WNK1 is required for proliferation induced by hypotonic challenge in rat vascular smooth muscle cells

Authors: Ya-juan ZHANG1, Hua-qing ZHENG1, Bao-yi CHEN1, Lu SUN1, Ming-ming MA1, Guan-lei WANG1, Yong-yuan GUAN1
1 Department of Pharmacology, Cardiac and Cerebral Vascular Research Center, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou 510080, China
Corresponding to: Yong-yuan GUAN:,
DOI: 10.1038/aps.2017.56
Received: 4 January 2017
Accepted: 7 April 2017
Advance online: 3 August 2017


Hypotonic challenge evoked vascular cell proliferation through activation of volume-regulated Cl channel (VRCC), leading to a decrease in the intracellular Cl concentration ([Cl]i). We hypothesize that the decrease in [Cl]i may activate one or several Cl-sensitive kinases, resulting in a subsequent signaling cascade. In this study we demonstrated that WNK1, a Cl-sensitive kinase, was involved in VRCCinduced proliferative signaling pathway in A10 vascular smooth muscle cells in vitro. A10 cells were exposed to a hypotonic challenge (225 mosmol·kg-1·H20), which caused significantly increase in WNK1 phosphorylation without altering WNK1 protein expression. WNK1 overexpression significantly increased hypotonic-induced A10 cell proliferation, whereas silencing of WNK1 caused an opposite action. WNK1 mutation did not affect hypotonic-induced WNK1 phosphorylation and cell proliferation. Silencing of WNK1 caused cell cycle arrest at G0/G1 phase and prevented transition from G1 to S phase, whereas the WNK1 overexpression accelerated cell cycle transition from G1 to S phase. Silencing of WNK1 significantly inhibited cyclin D1/cyclin E1 expression and increased p27KIP/p21CIP expression. WNK1 overexpression significantly increased cyclin D1/cyclin E1 expression and reduced p27KIP/p21CIP expression. In addition, WNK1 knockdown or overexpression significantly attenuated or increased the hypotonic-induced phosphorylation of Akt and PI3K respectively. In conclusion, the reduction in [Cl]i caused by hypotonic challenge-induced VRCC opening evokes WNK1 phosphorylation in A10 VSMCs, which mediates cell cycle transition from G0/G1 to S phase and proliferation through the PI3K-Akt signaling pathway.
Keywords: vascular smooth muscle cells; A10 cells; cell proliferation; hypotonic solution; WNK1; cell cycle regulators; PI3K-Akt signaling