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Molecular Endocrinology, doi:10.1210/me.2003-0285
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Molecular Endocrinology 18 (4): 851-862
Copyright © 2004 by The Endocrine Society

Identification of a Liver-Specific Uridine Phosphorylase that Is Regulated by Multiple Lipid-Sensing Nuclear Receptors

Yuan Zhang, Joyce J. Repa, Yusuke Inoue, Graham P. Hayhurst, Frank J. Gonzalez and David J. Mangelsdorf

Department of Pharmacology and Howard Hughes Medical Institute (Y.Z., D.J.M.), University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9050; Departments of Physiology and Internal Medicine (J.J.R.), Touchstone Center for Diabetes Research, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-8854; and Laboratory of Metabolism (Y.I., G.P.H., F.J.G.), Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: David J. Mangelsdorf, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, Texas 75390-9050. E-mail: Davo.Mango{at}utsouthwestern.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In this work, we report the characterization of a novel liver-specific gene (L-UrdPase), whose expression is regulated by a number of hepatic nuclear receptors (including liver X receptors, peroxisome proliferator-activated receptor {alpha}, farnesoid X receptor, and hepatic nuclear factor-4{alpha}), which have been shown to be involved in lipid metabolism. L-UrdPase encodes a previously uncharacterized protein with similarity to an intestine-specific uridine phosphorylase. Enzymatic assays confirmed that L-UrdPase has uridine phosphorylase activity. However, L-UrdPase has a highly restricted, nonoverlapping pattern of expression with its intestinal counterpart and is regulated in a distinct manner by several different nuclear receptors. The identification of the liver uridine phosphorylase and its characterization as a target of lipid-sensing nuclear receptors implies the existence of a previously unknown nuclear receptor signaling pathway that links lipid and uridine metabolism.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
NUCLEAR RECEPTORS COMPRISE a superfamily of transcription factors that play important roles in virtually every developmental and metabolic pathway (1). Historically, this protein family has been subdivided into two categories. The first includes the known hormone receptors, which become trans-activated after binding to their cognate endocrine ligands. The second category includes the orphan receptors, so-named because regulatory ligands for these proteins either do not exist or are initially not known. Most of these receptors are composed of a DNA-binding domain, a ligand-binding domain, and two activation functions at the N terminus and C terminus of the protein (AF1 and AF2). Some atypical members of the family lack either DNA-binding domain or ligand-binding domain.

Several recent studies have focused on a subset of the orphan receptors, which are called the "adopted" orphan receptors (2). These receptors became adopted once their physiological ligands were discovered. Adopted orphan receptors include peroxisome proliferator-activated receptors (PPARs), liver X receptors (LXRs), farnesoid X receptor (FXR), steroid xenobiotic receptor/pregnane X receptor (SXR/PXR), and constitutive androstane receptor (CAR). These receptors function as heterodimers with the retinoid X receptor (RXR) and respond to dietary lipid metabolites as their ligands. PPAR{alpha}, {gamma}, and {delta} are activated by natural fatty acids and various synthetic compounds and are involved in fatty acid metabolism and adipocyte differentiation (3). LXR{alpha} and ß are activated by naturally occurring oxysterols and several nonsteroidal synthetic agonists (4, 5, 6). LXRs function as sterol sensors and regulate cholesterol and fatty acid metabolism (7). FXR is activated by bile acids and thereby regulates bile acid homeostasis. SXR/PXR and CAR function as xenobiotic sensors and regulate detoxification and elimination of foreign chemicals and toxic lipids. Together, these lipid-sensing receptors regulate a metabolic cascade that maintains whole body lipid homeostasis (2).

Previous work from our laboratory and others has revealed the existence of multiple orphan receptor regulatory networks that often converge on one specific pathway or target gene. One such pathway is bile acid synthesis, which in mice is governed by the rate-limiting enzyme, cholesterol 7{alpha}-hydroxylase (Cyp7a1). LXRs, which sense high levels of liver cholesterol, up-regulate the expression of CYP7A1, increase the conversion of cholesterol into bile acids (4, 8). FXR responds to elevated levels of bile acids and down-regulates the expression of CYP7A1, thereby counteracting the function of LXRs in liver. This repression is mediated by a nuclear receptor called small heterodimer partner (SHP) (9, 10). PPAR{alpha} is also expressed in the liver and regulates the transcription of genes involved in fatty acid ß-oxidation. PPAR{alpha} also has been shown to repress the expression of CYP7A1 gene in vivo (4, 11), although the molecular mechanism of PPAR{alpha}-mediated repression remains controversial. It has been suggested that the repression occurs by reducing the availability of hepatic nuclear factor-4{alpha} (HNF-4{alpha}), which is one of the liver-specific competence factors required for CYP7A1 basal expression (11). HNF-4{alpha} is one of the liver-enriched orphan receptors that is essential for the expression of genes involved in a variety of hepatic metabolic functions, including lipid, amino acid, and glucose metabolism (12, 13).

