GEF-H1調控RhoA訊息傳遞對細胞命運決定及移動力之探討

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

張元貞(Yuan-Chen Chang)

指導教授 ： 張智芬

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/博士(2008年)

https://doi.org/10.6342/NTU.2008.00147

摘要

RhoA是多種訊息傳遞的中樞，而懸浮性的骨髓細胞常含有高量的活化態RhoA。當D2細胞受PMA刺激時，可誘使半數細胞RhoA活性降低並貼附於培養皿而分化成類單核球/巨噬細胞球樣的形態，另一部分維持懸浮狀態的細胞則仍具有高的RhoA活性但卻走向凋亡途徑。由於D2細胞在受到PMA誘發而走向分化或凋亡命運，與RhoA活性高低息息相關，故此研究旨在探討PMA誘發的分化及凋亡的細胞中，調控RhoA活性的機制。我在D2細胞中找到具有和RhoA專一性結合的GEF-H1蛋白，此具有活化RhoA的GEF-H1會和細胞骨架微管結合，當GEF-H1從微管上釋放後才具有活化RhoA的能力。在PMA誘發的貼附細胞中，GEF-H1會和微管結合而不被活化；至於PMA誘發的懸浮細胞，GEF-H1則分布在細胞質中可活化RhoA。當細胞中GEF-H1的表現受RNAi抑制時，大幅減少了PMA所引發的細胞凋亡；而過量表現不具和微管結合能力之持續活化態GEF-H1，則促進了細胞在PMA誘發下走向凋亡。因此，PMA刺激影響了細胞中GEF-H1的分布情形，進而調控RhoA活性的高低使細胞走向不同的命運。
在單層培養的附著性細胞中，藉由微管和肌動蛋白間交互作用產生極性而移動，RhoA亦調控著具收縮力的stress fibers之形成。藥物nocodazole會促使細胞中的微管瓦解而釋放GEF-H1。以siRNA抑制內生性GEF-H1表現時，會抑制nocodazole所誘發之HeLa細胞收縮及由RhoA/ROCK所調控的肌凝蛋白調節輕鏈(MLC)之磷酸化；而加入抗siRNA抑制的質體使再度表現GEF-H1，則可恢復nocodazole誘導之RhoA活化及細胞收縮能力。這些結果確立了GEF-H1/RhoA/ROCK/MLC為nocodazole誘發HeLa細胞收縮之必要訊息傳遞途徑。此外，抑制GEF-H1表現會經由影響活化RhoA情形，而降低HeLa細胞方向性移動的能力。移動中細胞前緣則因缺乏GEF-H1而產生不規則的膜波動(membrane ruffling)，並影響focal adhesion的動態變化。當GEF-H1表現受抑制時，移動中的細胞其focal adhesion kinase(FAK)的活化及下游paxillin的酪胺酸磷酸化亦隨之下降。因此，GEF-H1可藉由RhoA訊息傳遞，影響細胞前緣之focal adhesion的動態變化而影響移動力。
總括本研究證實，藉由微管的動態變化調控GEF-H1活化RhoA訊息的能力，進而影響細胞的命運決定與移動力。

關鍵字

細胞命運決定 ； 移動力

並列摘要

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.

並列關鍵字

cell fate ； migration

參考文獻

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