HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 281, Issue 15, 10081-10088, April 14, 2006

This Article Abstract Full Text (PDF) All Versions of this Article: 281/15/10081    most recent M513420200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Song, K.-H. Articles by Chiang, J. Y. L. Search for Related Content PubMed PubMed Citation Articles by Song, K.-H. Articles by Chiang, J. Y. L. Social Bookmarking                      What's this?

A Prospero-related Homeodomain Protein Is a Novel Co-regulator of Hepatocyte Nuclear Factor 4 That Regulates the Cholesterol 7-Hydroxylase Gene^*

From the Department of Microbiology, Immunology and Biochemistry, Northeastern Ohio Universities College of Medicine, Rootstown, Ohio 44272

Received for publication, December 16, 2005 , and in revised form, February 16, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Prox1, an early specific marker for developing liver and pancreas in foregut endoderm has recently been shown to interact with -fetoprotein transcription factor and repress cholesterol 7-hydroxylase (CYP7A1) gene transcription. Using a yeast two-hybrid assay, we found that Prox1 strongly and specifically interacted with hepatocyte nuclear factor (HNF)4, an important transactivator of the human CYP7A1 gene in bile acid synthesis and phosphoenolpyruvate carboxykinase (PEPCK) gene in gluconeogenesis. A real time PCR assay detected Prox1 mRNA expression in human primary hepatocytes and HepG2 cells. Reporter assay, GST pull-down, co-immunoprecipitation, and yeast two-hybrid assays identified a specific interaction between the N-terminal LXXLL motif of Prox1 and the activation function 2 domain of HNF4. Prox1 strongly inhibited HNF4 and peroxisome proliferators-activated receptor coactivator-1 co-activation of the CYP7A1 and PEPCK genes. Knock down of the endogenous Prox1 by small interfering RNA resulted in significant increase of CYP7A1 and PEPCK mRNA expression and the rate of bile acid synthesis in HepG2 cells. These results suggest that Prox1 is a novel co-regulator of HNF4 that may play a key role in the regulation of bile acid synthesis and gluconeogenesis in the liver.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
CYP7A1² catalyzes the first and rate-limiting step in the conversion of cholesterol to bile acids and plays an important role in maintaining whole body lipid homeostasis (1). Bile acids are physiological detergents that facilitate absorption, transport and distribution of sterols and lipid-soluble vitamins, and disposal of toxic metabolites and xenobiotics. Bile acid synthesis and CYP7A1 gene transcription is feedback inhibited by bile acids returning to the liver via enterohepatic circulation of bile (1). Recent studies have identified farnesoid X receptor (NR1H4) as a bile acid-activated receptor that induces an atypical nuclear receptor small heterodimer partner (SHP, NR0B2), which interacts with FTF (NR5A2) and HNF4 (NR2A1) bound to an overlapping sequence located in the bile acid response element II (–144/–126) and represses CYP7A1 gene transcription (2). However, the molecular mechanism by which FTF and HNF4 regulate the CYP7A1 gene is not completely understood.

HNF4 is the most abundant nuclear receptor expressed in the liver and is involved in early liver development (3). Conditional knock-out of the HNF4 gene in mouse liver caused accumulation of lipids in the liver, markedly reduced serum cholesterol and triglycerides, and increased serum bile acids (4). CYP7A1, Na⁺-taurocholate co-transport peptide, organic anion transporter 1, apolipoprotein B100, and scavenger receptor B-1 expression are reduced in these mice (4). It appears that HNF4 is a key regulator of bile acid and lipoprotein metabolism and plays a central role in lipid homeostasis (5). HNF4 is involved in diabetes; mutation of the HNF4 gene causes maturity onset diabetes of the young type 1 (MODY1) (6). HNF4 regulates the HNF1 gene, a MODY 3 gene (7).

The transcriptional activities of nuclear receptors are largely dependent on ligand binding and activation. Nuclear receptors interact with co-regulators and regulate their target genes in a tissue- and gene-specific manner (8). Upon ligand binding, the helix 12 of nuclear receptor is exposed and binds to the co-activators and activates nuclear receptor activity. Recently, PGC-1 has been identified as a co-activator of HNF4 (9). PGC-1 is highly induced during starvation by glucocorticoids and glucagon to induce PEPCK, a rate-limiting enzyme in gluconeogenesis (10). It has been reported that PGC-1 co-activates HNF4 and induces CYP7A1 gene transcription during starvation in mice (11). It has been suggested that bile acid synthesis and gluconeogenesis may be coordinately regulated in fasted-to-fed cycle (12). Our recent study (13) shows that glucagon and cAMP inhibit CYP7A1 by inducing phosphorylation of HNF4.

