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【关键词】 Regulation
Nicotinamide adenine dinucleotide (NAD) plays a critical role in the maintenance of cellular energy homeostasis. -Amino-β-carboxymuconate--semialdehyde decarboxylase (ACMSD) is the key enzyme regulating de novo synthesis of NAD from L-tryptophan (Trp), designated the Trp-NAD pathway. Acmsd gene expression was found to be under the control of both hepatocyte nuclear factor 4 (HNF4) and peroxisome proliferator-activated receptor (PPAR). Constitutive expression of ACMSD mRNA levels were governed by HNF4 and downregulated by activation of PPAR by the ligand Wy-14,643 ([4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid]), as revealed by studies with hepatic HNF4-null mice and PPAR-null mice, respectively. Transient transfection and electrophoretic mobility shift analyses showed an HNF4 binding site in the Acmsd gene promoter that directed transactivation of reporter gene constructs by HNF4. The Acmsd promoter was not responsive to PPAR in transactivation assays. Wy-14,643 treatment decreased HNF4 protein levels in wild-type, but not PPAR-null, mouse livers, with no changes in HNF4 mRNA. These results show that Wy-14,643, through PPAR, post-transcriptionally down-regulates HNF4 protein levels, leading to reduced expression of the HNF4 target gene Acmsd.Nicotinamide adenine dinucleotide (NAD) plays a critical role as a cofactor for oxidation-reduction enzymes and histone deacetylase, and as a substrate for second messenger cADP-ribose production and poly(ADP-ribosylation). Exposure to xenobiotic chemicals can result in altered total pyridine nucleotide levels, notably decreases in NAD caused by increased degradation or decreased biosynthesis of NAD; at the extreme, this can lead to apoptosis and cell death. The biosynthesis of NAD is under tight regulation by two routes in mammalian livers: de novo synthesis from L-tryptophan (Trp) and from nicotinic acid (see Fig. 1).
Fig. 1. Metabolic pathway of Trp-NAD biosynthesis in mammalian liver. NAD is biosynthesized by catalysis of QAPRT from quinolinic acid that is formed nonenzymatically from ACMS. ACMS is produced from Trp and completely oxidized through the glutarate pathway by ACMSD.
-Amino-β-carboxymuconate--semialdehyde (ACMS), generated from 3-hydroxyanthranilic acid and catalyzed by 3-hydroxyanthranilic acid oxygenase, is metabolized by -amino-β-carboxymuconate--semialdehyde decarboxylase (ACMSD) (E.C.4.1.1.45) or nonenzymatically to quinolinic acid, which is finally converted to NAD by quinolinate phosphoribosyltransferase (QAPRT) (E.C.2.4.2.19). ACMSD activity is found in the liver and kidney, although the activity in liver is much lower than that in kidney (Ikeda et al., 1965). On the other hand, L-Trp 2,3-dioxygenase (TDO) (E.C.1.13.11.11), the enzyme that initiates the Trp-NAD pathway, is only detectable in liver. Therefore, in mammals, liver is the sole organ having the complete Trp-NAD pathway, and hepatic ACMSD and QAPRT play critical roles in NAD biosynthesis, especially in the case of restricted niacin availability (Bender, 1983).
Several steps of NAD biosynthesis from Trp have been reported to be up- or down-regulated by various factors, including hormones (Mehler et al., 1958), nutrients (Sanada and Miyazaki, 1984; Egashira et al., 2004), and drugs (Shin et al., 1999); however, the molecular mechanisms governing these changes are largely unknown. Clofibrate, a hypolipidemic drug that stimulates peroxisome proliferation and fatty acid β-oxidation, significantly increases hepatic NAD and total pyridine nucleotide levels in rats (Loo et al., 1995; Shin et al., 1999). From the study of Trp fluxes in rat liver, the Trp-NAD pathway was increased by decreasing the flux via the glutarate pathway in hepatocytes prepared from rats fed a clofibrate diet (Shin et al., 1996). The activities of key enzymes such as ACMSD and QAPRT changed in concert with the increase in hepatic NAD (Shin et al., 1999). Peroxisome proliferators (PP) such as Wy-14,643, plasticizer phthalate esters, and dehydroepiandrosterone showed the same effect on ACMSD and QAPRT activities as clofibrate (Shin et al., 1999). Because most of these drugs are known activators of peroxisome proliferator-activated receptor (PPAR), a member of the nuclear receptor superfamily, the possibility exists that expression of the Acmsd and Qaprt genes are regulated by PPAR. PPAR is predominantly expressed in liver, heart, and kidney, which are tissues that carry out fatty acid oxidation.
