Msx2 promotes cardiovascular calcification by activating paracrine Wnt signals
Jian-Su Shao, Su-Li Cheng, Joyce M. Pingsterhaus, Nichole Charlton-Kachigian, Arleen P. Loewy, Dwight A. Towler
Jian-Su Shao, Su-Li Cheng, Joyce M. Pingsterhaus, Nichole Charlton-Kachigian, Arleen P. Loewy, Dwight A. Towler
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Article Cardiology

Msx2 promotes cardiovascular calcification by activating paracrine Wnt signals

Abstract

In diabetic LDLR–/– mice, an ectopic BMP2-Msx2 gene regulatory program is upregulated in association with vascular calcification. We verified the procalcific actions of aortic Msx2 expression in vivo. CMV-Msx2 transgenic (CMV-Msx2Tg+) mice expressed 3-fold higher levels of aortic Msx2 than nontransgenic littermates. On high-fat diets, CMV-Msx2Tg+ mice exhibited marked cardiovascular calcification involving aortic and coronary tunica media. This corresponded to regions of Msx2 immunoreactivity in adjacent adventitial myofibroblasts, suggesting a potential paracrine osteogenic signal. To better understand Msx2-regulated calcification, we studied actions in 10T1/2 cells. We found that conditioned media from Msx2-transduced 10T1/2 cells (Msx2-CM) is both pro-osteogenic and adipostatic; these features are characteristic of Wnt signaling. Msx2-CM stimulated Wnt-dependent TCF/LEF transcription, and Msx2-transduced cells exhibited increased nuclear β-catenin localization with concomitant alkaline phosphatase induction. Msx2 upregulated Wnt3a and Wnt7a but downregulated expression of the canonical inhibitor Dkk1. Dkk1 treatment reversed osteogenic and adipostatic actions of Msx2. Teriparatide, a PTH1R agonist that inhibits murine vascular calcification, suppressed vascular BMP2-Msx2-Wnt signaling. Analyses of CMV-Msx2Tg+ mice confirmed that Msx2 suppresses aortic Dkk1 and upregulates vascular Wnts; moreover, TOPGAL+ (Wnt reporter); CMV-Msx2Tg+ mice exhibited augmented aortic LacZ expression. Thus, Msx2-expressing cells elaborated an osteogenic milieu that promotes vascular calcification in part via paracrine Wnt signals.

Authors

Jian-Su Shao, Su-Li Cheng, Joyce M. Pingsterhaus, Nichole Charlton-Kachigian, Arleen P. Loewy, Dwight A. Towler

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Teriparatide, a PTH1R agonist that suppresses vascular calcification, my...
Teriparatide, a PTH1R agonist that suppresses vascular calcification, myofibroblast Wnt expression, and BMP2-Msx2-Wnt signaling. (A) Aortic mRNA levels for Msx2, Wnt3a, and Wnt7a were suppressed in diabetic dyslipidemic animals treated for 1 month with daily teriparatide (i.e., PTH1–34). PTH1–34 treatment suppresses cardiovascular calcification in this model (19). (B) mRNA levels for Msx2, Wnt3a, and Wnt7a are suppressed in cultured primary aortic myofibroblasts treated with PTH1–34. (C) Regulation of SMAD-LUC reporter activity by 100 ng/ml BMP2 and 200 nM PTH1–34 in A7r5 cells. (D) Regulation of the Msx2-enhancer activity by Wnt3a CM (50% v/v) and PTH1–34 in C2C12 cells. The Wnt3a- and BMP2-responsive Msx2 enhancer fragment –4104 to –3143 was cloned upstream of the RSVLUC minimal promoter-reporter. Note that PTH1–34 inhibits Wnt3a activation of the Msx2 enhancer. *P = 0.02; **P = 0.006.