Effect of mitofusin 2 gene with protein kinase A phosphorylation site deletion on the proliferation of vascular smooth muscle cells / 中国组织工程研究

ABSTRACT

BACKGROUND: The mitofusin 2 (Mfn2) may affects vascular smooth muscle cell Ras protein and suppress cellular proliferation through inhibition of extracellular signal-regulated protein kinase signaling pathway, which plays an important role in pathogenesis of vascular disorders such as hypertension, atherosclerosis and post-angioplasty restenosis. Mfn-2 gene amino acid sequence of the first 442 serine serves as protein kinase A (PKA) phosphorylation site, which is closely related to its phosphorylation status and may be involved in its functional regulation.OBJECTIVE: To investigate the effect of Mfn2 gene with PKA phosphorylation site deletion[Mfn2-PKA (△)] on inhibiting the proliferation of vascular smooth muscle cells and related signaling pathway.METHODS: Vascular smooth muscle cells of rats infected by recombinational adenovirus carrying green fluorescent protein,Mfn2 gane and Mfn2-PKA (△), were subcultured for 3-10 passages and randomly divided into 4 groups ① Control group without intervention. ② Control group infected with adenovirus carrying green fluorescent protein. ③ Experiment group infected with adenovirus carrying Mfn-2 gene.④ Experiment group infected with adenovirus carrying Mfn2-PKA (△). Laser confocal microscopy was used to observe the locations of Mfn2 gene with and without PKA in cells. The expressions of extracallular signal-regulated protein kinase, Mfn2 gone and Mfn2-PKA (△) were determined by Western blot analysis. The growth curve of the vascular smooth muscle cells was explored by MTT.RESULTS AND CONCLUSION: The Mfn-2 and Mfn2-PKA (△) both expressed protein-specific bands in vascular smooth muscle cells. Two kinds of gone expression products were mainly located at the out membrane of mitochondria. Compared with the control group and adenovirus carrying green fluorescent protein group, the absorbance values at 3, 4, 5, 6 days were significantly reduced in adenovirus carrying Mfn2 group (P ＜ 0.01), and no obvious changes were observed in adenovirus carrying Mfn2-PKA (△) group. Compared with the control group and adenovirus carrying green fluorescent protein group, the extracellular signal-regulated protein kinase expression was significantly reduced in adenovirus carrying Mfn2 group (P ＜ 0.01), and no obvious changes were observed in adenovirus carrying Mfn2-PKA (△) group. Mfn2-PKA (△) located at the out membrane of mitochondria, has no effect on suppressing the proliferation of vascular smooth muscle cells, and no inhibition effect on extracellular signal-regulated protein kinase signaling pathway.