Prox1 has recently been identified as a co-repressor of FTF/LRH-1 by yeast two-hybrid screening (14, 15). Prox1 was originally cloned by homology to the Drosophila melanogaster gene prospero (16). Prox1 is expressed in lens, heart, liver, kidney, skeletal muscle, pancreas, and central nervous system (16, 17). Earlier studies have linked Prox1 function to lens and lymphatic system development (18, 19). More recent studies (20, 21) indicate that Prox1 is required for hepatocyte migration in developing liver and pancreas in the mammalian foregut endoderm. Prox1 interacts with the NR5 subfamily of nuclear receptors including Ff1b (NR5A4), a zebrafish homologue of nuclear receptor, steroidogenic factor 1 (NR5A1) (22), and FTF (14, 15) and represses their transactivation activity. We hypothesized that Prox1 may interact with HNF4 and suppressed CYP7A1 gene transcription. To test this hypothesis, we used yeast two-hybrid assay to study the interaction between Prox1 and HNF4 and studied the effect of Prox1 on the HNF4 transactivation of the human CYP7A1 gene. Our findings provide a novel molecular mechanism for Prox1 inhibition of bile acid synthesis and gluconeogenesis.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell Culture—Primary human hepatocytes were isolated from human donors (HH1201, 69-year-old male; HH1205, 45-year-old male; HH1209 50-year-old female; HH1234, 56-year-old male; HH1247, 3-year-old male; HH1248, 42-year-old female) and were obtained from the Liver Tissue Procurement and Distribution System of National Institutes of Health (S. Strom, University of Pittsburgh, PA). The HepG2 cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were maintained as described previously (13).

Plasmids—The mammalian expression plasmids for HNF4, Nur77, PGC-1, Flag-Prox1, Gal4-HNF4, pHNF4-tk-Luc reporter, NurRE-Luc, 5x upsteam activating sequence-Luc and human CYP7A1 promoter luciferase reporters were as described previously (14, 23, 24). Human PEPCK promoter luciferase reporter was generously provided by Dr. Richard Hanson (Case Western Reserve University, Cleveland, OH). The various deletion constructs of Prox1 (Prox1-NT-WT, amino acids 1–312; Prox1-NT-MT, amino acids 1–312, alanine mutations were introduced to convert LRKLL (amino acids 70–74) to ARKAL; Prox1-Homeo (amino acids 313–736)) and HNF4 (HNF4-NT, amino acids 1–128; HNF4-LBD, amino acids 129–455; HNF4-AF2, amino acids 1–352) were made by PCR with suitable restriction endonucleases and inserted into the pcDNA3-HA or the yeast LexA or B42 expression vector (Clontech Laboratories, Inc.). For bacterial expression, GST-fused Prox1 was constructed by inserting EcoRI-XhoI fragments of Prox1 from B42-Prox1 into pGEX4T-1 vector (Amersham Biosciences). All the clones were confirmed by sequencing analysis.

GST Pull-down Assay—[³⁵S]Methionine-labeled proteins were prepared by in vitro translation using the TNT-coupled transcriptional translation system with conditions as described by the manufacturer (Promega). GST-fused Prox1 (GST-Prox1) was expressed in the Escherichia coli BL21 (DE3) strain and purified using glutathione-Sepharose 4B beads (Pharmacia, Piscataway, NJ). In vitro protein-protein interaction assays were carried out as described (23).

Co-immunoprecipitation Assay—The cell extracts of human primary hepatocytes were precleared with whole rabbit serum adsorbed on Protein A-Sepharose beads (Amersham Biosciences) and subsequently subjected to immunoprecipitation with 10 µg of anti-HNF4 or non-immune serum (IgG) overnight at 4 °C. The beads were washed several times with ice-cold modified radioimmunoprecipitation assay buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% Na⁺-deoxycholate, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 1 mM ethylendiamine tetraacetic acid, 1 mM sodium orthovanadate, 1 mM NaF), and the immune complexes were subjected to electrophoresis on a 10% SDS-polyacrylamide gel and then transferred to a nitrocellulose membrane (Amersham Biosciences). Enhanced chemiluminescence Western blotting (Amersham Biosciences) was performed according to the manufacturer's instructions. Prox1 and HNF4 proteins were detected by incubation of blots with an anti-Prox1 antibody (1:2000 dilution; Upstate, Lake Placid, NY) and anti-HNF4 antibody (1:2000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), respectively.

Yeast Two-hybrid Assay—For the yeast two-hybrid system, LexA and B42 fusion plasmids for nuclear receptors or co-activators were co-transformed into Saccharomyces cerevisiae EGY48 strain containing the LacZ reporter plasmid (p80p-lac Z) under the control of the LexA binding site. -Galactosidase activity expressed on the plates was assayed as described previously (23). For assay of thyroid hormone receptor, glucocorticoid receptor, retinoid X receptor, and retinoic acid receptor, 100 µl of 1 µM stock solution of the appropriate ligands (T3, dexamethasone, all-trans-retinoic acid and 9-cis-retinoid) was added before plating to test the effect of ligand activation on interaction. Assays were repeated at least three times.

Transient Transfection and Luciferase Reporter Assay—For luciferase reporter assay, HepG2 cells were plated in 24-well plates 24 h before transfection with reporter or expression plasmids using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. The total DNA used in each transfection was adjusted by adding the appropriate amount of pcDNA3 vector. Luciferase activities are expressed as relative luciferase unit/-galactosidase activity as described previously (23).

