Abstract
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.
Copyright 2004 S. Karger AG, Basel
MeSH terms
-
Acetyltransferases / genetics
-
Alkaline Phosphatase / genetics
-
Alkaline Phosphatase / metabolism
-
Animals
-
Bone Morphogenetic Protein 7
-
Bone Morphogenetic Proteins / pharmacology*
-
Bone Morphogenetic Proteins / physiology
-
CCAAT-Enhancer-Binding Protein-delta
-
CCAAT-Enhancer-Binding Proteins / genetics
-
Cell Cycle Proteins / genetics
-
Cell Differentiation / genetics
-
Cell Differentiation / physiology
-
Cell Line
-
Cluster Analysis
-
Collagen Type II / genetics
-
Core Binding Factor Alpha 1 Subunit
-
DNA-Binding Proteins / genetics
-
Forkhead Transcription Factors
-
Gene Expression / drug effects
-
Gene Expression Profiling*
-
High Mobility Group Proteins / genetics
-
Histone Acetyltransferases
-
Homeodomain Proteins / genetics
-
Insulin-Like Growth Factor Binding Protein 4 / genetics
-
Intracellular Signaling Peptides and Proteins
-
Mice
-
Myoblasts / cytology
-
Myoblasts / drug effects
-
Myoblasts / metabolism*
-
Neoplasm Proteins / genetics*
-
Neoplasm Proteins / physiology
-
Oligonucleotide Array Sequence Analysis
-
Osteoblasts / cytology
-
Osteocalcin / genetics
-
Peptides / genetics
-
SOX9 Transcription Factor
-
Trans-Activators / genetics
-
Transcription Factors / genetics*
-
Transcription Factors / physiology
-
Transforming Growth Factor beta / pharmacology*
-
Transforming Growth Factor beta / physiology
-
Up-Regulation / genetics
-
Up-Regulation / physiology
-
p300-CBP Transcription Factors
Substances
-
BMP7 protein, human
-
Bone Morphogenetic Protein 7
-
Bone Morphogenetic Proteins
-
CCAAT-Enhancer-Binding Proteins
-
Cebpd protein, mouse
-
Cell Cycle Proteins
-
Collagen Type II
-
Core Binding Factor Alpha 1 Subunit
-
DNA-Binding Proteins
-
Distal-less homeobox proteins
-
Forkhead Transcription Factors
-
Foxf2 protein, mouse
-
High Mobility Group Proteins
-
Homeodomain Proteins
-
Insulin-Like Growth Factor Binding Protein 4
-
Intracellular Signaling Peptides and Proteins
-
LUN protein, vertebrate
-
MSX2 protein
-
Neoplasm Proteins
-
Peptides
-
SOX9 Transcription Factor
-
SOX9 protein, human
-
Sox9 protein, mouse
-
Trans-Activators
-
Transcription Factors
-
Transforming Growth Factor beta
-
osteoclast stimulating factor
-
Osteocalcin
-
CCAAT-Enhancer-Binding Protein-delta
-
Acetyltransferases
-
Histone Acetyltransferases
-
p300-CBP Transcription Factors
-
p300-CBP-associated factor
-
Alkaline Phosphatase