FIG. 2.

PTHrP inhibits expression of a MEF2-lacZ transgene in developing cartilage and the transcriptional activity of both endogenous and exogenous MEF2C, but not that of MEF2-VP16. (A) USCs were cotransfected with a 3X(MEF2)-firefly luciferase reporter and an SV40-renilla luciferase reporter plus MEF2C-WT or MEF2C-VP16 expression vehicles in either the absence or presence of PTHrP or forskolin as indicated. The insert shows a Western blot of extracts from USCs transfected with wild-type MEF2C-Flag and cultured in either the absence or presence of PTHrP or forskolin as indicated. RLU, relative luciferase units. (B) USCs were cotransfected with a −4kb ColX-renilla luciferase reporter and an SV40-firefly luciferase reporter plus Runx2, Smad1, MEF2C-WT, or MEF2C-VP16 expression vehicles in either the absence or presence of PTHrP or forskolin as indicated. (C and D) Metatarsals were isolated from 15.5-dpc mouse embryos containing a lacZ transgene driven by three MEF2 binding sites (24) cultured in either the absence or presence of 10⁻⁶ M PTHrP for 3 days and stained for beta-galactosidase expression. Arrowheads, regions of the explants showing beta-galactosidase activity. (E) Gene expression from three pooled metatarsal explants treated as described in the legends for panels C and D was measured by RT-qPCR and normalized to Tbp expression.