去除蛋白激酶A磷酸化位点的线粒体融合素2基因对血管平滑肌细胞凋亡的影响

Effect of Removal of Protein Kinase A Phosphorylation Site from Mfn2 Gene on the Apoptosis of Vascular Smooth Muscle Cell

目的研究大鼠线粒体融合素2基因在去除蛋白激酶A磷酸化位点后对大鼠血管平滑肌细胞凋亡的影响及其相关的信号通路。方法利用携带去除蛋白激酶A磷酸化位点的线粒体融合素2基因重组腺病毒(Adv-Mfn2-PKA(△))和携带线粒体融合素2基因的重组腺病毒(Adv-Mfn2),感染培养的大鼠血管平滑肌细胞。免疫印迹分析相关蛋白的表达;激光共聚焦显微镜观察其亚细胞定位;细胞凋亡酶联免疫吸附法比较其对细胞凋亡的影响;免疫印迹法分析磷酸化蛋白激酶B蛋白表达变化。结果外源基因转染后可表达特异性蛋白产物;激光共聚焦显微镜显示去除蛋白激酶A磷酸化位点后的线粒体融合素2基因表达产物主要分布于线粒体外膜;酶联免疫吸附法检测显示去除蛋白激酶A磷酸化位点后,线粒体融合素2基因诱导大鼠血管平滑肌细胞凋亡作用显著减弱(P<0.01),与空白对照组差异无显著性;免疫印迹检测表明Mfn2-PKA(△)组磷酸化蛋白激酶B表达较Mfn2组显著升高(P<0.01),与空白对照组差异无显著性。结论去除蛋白激酶A磷酸化位点的线粒体融合素2基因表达产物仍主要分布于线粒体外膜,但其诱导血管平滑肌细胞凋亡的作用丧失,对蛋白激酶B信号通路也无抑制作用。这表明蛋白激酶A磷酸化位点是调控线粒体融合素2基因诱导血管平滑肌细胞凋亡的重要功能位点。

Aim To investigate the effect of mitofusin2 （Mfn2） with the removal of protein kinase A （PKA） phosphorylation site （Mfn2-PKA（ A ） ） on promoting the apoptosis of vascular smooth muscle cell （VSMC） and related sig- naling pathway. Methods VSMC were transfected with adenovirus vector encoding Mfn2-PKA （ A ） or Mfn2 （ Adv- Mfn2-PKA （ a ） , Adv-Mfn2）. The abundance of Mfn2-PKA （ A ） protein and Mfn2 protein were determined by Western Blot analysis using Mfn2 phosphorylated antibody. Laser confocal microscopy（LCMS） was used to observe the location of proteins. The effect of Adv-Mfn2-PKA（ A ） on the apoptosis of VSMC was explored by cell death ELISA. Western Blot were used to detect the expression of p-Akt. Results Both Mfn2-PKA （ A ） and Mfn2 were located at the outmem- brahe of mitochondria. ELISA assay showed Mfn2-PKA（ A ） had no effect on promoting the apoptosis of VSMC compared with Mfn2 （ P 〈 0.01 ）. Western Blot indicated that overexpression of Mfn2-PKA （ A ） gene could not down-regulate the phosphorylation of Akt （P 〈 0. 01 ）. Conclusion Mfn2-PKA （ A ） could not induce the apoptosis of VSMC. PKA phosphorylation site plays an important role in regulating the function of Mfn2 gene.