RESUMO
Cyclic stretch is an artificial model of mechanical force loading, which induces the reorientation of vascular endothelial cells and their stress fibers in a direction perpendicular to the stretch axis. Rho family GTPases are crucial for cyclic-stretch-induced endothelial cell reorientation; however, the mechanism underlying stretch-induced activation of Rho family GTPases is unknown. A screen of short hairpin RNAs targeting 63 Rho guanine nucleotide exchange factors (Rho-GEFs) revealed that at least 11 Rho-GEFs Abr, alsin, ARHGEF10, Bcr, GEF-H1 (also known as ARHGEF2), LARG (also known as ARHGEF12), p190RhoGEF (also known as ARHGEF28), PLEKHG1, P-REX2, Solo (also known as ARHGEF40) and α-PIX (also known as ARHGEF6) which specifically or broadly target RhoA, Rac1 and/or Cdc42, are involved in cyclic-stretch-induced perpendicular reorientation of endothelial cells. Overexpression of Solo induced RhoA activation and F-actin accumulation at cell-cell and cell-substrate adhesion sites. Knockdown of Solo suppressed cyclic-stretch- or tensile-force-induced RhoA activation. Moreover, knockdown of Solo significantly reduced cyclic-stretch-induced perpendicular reorientation of endothelial cells when cells were cultured at high density, but not when they were cultured at low density or pretreated with EGTA or VE-cadherin-targeting small interfering RNAs. These results suggest that Solo is involved in cell-cell-adhesion-mediated mechanical signal transduction during cyclic-stretch-induced endothelial cell reorientation.
Assuntos
Células Endoteliais da Veia Umbilical Humana/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Estresse Mecânico , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Antígenos CD/metabolismo , Caderinas/metabolismo , Adesão Celular/efeitos dos fármacos , Contagem de Células , Ácido Egtázico/farmacologia , Ativação Enzimática/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , RNA Interferente Pequeno/metabolismo , Fibras de Estresse/efeitos dos fármacos , Fibras de Estresse/metabolismo , Resistência à Tração/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Short hairpin RNAs targeting 66 Rho-GEFs were screened for inhibition of chemotaxis. Six Rho-GEFs (p63RhoGEF, Trio, Duet, Net1, Frabin/Fgd4, and AAH33666) were found to be required for the serum-induced chemotactic migration of MDA-MB-231 human breast carcinoma cells. Knockdown of p63RhoGEF suppressed serum-induced RhoA activation and chemotaxis and caused the aberrant formation of multiple lamellipodial protrusions after serum stimulation while control cells formed a single polarized lamellipodium. These results indicate that p63RhoGEF plays a crucial role in serum-induced chemotaxis by limiting lamellipodial protrusion to one direction via RhoA activation.
