Figure 1.

MINT stimulates Runx2- and FGF2-dependent transcriptional activation of the OCFRE via the MID domain. A, C3H10T1/2 cells were transiently transfected OC promoter, RANKL promoter, and NFAT response element-LUC reporter vectors. Plasmids for CMV vector or CMV-MINT expression (full length) were cotransfected as indicated, and cultures were treated with either vehicle or FGF2 (50 ng/ml) as described in Materials and Methods. Runx2 expression plasmid was included in all transfections. B, The OCFRE reporter OCFRE7-RSVLUC was used as a reporter to assess a systematic series of CMV-MINT variants in transient transfection assay. All transfections included Runx2 expression plasmid. As compared with FGF2-treated vector control (dotted vertical line) all C-truncated MINT expression constructs except for CMV-MINT(1–2180) significantly stimulated OCFRE activity. Comparisons were made vs. FGF2-treated vector control. One-way ANOVA with post hoc testing was carried out as described in Materials and Methods. Inset, A depiction of expressed MINT protein fragments and domains. C, A schematic representation of MINT C-truncated, N-truncated, and EYFP-fusion protein variants with OCFRE/Runx2 activation summary. D, MINT transcriptional regulatory domains were expressed as EYFP fusion proteins, with a uniform C-terminally located NLS and assayed in cotransfection assays with OCFRE7-RSVLUC. Only the MINT MID domain significantly stimulated OCFRE activity (asterisks). E, Coexpression of full-length MINT augments the TAF of the G4-Runx2, but not G4-USF1, in the mammalian one-hybrid assay. Inset, A schematic representation of G4-Runx2 fusion protein. See text for details. *, P < 0.05; **, P < 0.01; ***, P < 0.001. NS, Not significant; G4, Gal4 DNA binding domain. Figure continued on next two pages.