FIG 5.

Fra-2 transcriptionally induces osteopontin (OPN) expression, which regulates mesenchymal stem cell (MSC) expansion. (A) Quantitative PCR analyses of OPN in fat pads, livers, lungs, spleens, brains, bone marrow, and long bones isolated from Fra-2^Ob-tet and littermate mice (n = 6). (B) Quantitative PCR analyses of OPN in mesenchymal stem cells and derived osteoblasts or adipocytes isolated from 10-week-old Fra-2^Ob-tet and littermate mice (n = 4). (C) OPN mRNA and protein levels in calvaria-derived osteoblast culture and supernatant isolated from Fra-2^Ob-tet and littermate mice at days 0 and 15 postdifferentiation in vitro (n = 6). (D) ChIP for the OPN promoter. Arrows indicate primers amplifying fragments of the promoter. Chromatin of the indicated genotypes was immunoprecipitated with AP-1 antibodies. Endpoint PCR-amplified fragments are shown (n = 3). (E) OPN-luc reporter assay for the OPN promoter fragments in the presence of Fra-2- or c-Jun-expressing vectors (n = 3). (F) FACS dot plots and quantification of MSC proliferation after OPN (15 ng/ml) treatment for 48 h in vitro (n = 6). (G) CFU-MSC pictures and quantification after OPN (15 ng/ml) treatment for 48 h in vitro (n = 6). (H) RT-PCR analyses of ang-1, jag-1, sdf-1, kit-l, mcp-1 and ccr2 gene expression in MSCs (Ter119⁻ CD45⁻ Sca-1⁺) after OPN (15 ng/ml) treatment for 48 h (n = 6). Data represent the mean values ± SEM. *, P < 0.05; **, P < 0.01 (by unpaired t test). In vitro experiments were performed 3 times in triplicate for each sample.