title: Cyclooxygenase inhibition is associated with downregulation of apolipoprotein AI promoter activity in cultured hepatoma cell line HepG2.
abstract: Prostanoids have been implicated in the transcriptional control of several genes. Since prostanoid synthesis inhibitors are commonly used in subjects with coronary heart disease we studied the effect of cyclooxygenase (COX) inhibition on apolipoprotein AI (apoAI) expression in a human hepatoma cell line (HepG2) transfected with full-length apoAI promoter attached to the chloramphenicol acetyl transferase (CAT) reporter gene. To control for transfection efficiency, the cells were cotransfected with the plasmid pCMV.SPORT-beta-gal containing the beta-galactosidase gene driven by the cytomegalovirus promoter. Treatment of these cells with varying concentrations of indomethacin (INDO, 0, 50, 100, and 300 micromol/L) resulted in a dose-dependent decrease in apoAI promoter activity (% acetylation corrected for beta-galactosidase activity: were 46.1 +/- 2.6, 29.9 +/- 1.2, 25.2 +/- 2.9, and 17.2 +/- 2.8, respectively, P <.001). INDO treatment did not cause significant changes in beta-galactosidase activity. A similar reduction in apoAI promoter activity was found after treating the cells with 50 micromol/L acetylsalicylic acid (ASA) (31.8 +/- 1.8%, P <.001), suggesting that the effect of INDO is related to COX inhibition rather than a peculiar effect of INDO. Nuclear run-off assays indicated that treatment of cells with 50 micromol/L INDO resulted in 31.4% reduction in apo A1 transcription rate (P <.0002). Northern blot analysis of RNA from HepG2 cells treated with 50 micromol/L of INDO for 72 hours showed that the apoAI mRNA concentration relative to G3PDH mRNA was 4,043.0 +/- 84.6 and 3,064.0 +/- 49.8 in control and INDO-treated cells, respectively (P <.0006). Kinetic studies of apoAI mRNA in HepG2 cells indicated that the half-life of apoAI mRNA was not significantly altered with 50 micromol/L INDO treatment. Apo AI mRNA half-life was 25.3 hours in control cells and 26.9 hours in INDO-treated cells. Western blot analysis of culture media of HepG2 cells treated with 50 micromol/L of INDO for 72 hours showed a significant reduction in apoAI protein (6,760.0 +/- 318.1 v 4,773.0 +/- 112.0 arbitrary units, P <.004). Treatment of cells with either arachidonic acid (COX substrate) or various prostanoids including prostaglandin I(2), thromboxane B(2), (+/-)5-HETE, or (+/-)12-HETE did not significantly alter apoAI promoter activity. However, prostaglandin E(1) and E(2) at the highest concentration tested (50 nmol/L) significantly repressed apoAI promoter activity. COX activity measurements in HepG2 cells verified the efficacy of COX inhibition by INDO. It is concluded that COX inhibition with INDO or ASA downregulates apoAI expression at the transcriptional level. This effect could not be attributed to either arachidonic acid excess or to a deficiency in various prostanoids tested.
authors.affiliation: Division of Endocrinology, Diabetes and Metabolism, Department of Internal Medicine, St. Louis University School of Medicine, St Louis, MO, USA.
authors.forename: Mohamad H
authors.initials: MH
authors.lastname: Horani
authors.forename: Freij
authors.initials: F
authors.lastname: Gobal
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Haas
authors.forename: Norman C W
authors.initials: NC
authors.lastname: Wong
authors.forename: Arshag D
authors.initials: AD
authors.lastname: Mooradian
mesh_terms: D016632:Apolipoprotein A-I / Q000096:biosynthesis*; D016718:Arachidonic Acid / Q000494:pharmacology; D001241:Aspirin / Q000494:pharmacology; D015152:Blotting, Northern; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D051546:Cyclooxygenase 2; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology*; D015536:Down-Regulation / Q000187:drug effects*; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000494:pharmacology; D007213:Indomethacin / Q000494:pharmacology; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics; D008113:Liver Neoplasms / Q000378:metabolism*; D008565:Membrane Proteins; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis / Q000235:genetics; D011453:Prostaglandins / Q000096:biosynthesis / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D013929:Thromboxane B2 / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D016632:Apolipoprotein A-I; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors; D006893:Hydroxyeicosatetraenoic Acids; D007527:Isoenzymes; D008565:Membrane Proteins; D011453:Prostaglandins; D012333:RNA, Messenger; D016718:Arachidonic Acid; D013929:Thromboxane B2; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D001241:Aspirin; D007213:Indomethacin