Msx2 promotes cardiovascular calcification by activating paracrine Wnt signals
Jian-Su Shao, … , Arleen P. Loewy, Dwight A. Towler
Jian-Su Shao, … , Arleen P. Loewy, Dwight A. Towler
Published May 2, 2005
Citation Information: J Clin Invest. 2005;115(5):1210-1220. https://doi.org/10.1172/JCI24140.
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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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Figure 3

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CM from Msx2-expressing 10T1/2 cells enhances ALP activity and suppresse...
CM from Msx2-expressing 10T1/2 cells enhances ALP activity and suppresses adipogenesis. C3H10T1/2 cells were transduced with retroviruses expressing either Msx2 or LacZ, and CM was harvested. Subsequently, naive 10T1/2 cells were induced to undergo either osteogenic or adipogenic differentiation in the presence of CM from either control LacZ cells or Msx2-expressing cells. (A) CM from Msx2-expressing cells supports significantly higher levels of ALP activity than CM from control cells. Percentages indicate the percent of the total cultured cell population that stains with oil red O. (B) CM from Msx2-expressing cells suppresses adipogenesis as measured by oil red O staining. (C) 10T1/2 cells were transiently transfected with expression vectors encoding either EGFP or EGPF-Msx2 fusion protein. Four days later, cells were fixed, and dual immunofluoresence was carried out as previously described, using Vector Red to image ALP. Arrows indicate cells expressing either EGFP or EGFP-Msx2 fusion proteins (green, EGFP-Msx2 in nuclear compartment). Intense ALP staining appears adjacent to cells expressing EGFP-Msx2. *P < 0.01