Because the present study revealed that only ACMSD mRNA was down-regulated by activation of PPAR, it was examined in detail in the present study. The molecular mechanism for regulation of Acmsd by PPAR is not known. Acmsd is transcriptionally down-regulated by dietary polyunsaturated fatty acids (PUFA) (Egashira et al., 2004). Expression of many other genes is also regulated by PUFA, for which PPAR-dependent or -independent mechanisms have been proposed (Jump et al., 1999; Jump, 2002; Pegorier et al., 2004). Fatty acyl-CoA, putative endogenous PPAR ligands, are associated with hepatocyte nuclear factor 4 (HNF4) (Hertz et al., 2001; Petrescu et al., 2002; Hostetler et al., 2005), another member of the nuclear receptor superfamily that is expressed in the liver, kidney, intestine, and pancreas (Sladec and Seidel, 2001). In mammalian liver, HNF4 functions as a homodimer and plays an important role in regulating genes involved in gluconeogenesis (Rhee et al., 2003), ureagenesis (Inoue et al., 2002), coagulation (Inoue et al., 2006a), amino acid synthesis (Kamiya et al., 2004), and bile acid synthesis (Inoue et al., 2006b).
The current study was initiated to determine whether PP affects Trp-NAD metabolism through altering the expression or activity of ACMSD and whether this regulation is mediated by PPAR and/or HNF4. The results revealed that HNF4 directly activates the Acmsd gene by binding to a specific binding site located in the promoter, whereas PPAR reduces Acmsd expression by suppressing HNF4 protein levels.
Animals. PPAR-null mice and liver-specific HNF4-null mice used in this study were as described previously (Lee et al., 1995; Hayhurst et al., 2001). The PPAR-null mice and their controls were of a 129/Sv genetic background, whereas the liver-specific HNF4-null mice and their littermate controls were of mixed 129/Sv, C57BL/6, FVB genetic background. Six-week-old PPAR-null mice (PPAR-/-) and their littermates (PPAR+/+) or 45-day-old male mice with HNF4flox/flox without the albumin-Cre transgene (HNF4F/F) and HNF4flox/flox with the albumin-Cre transgene (HNF4L) were housed in a pathogen-free facility under standard 12-h light/dark cycle with water and diet ad libitum. Experiments were carried out in accordance with animal study protocols approved by the National Cancer Institute, Animal Care and Use Committee. Diets containing 0.1% (w/w) Wy-14,643 were prepared by Bioserv (Frenchtown, NJ) and fed to mice ad libitum for 3 days (HNF4 mice) or 2 weeks (PPAR mice). Livers were removed, weighed, quickfrozen in liquid nitrogen, and stored at -80°C until analysis.
Crude Enzyme Preparation and Measurement of Enzyme Activity. A 25% liver homogenate was prepared using a Polytron homogenizer in 50 mM potassium phosphate buffer, pH 7.0, containing 140 mM potassium chloride, 5 mM 2-mercaptoethanol, 1 mM dithiothreitol, 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride. The homogenate was centrifuged at 105,000g for 1 h at 4°C, and the supernatant was used for the determination of ACMSD activity. ACMSD activity was determined by the decrease in absorbance at 360 nm that monitors the decrease of ACMS produced from 3-hydroxyanthranilic acid. 3-Hydroxyanthranilic acid oxygenase was partially purified from acetone powder of mouse liver and used for the assay of ACMSD (Mehler, 1956). The BCA Protein Assay Kit (Pierce, Rockford, IL) was used to measure total protein content.
Reverse-Transcription and Real-Time Polymerase Chain Reaction. Total RNA (2 µg) prepared from each mouse liver with TRIzol reagent (Invitrogen, Carlsbad, CA) was reverse-transcribed in a final volume of 40 µl of first-strand buffer including oligo(dT) primer and SuperScript II Reverse Transcriptase (Invitrogen). For real-time polymerase chain reaction (PCR) analyses of reverse-transcribed cDNA, 1 µl of product was used as template with specific primers for each gene and SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA), and analysis was done with an ABI PRISM, 7900HT Sequence Detection System (Applied Biosystems). The PCR conditions were 95°C for 10 min, followed by 40 cycles of 15 s at 95°C and 60 s at 60°C. Serial dilution of each PCR product was used to draw a standard curve, and mRNA levels were determined and normalized for ribosomal protein 36B4.
Northern Blot Analysis. Hepatic total RNA (10 µg) was fractionated by electrophoresis on a 1% agarose gel containing 0.7% formaldehyde and transferred to GeneScreen Plus membranes (DuPont, Wilmington, DE). Blots were hybridized at 65°C with [32P]dCTP labeled cDNA probes generated by random primer labeling kit (Ready-To-Go DNA labeling beads; Amersham Biosciences, Piscataway, NJ) in PerfectHyb Plus hybridization buffer (Sigma, St. Louis, MO). After washing, blots were exposed to a PhosphorImager screen, followed by visualization of signal using a Molecular Dynamics Storm 860 PhosphorImager system (Molecular Dynamics, Sunnyvale, CA). cDNA used as a probe were amplified from a mouse hepatic cDNA library using gene-specific primers, which were then cloned into the pCR TOPOII vector (Invitrogen). The sequence was confirmed by sequencing with a CEQ 2000XL DNA Analysis system and CEQ 2000 Dye Terminator cycle sequencing kit (Beckman Coulter, Fullerton, CA).
