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Down-regulation of Cholesterol 7α-Hydroxylase (CYP7A1) Gene Expression by Bile Acids in Primary Rat Hepatocytes Is Mediated by the c-Jun N-terminal Kinase Pathway

2001; Elsevier BV; Volume: 276; Issue: 19 Linguagem: Inglês

10.1074/jbc.m010878200

ISSN

1083-351X

Autores

Seema Gupta, R. Todd Stravitz, Paul Dent, Phillip B. Hylemon,

Tópico(s)

Pharmacogenetics and Drug Metabolism

Resumo

Cholesterol 7α-hydroxylase (CYP7A1), the rate-limiting enzyme in the neutral pathway of bile acid biosynthesis, is feedback-inhibited at the transcriptional level by hydrophobic bile acids. Recent studies show that bile acids are physiological ligands for farnesoid X receptor (FXR). Activated FXR indirectly repressesCYP7A1 transcription through induction of small heterodimer protein (SHP-1). In this study, we provide evidence that bile acids rapidly down-regulate CYP7A1 transcription via activation of the JNK/c-Jun pathway. Furthermore, we demonstrate that SHP-1 is also a direct target of activated c-Jun. In primary rat hepatocyte cultures, taurocholate (TCA) strongly activated JNK in a time- and concentration-dependent manner. Tumor necrosis factor-α, a potent activator of JNK, also rapidly activated JNK and down-regulated CYP7A1 mRNA levels. Overexpression of dominant-negative JNK1 or a transactivating domain mutant of c-Jun significantly blocked the ability of TCA to down-regulateCYP7A1 mRNA. In contrast, overexpression of wild-type c-Jun (c-Junwt) enhanced the repression ofCYP7A1 by TCA. Moreover, overexpression of c-Junwt resulted in increased SHP-1 promoter activity. Mutation of a putative AP-1 (c-Jun) element suppressed c-Jun-mediated activation of the SHP-1 promoter construct. These results indicate that the bile acid-activated JNK pathway plays a pivotal role in regulatingCYP7A1 levels in primary rat hepatocytes. Cholesterol 7α-hydroxylase (CYP7A1), the rate-limiting enzyme in the neutral pathway of bile acid biosynthesis, is feedback-inhibited at the transcriptional level by hydrophobic bile acids. Recent studies show that bile acids are physiological ligands for farnesoid X receptor (FXR). Activated FXR indirectly repressesCYP7A1 transcription through induction of small heterodimer protein (SHP-1). In this study, we provide evidence that bile acids rapidly down-regulate CYP7A1 transcription via activation of the JNK/c-Jun pathway. Furthermore, we demonstrate that SHP-1 is also a direct target of activated c-Jun. In primary rat hepatocyte cultures, taurocholate (TCA) strongly activated JNK in a time- and concentration-dependent manner. Tumor necrosis factor-α, a potent activator of JNK, also rapidly activated JNK and down-regulated CYP7A1 mRNA levels. Overexpression of dominant-negative JNK1 or a transactivating domain mutant of c-Jun significantly blocked the ability of TCA to down-regulateCYP7A1 mRNA. In contrast, overexpression of wild-type c-Jun (c-Junwt) enhanced the repression ofCYP7A1 by TCA. Moreover, overexpression of c-Junwt resulted in increased SHP-1 promoter activity. Mutation of a putative AP-1 (c-Jun) element suppressed c-Jun-mediated activation of the SHP-1 promoter construct. These results indicate that the bile acid-activated JNK pathway plays a pivotal role in regulatingCYP7A1 levels in primary rat hepatocytes. cholesterol 7α-hydroxylase farnesoid X receptor retinoid X receptor small heterodimer partner 1 liver receptor homolog-1 CYP7A1 promoter binding factor α1-fetoprotein transcription factor protein kinase C extracellular signal-regulated kinase c-Jun N-terminal kinase tumor necrosis factor α taurocholate deoxycholate mitogen-activated protein kinase kinase 4 