Small Interfering RNA (siRNA) Experiments—The SMART pool siRNAs for human Prox1 were purchased from Dharmacon Research (Lafayette, Co) and transfected into HepG2 cells using Lipofectamine 2000 reagent according to the manufacturer's instructions. Forty-eight hours after transfection, cells were extracted and analyzed. The SMART pool is a mixture of four sequences located at different regions of mRNA. Two of them were tested to be effective in knockdown Prox1 in HepG2 cells. The siRNA sequences are: siRNA#1, nucleotides 1009–1027, GGGCCAAACTCCTTACAAC; siRNA#2 nucleotides 2096–2114, GCAAAGATGTTGATCCTTC. The control siRNA probe is a scrambled siRNA that was designed to have the same G-C content as the siRNA#2 but did not display sequence identity with Prox1: GATCGTGTGTAGTTCATAACC.

RNA Isolation and Real Time Quantitative PCR—HepG2 cells was transfected with synthesized siRNA of human Prox1 and total RNA was isolated using Tri-reagent (Sigma, St. Louis, MO) according to the manufacturer's instruction. Reverse-transcription and real time quantitative PCR were performed to detect Prox1, CYP7A1, PEPCK, HNF4, and cyclophilin B mRNAs as described previously (13).

Chromatin Immunoprecipitation (ChIP) Assay—ChIP assays were performed using a ChIP Assay kit (Upstate Cell Signaling Solutions, Lake Placid, NY) according to the manufacturer's instructions. HepG2 were transfected with pcDNA3 empty vector or Flag-Prox1, and chromatin was cross-linked in 1% formaldehyde and sonicated as reported previously (24). Cell lysate solution (5%) in ChIP dilution buffer was kept aside as "input." Ten µg of HNF4 antibody (Santa Cruz Biotechnology) or anti-FLAG antibody (Sigma) was added to precipitate DNA-protein complexes, and non-immune IgG was used as a control. A 391-bp DNA fragment (–432 to –41) containing the BARE-I and BARE-II of the CYP7A1 promoter was PCR-amplified for 30 cycles using 5 µl of the DNA as template and analyzed on a 1.5% agarose gel. PCR primers for amplification were as follows: 5'-ATCACCGTCTCTCTGGCAAAGCAC-3'; reverse primer, 5'-CCATTAACTTGAGCTTGGTTGACAAAG-3'.

Bile Acid Analysis—The siRNA for human Prox1 and control siRNA were transfected into HepG2 cells using Lipofectamine 2000 reagent according to the manufacturer's instructions. Forty-eight hours after transfection, cells were washed and then incubated with serum-free medium for a period of time from 3 to 24 h. The medium was collected at indicated time points and frozen at –80 °C for later analysis of bile acids. At the end of the incubation, the cells were harvested and stored at –80 °C until use. A Sep-Pak C18 reversed phase cartridge (Waters Associates, Inc., Milford, MA) was used for bile acid extraction from media as described previously (25). Total bile acid concentration was analyzed by enzymatic 3-hydroxysteroid dehydrogenase method using total bile acid assay kit (Bio-quant Inc., San Diego, CA) according to the manufacturer's instruction.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Prox1 Interacts with HNF4 in Vivo and in Vitro—The nuclear receptors that have been identified to interact with Prox1 all belong to the NR5A subfamily (14, 15, 22). To identify other potential interaction partners of Prox1, we performed yeast two-hybrid protein interaction assays using LexA and B42 constructs available in our laboratory. Table 1 shows that Prox1 interacted with HNF4. This interaction is stronger than Prox1 interaction with steroidogenic factor 1. On the other hand, Prox1 did not interact with thyroid hormone receptor , thyroid hormone receptor , retinoid X receptor, retinoic acid receptor, glucocorticoid receptor, SHP, dosage-sensitive sex reversal, AHC critical region on the X chromosome, gene 1 (DAX-1), and Nur77 regardless of the presence or absence of their respective ligands (Table 1). Transcriptional repressors N-CoR and SMRT did not interact with Prox1. These results showed that Prox1 interacts with HNF4 and NR5A family nuclear receptors.

View larger version (23K): [in this window] [in a new window]   FIGURE 1. GST pull-down and co-immunoprecipitation assays of Prox1 interaction with HNF4. A, GST pull-down assay. Purified GST alone (negative control) or GST-Prox1 bound to glutathione-Sepharose beads were incubated with 35S-labeled HNF4, retinoid X receptor (RXR), retinoic acid receptor (RAR), and FTF (positive control). The reactions were analyzed by SDS-polyacrylamide gel electrophoresis, and bound proteins were visualized by autoradiography. The input represents 10% of the labeled proteins used for the pull-down assay. B, co-immunoprecipitation assay. Protein extracts were prepared from human primary hepatocytes and immunoprecipitated (IP) with anti-HNF4 antibody or non-immune serum (IgG, as control). Immunoprecipitated proteins were resolved on SDS-polyacrylamide gel and analyzed by immunoblotting with anti-Prox1 and anti-HNF4 antibodies. Data represent one of three separate experiments. WB, Western blot.

