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.

Fig. 1.

TNF up-regulates Msx2 mRNA accumulation in primary aortic adventitial myofibroblasts. A, Upper left panel, Primary aortic myofibroblasts cultures were treated with TNF for 3 h and total cellular RNA harvested for gene expression analysis by RT-qPCR. Unlike Msx1, Msx2 is up-regulated by TNF (5 ng/ml; n = 3 per group; P < 0.05). Upper right and bottom panels, Msx2 protein levels as assessed by Western blot were also up-regulated by TNF after 4 h of treatment (1.8-fold, P < 0.05; n = 3 per group; see also Supplemental Fig. 1). B, Other inflammatory polypeptides and cytokines such as endothelin, AngII, IL-6, TRAIL, and RANKL do not increase Msx2 message, but IL-1β is active (n = 3 per treatment, P < 0.05).

Fig. 2.

TNF induction of Msx2 gene expression requires intact Nox and mitochondrial redox signaling. A, Treatment of primary aortic myofibroblasts with the Nox inhibitor apocynin partially reduced Msx2 while completely preventing TNF induction of Nox2. DPI, a dual inhibitor of Nox and mitochondrial respiration, abrogated TNF induction of both Msx2 and Nox2 (n = 3 per group; replicated > 10 times). Wnt7b responses parallel those of Msx2. Lower panel, Myofibroblasts from mice deficient for p47phox^−/−, an obligatory coregulator of Nox1 and Nox2 activity and target of apocynin inhibition, exhibit significantly reduced Msx2 induction (n = 3 per treatment; P < 0.05). ASO directed toward Nox1 and Nox2 reduced TNF-induced oxidative stress (B) and Msx2 induction (C; n = 4 / group; replicated more than three times). Nox1 and Nox2 ASO concentrations were each at 0.25 μm and the control ASO (CON ASO) at 0.5 μm. B, inset, Western blot analyses confirm reductions in Nox1 and Nox2 protein levels with ASO treatment. Pharmacological inhibitors were introduced 30 min before treatment with TNF, and cultures were incubated overnight with the indicated ASO before TNF treatment. D, Mice transgenic for SM22-TNF express elevated aortic Msx2 but not Msx1 (n = 6–8 per genotype; P = 0.05). E, As in vitro, administration of Nox1+Nox2 ASO (n = 8) significantly reduces aortic Msx2 in vivo in SM22-TNF transgenic mice vs. control ASO (n = 6). OPN, another TNF target, is also reduced, whereas macrophage CD68 is not. *, P < 0.05 vs. TNF; **, P < 0.01 vs. TNF; ***, P < 0.001 vs.TNF.

Fig. 3.

The mitochondrial respiratory chain inhibitor rotenone reduces aortic adventitial Msx2 expression in vitro and in vivo. A, Rotenone inhibits TNF induction of Msx2-Wnt signaling in primary aortic myofibroblast cultures (n = 6 per treatment). Msx1 and GAPDH (glyceraldehyde phosphate dehydrogenase) were not altered. Rotenone treatment was initiated 30 min before challenge with TNF. B, Conversely, pyruvic acid, an anapleurotic substrate for mitochondrial tricarboxylic cycle metabolism, significantly enhances TNF induction of Msx2 (n = 6 per treatment). C, In vivo rotenone treatment reduces aortic Msx2, but not Msx1, in SM22-TNF transgenic mice (n = 16 animals per treatment group).

Fig. 4.

Hydrogen peroxide (H₂O₂) supports Msx2 induction in aortic adventitial myofibroblasts. A, The glutathione peroxidase-mimetic ebselen inhibits TNF induction of Msx2 (n = 4 per treatment group). Cultures were pretreated with ebselen for 30 min before treatment with TNF. B, Three hours of exogenous H₂O₂ treatment dose dependently increases Msx2 expression, phenocopying TNF (n = 3 per treatment group). C, Msx2 protein levels were also increased by H₂O₂ treatment (n = 4 per treatment group; P < 0.047; see also Supplemental Fig. 3).

Fig. 5.

Hydrogen peroxide up-regulates Msx2 promoter activity. A, upper panel, TNF treatment stimulates the Msx2 promoter-luciferase reporter activity in transiently transfected C3H10T1/2 mesenchymal cells via proximal promoter elements. A, lower panel, peroxide treatment also stimulates Msx2 promoter-luciferase reporter activity via proximal promoter elements. By contrast, the RSV (Rous sarcoma virus) proximal promoter was not stimulated by peroxide. Treatments were for 6 h, n = 3 per treatment group, and replicated more than three times. B, Upper-strand sequences of the Msx2 proximal promoter duplex oligos radiolabeled and used as EMSA probes. C, H₂O₂ treatment up-regulates DNA binding activity recognizing the proximal Msx2 promoter regions −69/−40 and −11/+26 detected by gel shift assay. Brackets indicate the EMSA complexes visualized by autoradiography. The unbound/unshifted radiolabeled duplex probes have been run off the bottom of the gel. Treatments were for 4 h. D, Upper-strand sequences of the native (WT) and mutant (M1–M5) Msx2 proximal promoter duplex oligos used for cold competition studies. E, Cold competition studies assessing binding specificity of complexes recognizing the Msx2 promoter region −69 to −40 (lanes 1–5) and −11 to +36 (lanes 6–10). The unbound, unshifted radiolabeled duplex probes are seen at the bottom of this gel (arrow). **, P < 0.01 vs. vehicle.

Fig. 6.

Intact TNFR1 is required for Msx2 expression and robust mineralization in primary aortic myofibroblasts. Primary aortic myofibroblast cultures were established from WT, TNFR1^−/−, and TNFR2^−/− mice at high cell densities (6 × 10⁵ per well), treated with either vehicle or 10 ng/ml TNF for 3 h, and RNA extracted for gene expression analysis. A, Loss of TNFR1 but not TNFR2 expression markedly reduced both basal and TNF induced Msx2 mRNA accumulation. Expression of the related homeodomain protein Msx1 was dependent on TNFR2, not TNFR1. The expression of osteogenic factor Wnt7b (84) parallels the expression of Msx2 (n = 3 per group; replicated more than two times). B, The dephosphorylated form of β-catenin, an index of activated canonical Wnt signaling (85), was reduced in TNFR1^−/− aortic myofibroblasts as assessed by Western blot (n = 3 per group, P = 0.0002). C, Calcium deposition was assessed after treatment with vehicle, 10 ng/ml TNF, 2.5 mm supplemental NaPi, or TNF + NaPi in mineralization media (n = 3 per treatment, replicated more than three times). Alizarin Red calcium staining demonstrated exhibited significantly reduced calcification in TNFR1^−/− cells compared with wild-type controls and TNFR2^−/− cultures.

Fig. 7.

A working model for regulation of vascular Msx2 gene expression via TNF and oxidative stress signaling. Inflammatory cytokine signaling initiated by TNF/TNFR1 engagement or IL-1β stimulation (not depicted) activates NAPDH oxidase (Nox) and mitochondrial ROS production. DPI-sensitive flavoenzymes in Nox and mitochondrial complexes generate ROS signals that activate Msx2 gene expression and downstream osteogenic Wnt/β-catenin cascades in vascular mesenchymal cells. Glutathione peroxidase mimetics such as ebselen (55) inhibit peroxide (H₂O₂)-dependent activation upstream of vascular osteogenic Wnt/β-catenin programs. Not depicted is the role for ROS-dependent vascular Runx2 activation, a transcription factor vital to the robust elaboration of osteogenic gene-regulatory programs during bone (86) and vascular (69) mineralization.