TY - JOUR AU - Vaes, B.L.T. AU - Dechering, K.J. AU - Someren, E.P. van AU - Hendriks, J.M. AU - Ven, C.J. van de AU - Feijen, A. AU - Mummery, C.L. AU - Reinders, M.J. AU - Olijve, W. AU - Zoelen, E.J.J. van AU - Steegenga, W.T. PY - 2005 UR - https://hdl.handle.net/2066/32739 AB - 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. TI - Microarray analysis reveals expression regulation of Wnt antagonists in differentiating osteoblasts EP - 811 SN - 8756-3282 IS - iss. 5 SP - 803 JF - Bone VL - vol. 36 DO - https://doi.org/10.1016/j.bone.2005.02.001 ER - TY - JOUR AU - Jong, D.S. de AU - Vaes, B.L.T. AU - Dechering, K.J. AU - Feijen, A. AU - Hendriks, J.M. AU - Wehrens, H.R.M.J. AU - Mummery, C.L. AU - Zoelen, E.J.J. van AU - Olijve, W. AU - Steegenga, W.T. PY - 2004 UR - https://hdl.handle.net/2066/58213 AB - 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. TI - Identification of novel regulators associated with early-phase osteoblast differentiation. EP - 958 SN - 0884-0431 IS - iss. 6 SP - 947 JF - Journal of Bone and Mineral Research VL - vol. 19 DO - https://doi.org/10.1359/JBMR.040216 ER - TY - JOUR AU - Jong, D.S. de AU - Steegenga, W.T. AU - Hendriks, J.M. AU - Zoelen, E.J.J. van AU - Olijve, W. AU - Dechering, K.J. PY - 2004 UR - https://hdl.handle.net/2066/57750 AB - The bone morphogenetic protein (BMP)-induced Smad signal transduction pathway is an important positive regulator of osteoblast differentiation. BMP and other members of the transforming growth factor-beta (TGF-beta) family have distinct effects on osteoblast differentiation, depending on cell type and cell differentiation status. In C2C12 mesenchymal cells, BMP-induced osteoblast differentiation can be blocked by TGF-beta. In a search for key regulators of osteoblast differentiation we have used microarray analysis to identify genes which are differentially regulated by BMP2 and TGF-beta. Within the first 24 h following the onset of differentiation, 61 BMP2-regulated genes were identified of which the BMP2 effect was counteracted by TGF-beta. The majority of these differentially expressed transcripts are related to signal transduction. Notably, our data show that three Notch signal transduction pathway genes, Lfng, Hey1, and Hes1, are differentially regulated by BMP2 and TGF-beta. This suggests that these genes might function as the focal point for interaction of Smad and Notch signaling during osteoblast differentiation. TI - Regulation of Notch signaling genes during BMP2-induced differentiation of osteoblast precursor cells. EP - 107 SN - 0006-291X IS - iss. 1 SP - 100 JF - Biochemical and Biophysical Research Communications VL - vol. 320 DO - https://doi.org/10.1016/j.bbrc.2004.05.150 ER - TY - JOUR AU - Vaes, B.L.T. AU - Dechering, K.J. AU - Feijen, A. AU - Hendriks, J.M.A. AU - Lefevre, C. AU - Mummery, C.L. AU - Olijve, W. AU - Zoelen, E.J.J. van AU - Steegenga, W.T. PY - 2002 UR - https://hdl.handle.net/2066/129307 TI - Comprehensive microarray analysis of bone morphogenetic protein 2-induced osteoblast differentiation resulting in the identification of novel markers for bone development EP - 2118 SN - 0884-0431 SP - 2106 JF - Journal of Bone and Mineral Research VL - vol. 17 DO - https://doi.org/10.1359/jbmr.2002.17.12.2106 ER -