Western Blot Analysis. Frozen livers were gently homogenized in a glass tube with a manual pestle, and nuclear and cytoplasmic extracts were prepared using NE-PER nuclear and cytoplasmic extraction reagents (Pierce), with the addition of proteinase inhibitors (Roche inhibitor mixture set I and 1 mM phenylmethylsulfonyl fluoride). Nuclear or cytoplasmic protein (15-50 µg) was subjected to SDS-polyacrylamide gel electrophoresis (10-12.5%), followed by transfer to a polyvinylidene difluoride membrane (Amersham Biosciences). The membrane was incubated with phosphate-buffered saline containing 0.1% Tween 20 and 5% dry milk for 1 h and then overnight with a primary antibody against HNF4 (dilution 1:500) (Santa Cruz Biotechnology, Santa Cruz, CA), HMGB1 (dilution 1:3000) (gift from Michael Bustin, National Cancer Institute), ornithine transcarbamylase (OTC) (dilution 1:3000) (gift from Masataka Mori, Japan), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (dilution 1:20,000) (gift from Kyung Lee, National Cancer Institute). After washing, the membrane was incubated with a 1:5000 diluted peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology), and the product was visualized using a chemiluminescent system (Super Signal West Pico Chemiluminescent Substrate; Pierce). The gels were scanned, and the bands were quantified by analysis of tagged image files using Image/J 1.36b software (Research Services Branch, National Institute of Mental Health, National Institutes of Health). The HMGB1 and GAPDH signals were used as loading controls for quantifying expression of HNF4 and OTC proteins, respectively.
Determination of the Transcription Start Site of Acmsd. The transcription start site of the mouse Acmsd gene was determined using mouse liver total RNA and the rapid amplification of 5'-cDNA end (RACE) method with the GeneRacer Kit (Invitrogen). After first-strand cDNA synthesis, PCR was performed with GeneRacer 5'-primer and a reverse gene-specific primer located within Acmsd exon 1. To generate a gene-specific RACE PCR product, nested PCR was performed with GeneRacer 5'-nested primer and reverse gene-specific nested primer. The transcription start site was determined by sequencing cloned PCR products.
Construction of Mouse ACMSD-Luciferase Reporter Plasmids and Site-Directed Mutagenesis. The -685, -295, -220, and -52/+66 fragments from the transcription start site of the mouse Acmsd gene were amplified by PCR using a common ACMSD-specific 3'-primer and a 5'-primer and cloned into the luciferase reporter vector, pGL3-basic (Promega, Madison, WI). Mutations were introduced into the HNF4-response element in the ACMSD-luciferase constructs (-685 and -295) using PCR-based, site-directed mutagenesis with QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). All of the plasmids were confirmed by DNA sequencing.
Cell Culture and Transient Transfection Assay. HepG2 or CV-1 cells were cultured in Dulbecco's modified Eagle's medium (BioSource, Camarillo, CA) containing 10% fetal bovine serum (Gemini Bio-products, Woodland, CA) and 100 units/ml antibiotics/antimycotic (Invitrogen). For transfection, cells were grown to 80 to 90% confluence in 24-well tissue culture plates without antibiotics. Transfection was performed using Lipofectamine 2000 (Invitrogen) with Opti-MEM I according to the manufacturer's instruction with ACMSD reporter constructs (500 ng/well), plasmid pRL/TK (Promega) containing the Renilla luciferase gene as an internal control (100 ng/well), and expression vectors pSG5/HNF4, pCMV/rHNF1, pSG5/mPPAR, or pSG5/hRXR (20, 100, or 150 ng each/well). After incubation for 4 to5hat 37°C, medium was changed to Dulbecco's modified Eagle's medium containing fetal bovine serum and antibiotics and subsequently cultured for 40 to 44 h. Cells were then washed with phosphate-buffered saline, and the lysates were prepared using Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instruction. Luciferase activity was determined using Monolight 3010 Luminometer (BD Biosciences PharMingen, San Diego, CA), and the relative luciferase activity was expressed as a -fold induction based on the activity of pGL3-basic.
Electrophoretic Mobility Shift Assay. Nuclear extracts were prepared from livers of PPAR+/+, PPAR-/-, HNF4F/F, and HNF4L mice fed control or Wy-14,643 diet. Nuclear extract (5 µg) was preincubated at room temperature for 20 min in 20 mM HEPESKOH, pH 7.9, 60 mM KCl, 0.3 mM MgCl2, 0.5 mM EDTA, 1.5 µg of poly(dI-dC), 10% glycerol, and 1 mM dithiothreitol. For competition experiments, a 50-fold excess of unlabeled oligonucleotide was added to the reaction mixture, and incubation was continued for additional 20 min. 32P end-labeled double-stranded oligonucleotide was then added and incubated at room temperature for 20 min. For supershift analysis, 1 µg of anti-HNF4 antibody (Santa Cruz Biotechnology) was added to the reaction mixture. DNA-protein complexes were resolved on a 5% polyacrylamide gel in 0.5x Tris borate/EDTA at room temperature, and a dried gel was exposed to a PhosphorImager screen.
