[BI019] Molecular Cloning And Characterisation Of The 5-Untranslated Region And Promoters In Human Peroxisome Proliferator-Activated Receptor Alpha (hPPARα) (original) (raw)
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Biochemical and Biophysical Research Communications, 2003
Peroxisome proliferator-activated receptor a (PPARaÞ is a ligand-activated transcriptional factor that governs many biological processes, including lipid metabolism, inflammation, and atherosclerosis. We demonstrate here the existence of six variants and multiple transcriptional start sites of the 5 0 untranslated region (UTR) of hPPARa gene, originating from the use of alternative splicing mechanisms and four different promoters. Three new novel exons at the 5 0-untranslated region of human PPARa gene were also identified and designated as Exon A, Exon B, and Exon 2b. In addition, 1.2 kb promoter fragment which drives the transcription of 2 variants with Exon B (hPPARa4 and 6) was successfully cloned and characterised. Sequencing results revealed promoter B did not contain a conservative TATA box within the first 100 nucleotides from transcriptional start site but has several GC-rich regions and putative Sp1 sites. Using luciferase reporter constructs transfected into HepG2 and Hep3B cell lines, promoter B was shown to be functionally active. Basal transcriptional activity was significantly high in the promoter fragment)341/+34, but lower in the region)341/)1147 as compared to the fragment)341/+34, indicating the presence of an element conferring transcriptional activation between positions)341 and +34 or alternatively, the presence of transcriptional repression between positions)341 and)1147 in the promoter B of hPPARa.
The Organization, Promoter Analysis, and Expression of the Human PPARγ Gene
Journal of Biological Chemistry, 1997
PPAR␥ is a member of the PPAR subfamily of nuclear receptors. In this work, the structure of the human PPAR␥ cDNA and gene was determined, and its promoters and tissue-specific expression were functionally characterized. Similar to the mouse, two PPAR isoforms, PPAR␥1 and PPAR␥2, were detected in man. The relative expression of human PPAR␥ was studied by a newly developed and sensitive reverse transcriptasecompetitive polymerase chain reaction method, which allowed us to distinguish between PPAR␥1 and ␥2 mRNA. In all tissues analyzed, PPAR␥2 was much less abundant than PPAR␥1. Adipose tissue and large intestine have the highest levels of PPAR␥ mRNA; kidney, liver, and small intestine have intermediate levels; whereas PPAR␥ is barely detectable in muscle. This high level expression of PPAR␥ in colon warrants further study in view of the well established role of fatty acid and arachidonic acid derivatives in colonic disease. Similarly as mouse PPAR␥s, the human PPAR␥s are activated by thiazolidinediones and prostaglandin J and bind with high affinity to a PPRE. The human PPAR␥ gene has nine exons and extends over more than 100 kilobases of genomic DNA. Alternate transcription start sites and alternate splicing generate the PPAR␥1 and PPAR␥2 mRNAs, which differ at their 5-ends. PPAR␥1 is encoded by eight exons, and PPAR␥2 is encoded by seven exons. The 5-untranslated sequence of PPAR␥1 is comprised of exons A1 and A2, whereas that of PPAR␥2 plus the additional PPAR␥2-specific N-terminal amino acids are encoded by exon B, located between exons A2 and A1. The remaining six exons, termed 1 to 6, are common to the PPAR␥1 and ␥2. Knowledge of the gene structure will allow screening for PPAR␥ mutations in humans with metabolic disorders, whereas knowledge of its expression pattern and factors regulating its expression could be of major importance in understanding its biology.
2002
PPAR␣ is a nuclear receptor that controls lipid and glucose metabolism and exerts antiinflammatory activities. The factors regulating human PPAR␣ (hPPAR␣) gene expression remain largely unexplored. To study the mechanisms controlling hPPAR␣ expression, the hPPAR␣ gene promoter was identified and characterized. First, an alternatively spliced exon within the 5-untranslated region of the hPPAR␣ gene was identified by RT-PCR. Next, the transcription start site was mapped and the hPPAR␣ gene promoter was cloned and functionally analyzed. Because PPAR␣ levels are elevated in tissues expressing the hepatocyte nuclear factor-4 (HNF4), such as liver, the regulation of hPPAR␣ by HNF4 was examined. Transient transfections in HepG2 and Cos cells showed that HNF4 enhances hPPAR␣ promoter activity. 5-Deletion and mutation analysis of the hPPAR␣ promoter identified a regulatory element (RE) consist-ing of a degenerate hexamer repeat with a single nucleotide spacer (direct repeat 1), termed ␣HNF4-RE. Gel shift assays demonstrated that HNF4 binds to this ␣HNF4-RE. Furthermore, HNF4 increased the activity of a heterologous promoter driven by two copies of the ␣HNF4-RE. The nuclear receptor COUP-TFII also bound this site and down-regulated basal as well as HNF4-induced hPPAR␣ promoter activity. Finally, PPAR␣ was shown to bind the ␣HNF4-RE, leading to an induction of PPAR␣ expression in hepatocytes. In summary, the organization of the 5-flanking and untranslated region of the hPPAR␣ gene was characterized and the hPPAR␣ promoter region has been identified.
