The muscle transcription factor MyoD promotes osteoblast differentiation by stimulation of the Osterix promoter.
Transcription factors regulate tissue-specific differentiation of pluripotent mesenchyme to osteoblast (OB), myoblast (MB), and other lineages. Osterix (Osx) is an essential transcription factor for bone development because knockout results in lack of a mineralized skeleton. The proximal Osx promoter contains numerous binding sequences for MyoD and 14 repeats of a binding sequence for Myf5. These basic helix-loop-helix (bHLH) transcription factors have a critical role in MB differentiation and muscle development. We tested the hypothesis that bHLH transcription factors also support OB differentiation through regulation of Osx. Transfection of a MyoD expression vector into two primitive mesenchymal cell lines, C3H/10T1/2 and C2C12, stimulated a 1.2-kb Osx promoter-luciferase reporter 70-fold. Myf5 stimulated the Osx promoter 6-fold. Deletion analysis of the promoter revealed that one of three proximal bHLH sites is essential for MyoD activity. The Myf5 repeat conferred 60% of Myf5 activity with additional upstream sequence required for full activity. MyoD bound the active bHLH sequence and its 3'-flanking region, as shown by EMSA and chromatin immunoprecipitation assays. Real-time PCR revealed that primitive C2C12 and C3H/10T1/2 cells, pre-osteoblastic MC3T3 cells, and undifferentiated primary marrow stromal cells express the muscle transcription factors. C2C12 cells, which differentiate to MB spontaneously and form myotubules, were treated with bone morphogenetic protein 2 (BMP-2) to induce OB differentiation. BMP-2 stimulated expression of Osx and the differentiation marker alkaline phosphatase and blocked myotubule development. BMP-2 suppressed the muscle transcription factor myogenin, but expression of MyoD and Myf5 persisted. Silencing of MyoD inhibited BMP-2 stimulation of Osx and blocked the later appearance of bone alkaline phosphatase. MyoD support of Osx transcription contributes to early OB differentiation.
Hewitt
Jocelyn
J
Division of Endocrinology, Metabolism, and Lipids, Department of Medicine, Emory University, Atlanta, GA 30033, USA.
Lu
Xiaghuai
X
Gilbert
Linda
L
Nanes
Mark S
MS
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002454:Cell Differentiation; D047369:Chromatin Immunoprecipitation; D024202:Electrophoretic Mobility Shift Assay; D018257:Helix-Loop-Helix Motifs / Q000235:genetics; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D017570:MyoD Protein / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D051787:Myogenic Regulatory Factor 5 / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000076182:Sp7 Transcription Factor; D014157:Transcription Factors / Q000235:genetics*
D017570:MyoD Protein; D051787:Myogenic Regulatory Factor 5; D034741:RNA, Small Interfering; D000076182:Sp7 Transcription Factor; C479466:Sp7 protein, mouse; D014157:Transcription Factors