We then investigated which region of HNF4 was required for interaction with Prox1. A series of deletion constructs of HNF4 were used to map the HNF4 interaction domain in yeast-two hybrid assay. Fig. 2B shows that the full-length HNF4 (HNF4-full) and LBD, which contains activation function 2 (AF2) domain (HNF4-LBD), interacted with Prox1. However, the N-terminal DBD region and a construct without AF2 (HNF4-AF2) did not interact with Prox1. These results indicate that HNF4 interacts with Prox1 through the AF2 domain of HNF4.

Prox1 Is Expressed in Human Hepatocytes—Although it has been reported that Prox1 is highly expressed in liver and pancreas (14), the expression of Prox1 in human hepatocytes has not been reported before. Using real time quantitative PCR, we were able to detect the mRNA expressions of CYP7A1, Prox1, PGC-1, and several nuclear receptors that are known to regulate CYP7A1 in five donor human primary hepatocytes and HepG2 cells. Table 2 shows Ct, the threshold cycle number, for each mRNA transcripts assayed in these hepatocytes. The mRNA expression levels were normalized to internal reference gene UBC and the Ct values and S.D. are shown in the Table 2. The expression patterns of these mRNA transcripts are similar in five donor hepatocytes and HepG2 cells. The Ct and Ct values of CYP7A1 and PGC-1 are high, reflecting low levels of mRNA expression. Those values for Prox1, HNF4, and SHP are low, indicating a relatively abundant expression of these three mRNA transcripts.

View larger version (23K): [in this window] [in a new window]   FIGURE 2. Mapping of interaction domains between Prox1 and HNF4. A, Prox1 N-terminal LRKLL motif (NR1) of Prox1 is required for HNF4 interaction. Various LexA-Prox1 constructs, as indicated in the upper panel, were co-transformed with B42 (–) or B42-HNF4 (+) into EGY48 yeast cells. B, the ligand binding domain (LBD) of HNF4 interacts with Prox1. LexA (–) or LexA-Prox1 (+) and various B42-HNF4 constructs, as indicated upper panel were co-transformed into EGY48 yeast cells. Transformants were selected on plates with appropriate selection marker, and the -galactosidase activity was measured. The results shown are the mean of -galactosidase value from five independent transformant colonies. DBD, DNA binding domain.

Prox1 Suppresses HNF4 Transactivation of the Human CYP7A1 Gene—Recent studies have provided substantial evidence that HNF4 is an important transcription factor that regulates liver-specific expression of CYP7A1 (4). HNF4 is the only nuclear receptor that activates the human CYP7A1 gene in transfection assays in HepG2 cells (24, 26). We thus investigated the effect of Prox1 on CYP7A1 transcription. As shown in Fig. 4A, ectopically expressed HNF4 modestly induced CYP7A1 reporter activity in HepG2 cells. This may be because of inhibition of HNF4 transactivation of CYP7A1 activity by endogenous Prox1. Co-expression of increasing amounts of Prox1 expression vectors caused marked repression of human CYP7A1 promoter reporter activity to a level below the basal activity. This suggests that exogenous Prox1 could suppress CYP7A1 basal reporter activity induced by endogenous HNF4.

View larger version (27K): [in this window] [in a new window]   FIGURE 3. Prox1 abrogates HNF4-mediated transactivation. A, HepG2 cells were co-transfected with the pHNF4-tk-Luc reporter (200 ng) along with the HNF4 expression plasmid (200 ng) and increasing amount of Prox1 expression plasmid (10, 50, and 100 ng). B, HepG2 cells were co-transfected with the Nur77 response element-Luc reporter (NurRE-Luc reporter, 200 ng) along with the Nur77 plasmid (100 ng) and increasing amount of Prox1 plasmid (10, 50, and 100 ng). C, HepG2 cells were co-transfected with the pHNF4-tk-Luc reporter (200 ng) along with the HNF4 plasmid (200 ng) and increasing amounts of Prox1-Full, Prox1-NT-WT (amino acids 1–312), and Prox1-NT-MT ((amino acids 1–312), mutations that convert LRKLL (amino acids 70–74) to ARKAL)) expression plasmids (10, 50, and 100 ng). Luciferase activity was normalized to -galactosidase activity. All experiments were done in duplicate, and data represent the mean ± S.D. of three individual experiments. RLU, relative luciferase units.

View larger version (15K): [in this window] [in a new window]   FIGURE 4. Prox1 represses CYP7A1 promoter activity through the HNF4 response element. A, HepG2 cells were co-transfected with the human CYP7A1 promoter reporter (hCYP7A1-Luc, 200 ng) along with the HNF4 plasmid (200 ng) and increasing amounts of Prox1 plasmid (10, 50, and 100 ng). B, HepG2 cells were transfected with a human CYP7A1 promoter reporter with HNF4 binding site mutation (mHNF4-hCYP7A1-Luc, 200 ng) and increasing amounts of Prox1 plasmid (10, 50 and 100 ng). Luciferase activity was normalized to -galactosidase activity. All experiments were done in duplicate, and data represent the mean ± S.D. of three individual experiments.

