SAT0015 The negative effects of glucocorticoids on bone are primarily mediated by genes involved in osteoblast differentiation and bone remodelling

Background Long-term and high-dose treatment with glucocorticoids causes bone loss and can lead to secondary osteoporosis in both human and mice. The molecular mechanisms underlying the adverse effects of glucocorticoids on bone are not well understood. We recently demonstrated that osteoblast-targeted disruption of glucocorticoid signalling attenuates glucocorticoid-induced bone loss in mice, indicating that the detrimental skeletal effects of glucocorticoids are predominantly mediated by osteoblasts. Objectives To further determine the role of the osteoblast in glucocorticoid-induced bone loss, we aimed to elucidate the effects of glucocorticoids on the gene expression profile in bone cells. Methods Seven-week old male CD1 outbred mice were subcutaneously implanted with slow-release pellets containing either 1.5 mg corticosterone (CS) or placebo (PLC) over 28 days. Blood was obtained weekly and osteocalcin serum levels were measured by IRMA. RNA was isolated from tibia at endpoint (day 28) and Affymetrix Gene Array analysis (GeneSpring GX) was performed to assess changes in expression of genes associated with osteoblast differentiation and the regulation of bone remodelling. Genes considered to be regulated were at least 1.5-fold differentially expressed. Quantitative RT-PCR was employed to validate the gene array results on a selection of genes. Results By comparing gene expression profiles in RNA from mice treated with corticosterone or placebo, we found that corticosterone specifically regulated 391 genes. To further investigate the genes targeted by corticosterone treatment we performed gene ontology analysis with the aid of heat maps. We found that the expression of genes implicated in osteoblast differentiation and the regulation of bone remodelling was downregulated in mice treated with corticosterone compared to placebo. We observed a downregulation of the osteoblast markers Runx2, Colα1, osteocalcin and sclerostin in corticosterone-treated mice compared to placebo. In addition BMP4 and BMP7 followed the same pattern. Genes that were most profoundly downregulated in the array analysis were validated by qRT-PCR. Consistent with mRNA levels, osteocalcin serum levels were suppressed to almost undetectable levels. Conclusions These results confirm that glucocorticoids primarily target genes involved in osteoblast differentiation and the regulation of bone remodelling. Gene expression profile analyses may point to the pathways involved in the negative