Insulin induction of apolipoprotein AI, role of Sp1.
Apolipoprotein AI (apo AI) is the major protein component of serum high-density lipoproteins. The abundance of apo AI correlates inversely with the risk of ischemic heart disease (IHD) and thus enhanced expression of the protein is expected to reduce the risk of IHD. Our previous studies show that insulin enhances apo AI promoter activity and this action requires the GC-rich insulin response core element (IRCE, -411 to -404). The motif binds to a ubiquitous transcription factor Sp1. We have extended studies that examine insulin induction of apo AI using a 41 bp (-425 to -385) fragment of apo AI DNA linked to the trout metallothionein TATA box and fused to luciferase (pIRCE-Luc). Luc activity in Hep G2 cells transfected with pIRCE-Luc was stimulated by insulin, an insulin mimetic bisperoxo (1,10-phenanthroline) oxovanadate (bpv) and the phorbol ester (PDBu). Our previous studies showed that insulin action on apo AI gene transcription flowed down two signaling pathways: Ras-raf and PI3K, leading to activation of the MAPK and PKC kinases, respectively. In contrast, PDBu activates only the PKC pathway. Although insulin and PDBu activation of apo AI were distinct, the cascades involved all appeared to target Sp1. Furthermore, exposure of transfected cells to okadaic acid or a phosphatase inhibitor also increased Luc activity and suggested a potential role for phosphorylation, likely involving Sp1. If true, then changes in the IRCE binding activity of Sp1 should be detected following exposure to MAPK, PKC, or the protein phosphatase I (PPI) alone and in various combinations followed by assaying the ability of Sp1 to bind the IRCE. Sp1 binding activity increased with either MAPK or PKC. Although exposure to PPI also affected IRCE binding activity of Sp1, whether it increased or decreased was dependent on the order of exposure to the protein. In summary, the IRCE alone can mediate the stimulatory effects of insulin, bpv, and PDBu, and Sp1 enhances these responses that may arise from phosphorylation of the protein.
Lam
Johnny K
JK
Endocrine research group, Department of Medicine, the Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada, T2N 4N1.
Matsubara
Shuji
S
Mihara
Koichiro
K
Zheng
Xi-long
XL
Mooradian
Arshag D
AD
Wong
Norman C W
NC
D000818:Animals; D016632:Apolipoprotein A-I / Q000096:biosynthesis* / Q000235:genetics; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D004789:Enzyme Activation; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D007328:Insulin / Q000235:genetics / Q000502:physiology*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D048728:MAP Kinase Kinase Kinase 1; D020935:MAP Kinase Signaling System / Q000187:drug effects; D019950:Mitogen-Activated Protein Kinase 1 / Q000502:physiology; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology; D019319:Okadaic Acid / Q000494:pharmacology; D009942:Organometallic Compounds / Q000494:pharmacology; D010618:Phenanthrolines / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000502:physiology; D010766:Phosphorylation; D010957:Plasmids; D011485:Protein Binding / Q000235:genetics; D011493:Protein Kinase C / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology; D051381:Rats; D020218:Response Elements / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D016329:Sp1 Transcription Factor / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D014162:Transfection; D014407:Tumor Cells, Cultured
D016632:Apolipoprotein A-I; D004268:DNA-Binding Proteins; D007328:Insulin; D009942:Organometallic Compounds; D010618:Phenanthrolines; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; C484091:insulin response element binding protein, rat; C075372:insulin response element binding protein, human; D051705:Sp3 Transcription Factor; D019319:Okadaic Acid; D015240:Phorbol 12,13-Dibutyrate; C102120:bisperoxo(1,10-phenanthroline)oxovanadate(1-); D008156:Luciferases; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; D019950:Mitogen-Activated Protein Kinase 1; D020928:Mitogen-Activated Protein Kinases; D048728:MAP Kinase Kinase Kinase 1; C482576:MAP3K1 protein, human; D010749:Phosphoprotein Phosphatases