Prox1 Blocks HNF4 Recruitment of PGC-1 to CYP7A1 Chromatin—To investigate the molecular mechanism of Prox1 inhibition of the HNF4 transactivation of CYP7A1, we first performed an in vivo ChIP assay to study the effect of Prox1 on the HNF4 binding to CYP7A1 chromatin. HepG2 cells were transfected with pcDNA3 or Flag-Prox1 plasmid, and the DNA-protein complex was immunoprecipitated with an anti-FLAG or anti-HNF4 antibody as indicated for PCR amplification of the CYP7A1 promoter sequence from –432 to –41, which contains an HNF4 binding site. Fig. 5A shows that overexpressing Prox1 in HepG2 cells does not affect HNF4 binding to CYP7A1 chromatin (lane 2 versus lane 3). CYP7A1 chromatin was also detected by PCR when anti-FLAG antibody was used to immunoprecipitated Flag-Prox1 (lane4). Because Prox1 does not bind to DNA, Prox1 must be recruited to CYP7A1 chromatin by HNF4. This data is consistent with the results in Fig. 1. No promoter sequence was amplified in control experiments using none-immune rabbit IgG (lane 1). The negative control sequence from +860 to +1160 of hCYP7A1 was not amplified by immunoprecipitation with anti-HNF4 antibody (data not shown). These results suggested that Prox1 was associated with hCYP7A1 promoter via interaction with HNF4 and that Prox1 did not affect HNF4 binding to the CYP7A1 chromatin.

Previously we have reported that HNF4 recruits PGC-1 to CYP7A1 chromatin (24). We thus studied the effect of Prox1 on HNF4 recruitment of PGC-1 to CYP7A1 chromatin. HepG2 cells were co-transfected with HA-PGC-1 and Flag-Prox1 or pcDNA3 empty vector (negative control) for ChIP assay using an antibody against HA. Fig. 5B shows that HA-PGC-1 was recruited to CYP7A1 chromatin (lane 2) via interaction with HNF4, because PGC-1 does not bind to DNA. Co-expression of Flag-Prox1 together with HA-PGC-1 markedly reduced the DNA fragment immunoprecipitated with anti-HA antibody (lane 3) indicating that Flag-Prox1 blocked HNF4 recruitment of HA-PGC-1 to CYP7A1 chromatin. These data suggested that Prox-1 and PGC-1 might compete for binding to HNF4 in CYP7A1 chromatin.

Prox1 and PGC-1 Compete for Interaction with HNF4 and Regulate CYP7A1 and PEPCK Genes—We then performed transfection assay to study the effect of Prox1 on HNF4 and PGC-1 co-activation of a heterologous reporter, 5x UAS-Luc and native human CYP7A1-luc and PEPCK-Luc reporters. As shown in Fig. 6A, Gal4-HNF4 alone slightly stimulates Gal4 reporter activity. Addition of PGC-1 drastically stimulated Gal4 reporter activity. Addition of Prox1 dose-dependently inhibited Gal4 reporter activity. These results suggest that Prox1 strongly inhibited HNF4 and PGC-1 transactivation activity. Fig. 6B shows that the Prox1 inhibition of HNF4-mediated hCYP7A1-Luc reporter activity could be reversed by expression of PGC-1 in a dose-dependent manner. The PEPCK gene is known to be induced by HNF4 and PGC-1 (9). We thus performed similar experiments to study the effect of Prox1 on PEPCK-Luc reporter activity. Fig. 6C shows that HNF4 stimulation of PEPCK-Luc reporter activity was repressed by Prox1 and increasing amounts of PGC-1 reversed the repressive effect of Prox1. These results further support the idea that direct interaction between Prox1 and HNF4 prevented the recruitment of co-activator, PGC-1 to stimulate HNF4 transactivation of the CYP7A1 and PEPCK genes.

View larger version (61K): [in this window] [in a new window]   FIGURE 5. Chromatin immunoprecipitation assays of Prox1, HNF4, and PGC-1 binding to the human CYP7A1 gene. A, HepG2 cells were transfected with pcDNA3 empty vector (negative control) or Flag-Prox1 expression plasmid and subjected to formaldehyde cross-linking. Chromatin fragments were prepared by sonication and immunoprecipitated (IP) with anti-HNF4 or anti-FLAG antibody, and promoter sequence containing HNF4 binding element was analyzed by PCR using primer sets specific for the CYP7A1 promoter. Cell lysate solution (5%) in ChIP dilution buffer was kept aside as Input. B, HepG2 cells were transfected with HA-PGC-1 alone or HA-PGC-1 together with Flag-Prox1 expression plasmid. Chromatin was immunoprecipitated with anti-hemagglutinin (HA) antibody, and the DNA sequence containing HNF4 binding element was analyzed by PCR. Data represent one of three separate experiments.

