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

• Main Authors: Wen-Ping Cheng, 鄭文斌
• Other Authors: 徐國基
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/80259689308968661062

博士 === 臺北醫學大學 === 醫學科學研究所 === 96 === 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.