Fig. 2. Hepatic mRNA levels of genes related to Trp-NAD metabolism (A) and ACMSD activity (B) in the PPAR-/-, PPAR+/+, liver-specifically HNF4-disrupted (HNF4L), and HNF4F/F mice fed control or Wy-14,643-containing (0.1%, w/w) diet. A, total RNA extracted from livers of these mice were subjected to real-time PCR analysis, and the results are shown as a relative expression to ribosomal protein 36B4 mRNA levels as normalization control. B, ACMSD activity is shown as the decreased ACMS amount/h in assay condition. Data are expressed as the mean ± S.D. (n = 5 for PPAR-/-, PPAR+/+, and HNF4F/F mice and n = 3 for HNF4L mice). Significant differences were based on the values with wild-type mice fed control diet. ****, p < 0.001; ***, p < 0.005; **, p < 0.01; *, p < 0.05.
Statistical Analysis. All of the values were expressed as the mean ± S.D. All of the data were analyzed by the unpaired Student's t test for significant differences; p < 0.05 was considered significant.
Genes Involved in Trp-NAD Metabolism Are Regulated by PPAR and HNF4. To understand the regulatory control of Trp-NAD metabolism in rodents by PP chemicals, wild-type (PPAR+/+), PPAR-null (PPAR-/-), or liver-specific HNF4-null mice (HNF4L) and their floxed (HNF4F/F) littermates were fed Wy-14,643, a PPAR agonist. Real-time PCR analysis revealed that in PPAR+/+, HNF4F/F, and HNF4L mice, but not in PPAR-/- mice, the expression of peroxisomal acyl-CoA oxidase (ACOX) mRNA was up-regulated by Wy-14,643 (Fig. 2A). These results were in agreement with the fact that Acox is a prototypical PPAR target gene. On the other hand, mRNA encoding Acmsd, one of the key enzymes involved in Trp-NAD metabolism, was down-regulated by administration of Wy-14,643 in livers of PPAR+/+ mice and in both HNF4F/F and HNF4L mice, but not in PPAR-/- mice; the increased expression in the latter mice could be caused by the elevated hepatic HNF4 protein (see below, Fig. 6). However, ACMSD mRNA expression levels in all of the samples were very low compared with the other mRNAs examined. Under control diets, PPAR-/- mice exhibited higher expression levels of ACMSD mRNA than PPAR+/+ mice, whereas the HNF4L mice exhibited lower expression compared with the HNF4F/F mice. These results suggest that HNF4 and PPAR regulate ACMSD expression positively and negatively, respectively. Gene expression patterns of QAPRT and TDO mRNA were similar and not substantially affected by Wy-14,643 treatment. There was a small but statistically significant lower QAPRT mRNA in PPAR+/+ mice after Wy-14,643 treatment that was not observed in the HNF4L mice; we have no explanation for this discrepancy other than possible strain differences. In HNF4L mice, however, the expression of these two genes was down-regulated; in particular, QAPRT mRNA was markedly lower compared with HNF4F/F mice. These results suggest that the expression of Qaprt and Tdo2 may be regulated by HNF4, but not by PPAR. When ACMSD enzyme activities were examined, a pattern similar to mRNA expression was obtained in either genotype with or without Wy-14,643 (Fig. 2B). A suppression of ACMSD activity in PPAR+/+ mice and increased activity in PPAR-/- mice was observed on Wy-14,643 treatment. In the case of HNF4F/F mice, however, the reduction of enzyme activity was similar to the mRNA levels, but not significantly different in Wy-14,643-treated HNF4F/F mice, suggesting that the difference may be caused by the shorter period of Wy-14,643 dosing. In the following experiments, the regulation of ACMSD by HNF4 and PPAR was examined in more detail.
Fig. 6. Protein and mRNA levels of HNF4, PPAR, and/or their target genes. A, Western blot analysis of HNF4 protein levels in the nuclear extract of livers from PPAR+/+, PPAR-/-, HNF4F/F, and HNF4L mice fed control or Wy-14,643 diet. HMGB1 (33) and GAPDH were used as loading controls for nuclear and non-nuclear fractions, respectively. The quantitated ratios of HNF4/HMGB1 and HNF4/OTC, with the PPAR+/+ set at 1.0, are shown. B, Northern blot analysis of RNA obtained from the same mice livers used for A. ACOX is a PPAR target gene. OTC, L-PK, and apoCIII are HNF4 target genes.
Fig. 3. Nucleotide sequence of the mouse Acmsd promoter. Numbering of nucleotides is based on the transcription start site as +1 (arrow). Translational start site is marked with asterisk. Several putative HNF4 binding sites and a PPAR binding site with low score that overlaps with the -224- to -243-bp HNF4 binding site are indicated. The nucleotide position of luciferase constructs used for transfection analysis in Fig. 4 is also shown.
Fig. 4. Transfection analysis of mouse ACMSD promoter activity. A, schematic diagram of five luciferase constructs containing various lengths of mouse Acmsd promoter sequence (-685, -295, -220, and -52/+66 bp from the transcription start site) used for the analysis. Mutations (m1, m2, m3) introduced into the -224- to -243-bp putative HNF4 binding site (ACMSD-HNF4) are also shown. B, Acmsd promoter activities obtained by transfecting five Acmsd reporter constructs (top) and the mutated constructs of -685 and -295, each containing m1, m2, or m3 mutation (bottom) into HepG2 cells. C, cotransfection analysis of five Acmsd reporter constructs (top) and -295 constructs containing m1, m2, or m3 mutation (bottom) with and without HNF4 expression plasmid in CV-1 cells. All of the values are expressed as -fold induction based on the activity obtained with the pGL3-basic plasmid as 1. Data are the mean ± S.D. from three separate experiments, each done in triplicate.
