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• W2003649068 abstract "Prox1, an early specific marker for developing liver and pancreas in foregut endoderm has recently been shown to interact with α-fetoprotein transcription factor and repress cholesterol 7α-hydroxylase (CYP7A1) gene transcription. Using a yeast two-hybrid assay, we found that Prox1 strongly and specifically interacted with hepatocyte nuclear factor (HNF)4α, 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 HNF4α. Prox1 strongly inhibited HNF4α and peroxisome proliferators-activated receptor γ coactivator-1α 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 HNF4α that may play a key role in the regulation of bile acid synthesis and gluconeogenesis in the liver. Prox1, an early specific marker for developing liver and pancreas in foregut endoderm has recently been shown to interact with α-fetoprotein transcription factor and repress cholesterol 7α-hydroxylase (CYP7A1) gene transcription. Using a yeast two-hybrid assay, we found that Prox1 strongly and specifically interacted with hepatocyte nuclear factor (HNF)4α, 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 HNF4α. Prox1 strongly inhibited HNF4α and peroxisome proliferators-activated receptor γ coactivator-1α 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 HNF4α that may play a key role in the regulation of bile acid synthesis and gluconeogenesis in the liver. CYP7A1 2The abbreviations used are: CYP7A1, cholesterol 7α-hydroxylase; Prox1, prospero-related homeodomain protein; CYP8B1, sterol 12α-hydroxylase; PEPCK, phosphoenolpyruvate carboxykinase; SHP, small heterodimer partner; FTF, α-fetoprotein transcription factor; HA, hemagglutinin; GST, glutathione S-transferase; LRH-1, liver related homologue; HNF4α, hepatocyte nuclear factor 4α; DAX-1, dosage-sensitive sex reversal, AHC critical region on the X chromosome, gene 1; Luc, luciferase; PGC-1α, peroxisome proliferators-activated receptor γ co-activator 1α; siRNA, small interfering RNA; SRC-1, steroid receptor co-activator 1; ChIP, chromatin immunoprecipitation; AF2, activation function 2; NR, nuclear receptor. 2The abbreviations used are: CYP7A1, cholesterol 7α-hydroxylase; Prox1, prospero-related homeodomain protein; CYP8B1, sterol 12α-hydroxylase; PEPCK, phosphoenolpyruvate carboxykinase; SHP, small heterodimer partner; FTF, α-fetoprotein transcription factor; HA, hemagglutinin; GST, glutathione S-transferase; LRH-1, liver related homologue; HNF4α, hepatocyte nuclear factor 4α; DAX-1, dosage-sensitive sex reversal, AHC critical region on the X chromosome, gene 1; Luc, luciferase; PGC-1α, peroxisome proliferators-activated receptor γ co-activator 1α; siRNA, small interfering RNA; SRC-1, steroid receptor co-activator 1; ChIP, chromatin immunoprecipitation; AF2, activation function 2; NR, nuclear receptor. 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 (1Chiang J.Y. Front Biosci. 1998; 3: d176-d193Crossref PubMed Scopus (252) Google Scholar). 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 (1Chiang J.Y. Front Biosci. 1998; 3: d176-d193Crossref PubMed Scopus (252) Google Scholar). 