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

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·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 p27^KIP/p21^CIP 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.