To further elucidate the functions of lipid-sensing nuclear receptors, we used an unbiased, PCR-based gene expression assay to identify unique target genes in the liver. Here we report the cloning and characterization of a novel liver-specific gene (L-UrdPase) that encodes a protein that is homologous to the intestinal uridine phosphorylase (UrdPase, EC2.4.2.3). However, in contrast to the known mouse UrdPase, L-UrdPase has a markedly different pattern of expression and regulation by hepatic nuclear receptors. The identification of this lipid regulated, liver-specific UrdPase suggests a previously unsuspected link between lipid and uridine metabolism.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cloning of Novel LXR{alpha} Regulated Genes by PCR-Select cDNA Subtraction
In an initial attempt to identify unique LXR target genes in liver, a PCR-based subtractive hybridization technique was performed between wild-type and Lxr{alpha} knockout mouse liver cDNAs. Single cDNA clones were isolated and sequenced for analysis and their regulation by LXR was verified by Northern blot. Several previously identified LXR target genes were isolated [e.g. CYP7A1, stearoyl-coenzyme A (CoA) desaturase 1], confirming the efficacy of the method (Fig. 1BGo). In addition, a novel 900-bp cDNA (called L-UrdPase) was also isolated that was regulated by a high cholesterol diet and a potent synthetic LXR agonist in wild-type mice, but not in Lxr{alpha}-null mice (see Figs. 1BGo and 4Go).



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  		Fig. 1. Cloning of L-UrdPase and Its Regulation by LXR{alpha}

A, The in-frame stop codons in the 5' UTR and the putative polyadenylation sites in the 3' UTR are underlined. The conserved phosphorylase motif residues are shown in underlined bold letters. B, Wild-type (+/+) and Lxr{alpha} knockout (-/-) mice were fed chow or chow containing 2% cholesterol (XOL) diets for 7 d. Liver mRNA was pooled from five mice per group and analyzed by Northern blot. The probes used were generated from cDNA fragments of L-UrdPase and SCD-1 (stearoyl-CoA desaturase 1) gene isolated from PCR-select cDNA subtraction. Actin was used as a loading control.

 


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  		Fig. 4. Northern Analysis of L-UrdPase and mUrdPase

A, Tissue distribution of L-UrdPase and mUrdPase. Male A129 mouse tissues were obtained and mRNA was isolated. The same Northern blot was probed with L-UrdPase cDNA and then stripped and reprobed with mUrdPase cDNA. Cyclophilin mRNA was monitored as a loading control. B, Response profiles to nuclear receptor agonists. Mice were fed diets containing 0.2% cholesterol plus vehicle (Veh) or the following nuclear receptor agonists for 12 h: 30 mpk LG268 (RXR), 0.5% fenofibrate (PPAR{alpha}), 150 mpk troglitazone (PPAR{gamma}), 0.05% pregnenolone {alpha}-carbonitrile (PXR), 0.5% chenodeoxycholic acid (FXR), or 50 mpk T0901317 (LXR). Liver and mucosa of small intestine were collected. Northern analyses were performed on mRNA pooled from four mice each group. The results were quantified by PhosphorImager (Amersham Biosciences, Piscataway, NJ) and standardized against actin. The mRNA level of the vehicle-treated group was defined as one unit. C, Real-time PCR analysis of L-UrdPase expression in livers from individual mice treated as described in panel B. Results represent mean ± SEM (n =4). The mRNA level in the vehicle-treated group was defined as one unit. Statistical significance was analyzed using ANOVA followed by multiple comparison testing using Dunnett’s test. *, P < 0.05 compared with vehicle-treated group. D, L-UrdPase gene expression exhibits circadian rhythm. Male A129 mice were housed under normal light cycle (light: 0700–1900 h; dark: 1900–0700 h). Livers were obtained every 4 h during a 24-h time course. Liver mRNA was isolated and pooled from three mice for each group. Northern analysis was performed using cDNAs to L-UrdPase and CYP7A1 as probes. ß-Actin was used as a loading control.

 
The 900-bp L-UrdPase contained a partial open reading frame that matched two expressed sequence tag clones (GenBank accession nos. AA270935 and AA212729), which are similar to uridine phosphorylase (see Fig. 2Go). To obtain the full-length of the cDNA, 5'-rapid amplification of cDNA ends was performed. The resultant L-UrdPase cDNA contains 1627 nucleotides with the predicted translation start codon at nucleotide 177 (Fig. 1AGo). This sequence harbors an open reading frame of 320 amino acids (Mr~35,731); the protein product was confirmed by in vitro transcription and translation (see supplemental Fig. 1Go published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). The 5'-untranslated region (UTR) contains several in-frame termination codons, and the transcription start site was confirmed by primer extension (supplemental Fig. 1Go). The cDNA also contains 488 nucleotides of 3'-UTR that includes several canonical polyadenylation signals. These features indicate that the cDNA encodes the full-length L-UrdPase.



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  		Fig. 2. L-UrdPase Is Homologous to UrdPase

A basic local alignment and search tool search revealed that L-UrdPase shares 60% identity with mUrdPase, 61% identity with human uridine phosphorylase (hUrdPase), and 83% identity with a similar human clone (AAD12227.1). The phosphorylase motif is underlined, and the conserved residues are indicated by asterisks. The alignment was performed by MegAlign software using the Clustal method (DNASTAR Inc.).

