Department of Cardiovascular Diseases, Katholieke Universiteit Leuven, Belgium.
Am J Hypertens. 2010 Oct;23(10):1128-35. doi: 10.1038/ajh.2010.128. Epub 2010 Jul 1.
The aim of this study was to determine whether angiotensin II (ANG II) affects the protein and mRNA expressions of the mitochondrial antioxidant manganese superoxide dismutase (Mn-SOD) in cardiac fibroblasts of rats through inducing the phosphorylation of the proteins Akt and FOXO3a, thereby contributing to the oxidative stress in the myocardium.
Cardiac fibroblasts (passage 2) from normal male adult rats were cultured to confluency and incubated in serum-free Dulbecco's modified Eagle's medium for 24 h. The cells were then preincubated with/without the tested inhibitors for 1 h and then further incubated with/without ANG II (1 µmol/l) for 24 h.
ANG II increased the production of superoxide ions in the cardiac fibroblasts, and decreased the activity levels of both Mn-SOD and CuZn-SOD, but not the activity levels of catalase and glutathione peroxidase. ANG II also decreased the mRNA and protein expressions of Mn-SOD, but not those of CuZn-SOD, catalase, and glutathione peroxidase. The likely mechanism through which ANG II produces the effect of reducing Mn-SOD activity is by reducing the extent of binding of FOXO3a to the Mn-SOD promoter. In control fibroblasts, inhibition of FOXO3a transcription with small-interfering RNA (siRNA) led to a reduction in the binding of FOXO3a to the Mn-SOD promoter, and a concomitant reduction in Mn-SOD gene expression. Our data suggest that when Akt is phosphorylated by ANG II, P-Akt is translocated from the cytoplasm to the nucleus; subsequently, nuclear phosphorylation of FOXO3a by P-Akt leads to relocalization of FOXO3a from the nucleus to the cytosol, resulting in a decrease in its transcriptional activity, and consequently in Mn-SOD expression. The likelihood of such a mechanism of action is further strengthened by the fact that inhibition of phosphoinositide 3-kinase with wortmannin or LY 294002, and Akt inhibition, were shown to lead to a decrease in P-AKT and to a consequent increase in Mn-SOD mRNA expression.
Our data indicate that ANG II inactivates FOXO3a by activating Akt, leading to a reduction in the expression of the antioxidant Mn-SOD, and thereby potentially contributing to oxidative stress in the myocardium.
本研究旨在探讨血管紧张素 II（ANG II）是否通过激活蛋白激酶 B（Akt）和叉头框转录因子 O3a（FOXO3a）使心肌成纤维细胞内的锰超氧化物歧化酶（Mn-SOD）发生磷酸化，从而影响其蛋白和 mRNA 表达，导致心肌发生氧化应激。
培养雄性成年大鼠的心肌成纤维细胞（第 2 代），使其融合并在无血清的杜氏改良伊格尔培养基中孵育 24 小时。然后，将细胞用/不用测试抑制剂孵育 1 小时，再用/不用 ANG II（1 μmol/L）孵育 24 小时。
ANG II 增加了心肌成纤维细胞中超氧阴离子的产生，降低了 Mn-SOD 和 CuZn-SOD 的活性，但对过氧化氢酶和谷胱甘肽过氧化物酶的活性没有影响。ANG II 还降低了 Mn-SOD 的 mRNA 和蛋白表达，但对 CuZn-SOD、过氧化氢酶和谷胱甘肽过氧化物酶的表达没有影响。ANG II 降低 Mn-SOD 活性的可能机制是通过减少 FOXO3a 与 Mn-SOD 启动子的结合。在对照成纤维细胞中，用小干扰 RNA（siRNA）抑制 FOXO3a 转录导致 FOXO3a 与 Mn-SOD 启动子的结合减少，同时 Mn-SOD 基因表达减少。我们的数据表明，当 Akt 被 ANG II 磷酸化时，P-Akt 从细胞质转移到细胞核；随后，P-Akt 使 FOXO3a 在核内磷酸化，导致 FOXO3a 从核内重新定位到细胞质，从而降低其转录活性，进而降低 Mn-SOD 的表达。这种作用机制的可能性进一步得到了以下事实的支持：用渥曼青霉素或 LY 294002 抑制磷酯酰肌醇 3-激酶，以及 Akt 抑制，都导致 P-AKT 减少，Mn-SOD mRNA 表达增加。
我们的数据表明，ANG II 通过激活 Akt 使 FOXO3a 失活，导致抗氧化剂 Mn-SOD 的表达减少，从而可能导致心肌发生氧化应激。