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 HNF4α (NR2A1) bound to an overlapping sequence located in the bile acid response element II (–144/–126) and represses CYP7A1 gene transcription (2Stroup D. Chiang J.Y. J. Lipid Res. 2000; 41: 1-11Abstract Full Text Full Text PDF PubMed Google Scholar). However, the molecular mechanism by which FTF and HNF4α regulate the CYP7A1 gene is not completely understood. HNF4α is the most abundant nuclear receptor expressed in the liver and is involved in early liver development (3Li J. Ning G. Duncan S.A. Genes Dev. 2000; 14: 464-474PubMed Google Scholar). Conditional knock-out of the HNF4α gene in mouse liver caused accumulation of lipids in the liver, markedly reduced serum cholesterol and triglycerides, and increased serum bile acids (4Hayhurst G.P. Lee Y.H. Lambert G. Ward J.M. Gonzalez F.J. Mol. Cell. Biol. 2001; 21: 1393-1403Crossref PubMed Scopus (841) Google Scholar). CYP7A1, Na+-taurocholate co-transport peptide, organic anion transporter 1, apolipoprotein B100, and scavenger receptor B-1 expression are reduced in these mice (4Hayhurst G.P. Lee Y.H. Lambert G. Ward J.M. Gonzalez F.J. Mol. Cell. Biol. 2001; 21: 1393-1403Crossref PubMed Scopus (841) Google Scholar). It appears that HNF4α is a key regulator of bile acid and lipoprotein metabolism and plays a central role in lipid homeostasis (5Sladek R. Giguere V. Adv. Pharmacol. 2000; 47: 23-87Crossref PubMed Scopus (48) Google Scholar). HNF4α is involved in diabetes; mutation of the HNF4α gene causes maturity onset diabetes of the young type 1 (MODY1) (6Navas M.A. Munoz-Elias E.J. Kim J. Shih D. Stoffel M. Diabetes. 1999; 48: 1459-1465Crossref PubMed Scopus (51) Google Scholar). HNF4α regulates the HNF1α gene, a MODY 3 gene (7Kuo C.J. Conley P.B. Chen L. Sladek F.M. Darnell Jr., J.E. Crabtree G.R. Nature. 1992; 355: 457-461Crossref PubMed Scopus (367) Google Scholar). 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 (8Glass C.K. Rosenfeld M.G. Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar). Upon ligand binding, the helix 12 of nuclear receptor is exposed and binds to the co-activators and activates nuclear receptor activity. Recently, PGC-1α has been identified as a co-activator of HNF4α (9Yoon 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. Spiegelman B.M. Nature. 2001; 413: 131-138Crossref PubMed Scopus (1482) Google Scholar). PGC-1α is highly induced during starvation by glucocorticoids and glucagon to induce PEPCK, a rate-limiting enzyme in gluconeogenesis (10Chakravarty K. Cassuto H. Reshef L. Hanson R.W. Crit. Rev. Biochem. Mol. Biol. 2005; 40: 129-154Crossref PubMed Scopus (173) Google Scholar). It has been reported that PGC-1α co-activates HNF4α and induces CYP7A1 gene transcription during starvation in mice (11Shin D.J. Campos J.A. Gil G. Osborne T.F. J. Biol. Chem. 2003; 278: 50047-50052Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). It has been suggested that bile acid synthesis and gluconeogenesis may be coordinately regulated in fasted-to-fed cycle (12De Fabiani E. Mitro N. Gilardi F. Caruso D. Galli G. Crestani M. J. Biol. Chem. 2003; 278: 39124-39132Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Our recent study (13Song K.H. Chiang J.Y. Hepatology. 2006; 43: 117-125Crossref PubMed Scopus (46) Google Scholar) shows that glucagon and cAMP inhibit CYP7A1 by inducing phosphorylation of HNF4α. Prox1 has recently been identified as a co-repressor of FTF/LRH-1 by yeast two-hybrid screening (14Steffensen K.R. Holter E. Bavner A. Nilsson M. Pelto-Huikko M. Tomarev S. Treuter E. EMBO Rep. 2004; 5: 613-619Crossref PubMed Scopus (53) Google Scholar, 15Qin J. Gao D.M. Jiang Q.F. Zhou Q. Kong Y.Y. Wang Y. Xie Y.H. Mol. Endocrinol. 