View larger version (24K): [in this window] [in a new window]   FIGURE 6. Effects of Prox1 on HNF4 and PGC-1 co-activation of gene transcription. A, pGAL4-HNF4 (200 ng) was co-transfected with PGC-1 (100 ng) and increasing amounts of Prox1 plasmid (50 and 100 ng) into HepG2 cells as indicated. B, HepG2 cells were co-transfected with a human CYP7A1 promoter reporter (hCYP7A1-Luc) and increasing amounts of Prox1 plasmid (10, 50, and 100 ng; +, 50 ng) or PGC-1 plasmid (50, 100, and 200 ng) as indicated. C, HepG2 cells were transfected with a human PEPCK promoter reporter (PEPCK-Luc) (200 ng) along with the HNF4 plasmid (200 ng) and increasing amounts of Prox1 plasmid (10, 50, and 100 ng; +, 50 ng) or PGC-1 (50, 100, and 200 ng) as indicated. Luciferase activity was normalized to -galactosidase activity. All experiments were done in duplicate, and data represent the mean ± S.D. of three individual experiments. RLU, relative luciferase units.

View larger version (31K): [in this window] [in a new window]   FIGURE 7. The siRNA knock down of Prox1 increases the expression of CYP7A1 and PEPCK mRNA in HepG2 cells. A, HepG2 cells were transfected with the Prox1 siRNA #1, #2, and control siRNA. The effects of siRNAs on the Prox1 expression were measured by Western blot analysis. Actin expression was used as a loading control. Data represent one of three separate experiments and vary from 35 to 88% reduction of protein expression. B, HepG cells were transfected with the Prox1 siRNA #1, #2, and control siRNA. Total RNA was isolated for real time quantitative PCR analysis of Prox1, CYP7A1, PEPCK, HNF4, and cyclophilin B (CycB) mRNA levels. Data show relative mRNA expression of Prox1 siRNA treated to the control siRNA treated samples. Data represent the mean ± S.D. of at least three individual experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Bile acid synthesis is tightly regulated under physiological conditions to protect the liver from accumulation of highly toxic bile acids. Under normal physiological conditions, expression of CYP7A1 must be suppressed by various factors including insulin, glucagon, bile acids, and cholesterol in the human (13). Nuclear receptors and co-regulators apparently play important roles in regulation of CYP7A1 gene transcription (27). Prox1 is constitutively expressed at high levels in adult livers and may be the major repressor of CYP7A1 gene transcription in human hepatocytes.

This study revealed a strong and specific interaction between Prox1 and HNF4 by yeast two-hybrid assay, in vivo coimmunoprecipitation assay using human primary hepatocytes, and in vitro GST pull-down assay. Our results indicate that Prox1 directly interacts with HNF4 via the N-terminal LXXLL motif of Prox1 and C-terminal AF2 domain of HNF4. Co-regulators are known to interact with nuclear receptors via interaction of the LXXLL motif of co-regulators and the AF2 domain of nuclear receptor (8, 28). However, the interaction of Prox1 with FTF also requires helices 2 and 10 of the LBD of FTF (15). Thus, it is likely that the interaction domain of Prox1 might be quite diverse compared with other nuclear receptors to allow interaction with various co-regulators such as SHP (29, 30). It is noted that Prox1 may inhibit HNF4 and FTF by somewhat different mechanisms. Prox1 impairs the FTF binding to DNA (15), whereas our data show that Prox1 does not affect HNF4 binding to CYP7A1 chromatin as demonstrated by ChIP assays (Fig. 5). Our results suggest that Prox1 competes with PGC-1 for interaction with HNF4 and thus counteracts PGC-1 co-activating activity. This is because both Prox1 and PGC-1 interacts with the AF2 domain of HNF4. Competition for binding and squelching of the limited co-activators could be a common mechanism for the negative regulation of gene transcription by nuclear receptors. In accordance with this scenario, increasing the amounts of PGC-1 could counteract Prox1-mediated repression of HNF4 activity. Conversely, increasing the amounts of Prox1 may interfere with PGC-1 co-activation of HNF4 activity. Thus, our finding that Prox1 interferes with the co-activator recruitment of HNF4 provides a novel mechanism for Prox1 repression of CYP7A1 gene transcription. Moreover, knock down of Prox1 expression in a hepatocyte cell line increased CYP7A1 mRNA expression and a corresponding increase in bile acid synthesis (Figs. 7 and 8). This study reveals a new biological function of Prox1, which has previously been shown to play an essential role for lymphatic system (19, 31) and lens (18) development in hepatocytes.

View larger version (10K): [in this window] [in a new window]   FIGURE 8. Knock down of Prox1 increases the rate of bile acid synthesis. HepG2 cells were transfected with Prox1 siRNA and control siRNA, and total bile acid was extracted from the medium at the indicated times. The amount of total bile acid was determined by enzymatic 3-hydroxysteroid dehydrogenase method and expressed as nmol/µg protein. The data represent one of three independent experiments.

In conclusion, here we provide direct experimental evidences that Prox1 acts as a novel co-repressor of nuclear receptor HNF4. Prox1-mediated repression of HNF4 transactivation may play an important role in the regulation of HNF4 target genes. The intricate regulatory circuitry of Prox1, PGC-1, HNF4, SHP, and FTF may maintain lipid and glucose homeostasis and prevent diabetes and obesity.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grants DK58379 and DK44442. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Microbiology, Immunology and Biochemistry, Northeastern Ohio University College of Medicine, 4209 State Route 44, P.O. Box 95, Rootstown, OH 44272. Tel.: 330-325-6694; Fax: 330-325-5910; E-mail: jchiang{at}neoucom.edu.