 
L-UrdPase Is Homologous to Uridine Phosphorylase (UrdPase)
Database search revealed that L-UrdPase is homologous to mouse uridine phosphorylase (mUrdPase), an enzyme that has previously been shown to be expressed in the intestine (Fig. 2Go). L-UrdPase shares 60% identity with mouse UrdPase, 61% identity with human UrdPase, and 83% identity with an uncharacterized, but similar human clone (GenBank accession no. AAD12227.1). L-UrdPase contains the sequence SHGMGIPSISIMLHEL (amino acids 113–128) that represents the signature motif for the phosphorylase family 1 (14) and is conserved in uridine phosphorylases throughout different species as shown in Fig. 2Go.

L-UrdPase Exhibits Uridine Phosphorylase Activity
To compare the potential uridine phosphorylase activity of L-UrdPase and mUrdPase, full-length cDNAs of each were cloned into a pCMV-Myc vector and transfected into human embryonic kidney (HEK) 293 cells to make Myc-tagged fusion protein (Fig. 3AGo). Enzyme activities were then examined in cell extracts. Figure 3BGo shows that L-UrdPase and mUrdPase have similar phosphorylase activities using uridine as substrate, whereas vector-transfected cell extracts contain a lower background activity, which may be explained by the expression of UrdPase in human kidney (15). Enzyme specificity was further examined by comparing uridine and thymidine as substrates. As summarized in Table 1Go, L-UrdPase shares almost identical kinetics to UrdPase, being over an order of magnitude more selective for uridine than thymidine. These studies showed that L-UrdPase-transfected lysates have a Michaelis constant (Km) value of 241.7 ± 51.54 µM using uridine as a substrate. A similar Km was observed for mUrdPase (Table 1Go). These values are comparable to those obtained with purified human UrdPase (189 ± 17 µM) (14). Consistent with the mRNA expression data (see Fig. 4Go), liver uridine phosphorylase activity was significantly induced by LXR agonist T0901317 in wild-type mice (Fig. 3CGo). In Lxr{alpha}/ß knockout mice, LXR agonist induced a slight, but nonspecific increase in uridine phosphorylase activity that was significantly lower than that observed in wild-type mice.



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  		Fig. 3. L-UrdPase Encodes a Protein with Uridine Phosphorylase Enzyme Activity

A, HEK293 cells were transfected with empty vector (Myc), Myc-L-UrdPase, or Myc-mUrdPase, and protein levels in the S100 fractions were examined by Western blot against the Myc-tag. B, HEK 293 cell extracts were used for enzyme analysis. The enzyme reaction mixture containing 50 mM potassium phosphate (pH 7.4), 1 mM DTT, 1 mM EDTA, 200 µM [2-14C]uridine, and 32 µl of cell extracts were incubated at 37 C. Data were analyzed as described in Materials and Methods. C, Male mixed-strain (A129/C57BL/6) wild-type (WT) and Lxr{alpha}/ß knockout (DKO) mice were fed 0.2% cholesterol diets containing vehicle (Veh), or 50 mpk of LXR agonist (T0901317) for 12 h. Liver uridine phosphorylase activity was measured as described in the Materials and Methods. The results represent mean ± SEM (n =3).

 

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  		Table 1. Substrate Specificity and Km Values of L-UrdPase and mUrdPase

 
Expression of L-UrdPase Is Liver Specific and Regulated by Multiple Orphan Nuclear Receptors
Various tissues were taken from male A129 mice and tissue distribution of L-UrdPase and mUrdPase was examined (Fig. 4AGo). By Northern analysis, the L-UrdPase gene transcribed a 2.4-kb RNA that was specifically expressed in liver. In contrast, the previously described mUrdPase is 1.4 kb and strongly expressed only in intestine. When mice were treated with different nuclear receptor agonists, L-UrdPase and mUrdPase displayed distinct responses (Fig. 4BGo). Consistent with our initial results, L-UrdPase expression in liver was markedly up-regulated by the LXR agonist (~3-fold), and slightly increased by a PXR agonist (~1.5-fold). Interestingly, L-UrdPase expression was strongly repressed by a PPAR{alpha} agonist, and to a lesser extent by FXR agonist (Fig. 4CGo). Although the repression by chenodeoxycholic acid does not appear significant in this experiment, an independent experiment using a more potent synthetic FXR-specific agonist showed significant repression of L-UrdPase (supplemental Fig. 2Go). Nevertheless, the basal level of L-UrdPase was not changed in PPAR{alpha} or FXR null mice (supplemental Fig. 2Go and data not shown). Conversely, expression of UrdPase in intestine was either not regulated or regulated only marginally by the tested receptor agonists. Taken together, these differences indicate that L-UrdPase and mUrdPase have unique regulatory requirements for their expression and thus are likely to have different functions.

