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研究生(外文):Chun-Chin Chen
論文名稱(外文):Functional studies on the role of p53 downstream gene Rad in cell migration
指導教授(外文):Fung-Fang Wang
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Rad屬於RGK small GTPase家族成員之一,其功能被廣泛認為與調控鈣離子通道及細胞骨架重組有關。先前文獻已指出Rad具有抑制Rho-ROK訊號傳遞的能力,亦能降低血管平滑肌細胞的移動力。因此在本研究中,我們意圖探討Rad影響細胞移動的可能機制。我們發現在U2OS細胞中表現Rad能誘導細胞轉變成多極性外觀,同時會減少細胞中stress fiber的生成,代表Rad可能對actin細胞骨架產生某些影響。在U2OS及CL1-5細胞中大量表現Rad時能抑制細胞對血清產生的趨化性移動,且此能力需透過Rad對14-3-3蛋白的結合來達成。我們進一步發現Rad會全面性減少actin調節因子cofilin上的抑制性磷酸化,可能因此干擾了它在細胞中活化所需的分布特異性。由於文獻已指出cofilin pathway主要調控方向性感知而非移動速度,因此我們的實驗顯示Rad的抑制效果主要在對血清的趨化性移動上而非隨機性移動,且在wound-healing實驗中不會影響癒合速率。此外在先前的研究中,我們已得知Rad為一p53下游基因,因此我們欲深入探討p53是否可能透過Rad來調控細胞移動。實驗結果顯示,在U2OS細胞中大量表現p53確實會造成cofilin的去磷酸化及抑制細胞的趨化性移動,而將內生性Rad 同時knockdown後則可以抗衡這些現象。在H1299細胞中活化一溫度敏感性p53突變蛋白亦能造成cofilin的去磷酸化,且與p53誘導的轉錄作用有關。這些結果證明,p53可能藉由轉錄出Rad GTPase來調控細胞中的cofilin pathway,進而影響了腫瘤細胞的方向性感知與趨化性移動能力。
Rad, a member of RGK small GTPase family, is known to participate in the regulation of cytoskeletal remodeling, and has been identified as a p53-upregulated gene in our lab. Rad is capable of suppressing Rho-ROK pathway and inhibiting migration of vascular smooth muscle cells. In this study, we elucidate the underlying mechanism of Rad-mediated migration inhibition. Expression of Rad in U2OS cells induced a multipolar phenotype and attenuated stress fiber formation, suggesting its involvement in actin cytoskeleton regulation. Overexpression of Rad suppressed directional migration of U2OS as well as CL1-5 cells in transwell assay, and this function was partially dependent on its 14-3-3 binding activity. Moreover, Rad globally downregulated the inhibitory phosphorylation of cofilin, which is a known regulator of actin dynamics. Consistent with previous findings that disrupting the cofilin pathway impaired directional sensing but not moving speed, Rad inhibited serum-induced chemotaxis without significantly affecting random migration in transwell assay, and had no impact on gap closing in wound-healing assay. Overexpression of p53 in U2OS cells promoted cofilin dephosphorylation and inhibited directional migration, whereas concomitant knockdown of endogenous Rad counteracted the p53-mediated effects. The cofilin dephosphorylation due to functional activation of tsp53 in H1299 stable clones was abolished by treatment with actinomycin D, suggesting that transcriptional activity was required. These results indicate that p53 is involved in the regulation of cell motility through transcriptional induction of Rad, which, by interfering with the cofilin pathway, inhibits directional sensing and chemotaxis of tumor cells.
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