地塞米松通过PKA信号通路调控水通道蛋白2的分子机制

目的：研究地塞米松（Dex）对水通道蛋白2（AQP2）的体外直接调控效应，明确该作用是否依赖于PKA信号通路。方法：制备AQP2 野生型及突变型质粒，突变型质粒通过突变AQP2 C末端4 个PKA磷酸化位点（S256、S261、S264 S269）阻断PKA信号通路。AQP2 野生型及突变型质粒分别转染HEK293 细胞和显微注射爪蟾卵母细胞，再予共转染PKA质粒或Dex 0.1 μmol/L干预，采用蛋白质印迹（Western blot）法检测AQP2 总蛋白表达，通过细胞表面生物素化评估AQP2 膜蛋白表达，比较各组爪蟾卵母细胞的水渗透性差别。结果：Dex及PKA均显著上调HEK293 细胞的AQP2 膜蛋白及总蛋白表达（均P ＜0.01）；与野生型相 比，PKA磷酸化位点的突变显著抑制了体外PKA直接诱导的AQP2 磷酸化；突变后AQP2 的膜蛋白及总蛋白表达均显著下调（均P ＜0.01）；PKA磷酸化位点突变后，Dex及PKA对AQP2 膜蛋白及总蛋白表达的上调效应均被显著抑制（均P ＜0.01）；Dex及PKA均显著增加爪蟾卵母细胞水通透活性，PKA磷酸化位点突变后，该效应被显著抑制（均P ＜0.01）。结论：Dex对AQP2 的总蛋白及膜蛋白表达具有显著的上调效应，该作用依赖于PKA信号通路实现。

Molecular mechanism of dexamethasone in regulating aquaporin 2 through PKA signal pathway

Objective: To observe the direct regulation effect of dexamethasone (Dex) on aquaporin 2 (AQP2) in vitro, and to clarify whether this effect depends on PKA signal pathway, using protein kinase A (PKA) as a reference. Methods: PKA, AQP2 wild-type and mutant plasmids were prepared. The mutant plasmids blocked the PKA signaling pathway by mutating the 4 PKA phosphorylation sites (S256, S261, S264, and S269) at the C-terminal of AQP2. AQP2 wild-type and mutant plasmids were transfected into HEK293 cells and Xenopus oocytes by microinjection and then co-transfected with PKA plasmid or Dex 0.1 μmol/L intervention.Western blot was used to detect the total protein expression of AQP2, and cell surface biotin was used to detect AQP2 total protein expression. The AQP2 membrane protein expression was evaluated chemically, and water permeability difference in oocytes was compared between each group. Results: Both Dex and PKA significantly up-regulated the expression of AQP2 membrane protein and total protein in HEK 293 cells (both P<0.01).Compared with the wild type, the mutation of PKA phosphorylation sites significantly inhibited the phosphorylation of AQP2, which was direct induced by PKA in vitro; AQP2 membrane protein and total protein expression were significantly down-regulated after mutation (all P<0.01). After the PKA phosphorylation sites mutated, the upregulating effect on the expression of AQP2 membrane protein and total protein by Dex and PKA was significantly inhibited (all P<0.01). Both Dex and PKA significantly increased the water permeability of oocytes; after mutation of the PKA phosphorylation sites, the effect was significantly inhibited (all P<0.01). Conclusion: Dex and PKAact in the same direction and have significant up-regulation effect on the total protein and membrane protein expression of AQP2. This effect depends on the normal PKA signaling pathway.