在機械性壓力下探討血管平滑肌細胞與心肌細胞凋亡 ：GADD153所扮演的角色

The Role of GADD153 in Apoptosis of Vascular Smooth Muscle Cells and Cardiomyocytes under Mechanical Stress

鄭文斌(Wen-Ping Cheng)

指導教授 ： 徐國基

臺北醫學大學/醫學院/醫學科學研究所/博士(2008年)

摘要

細胞凋亡與動脈粥狀硬化及心肌肥大之間的關係非常密切。當細胞發生內質網壓力時，與細胞凋亡有關的基因GADD153之表現會增加。文獻已證實，機械性壓力會誘發血管平滑肌細胞與心肌細胞之凋亡。然而機械性壓力是否可透過GADD153，影響血管平滑肌細胞與心肌細胞之細胞凋亡，目前仍然充滿爭議。本論文之目的即在於釐清機械性壓力是否能經由GADD153，誘發血管平滑肌細胞與心肌細胞之凋亡，並探討其中的分子調控機制。
第一部分，我們使用小片段干擾RNA去探討週期性機械展延誘發GADD153的機制與訊號傳遞路徑。GADD153的蛋白質與mRNA表現經過十八小時的週期性展延後皆會增加。週期性展延前三十分鐘加入JNK的抑制劑SP600125、JNK的小片段干擾RNA、腫瘤壞死因子-?悀峔鋮?體的單株抗體，皆可明顯的抑制展延所誘發之GADD153蛋白質表現。膠體位移實驗的結果顯示血管平滑肌細胞中，週期性展延可以增加轉錄因子AP-1與DNA的結合活性；而SP600125、JNK的小片段干擾RNA與腫瘤壞死因子-?悛熙畾閫凗擃h會抑制由展延所增加之AP?{?△畢X活性。GADD153的突變質體、SP600125與c-jun的單株抗體會抑制週期性展延所增加之GADD153促進子活性。取經過展延後的培養基與外因性腫瘤壞死因子-?悒[入血管平滑肌細胞中，同樣可以誘發GADD153的蛋白質表現，而且此蛋白表現量與週期性展延所誘發的表現量相似。接著進一步探討週期性展延所誘發之GADD153，對血管平滑肌細胞凋亡的影響。結果顯示，GADD153在週期性展延所造成的血管平滑肌細胞凋亡中扮演了重要的角色。活體內之動靜脈廔管手術中亦發現，容積性壓力超載可增加主動脈組織中GADD153的蛋白質表現量。
第二部份我們試圖透過活體內外的實驗，釐清GADD153在心肌肥大中扮演的角色。為了測試GADD153於衰竭的心肌中扮演之角色，對wistar大鼠施以三天的動靜脈廔管或三週的動脈縮小手術，造成其容積或壓力性超載之心肌肥大與心臟衰竭。此外，伴隨分子PBA可以改善內質網的折疊能力，並加速突變蛋白的運送。因此我們給予手術後的wistar大鼠，每公斤體重五百毫克的PBA，希望能改善其心肌肥大的狀況。超音波的結果顯示動脈縮小手術可以造成同心圓型的心肌肥大，而動靜脈廔管手術則是會產生不同圓心的心肌肥大。動靜脈廔管與動脈縮小組中，PBA的治療皆可以還原大鼠被誘發的心臟重量與心跳。動脈縮小組中原本被增厚的左心室後壁，在經過PBA治療後，會縮小至正常的厚度；而動靜脈廔管組中被增大的左心室體積，以PBA治療後，會減小至正常的大小。動脈縮小組中的平均動脈壓會上升，動靜脈廔管組中的平均動脈壓則是會下降；經過PBA治療後兩者皆會回歸正常值。西方點墨法的結果顯示，GADD153與BNP的蛋白質表現在shunt與banding的組別中皆會增加，然而NGF-?猁熙J白質表現則是會下降。即時聚合鏈反應的結果亦顯示，GADD153的mRNA表現經過動脈縮小與動靜脈廔管後會提高。經過PBA的治療後，GADD153 、BNP與NGF-?猁熙J白質表現皆會恢復正常。TUNEL測試的結果顯示給予大鼠PBA，可降低其被容積或壓力超載所誘發之細胞凋亡。接著我們研究週期性展延所誘發的GADD153，對心肌細胞凋亡的影響。活體外的實驗中，十四小時的週期性展延會增加心肌細胞中GADD153的蛋白質表現。週期性展延前三十分鐘加入JNK的抑制劑：SP600125、JNK的小片段干擾RNA、腫瘤壞死因子-?悛熙畾閫凗憿A都能顯著的抑制機械展延所誘發之GADD153蛋白質表現。膠體位移實驗的結果顯示，週期性展延可以增加心肌細胞中轉錄因子AP-1與DNA的結合活性；而SP600125、JNK的小片段干擾RNA與腫瘤壞死因子-?悛熙畾閫凗攳鄑磻謢馱@現象。此外，GADD153的突變質體、SP600125與腫瘤壞死因子-?悛熙畾閫凗攳鈶ㄖ穚g期性機械展延所提高的GADD153促進子活性。GADD153的小片段干擾RNA、GADD153的突變質體與伴隨分子PBA，皆可抑制機械展延經由誘發GADD153所造成的心肌細胞凋亡。
總括來說，這篇論文確認了不論在活體內外，GADD153皆會參與在機械性壓力所誘發之血管平滑肌細胞與心肌細胞的凋亡中。活體外，GADD153的小片段干擾RNA可以抑制機械性展延所誘發的GADD153的表現，並抑制GADD153在血管平滑肌細胞與心肌細胞所造成的細胞凋亡。活體內，容積與壓力超載能誘發主動脈與心肌組織中GADD153的表現及細胞凋亡。期許對於GADD153有更深入的了解及研究後，能發展為治療標的，以提供對動脈粥狀硬化與心肌肥大疾病治療上之另一選擇。