L-UrdPase Gene Expression Is Diurnally Regulated
It has been reported that uridine phosphorylase activity has a circadian rhythm (16). The peak enzyme activity occurs at 15 h after light onset and the nadir enzyme activity occurs at 3 h after light onset. Because L-UrdPase is homologous to other known UrdPases, the expression level of L-UrdPase gene was examined over a 24 h period, with samples taken every 4 h. Figure 4DGo shows that L-UrdPase expression has a similar circadian rhythm. The highest mRNA level occurred at 1900 h, 12 h after light onset and the lowest level at 0700 h, 0 h after light onset. The reported enzyme activity for liver uridine phosphorylase (16) reaches peak (and nadir) 3 h after the transcription of L-UrdPase does, which is expected because it takes time for protein synthesis. L-UrdPase’s pattern of diurnal expression, as well as its regulatory response profile to nuclear receptors, are strikingly similar to that of cholesterol 7{alpha}-hydroxylase (CYP7A1), the rate-limiting enzyme in conversion of cholesterol to bile acid in liver (8, 17, 18).

The L-UrdPase Gene Promoter Contains a Functional HNF-4{alpha} Binding Site
To obtain the potential upstream regulatory elements of the L-UrdPase gene promoter, the first 200 bp of cDNA were used as a probe to screen a 129 mouse P1 artificial chromosome library RPCI-21 (Roswell Park Cancer Institute). A positive PAC clone was identified and digested with restriction enzymes and the fragments containing up to 4 kb of the 5' flanking region of the L-UrdPase coding region were cloned and sequenced. A computer-assisted search revealed several putative nuclear receptor binding sites in this region, several of which are similar to direct repeat (DR)1-like elements. Interestingly, however, there was no response or binding to any of the liganded nuclear receptors shown in Fig. 4Go that activated or repressed L-UrdPase gene expression in vivo, including the LXRs, FXR, RXR, PXR, or the PPARs (data not shown). These results suggest that L-UrdPase regulation by these receptors is either indirect or that their regulatory elements are in other distal or intronic regions of the gene.

Nevertheless, the DR1-like nature of the sequences that were found prompted us to look at the orphan nuclear receptor, HNF-4{alpha} as a potential regulator of L-UrdPase expression. HNF-4{alpha} is known to bind to DR1 sequences and regulate the expression of a number of liver-specific genes. To that end, a series of deletions of the L-UrdPase promoter were cloned into a luciferase vector and tested for HNF-4{alpha} responsiveness in a HEK 293 cell cotransfection assay. In these initial response element mapping experiments, we used a constitutively active form of HNF-4{alpha} that contains the potent trans-activation domain of VP16 fused to the amino terminus of the receptor (9) because regulatory ligands for HNF-4{alpha} are not known. Preliminary data narrowed the region of HNF-4{alpha} responsiveness to a 481-bp proximal promoter fragment. As shown in Fig. 5AGo, the HNF-4{alpha} response element maps between -424 and -411, and is responsible for trans-activation by the VP16-HNF-4{alpha} construct. Mutation of this site abolished the activity. An experiment using wild-type HNF-4{alpha} produced similar results (see Fig. 7CGo). To further confirm that this site is sufficient for HNF-4{alpha} responsiveness, a heterologous reporter gene containing two copies of the DR1 response element was also tested for trans-activation by HNF-4{alpha} (Fig. 5BGo). The L-UrdPase DR1 was trans-activated as well as the known HNF-4{alpha} response element from the ApoCIII gene promoter (19).



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  		Fig. 5. The L-UrdPase Promoter Contains a Functional HNF-4{alpha} Response Element

A, A series of deletions of L-UrdPase promoter were fused to a TK-luciferase reporter gene and tested for responsiveness to VP16-HNF-4{alpha}. The DR1 response element between -424 and -411 was mutated in 635m-luc and 481m-luc (mutated nucleotides are shown in bold letters). Reporter genes were cotransfected into HEK 293 cells with empty vector (no receptor) or CMX-VP16-HNF-4{alpha}. B, HNF-4{alpha} response elements mediate the transcriptional activity of HNF-4{alpha} in the absence of ligand. Two copies of wild-type or mutated HNF-4{alpha} RE from L-UrdPase promoter, respectively were assayed for HNF-4{alpha} responsiveness in TK-luciferase reporter gene assays as in (A) above. Luciferase activities were measured as described in Materials and Methods. Results represent the mean ± SD of triplicate assays.

 


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  		Fig. 7. Expression of L-UrdPase Is Dependent on HNF-4{alpha} and SHP in Vivo and in Vitro

A, Liver-specific HNF-4{alpha} conditional knockout mice were analyzed for L-UrdPase expression. Total liver RNA for Northern blots was isolated from individual wild-type (WT), albumin-Cre control (AlbCre), HNF-4{alpha} liver-specific knockout (H4LivKO), or floxed HNF-4{alpha} control (H4Flox) mice. Northern blot was hybridized with L-UrdPase cDNA probes as in Fig. 1Go. ß-Actin was used as a loading control (20 ). B, H4Flox control and H4LivKO mice were treated with 100 mpk PCN (PXR agonist) or 50 mpk T0901317 (LXR agonist) for 3 d. Liver total RNA from individual mice (n = 2 or 3) was isolated and analyzed by real-time PCR. Results represent mean ± SEM. The mRNA level in the vehicle-treated control group was defined as one unit. C, SHP represses HNF-4{alpha} activation of L-UrdPase. HEK 293 cells were transfected with 15 ng of empty vector, CMX-SHP, or CMX-HNF-4{alpha} in combination with p635-luc reporter. Increasing amounts of SHP (1.5, 5, 15, or 30 ng) was cotransfected with HNF-4{alpha}. Luciferase activities were measured as described in Fig. 5Go.

