Identification of a functional peroxisome proliferator-activated receptor (PPAR) response element (PPRE) in the human apolipoprotein A-IV gene.
Peroxisome proliferator-activated receptor-alpha (PPARalpha) is a key regulator in hepatic lipid metabolism and is a potential therapeutic target for dyslipidaemia. We reported previously that human hepatic apoA-IV is a highly sensitive gene up-regulated by the PPARalpha agonist KRP-101 (KRP), suggesting that induction of apoA-IV expression is one of the mechanisms underlying the decrease in triglycerides and elevation of HDL observed with PPARalpha agonist treatment. However, the mechanism of transcriptional regulation of apoA-IV by PPARalpha activation remains unclear. To clarify whether the apoA-IV promoter is regulated directly by PPARalpha, we analysed the apoA-IV promoter region by transient transfection assay in the human hepatocellular carcinoma cell line, HepG2. Co-transfection assay of unilateral deletions of apoA-IV promoter construct with human PPARalpha/RXRalpha showed that the region from -3279 to -2261 of the apoA-IV promoter includes key sites for transactivation by PPARalpha/RXRalpha. Sequence analysis suggested three putative PPAR response elements (PPREs) in this region. Electrophoretic mobility shift assay (EMSA) showed that a PPRE located from -2979 to -2967 can bind to PPARalpha/RXRalpha. Moreover, site-directed mutagenesis experiments indicated that the -2979/-2967 PPRE plays an essential role in transcriptional regulation of apoA-IV by PPARalpha. Chromatin immunoprecipitation (ChIP) assay confirmed that ligand-induced binding of PPARalpha to endogenous -2979/-2967 PPRE. These results indicate that human apoA-IV is regulated directly by PPARalphavia the -2979/-2967 PPRE.
Discovery Research Laboratories, Kyorin Pharmaceutical Co., Ltd., 2399-1 Nogi-machi, Shimotsuga-gun, Tochigi 329-0114, Japan. michiaki.nagasawa@mb.kyorin-pharm.co.jp
Michiaki
M
Nagasawa
Tomoko
T
Hara
Ai
A
Kashino
Yunike
Y
Akasaka
Tomohiro
T
Ide
Koji
K
Murakami
D001054:Apolipoproteins A / Q000235:genetics*; D001483:Base Sequence; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D017931:DNA Primers; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D016297:Mutagenesis, Site-Directed; D047492:Peroxisome Proliferator-Activated Receptors / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic
D001054:Apolipoproteins A; D017931:DNA Primers; D047492:Peroxisome Proliferator-Activated Receptors; C022205:apolipoprotein A-IV