Coordinate expression of novel genes during osteoblast differentiation

To achieve new insights into the coordinate regulation of gene expression d uring osteoblast differentiation we utilized an approach involving global a nalysis of gene expression to obtain the identities of messenger RNAs (mRNA s) expressed using an established in vitro model of bone development. MC3T3 -E1 osteoblast-like cells were induced to differentiate by the addition of beta-glycerophosphate (beta-GP) and ascorbic acid, RNA samples derived from induced and uninduced control MC3T3-E1 cells were used to prepare compleme ntary DNA (cDNA) for serial analysis of gene expression (SAGE), A prelimina ry SAGE database was produced and used to prepare a hybridization array to further facilitate the characterization of changes in the expression levels of 92 of the SAGE-mRNA assignments after induction of osteoblast different iation, specifically after 6 days and 14 days of ascorbate treatment. SAGE- array hybridization analysis revealed coordinate induction of a number of m RNAs including Rab24, calponin, and calcyclin, Levels of MSY-1, SH3P2, fibr onectin, cw-collagen, procollagen, and LAMP1 mRNAs, present at day 6 postin duction, were markedly reduced by day 14 postinduction, A number of unantic ipated and potentially important developmental genes were identified includ ing the transforming growth factor beta (TGF-beta) superfamily member Lefty -1, Lefty-1 transcript and translation product were found to be induced dur ing the course of MC3T3-E1 cell differentiation. We present evidence, using transient transfection and antibody neutralization approaches, that Lefty- 1 modulates the induction of alkaline phosphatase (ALP) after treatment of MC3T3-E1 cells with ascorbate and beta-GP. These data should provide useful new information for future analysis of transcriptional events in osteoblas t differentiation and mineralization.