FIG. 3.

Binding of Runx2 to the Runx binding site is essential for the TGF-β1 responsiveness of the TβRE. (A) Diagrams of luciferase reporter constructs. Two reporters for the TβRE of the Ig Cα promoter were constructed using the pGL3-promoter plasmid (Promega) containing the simian virus 40 promoter (SV40 Pr) as a backbone, i.e., one with the wild-type (WT) TβRE (pGL3-TβRE) DNA and the other with the TβRE mutated at the Runx binding site (pGL3-M2). Two copies of the element were inserted for each plasmid construct. (B) pGL3, pGL3-M2, and pGL3-TβRE reporters were transfected into C2C12 cells with the Runx2 expression plasmid (Runx2) or the constitutively active TGF-β receptor I expression plasmid (TβR-I) or with both. Cells were harvested 48 h after transfection, and luciferase activities were assayed. (C and D) The same reporters were transfected into MC3T3-E1 and H1-127-30 cells with or without the Runx2 expression plasmid (Runx2) and cultured in the presence or absence of TGF-β1 for 24 h. Luciferase activities were measured, and relative activities are shown.