PPARγ3 mRNA: a distinct PPARγ mRNA subtype transcribed from an independent promoter
FEBS Letters, 1998
PPARQ Q is a member of the peroxisome proliferator activated receptors (PPAR) subfamily of nuclear receptors. So far two PPARQ Q isoforms, PPARQ Q1 and PPARQ Q2, were known in mammals. We describe the structure of a novel human PPARQ Q subtype, PPARQ Q3. The PPARQ Q3 mRNA is transcribed from a novel promoter localized 5P of exon A2. PPARQ Q3 mRNA expression was restricted to adipose tissue and large intestine. Similar to human PPARQ Q1 and -2, PPARQ Q3 is activated by thiazolidinediones and prostaglandin J derivatives and binds with high affinity to a PPRE.
PPAR Could Contribute to the Pathogenesis of Hepatocellular Carcinoma
PPAR Research, 2012
Viral hepatitis with hepatitis C virus or hepatitis B virus and chronic liver disease such as alcoholic or nonalcoholic steatohepatitis are critical factors in the development of hepatocellular carcinoma (HCC). Furthermore, diabetes is known as an independent risk factor for HCC. Peroxisome proliferator-activated receptor (PPAR) is known to have an important role in fatty liver, and the mechanism of carcinogenesis has been clarified. PPAR controls ligand-dependent transcription, and three subtypes (α, δ, and γ) in humans are known. PPARs could contribute to the mechanisms of cell cycling, anti-inflammatory responses, and apoptosis. Therefore, to clarify the pathogenesis of HCC, we should examine PPAR signaling. In this paper, we have summarized the relevance of PPARs to the pathogenesis of HCC and cancer stem cells and possible therapeutic options through modifying PPAR signaling.
Pharmacogenetics, 2000
Peroxisome proliferator-activated receptor (PPAR)á-null mice have a defect in fatty acid metabolism but reproduce normally. The lack of a detrimental effect of the null phenotype in development and reproduction opens up the possibility for null or variant PPARá gene (PPARA) alleles in humans. To search the coding region and splice junctions for mutant and variant PPARá alleles, the human PPARá gene was cloned and characterized and sequencing by polymerase chain reaction was carried out. Two point mutations in the human gene were found in the DNA binding domain at codons for amino acids 131 and 162. The allele containing the mutation in codon 162 (CTT to GTT, L162V) designated PPARA Ã 3, was found at a high frequency in a Northern Indian population. Transfection assays of this mutant showed that the non-ligand dependent transactivation activity was less than one-half as active as the wild-type receptor. PPARA Ã 3 was also unresponsive to low concentrations of ligand as compared to the wild-type PPARA Ã 1 receptor. However, the difference is ligand concentration dependent; at concentrations of the peroxisome proliferator Wy-14 643 > 25 ìM, induction activity was restored in this variant's transactivation activity to a level ®vefold greater as compared with wild-type PPARA Ã 1 with no ligand. The mutation in codon 131 (CGA to CAA, R131Q), designated PPARA Ã 2 is less frequent than PPARA Ã 3, and the constitutive ligand independent activity was slightly higher than PPARA Ã 1. Increasing concentrations of Wy-14 643 activated PPARA Ã 2 similar to that observed with PPARA Ã 1. The biological signi®cance of these novel PPARá alleles remains to be established. It will be of great interest to determine whether these alleles are associated with differential response to ®brate therapy.
