From the Department of Pathology, Metabolic Diseases Institute, University of Cincinnati, OH.
Arterioscler Thromb Vasc Biol. 2014 Jul;34(7):1429-36. doi: 10.1161/ATVBAHA.114.303439. Epub 2014 May 1.
Mitogen-activated protein kinase pathways play an important role in neointima formation secondary to vascular injury, in part by promoting proliferation of vascular smooth muscle cells (VSMC). Mixed-lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase that activates multiple mitogen-activated protein kinase pathways and has been implicated in regulating proliferation in several cell types. However, the role of MLK3 in VSMC proliferation and neointima formation is unknown. The aim of this study was to determine the function of MLK3 in the development of neointimal hyperplasia and to elucidate the underlying mechanisms.
Neointima formation was analyzed after endothelial denudation of carotid arteries from wild-type and MLK3-deficient mice. MLK3 deficiency promoted injury-induced neointima formation and increased proliferation of primary VSMC derived from aortas isolated from MLK3-deficient mice compared with wild-type mice. Furthermore, MLK3 deficiency increased the activation of p63Rho guanine nucleotide exchange factor, RhoA, and Rho kinase in VSMC, a pathway known to promote neointimal hyperplasia, and reconstitution of MLK3 expression attenuated Rho kinase activation. Furthermore, cJun NH2-terminal kinase activation was decreased in MLK3-deficient VSMC, and proliferation of wild-type but not MLK3 knockout cells treated with a cJun NH2-terminal kinase inhibitor was attenuated.
We demonstrate that MLK3 limits RhoA activation and injury-induced neointima formation by binding to and inhibiting the activation of p63Rho guanine nucleotide exchange factor, a RhoA activator. In MLK3-deficient cells, activation of p63Rho guanine nucleotide exchange factor proceeds in an unchecked manner, leading to a net increase in RhoA pathway activation. Reconstitution of MLK3 expression restores MLK3/p63Rho guanine nucleotide exchange factor interaction, which is attenuated by feedback from activated cJun NH2-terminal kinase.
丝裂原活化蛋白激酶途径在血管损伤引起的新生内膜形成中起着重要作用，部分通过促进血管平滑肌细胞（VSMC）增殖。混合谱系激酶 3（MLK3）是一种丝裂原活化蛋白激酶激酶激酶，可激活多种丝裂原活化蛋白激酶途径，并被认为在调节多种细胞类型的增殖中起作用。然而，MLK3 在 VSMC 增殖和新生内膜形成中的作用尚不清楚。本研究旨在确定 MLK3 在新生内膜增生中的作用，并阐明其潜在机制。
从野生型和 MLK3 缺陷型小鼠的颈动脉内皮剥脱后分析新生内膜形成。与野生型小鼠相比，MLK3 缺陷促进了损伤诱导的新生内膜形成，并增加了源自 MLK3 缺陷型小鼠主动脉的原代 VSMC 的增殖。此外，MLK3 缺陷增加了 VSMC 中 p63Rho 鸟嘌呤核苷酸交换因子、RhoA 和 Rho 激酶的激活，该途径已知可促进新生内膜增生，并且 MLK3 表达的重建减弱了 Rho 激酶的激活。此外，MLK3 缺陷型 VSMC 中 cJun N 末端激酶的激活减少，并且用 cJun N 末端激酶抑制剂处理的野生型而非 MLK3 敲除细胞的增殖受到抑制。
我们证明 MLK3 通过与 p63Rho 鸟嘌呤核苷酸交换因子结合并抑制其激活来限制 RhoA 的激活和损伤诱导的新生内膜形成，p63Rho 鸟嘌呤核苷酸交换因子是 RhoA 的激活剂。在 MLK3 缺陷型细胞中，p63Rho 鸟嘌呤核苷酸交换因子的激活以不受控制的方式进行，导致 RhoA 途径的激活净增加。MLK3 表达的重建恢复了 MLK3/p63Rho 鸟嘌呤核苷酸交换因子的相互作用，该作用被激活的 cJun N 末端激酶的反馈减弱。