關鍵字

粥狀動脈硬化 ； 心肌肥大 ； 細胞凋亡 ； GADD153 ； 週期性機械延展 ； 容積超載 ； 壓力超載 ； 小片段干擾RNA

並列摘要

There is a very close relationship between apoptosis with atherosclerosis and heart failure. Growth arrest and DNA damage-inducible gene 153 (GADD153), an apoptosis regulated gene, increased during endoplasmic reticulum (ER) stress. It has been demonstrated that mechanical stress could induce apoptosis in vascular smooth muscle cells (VSMCs) and cardiomyocytes. However, the mechanism how cyclic mechanical stress affected GADD153-mediated apoptosis in VSMCs and cardiomyocytes is still a controversy issue. We aimed to test the hypothesis that mechanical stress induces GADD153 expression in VSMCs and cardiomyocytes apoptosis.
In the first part of the study, we used siRNA to investigate the mechanism and signal pathways of GADD153 by cyclical mechanical stretch. Then we investigated the effect of GADD153 induce by cyclical stretch on the apoptosis in VSMCs. Cyclic stretch significantly increased GADD153 protein and mRNA expression after 18 h of stretch. Addition of c-jun N-terminal kinase (JNK) inhibitor SP600125, JNK siRNA, tumor necrosis factor-?? (TNF-??) and TNF-?? receptor antibody 30 min before stretch, inhibited the induction of GADD153 protein. Gel shift assay showed that DNA-binding activity of activating factor 1 (AP-1) increased after stretch. SP600125, JNK siRNA and TNF-?? antibody abolished the binding activity induced by stretch. Stretch increased, while GADD153-Mut plasmid, SP600125 and c-jun antibody abolished the promoter activity. Both conditioned media from stretched VSMCs and exogenous administration of TNF-?? recombinant protein to the non-stretch VSMCs increased GADD153 protein expression similar to that seen after stretch. An in vivo model of aorta-caval shunt in adult rats also demonstrated the increased GADD153 protein expression in the aorta. These finding suggested that GADD153 plays a role in stretch-induced VSMCs apoptosis.
In the second part of the study, we identified the role of GADD153 on the cardiac hypertrophy in vivo and in vitro. To test the hypothesis that GADD153 play a role in the failing myocardium because of volume and pressure overload. Aorta-caval shunt (AV-shunt) and aortic banding was performed for 3 day and 3 weeks in adult Sprague-Dawley rats to induce volume and pressure overload heart failure. 4-phenylbutyric acid (PBA) is one of the chemical chaperon can improve ER folding capacity, and facilitate the trafficking of mutant proteins. PBA at 500mg/kg body weight per day after surgery was given. Echocardiography showed concentric hypertrophy after aortic banding but eccentric hypertrophy after aorta-caval shunt. Treatment with PBA in the shunt and banding group reversed the heart weight and heart rate. The increased wall thickness in the banding group and the increased ventricular dimension in the AV-shunt were reverse to normal by PBA. Mean arterial pressure increased in the shunt group but decreased in the banding group after treatment with PBA. Western blot showed that GADD153 and brain natriuretic peptide (BNP) proteins were up-regulated and nerve growth factor-?? (NGF-??) down-regulated in the shunt and banding group. Real-time polymerase chain reaction showed that mRNA of GADD153 increased in the shunt and banding group. Treatment of PBA reversed the protein of GADD153, BNP and NGF-?? to the baseline values. The TUNEL assay showed that PBA reduced the apoptosis induced by the volume and pressure overload. Then we investigated the effect of GADD153 induced by cyclical stretch on the apoptosis in cardiomyocytes. Cyclic stretch significantly increased GADD153 protein and mRNA expression after 14 h of stretch. Addition of c-jun N-terminal kinase (JNK) inhibitor SP600125, JNK siRNA and tumor necrosis factor-?? (TNF-??) antibody 30 min before stretch, reduced the induction of GADD153 protein. Gel shift assay showed that SP600125, JNK siRNA and TNF-?? antibody abolished the activating protein 1 (AP-1) binding activity induced by stretch in cardiomyocytes. Stretch increased, while GADD153-Mut plasmid, SP600125 and TNF-?? antibody abolished the promoter activity. GADD153 mediated apoptosis induced by stretch was reverse by GADD153 siRNA , GADD153-Mut plasmid and PBA.
In conclusion, our study confirmed that GADD153 involved in the VSMCs and cardiomyocytes apoptosis induced by mechanical stress in vitro and in vivo. Treatment of GADD153siRNA reduced the GADD153 expression and GADD153-mediated apoptosis induced by stretch in VSMCs and cardioyocytes. Moreover, volume and pressure overload could induce GADD153 expression and apoptosis in aorta and myocardium. Therefore, GADD153 might serve as therapeutic target for atherosclerosis and hypertrophy in the nearly future.

並列關鍵字

atherosclerosis ； cardiac hypertrophy ； apoptosis ； GADD153 ； cyclic mechanical stretch ； volume overload ； pressure overload ； small interference RNA

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