TNFR1-Activated Reactive Oxidative Species Signals Up-Regulate Osteogenic Msx2 Programs in Aortic Myofibroblasts

Abstract

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.

Cross-Talk of Receptor Activator of Nuclear Factor-κB Ligand Signaling With Renin–Angiotensin System in Vascular Calcification

“…15 In this study, we also showed that Ang II increased RANKL mRNA level and RANK expression in VSMC ( Figure 1C-1E), whereas the blockade of RANKL by neutralizing anti-RANKL antibody significantly inhibited Ang II-induced cbfa1 expression ( Figure 1F and 1G). In contrast with other studies that show no direct effect of RANKL in the dedifferentiation of VSMC into osteoblast-like cells, 12,16 we have reported the induction of cbfa1 expression under RANKL stimulation. 2 The discrepancy may be because of the addition of anti-OPG neutralizing antibody that by binding to the abundant endogenous OPG allowed the proper stimulation by RANKL in the VSMC and consequent induction of cbfa1 expression.…”

AMPK dysregulation promotes diabetes-related reduction of superoxide and mitochondrial function

“…Indeed, AMPK activity was reduced in the diabetic mouse kidney and glomeruli, as demonstrated by both immunostaining and a quantitative assay ( Figure 4F and Supplemental Figure 3B To establish a link between reduced mitochondrial superoxide production and AMPK regulation, we administered rotenone to wild-type mice. Short-term rotenone has been previously demonstrated to reduce superoxide production (20,21), and we found that rotenone administration led to a marked reduction in overall renal and heart DHE fluorescence consistent with a reduction in mitochondrial superoxide production ( Figure 5, A and B). Although no effects were seen in blood glucose levels, rotenone reduced p-AMPK in both the kidney and heart ( Figure 5, C-E, and G), indicating that reduced mitochondrial superoxide is sufficient to lower AMPK activity.…”

Activating transcription factor-4 promotes mineralization in vascular smooth muscle cells

“…Many key transcriptional regulators involved in skeletal osteogenesis, such as Msx-2, osterix, and Runx-2, are expressed in both calcified medial arterial layers and atherosclerotic plaques (22,23). In addition, many causative hormonal factors (TNF-α, FGF23/Klotho, and phosphorus) and lipid-derived factors (saturated fats and oxidized lipids) have been identified in vascular calcification (7)(8)(9)(10)(24)(25)(26)(27)(28)(29). We recently reported that these factors induce ATF4 activation through the ER stress response, resulting in osteogenic differentiation and mineralization of vascular smooth muscle cells (VSMCs) in vitro (8,9).…”