2004; 18: 2424-2439Crossref PubMed Scopus (80) Google Scholar). Prox1 was originally cloned by homology to the Drosophila melanogaster gene prospero (16Oliver G. Sosa-Pineda B. Geisendorf S. Spana E.P. Doe C.Q. Gruss P. Mech. Dev. 1993; 44: 3-16Crossref PubMed Scopus (300) Google Scholar). Prox1 is expressed in lens, heart, liver, kidney, skeletal muscle, pancreas, and central nervous system (16Oliver G. Sosa-Pineda B. Geisendorf S. Spana E.P. Doe C.Q. Gruss P. Mech. Dev. 1993; 44: 3-16Crossref PubMed Scopus (300) Google Scholar, 17Zinovieva R.D. Duncan M.K. Johnson T.R. Torres R. Polymeropoulos M.H. Tomarev S.I. Genomics. 1996; 35: 517-522Crossref PubMed Scopus (78) Google Scholar). Earlier studies have linked Prox1 function to lens and lymphatic system development (18Wigle J.T. Chowdhury K. Gruss P. Oliver G. Nat. Genet. 1999; 21: 318-322Crossref PubMed Scopus (340) Google Scholar, 19Wigle J.T. Oliver G. Cell. 1999; 98: 769-778Abstract Full Text Full Text PDF PubMed Scopus (1191) Google Scholar). More recent studies (20Sosa-Pineda B. Wigle J.T. Oliver G. Nat. Genet. 2000; 25: 254-255Crossref PubMed Scopus (287) Google Scholar, 21Burke Z. Oliver G. Mech. Dev. 2002; 118: 147-155Crossref PubMed Scopus (116) Google Scholar) 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) (22Liu Y.W. Gao W. Teh H.L. Tan J.H. Chan W.K. Mol. Cell. Biol. 2003; 23: 7243-7255Crossref PubMed Scopus (59) Google Scholar), and FTF (14Steffensen K.R. Holter E. Bavner A. Nilsson M. Pelto-Huikko M. Tomarev S. Treuter E. EMBO Rep. 2004; 5: 613-619Crossref PubMed Scopus (53) Google Scholar, 15Qin J. Gao D.M. Jiang Q.F. Zhou Q. Kong Y.Y. Wang Y. Xie Y.H. Mol. Endocrinol. 2004; 18: 2424-2439Crossref PubMed Scopus (80) Google Scholar) and represses their transactivation activity. We hypothesized that Prox1 may interact with HNF4α and suppressed CYP7A1 gene transcription. To test this hypothesis, we used yeast two-hybrid assay to study the interaction between Prox1 and HNF4α and studied the effect of Prox1 on the HNF4α transactivation of the human CYP7A1 gene. Our findings provide a novel molecular mechanism for Prox1 inhibition of bile acid synthesis and gluconeogenesis. 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 (13Song K.H. Chiang J.Y. Hepatology. 2006; 43: 117-125Crossref PubMed Scopus (46) Google Scholar). Plasmids—The mammalian expression plasmids for HNF4α, Nur77, PGC-1α, Flag-Prox1, Gal4-HNF4α, pHNF4α-tk-Luc reporter, NurRE-Luc, 5× upsteam activating sequence-Luc and human CYP7A1 promoter luciferase reporters were as described previously (14Steffensen K.R. Holter E. Bavner A. Nilsson M. Pelto-Huikko M. Tomarev S. Treuter E. EMBO Rep. 2004; 5: 613-619Crossref PubMed Scopus (53) Google Scholar, 23Song K.H. Park Y.Y. Park K.C. Hong C.Y. Park J.H. Shong M. Lee K. Choi H.S. Mol. Endocrinol. 2004; 18: 1929-1940Crossref PubMed Scopus (77) Google Scholar, 24Li T. Chiang J.Y. Am. J. Physiol. 2005; 288: G74-G84Crossref PubMed Scopus (175) Google Scholar). 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 HNF4α (HNF4α-NT, amino acids 1–128; HNF4α-LBD, amino acids 129–455; HNF4α-ΔAF2, 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 (23Song K.H. Park Y.Y. Park K.C. Hong C.Y. Park J.H. Shong M. Lee K. Choi H.S. Mol. Endocrinol. 2004; 18: 1929-1940Crossref PubMed Scopus (77) Google Scholar). 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-HNF4α 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 HNF4α proteins were detected by incubation of blots with an anti-Prox1 antibody (1:2000 dilution; Upstate, Lake Placid, NY) and anti-HNF4α 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. β-Galactosidase activity expressed on the plates was assayed as described previously (23Song K.H. Park Y.Y. Park K.C. Hong C.Y. Park J.H. Shong M. Lee K. Choi H.S. Mol. Endocrinol. 