Suppression of soluble adenylyl cyclase protects smooth muscle cells against oxidative stress-induced apoptosis
Suppression of soluble adenylyl cyclase protects smooth muscle cells against oxidative stress-induced apoptosis
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DOI：
10.1007/s10495-014-0989-9
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发表时间：
2014-07-01
期刊：
影响因子：
7.2
通讯作者：
Ladilov, Yury
中科院分区：
文献类型：
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作者：
Kumar, Sanjeev;Appukuttan, Avinash;Ladilov, Yury
血管平滑肌细胞（VSMC）凋亡是导致动脉粥样硬化斑块不稳定的重要因素。氧自由基是导致VSMC死亡的重要原因。然而，氧化应激诱导VSMC凋亡的确切机制仍然知之甚少。在这里，我们的目的是分析可溶性腺苷酸环化酶（sAC）的作用。用300 μ mol/L的H_2O_2或30 μ mol/L的DMNQ处理大鼠主动脉VSMC 6 h。氧化应激诱导的细胞凋亡可以通过30 A μ mol/L KH 7（sAC的特异性抑制剂）或稳定的sAC敲除（shRNA转染）来预防。抑制蛋白激酶A（PKA）后，发现类似的效果。sAC/PKA轴的抑制导致氧化应激下p38促分裂原活化蛋白激酶的磷酸化显著增加，伴随着促凋亡Bcl-2家族蛋白Bad的p38依赖性磷酸化/失活。p38的药理学抑制逆转了sAC敲低对细胞凋亡和Bad磷酸化的这些影响，表明p38是sAC和细胞凋亡之间的联系。蛋白磷酸酶1和2A活性的分析揭示了磷酸酶1的激活，但不是磷酸酶2A，在氧化应激下的sAC/PKA依赖的方式和其在控制p38磷酸化的作用。抑制蛋白磷酸酶1，但不是2A，防止氧化应激的促凋亡作用。总之，sAC/PKA信号通路在氧化应激诱导的VSMC凋亡中起关键作用。细胞机制包括sAC促进和蛋白磷酸酶1介导的p38磷酸化抑制，导致线粒体凋亡途径激活。
Apoptosis of vascular smooth muscle cells (VSMC) significantly contributes to the instability of advanced atherosclerotic plaques. Oxygen radicals are an important cause for VSMC death. However, the precise mechanism of oxidative stress-induced VSMC apoptosis is still poorly understood. Here, we aimed to analyse the role of soluble adenylyl cylclase (sAC). VSMC derived from rat aorta were treated with either H2O2 (300 A mu mol/L) or DMNQ (30 A mu mol/L) for 6 h. Oxidative stress-induced apoptosis was prevented either by treatment with 30 A mu mol/L KH7 (a specific inhibitor of sAC) or by stable sAC-knockdown (shRNA-transfection). A similar effect was found after inhibition of protein kinase A (PKA). Suppression of the sAC/PKA-axis led to a significant increase in phosphorylation of the p38 mitogen-activated protein kinase under oxidative stress accompanied by a p38-dependent phosphorylation/inactivation of the pro-apoptotic Bcl-2-family protein Bad. Pharmacological inhibition of p38 reversed these effects of sAC knockdown on apoptosis and Bad phosphorylation, suggesting p38 as a link between sAC and apoptosis. Analysis of the protein phosphatases 1 and 2A activities revealed an activation of phosphatase 1, but not phosphatase 2A, under oxidative stress in a sAC/PKA-dependent manner and its role in controlling the p38 phosphorylation. Inhibition of protein phosphatase 1, but not 2A, prevented the pro-apoptotic effect of oxidative stress. In conclusion, sAC/PKA-signaling plays a key role in the oxidative stress-induced apoptosis of VSMC. The cellular mechanism consists of the sAC-promoted and protein phosphatase 1-mediated suppression of p38 phosphorylation resulting to activation of the mitochondrial pathway of apoptosis.