Molecular and Cellular Biochemistry, 2001
Two alternatively spliced forms of human PPARα mRNA, PPARα1 and PPARα2, have been identified. PPARα1 mRNA gives rise to an active PPARα protein while PPARα2 mRNA gives rise to a form of PPAR which lacks the ligand-binding domain. PPARα2 is unable to activate a peroxisome proliferator response element (PPRE) reporter gene construct in transient transfection assays. Both PPARα1 and PPARα2 mRNA are present in human liver, kidney, testes, heart, small intestine, and smooth muscle. In human liver, PPARα2 mRNA abundance is approximately half that of PPARα1 mRNA; a correlation analysis of PPARα1 and PPARα2 mRNA mass revealed an r-value of 0.75 (n = 18). Additional studies with intact liver from various species, showed that the PPARα2/PPARα1 mRNA ratios in rat, rabbit, and mouse liver were less than 0.10; significantly lower than the 0.3 and 0.5 ratios observed in monkey and human livers, respectively. To determine if a high PPARα2/PPARα1 mRNA ratio was associated with insensitivity to peroxisome proliferators, we treated human, rat, and rabbit hepatocytes with WY14643, a potent PPARa activator, and measured acyl CoA oxidase (ACO) mRNA levels. Rat ACO mRNA levels increased markedly in response to WY14643 while human and rabbit hepatocytes were unresponsive. Thus, although the PPARα2/PPARα1 mRNA ratio is low in rabbits, this species is not responsive to peroxisome proliferators. Further studies with male and female rats, which vary significantly in their response to peroxisome proliferators, showed little difference in the ratio of PPARα2/PPARα1 mRNA. These data suggest that selective PPARα2 mRNA expression is not the basis for differential species or gender responses to peroxisome proliferators.
Biochemical and Biophysical Research Communications, 1997
activating target genes depending on respective liThe strain difference, peroxisome proliferator specigands (9,10). ficity and role of PPARa in peroxisome proliferator-In the rat liver, PPs not only induce transcription of induced transcriptional repression of nonperoxisomal the genes encoding lipid metabolizing enzymes, but transthyretin and a 2u-globulin genes were examined. also repress several other genes. We found that apoE The genes were repressed by four peroxisome proliferis transiently down regulated by various proliferators ators in all seven mouse strains tested. The extent of (11). ApoAI, apoAIV (8) and apoCIII (12) have been repression was strongly dependent on both the mouse reported to be down regulated by the proliferators. In strains and type of proliferator, although the mRNA addition to these proteins related to lipid metabolism, levels of PPARa and its partner in heterodimerization, we have shown the immediate down regulation of RXRa were not different. The role of PPARa in represtransthyretin (13) and the endoplasmic protein, BiP/ sion was confirmed by the finding that PPARa-null GRP78 (14), both of which are not apparently involved mice were not responsive to transcriptional represin lipid homeostasis. Alvares et al. reported that a 2usion. These results indicate that PPARa plays an obligglobulin is down-regulated by ciprofibrate in the rat atory role in transcription of various genes, some of liver (15), whereas we detected little effect of clofibrate which are not related to lipid metabolism.
European Journal of Biochemistry, 1995
Three murine peroxisome-proliferator-activated-receptor (PPAR) genes were localised to chromosome 15 (PPARa), chromosome 17 (PPARP) and chromosome 6 (PPARy). The expression of the three PPAR RNAs was determined using a specific RNase protection assay. In liver RNA, PPARa was expressed at the highest level, with 20-fold lower levels of PPARj?, and very low levels of PPARy. The three PPAR RNAs showed no sex-specific differences in expression, and the levels of these transcripts were unaffected by treatment of mice with testosterone or the potent peroxisome proliferator, methylclofenapate. In agreement with this data, the level of PPARa protein in liver was unchanged after treatment of mice with methylclofenapate. Investigation of the tissue-specific distribution revealed that the PPARa RNA was expressed at highest levels in liver, to moderate levels in kidney and brown adipose tissue, and at low levels elsewhere. PPARj? was expressed at moderate levels in liver, and lower levels in other tissues, including brown adipose tissue. In contrast, PPARg RNA was expressed at low levels in liver or epididy-ma1 white adipose tissue and at very low levels elsewhere, but was expressed at high levels in brown adipose tissue. The tissue distribution of these receptors suggests an important role in lipid metabolism and toxicity for individual members of the PPAR family. The expression of PPARa and PPARP RNAs was examined in 13 strains of mice, and the levels of expression varied within a fourfold range. Polymorphism in the size of PPARa RNA from Swiss-Webster mice was detected, and shown to be due to a 2-bp mutation in the 3' non-coding region of PPARa in Swiss Webster mice.