activator protein-1 sterol 12α-hydroxylase glutathione S-transferase cytomegalovirus mitogen-activated protein kinase/ERK kinase dominant-negative base pair(s) bile acid-responsive element inverted repeat-1 The metabolism of cholesterol to bile acids represents a major pathway for its elimination from the body and accounts for ∼50% of total output in humans. Bile acid biosynthesis can proceed via either the “neutral” (classic) or the “acidic” (alternative) pathway (1Vlahcevic Z.R. Pandak W.M. Stravitz R.T. Gastroenterol. Clin. North Am. 1999; 28: 1-25Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). The end products of cholesterol degradation via these pathways are the two primary bile acids, cholic and chenodeoxycholic acid. It is well established that bile acids undergoing enterohepatic circulation feedback repress their own biosynthesis (2Chiang J.Y.L. Front. Biosci. 1998; 3: D176-D193Crossref PubMed Scopus (262) Google Scholar). In experimental animals and in humans, the expansion of the bile acid pool by bile acid feeding suppresses bile acid biosynthesis, whereas the depletion of the bile acid pool by biliary diversion or bile acid sequestrant feeding increases bile acid biosynthesis. The first and rate-limiting enzyme in the neutral bile acid biosynthetic pathway, cholesterol 7α-hydroxylase (CYP7A1),1 is the most highly regulated step in this feedback inhibitory loop (3Russell D.W. Setchell K.D. Biochemistry. 1992; 31: 4737-4749Crossref PubMed Scopus (659) Google Scholar); however, other genes encoding bile acid biosynthetic enzymes are also under dynamic control by the bile acid pool (4Vlahcevic Z.R. Eggertsen G. Bjorkhem I. Hylemon P.B. Redford K. Pandak W.M. Gastroenterology. 2000; 118: 599-607Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 5Ghosh S. Natarajan R. Pandak W.M. Hylemon P.B. Grogan W.M. Am. J. Physiol. 1998; 274: G662-G668PubMed Google Scholar, 6Vlahcevic Z.R. Jairath S.K. Heuman D.M. Stravitz R.T. Hylemon P.B. Avadhani N.G. Pandak W.M. Am. J. Physiol. 1996; 270: G646-G652PubMed Google Scholar, 7Sinal C.J. Tohkin M. Miyata M. Ward J.M. Lambert G. Gonzalez F.J. Cell. 2000; 102: 731-744Abstract Full Text Full Text PDF PubMed Scopus (1426) Google Scholar, 8Gupta S. Stravitz R.T. Pandak W.M. Muller M. Vlahcevic Z.R. Hylemon P.B. Hepatology. 2000; 32: 341-347Crossref PubMed Scopus (39) Google Scholar). Studies from our laboratory have demonstrated that bile acids repress CYP7A1 at the level of gene transcription and that the degree of repression paralleled both the concentration and relative hydrophobicity of bile acids added to the diets of intact animals (9Horton J.D. Cuthbert J.A. Spady D.K. J. Biol. Chem. 1995; 270: 5381-5387Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 10Jelinek D.F. Andersson S. Slaughter C.A. Russell D.W. J. Biol. Chem. 1990; 265: 8190-8197Abstract Full Text PDF PubMed Google Scholar, 11Pandak W.M. Vlahcevic Z.R. Heuman D.M. Redford K.S. Chiang J.Y. Hylemon P.B. Hepatology. 1994; 19: 941-947Crossref PubMed Scopus (66) Google Scholar) and to primary rat hepatocyte cultures (12Twisk J. Lehmann E.M. Princen H.M. Biochem. J. 1993; 290: 685-691Crossref PubMed Scopus (52) Google Scholar). Bile acids have been shown to bind and activate the orphan nuclear receptor, farnesoid X receptor (FXR) (13Wang H. Chen J. Hollister K. Sowers L.C. Forman B.M. Mol. Cell. 1999; 3: 543-553Abstract Full Text Full Text PDF PubMed Scopus (1298) Google Scholar, 14Makishima M. Okamoto A.Y. Repa J.J. Tu H. Learned R.M. Luk A. Hull M.V. Lustig K.D. Mangelsdorf D.J. Shan B. Science. 1999; 284: 1362-1365Crossref PubMed Scopus (2164) Google Scholar, 15Parks D.J. Blanchard S.G. Bledsoe R.K. Chandra G. Consler T.G. Kliewer S.A. Stimmel J.B. Willson T.M. Zavacki A.M. Moore D.D. Lehmann J.M. Science. 