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 center dot kg(-1)center dot 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 G(0)/G(1) phase and prevented transition from G(1) to S phase, whereas the WNK1 overexpression accelerated cell cycle transition from G(1) 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 G(0)/G(1) to S phase and proliferation through the PI3K-Akt signaling pathway.