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 microm 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 microm 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 microm 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.

Highlights

  • Apolipoprotein AI is the dominant and most important structural protein component of the antiatherogenic high density

  • Human hepatoma, Hep G2 cells stably transfected with pAI.474-CAT, a reporter construct spanning ؊474 to ؊7 of apolipoprotein AI (apoAI) DNA fused to chloramphenicol acetyltransferase (CAT) were treated with 10 ␮M forskolin (FSK) or 50 nM phorbol dibutyrate (PDBu) to activate protein kinase A (PKA) and PKC, respectively

  • The stimulatory effects of both pathways are mediated by a single transcription factor that converges on the insulin-responsive core element, IRCE [9]

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Summary

EXPERIMENTAL PROCEDURES

Plasmid Constructs—Construction of the reporter, pAI.474-CAT, was described previously [12]. The deletional constructs; pAI.425-, pAI.375-, pAI.325-, and pAI.235-CAT that contained rat apoAI DNA spanning Ϫ425, Ϫ375, Ϫ325, and Ϫ235 to Ϫ7 were synthesized using the parent pAI.474-CAT as template in separate PCR as described previously [12]. The wild-type IRCE (Ϫ411 to Ϫ404), GAGGCGGG, was mutated to TCTTATTT by using a primer containing these transverse mutations in a PCR. The construct containing an internal deletional of.

Regulation of ApoAI Promoter by PKC and PKA
RESULTS
DISCUSSION
Treatment Transfection None

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