title: 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 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.
authors.affiliation: Endocrine Research Group, Departments of Medicine and Biochemistry & Molecular Biology and Pharmacology & Therapeutics, the Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
authors.forename: X L
authors.initials: XL
authors.lastname: Zheng
authors.forename: S
authors.initials: S
authors.lastname: Matsubara
authors.forename: C
authors.initials: C
authors.lastname: Diao
authors.forename: M D
authors.initials: MD
authors.lastname: Hollenberg
authors.forename: N C
authors.initials: NC
authors.lastname: Wong
mesh_terms: D016632:Apolipoprotein A-I / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D005576:Colforsin / Q000037:antagonists & inhibitors / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020218:Response Elements / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D016632:Apolipoprotein A-I; D007328:Insulin; D016329:Sp1 Transcription Factor; D003609:Dactinomycin; D005576:Colforsin; D015240:Phorbol 12,13-Dibutyrate; D003513:Cycloheximide; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D020962:Heterotrimeric GTP-Binding Proteins