Fig. 3.

ATP-induced, PKA-mediated activation of T-cell factor (TCF)-dependent gene transcription in VSMC. A: PKA-dependent activation of TCF reporter by ATP. VSMC were transfected with cDNA for TCF-luciferase (Luc) reporter (TOPflash) or the mutated control reporter (FOPflash) along with a renilla reporter driven by thymidine kinase promoter (TK-RL), and with an empty vector or cDNA for PKA dominant negative mutant (dnPKA). Following the stimulation of cells with 30 μM ATP for 12 h, luciferase activity was measured and normalized to a corresponding renilla activity. The TOP-Luc/TK-RL values were subtracted from the FOP-Luc/TK-RL values. Data represent means ± SD from a representative of three experiments performed in triplicate. *P < 0.01. B: PKA-dependent interaction between endogenous TCF-4 and β-catenin in response to ATP. VSMC were transduced with Ad-PKI, stimulated with ATP for 5 min, and lysed. Endogenous TCF-4 was immunoprecipitated from cell lysates, and the immune complexes or total cell lysates were examined by Western blotting with desired antibodies as indicated. The densitometry of a selected blot is shown as % of maximal response to ATP.