中間絲巢蛋白於大鼠血管平滑肌細胞之表現、調控與功能之研究

血管平滑肌細胞之型態由收縮型轉變為合成型為動脈粥狀硬化病變過程一重要特徵之ㄧ。雖然目前已有許多研究報導指出可能的細胞激素或是生長因子參與在此一路徑中。但是，由於缺乏早期的去分化指標，因此確切的影響分子仍不清楚。於本篇論文中，我們發現一個中間絲巢蛋白會表現於大鼠初級培養之血管平滑肌細胞，並代表著分泌型之細胞型態。而其表現會隨著這些細胞經去除血清後所造成的細胞重新分化而消失。雖然巢蛋白常被利用來當作細胞早期分化指標，但其基因調控機制與其功能仍不清楚。因此，本篇論文的目的在於研究巢蛋白在大鼠初級培養之血管平滑肌細胞中的表現、基因調控方式與功能。首先，我們利用西方墨點法分析，發現表皮生長因子可經由新核醣核酸與新蛋白質形成之路徑來調控巢蛋白表現。進一步的訊息傳遞路徑分析結果顯示，表皮生長因子可經由Ras-Raf-ERK此一路徑來誘導巢蛋白表現。另一方面，我們亦發現凝血酶經活化其受器PAR-1所轉活化之表皮生長因子受器路徑亦可調控大鼠血管平滑肌細胞巢蛋白之表現。而由PAR-1所活化之c-Src在此一路徑中扮演著兩種重要之角色︰(一) 經由細胞內的路徑直接磷酸化表皮生長因子受器；(二) 經由細胞外路徑活化MMP-2促使HB-EGF之切割而活化表皮生長因子受器。經轉活化誘導之表皮生長因子受器路徑會接著活化下游Ras-Raf-ERK路徑，來調控大鼠巢蛋白之表現。此外，電泳遷移率檢測實驗結果顯示，凝血酶需透過活化一個轉錄因子Sp1來誘導大鼠巢蛋白之形成。最後一部份，我們探討巢蛋白在大鼠血管平滑肌細胞之功能。巢蛋白經干擾性核醣核酸技術去除後，會減緩由凝血酶所誘導之細胞增生，此一結果顯示，凝血酶所誘導之巢蛋白形成與細胞增生兩項特徵皆會經由轉活化表皮生長因子受器路徑。進一步的去氧核醣核酸階梯化分析與流式細胞技術結果顯示，去除巢蛋白會強化經過氧化氫所導致之細胞凋零。此外，在250 μM的過氧化氫處理下，只有去除巢蛋白的血管平滑肌細胞其caspase-9、caspase-3及PARP會被活化。因此，顯示巢蛋白抑制在caspase活化的上游。目前已知，表皮生長因子在大鼠血管平滑肌細胞可當作一存活因子。在本篇論文中，我們亦證實去除巢蛋白會消除表皮生長因子的細胞保護作用。總而言之，這些結果顯示在大鼠血管皮滑肌細胞中，巢蛋白可能在表皮生長因子受器活化所傳遞的細胞增生與細胞保護作用扮演著一極重要之角色。

關鍵字

巢蛋白；表皮生長因子；凝血酶血管平滑肌細胞；轉活化表皮生長因子受器；細胞增生；細胞凋零； caspase

並列摘要

The contractile-synthetic phenotypic modulation of vascular smooth muscle cells (VSMCs) is a key event during the progression of atherosclerosis. Although many studies possibly implicated cytokines and growth factors in this process, critical factors affecting the VSMC phenotype remain unclear due to a lack of identification of early de-differentiation markers. In this study, we show that nestin, an intermediate filament protein, is expressed in primary cultures of rat VSMCs, which represents the synthetic phenotype, and its expression is diminished as these cells re-differentiate following serum deprivation. However, regulation of nestin gene expression and its function have never been reported despite its common usage as an early differentiation marker. Thus, the purposes of this study were to investigate the expression, gene regulation, and function of nestin in primary cultures of rat VSMCs. By immunoblotting, we show that nestin expression is regulated by epidermal growth factor (EGF) via de novo RNA and protein syntheses. Furthermore, signaling analyses revealed that EGF-induced nestin re-expression is mediated by activation of the Ras-Raf-ERK signaling axis. On the other hand, we also show that nestin can be regulated by thrombin/PAR-1-mediated EGFR transactivation in serum-deprived primary cultures of rat VSMCs. PAR-1-induced c-Src plays a critical role through two routes: direct intracellular phosphorylation of the EGFR and extracellular activation of the matrix metalloproteinase (MMP)-2-mediated shedding of HB-EGF. The transactivated EGFR then leads to the downstream Ras-Raf-ERK signaling axis. In addition, EMSA experiments showed that the transcriptional factor, Sp1, is critical for thrombin-induced nestin expression in rat VSMCs. Furthermore, RNA interference (RNAi) of nestin attenuated thrombin-induced cell proliferation, indicating that thrombin-induced nestin expression and cell proliferation share the same EGFR transactivation mechanism. Further, DNA laddering analysis and flow cytometric results demonstrated that apoptotic activity is enhanced in nestin-depleted cells after H2O2 treatment. In addition, caspase-9, caspase-3, and PARP were activated in nestin-depleted rat VSMCs following treatment with 250 μM H2O2, indicating that nestin has an upstream inhibitory effect on caspase activation. It is well-known that the EGF serves as a survival factor in rat VSMCs. Herein, we showed that RNAi of nestin abolished the cytoprotective effect of the EGF. Taken together, these results indicate that nestin may play essential roles in EGFR-mediated cell proliferation and cytoprotective effects in rat VSMCs.

並列關鍵字

nestin ； EGF ； thrombin ； vascular smooth muscle cells ； EGFR transactivation ； proliferation ； apoptosis ； caspase

參考文獻

Abouhamed M, Reichenberg S, Robenek H, Plenz G (2003) Tropomyosin 4 expression is enhanced in dedifferentiating smooth muscle cells in vitro and during atherogenesis. Eur J Cell Biol 82(9): 473-482

Barnes JA, Singh S, Gomes AV (2004) Protease activated receptors in cardiovascular function and disease. Mol Cell Biochem 263(1-2): 227-239

Baykal D, Schmedtje JF Jr, Runge MS (1995) Role of the thrombin receptor in restenosis and atherosclerosis. Am J Cardiol 75(6): 82B-87B

Biscardi JS, Maa MC, Tice DA, Cox ME, Leu TH, Parsons SJ (1999) c-Src-mediated phosphorylation of the epidermal growth factor receptor on Tyr845 and Tyr1101 is associated with modulation of receptor function. J Biol Chem 274(12): 8335-8343

Brunt KR, Fenrich KK, Kiani G, Tse MY, Pang SC, Ward CA, Melo LG (2006) Protection of human vascular smooth muscle cells from H2O2-induced apoptosis through functional codependence between HO-1 and AKT. Arterioscler Thromb Vasc Biol 26(9): 2027-2034

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中間絲巢蛋白於大鼠血管平滑肌細胞之表現、調控與功能之研究

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