 
EMSAs were performed to verify that HNF-4{alpha} binds to the L-UrdPase DR1 element (Fig. 6Go). In vitro synthesized HNF-4{alpha} protein bound to the -424/-411 site (Fig. 6BGo, lane 2) and this binding was competed by an excess of unlabeled probe (lanes 3 and 4) and the HNF-4{alpha} response element from the ApoCIII promoter (lanes 7 and 8), but not by a mutated version of the L-UrdPase element (lanes 5 and 6). The same results were obtained using HepG2 nuclear extracts (Fig. 6CGo). To confirm that HNF-4{alpha} was present in the HepG2 nuclear extracts, anti-HNF-4{alpha} antibody was used to supershift the DNA-protein complex (Fig. 6DGo, lane 3). As a control, no supershift was observed with nonspecific antibody (lane 4). These results demonstrated that the DNA/protein complex formed with the L-UrdPase DR1 site contains a bona fide HNF-4{alpha} binding site. The identification of a functional HNF-4{alpha} binding site in L-UrdPase promoter is further evidence that the liver-specific expression of L-UrdPase is regulated by HNF-4{alpha}.



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  		Fig. 6. HNF-4{alpha} Binds to the DR1 Site in the L-UrdPase Promoter

A, Oligonucleotide sequences of the DR1 HNF-4{alpha} response element from the ApoCIII, and L-UrdPase (L-DR1) promoters are shown. L-DR1m represents the mutated version of the L-UrdPase DR1 element. The DR1 sequences are indicated in bold letters and by arrows. The nucleotides mutated in L-DR1m are underlined. B–D, Gel mobility shift assays were performed as described in Methods and Materials. Double-stranded 32P-labeled L-UrdPase DR1 oligonucleotides were incubated with in vitro synthesized HNF-4{alpha} (B) or HepG2 nuclear extracts (C and D). Unlabeled competitor oligonucleotides at 10- and 50-fold molar excesses were used to demonstrate specific HNF-4{alpha} binding in panels B and C (lanes 3–8). Anti-HNF-4{alpha} antibody (H4) (panel D, lane 3) or a nonspecific antibody (ns) (panel D, lane 4) was incubated with the L-UrdPase oligonucleotide and HepG2 nuclear extracts as in (C). HNF-4{alpha} protein/DNA complexes (arrow), antibody supershifted complexes (arrowhead), and nonspecific bands (n.s.) are indicated.

 
Expression of L-UrdPase Is Reduced in HNF-4{alpha} Liver Knockout Mice
Previous work has demonstrated that expression of several genes involved in lipid metabolism and transport are altered in HNF-4{alpha} liver-specific conditional knockout mice (20). Therefore, these mice were also tested for altered expression of L-UrdPase. As expected, Northern analysis showed that L-UrdPase expression was significantly decreased in the HNF-4{alpha} liver-specific knockout animals compared with control animals (Fig. 7AGo). These results are similar to those observed with several known HNF-4{alpha} target genes, such as ApoAII and ApoCIII (20) and supported the conclusion that L-UrdPase is a liver-specific HNF-4{alpha} target gene. To further examine whether the induction of L-UrdPase by LXR and PXR is dependent on HNF-4{alpha}, floxed HNF-4{alpha} control and HNF-4{alpha} liver-specific knockout mice were treated with agonists to these receptors. As expected, knockout of HNF-4{alpha} in the liver completely abolished the induction by LXR and PXR (Fig. 7BGo).

SHP Represses HNF-4{alpha} Trans-Activation of L-UrdPase Promoter
Because the ability of FXR agonists to repress L-UrdPase was similar to the regulation of CYP7A1 by FXR (9), we investigated whether this repression is mediated by SHP, whose expression is known to be induced by RXR/FXR heterodimers (Refs.9 and 10 and supplemental Fig. 2Go). It has been shown that SHP can bind to HNF-4{alpha} and inhibit its transcriptional activity (21). Figure 7CGo showed that wild-type HNF-4{alpha} activated L-UrdPase promoter in HEK 293 cells in the absence of SHP. Cotransfection of increasing concentrations of SHP resulted in complete repression of HNF-4{alpha} activity in a dose-dependent manner.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In this study, we report the identification of a novel uridine phosphorylase (L-UrdPase) that is regulated by a class of lipid-sensing nuclear receptors. In addition to its liver-specific expression, L-UrdPase shares several other features in common with another crucial metabolic enzyme, CYP7A1. Like CYP7A1, L-UrdPase expression is restricted to liver and has an exquisitely sensitive circadian pattern. Also similar to CYP7A1, L-UrdPase expression is regulated by several hepatic nuclear receptor agonists, including LXR, FXR, and PPAR{alpha}. In the case of CYP7A1, previous studies have shown that it is directly regulated by LXR and repressed by RXR, FXR, and PPAR{alpha} (4, 8). The basis for transcriptional repression by RXR and FXR is via the induction of the repressor protein, SHP (9, 10). SHP can repress a number of nuclear receptors, including HNF-4{alpha}, which is a direct activator of L-UrdPase and which may in part explain L-UrdPase’s liver-specific expression (22). Our results revealed that SHP can also repress HNF-4{alpha} activation of the L-UrdPase promoter, which likely explains how FXR represses L-UrdPase gene expression. PPAR{alpha} has also been reported to repress CYP7A1 by reducing the trans-activation ability of HNF-4{alpha}, which is one of the liver-specific competence factors required for CYP7A1 basal expression (11).