Determination of the Transcription Start Site of the Mouse Acmsd Gene. To understand whether the Acmsd gene is transcriptionally regulated by HNF4 or PPAR, a DNA fragment containing the 5'-flanking region of the mouse Acmsd gene was isolated and the transcription start site determined by 5'-RACE. Two major start sites were identified: one at -74 base pair (bp) and the other at -53 bp upstream from the translation start site. The RACE product with the -74 bp as a start site was found at a higher level (54%) compared with the -53-bp (35%) product. Thus, the former fragment was determined to represent the major transcription start site. Moreover, a TATA motif (TAAA) was located at -28 bp from the -74-bp transcription start site. Therefore, the -74 bp from the translation start site was determined to be the major transcription initiation point of the mouse Acmsd gene (Fig. 3).
HNF4 Activates the Mouse ACMSD Promoter. Because Acmsd gene expression seems to be regulated by both HNF4 and PPAR, the Acmsd gene promoter sequence was analyzed for potential consensus binding sites for these transcription factors. Several putative binding sites were identified by MOTIF search (http://motif.genome.jp) with scores higher than 70 for HNF4 within -690 bp upstream of the mouse Acmsd gene transcription start site. Although no high-score PPAR binding sites were found, one low-score site was found that partially overlapped with a -224- to -243-bp HNF4 binding site (Fig. 3). Based on this information, several ACMSD promoter-luciferase reporter plasmids were constructed (Fig. 3 and Fig. 4A) and subjected to transient transfection analysis using human hepatocellular carcinoma-derived HepG2 cells that express endogenous HNF4 (Inoue et al., 2002) (Fig. 4B, top). The promoter activity of constructs with -295- and -685-bp fragments was significantly higher than those with the -52- or -220-bp fragment. These results suggested that one or more elements important for hepatic transcription of Acmsd gene are located between -295 and -220 bp of the Acmsd gene promoter. In fact, a proximal HNF4 binding site was located at -224 to -243 bp of the Acmsd gene promoter. Next, monkey kidney-derived CV-1 cells that do not express endogenous HNF4 (Inoue et al., 2002) were used for cotransfection analysis (Fig. 4C, top). In these cells, cotransfection with an HNF4 expression plasmid significantly increased promoter activity of the -295- and -685-bp construct, but promoter activity of the -685-bp construct never exceeded the activity of the -295-bp construct. Cotransfection of these constructs with an HNF1 expression vector had no effect on this promoter activity (data not shown). These results suggest that the putative HNF4 binding site at -224- to -243-bp identified by MOTIF may be responsible for the increased promoter activity.
Identification of HNF4 Binding Site in the Mouse ACMSD Promoter. To prove that the putative HNF4 binding site located at -224 to -243 bp directly binds to HNF4, leading to the transcriptional activation of Acmsd gene, electrophoretic mobility shift assays (EMSA) were performed (Fig. 5). The protein in hepatic nuclear extracts from HNF4F/F mice bound to the ACMSD-HNF4 binding sequence (Fig. 5B, lanes 3 and 6), and the shifted band corresponded in mobility to a band obtained when an HNF4-consensus sequence was used as a probe (Fig. 5B, lane 1 versus 3). Both bands obtained with the HNF4-consensus and ACMSD-HNF4 binding sequence were supershifted by the addition of HNF4 antibody (Fig. 5B, lanes 2, 5, and 7). The ACMSD-HNF4 band was completely lost by the addition of a 50-fold molar excess of unlabeled probe (Fig. 5B, lanes 4 and 8; Fig. 5D, lane 18) and by the HNF4 consensus sequence probe (Fig. 5D, lane 19). Furthermore, this band was not detected with extracts from HNF4L mouse liver nuclei (Fig. 5B, lanes 9 and 10). Another putative HNF4 binding site with a score higher than 60 located between -685 and -220 bp of the ACMSD promoter did not bind to HNF4 (data not shown).
