Exploration of the mouse osteoblast transcriptome

Contains fulltext : 30023.pdf (publisher's version ) (Open Access)The frequently occurring bone disorder osteoporosis is characterized by a strong increase in bone fracture risk, caused by a dramatically disturbed balance in the activity of the cells that degrade bone (osteoclasts) and cells that synthesize new bone (osteoblasts). Therapies against osteoporosis are currently based on inhibition of osteoclast activity in order to reduce the amount of bone resorption. An alternative strategy would be to stimulate new bone formation by enhancing the activity of osteoblasts. The main objective of this thesis was to identify genes of which expression and function are specifically associated with the differentiation of mesenchymal stem cells into osteoblasts. Therefore, we used microarray-based gene expression profiling to study the transcriptome of differentiating osteoblasts during in vitro cell culture as well as in their physiological context during skeletogenesis. The analyses revealed many genes of which expression in osteoblasts had not previously been detected. Novel markers of osteoblast differentiation and bone development were identified as well as putative in vivo target genes of the transcription factor Runx2 which is a key regulator of skeletal development. One of the markers identified encodes the protein Wif1 (Wnt Inhibitory Factor 1), which was shown to be specifically expressed in osteoblasts during embryonic development. Typically, genes encoding antagonists of the Wnt signalling pathway were strongly expressed during late stages of osteoblast differentiation and implicate an important role of Wnt antagonism in the maturation of osteoblasts. Given the role of Wnt signalling in the maintenance of bone mass, this class of proteins may be potential drug targets to treat bone disorders. This thesis shows that the combination of microarray technology and bioinformatic analyses has provided new possibilities to the understanding of bone biology. Consequently, our studies have contributed to a more detailed knowledge on osteoblast differentiation and provide focus points to develop strategies that will prevent and treat bone disorders.Radboud University, Applied Biology, 4 juni 2007Promotores : Olijve, W., Zoelen, E.J.J. van Co-promotor : Dechering, K.J.264 p

Least Absolute Regression Network Analysis of the murine osteoblast differentiation network

Item does not contain fulltextMOTIVATION: We propose a reverse engineering scheme to discover genetic regulation from genome-wide transcription data that monitors the dynamic transcriptional response after a change in cellular environment. The interaction network is estimated by solving a linear model using simultaneous shrinking of the least absolute weights and the prediction error. RESULTS: The proposed scheme has been applied to the murine C2C12 cell-line stimulated to undergo osteoblast differentiation. Results show that our method discovers genetic interactions that display significant enrichment of co-citation in literature. More detailed study showed that the inferred network exhibits properties and hypotheses that are consistent with current biological knowledge. AVAILABILITY: Software is freely available for academic use as a Matlab package, called GENLAB: http://genlab.tudelft.nl/genlab.html. SUPPLEMENTARY INFORMATION: Additional data, results and figures can be found at http://genlab.tudelft.nl/larna.html

Comprehensive microarray analysis of bone morphogenetic protein 2-induced osteoblast differentiation

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 reveals expression regulation of Wnt antagonists in differentiating osteoblasts

Item does not contain fulltextWnt 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

Inhibition of methylation decreases osteoblast differentiation via a non-DNA-dependent methylation mechanism

Contains fulltext : 83427.pdf (publisher's version ) (Closed access)10 p

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

Contains fulltext : 35643.pdf (publisher's version ) (Closed access