From the Center for Molecular and Translational Medicine, Georgia State University, Atlanta (Z.C., Y.D., Q.L., S.W., H.Z., P.S., M.-H.Z.); Department of Cardiology, Second Affiliated Hospital, Zhejiang University College of Medicine, Hangzhou, Zhejiang, China (Z.C.); and Department of Medicine, University of Oklahoma Health Sciences Center (M.Z.).
Circ Res. 2016 Jul 22;119(3):422-33. doi: 10.1161/CIRCRESAHA.116.308301. Epub 2016 Jun 2.
RATIONALE: Atherosclerotic calcification is highly linked with plaque rapture. How calcification is regulated is poorly characterized. OBJECTIVE: We sought to determine the contributions of AMP-activated protein kinase (AMPK) in atherosclerotic calcification. METHODS AND RESULTS: Aortic calcification was evaluated in aortic roots and brachiocephalic arteries of atherosclerotic prone ApoE(-/-) mice or in mice with dual deficiencies of ApoE and AMPKα isoforms in whole body (ApoE(-/-)/AMPKα1(-/-) and ApoE(-/-)/AMPKα2(-/-)) or vascular smooth muscle cell (VSMC)-specific or macrophage-specific knockout of AMPKα1 fed with Western diet for 24 weeks. Genetic deficiency of AMPKα1 but not of AMPKα2 promoted atherosclerotic calcification and the expression of Runx2 (Runt-related transcription factor). Conversely, chronic administration of metformin, which activated AMPK, markedly reduced atherosclerotic calcification and Runx2 expression in ApoE(-/-) mice but had less effects in ApoE(-/-)/AMPKα1(-/-) mice. Furthermore, VSMC-specific but not macrophage-specific ablation of AMPKα1 promoted aortic calcification in vivo. Ablation of AMPKα1 in VSMC prevented Runx2 from proteasome degradation in parallel with aberrant osteoblastic differentiation of VSMC, whereas AMPK activation promoted Runx2 post-translational modification by small ubiquitin-like modifier (SUMO, SUMOylation), which is associated with its instability. Mechanically, we found that AMPKα1 directly phosphorylated protein inhibitor of activated STAT-1 (PIAS1), the SUMO E3-ligase of Runx2, at serine 510, to promote its SUMO E3-ligase activity. Finally, mutation of protein inhibitor of activated STAT-1 at serine 510 suppressed metformin-induced Runx2 SUMOylation and subsequently prevented metformin's effect on reducing oxidized low-density lipoprotein-triggered Runx2 expression in VSMC. CONCLUSIONS: AMPKα1 phosphorylated protein inhibitor of activated STAT-1 to promote Runx2 SUMOylation and subsequently lead to its instability. AMPKα1 deficiency in VSMC increased Runx2 expression and promoted atherosclerotic calcification in vivo.
背景：动脉粥样硬化钙化与斑块破裂高度相关。然而，钙化的调控机制仍知之甚少。
目的：本研究旨在探讨 AMP 激活的蛋白激酶（AMPK）在动脉粥样硬化钙化中的作用。
方法和结果：通过对动脉粥样硬化易感 ApoE（-/-）小鼠的主动脉根部和头臂动脉，或全身（ApoE（-/-）/AMPKα1（-/-）和 ApoE（-/-）/AMPKα2（-/-））或血管平滑肌细胞（VSMC）特异性或巨噬细胞特异性 AMPKα1 基因敲除小鼠，以及给予西方饮食 24 周的 ApoE（-/-）小鼠，评估动脉粥样硬化钙化和 Runx2（Runt 相关转录因子）的表达情况。AMPKα1 而不是 AMPKα2 的遗传缺失促进了动脉粥样硬化钙化和 Runx2 的表达。相反，AMPK 的激活剂二甲双胍可显著减少 ApoE（-/-）小鼠的动脉粥样硬化钙化和 Runx2 的表达，但对 ApoE（-/-）/AMPKα1（-/-）小鼠的作用较小。此外，VSMC 特异性而非巨噬细胞特异性 AMPKα1 缺失促进了体内主动脉钙化。VSMC 中 AMPKα1 的缺失可防止 Runx2 被蛋白酶体降解，同时伴随着 VSMC 的异常成骨样分化，而 AMPK 的激活可促进 Runx2 的翻译后修饰（小泛素相关修饰物 SUMO，SUMOylation），从而导致其不稳定。从机制上讲，我们发现 AMPKα1 可直接在丝氨酸 510 位磷酸化 Runx2 的蛋白抑制因子激活 STAT-1（PIAS1），即 Runx2 的 SUMO E3 连接酶，以促进其 SUMO E3 连接酶活性。最后，丝氨酸 510 位的蛋白抑制因子激活 STAT-1 的突变抑制了二甲双胍诱导的 Runx2 SUMOylation，进而阻止了二甲双胍对降低氧化型低密度脂蛋白诱导的 VSMC 中 Runx2 表达的作用。
结论：AMPKα1 磷酸化蛋白抑制因子激活 STAT-1 以促进 Runx2 SUMOylation，并随后导致其不稳定。VSMC 中的 AMPKα1 缺失增加了 Runx2 的表达，并促进了体内动脉粥样硬化钙化。
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