Because phosphorylation influences PPARĪ± activity [18], we assessed whether the PKA pathway activator forskolin, alone or in combination with GW7647, affects CAC gene expression. When
3X FLAG tag were transfected with the wtPPRE-containing pGL3 basic-LUC vector (Fig. 3A, black bars), forskolin and GW7647 stimulated gene reporter activity by 49% and 138%, respectively, compared to control. Combined forskolin + GW7647 treatment further enhanced LUC activity by 1.9-fold as compared to cells treated with GW7647 alone. PKA pathway involvement in CAC gene expression was further demonstrated by adding H89, a specific inhibitor of PKA [19]. H89 repressed LUC activity which was increased by forskolin and the GW7647 + forskolin combination (Fig. 3A, black bars). In the latter case, LUC activity was diminished to the level found with GW7647 alone. Of note, forskolin and GW7647 treatment, alone or in combination, did not affect the gene reporter activity in
cells transfected with the mutPPRE-containing pGL3 basic vector (Fig. 3A, gray bars). In agreement with these results, Fig. 3B and C show that forskolin and GW7647 also enhanced CAC transcript and protein levels, whereas H89 abolished the forskolin-dependent increase in CAC transcript and protein levels. These results demonstrate that the PKA pathway plays a role in the activation of CAC gene expression.
