Identification of potential modifiers of Runx2/Cbfa1 activity in C2C12 cells in response to bone morphogenetic protein-7.
Treatment with BMP-7 causes a shift in the differentiation pathway from myoblastic to osteoblastic in C2C12 mouse myoblast precursor cells in vitro. The underlying molecular mechanism is largely unknown. BMP-7 at 200 ng/ml completely inhibited myotube formation in C2C12 cells and dramatically induced alkaline phosphatase activity up to 20-fold when compared to untreated cells by day 12 in culture. The level of Runx2/Cbfa1 mRNA, a bone-specific transcription factor, was also stimulated up to 6-fold by BMP-7 with a peak at 24 h. In addition BMP-7 treatment stimulated a 55-fold increase in osteocalcin mRNA as early as 24 h after treatment. A novel finding was that the expression of the chondrocyte markers Sox9 and type II collagen was increased as well. Runx2/Cbfa1 is a molecular switch for osteoblast differentiation. To initiate the study of modulators of Runx2/Cbfa1, such as kinases and cofactors, during osteoblastic differentiation of C2C12 cells treated by BMP-7 in vitro, microarray analyses of gene expressions were performed. Microarray data suggested that a total of 882 transcripts were either up- or downregulated at least 2-fold. Cluster analyses revealed 76 genes (including ESTs) with expression patterns that paralleled Runx2/Cbfa1. Thirteen of these 76 genes were initially selected as potential transcription modulators for further study; including CCAAT/enhancer binding protein delta, distal- less homeobox 1, forkhead box F2, insulin-like growth factor binding protein 4, an ortholog of human osteoclast stimulating factor 1 and p300/CBP-associated factor. Some transcription modulators have been associated with osteoblastic differentiation or interacted with Runx2/Cbfa1. Most of them have not been extensively studied in osteoblastic differentiation and in relationship to Runx2/Cbfa1. Thus, these studies identify potential regulators for Runx2/Cbfa1 and osteoblast differentiation. In addition, our data revealed for the first time that BMP-7 not only induced the expression of osteoblastic differentiation markers but also stimulated the expression of chondroblastic markers in C2C12 cells.
Gu
Keni
K
Department of Cariology, Restorative Sciences and Endodontics, School of Dentistry,University of Michigan, Ann Arbor, Mich., USA. rongguke@umich.edu
Zhang
Lixin
L
Jin
Taocong
T
Rutherford
R Bruce
RB
D000123:Acetyltransferases / Q000235:genetics; D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D055419:Bone Morphogenetic Protein 7; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology* / Q000502:physiology; D051743:CCAAT-Enhancer-Binding Protein-delta; D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics; D018797:Cell Cycle Proteins / Q000235:genetics; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D016000:Cluster Analysis; D024043:Collagen Type II / Q000235:genetics; D050659:Core Binding Factor Alpha 1 Subunit; D004268:DNA-Binding Proteins / Q000235:genetics; D051858:Forkhead Transcription Factors; D015870:Gene Expression / Q000187:drug effects; D020869:Gene Expression Profiling; D006609:High Mobility Group Proteins / Q000235:genetics; D051548:Histone Acetyltransferases; D018398:Homeodomain Proteins / Q000235:genetics; D018974:Insulin-Like Growth Factor Binding Protein 4 / Q000235:genetics; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D032446:Myoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000235:genetics* / Q000502:physiology; D020411:Oligonucleotide Array Sequence Analysis; D010006:Osteoblasts / Q000166:cytology; D015675:Osteocalcin / Q000235:genetics; D010455:Peptides / Q000235:genetics; D055758:SOX9 Transcription Factor; D015534:Trans-Activators / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics* / Q000502:physiology; D016212:Transforming Growth Factor beta / Q000494:pharmacology* / Q000502:physiology; D015854:Up-Regulation / Q000235:genetics / Q000502:physiology; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor
D000123:Acetyltransferases; D000469:Alkaline Phosphatase; D055419:Bone Morphogenetic Protein 7; D019485:Bone Morphogenetic Proteins; D051743:CCAAT-Enhancer-Binding Protein-delta; D022762:CCAAT-Enhancer-Binding Proteins; D018797:Cell Cycle Proteins; D024043:Collagen Type II; D050659:Core Binding Factor Alpha 1 Subunit; D004268:DNA-Binding Proteins; D051858:Forkhead Transcription Factors; D006609:High Mobility Group Proteins; D051548:Histone Acetyltransferases; D018398:Homeodomain Proteins; D018974:Insulin-Like Growth Factor Binding Protein 4; D047908:Intracellular Signaling Peptides and Proteins; D009363:Neoplasm Proteins; D015675:Osteocalcin; D010455:Peptides; D055758:SOX9 Transcription Factor; D015534:Trans-Activators; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor; C525819:BMP7 protein, human; C497253:Cebpd protein, mouse; C088334:Distal-less homeobox proteins; C486412:Foxf2 protein, mouse; C114562:LUN protein, vertebrate; C084119:MSX2 protein; C527932:SOX9 protein, human; C527933:Sox9 protein, mouse; C408994:osteoclast stimulating factor