² The abbreviations used are: CYP7A1, cholesterol 7-hydroxylase; Prox1, prospero-related homeodomain protein; CYP8B1, sterol 12-hydroxylase; PEPCK, phosphoenolpyruvate carboxykinase; SHP, small heterodimer partner; FTF, -fetoprotein transcription factor; HA, hemagglutinin; GST, glutathione S-transferase; LRH-1, liver related homologue; HNF4, hepatocyte nuclear factor 4; DAX-1, dosage-sensitive sex reversal, AHC critical region on the X chromosome, gene 1; Luc, luciferase; PGC-1, peroxisome proliferators-activated receptor co-activator 1; siRNA, small interfering RNA; SRC-1, steroid receptor co-activator 1; ChIP, chromatin immunoprecipitation; AF2, activation function 2; NR, nuclear receptor.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Chiang, J. Y. (1998) Front Biosci. 3, d176–193[Medline] [Order article via Infotrieve]
2. Stroup, D., and Chiang, J. Y. (2000) J. Lipid Res. 41, 1–11[Abstract/Free Full Text]
3. Li, J., Ning, G., and Duncan, S. A. (2000) Genes Dev. 14, 464–474[Abstract/Free Full Text]
4. Hayhurst, G. P., Lee, Y. H., Lambert, G., Ward, J. M., and Gonzalez, F. J. (2001) Mol. Cell. Biol. 21, 1393–1403[Abstract/Free Full Text]
5. Sladek, R., and Giguere, V. (2000) Adv. Pharmacol. 47, 23–87[Medline] [Order article via Infotrieve]
6. Navas, M. A., Munoz-Elias, E. J., Kim, J., Shih, D., and Stoffel, M. (1999) Diabetes 48, 1459–1465[Abstract]
7. Kuo, C. J., Conley, P. B., Chen, L., Sladek, F. M., Darnell, J. E., Jr., and Crabtree, G. R. (1992) Nature 355, 457–461[CrossRef][Medline] [Order article via Infotrieve]
8. Glass, C. K., and Rosenfeld, M. G. (2000) Genes Dev. 14, 121–141[Free Full Text]
9. Yoon, J. C., Puigserver, P., Chen, G., Donovan, J., Wu, Z., Rhee, J., Adelmant, G., Stafford, J., Kahn, C. R., Granner, D. K., Newgard, C. B., and Spiegelman, B. M. (2001) Nature 413, 131–138[CrossRef][Medline] [Order article via Infotrieve]
10. Chakravarty, K., Cassuto, H., Reshef, L., and Hanson, R. W. (2005) Crit. Rev. Biochem. Mol. Biol. 40, 129–154[CrossRef][Medline] [Order article via Infotrieve]
11. Shin, D. J., Campos, J. A., Gil, G., and Osborne, T. F. (2003) J. Biol. Chem. 278, 50047–50052[Abstract/Free Full Text]
12. De Fabiani, E., Mitro, N., Gilardi, F., Caruso, D., Galli, G., and Crestani, M. (2003) J. Biol. Chem. 278, 39124–39132[Abstract/Free Full Text]
13. Song, K. H., and Chiang, J. Y. (2006) Hepatology 43, 117–125[CrossRef][Medline] [Order article via Infotrieve]
14. Steffensen, K. R., Holter, E., Bavner, A., Nilsson, M., Pelto-Huikko, M., Tomarev, S., and Treuter, E. (2004) EMBO Rep. 5, 613–619[CrossRef][Medline] [Order article via Infotrieve]
15. Qin, J., Gao, D. M., Jiang, Q. F., Zhou, Q., Kong, Y. Y., Wang, Y., and Xie, Y. H. (2004) Mol. Endocrinol. 18, 2424–2439[Abstract/Free Full Text]
16. Oliver, G., Sosa-Pineda, B., Geisendorf, S., Spana, E. P., Doe, C. Q., and Gruss, P. (1993) Mech. Dev. 44, 3–16[CrossRef][Medline] [Order article via Infotrieve]
17. Zinovieva, R. D., Duncan, M. K., Johnson, T. R., Torres, R., Polymeropoulos, M. H., and Tomarev, S. I. (1996) Genomics. 35, 517–522[CrossRef][Medline] [Order article via Infotrieve]
18. Wigle, J. T., Chowdhury, K., Gruss, P., and Oliver, G. (1999) Nat. Genet. 21, 318–322[CrossRef][Medline] [Order article via Infotrieve]
19. Wigle, J. T., and Oliver, G. (1999) Cell 98, 769–778[CrossRef][Medline] [Order article via Infotrieve]
20. Sosa-Pineda, B., Wigle, J. T., and Oliver, G. (2000) Nat. Genet. 25, 254–255[CrossRef][Medline] [Order article via Infotrieve]
21. Burke, Z., and Oliver, G. (2002) Mech. Dev. 118, 147–155[CrossRef][Medline] [Order article via Infotrieve]
