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Abstract: Bone marrow-derived human mesenchymal stem cells (hMSCs) have the in vitro capacity to differentiate into osteoblasts, chondrocytes or adipocytes, depending on the applied stimulus. In order to identify novel regulators of osteogenesis in hMSCs, osteo-transcriptomics was performed whereby differentiation induced by dexamethasone (DEX), DEX+ bone morphogenetic protein 2 (BMP2), and DEX+ Vitamin D3 (1,25(OH)2D3) was studied over a course of 12 days. Microarray analysis revealed that 2095 genes were significantly regulated by DEX+ 1,25(OH)2D3, of which 961 showed accelerated expression kinetics compared to treatment by DEX alone. The majority of these genes were accelerated 24–48 h after onset of osteogenic treatment. Gene ontology (GO) analysis of these 1,25(OH)2D3-accelerated genes indicated their involvement in biological processes related to cellular differentiation and cell cycle regulation. When compared to cells treated with DEX or DEX+BMP2, treatment with DEX+ 1,25(OH)2D3 clearly accelerated osteoprogenitor commitment and osteoblast maturation, as measured by alkaline phosphatase (ALP) activity and calcification of the matrix. Cell cycle progression, as observed after initial growth arrest, was not significantly accelerated by 1,25(OH)2D3 and was not required for onset and progression of osteogenesis. However, expression of c-Myc was accelerated by 1,25(OH)2D3, and binding sites for c-MYC were enriched in promoters of genes accelerated by 1,25(OH)2D3. Lentiviral overexpression of c-MYC strongly promoted DEX+ BMP2-induced osteoblast differentiation and matrix maturation. In conclusion, our studies show for the first time that 1,25(OH)2D3 strongly accelerates expression of genes involved in differentiation of hMSCs and, moreover, identify c-MYC as a novel regulator of osteogenesis. [Copyright &y& Elsevier]

Abstract: Wnt signaling has been implicated in regulating bone formation by controlling osteoblast proliferation and function. Although stabilization of β-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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]