2004; 18: 1929-1940Crossref PubMed Scopus (77) Google Scholar). 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/β-galactosidase activity as described previously (23Song K.H. Park Y.Y. Park K.C. Hong C.Y. Park J.H. Shong M. Lee K. Choi H.S. Mol. Endocrinol. 2004; 18: 1929-1940Crossref PubMed Scopus (77) Google Scholar). 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, HNF4α, and cyclophilin B mRNAs as described previously (13Song K.H. Chiang J.Y. Hepatology. 2006; 43: 117-125Crossref PubMed Scopus (46) Google Scholar). 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 (24Li T. Chiang J.Y. Am. J. Physiol. 2005; 288: G74-G84Crossref PubMed Scopus (175) Google Scholar). Cell lysate solution (5%) in ChIP dilution buffer was kept aside as “input.” Ten μg of HNF4α 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 (25Feldmann D. Fenech C. Cuer J.F. Clin. Chem. 1983; 29: 1694Crossref PubMed Scopus (12) Google Scholar). Total bile acid concentration was analyzed by enzymatic 3α-hydroxysteroid dehydrogenase method using total bile acid assay kit (Bio-quant Inc., San Diego, CA) according to the manufacturer's instruction. Prox1 Interacts with HNF4α in Vivo and in Vitro—The nuclear receptors that have been identified to interact with Prox1 all belong to the NR5A subfamily (14Steffensen K.R. Holter E. Bavner A. Nilsson M. Pelto-Huikko M. Tomarev S. Treuter E. EMBO Rep. 2004; 5: 613-619Crossref PubMed Scopus (53) Google Scholar, 15Qin J. Gao D.M. Jiang Q.F. Zhou Q. Kong Y.Y. Wang Y. Xie Y.H. Mol. Endocrinol. 2004; 18: 2424-2439Crossref PubMed Scopus (80) Google Scholar, 22Liu Y.W. Gao W. Teh H.L. Tan J.H. Chan W.K. Mol. Cell. Biol. 2003; 23: 7243-7255Crossref PubMed Scopus (59) Google Scholar). 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 HNF4α. This interaction is stronger than Prox1 interaction with steroidogenic factor 1. On the other hand, Prox1 did not interact with thyroid hormone receptor α, thyroid hormone receptor β, 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 HNF4α and NR5A family nuclear receptors.TABLE 1Interaction of Prox1 with nuclear receptors in yeast two-hybrid interaction assayLEXAB42Interaction---Prox1---Prox1-Thyroid hormone receptor αProx1-Thyroid hormone receptor βProx1-Glucocorticoid receptorProx1-Retinoic acid receptor αProx1-Retinoid X receptor αProx1-SHPProx1-DAX-1Prox1-N-CORProx1-SMRTProx1-Prox1NUR77-Prox1SF-1+++Prox1HNF4α++++SHPHNF4α+++ Open table in a new tab To further confirm the results of the yeast two-hybrid assay, we performed in vitro GST pull-down assays to study Prox1 and HNF4α interaction. Consistent with yeast two-hybrid assay results, GST-Prox1 interacted with HNF4α but not retinoid X receptor and retinoic acid receptor (Fig. 1A). The physical interaction between GST-Prox1 and 35S-labeled FTF was used as a positive control (Fig. 1A). To further verify the interaction between Prox1 and HNF4α, we performed a co-immunoprecipitation assay using human primary hepatocyte extracts. Cell extracts were immunoprecipitated with an anti-HNF4α antibody. The immunoprecipitated complexes were then analyzed on an immunoblot with anti-Prox1 antibody. As shown in Fig. 1B, the anti-Prox1 antibody detected Prox1 in the immunoprecipitates, whereas