A case report demonstrated that a male with CLCN2 mutation had azoospermia. Blood-testis barrier is formed by Sertoli cells, which is important for spermatogenesis. We thus used mouse Sertoli cells TM4 to investigate the effect of ClC-2 on barrier integrity. We used three treatments in H295R cells, including ClC-2 knockdown, ClC-2 inhibitor GaTx2 and ClC-2 activator lubiprostone to test the effects on the expression of genes for aldosterone synthesis. The results revealed that knockdown of ClC-2 decreased the mRNA levels of CYP11B2, StAR and CYP11A1. However, these effects were not observed by GaTx2 treatment. Conversely, lubiprostone could promote the expression levels of CYP11B2, StAR and CYP21A2 mRNA and also StAR protein, but no effect on CYP11A1 and HSD3B2. These results indicated that ClC-2 plays a critical role in aldosterone synthesis.