Fig. 5. Examination of HNF4 and PPAR binding sites in the mouse Acmsd promoter by EMSA. A, list of sequences used for EMSA experiments. B, nuclear extracts from the liver of HNF4F/F (lanes 1-8) or HNF4L mice (lanes 9 and 10) were incubated with labeled HNF4-consensus (lanes 1 and 2) or ACMSD-HNF4 probe (lanes 3-10); lanes 4 and 8 in the presence of excess unlabeled ACMSD-HNF4 oligonucleotide, and lanes 2, 5, 7, and 10 with the addition of anti-HNF4 antibody. C, the effect of mutation introduced into the HNF4 binding site was examined using ACMSD-HNF4 as a probe and the following sequences as competitors: own oligonucleotide (lane 3), m1 (lane 4), m2 (lane 5), and m3 (lane 6) (see Fig. 4A). Lanes 7 through 12, oligonucleotides m1 (lanes 7 and 8), m2 (lanes 9 and 10), and m3 (lanes 11 and 12) were also used as a probe. Nuclear extracts from the liver of HNF4F/F were used for all of the lanes. D, ACMSD-HNF4 (lanes 1, 2, and 16-22), ACOX-PPRE (lanes 3-8), and ACMSD-PPRE sequence (lanes 9-15) were used as a probe for EMSA with the following competitors; ACMSD-HNF4 own oligonucleotide (1) (lanes 5, 11, and 18), ACOX-PPRE (4) (lanes 4, 7, and 22), ACMSD-PPRE (2) (lanes 10, 13, 15, and 20), HNF4-consensus (3) (lane 19), or PPRE-consensus (5) (lane 21). Nuclear extracts from the liver of HNF4F/F, control diet (lanes 1, 2, and 16-22), HNF4F/F, Wy-14,643 diet (lanes 6, 7, 12, and 13), PPAR+/+, Wy-14,643 diet (lanes 3-5 and 9-11), and PPAR-/-, control diet (lanes 8, 14, and 15) were used. An arrow denotes the HNF4-specific band and its supershifted band.
To confirm that the -224- to -243-bp HNF4 binding site is functionally active, three different mutations were introduced to disrupt binding (Fig. 4A), and EMSA (Fig. 5C) and transfection assays (Fig. 4, B and C) were performed. When examined by EMSA, these mutated probes did not seem to bind to HNF4 (Fig. 5C, lanes 7, 9, and 11), nor did they compete with the ACMSD-HNF4 probe by the addition of unlabeled competitor oligonucleotide probes (Fig. 5C, lanes 4-6). However, a very faint band corresponding in mobility to the HNF4 antibody supershifted band (Fig. 5C, lane 2) was detected with the m1 oligo (Fig. 5C, lane 8), suggesting that this oligo might still have low affinity binding to HNF4. In the reporter gene assay, in all of the mutated constructs, the increased ACMSD promoter activity observed with HNF4 decreased to levels of activity obtained with the pGL3-basic in both HepG2 and CV-1 cells (Fig. 4B, bottom and Fig. 4C, bottom, respectively). These results show that HNF4 binds to the -224- to -243-bp binding site and increases promoter activity of the ACMSD.
Because a low-score putative PPAR binding site (-212 to -231 bp) is present that partially overlaps with the -224- to -243-bp HNF4 binding site in the Acmsd gene promoter, EMSA was carried out to examine whether PPAR binds to this binding site. With an ACOX-peroxisome-proliferator responsive element (PPRE) sequence as a probe and nuclear extracts prepared from PPAR+/+ in the presence of Wy-14,643 (Fig. 5D, lanes 3-5), HNF4F/F in the presence of Wy-14,643 (Fig. 5D, lanes 6 and 7), or PPAR-/- fed control diet (Fig. 5D, lane 8), a clear shifted band was obtained that corresponded to HNF4 (Fig. 5D, lanes 3, 6, and 8 versus 1); this band was lost by the addition of a 50-fold excess of unlabeled oligonucleotide (Fig. 5D, lanes 4 and 7) or the ACMSD-HNF4 oligonucleotide (Fig. 5D, lane 5). Indeed, the ACOX-PPRE contains a sequence similar to the consensus HNF4 binding site (Fig. 5A), thus accounting for this result (Sladek, 1994). When ACMSD-HNF4 was used as a probe with HNF4F/F nuclear extracts, a shifted band corresponding to HNF4 was lost with a 50-fold excess of unlabeled probes of ACMSD-HNF4, HNF4-consensus, or ACOXPPRE (Fig. 5D, lanes 18, 19, and 22, respectively), but not with ACMSD-PPRE or a consensus PPRE (Fig. 5D, lanes 20 and 21). Furthermore, with the ACMSD-PPRE sequence as a probe, no bands were obtained with any nuclear extracts (Fig. 5D, lanes 9-15). These results suggest that ACMSDHNF4 sequence does not compete with ACMSD-PPRE sequence and that PPAR may not directly bind the ACMSDPPRE site located -212 to -231 bp of the ACMSD promoter.
HNF4 Protein Level Is Decreased in Livers from Mice Treated with Wy-14,643. To further investigate the mechanism by which PPAR represses the transcription of Acmsd, the effect of Wy-14,643 and/or PPAR on the expression of HNF4 in mouse liver was studied by Western blot analysis (Fig. 6A). The chromatin protein HMGB1 (Bustin, 2002) and GAPDH were used as controls for nuclear and cytoplasmic proteins, respectively. Interestingly, HNF4 protein level was decreased in livers of Wy-14,643-treated PPAR+/+ and HNF4F/F mice but not PPAR-/- livers; the latter mice had increased levels of HNF4 compared with PPAR+/+ with and without Wy-14,643 treatment. The reason for this increase is not known, but it is correlated with the increase in ACMSD mRNA in the livers of PPAR-/- mice (Fig. 2A). As expected, no HNF4 protein was detected in HNF4L liver. Mature OTC located in mitochondria and a positive control product for a bonafide HNF4 target gene also showed decreased amounts after Wy-14,643 treatment in both PPAR+/+ and HNF4F/F mice. This protein was not detected in the extract from HNF4L mouse liver. These data suggest that the decreased protein level of OTC may be caused by the lower hepatic levels of HNF4 protein.