non-immune IgG did not. These results indicated that Prox1 interacted with HNF4α in primary human hepatocytes, consistent with the results from yeast two-hybrid assay and GST pull-down assay. Mapping of the Interaction Regions of Prox1 and HNF4α—Co-regulators have conserved LXXLL motifs that are known to interact with the ligand binding domain (LBD) of nuclear receptors. Prox1 has two LXXLL motifs located in the N terminus and another motif located in the C terminus region. It has been reported that the N-terminal nuclear receptor box 1 (NR1, LRKLL) is critical for interaction with FTF/LRH-1, whereas NR2 (ISQLL) and NR3 are not essential for interaction (14Steffensen K.R. Holter E. Bavner A. Nilsson M. Pelto-Huikko M. Tomarev S. Treuter E. EMBO Rep. 2004; 5: 613-619Crossref PubMed Scopus (53) Google Scholar, 15Qin J. Gao D.M. Jiang Q.F. Zhou Q. Kong Y.Y. Wang Y. Xie Y.H. Mol. Endocrinol. 2004; 18: 2424-2439Crossref PubMed Scopus (80) Google Scholar, 22Liu Y.W. Gao W. Teh H.L. Tan J.H. Chan W.K. Mol. Cell. Biol. 2003; 23: 7243-7255Crossref PubMed Scopus (59) Google Scholar). The yeast two-hybrid assay revealed that the full-length and N-terminal amino acid residues from 1 to 312 (Prox1-NT-WT) interacted with HNF4α (Fig. 2A). However, the C-terminal homeo and prospero domains (Prox1-Homeo) did not interact with HNF4α. When the LRKLL sequence (NR1) was mutated to ARKAL (Prox-NT-MT), this mutant did not interact with HNF4α. These results demonstrated that the N-terminal region of Prox1 interacted with HNF4α and the LRKLL motif is critical for Prox1 to interact with HNF4α. We then investigated which region of HNF4α was required for interaction with Prox1. A series of deletion constructs of HNF4α were used to map the HNF4α interaction domain in yeast-two hybrid assay. Fig. 2B shows that the full-length HNF4α (HNF4α-full) and LBD, which contains activation function 2 (AF2) domain (HNF4α-LBD), interacted with Prox1. However, the N-terminal DBD region and a construct without AF2 (HNF4α-ΔAF2) did not interact with Prox1. These results indicate that HNF4α interacts with Prox1 through the AF2 domain of HNF4α. Prox1 Is Expressed in Human Hepatocytes—Although it has been reported that Prox1 is highly expressed in liver and pancreas (14Steffensen K.R. Holter E. Bavner A. Nilsson M. Pelto-Huikko M. Tomarev S. Treuter E. EMBO Rep. 2004; 5: 613-619Crossref PubMed Scopus (53) Google Scholar), 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-1α, 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 ΔCt 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 ΔCt values of CYP7A1 and PGC-1α are high, reflecting low levels of mRNA expression. Those values for Prox1, HNF4α, and SHP are low, indicating a relatively abundant expression of these three mRNA transcripts.TABLE 2Quantitative real time PCR analysis of mRNA expression levels of CYP7A1, nuclear receptors, and co-regulators in human primary hepatocytes and HepG2 cellsHH1201HH1205HH1209HH1247HH1248HepG2UBCCt ± S.D.Ct20.6 ± 0.319.8 ± 0.319.9 ± 0.0522.7 ± 0.0321.6 ± 0.0820.9 ± 0.07CYP7A1Ct ± S.D.Ct30.4 ± 0.0925.0 ± 0.1929.2 ± 0.0535.9 ± 0.4935.5 ± 0.3131.2 ± 0.03ΔCt ± S.D.ΔCt9.7 ± 0.314.9 ± 0.369.3 ± 0.0713.1 ± 0.4913.4 ± 0.3210.2 ± 0.076PGC-1αCt ± S.D.Ct27.7 ± 0.0924.4 ± 0.0127.4 ± 0.0228.2 ± 0.0228.3 ± 0.0327.8 ± 0.09ΔCt ± S.D.ΔCt7.0 ± 0.314.3 ± 0.37.5 ± 0.055.5 ± 0.046.7 ± 0.096.8 ± 0.11FXRCt ± S.D.Ct26.1 ± 0.422.8 ± 0.423.3 ± 0.0726.1 ± 0.0524.8 ± 0.0427.8 ± 0.3ΔCt ± S.D.