The regulatory role of RanBPM in renal tubular sodium chloride cotransporter and its effect on ERK1/2 signaling pathway
ZHANG Yiqian1, ZHUANG Zhizhi1, SHEN Meng1, ZHUANG Jieqiu2, CAI Hui1
1.Department of Nephrology, the Second Affiliated Hospital & Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou 325027, China; 2.Department of Pediatric Nephrology, the Second Affiliated Hospital & Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou 325027, China
ZHANG Yiqian,ZHUANG Zhizhi,SHEN Meng, et al. The regulatory role of RanBPM in renal tubular sodium chloride cotransporter and its effect on ERK1/2 signaling pathway[J]. JOURNAL OF WEZHOU MEDICAL UNIVERSITY, 2020, 50(7): 553-556，562.
摘要目的：观察Ran结合蛋白M（RanBPM）对肾小管钠-氯共同转运子（NCC）蛋白表达及相关ERK1/2信号通路蛋白表达的影响。方法：免疫共沉淀检测RanBPM蛋白与WNK4蛋白是否存在直接相互作用；观察转染RanBPM质粒对EGF诱导细胞ERK1/2磷酸化的影响，通过转染质粒过表达RanBPM蛋白或siRNA抑制RanBPM基因，免疫蛋白印迹法检测细胞内源性NCC蛋白含量和ERK1/2磷酸化水平的变化。结果：免疫共沉淀实验证实WNK4和RanBPM之间存在直接的相互作用。RanBPM可以抑制EGF诱导的ERK1/2磷酸化。RanBPM过表达可使细胞ERK1/2磷酸化减少（1.000±0.074 vs. 0.275±0.041，P＜0.01），而NCC蛋白表达增加（1.000±0.115 vs. 1.470±0.105，P＜0.01）。siRNA沉默RanBPM基因后，ERK1/2磷酸化增加（1.000±0.194 vs. 2.301±0.220，P＜0.01），NCC蛋白表达下降（1.000±0.223 vs. 0.556±0.132，P＜0.01）。转染RanBPM可阻止WNK4对ERK1/2信号通路的影响和对NCC蛋白表达的抑制作用。结论：RanBPM通过与WNK4相互作用，影响ERK1/2信号通路对NCC蛋白表达的调控作用。
Abstract：Objective: To observe the effect of Ran binding protein M (RanBPM) on the expression of NCC protein in renal tubules and the change of ERK1/2 signaling pathway protein. Methods: Immunocoprecipitation was used to detect whether there was direct interaction between RanBPM and WNK4 protein. The effect of transfection of RanBPM plasmid on ERK1/2 phosphorylation induced by EGF was observed. RanBPM gene was inhibited by overexpression of RanBPM protein or siRNA. The content of endogenous NCC protein and the level of ERK1/2 phosphorylation were detected by Western blot. The effect of RanBPM on WNK4 regulation of NCC by ERK1/2 phosphorylation was explored before and after transfection. Results: Immunocoprecipitation confirmed that there was a direct interaction between WNK4 and RanBPM. RanBPM could inhibit EGF induced ERK1/2 phosphorylation. Overexpression of RanBPM reduced ERK1/2 phosphorylation (1.000±0.074 vs. 0.275± 0.041, P<0.01) and increased NCC protein expression (1.000±0.115 vs. 1.470±0.105, P<0.01). After siRNA silenced RanBPM gene, ERK1/2 phosphorylation increased (1.000±0.194 vs. 2.301±0.220, P<0.01), resulting in decreased NCC protein expression (1.000±0.223 vs. 0.556±0.132, P<0.01). When RanBPM was transfected, WNK4 inhibited NCC protein expression and no longer affected ERK1/2 phosphorylation. Conclusion: RanBPM interacted with WNK4 affects the regulation of ERK1/2 signaling pathway in NCC protein expression.