The effect of Wy-14,643 on HNF4 mRNA levels and its target genes was also examined by Northern blotting (Fig. 6B). Levels of PPAR mRNA increased in livers of PPAR+/+, HNF4F/F, and HNF4L mice treated with Wy-14,643. ACOX mRNA was also markedly induced in these mice. HNF4 mRNA levels were similar with or without Wy-14,643 in PPAR+/+ and HNF4F/F mouse livers, in contrast to the results found with protein expression. However, the expression of several HNF4 target genes, such as Otc, liver-type pyruvate kinase Pklr), and apolipoprotein C-III (ApoC3) was significantly decreased in the liver of PPAR+/+ and HNF4F/F mice treated with Wy-14,643, but not PPAR-/- mice, suggesting that the decrease in expression of these genes is PPAR-dependent. Taken together, these results show that Wy-14,643, through PPAR, regulates HNF4 levels post-transcriptionally, resulting in decreased expression of HNF4 target genes, including Acmsd, by decreasing the cellular levels of HNF4.
ACMSD and QAPRT are the key enzymes in Trp-NAD metabolism and regulate the flux to NAD from Trp in rodents. Alteration in ACMSD activity is correlated with hepatic NAD levels (Shin et al., 1999) and the NAD metabolites N1-methylnicotinamide, N1-methyl-4-pyridone-3-carboxamide, and N1-methyl-2-pyridone-5-carboxamide excreted in urine as biomarkers (Delaney et al., 2005). ACMSD activity was reported to be down-regulated by dietary PUFA (Egashira et al., 2004), low protein (Fukuoka et al., 2002), and PP (Loo et al., 1995; Shin et al., 1999) and up-regulated by glucocorticoids (Fukuoka et al., 2002), high protein diets, and in streptozotocin-induced diabetic rats (Tanabe et al., 2002). The mechanisms mediating these activity changes are not known.
By using PPAR-null or liver-specific HNF4-null mice in the current study, the expression of ACMSD was found to be under positive and negative regulation of HNF4 and PPAR, respectively. ACMSD mRNA is suppressed in wildtype mice and not in PPAR-null mice administered the ligand Wy-14,643, indicating that PPAR attenuates Acmsd gene expression. Levels of ACMSD mRNA and activity are markedly lower in mice lacking hepatic expression of HNF4, revealing that HNF4 controls constitutive expression of the gene. Indeed, a functional HNF4 binding site was located in the ACMSD gene promoter that is responsible for the increased promoter activity by HNF4 as assessed by reporter gene transfection studies. Activation of PPAR also results in post-transcriptional suppression of HNF4 protein levels that correlates with decreased expression of ACMSD. However, ACMSD expression is also suppressed in HNF4L mice fed Wy-14,643, suggesting that there may be another indirect mechanism by which this gene is suppressed by PPAR. HNF4 is constitutively expressed in liver and is active in the absence of exogenous ligand, whereas PPAR is a ligand-activated transcription factor. HNF4 and PPAR share similar hexanucleotide direct repeat 1 (DR1) consensus binding sequences (Sladek, 1994; Hertz et al., 1998). Indeed, a DR1 was found in the mouse Acmsd promoter, and activation of the promoter was mediated by HNF4 binding similar to other HNF4 target genes (Rajas et al., 2002). DR1 elements are quite promiscuous, thus allowing binding of other transcription factors, including HNF4, PPAR/RXR, RXR homodimers, retinoic acid receptor /RXR heterodimers, and chicken ovalbumin upstream promoter transcription factor I and II (Nakshatri and Bhat-Nakshatri, 1998), suggesting that these factors potentially could compete for binding to specific DR1 elements. Competitive binding of HNF4 and PPAR/RXR to DR1 elements as a mechanism for modulating transcriptional activation of target genes has been proposed (Hertz et al., 1995, 1996; Marrapodi and Chiang, 2000). For example, HNF4 was reported to bind to the ACOX-PPRE (Sladek, 1994), and in HepG2 cells, the apoC-III promoter activity was affected by hypolipidemic drugs (Hertz et al., 1995). PPAR and RXR specifically counteracted HNF4-activated transcription rather than inhibiting basal transcription of apoC-III. This result is similar to what was observed for the Acmsd gene in the current study. By EMSA using in vitro translated or COS cell-expressed transcription factors, HNF4 and PPAR/RXR heterodimers bound strongly to the C3P elements (Hertz et al., 1995), previously shown to be the binding site of nuclear proteins to the rat and human apoC-III gene promoter (Ladias et al., 1992, Mietus-Snyder et al., 1992). However, the binding of PPAR/RXR to the Acmsd gene HNF4 binding site was not observed in the current studies. These data indicate that PPAR reduces Acmsd gene expression indirectly through decreasing cellular contents of HNF4 protein. However, it should be noted that a small but significant suppression of residual ACMSD activity was also found in the HNF4L liver fed Wy-14,643, suggesting the possibility of another mechanism for attenuation of expression in this mouse model that is independent of HNF4 protein. In another study, cholesterol 7-hydroxylase was reduced by treatment with fibrates, and the reporter activity of a human cholesterol 7-hydroxylase-luciferase construct was downregulated by Wy-14,643 (Marrapodi and Chiang, 2000). Similar to the current results with Acmsd, no direct binding of in vitro synthesized PPAR/RXR heterodimer to the DR1 element of this gene was detected by EMSA. Therefore, it was suggested that attenuation of the transactivation of this gene is caused by altered availability of HNF4 in the presence of PPAR and its ligand (Marrapodi and Chiang, 2000). The effects of fibrates on HNF4 protein were not examined in this study.
