Smad5 and DPC4 Are Key Molecules in Mediating BMP-2-induced Osteoblastic Differentiation of the Pluripotent Mesenchymal Precursor Cell Line C2C12*
- Riko Nishimura‡§,
- Yoichi Kato‡,
- Di Chen‡,
- Stephen E. Harris‡,
- Gregory R. Mundy‡ and
- Toshiyuki Yonedaद
- From the ‡Division of Endocrinology and Bone Metabolism, Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284 and the§Department of Biochemistry, Faculty of Dentistry, Osaka University, 565-0871 Osaka, Japan
Abstract
Since the bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β (TGF-β) superfamily that induce the differentiation of mesenchymal precursor cells into the osteogenic cells, we identified the relevant signaling molecules responsible for mediating BMP-2 effects on mesenchymal precursor cells. BMP-2 induces osteoblastic differentiation of the pluripotent mesenchymal cell line C2C12 by increasing alkaline phosphatase activity and osteocalcin production. As recent studies have demonstrated that cytoplasmic Smad proteins are involved in TGF-β superfamily signaling, we plan to isolate the relevant Smad family members involved in osteoblastic differentiation. We identified human Smad5, which is highly homologous to Smad1. BMP-2 caused serine phosphorylation of Smad5 as well as Smad1. In contrast, TGF-β failed to cause serine phosphorylation of Smad1 and Smad5. We found Smad5 is directly activated by BMP type Ia or Ib receptors through physical association with these receptors. Following phosphorylation, Smad5 bound to DPC4, another Smad family member, and the complex was translocated to the nucleus. Overexpression of point-mutated Smad5 (G419S) or a C-terminal deletion mutant DPC4 (DPC4ΔC) blocked the induction of alkaline phosphatase activity, osteocalcin production, and Smad5-DPC4 signaling cascades upon BMP-2 treatment in C2C12 cells. These data suggest that activation of Smad5 and subsequent Smad5-DPC4 complex formation are key steps in the BMP signaling pathway, which mediates BMP-2-induced osteoblastic differentiation of the C2C12 mesenchymal cells.
Footnotes
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↵* This work was supported in part by National Institutes of Health Grant RO1-DK45229-05.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵¶ To whom correspondence should be addressed: Dept. of Medicine, Div. of Endocrinology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78284-7877. Tel.: 210-567-4900; Fax: 210-567-6693; E-mail: yoneda{at}uthscsa.edu.
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↵1 The abbreviations used are: BMP, bone morphogenetic protein; TGF-β, transforming growth factor-β; BMPIR, BMP type I receptor; BMPIaR, BMP type Ia receptor; BMPIbR, BMP type Ib receptor; BMPRII, BMP type II receptor; TGF-βRI, TGF-β type I receptor; TGF-βRII, TGF-β type II receptor; Mad, mother against dpp; Smad1 G419S, point mutant Smad1 (glycine 419 replaced by serine); Smad5 G419S, point mutant Smad5 (glycine 419 replaced by serine); DPC4ΔC, C-terminal deletion mutant of DPC4; Smad3 G379S, point mutant Smad3 (glycine 379 replaced by serine); ALP, alkaline phosphatase; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; GST, glutathioneS-transferase.
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↵2 R. Nishimura, Y. Kato, D. Chen, S. E. Harris, G. R. Mundy, and T. Yoneda, unpublished observation.
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- Received June 25, 1997.
- Revision received October 17, 1997.
- The American Society for Biochemistry and Molecular Biology, Inc.
