Identification of Potential Modifiers of Runx2/Cbfa1 Activity in C2C12 Cells in Response to Bone Morphogenetic Protein-7

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.