The current finding, that Wy-14,643 treatment resulted in a PPAR-dependent suppression of HNF4 protein levels, is similar to that observed in HepG2 cells treated with Wy-14,643 (Marrapodi and Chiang, 2000). Of interest, activation of AMP-activated protein kinase (AMPK) by 5-amino-4-imidazolecarboxamide riboside in hepatocytes decreased HNF4 protein levels, and consequently the expression of HNF4 target genes was down-regulated, but HNF4 mRNA was unchanged (Leclerc et al., 2001). Indeed, in the liver of PPAR+/+ and HNF4F/F mice treated with Wy-14,643, expression of HNF4 target genes encoding Otc1, Pklr, and ApoC3 were also down-regulated. Phosphorylation was suggested to be one of regulatory mechanisms of HNF4 transactivation activity. HNF4 can be phosphorylated by protein kinase A (Viollet et al., 1997), extracellular signal-regulated kinase (Reddy et al., 1999), AMPK (Leclerc et al., 2001; Hong et al., 2003), and c-Jun NH2-terminal kinase 1 (Jahan and Chiang, 2005), and phosphorylation suppresses the DNA-binding affinity and trans-activation of HNF4. Modulation of kinase activity can also alter target gene expression by altering HNF4 protein levels (Reddy et al., 1999; Leclerc et al., 2001; Hong et al., 2003; Jahan and Chiang, 2005). Inhibition of extracellular signal-regulated kinase increased HNF4 mRNA and protein level (Reddy et al., 1999), whereas activation of AMPK was shown to promote degradation of HNF4 protein without changing HNF4 mRNA expression (Leclerc et al., 2001; Hong et al., 2003). However, preliminary studies in vivo revealed that activation of PPAR by Wy-14,643 had no effect on AMPK activity (date now shown). In any case, it still remains to be determined whether phosphorylation is the underlying mechanism of the reduced HNF4 protein level and ACMSD expression in Wy-14,643-treated mouse liver, and which kinase is responsible. Nevertheless, it is interesting that the activation of PPAR represses HNF4 protein expression, as well as transcription of its target genes, suggesting that some phenotypes resulting from PPAR activation in liver may be through HNF4 repression. Given that PPAR target gene expression was induced in HNF4-null mouse liver (Hayhurst et al., 2001), it is of interest to investigate cross-talk between PPAR and HNF4 in normal and disease states.
In conclusion, the role of transcriptional factors HNF4 and PPAR in the biosynthesis of Trp-NAD was examined. The ACMSD promoter activity was found to be under direct transcriptional regulation by HNF4. Wy-14,643 activation of PPAR indirectly results in suppression of HNF4 protein levels, leading to the negative regulation of ACMSD activity. These results will provide a better understanding of the Trp-NAD pathway and NAD homeostasis.
Acknowledgements
We thank Connie Cheung, Masamichi Yamashita, Keiichirou Morimura, and Yatrik Shah for providing animals and technical advice.
ABBREVIATIONS: NAD, nicotinamide adenine dinucleotide; Trp, L-tryptophan; ACMS, -amino-β-carboxymuconate--semialdehyde; ACMSD, -amino-β-carboxymuconate--semialdehyde decarboxylase; QAPRT, quinolinate phosphoribosyltransferase; TDO2, L-tryptophan 2,3-dioxygenase; PP, peroxisome proliferator(s); PPAR, peroxisome proliferator-activated receptor ; PUFA, polyunsaturated fatty acids; HNF4, hepatocyte nuclear factor 4; PPAR-/-, PPAR-null mice; PPAR+/+, PPAR wild-type mice; HNF4F/F, HNF4-floxed mice; HNF4L, liver-specific HNF4-null mice; PCR, polymerase chain reaction; OTC, ornithine transcarbamylase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RACE, rapid amplification of 5'-cDNA end; ACOX1, peroxisomal acyl-CoA oxidase; bp, base pair; EMSA, electrophoretic mobility shift assay(s); PPRE, peroxisome-proliferator responsive element; L-PK, liver-type pyruvate kinase; apoC-III, apolipoprotein C-III; RXR, retinoic X receptor ; DR1, hexanucleotide direct repeat 1; Wy-14,643, [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid; AMPK, AMP-activated protein kinase.
1 Current affiliation: School of Pharmacy, Kobe-Gakuin University, Nishiku, Kobe, 651-2180, Japan.
2 Current affiliation: Faculty of Engineering, Gunma University, Gunma, Japan.
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作者单位：Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, Maryland