Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL, USA.
Arterioscler Thromb Vasc Biol. 2011 Jun;31(6):1387-96. doi: 10.1161/ATVBAHA.110.222547. Epub 2011 Mar 31.
Clinical and experimental studies demonstrate the important roles of vascular smooth muscle cells (VSMC) in the pathogenesis of atherosclerosis. We have previously determined that the osteogenic transcription factor Runx2 is essential for VSMC calcification. The present study characterized Runx2-regulated signals and their potential roles in vascular calcification.
In vivo studies with atherogenic apolipoprotein E(-/-) mice demonstrated that increased oxidative stress was associated with upregulation of Runx2 and receptor activator of nuclear factor κB ligand (RANKL), which colocalized in the calcified atherosclerotic lesions and were juxtaposed to infiltrated macrophages and osteoclast-like cells that are positively stained for an osteoclast marker, tartrate-resistant acid phosphatase. Mechanistic studies using RNA interference, a luciferase reporter system, chromatin immunoprecipitation, and electrophoretic mobility shift assays indicated that Runx2 regulated the expression of RANKL via a direct binding to the 5'-flanking region of the RANKL. Functional characterization revealed that RANKL did not induce VSMC calcification, nor was RANKL required for oxidative stress-induced VSMC calcification. Using a coculture system, we demonstrated that VSMC-expressed RANKL induced migration as well as differentiation of bone marrow-derived macrophages into multinucleated, tartrate-resistant acid phosphatase-positive osteoclast-like cells. These effects were inhibited by the RANKL antagonist osteoprotegerin and with VSMC deficient in Runx2 or RANKL.
We demonstrate that Runx2 directly binds to the promoter and controls the expression of RANKL, which mediates the crosstalk between calcifying VSMC and migration and differentiation of macrophages into osteoclast-like cells in the atherosclerotic lesions. Our studies provide novel mechanistic insights into the regulation and function of VSMC-derived RANKL in the pathogenesis of atherosclerosis and vascular calcification.
临床和实验研究表明血管平滑肌细胞（VSMC）在动脉粥样硬化的发病机制中起着重要作用。我们之前已经确定了成骨转录因子 Runx2 是 VSMC 钙化所必需的。本研究描述了 Runx2 调节的信号及其在血管钙化中的潜在作用。
用动脉粥样硬化的载脂蛋白 E（-/-）小鼠进行的体内研究表明，氧化应激的增加与 Runx2 和核因子 κB 受体激活剂配体（RANKL）的上调有关，RANKL 与钙化的动脉粥样硬化病变共定位，并与浸润的巨噬细胞和破骨细胞样细胞相邻，这些细胞对破骨细胞标志物抗酒石酸酸性磷酸酶呈阳性染色。使用 RNA 干扰、荧光素酶报告系统、染色质免疫沉淀和电泳迁移率变动分析的机制研究表明，Runx2 通过直接结合 RANKL 的 5'-侧翼区域来调节 RANKL 的表达。功能特征表明，RANKL 本身不会诱导 VSMC 钙化，也不是氧化应激诱导的 VSMC 钙化所必需的。使用共培养系统，我们证明了 VSMC 表达的 RANKL 诱导骨髓源性巨噬细胞迁移，并分化为多核、抗酒石酸酸性磷酸酶阳性的破骨细胞样细胞。这些作用被 RANKL 拮抗剂骨保护素抑制，并且在缺乏 Runx2 或 RANKL 的 VSMC 中也被抑制。
我们证明 Runx2 直接结合启动子并控制 RANKL 的表达，RANKL 介导了钙化的 VSMC 与迁移和分化的巨噬细胞之间的串扰，进入动脉粥样硬化病变中的破骨细胞样细胞。我们的研究为 VSMC 衍生的 RANKL 在动脉粥样硬化和血管钙化发病机制中的调节和功能提供了新的机制见解。
