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 VRCC-induced proliferative signaling pathway in A10 vascular smooth muscle cells in vitro.A10 cells were exposed to a hypotonic challenge (225 mosmol·kg-1·H2O),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.