Regulation of RhoA signaling by GEF-H1 in cell fate and migration

• Main Authors: Yuan-Chen Chang, 張元貞
• Other Authors: 張智芬
• Format: Others
• Language: en_US
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/04682958075538475093

博士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 96 === RhoA is known as a signaling node to regulate diverse functions. Myeloid cells in suspension often contain elevated level of RhoA activity. In the erythroblastic D2 cell line, phorbol ester PMA treatment induces half of D2 cells adhesion and differentiates into monocyte/macrophage-like morphology with downregulated RhoA activity, while the other population remains in suspension with elevated RhoA activity and undergoes apoptosis. Since RhoA activity is closely associated with PMA-induced cell death and differentiation, I aimed to clarify the mechanism responsible for differential regulation of RhoA in D2 cells upon PMA stimulation. I found GEF-H1 specifically interacted with RhoA in D2 cells. GEF-H1 is a microtubule-associated guanine nucleotide exchange factor activating RhoA upon release from microtubules. In PMA-induced attached cells, GEF-H1 became associated with organized microtubules as an inactive form; whereas in PMA-induced suspension cells, GEF-H1 remained in the cytosol freely to activate RhoA. Knockdown of GEF-H1 significantly decreased PMA-induced apoptosis, while overexpression of active form of GEF-H1 increased it in D2 cells. Thus, subcellular localization of GEF-H1 specifies differential RhoA activity in determining cell fate upon PMA stimulation. Reorganized interactions between microtubules and actin attribute to motility of polarized cells in adherent monolayer culture. RhoA also regulates the assembly of contractile stress fibers. Nocodazole can induce microtubule disassembly to release GEF-H1. siRNA-mediated depletion of GEF-H1 prevented nocodazole-induced HeLa cell contraction and RhoA/ROCK-mediated phosphorylation of MLC. Reintroduction of siRNA-resistant GEF-H1 into GEF-H1 depleted cells rescued nocodazole-induced RhoA activation and contractility. Therefore, GEF-H1/RhoA/ROCK/MLC signaling is required for nocodazole-induced HeLa cell contraction. In addition, depletion of GEF-H1 reduced directional cell motility depending on RhoA. GEF-H1 depletion not only resulted in the irregular behavior of membrane ruffling with differential dynamics of focal adhesion at the leading edge of migrating cells but also reduced tyrosine phosphorylation of FAK and paxillin during migration. Thus, GEF-H1 might control cell motility via RhoA to influence focal adhesion turnover at the front. In summary, the present study reveals the role of microtubule-regulated GEF-H1 in controlling RhoA activation, which in turn affects cell fate and migration behaviors.