Activation of the Piezo1 channel stimulates protein kinase D and migration in human aortic endothelial cells

Piezo 1 is an evolutionally conserved mechanosensitive ion channel implicated in the regulation of development, differentiation, and growth of multiple tissues. Despite its importance, the pathways induced downstream of Piezo1 activation remain incompletely defined. Here, we report that the selective Piezo1 agonists, Yoda1, Yaddle1 and Yoda2 stimulate PKD family activation in a concentration-dependent manner, in human aortic endothelial cells (ECs), as shown by increase in PKD phosphorylation at Ser 910 , an auto-phosphorylation site in the C-terminus, and Ser 738/742 located in the activation loop. Depletion of extracellular Ca 2+ by EGTA abolished PKD phosphorylation stimulated by Piezo1 agonists and exposure to the Ca 2+ ionophore ionomycin potently stimulated PKD. The PKD activation induced by Yoda1, Yaddle1 or Yoda2 was prevented by inhibitors of PKDs (CRT0066101) and PKCs (Go6983) and abrogated by siRNA-mediated knock down of Piezo1. Treatment of ECs with H-067047 or apyrase did not alter the increase in the phosphorylation of PKD at either Ser 910 or Ser 738/742 induced by Yoda1, implying that stimulation of Piezo1 induces PKD activation independently from TRPV4 and autocrine ATP signaling. Exposure of ECs to Yoda1 increased the histone deacetylase (HDAC)7 phosphorylation and migration of ECs into the denuded area of the monolayer, as shown using a scratch wound assay. Treatment of ECs with CRT0066101 prevented HDAC7 phosphorylation and migration of these cells induced by Yoda1, suggesting that Yoda1-stimulated Piezo1 promotes EC migration through a PKD. Our results identify PKD as a novel downstream mediator of effects produced by agonist-induced activation of the Piezo1 mechanosensitive channel in ECs.