蛋白激酶C对低氧诱导小鼠肺动脉平滑肌细胞增殖及蛋白表达的影响

Effects of typical PKC subtypes on the proliferation of mouse pulmonary artery smooth muscle cells and the expression of ERK1/2 and Akt induced by hypoxia

目的探讨经典型蛋白激酶C(PKC)对低氧诱导的小鼠远端肺动脉平滑肌细胞(PASMC)增殖及细胞外信号调节激酶1/2(ERK1/2)、丝氨酸苏氨酸蛋白激酶(Akt)表达的影响。方法利用Dynal MPC-1磁分离器分离含铁粉的远端肺小动脉,原代培养并鉴定肺动脉平滑肌细胞。应用PKC激动剂(PMA)、PKCα抑制剂(safingol)、PKCβⅠ抑制剂(Go6976)、PKCβⅡ抑制剂(LY333531)分别干预细胞。在常氧和低氧条件下,采用免疫印迹法观察cPKC亚型蛋白表达变化以及ERK1/2、Akt磷酸化水平变化。应用PKCα、PKCβ的慢病毒载体,通过病毒转染技术特异性敲减目标基因活性,采用免疫印迹法观察低氧诱导小鼠PASMC中cPKC亚型蛋白表达变化,以及ERK1/2、Akt磷酸化水平变化。结果采用Brdu法,检测到用safingol、Go6976、LY333531分别抑制cPKCα、βⅠ和βⅡ时,低氧诱导的PASMC增殖能力受到明显抑制,与低氧对照组相比,Brdu阳性细胞率分别为(7.35±0.26)%vs(11.28±0.43)%,(3.76±0.25)%vs(7.98±0.28)%和(4.12±0.46)%vs(7.78±0.53)%;且PMA在常氧条件下可显著促进PASMC的增殖能力,与常氧对照组相比,Brdu阳性细胞率分别为(9.65±0.47)%vs(6.34±0.52)%,(9.34±0.38)%vs(5.42±0.21)%和(7.78±0.53)%vs(4.12±0.46)%;此外经过PKCα、PKCβ的慢病毒载体转染细胞后,也发现低氧转染组的PASMC增殖能力较空载对照组显著下降,Brdu阳性细胞率分别为(3.58±0.54)%vs(5.97±0.63)%。用safingol、Go6976、LY333531分别抑制cPKCα、βⅠ和βⅡ表达后,低氧诱导的PASMC中ERK1/2、Akt的磷酸化水平较对照组显著降低,使用safingol之后,ERK1/2、Akt的磷酸化水平分别为:0.56±0.07 vs 1.08±0.13和0.49±0.04 vs 0.97±0.08,使用Go6976之后,ERK1/2、Akt的磷酸化水平分别为:0.41±0.09 vs 0.79±0.10和0.48±0.09 vs 0.82±0.16,使用LY333531之后,ERK1/2、Akt的磷酸化水平分别为:0.42±0.03 vs 0.87±0.06和0.34±0.07 vs 0.78±0.05;PMA可使常氧条件下PASMC中ERK1/2、Akt的磷酸化水平增高,分别为1.25±0.12 vs 0.41±0.07和0.98±0.06 vs 0.37±0.08;利用病毒转染技术对cPKCα、β的表达进行特异性敲底,发现在低氧条件下,转染细胞后均可使细胞中ERK1/2磷酸化水平显著降低,其磷酸化水平为0.29±0.06 vs 0.76±0.05,而Akt磷酸化水平较对照组无明显变化。结论激动剂及抑制剂干预或病毒转染可使PASMC中cPKCα、βⅠ和βⅡ表达下降,可显著抑制低氧诱导的小鼠远端PASMC异常增殖,其作用机制可能与cPKCα、βⅠ和βⅡ蛋白表达上调有关。低氧可能通过上调cPKCα、βⅠ和βⅡ的蛋白表达,使得ERK1/2磷酸化水平增高,最终引起小鼠远端PASMC的异常增殖,参与低氧性肺动脉高压的形成过程。

