- 學位論文
葉酸在以同半胱胺酸處理過的老鼠主動脈平滑肌細胞的 抗細胞遷移作用的分子機轉
Molecular Mechanisms Underlying Folate’s Antimigratory Effects on Homocysteine-Challenged Rat Aortic Smooth Muscle Cells
摘要
同半胱胺酸 (homocysteine) 是一含硫的氨基酸，它少量存在血液中。當攝取含有甲硫胺酸 (methionine) 的動物性蛋白質時，甲硫胺酸會去甲基形成同半胱胺酸。過多的同半胱胺酸會去硫基代謝成胱硫醚 (cystathionine) 或再甲基化形成甲硫胺酸。自1969年McCully等人在兩位患有同半胱胺酸尿症 (homocystinuria) 的病童發現早發性的粥狀動脈硬化 (premature atherosclerosis) ，並認為其與體內較高的同半胱胺酸濃度有關。從此同半胱胺酸與血管硬化的疾病就被廣泛的研究。近二十年來高同半胱胺酸血症(hyperhomocystenemia) 已被認為是血管疾患的獨立危險因子，它發生的原因最常見的為葉酸 (folic acid) 缺乏、基因異常及慢性腎衰竭。目前臨床上是以補充葉酸來治療高同半胱胺酸血症。 血管壁及血管平滑肌細胞型態的改變是粥狀動脈瘤 (atherosis) 形成發展的重點。粥狀動脈瘤的發展過程中，血管平滑肌細胞的一個主要的功能是細胞遷移 (cell migration) 。 細胞遷徙是一個複雜及統合的現象，在許多生理或病理的步驟中扮演重要角色。因此，我們從與細胞遷徙相關分子作用機轉著手，探討葉酸在加入同半胱胺酸培養的主動脈平滑肌細胞(aortic smooth muscle cell) 的抗趨化性的分子機轉，嘗試釐清葉酸在治療同半胱胺酸引起的血管疾患所扮演的角色。 AKT 亦被稱為 PKB，調節重要的細胞功能，例如：細胞遷移、細胞繁殖、細胞分化、凋亡及代謝。AKT 影響各種促血管增生或抗血管增生因子的表現及活性。微小 GTP 組合蛋白- Rho，在生長因子 (growth factors)刺激下會調節粘著斑 (focal adhesion) 與肌動應力纖維 (actin stress fibers) 的組裝。活化的AKT會藉由磷酸化細胞週期蛋白激酶抑制劑(cyclin dependent kinase inhibitors) p21 及 p27，促進細胞週期的進展。p21 及 p27被磷酸化後會離開細胞核進入細胞質，經由 Rho 路徑調節細胞遷移。Paxillin 是一種 integrin 的組合蛋白，當在酪胺酸31及118 (Tyr31/118) 發生磷酸化時，會抑制 RhoA 與細胞附著及遷移相關的活性。粘著斑激素 (focal adhesion kinase，FAK) 一種非接受器蛋白，一種可見於粘著斑的酪胺酸激素，策動粘著斑複合物的形成。FAK 可藉由活化 p190RhoGAP 抑制 RhoA 的活性。 我們在同半胱胺酸處理過的老鼠主動脈平滑肌細胞中，對 p21/p27，AKT 及 RhoA 以功能獲得(gain-of-function) 或功能失去 (loss-of-function) 的實驗方法，評估上述與細胞遷移相關的蛋白質，探討葉酸對同半胱胺酸挑戰的保護機轉。 在老鼠的主動脈平滑肌細胞，葉酸降低同半胱胺酸促進細胞遷徙的作用，來自降低 AKT1、FAK、paxillin 及 p190RhoGAP 的活化及磷酸化，與增加 RhoA 在細胞膜上的分布。是 AKT1 並非 AKT2 的功能獲得，引發 p21 與 p27 的磷酸化，並增加 p21 與 p27 在細胞質中的分布，如同同半胱胺酸的作用。DNAKT 則類似葉酸，會抵消上述的作用。同時，我們也發現 AKT 是 FAK 活化的上游標的。並藉由過度表現 CARhoA，證實 RhoA 的活化參與葉酸在老鼠的主動脈平滑肌細胞的抗細胞遷徙的作用；加入 Y27632 及 DNRhoA 則抵消了抗細胞遷徙的作用，再度證實RhoA 的參與。 這個研究是第一次證實葉酸在同半胱胺酸處理過的老鼠主動脈平滑肌細胞的抗細胞遷徙的作用。並描繪出葉酸在保護同半胱胺酸引起的血管硬化的分子機轉。
並列摘要
Homocysteine is a sulfur-containing amino acid , it exits in the blood in small quantities. It is derived from Methionine in diets. It is either further metabolized to cystathionine by transsulfuration or remethylated to form methionine again . In 1969, McCully reported premature atherosclerosis in two children with homocysteinuria and first linked elevated hyperhomocysteinemia to atherosclerosis. Since then, an association between homocysteine and atherosclerotic vascular diseases has been extensively studied. In the last two decades, hyperhomocysteinaemia has been proven to be an independent risk factor of many cardiovascular diseases, such as AMI, CVA, and deep vein thrombosis. Hyperhomocysteinaemia can be induced by the congenital deficiency of cystathionine ?? synthase, or the acquired deficiencies of vitamins B6, B12, and folate, which are the co-factors for homocystein metabolism. Therapeutically, plasma homocysteine levels are readily managed by the administration of folic acid. Changes in the vessel wall and in vascular smooth muscle cell biology are keys to plaque formation and development. One primary VSMC function in plaque development is cell migration. Cell migration is a complex and integrated phenomenon that plays a central role in a variety of physiological and pathological processes. Therefore, we look into how folic acid can help protect vessels by reversing the molecular mechanisms of cell migration caused by Homocystinemia. AKT, also known as Protein kinase B (PKB), regulates essential cellular functions such as migration, proliferation, differentiation, apoptosis, and metabolism. AKT influences the expression and/or activity of various pro- and anti-angiogenic factors. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Active AKT promates cell cycle progression via the phosphorylation of cyclin dependent kinase inhibitors p21 and p27.Phosphrylated p21 and p27 leaves nucleus into cytoplasma and regulates cell migration via the Rho pathway. Paxillin, an integrin-assembly protein, has four major tyrosine phosphorylation sites, and the phosphorylation of Tyr31 and Tyr118 correlates with cell migration and suppresses Rho A activity . Focal adhesion kinase (FAK), a non-receptor protein-tyrosine kinase found in focal adhesions, mediates the formation of the focal adhesion complex. Suppression of RhoA activity by focal adhesion kinase-induced activation of p190RhoGAP. Rat aortic smooth muscule cells (RASMCs) were cultured in vitro. Linear wound and microchemotaxis assays of migration were performed in the presence of homocysteine with and without folate. Then we evaluated the protective mechanisms of folate against homocysteine challenge according to the gain-of-function and loss-of-function of p21/p27, AKT, and RhoA in homocysteine-treated RASMCs. Folic acid can reverse the effect of cytoplasmic P21 and P27 increased by AKT, the effect of integrin-assembly proteins (FAK, paxillin, and p190RhoGAP) induced by AKT and promotes the activity of Rho A in homocysteine-treated aortic SMCs, so folic acid suppresses cell migration in homocysteine-treated aortic SMCs. The study is the first to demonstrate the antimigratory effect of folate on homocysteine-challenged RASMC and to delineate the mechanisms underlying folate-mediated protection against the proatherosclerotic effects of homocysteine.
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