Activation of Apolipoprotein AI Gene Expression by Protein Kinase A and Kinase C through Transcription Factor, Sp1

Abstract

Our previous finding that insulin induces apolipoprotein AI (apoAI) transcription points to the participation of intracellular signaling. This finding prompted us to ask whether two classical G-protein-coupled signaling pathways requiring activated protein kinase A (PKA) or kinase C (PKC) may also regulate apoAI. Therefore, human hepatoma, Hep G2 cells stably transfected with pAI.474-CAT, a reporter construct spanning ؊474 to ؊7 of apoAI DNA fused to chloramphenicol acetyltransferase (CAT) were treated with 10 M forskolin (FSK) or 50 nM phorbol dibutyrate (PDBu) to activate PKA and PKC, respectively. Results showed that the apoAI promoter activity increased 4 -5-fold following 24 h of treatment with either FSK or PDBu. Induction by either agent was blocked with actinomycin D but not the protein synthesis inhibitor, cycloheximide. The PKA inhibitor, PKI 14 -22 amide, abrogated induction by FSK, 100 M 8-bromo-cAMP, or 100 ng/ml cholera toxin, but it had no effect on activation via PKC. Similarly, PDBu induction was attenuated by 2 M of the PKC inhibitor, GF109203X, but it did not affect FSK activity. Next we used deletional constructs to show that the actions of FSK and PDBu required the insulin-responsive core element (IRCE). This motif matched the consensus binding site for the transcription factor, Sp1. The binding of Sp1 to the IRCE was confirmed by gel-retardation and supershift analysis. Site-directed mutagenesis of the IRCE eliminated Sp1 action and induction by FSK or PDBu. Whereas overexpression of Sp1 enhanced basal and FSK or PDBu induced promoter activity, transfection of an antisense oligomer against Sp1 mRNA attenuated both parameters. In summary, activation of PKA or PKC increases apoAI promoter activity. The activity of both signaling pathways is mediated by the IRCE, a motif that binds the transcription factor, Sp1.

Regulation of apoA1 gene expression with acidosis: requirement for a transcriptional repressor

“…However, our results with PMA indicate that perturbation of PKC activity affects basal apoA 1 expression (Table 2). Similar results were recently demonstrated by Zheng et al (2000), who showed that PKC modulates apoA 1 promoter through the IRCE and the transcription factor SP1.…”

Transcriptional Regulation of Plasminogen Activator Inhibitor Type 1 Gene by Insulin

“…The stimulation of Sp1-mediated vascular permeability factor/ vascular endothelial growth factor transcription required an interaction between Sp1 and PKC (58). More recently, Sp1 was shown to be essential in the 12-O-tetradecanoylphorbol-13-acetate stimulation of human lysosomal acid lipase gene activity in monocytes (59) and in the regulation of apoAI gene expression by PKA and PKC (60).…”

Transcriptional Control of Apolipoprotein A-I Gene Expression in Diabetes

“…However, insulin sensitization with thiazolidendiones may not always be sufficient to induce apo A-I expression (18,19). It appears that insulin acts on the apo A-I promoter through at least two signaling pathways: Ras-raf and phosphatidylinositol 3-kinase (PI3-K), leading to activation of the mitogen-activated protein kinase (MAPK) and PKC kinases, respectively (20). Despite these differences, all these pathways ultimately target the transcriptional factor Sp1 (20,21).…”

“…It appears that insulin acts on the apo A-I promoter through at least two signaling pathways: Ras-raf and phosphatidylinositol 3-kinase (PI3-K), leading to activation of the mitogen-activated protein kinase (MAPK) and PKC kinases, respectively (20). Despite these differences, all these pathways ultimately target the transcriptional factor Sp1 (20,21). Cotransfection of Sp1 expression vector with an apo A-I IRCE reporter construct augments the actions of insulin, while reducing Sp1 levels with an antisense RNA impairs the insulin response (17).…”