ΔCt5.5 ± 0.52.7 ± 0.53.4 ± 0.073.4 ± 0.063.2 ± 0.096.7 ± 0.3FTFCt ± S.D.Ct24.4 ± 0.0324.2 ± 0.524.2 ± 0.0327.2 ± 0.0306.3 ± 0.0825.8 ± 0.08ΔCt ± S.D.ΔCt3.7 ± 0.34.1 ± 0.584.3 ± 0.064.5 ± 0.044.7 ± 0.114.9 ± 0.11Prox1Ct ± S.D.Ct24.8 ± 0.2323.9 ± 0.224.2 ± 0.0826.1 ± 0.0825.3 ± 0.0824.1 ± 0.03ΔCt ± S.D.ΔCt4.2 ± 0.383.8 ± 0.364.3 ± 0.093.4 ± 0.093.7 ± 0.113.1 ± 0.08HNF4αCt ± S.D.Ct24.6 ± 0.0121.4 ± 0.0821.9 ± 0.0125.2 ± 0.0223.4 ± 0.0423.8 ± 0.05ΔCt ± S.D.ΔCt3.9 ± 0.31.3 ± 0.312.0 ± 0.052.5 ± 0.041.8 ± 0.092.8 ± 0.08SHPCt ± S.D.Ct24.4 ± 0.0321.7 ± 0.423.4 ± 0.125.7 ± 0.0723.9 ± 0.1421.8 ± 0.07ΔCt ± S.D.ΔCt3.7 ± 0.31.6 ± 0.53.5 ± 0.113.0 ± 0.082.3 ± 0.160.9 ± 0.10 Open table in a new tab Prox1 Is a Transcriptional Repressor of HNF4α—We then studied the transcriptional activity of Prox1 in reporter assays in HepG2 cells. As shown in Fig. 3A, ectopic expression of HNF4α increased a heterologous HNF4α-tk-luciferase reporter activity. Addition of Prox1 substantially repressed HNF4α transactivation activity in a dose-dependent manner. As a negative control, a reporter construct containing 3 copies of Nur77 response element (NurRE-Luc) was not affected by Prox1 (Fig. 3B). Because the N-terminal domain that contains an NR1 motif is important for Prox1 to interact with HNF4α, we performed transfection assays to test the effect of wild-type and mutant Prox1 constructs on HNF4α reporter activity. Fig. 3C shows that wild-type Prox1-Full and Prox1-NT-WT represses HNF4α-mediated transactivation but Prox-1-NT-MT failed to repress the activity suggesting that the N-terminal region of the LXXLL motif of Prox1 is involved in the interaction and repression of HNF4α transactivation activity. Prox1 Suppresses HNF4α Transactivation of the Human CYP7A1 Gene—Recent studies have provided substantial evidence that HNF4α is an important transcription factor that regulates liver-specific expression of CYP7A1 (4Hayhurst G.P. Lee Y.H. Lambert G. Ward J.M. Gonzalez F.J. Mol. Cell. Biol. 2001; 21: 1393-1403Crossref PubMed Scopus (841) Google Scholar). HNF4α is the only nuclear receptor that activates the human CYP7A1 gene in transfection assays in HepG2 cells (24Li T. Chiang J.Y. Am. J. Physiol. 2005; 288: G74-G84Crossref PubMed Scopus (175) Google Scholar, 26Chen W. Owsley E. Yang Y. Stroup D. Chiang J.Y. J. Lipid Res. 2001; 42: 1402-1412Abstract Full Text Full Text PDF PubMed Google Scholar). We thus investigated the effect of Prox1 on CYP7A1 transcription. As shown in Fig. 4A, ectopically expressed HNF4α modestly induced CYP7A1 reporter activity in HepG2 cells. This may be because of inhibition of HNF4α 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 HNF4α. To further confirm the involvement of HNF4α in transactivation of CYP7A1 and its inhibition by Prox1, we studied the effect of Prox1 on a reporter with a mutation in the HNF4α binding site (mHNF4-hCYP7A1-Luc). This reporter has a basal activity that is ∼1,000-fold lower than the wild-type reporter. Prox1 only slightly inhibited the HNF4α site mutant reporter (Fig. 4B). Taken together, these results suggest that Prox1 interacts with HNF4α and represses HNF4α-mediated human CYP7A1 gene expression. Prox1 Blocks HNF4α Recruitment of PG" @default.
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• W2003649068 title "A Prospero-related Homeodomain Protein Is a Novel Co-regulator of Hepatocyte Nuclear Factor 4α That Regulates the Cholesterol 7α-Hydroxylase Gene" @default.
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