Piezo2 mechanically regulate scleral fibroblast differentiation by activating relA/RhoA pathway.

Abstract

PURPOSE: Biomechanical property changes and fibroblasts differentiation contribute to myopia scleral remodeling. This study aimed to investigate the role of Piezo2 in form-deprivation myopia (FDM) and delineat its mechanism in regulating α-SMA expression. METHODS: FDM model was established in 2-week-old pigmented guinea pigs. Piezo2 expressions in the sclera were analyzed using qRT-PCR and western blotting. To elucidate the underlying mechanism, scleral fibroblasts were subjected to a cyclic stretch regimen (sine wave, 4% equibiaxial stretching amplitude, 0.5 Hz, 24-h duration) using FX-5000™ system. Levels of RhoA, relA, Calpain and α-SMA were assessed by western blotting following Piezo2 or relA siRNA knockdown. α-SMA-positive myofibroblasts were visualized by immunofluorescence. Fibroblast contraction behaviors were determined by three-dimensional collagen gels. The transcriptional regulation of RhoA by relA was examined with a dual-luciferase reporter assay. RESULTS: Guinea pig samples revealed an increased Piezo2 in myopia sclera. In cultured scleral fibroblast, Piezo2 expression was increased in response to 4% cyclic stretch, along with the increased expressions of RhoA, relA, α-SMA compared to the static controls. Additionally, Cainflux and calpain activity showed significantly increased in response to 4% cyclic stretch. Conversely, inhibition of Piezo2 or Cainflux abolished the stretch-induced upregulation of RhoA, relA and α-SMA. SiRelA reduced the expression of RhoA, and luciferase reporter assay further confirmed relA binding to the promoter region of RhoA. CONCLUSIONS: Piezo2 acts as a mechanotransduction ion channel in myopia sclera, by modulating Ca-dependent relA/RhoA pathway. This mechanoresponsive signaling is essential for fibroblast to myofibroblast differentiation.