| 摘要: | 中文摘要
更年期前，婦女罹患動脈粥狀硬化心臟血管疾病死亡率較之同年紀的男生為低，而在更年期後其患病率則直追同年齡的男性。更年期後的婦女若服用動情素則其心血管疾病的患病率則有改善的現象，且同時服用動情素 (estrogen; E2) 與助孕酮 (progesterone; P4) 者，又較之只用動情素者死亡率低。此現象意味著助孕酮在抗動脈粥狀硬化中可能具有保護作用。
動脈粥狀硬化的形成關鍵步驟是血管平滑肌細胞的增生與移行。過去本實驗室已經證明助孕酮在生理濃度下會抑制培養中的老鼠動脈平滑肌細胞的細胞增生與細胞移行。助孕酮會藉由增加p21及p27蛋白的表現，而增加cyclin-dependent kinase (CDK)2-p21與CDK2-p27複合體的形成，進而抑制CDK2激酶的活性，最後造成細胞週期停滯而 抑制老鼠動脈平滑肌細胞細胞增生。助孕酮誘導老鼠動脈平滑肌細胞的細胞移行抑制作用，導因於活化cSrc所引發的RhoA活性受到抑制。而本論文的研究主題為探討助孕酮透過何種訊息傳導路徑而使RhoA活性降低，進而誘導老鼠動脈平滑肌細胞的細胞移行抑制。
本論文研究中, 助孕酮在生理濃度下 (5-500 nmole/L)，隨著加藥時間增長會持續誘導老鼠動脈平滑肌細胞的p27蛋白質量增加，然而利用p27小干擾型RNA (p27 siRNA) 刪除 (knockdown) p27的基因表現時，則助孕酮所誘導抑制老鼠動脈平滑肌細胞移行的現象消失，顯示助孕酮可能是透過增加p27蛋白的表現，進而抑制血管平滑肌細胞的移行作用。利用西方點墨法(Western blotting)來探討參與助孕酮誘導動脈平滑肌細胞移行 抑制的分子機轉。我們的實驗證實助孕酮可透過活化cSrc/AKT/ERK2/p38/NF-κB訊息路徑，誘導p27蛋白質量增加。
利用免疫沉澱技術 (immunoprecipitation)，我們觀察到助孕酮可增加老鼠動脈平滑肌細胞內p27與RhoA複合體的形成。我們的實驗同時證明了助孕酮會增加細胞核中KIS的酵素表現，而促進p27第十個位置胺基酸絲氨酸磷酸化 (p-p27S10)，此磷酸化會誘導p27由細胞核移至細胞質中，接著增加了p27與RhoA複合體的形成，接著使p27 與RhoA產生泛素化 (ubiquitination)，最後造成p27 與RhoA的分解，而降低了血管平滑肌細胞的爬行能力。助孕酮對於血管平滑肌細胞移行的抑制，是組合細胞內連續事件機制，包括1) 啟動cSrc/AKT/ERK2/p38/NF-κB訊息路徑活化，2) 增加細胞核中p27與KIS複合體形成，3) 誘導細胞核中p27第十個位置胺基酸絲氨酸磷酸化，4) 增加p27移至細胞質中，且在細胞質中形成p27與RhoA的複合體，5) 透過泛素蛋白瓦解體路徑 (ubiquitin proteasome pathway)瓦解p27與RhoA。這些發現解釋助孕酮誘導老鼠動脈平滑肌細胞移行抑制的分子作用機制。
Abstract
Mortality from atherosclerotic cardiovascular disease is lower in premenopausal women than in age-matched men. However, the incidence of cardiovascular disease in postmenopausal women gradually approaches that in age-matched men, suggesting that female sex hormones might have a cardiovascular protective effect in premenopausal women. A prospective study supports a cardiovascular protective effect for estrogens by showing that estrogen replacement therapy reduces both the incidence and mortality from cardiovascular disease in postmenopausal women. Moreover, it has been shown that the mortality is also lower in postmenopausal women who take E2 and P4 together rather than E2 alone, suggesting that P4 may participate in the hormonal protective effect against atherosclerosis.
Previously, our laboratory has demonstrated that P4 at physiologic levels inhibited proliferation and migration of cultured rat artic smooth muscle cells (RASMCs). P4 increases the levels of p21and p27 protein and the formations of CDK2-p21and CDK2-p27complex of induces cell cycle arrest in RASMCs through inhibiting the CDK2 activity, which in turn causes reduction of the CDK2 activity, finally inducing cell cycle arrest. Moreover, P4-induced migration inhibition in RASMCs results from Ras homolog gene family, memberA (RhoA) inactivation mediated by cSrc activation. The aim of this dissertation study is to investigate the molecular mechanisms underlying P4-induced RhoA inactivation in RASMCs.
In this dissertation study, our data show that P4 at physiologic levels (5-500 n mole/L) time-dependently induced increases on the levels of p27 protein in RASMCs. Knock-down of the p27 expression using the p27-siRNA abolished the P4- induced p27decreases of migration in RASMCs. Using Western blot analyses to illustrate the signaling pathway involved in the P4-induced migration inhibition in RASMCs, our data suggest that P4 increased the level of p27 protein through activating the cSrc/AKT/ERK2/p38/NF-κB signaling pathway.
Using immunoprecipitation technique, we observed that P4 increased the formation of p27-RhoA complex in RASMCs. We also demonstrated that P4 increased phosphorylation of p27 at Ser10 in the nucleus, subsequently inducing p27 translocation from the nucleus to the cytosol, which in turn increased formation of the p27-RhoA complex and ubiquitination of p27and RhoA, eventually causing degradation of p27 and RhoA and migration inhibition in RASMCs.
Taken together, our investigation of P4-induced migration inhibition in RASMCs showed a sequence of associated intracellular events that included (1) activating the cSrc/AKT/ERK2/p38/NF-κB signaling pathway, (2) increasing the formation of KIS-p27 complex in the nucleus, (3) increasing the phosphorylation of nuclear p27 at Ser10, (4) inducing cytosolic translocation of p27 and formation of the p27-RhoA complex in the cytosol, and (5) causing degradation of p27 and RhoA through the ubiquitin-proteasome pathway. These findings highlight the molecular mechanisms underlying P4-induced migration inhibition in RASMCs. |
