Figure 8.

Activation of the cyclin D1 CRE by TGFβ and PTHrP is mediated by ATF-2 and CREB. (A) RCS cells were serum-starved for 3 d and stimulated for 8 h with control medium, TGFβ (1 ng/ml), PTHrP (10⁻⁸ M), or both. Cells were harvested for the analyses of protein expression of ATF-2 and of phosphorylated forms of ATF-2 and CREB. (B) The plasmids pFA-ATF-2 and pFA-CREB (encoding Gal4-ATF-2 and Gal4-CREB fusion proteins, respectively) were transfected into RCS cells together with the Gal4 reporter plasmid pFRluc and pRlSV40. After transfection, cells were serum-starved for 3 d and stimulated with control medium, TGFβ (1 ng/ml), or PTHrP (10⁻⁸ M) for 8 h. Cells were harvested, firefly luciferase activity was measured and standardized to Renilla luciferase activity to yield the relative luciferase activity. (C) The plasmid −1745 CD1Luc was transfected into RCS cells together with pRlSV40 and empty expression vector or expression vectors for dominant-negative forms of ATF-2 and CREB. After transfection, cells were serum-starved for 3 d and stimulated with control medium, TGFβ (1 ng/ml), PTHrP (10⁻⁸ M), or both for 8 h. Cells were harvested, and firefly luciferase activity was measured and standardized to Renilla luciferase activity to yield the relative luciferase activity.