Although first identified as an LXR target gene, our promoter analysis of the L-UrdPase gene has not yet revealed the basis for regulation by LXRs. Whether this is a direct or indirect target remains to be determined. Like other known LXR target genes, the LXR-responsive element could be several kilobases further upstream of the promoter or in the intronic regions of the gene (23, 24, 25, 26, 27, 28). It is also feasible that the regulation by LXRs could be mediated indirectly through another transcription factor. One such transcription factor may be HNF-4{alpha}. Previous work has shown that HNF-4{alpha} activity can be modulated by phosphorylation (29, 30) and by coactivators, such as PPAR{gamma} coactivator-1 (31). Ongoing experiments are addressing whether LXR and other ligand-dependent receptors regulate L-UrdPase expression by modulating HNF-4{alpha} activity in this way. Taken together, the present work suggests a common circadian and lipid regulatory cascade has evolved to govern the expression of L-UrdPase and other liver metabolic enzymes, a conclusion that fits the predicted model for regulation by the lipid sensing nuclear receptors (2).

L-UrdPase shares high sequence identity to uridine phosphorylases and exhibits a similar enzymatic activity. UrdPase is one of two enzymes in the salvage pathway of nucleotide biosynthesis and is involved in the regulation of plasma uridine levels (16, 32). Circulating uridine is taken up and used by bone marrow, kidney, lung, spleen, and intestine; however, it is rapidly degraded in the liver (32, 33, 34). The data presented here show that the expression of previously characterized mUrdPase mRNA is restricted almost entirely to the intestine, whereas L-UrdPase is only expressed in liver. This brings up the question of why there are two distinctly regulated, tissue-specific forms of UrdPase present. Previous studies have suggested that diets can influence the circulating levels of uridine (34, 35). Thus, one explanation for the presence of L-UrdPase is that it encodes the liver-specific isoform of UrdPase that is responsible for the diet-induced clearance of plasma uridine to maintain uridine homeostasis. This idea is supported further by the finding that L-UrdPase (but not its intestinal counterpart) is regulated by nuclear receptors that function as sensors of lipids derived from the diet.

Although the exact biological function of L-UrdPase remains unsolved, its regulation by the lipid-sensing nuclear receptors leads to an interesting hypothesis that is being addressed by ongoing studies. In a previous study using large-scale gene expression analysis, UrdPase mRNA was shown to be up-regulated in cholesterol-loaded macrophages (36). Given the predominant role of LXRs in macrophage cholesterol metabolism (7), it is tempting to speculate that there is more than just a casual link between uridine metabolism and reverse cholesterol transport. The liver-specific expression of L-UrdPase may provide another important link. A crucial metabolic product of uridine catabolism in the liver is ß-alanine, which serves as a precursor to fatty acid synthesis. It has been shown that LXRs induce lipogenesis (5, 37), whereas PPAR{alpha} regulates genes that are involved in peroxisomal and mitochondrial ß-oxidation and {omega}-oxidation of fatty acids (38), and activation of FXR reduces triglyceride content (39). In keeping with a predicted role for UrdPase in hepatic lipogenesis, these findings may explain why LXRs up-regulate L-UrdPase expression, whereas PPAR{alpha} and FXR repress it. Finally, the finding that L-UrdPase is a target gene of HNF-4{alpha} is consistent with the critical role of HNF-4{alpha} in regulating expression of genes involved in a variety of hepatic metabolic functions, including lipid, amino acid, and glucose metabolism (12, 13). Interestingly, mice carrying a liver-specific disruption of HNF-4{alpha} show increased expression of genes involved in the uptake (scavenger receptor class B type I) and catabolism of lipids (medium-chain acyl-CoA dehydrogenase, carnitine palmitoyltransferase II, and hydroxymethylglutaryl CoA synthase), whereas genes involved in very low-density lipoprotein synthesis and secretion (a prerequisite for the export and subsequent storage of lipid in the adipose tissue) are strongly down-regulated (20). The corollary of these observations is that the net effect of HNF-4{alpha} activity in the liver is to oppose the burning of, and to promote the storage of, lipid as an energy source. We note that the HNF-4{alpha} response element identified in the mouse L-UrdPase is conserved in the homologous human gene, suggesting that a similar regulatory pathway may exist in humans. The identification of L-UrdPase as a novel HNF-4{alpha} target gene suggests that HNF-4{alpha} may also promote lipogenesis and, thus, oppose the action of PPAR{alpha}. In conclusion, the identification of L-UrdPase and its regulation by nuclear receptors suggests the existence of a new signaling pathway. The further elucidation of this pathway and the function of L-UrdPase may provide an unsuspected link between two crucial metabolic processes, lipid and uridine metabolism.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
[2-14C]uridine (54 Ci/mol) and [2-14C]thymidine (53 Ci/mol) were obtained from Moravek Biochemicals, Inc. (Brea, CA). Silica gel thin-layer chromatography plates were obtained from Whatman Ltd. (Maidstone, Kent, UK). Uridine was obtained from Sigma Chemical Co. (St. Louis, MO). Rabbit polyclonal anti-HNF-4{alpha} (H-171)X IgG was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Fenofibrate, pregnenolone {alpha}-carbonitrile, and chenodeoxycholic acid were purchased from Sigma Chemical Co. LG268, T0901317, and troglitazone were gifts of Richard Heyman (Ligand Pharmaceuticals, San Diego, CA), and Roger Unger (University of Texas Southwestern Medical Center).

