Microarray analysis on Runx2-deficient mouse embryos reveals novel Runx2 functions and target genes during intramembranous and endochondral bone formation.

A major challenge in developmental biology is to correlate genome-wide gene expression modulations with developmental processes in vivo. In this study, we analyzed the role of Runx2 during intramembranous and endochondral bone development, by comparing gene expression profiles in 14.5 dpc wild-type and Runx2 (-/-) mice. A total of 1277, 606 and 492 transcripts were found to be significantly modulated by Runx2 in calvaria, forelimbs and hindlimbs, respectively. Bioinformatics analysis indicated that Runx2 not only controls the processes of osteoblast differentiation and chondrocyte maturation, but may also play a role in axon formation and hematopoietic cell commitment during bone development. A total of 41 genes are affected by the Runx2 deletion in both intramembranous and endochondral bone, indicating common pathways between these two developmental modes of bone formation. In addition, we identified genes that are specifically involved in endochondral ossification. In conclusion, our data show that a comparative genome-wide expression analysis of wild-type and mutant mouse models allows the examination of mutant phenotypes in complex tissues.

Microarray analysis reveals expression regulation of Wnt antagonists in differentiating osteoblasts.

Wnt signaling has been implicated in regulating bone formation by controlling osteoblast proliferation and function. Although stabilization of beta-catenin by Wnt has been shown to increase alkaline phosphatase expression and osteoblast differentiation, the precise role of Wnt signaling during the process of osteoblast differentiation is largely unknown. In this study, we used microarray technology to investigate expression regulation of Wnt signaling components during in vitro osteoblast differentiation. Expression was analyzed during bone morphogenetic protein 2 (BMP2)-induced osteoblast differentiation of murine C2C12 and MC3T3 cells and data were compared with expression in BMP2-treated NIH3T3 fibroblasts. During osteoblast differentiation, particularly strong expression regulation of the Wnt antagonists Sfrp2 (secreted frizzled related protein 2) and Wif1 (Wnt inhibitory factor 1) was observed in the late phase of differentiation. In situ expression analysis in murine tail vertebrae supported Wif1 expression during late phase bone cell differentiation, since Wif1 was found to be expressed in vivo in trabecular, but not in cortical bone. We further analyzed the effects of continuous activation of Wnt signaling by lithium chloride and observed that osteoblast differentiation was reduced, as measured by expression of osteoblast marker genes encoding alkaline phosphatase, osteocalcin, and osterix, as well as by the amount of calcium release. Taken together, our data indicate that endogenous expression of Wnt antagonists by osteoblasts provides a negative Wnt feedback loop which is essential in controlling osteoblast maturation.

Identification of novel regulators associated with early-phase osteoblast differentiation.

UNLABELLED: 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. INTRODUCTION: 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. MATERIALS AND 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. RESULTS AND 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.

Gene array analysis of bone morphogenetic protein type I receptor-induced osteoblast differentiation.

UNLABELLED: 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. INTRODUCTION: 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. MATERIALS AND 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%. RESULTS AND 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.

Comprehensive microarray analysis of bone morphogenetic protein 2-induced osteoblast differentiation resulting in the identification of novel markers for bone development.

Osteoblasts are cells responsible for matrix deposition during bone development and although temporal expression of many genes has been related to osteoblast differentiation, a complete description of osteoblast-specific gene regulation will lead to a better understanding of osteoblast function. In this study, microarray technology was used to analyze gene expression on a broad scale during osteoblast differentiation. Expression analysis of 9596 sequences revealed 342 genes and expressed sequence tags (ESTs) to be modulated differentially during a time course experiment in which murine C2C12 mesenchymal progenitor cells were induced to differentiate into mature osteoblasts by treatment with bone morphogenetic protein 2 (BMP-2). By means of hierarchical clustering, these genes were grouped by similarities in their expression profiles, resulting in subsets of early, intermediate, and late response genes, which are representative of the distinct stages of osteoblast differentiation. To identify new bone markers, the bone specificity of the late response genes was determined by comparing BMP-induced expression in C2C12 and MC3T3 osteoblasts with that in NIH3T3 fibroblasts. This resulted in the identification of nine novel genes and ESTs that were induced specifically in osteoblasts, in addition to the well-known markers ALP and osteocalcin. For at least one of these novel genes, Wnt inhibitory factor 1, and two of the ESTs, expression in developing bone was verified in vivo by in situ hybridization of E16.5 mouse embryos. In conclusion, by a combination of in vitro and in vivo screening approaches, a set of new genes related to osteoblast differentiation and skeletal development has been identified.

Microarray analysis of bone morphogenetic protein, transforming growth factor beta, and activin early response genes during osteoblastic cell differentiation.

Bone morphogenetic protein (BMP) 2, a member of the transforming growth factor (TGF) beta family, is a potent regulator of osteoblast differentiation. In addition, both TGF-beta and activin A can either induce bone formation or inhibit bone formation depending on cell type and differentiation status. Although much is known about the receptors and intracellular second messengers involved in the action of TGF-beta family members, little is known about how selectivity in the biological response of individual family members is controlled. In this study, we have investigated selective gene induction by BMP-2, TGF-beta1 and activin A in relation to their ability to control differentiation of mouse mesenchymal precursor cells C2C12 into osteoblastic cells. TGF-beta1 can inhibit BMP-2-induced differentiation of these cells, whereas activin A was found to be without morphogenetic effect. Using a gene expression microarray approach covering 8636 sequences, we have identified a total of 57 established genes and expressed sequence tags (ESTs) that were either up-regulated or down-regulated 2 h after treatment with at least one of these three stimuli. With respect to the established genes, 15 new target genes for TGF-beta family members thus were identified. Furthermore, a set of transcripts was identified, which was oppositely regulated by TGF-beta1 and BMP-2. Based on the inverse biological effects of TGF-beta1 and BMP-2 on C2C12 cells, these genes are important candidates for controlling the process of growth factor-induced osteoblast differentiation.