Objective To study the effects of specific isoforms of classic protein kinase C(cPKCs)on hypoxia-induced proliferation and the expression of ERK1/2 and Akt using drug intervention or virus transfection in vitro.Methods Dynal MPC-1 magnetic particle concentrator was used to separate iron-containing pulmonary arterioles fragments,and the pulmonary artery smooth muscle cells(PASMCs)were primary cultured and identified.The cells were intervened by PKC agonist(PMA),PKCαinhibitor(safingol),PKCβⅠinhibitor(Go6976)and PKCβⅡinhibitor(LY333531)respectively,and the changes in protein expressions of cPKCs,and the phosphorylation levels of ERK1/2 and Akt were observed by immunoblotting under the condition of normal oxygen or hypoxia.The lentiviral vectors of PKCαand PKCβwere used to specifically knock-down the activity of target genes by virus transfection techniques,and Western blotting was used to observe the protein expressions of cPKCs,and the phosphorylation levels of ERK1/2 and Akt in hypoxia-induced PASMCs in mice.Results With Brdu method,the proliferation of PASMCs induced by hypoxia was significantly inhibited by safingol,Go6976 and LY333531 by inhibiting cPKCα,βⅠandβⅡrespectively.Compared with the hypoxic control group,the rates of Brdu positive cells were(7.35±0.26)%vs(11.28±0.43)%,(3.76±0.25)%vs(7.98±0.28)%and(4.12±0.46)%vs(7.78±0.53)%.We also observed that PMA could significantly promote the proliferation of PASMCs under normoxic condition.Compared with the normoxia control group,the Brdu-positive cell rates were(9.65±0.47)%vs(6.34±0.52)%,(9.34±0.38)%vs(5.42±0.21)%and(7.78±0.53)%vs(4.12±0.46)%.In addition,after transfection with PKCαor PKCβlentiviral vector,the proliferation of PASMCs was significantly lower in hypoxia transfection group than in the control group.The rates of Brdu positive cells were(3.58±0.54)%vs(5.97±0.63)%,respectively.Using Western blotting,we also observed that after being inhibited by safingol,Go6976 and LY333531 respectively,the phosphorylation levels of ERK1/2 and Akt in PASMCs induced by hypoxia was significantly lower than the control group.After using safingol,the phosphorylation levels of ERK1/2 and Akt were(0.56±0.07)vs(1.08±0.13)and(0.49±0.04)vs(0.97±0.08).After using Go6976,the phosphorylation levels of ERK1/2 and Akt were(0.41±0.09)vs(0.79±0.10)and(0.48±0.09)vs(0.82±0.16),after using LY333531,the phosphorylation levels of ERK1/2 and Akt were(0.42±0.03)vs(0.87±0.06)and(0.34±0.07)vs(0.78±0.05).While PMA could promote the phosphorylation levels of ERK1/2 and Akt under normoxic condition,1.25±0.12 vs 0.41±0.07 and 0.98±0.06 vs 0.37±0.08,respectively.Using transfection technique to specifically knock down the expression of cPKCαandβ,we found that under hypoxic conditions,transfection of PASMCs could significantly lower the phosphorylation levels of ERK1/2,its phosphorylation level was 0.29±0.06 vs 0.76±0.05,with no evident change in the phosphorylation levels of Akt.Conclusions Hypoxia may lead to phosphorylation of ERK1/2 by promoting the protein expression of cPKCα,cPKCβⅠand cPKCβⅡrespectively,which eventually induces abnormal proliferation of PASMCs from the distal pulmonary arteries,participating in the development of hypoxic pulmonary hypertension(HPH)of the mice.Regulation of the expression of cPKCα,cPKCβⅠand cPKCβⅡmay help to attenuate the formation of pulmonary vascular remodeling.Target therapy based on cPKCs is expected to be a new direction for HPH therapy in the future.