PCR-Select cDNA Subtraction
Liver mRNA was isolated from wild-type (on 2% cholesterol diet) and Lxr{alpha} -/- (on chow diet) mice as described previously (8). Liver cDNA was generated and subtractive hybridization was performed after the protocol of a CLONTECH (Palo Alto, CA) PCR-select cDNA subtraction kit. Wild-type liver cDNA was digested with RsaI and ligated with adapters (defined as tester), whereas unligated Lxr{alpha}-/- liver cDNA (defined as driver) was used as the reference cDNA. The fragment obtained from subtraction of tester from driver was used as a probe to screen a mouse liver cDNA library (CLONTECH). Full-length cDNA was isolated using 5'-rapid amplification of cDNA ends (Invitrogen Life Technologies, Carlsbad, CA). Primer extension (Promega, Madison, WI) and in vitro transcription and translation (Promega) assays were performed to verify the cDNA.

Plasmids
Mouse uridine phosphorylase cDNA was amplified by RT-PCR using primers 5'-CCGGAATTCATGGCAGCTACAGGAACTGAAG-3' and 5'-CCCAAGCTTCCAGG-AGTTGCAGAGGCTTC-3' (GenBank accession no. D44464) and cloned into EcoRI/HindIII sites of pCMV-Myc vector and EcoRI sites of pCMX vector. L-UrdPase cDNA was amplified by RT-PCR using primers 5'-AGCCGATATCATGGCTTCCATTTTGCCTG-CTTC-3' and 5'-GCCATATTTCCCCGAGGATAATTCTTC-3', and then cloned into EcoRI(blunted)/HindIII sites of pCMV-Myc vector and EcoRV/HindIII sites of pCMX vector. L-UrdPase promoter constructs were amplified by PCR and cloned into BamHI/XhoI sites of TK-LUC vector. Mutations at DR1 site were created by splicing by overlap extension PCR (40) using primers 5'-GTTCAAATGGGGAACT-CGAGGATAAAGGA-3' and 5'-GGTTC-CTTTATCCT-CGAGTTCCCCATTTG-3' (mutated nucleotides are underlined). VP16-HNF-4{alpha} was made by cloning HNF-4{alpha} into EcoRV/BamHI sites of pCMX-VP16 vector. HNF-4{alpha} was PCR amplified using primers: 5'-AGCCGATATCATGGACATGGCTGACTAC-3'; 5'-CGCGGATCCCTAGATGGCTTCCTGCTTG-3'. The cloning and mutation sites were verified by sequencing.

Animal Studies
Mice were housed under the normal 12-h light, 12-h dark cycle and fed ad libitum rodent diet (Harlan-Teklad 7001, Madison, WI) supplemented with cholesterol and/or agonists or vehicle. Animal experiments were conducted as described previously (4). Care and use of animals were approved by the Institution Animal Care and Research Advisory Committee of the University of Texas Southwestern Medical Center.

Northern Analysis
RNA extraction and Northern analyses were performed as described (8). Five micrograms of mRNA were loaded in each lane of a 1% formaldehyde agarose gel and transferred to Nylon membrane (41). cDNAs of mUrdPase, L-UrdPase, and CYP7A1 were 32P-labeled and used to hybridize to Northern membranes.

Real-Time PCR Analysis
Total RNA was extracted from tissues using RNA STAT-60 (Tel-Test, Inc., Friendswood, TX), treated with deoxyribonuclease I (ribonuclease-free, Roche Molecular Biochemicals, Indianapolis, IN), and reverse-transcribed with random hexamers using the SuperScript II First-Strand Synthesis System (Invitrogen, Carlsbad, CA) to generate cDNA. Primers for each gene were designed using Primer Express Software (Applied Biosystems, Foster City, CA) based on sequence data available through GenBank. The real-time PCR contained 25 ng of reverse-transcribed total RNA, 150 nM of each primer, and 5 µl of 2x Jump Start SYBR Green PCR Master Mix (Sigma) in a total volume of 10 µl. All PCRs were performed in triplicate on an Applied Biosystems Prism 7900HT Sequence Detection System, and relative mRNA levels were calculated using the comparative threshold cycle method (User Bulletin No. 2, Applied Biosystems). Primers were validated by analysis of a standard curve and dissociation curve for each primer pair in a template titration assay.

