Figure 1. Adam17, Adam10 and Adam9 gene expression is up regulated during early stage of commitment and differentiation to the osteoblast phenotype and stably expressed in human immature osteoblastic cells.

To identify genes that immediately respond to osteogenic stimuli, we retrieved microarray data were retrieved for experiments with mouse C2C12 mesenchymal cells that were treated with 300 ng/ml of BMP-2 and analysed at distinct time points (0, 4, 8, 12, 16, 20, and 24 h). Data on ADAM genes were filtered for genes that show more than a 2 fold change in gene expresseion. This analysis revealed that the Adam17, Adam10 and Adam9 genes are upregulated for more than 2-fold (A), while three others are downregulated (Adam8, Adam15, Adam19) (B). (C) The bar graph shows average expression values (in RPKM; STD as error bar for n=3 human donors) that were rank ordered for relative expression based on RNA-seq analysis of human osteoblastic bone-derived cells from three different donors. The pie chart presented in the inset shows that the six most highly expressed ADAM genes (including the BMP-responsive ADAMs ADAM17, ADAM10 and ADAM9, which are presented in color) account for almost all (~97%) ADAM-related transcripts. (D) Results of RNA-seq analysis for each individual donor and select osteosarcoma cell lines (SaOS-2, MG63 and U2OS) as indicated. (E) Visual presentation and validation of gene expression data using semi-quantitative RT-PCR and ethidium bromide staining. ADAM17, ADAM10 and ADAM9 gene expression was assessed as indicated in human SAOS-2, MG63, U2OS, HOS, G292 and 143B immature osteoblast cells. The data shown are representative of three experiments with similar outcomes. The graphs show quantification of the RT-PCR data relative to Gapdh mRNA (D). All data are presented as mean ± SEM from three independent experiments.