Regulation of Mouse Hepatic -Amino- -Carboxymuconate- - Semialdehyde Decarboxylase, a Key Enzyme in the Tryptophan-Nicotinamide Adenine Dinucleotide Pathway, by Hepatocyte Nuclear Factor 4 and Peroxisome Proliferator- Activated Receptor

Nicotinamide adenine dinucleotide (NAD) plays a critical role in the maintenance of cellular energy homeostasis. -Aminocarboxymuconate-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 , posttranscriptionally 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). -Amino-carboxymuconate-semialdehyde (ACMS), generated from 3-hydroxyanthranilic acid and catalyzed by 3-hydroxyanthranilic acid oxygenase, is metabolized by -amino-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 This work was supported by the National Cancer Institute Intramural Research Program. 1 Current affiliation: School of Pharmacy, Kobe-Gakuin University, Nishiku, Kobe, 651-2180, Japan. 2 Current affiliation: Faculty of Engineering, Gunma University, Gunma, Japan. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.106.026294. ABBREVIATIONS: NAD, nicotinamide adenine dinucleotide; Trp, L-tryptophan; ACMS, -amino-carboxymuconate-semialdehyde; ACMSD, -amino-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; HNF4 , HNF4 -floxed mice; HNF4 , 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. 0026-895X/06/7004-1281–1290 MOLECULAR PHARMACOLOGY Vol. 70, No. 4 U.S. Government work not protected by U.S. copyright 26294/3138132 Mol Pharmacol 70:1281–1290, 2006 Printed in U.S.A. 1281 at A PE T Jornals on O cber 9, 2017 m oharm .aspeurnals.org D ow nladed from 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 upor 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 -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. Materials and Methods 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 HNF4 flox/flox without the albumin-Cre transgene (HNF4 ) and HNF4 flox/flox with the albumin-Cre transgene (HNF4 ) 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. 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. 1282 Shin et al. at A PE T Jornals on O cber 9, 2017 m oharm .aspeurnals.org D ow nladed from 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 g) prepared from each mouse liver with TRIzol reagent (Invitrogen, Carlsbad, CA) was reverse-transcribed in a final volume of 40 l 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 l 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 g) 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 [P]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 g) 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 ACMSDspecific 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 ACMSDluciferase 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