Abstract：Objective To study whether the ERK1/2 pathway is involved in aortic vascular smooth muscle cells (VSMCs) phenotype transformation induced by low expression of Pkd2 gene and to explore its possible molecular mechanisms. Methods Mouse aortic VSMCs were primarily cultured, Pkd2+/- mutant vectors were transfected, and low expression cell model of Pkd2 gene was established. The experiment included 6 groups: the control group, Pkd2+/- group, blank vector group, Ets1 (Pkd2 agonist) group, PD98059 (ERK inhibition) group and EGF (ERK agonist) group. Western blot was used to detect the expression levels of polycystin 2 (PC2, Pkd2 encoding protein), a-SMA (VSMCs contraction marker), osteopontin (OPN, VSMCs proliferation marker), ERK1/2 and the phosphorylation of ERK protein (P-ERK1/2). The expression levels of PC2, a-SMA, OPN, ERK1 and ERK2 mRNAs were measured by real-time PCR. MTT assay was used to detect cell proliferation. Results The expression of PC2 protein and mRNA in Pkd2+/- group was significantly down-regulated, and the low expression model of Pkd2 gene was successfully constructed. The expression of a-SMA in Pkd2+/- group was down-regulated, while the expression of OPN was elevated. The proliferation of VSMCs was obvious, and cellular phenotype transformation occurred. The cell proliferation was significantly inhibited (P<0.05) after Ets1 was added. The expression of ERK1/2 and P-ERK1/2 in Pkd2+/- group was significantly elevated, but was inhibited after PD98059 was added, while the expression of PC2 remained unchanged (P<0.05). After PD98059 was added in Pkd2+/- group, the expression of a-SMA was elevated, while the expression of OPN was down-regulated. The proliferation of VSMCs and cellular phenotype transformation were both inhibited. Conclusions The low expression of Pkd2 gene can induce the phenotype transformation of aortic VSMCs in mice, and this process may be related to the abnormal activation of ERK1/2.
[1] Wang X, LeMaire SA, Chen L, et al. Increased collagen deposition and elevated expression of connective tissue growth factor in human thoracic aortic dissection[J]. Circulation,2006,114(1 Suppl):I 200-205. [2] Guo DC, Pannu H, Tran-Fadulu V, et al. Mutations in smooth muscle alpha-actin (ACTA2) lead to thoracic aortic aneurysms and dissections[J].Nat Genet, 2007,39(12):1488-1493. [3] Fukunaga N, Yuzaki M, Nasu M, et al. Dissecting aneurysm in a patient with autosomal dominant polycystic kidney disease [J]. Ann Thorac Cardiovasc Surg, 2012 ,18(4):375-378. [4] Hassane S, Claij N, Jodar M, et al. Pkd1-inactivation in vascular smooth muscle cells and adaptation to hypertension [J]. Lab Invest,2011,91(1):24-32. [5] Hassane S, Claij N, Leeuwen ISL, et al. Pathogenic sequence for dissecting aneurysm formation in a hypomorphic polycystic kidney disease 1 Mouse Model[J]. Arterioscler Thromb Vasc Biol,2007,27(10):2177-2183. [6] Fogelgren B, Lin SY, Zuo X, et al. The exocyst protein Sec10 interacts with Polycystin-2 and knockdown causes PKD-phenotypes[J]. PLoS Genet,2011,7(4):e1001361. [7] Mutlak M, KehatI.Extracellular signal-regulated kinases 1/2 as regulators of cardiac hypertrophy[J].Front Pharmacol, 2015,24(6):149. [8] Torres VE, Harris PC. Polycystic kidney disease:genes, proteins, animal models, disease mechanisms and therapeutic opportunities[J]. J Intern Med,2007,261(1):17-31. [9] Howard DP, Banerjee A, Fairhead JF,et al. Population-based study of incidence and outcome of acute aortic dissection and premorbid risk factor control:10-year results from the Oxford Vascular Study [J]. Circulation,2013,127(20):2031-2037. [10] Guo DC, Pannu H, Tran-Fadulu V, et al. Mutations in smooth muscle alpha-actin (ACTA2) lead to thoracic aortic aneurysms and dissections[J].Nat Genet,2007,39(12):1488-1493. [11] Spirli C, Okolicsanyi S, Fiorotto R, et al. ERK1/2-dependent vascular endothelial growth factor signaling sustains cyst growth in polycystin-2 defective mice[J]. Gastroenterology,2010,138(1):360-371. [12] Mebratu Y, Tesfaigzi Y. How ERK1/2 activation controls cell proliferation and cell death: is subcellular localization the answer?[J]. Cell Cycle,8(8),2009:1168-1175. [13] Tsiokas L, Kim S, Ong EC. Cell biology of polycystin-2[J].Cell Signal,2007, 19(3):444-453[14] Paavola J1, Schliffke S, Rossetti S, et al. Polycystin-2 mutations lead to impaired calcium cycling in the heart and predispose to dilated cardiomyopathy[J]. J Mol Cell Cardiol, 2013,58(5):199-208. [15] Qian Q, Hunter LW, Li M, et al. Pkd2 haploinsufficiency alters intracellular calcium regulation in vascular smooth muscle cells[J].Hum Mol Genet,2003,12(15):1875-1880.
2016, Vol. 23 
