Identification of novel regulators associated with early-phase osteoblast differentiation.
Key regulatory components of the BMP-induced osteoblast differentiation cascade remain to be established. Microarray and subsequent expression analyses in mice identified two transcription factors, Hey1 and Tcf7, with in vitro and in vivo expression characteristics very similar to Cbfa1. Transfection studies suggest that Tcf7 modulates BMP2-induced osteoblast differentiation. This study contributes to a better definition of the onset of BMP-induced osteoblast differentiation.


BACKGROUND
Elucidation of the genetic cascade guiding mesenchymal stem cells to become osteoblasts is of extreme importance for improving the treatment of bone-related diseases such as osteoporosis. The aim of this study was to identify regulators of the early phases of bone morphogenetic protein (BMP)2-induced osteoblast differentiation.


METHODS
Osteoblast differentiation of mouse C2C12 cells was induced by treatment with BMP2, and regulation of gene expression was studied during the subsequent 24 h using high-density microarrays. The regulated genes were grouped by means of model-based clustering, and protein functions were assigned. Real-time quantitative RT-PCR analysis was used to validate BMP2-induced gene expression patterns in C2C12 cells. Osteoblast specificity was studied by comparing these expression patterns with those in C3H10T1/2 and NIH3T3 cells under similar conditions. In situ hybridization of mRNA in embryos at embryonic day (E)14.5 and E16.5 of gestation and on newborn mouse tails were used to study in vivo expression patterns. Cells constitutively expressing the regulated gene Tcf7 were used to investigate its influence on BMP-induced osteoblast differentiation.


CONCLUSIONS
A total of 184 genes and expressed sequence tags (ESTs) were differentially expressed in the first 24 h after BMP2 treatment and grouped in subsets of immediate early, intermediate early, and late early response genes. Signal transduction regulatory factors mainly represented the subset of immediate early genes. Regulation of expression of these genes was direct, independent of de novo protein synthesis and independent of the cell type studied. The intermediate early and late early genes consisted primarily of genes related to processes that modulate morphology, basement membrane formation, and synthesis of extracellular calcified matrix. The late early genes require de novo protein synthesis and show osteoblast specificity. In vivo and in vitro experiments showed that the transcription factors Hey1 and Tcf7 exhibited expression characteristics and cell type specificity very similar to those of the osteoblast specific transcription factor Cbfa1, and constitutive expression of Tcf7 in C2C12 cells differentially regulated osteoblast differentiation marker genes.
de Jong
Diana S
DS
Department of Applied Biology, University of Nijmegen, Nijmegen, The Netherlands.
Vaes
Bart L T
BL
Dechering
Koen J
KJ
Feijen
Alie
A
Hendriks
Jose M A
JM
Wehrens
Ron
R
Mummery
Christine L
CL
van Zoelen
Everardus J J
EJ
Olijve
Wiebe
W
Steegenga
Wilma T
WT
D000818:Animals; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000502:physiology; D018797:Cell Cycle Proteins / Q000235:genetics; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics; D020224:Expressed Sequence Tags; D051538:Hepatocyte Nuclear Factor 1-alpha; D017403:In Situ Hybridization; D051821:Lymphoid Enhancer-Binding Factor 1; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D010006:Osteoblasts / Q000166:cytology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051822:T Cell Transcription Factor 1; D015534:Trans-Activators / Q000502:physiology; D014157:Transcription Factors / Q000235:genetics; D016212:Transforming Growth Factor beta / Q000502:physiology
C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D051538:Hepatocyte Nuclear Factor 1-alpha; C120420:Hey1 protein, mouse; C496488:Hnf1a protein, mouse; D051821:Lymphoid Enhancer-Binding Factor 1; D051822:T Cell Transcription Factor 1; D015534:Trans-Activators; D014157:Transcription Factors; D016212:Transforming Growth Factor beta