FIG. 1.

PTHrP signaling/forskolin blocks expression of a collagen X reporter in chondrocytes without repressing the transcriptional activity of either Runx2 or Smad1/4. (A) USCs were cotransfected with the −4kb ColX-renilla luciferase reporter and an SV40-firefly luciferase reporter plus Runx2, MEF2C, and Smad1 expression vehicles in either the absence or presence of PTHrP or forskolin as indicated. As shown in this and subsequent figures, renilla luciferase units were normalized to the expression of cotransfected SV40-firefly luciferase to obtain relative luciferase units (RLU). (B) The PTHrP/forskolin transcriptional target in the collagen X enhancer was determined by evaluating whether these signals block the expression of luciferase reporters driven by either the intact collagen X enhancer, 6 copies of a Runx2 binding element, 30 copies of a Smad1/4 binding element, or 3 copies of a MEF2 binding element. (C) USCs were transfected with a reporter driven by six copies of a Runx2 binding element [6x(Runx2)-firefly luciferase] and an SV40-renilla luciferase reporter plus a Runx2 expression vehicle in either the absence or presence of PTHrP or forskolin as indicated. (D) USCs were transfected with a reporter driven by 30 copies of a Smad1/4 binding element [30X(SBE)-renilla luciferase] and an SV40-firefly luciferase reporter plus Smad1/4 expression vehicles in either the absence or presence of PTHrP or forskolin as indicated.