Menin Is Required for Bone Morphogenetic Protein 2- and Transforming Growth Factor β-regulated Osteoblastic Differentiation through Interaction with Smads and Runx2

Kazuo Chihara,Hideaki Sowa,Hiroshi Kaji,Toshitsugu Sugimoto,Lucie Canaff,Toshihisa Komori,Geoffrey N. Hendy

doi:10.1074/jbc.m401312200

Kazuo Chihara, Hideaki Sowa + Show 5 more

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https://doi.org/10.1074/jbc.m401312200

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Abstract

Menin, the product of the multiple endocrine neoplasia type 1 (MEN1) gene, is required for commitment of multipotential mesenchymal stem cells to the osteoblast lineage, however, it inhibits their later differentiation (Sowa, H., Kaji, H., Canaff, L., Hendy, G.N., Tsukamoto, T., Yamaguchi, T., Miyazono, K., Sugimoto, T., and Chihara, K. (2003) J. Biol. Chem. 278, 21058-21069). Here, we have examined the mechanism of action of menin in regulating osteoblast differentiation using the mouse bone marrow stromal ST2 and osteoblast MC3T3-E1 cell lines. In ST2 cells, reduced menin expression achieved by transfection of menin antisense DNA (AS) antagonized bone morphogenetic protein (BMP)-2-induced alkaline phosphatase activity and osteocalcin and Runx2 mRNA expression. Menin was co-immunoprecipitated with Smad1/5 in ST2 and MC3T3-E1 cells, and inactivation of menin antagonized BMP-2-induced transcriptional activity of Smad1/5 in ST2 cells, but not MC3T3-E1 cells. Menin was co-immunoprecipitated with the key osteoblast regulator, Runx2, and AS antagonized Runx2 transcriptional activity and the ability of Runx2 to stimulate alkaline phosphatase activity only in ST2 cells but not in MC3T3-E1 cells. In the osteoblast MC3T3-E1 cells, transforming growth factor-beta and its signaling molecule, Smad3, negatively regulated Runx2 transcriptional activity. Menin and Smad3 were co-immunoprecipitated, and combined menin and Smad3 overexpression antagonized, whereas menin and the dominant-negative Smad3DeltaC together enhanced BMP-2-induced transcriptional activity of Smad1/5 and Runx2. Smad3 alone had no effect. Therefore, menin interacts physically and functionally with Runx2 in uncommitted mesenchymal stem cells, but not in well differentiated osteoblasts. In osteoblasts the interaction of menin and the transforming growth factor-beta/Smad3 pathway negatively regulates the BMP-2/Smad1/5- and Runx2-induced transcriptional activities leading to inhibition of late-stage differentiation.

Highlights

Menin, the product of the multiple endocrine neoplasia type 1 (MEN1) gene, is required for commitment of multipotential mesenchymal stem cells to the osteoblast lineage, it inhibits their later differentiation (Sowa, H., Kaji, H., Canaff, L., Hendy, G.N., Tsukamoto, T., Yamaguchi, T., Miyazono, K., Sugimoto, T., and Chihara, K. (2003) J
These results indicate that menin inactivation with antisense DNA (AS) antagonizes bone morphogenetic protein (BMP)-2-induced osteoblast differentiation of mesenchymal cells
Co-immunoprecipitation was not observed in the presence and absence of BMP-2, when cytoplasmic extracts were employed

Summary

Introduction

The product of the multiple endocrine neoplasia type 1 (MEN1) gene, is required for commitment of multipotential mesenchymal stem cells to the osteoblast lineage, it inhibits their later differentiation (Sowa, H., Kaji, H., Canaff, L., Hendy, G.N., Tsukamoto, T., Yamaguchi, T., Miyazono, K., Sugimoto, T., and Chihara, K. (2003) J. We have examined the interactions of menin with BMP-2 and TGF-␤ signaling molecules, the Smads, and the pivotal transcriptional regulator of osteogenesis, Runx2, in both a multipotential mesenchymal cell line and an osteoblast cell line. Overexpression of the dominantnegative mutant, Smad3⌬C [43], and menin, augmented BMP2-induced transcriptional activity of the Smad5 response element containing the 3GC2-lux reporter construct (Fig. 9A).

Results

Conclusion