22. Liu, Y. W., Gao, W., Teh, H. L., Tan, J. H., and Chan, W. K. (2003) Mol. Cell. Biol. 23, 7243–7255[Abstract/Free Full Text]
23. Song, K. H., Park, Y. Y., Park, K. C., Hong, C. Y., Park, J. H., Shong, M., Lee, K., and Choi, H. S. (2004) Mol. Endocrinol. 18, 1929–1940[Abstract/Free Full Text]
24. Li, T., and Chiang, J. Y. (2005) Am. J. Physiol. 288, G74–G84
25. Feldmann, D., Fenech, C., and Cuer, J. F. (1983) Clin. Chem. 29, 1694[Free Full Text]
26. Chen, W., Owsley, E., Yang, Y., Stroup, D., and Chiang, J. Y. (2001) J. Lipid Res. 42, 1402–1412[Abstract/Free Full Text]
27. Chiang, J. Y. (2002) Endocr. Rev. 23, 443–463[Abstract/Free Full Text]
28. McKenna, N. J., and O'Malley, B. W. (2002) Endocrinology 143, 2461–2465[Abstract/Free Full Text]
29. Lee, Y. K., Dell, H., Dowhan, D. H., Hadzopoulou-Cladaras, M., and Moore, D. D. (2000) Mol. Cell. Biol. 20, 187–195[Abstract/Free Full Text]
30. Lee, Y. K., and Moore, D. D. (2002) J. Biol. Chem. 277, 2463–2467[Abstract/Free Full Text]
31. Petrova, T. V., Makinen, T., Makela, T. P., Saarela, J., Virtanen, I., Ferrell, R. E., Finegold, D. N., Kerjaschki, D., Yla-Herttuala, S., and Alitalo, K. (2002) EMBO J. 21, 4593–4599[CrossRef][Medline] [Order article via Infotrieve]
32. del Castillo-Olivares, A., Campos, J. A., Pandak, W. M., and Gil, G. (2004) J. Biol. Chem. 279, 16813–16821[Abstract/Free Full Text]
33. Silander, K., Mohlke, K. L., Scott, L. J., Peck, E. C., Hollstein, P., Skol, A. D., Jackson, A. U., Deloukas, P., Hunt, S., Stavrides, G., Chines, P. S., Erdos, M. R., Narisu, N., Conneely, K. N., Li, C., Fingerlin, T. E., Dhanjal, S. K., Valle, T. T., Bergman, R. N., Tuomilehto, J., Watanabe, R. M., Boehnke, M., and Collins, F. S. (2004) Diabetes 53, 1141–1149[Abstract/Free Full Text]
34. Krylova, I. N., Sablin, E. P., Moore, J., Xu, R. X., Waitt, G. M., MacKay, J. A., Juzumiene, D., Bynum, J. M., Madauss, K., Montana, V., Lebedeva, L., Suzawa, M., Williams, J. D., Williams, S. P., Guy, R. K., Thornton, J. W., Fletterick, R. J., Willson, T. M., and Ingraham, H. A. (2005) Cell 120, 343–355[CrossRef][Medline] [Order article via Infotrieve]
35. Ortlund, E. A., Lee, Y., Solomon, I. H., Hager, J. M., Safi, R., Choi, Y., Guan, Z., Tripathy, A., Raetz, C. R., McDonnell, D. P., Moore, D. D., and Redinbo, M. R. (2005) Nat. Struct. Mol. Biol. 12, 357–363[CrossRef][Medline] [Order article via Infotrieve]
36. Petrescu, A. D., Hertz, R., Bar-Tana, J., Schroeder, F., and Kier, A. B. (2002) J. Biol. Chem. 277, 23988–23999[Abstract/Free Full Text]
37. Harvey, N. L., Srinivasan, R. S., Dillard, M. E., Johnson, N. C., Witte, M. H., Boyd, K., Sleeman, M. W., and Oliver, G. (2005) Nat. Genet. 37, 1072–1081[CrossRef][Medline] [Order article via Infotrieve]
38. Kakuma, T., Wang, Z. W., Pan, W., Unger, R. H., and Zhou, Y. T. (2000) Endocrinology 141, 4576–4582[Abstract/Free Full Text]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				X. Chen, J. R. Taube, V. I. Simirskii, T. P. Patel, and M. K. Duncan Dual Roles for Prox1 in the Regulation of the Chicken {beta}B1-Crystallin Promoter Invest. Ophthalmol. Vis. Sci., April 1, 2008; 49(4): 1542 - 1552. [Abstract] [Full Text] [PDF]

				T. Li, X. Kong, E. Owsley, E. Ellis, S. Strom, and J. Y. L. Chiang Insulin Regulation of Cholesterol 7{alpha}-Hydroxylase Expression in Human Hepatocytes: ROLES OF FORKHEAD BOX O1 AND STEROL REGULATORY ELEMENT-BINDING PROTEIN 1c J. Biol. Chem., September 29, 2006; 281(39): 28745 - 28754. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 281/15/10081    most recent M513420200v1 Alert me when this article is cited Alert me if a correction is posted