Cell Culture and Cotransfection Assays
HEK 293 cells, were incubated at 37 C, 5% CO2 in DMEM containing 10% FBS. Transfections were performed in 96-well plates in media containing 10% dextran-charcoal-stripped FBS by calcium phosphate coprecipitation technique as described previously (9). All transfection data were normalized using an internal ß-galactosidase marker and represent the mean ± SD of triplicate assays.

Nuclear Extracts
The human hepatocyte cell line, HepG2, was cultured at 37 C, 5% CO2 in DMEM/F12 containing 10% FBS. Nuclear proteins were isolated as described previously (42) and resuspended in 50 µl of buffer C. Protein concentrations were determined by the method of Bradford (43).

EMSA
EMSA was performed as described (44) using 32P-labeled oligonucleotides indicated in Fig. 6Go. After electrophoresis, the gel was dried at 80 C for 1.5 h and exposed on autoradiographic film at -80 C overnight with intensifying screens.

cDNA Expression in HEK 293 Cells
Ten micrograms of plasmid DNA containing either pCMV-Myc, pCMV-Myc-mUrdPase, or pCMV-Myc-L-UrdPase were transfected into HEK 293 cells in a 10-cm dish. After incubation for 24 h, cells were harvested and sonicated in buffer containing 50 mM potassium phosphate (pH 7.4), 1 mM EDTA, and 1 mM dithiothreitol (DTT). Cell extracts were centrifuged at 100,000 x g for 45 min and the supernatant was saved for assays. Protein concentrations were determined by the method of Bradford (43). Protein expression was verified by Western blot using anti-Myc antibody.

Uridine Phosphorylase Assay
All assays were run at 37 C under conditions where activity was linear with time and enzyme concentration. Nucleoside cleavage was measured isotopically by following the formation of uracil from uridine (16). The reaction mixture contained 50 mM potassium phosphate (pH 7.4), 1 mM EDTA, 1 mM DTT, 200 µM [2-14C]uridine or [2-14C]thymidine and 32 µl of cell lysate in a final volume of 80 µl. Protein levels were adjusted so that the activity was linear with time and the amount of enzyme. Samples were withdrawn from reaction mixture at each time point and stopped by boiling in a water bath for 2 min followed by freezing. Precipitated proteins were removed by centrifugation and 8 µl of the supernatant was spotted on a silica gel thin-layer chromatography plate. The plate was developed with chloroform:methanol:acetic acid (90:5:5 by vol). The amounts of radioactivity in uridine and uracil spots were determined by phosphorimager analysis. To measure Km values of L-UrdPase and mUrdPase enzyme activities, reaction mixtures containing 62.5 µM, 125 µM, 250 µM, 500 µM, and 1 mM uridine (9 Ci/mol) were incubated at 37 C for 25 min. Data were analyzed by Microsoft Excel and Prism Software (GraphPad, San Diego, CA).

To measure liver uridine phosphorylase activity, approximately 0.33 g of liver were taken and homogenized in 3 vol of the buffer described above. The homogenates were centrifuged at 100,000 x g for 45 min at 4 C. Twenty-five microliters of supernatant were used in a 50-µl reaction for uridine phosphorylase activity (16).

Sequence Analysis
Sequence identities were obtained by basic local alignment and search tool (National Center for Biotechnology Information). The alignment was obtained by MegAlign software using Clustal method with PAM250 residue weight table (DNASTAR Inc., Madison, WI). The cDNA sequence of L-UrdPase has been submitted to the GenBank database under accession no. AY152393. While this manuscript was in preparation, a study identifying a second human uridine phosphorylase that is distinguishing from previously cloned human UrdPase was reported (15). L-UrdPase appears to have high identity (~80%) with this second human gene, although at present it is not clear whether this second gene is the mouse homolog or not.


   ACKNOWLEDGMENTS
 
The authors thank the members of the Mango lab for help on animal experiments and critically reading the manuscript.


   FOOTNOTES
 
This work was supported by the Howard Hughes Medical Institute (to D.J.M.), the Robert A. Welch Foundation (to D.J.M.), the National Cancer Institute (to Y.Z.), and NIH Grant 1U19DK62434 (to D.J.M.).

Current address for G.P.H.: Unité de Génétique de la Différenciation, Département de Biologie du Développement, 25/28 rue du Dr. Roux, Institut Pasteur, 75724 Paris Cedex 15, France.

Abbreviations: CAR, Constitutive androstane receptor; CoA, coenzyme A; Cyp7a1, cholesterol 7{alpha}-hydroxylase; DR, direct repeat; DTT, dithiothreitol; FXR, farnesoid X receptor; HEK, human embryonic kidney; HNF-4{alpha}, hepatic nuclear factor-4{alpha}; Km, Michaelis constant; L-UrdPase, novel liver-specific UrdPase gene; LXR, liver X receptor; mUrdPase, mouse UrdPase; PPAR, peroxisome proliferator-activated receptor; PXR, pregnane X receptor; RXR, retinoid X receptor; SHP, small heterodimer partner; SXR, steroid xenobiotic receptor; UrdPase, uridine phosphorylase; UTR, untranslated region.

Received for publication July 18, 2003. Accepted for publication December 23, 2003.


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