Gene array analysis of bone morphogenetic protein type I receptor-induced osteoblast differentiation.
The genomic response to BMP was investigated by ectopic expression of activated BMP type I receptors in C2C12 myoblast using cDNA microarrays. Novel BMP receptor target genes with possible roles in inhibition of myoblast differentiation and stimulation of osteoblast differentiation were identified.


BACKGROUND
Bone morphogenetic proteins (BMPs) have an important role in controlling mesenchymal cell fate and mediate these effects by regulating gene expression. BMPs signal through three distinct specific BMP type I receptors (also termed activin receptor-like kinases) and their downstream nuclear effectors, termed Smads. The critical target genes by which activated BMP receptors mediate change cell fate are poorly characterized.


METHODS
We performed transcriptional profiling of C2C12 myoblasts differentiation into osteoblast-like cells by ectopic expression of three distinct constitutively active (ca)BMP type I receptors using adenoviral gene transfer. Cells were harvested 48 h after infection, which allowed detection of both early and late response genes. Expression analysis was performed using the mouse GEM1 microarray, which is comprised of approximately 8700 unique sequences. Hybridizations were performed in duplicate with a reverse fluor labeling. Genes were considered to be significantly regulated if the p value for differential expression was less than 0.01 and inverted expression ratios per duplicate successful reciprocal hybridizations differed by less than 25%.


CONCLUSIONS
Each of the three caBMP type I receptors stimulated equal levels of R-Smad phosphorylation and alkaline phosphatase activity, an early marker for osteoblast differentiation. Interestingly, all three type I receptors induced identical transcriptional profiles; 97 genes were significantly upregulated and 103 genes were downregulated. Many extracellular matrix genes were upregulated, muscle-related genes downregulated, and transcription factors/signaling components modulated. In addition to 41 expressed sequence tags without known function and a number of known BMP target genes, including PPAR-gamma and fibromodulin, a large number of novel BMP target genes with an annotated function were identified, including transcription factors HesR1, ITF-2, and ICSBP, apoptosis mediators DRP-1 death kinase and ZIP kinase, IkappaB alpha, Edg-2, ZO-1, and E3 ligase Dactylin. These target genes, some of them unexpected, offer new insights into how BMPs elicit biological effects, in particular into the mechanism of inhibition of myoblast differentiation and stimulation of osteoblast differentiation.
Korchynskyi
Olexander
O
Division of Cellular Biochemistry, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
Dechering
Koen J
KJ
Sijbers
Anneke M
AM
Olijve
Wiebe
W
ten Dijke
Peter
P
D000818:Animals; D015152:Blotting, Northern; D052005:Bone Morphogenetic Protein Receptors, Type I; D002454:Cell Differentiation; D002460:Cell Line; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D051379:Mice; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017978:Receptors, Growth Factor / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D012333:RNA, Messenger; D017978:Receptors, Growth Factor; D017346:Protein Serine-Threonine Kinases; D052005:Bone Morphogenetic Protein Receptors, Type I