A Kinase Domain-truncated Type I Receptor Blocks Bone Morphogenetic Protein-2-induced Signal Transduction in C2C12 Myoblasts

Mana Namiki,Shuichi Akiyama,Takenobu Katagiri,Atsushi Suzuki,Naoto Ueno,Noboru Yamaji,Vicki Rosen,John M Wozney,Tatsuo Suda

doi:10.1074/jbc.272.35.22046

Mana Namiki, Shuichi Akiyama + Show 7 more

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https://doi.org/10.1074/jbc.272.35.22046

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Abstract

Members of the transforming growth factor (TGF)-beta superfamily bind the transmembrane serine/threonine kinase complex consisting of type I and type II receptors. Their intracellular signals are propagated via respective type I receptors. Bone morphogenetic protein (BMP)-2, a member of the TGF-beta superfamily, induces ectopic bone formation when implanted into muscular tissues. Two type I receptors (BMPR-IA and BMPR-IB) have been identified for BMP-2. We have reported that BMP-2 inhibits the terminal differentiation of C2C12 myoblasts and converts their differentiation pathway into that of osteoblast lineage cells (Katagiri, T., Yamaguchi, A., Komaki, M., Abe, E., Takahashi, N., Ikeda, T., Rosen, V., Wozney, J. M., Fujisawa-Sehara, A. and Suda, T. (1994) J. Cell Biol. 127, 1755-1766). In the present study, we examined the involvement of functional BMP-2 type I receptors in signal transduction in C2C12 cells, which expressed mRNA for BMPR-IA, but not for BMPR-IB in Northern blotting. TGF-beta type I receptor (TbetaR-I) mRNA was also expressed in C2C12 cells. Subclonal cell lines of C2C12 that stably expressed a kinase domain-truncated BMPR-IA (DeltaBMPR-IA) differentiated into myosin heavy chain-expressing myotubes but not into alkaline phosphatase (ALP)-positive cells, even in the presence of BMP-2. In contrast, the differentiation of the DeltaBMPR-IA-transfected C2C12 cells into myotubes was suppressed by TGF-beta1, as in the parental C2C12 cells. BMP-2 did not efficiently suppress the mRNA expression of muscle-specific genes such as muscle creatine kinase, MyoD, and myogenin, nor did it induce the expression of ALP mRNA in the DeltaBMPR-IA-transfected C2C12 cells. In contrast, TGF-beta1 inhibited mRNA expression of the muscle-specific genes in those cells. When wild-type BMPR-IA was transiently transfected into the DeltaBMPR-IA-transfected C2C12 cells, a number of ALP-positive cells appeared in the presence of BMP-2. Transfection of wild-type BMPR-IB or TbetaR-I failed to increase the number of ALP-positive cells. These results suggest that the BMP-2-induced signals, which inhibit myogenic differentiation and induce osteoblast differentiation, are transduced via BMPR-IA in C2C12 myoblasts.

Highlights

Demineralized bone matrix can induce ectopic bone formation when implanted into muscular tissues [1]
C2C12 Myoblasts Express BMPR-IA but Not BMPR-IB— First, we examined the mRNA levels of the two Bone morphogenetic proteins (BMPs) type I receptors in C2C12 myoblasts by Northern blotting (Fig. 1)
In the C2C12 cells transfected with ⌬BMPR-IA, little alkaline phosphatase (ALP) activity was induced by 1000 ng/ml BMP-2 (Fig. 2B)

Summary

Introduction

Demineralized bone matrix can induce ectopic bone formation when implanted into muscular tissues [1]. The deduced amino acid sequence of BMPs indicates that they are members of the transforming growth factor-␤ (TGF-␤) superfamily (3, 6 – 8). The intracellular signals of members of the TGF-␤ superfamily are transduced via transmembrane serine/threonine kinase receptors [9, 11, 18, 19]. These are classified into two groups known as type I and type II receptors that are distinguishable by their amino acid sequences and functional features. This paper is available on line at http://www.jbc.org main This phosphorylated T␤R-I propagates the ligand-induced signals by phosphorylating downstream substrates. BMP-7 can interact with activin type I and type II receptors in addition to BMP-2/BMP-4 receptors [26, 28]

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