Department of Internal Medicine, Washington University in St. Louis, Missouri 63110, USA.
Endocrinology. 2012 Aug;153(8):3897-910. doi: 10.1210/en.2012-1216. Epub 2012 Jun 8.
In LDLR(-/-) mice fed high-fat diabetogenic diets, osteogenic gene-regulatory programs are ectopically activated in vascular myofibroblasts and smooth muscle cells that promote arteriosclerotic calcium deposition. Msx2-Wnt signaling pathways previously identified as important for craniofacial skeletal development are induced in the vasculature by TNF, a prototypic cytokine mediator of the low-grade systemic inflammation of diabesity. To better understand this biology, we studied TNF actions on Msx2 in aortic myofibroblasts. TNF up-regulated Msx2 mRNA 4-fold within 3 h but did not regulate Msx1. Although IL-1β could also induce Msx2 expression, TNF-related apoptosis inducing ligand, receptor activator of nuclear factor-κB ligand, and IL-6 were inactive. Inhibition of nicotinamide adenine dinucleotide phosphate oxidase (Nox) activity and genetically induced Nox deficiency (p47phox(-/-)) reduced Msx2 induction, indicating contributions of reactive oxygen species (ROS) and redox signaling. Consistent with this, rotenone, an antagonist of mitochondrial complex I, inhibited TNF induction of Msx2 and Nox2, whereas pyruvate, an anapleurotic mitochondrial metabolic substrate, enhanced induction. Moreover, the glutathione peroxidase-mimetic ebselen abrogated this TNF response. Treatment of aortic myofibroblasts with hydrogen peroxide up-regulated Msx2 mRNA, promoter activity, and DNA-protein interactions. In vivo, SM22-TNF transgenic mice exhibit increased aortic Msx2 with no change in Msx1. Dosing SM22-TNF mice with either 20 ng/g Nox1 + 20 ng/g Nox2 antisense oligonucleotides or low-dose rotenone reduced arterial Msx2 expression. Aortic myofibroblasts from TNFR1(-/-) mice expressed levels of Msx2 that were 5% that of wild-type and were not inducible by TNF. Wnt7b and active β-catenin levels were also reduced. By contrast, TNF-inducible Msx2 expression was not reduced in TNFR2(-/-) cells. Finally, when cultured under mineralizing conditions, TNFR1(-/-) aortic myofibroblasts exhibited reduced calcification compared with wild-type and TNFR2(-/-) cells. Thus, ROS metabolism contributes to TNF induction of Msx2 and procalcific responses in myofibroblasts via TNFR1. Strategies that reduce vascular Nox- or mitochondrially activated ROS signals may prove useful in mitigating arteriosclerotic calcification.
在喂食高脂肪致糖尿病饮食的 LDLR(-/-) 小鼠中，血管平滑肌成纤维细胞和血管平滑肌细胞中异位激活了成骨基因调控程序，促进了动脉粥样硬化性钙沉积。此前已确定 TNF 是颅面骨骼发育的重要 Msx2-Wnt 信号通路的原型细胞因子介质，可在血管中诱导。为了更好地了解这种生物学，我们研究了 TNF 对主动脉肌成纤维细胞中 Msx2 的作用。TNF 在 3 小时内将 Msx2 mRNA 上调了 4 倍，但不调节 Msx1。虽然白细胞介素-1β也可以诱导 Msx2 表达，但 TNF 相关凋亡诱导配体、核因子-κB 配体受体激活剂和白细胞介素-6 均无活性。烟酰胺腺嘌呤二核苷酸磷酸氧化酶 (Nox) 活性抑制和基因诱导的 Nox 缺乏 (p47phox(-/-)) 降低了 Msx2 的诱导，表明活性氧 (ROS) 和氧化还原信号的贡献。与此一致，鱼藤酮，一种线粒体复合物 I 的拮抗剂，抑制 TNF 诱导的 Msx2 和 Nox2，而丙酮酸，一种代谢性线粒体代谢底物，增强诱导作用。此外，谷胱甘肽过氧化物酶模拟物依布硒啉消除了这种 TNF 反应。用过氧化氢处理主动脉肌成纤维细胞可上调 Msx2 mRNA、启动子活性和 DNA-蛋白质相互作用。在体内，SM22-TNF 转基因小鼠表现出主动脉 Msx2 增加，而 Msx1 没有变化。用 20ng/g Nox1+20ng/g Nox2 反义寡核苷酸或低剂量鱼藤酮对 SM22-TNF 小鼠进行给药可降低动脉 Msx2 表达。来自 TNFR1(-/-) 小鼠的主动脉肌成纤维细胞表达的 Msx2 水平为野生型的 5%，且不能被 TNF 诱导。Wnt7b 和活性 β-连环蛋白水平也降低。相比之下，TNFR2(-/-) 细胞中 TNF 诱导的 Msx2 表达没有降低。最后，在矿化条件下培养时，与野生型和 TNFR2(-/-) 细胞相比，TNFR1(-/-) 主动脉肌成纤维细胞的钙化减少。因此，ROS 代谢通过 TNFR1 促进 TNF 诱导的 Msx2 表达和肌成纤维细胞中的促钙化反应。减少血管 Nox 或线粒体激活的 ROS 信号的策略可能有助于减轻动脉粥样硬化性钙化。
