Regulation of NHE3 by glucocorticoids, SGK1 and NHERF2

描述(申请人提供)：糖皮质激素对维持体液和电解质平衡的促进作用已有二十多年的历史。药理剂量的甲基强的松龙促进小动物对Na+的吸收。然而，这种激活背后的分子机制仍然难以捉摸。位于肠和肾刷状缘膜上的Na+/H+交换器NHE3在跨上皮钠离子吸收中起主要作用。我们先前证明，甲基强的松龙在兔体内特异性地刺激回肠NHE3的mRNA，而不影响NHE1的mRNA水平。其他研究表明，糖皮质激素特异性地激活回肠、近端结肠和肾近端小管中的NHE3mRNA。提示糖皮质激素通过NHE3基因表达激活NHE3活性。然而，我们最近在使用Caco-2细胞时发现，地塞米松激活了NHE3的转运，而不影响NHE3mRNA的表达。在OK细胞中，地塞米松对NHE3的这种非转录激活被证实，提示NHE3的转录激活可能不是糖皮质激素刺激NHE3的唯一决定因素。我们发现，地塞米松只有在NHE3调节蛋白NHERF2存在的情况下才能增强NHE3的活性。我们鉴定了血清和糖皮质激素诱导的蛋白激酶1，SGK1是一种与NHERF2的PDZ结构域相互作用的蛋白质。我们证明了SGK1激活NHE3的活性和在OK细胞中“死”的SGK1的表达明显地阻断了地塞米松的作用，从而证明了SGKI的重要性。我们还发现SGK1在体外直接磷酸化NHE3，提示对NHE3的调节依赖于磷酸化。在这个应用中，我们建议研究：(1)SGK1和NHERF2在糖皮质激素激活NHE3中的作用；(2)SGK1和NHERF2响应糖皮质激素激活NHE3的分子机制；(3)SGK1、NHERF2和NHE3之间的相互作用。这一建议将解决糖皮质激素对NHE3调控的复杂性，并加深我们对膜转运蛋白调控中蛋白质-蛋白质相互作用的理解。

DESCRIPTION (provided by applicant): The stimulative effect of glucocorticoids on the maintenance of fluid and electrolyte balance has been known for more than two decades. Pharmacological doses of methylprednisolone stimulated Na+ absorption in small animals. However, molecular mechanisms underlying this activation remain elusive. Na+/H+ exchanger NHE3 in the brush border membrane of intestine and kidney plays a major role in transepithelial Na+ absorption. We previously demonstrated that methylprednisolone in rabbits specifically stimulated NHE3 mRNA in ileum without affecting NHE1 mRNA levels. Others have shown that glucocorticoids specifically activated NHE3 mRNA in ileum, proximal colon and renal proximal tubules. These results suggest that glucocorticoids activate NHE3 activity by gene expression of NHE3. However, we recently found using Caco-2 cells that dexamethasone activates NHE3 transport without affecting NHE3 mRNA expression. This non-transcriptional activation of NHE3 by dexamethasone was demonstrated in OK cells, suggesting that transcriptional activation of NHE3 may not be the only determining factor in glucocorticoid-stimulation of NHE3. We found that dexamethasone enhanced NHE3 activity only in the presence of a NHE3 regulatory protein, NHERF2. We identified serum- and glucocorticoids-induced protein kinase 1, SGK1, as a protein interacting with PDZ domains of NHERF2. We demonstrated that SGK1 activated NHE3 activity and expression of "kinase-dead" SGK1 in OK cells markedly blocked the dexamethasone effect, demonstrating the importance of SGKI. We also showed that SGK1 directly phosphorylated NHE3 in vitro suggesting phosphorylation-dependent regulation of NHE3. In this application, we propose to investigate: (1) the roles of SGK1 and NHERF2 in activation of NHE3 by glucocorticoids; (2) molecular mechanisms underlying the activation of NHE3 by SGK1 and NHERF2 in response to glucocorticoid; and (3) the interaction between SGK1, NHERF2 and NHE3. This proposal will resolve complexity in NHE3 regulation by glucocorticoids, and enhance our understanding of protein-protein interaction in regulation of membrane transport proteins.