Suppression of apolipoprotein AI gene expression in HepG2 cells by TNF alpha and IL-1beta.
Plasma inflammatory cytokines are elevated in obese subjects as well as in those with type 2 diabetes. This presumably results in systemic insulin resistance, characterized by a pro-atherogenic plasma lipid profile and reduced apolipoprotein AI (apoAI) protein levels. To determine how cytokine-mediated insulin resistance suppresses apoAI gene expression, we investigated the effect of tumor necrosis factor alpha (TNF alpha) and interleukin-1beta (IL-1beta) on apoAI protein, mRNA, and transcriptional activity in the human hepatoma cell line HepG2. ApoAI secretion was suppressed in a dose-dependent manner in HepG2 cells treated with both cytokines. ApoAI protein levels were 2892+/-22.0, 2263+/-117, 2458+/-25.0, 3401+/-152, 2333+/-248, 1520+/-41.5 and 956.0+/-11.0 arbitrary units (AU) in cells treated with 0, 0.3, 1.0, 3.0, 10, 30, and 100 ng/ml TNF alpha, achieving statistical significance in the 30 and 100 ng/ml range (P<0.0009). ApoAI protein levels were 4055+/-360, 3697+/-101, 3347+/-327, 1561+/-33.0, 1581+/-182, 810.0+/-59.5, and 1766+/-717 AU in cells treated with similar doses of IL-1beta, achieving statistical significance within the range of 3-100 ng/ml (P<0.02). ApoAI mRNA levels were suppressed 50.8% in HepG2 cells treated with 30 ng/ml TNF alpha for 24 h (P<0.05), and remained suppressed for up to 96 h. Similarly, treatment of cells with 30 ng/ml IL-1beta for 24 h, resulted in 42.9% reduction in apoAI mRNA levels (P<0.05) and remained suppressed for up to 96 h. In order to determine if the effect of TNF alpha and IL-1beta occurs at the transcriptional level, HepG2 cells were transfected with a chloramphenicol acetyltransferase (CAT) reporter gene plasmid containing the full-length apoAI promoter, and after 24 h, treated with TNF alpha (30 ng/ml), IL-1beta (30 ng/ml), or both cytokines. CAT activity was suppressed by both cytokines (24.0+/-1.9% acetylation in control cells vs. 5.6+/-1.2% (P<0.0004), 10.2+/-1.5% (P<0.0006), and 3.9+/-0.9% acetylation (P<0.0002) in cells treated with TNF alpha, IL-1beta, and the combination of both cytokines, respectively) suggesting that cytokine-mediated suppression occurs at the transcriptional level. Using a series of apoAI deletion constructs, the cytokine response element was mapped between nucleotides -325 and -186 (relative to the transcriptional start site). This region contains a previously identified and characterized cis-element, site A, which binds several different transcription factors. Finally, electrophoretic mobility shift assays (EMSA) showed that TNF alpha treatment of HepG2 cells is associated with reduced nuclear factor binding to site A. These studies suggest that inflammatory cytokines down-regulate apoAI expression at least partly through inhibition of binding of the nuclear factors to site A of the apoAI promoter.
Department of Internal Medicine, School of Medicine, St. Louis University, Health Sciences Center, 1402 S. Grand Boulevard, St. Louis, MO 63104, USA.
Michael J
MJ
Haas
Mohammad
M
Horani
Amjad
A
Mreyoud
Brian
B
Plummer
Norman C W
NC
Wong
Arshag D
AD
Mooradian
D016632:Apolipoprotein A-I / Q000096:biosynthesis / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D016207:Cytokines / Q000378:metabolism / Q000494:pharmacology; D004247:DNA / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism / Q000494:pharmacology; D007333:Insulin Resistance / Q000235:genetics / Q000276:immunology; D007375:Interleukin-1 / Q000494:pharmacology*; D011401:Promoter Regions, Genetic / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D016632:Apolipoprotein A-I; D016207:Cytokines; D018836:Inflammation Mediators; D007375:Interleukin-1; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D004247:DNA