Robert M. Berne Cardiovascular Research Center, Charlottesville, VA 22908, USA.
Circ Res. 2012 Jul 6;111(2):201-11. doi: 10.1161/CIRCRESAHA.112.272302. Epub 2012 May 31.
Dedifferentiation of vascular smooth muscle cells (VSMC) leading to a proliferative cell phenotype significantly contributes to the development of atherosclerosis. Mitogen-activated protein kinase (MAPK) phosphorylation of proteins including connexin 43 (Cx43) has been associated with VSMC proliferation in atherosclerosis.
To investigate whether MAPK phosphorylation of Cx43 is directly involved in VSMC proliferation.
We show in vivo that MAPK-phosphorylated Cx43 forms complexes with the cell cycle control proteins cyclin E and cyclin-dependent kinase 2 (CDK2) in carotids of apolipoprotein-E receptor null (ApoE(-/-)) mice and in C57Bl/6 mice treated with platelet-derived growth factor-BB (PDGF). We tested the involvement of Cx43 MAPK phosphorylation in vitro using constructs for full-length Cx43 (Cx43) or the Cx43 C-terminus (Cx43(CT)) and produced null phosphorylation Ser>Ala (Cx43(MK4A)/Cx43(CTMK4A)) and phospho-mimetic Ser>Asp (Cx43(MK4D)/Cx43(CTMK4D)) mutations. Coimmunoprecipitation studies in primary VSMC isolated from Cx43 wild-type (Cx43(+/+)) and Cx43 null (Cx43(-/-)) mice and analytic size exclusion studies of purified proteins identify that interactions between cyclin E and Cx43 requires Cx43 MAPK phosphorylation. We further demonstrate that Cx43 MAPK phosphorylation is required for PDGF-mediated VSMC proliferation. Finally, using a novel knock-in mouse containing Cx43-MK4A mutation, we show in vivo that interactions between Cx43 and cyclin E are lost and VSMC proliferation does not occur after treatment of carotids with PDGF and that neointima formation is significantly reduced in carotids after injury.
We identify MAPK-phosphorylated Cx43 as a novel interacting partner of cyclin E in VSMC and show that this interaction is critical for VSMC proliferation. This novel interaction may be important in the development of atherosclerotic lesions.
血管平滑肌细胞（VSMC）去分化导致增殖细胞表型显著促进动脉粥样硬化的发展。有研究表明，包括缝隙连接蛋白 43（Cx43）在内的蛋白的丝裂原活化蛋白激酶（MAPK）磷酸化与动脉粥样硬化中的 VSMC 增殖有关。
研究 Cx43 的 MAPK 磷酸化是否直接参与 VSMC 增殖。
我们体内研究表明，MAPK 磷酸化的 Cx43 在载脂蛋白 E 受体缺失（ApoE(-/-)）小鼠和接受血小板衍生生长因子-BB（PDGF）处理的 C57Bl/6 小鼠的颈动脉中与细胞周期控制蛋白周期蛋白 E 和细胞周期依赖性激酶 2（CDK2）形成复合物。我们使用全长 Cx43（Cx43）或 Cx43 C 端（Cx43(CT)）构建体在体外测试了 Cx43 MAPK 磷酸化的参与，并产生了无磷酸化丝氨酸突变为丙氨酸（Cx43(MK4A)/Cx43(CTMK4A)）和磷酸化模拟丝氨酸突变为天冬氨酸（Cx43(MK4D)/Cx43(CTMK4D)）突变。从 Cx43 野生型（Cx43(+/+)）和 Cx43 缺失（Cx43(-/-)）小鼠分离的原代 VSMC 的共免疫沉淀研究和纯化蛋白的分析大小排阻研究表明，周期蛋白 E 与 Cx43 之间的相互作用需要 Cx43 MAPK 磷酸化。我们进一步证明 Cx43 MAPK 磷酸化是 PDGF 介导的 VSMC 增殖所必需的。最后，使用含有 Cx43-MK4A 突变的新型基因敲入小鼠，我们在体内表明，PDGF 处理后 Cx43 与 cyclin E 之间的相互作用丢失，VSMC 增殖不会发生，并且颈动脉损伤后新生内膜形成明显减少。
我们确定 MAPK 磷酸化的 Cx43 作为 VSMC 中 cyclin E 的新相互作用伙伴，并表明这种相互作用对于 VSMC 增殖至关重要。这种新的相互作用可能对动脉粥样硬化病变的发展很重要。
