Activation of apolipoprotein AI gene expression by protein kinase A and kinase C through transcription factor, Sp1

Our previous finding that insulin induces apolipoprotein AI (apoAI) transcr iption points to the participation of intracellular signaling. This finding prompted us to ask whether two classical G-protein-coupled signaling pathw ays requiring activated protein kinase A (PKA) or kinase C (PKC) may also r egulate apoAI. Therefore, human hepatoma, Rep G2 cells stably transfected w ith pAI.474-CAT, a reporter construct spanning -474 to -7 of apoAI DNA fuse d to chloramphenicol acetyltransferase (CAT) were treated with 10 mu M fors kolin (FSK) or 50 nM phorbol dibutyrate (PDBu) to activate PKA and PKC, res pectively, Results showed that the apoAI promoter activity increased 4-5-fo ld following 24 h of treatment with either FSK or PDBu. Induction by either agent was blocked with actinomycin D but not the protein synthesis inhibit or, cycloheximide. The PKA inhibitor, PKI 14-22 amide, abrogated induction by FSK, 100 mu M 8-bromo-cAMP, or 100 ng/ml cholera toxin, but it had no ef fect on activation via PKC. Similarly, PDBu induction was attenuated by 2 m u M Of the PKC inhibitor, GF109203X, but it did not affect FSK activity. Ne xt we used deletional constructs to show that the actions of FSK and PDBu r equired the insulin-responsive core element (IRCE). This motif matched the consensus binding site for the transcription factor, Spl. The binding of Sp l to the IRCE was confirmed by gel-retardation and supershift analysis. Sit e-directed mutagenesis of the IRCE eliminated Spl action and induction by F SK or PDBu. Whereas overexpression of Spl enhanced basal and FSK or PDBu in duced promoter activity, transfection of an antisense oligomer against Spl mRNA attenuated both parameters. In summary, activation of PKA or PKC incre ases apoAI promoter activity. The activity of both signaling pathways is me diated by the IRCE, a motif that binds the transcription factor, Spl.