Msx2 Stimulates Chondrocyte Maturation by Controlling Ihh Expression

Katsuhiko Amano,Fumitaka Ichida,Atsushi Sugita,Kenji Hata,Masahiro Wada,Yoko Takigawa,Masako Nakanishi,Mikihiko Kogo,Riko Nishimura,Toshiyuki Yoneda

doi:10.1074/jbc.m803681200

Katsuhiko Amano, Fumitaka Ichida + Show 8 more

Open Access

https://doi.org/10.1074/jbc.m803681200

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Journal: Journal of Biological Chemistry	Publication Date: Oct 1, 2008
Citations: 37	License type: cc-by

Affiliation: Osaka University

Abstract

Several studies indicated that a homeobox gene, Msx2, is implicated in regulation of skeletal development by controlling enchondral ossification as well as membranous ossification. However, the molecular basis by which Msx2 conducts chondrogenesis is currently unclear. In this study, we examined the role of Msx2 in chondrocyte differentiation using mouse primary chondrocytes and embryonic metatarsal explants. Treatment with BMP2 up-regulated the expression of Msx2 mRNA along with chondrocyte differentiation in murine primary chondrocytes. Overexpression of wild-type Msx2 stimulated calcification of primary chondrocytes in the presence of BMP2. We also found that constitutively active Msx2 (caMsx2) enhanced BMP2-dependent calcification more efficiently than wild-type Msx2. Consistently, caMsx2 overexpression up-regulated the expression of alkaline phosphatase and collagen type X induced by BMP2. Furthermore, organ culture experiments using mouse embryonic metatarsals indicated that caMsx2 clearly stimulated the maturation of chondrocytes into the prehypertrophic and hypertrophic stages in the presence of BMP2. In contrast, knockdown of Msx2 inhibited maturation of primary chondrocytes. The stimulatory effect of Msx2 on chondrocyte maturation was enhanced by overexpression of Smad1 and Smad4 but inhibited by Smad6, an inhibitory Smad for BMP2 signaling. These data suggest that Msx2 requires BMP2/Smad signaling for its chondrogenic action. In addition, caMsx2 overexpression induced Ihh (Indian hedgehog) expression in mouse primary chondrocytes. Importantly, treatment with cyclopamine, a specific inhibitor for hedgehogs, blocked Msx2-induced chondrogenesis. Collectively, our results indicated that Msx2 promotes the maturation of chondrocytes, at least in part, through up-regulating Ihh expression.

Highlights

Chondrocytes, and secret the matrix rich in type II collagen and aggrecan [1]
Msx2 clearly stimulated the calcification of the primary chondrocytes in the presence of BMP2 (Fig. 2B). constitutively active Msx2 (caMsx2) more efficiently enhanced the chondrogenic action of BMP2 than wild type Msx2 (Fig. 2B)
CaMsx2 failed to stimulate Col2␣1 expression and differentiation into Alcian blue-positive chondrocyte differentiation (Fig. 2, D and E). These results indicated that Msx2 is involved in chondrocyte maturation rather than initiation of chondrocyte differentiation

Summary

Introduction

Chondrocytes, and secret the matrix rich in type II collagen and aggrecan [1]. The chondrocytes stop to proliferate, further differentiate into hypertrophic chondrocytes, produce type X collagen, and calcify. Organ culture experiments using mouse embryonic metatarsals indicated that caMsx2 clearly stimulated the maturation of chondrocytes into the prehypertrophic and hypertrophic stages in the presence of BMP2. Our results indicated that Msx2 promotes the maturation of chondrocytes, at least in part, through up-regulating Ihh expression.

Results

Conclusion