Molecular mechanism of dexamethasone in regulating aquaporin 2 through PKA signal pathway
ZHU Lingli, YANG Jianhuan, WANG Dexuan, CHEN Minguang
Division of Pediatric Nephrology, Wenzhou Key Laboratory of Children Genitourinary Diseases, the Second Affiliated Hospital & Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou 325027, China
引用本文:
朱玲丽,杨建环,王德选,等.
地塞米松通过PKA信号通路调控水通道蛋白2的分子机制
[J]. 温州医科大学学报, 2022, 52(3): 186-193.
Cite this article:
ZHU Lingli,YANG Jianhuan,WANG Dexuan, et al. Molecular mechanism of dexamethasone in regulating aquaporin 2 through PKA signal pathway[J]. JOURNAL OF WEZHOU MEDICAL UNIVERSITY, 2022, 52(3): 186-193.
Abstract： 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.