1999; 284: 1365-1368Crossref PubMed Scopus (1843) Google Scholar). FXR binds to DNA as a heterodimer with retinoid X receptor (RXR), recognizing an inverted hexanucleotide repeat separated by a single base (an IR-1 motif) (16Forman B.M. Goode E. Chen J. Oro A.E. Bradley D.J. Perlmann T. Noonan D.J. Burka L.T. McMorris T. Lamph W.W. Cell. 1995; 81: 687-693Abstract Full Text PDF PubMed Scopus (973) Google Scholar). Although the activated FXR/RXR heterodimer repressed CYP7A1promoter activity (14Makishima M. Okamoto A.Y. Repa J.J. Tu H. Learned R.M. Luk A. Hull M.V. Lustig K.D. Mangelsdorf D.J. Shan B. Science. 1999; 284: 1362-1365Crossref PubMed Scopus (2164) Google Scholar, 17Chiang J.Y. Kimmel R. Weinberger C. Stroup D.J. J. Biol. Chem. 2000; 275: 10918-10924Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 18del Castillo-Olivares A. Gil G. Nucleic Acids Res. 2000; 28: 3587-3593Crossref PubMed Google Scholar), no IR-1 element has been identified in the CYP7A1 promoter. Recent studies show that activated FXR induces the expression of small heterodimer partner 1 (SHP-1) protein, an orphan nuclear receptor that lacks a DNA-binding domain (19Lu T.T. Makishima M. Repa J.J. Schoonjans K. Kerr T.A. Auwerx J. Mangelsdorf D.J. Mol. Cell. 2000; 6: 507-515Abstract Full Text Full Text PDF PubMed Scopus (1229) Google Scholar, 20Goodwin B. Jones S.A. Price R.R. Watson M.A. McKee D.D. Moore L.B. Galardi C. Wilson J.G. Lewis M.C. Roth M.E. Maloney P.R. Willson T.M. Kliewer S.A. Mol. Cell. 2000; 6: 517-526Abstract Full Text Full Text PDF PubMed Scopus (1515) Google Scholar). It has been proposed that elevated SHP-1 protein levels prevent activation of CYP7A1 transcription by heterodimerizing with LRH-1 (CPF or FTF), a positive transcription factor required for maximalCYP7A1 transcription. Finally, studies with FXR “knock-out” mice showed that these animals were defective in bile acid regulation of a number of genes involved in bile acid biosynthesis and transport, including CYP7A1 (7Sinal C.J. Tohkin M. Miyata M. Ward J.M. Lambert G. Gonzalez F.J. Cell. 2000; 102: 731-744Abstract Full Text Full Text PDF PubMed Scopus (1426) Google Scholar). However, several genes regulated by bile acids were not altered in the FXR null mice. In the current study, we provide evidence for a “cell signaling model” of bile acid regulation of CYP7A1. We have previously reported that bile acids activate different isoforms of protein kinase C (PKC) in a time (minutes)- and concentration (10–100 μm)-dependent manner (21Stravitz R.T. Rao Y.P. Vlahcevic Z.R. Gurley E.C. Jarvis W.D. Hylemon P.B. Am. J. Physiol. 1996; 271: G293-G303PubMed Google Scholar, 22Stravitz R.T. Vlahcevic Z.R. Gurley E.C. Hylemon P.B. J. Lipid Res. 1995; 36: 1359-1369Abstract Full Text PDF PubMed Google Scholar). Moreover, activation of PKC by phorbol esters repressed CYP7A1transcriptional activity (22Stravitz R.T. Vlahcevic Z.R. Gurley E.C. Hylemon P.B. J. Lipid Res. 1995; 36: 1359-1369Abstract Full Text PDF PubMed Google Scholar). It is well accepted that activated PKC contributes to the activation of downstream mitogen-activated protein kinase signaling cascades, including the extracellular-signal regulated (ERK) pathway and the c-Jun N-terminal kinase (JNK) pathway (23Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2286) Google Scholar). Indeed, it has been shown in vitro and in vivo, that both bile acids and phorbol esters can activate the PKC/Raf-1/ERK1,2 pathway and regulate expression of the low density lipoprotein receptor (LDLR) gene and the early growth response genes (24Kumar A. Chambers T.C. Cloud-Heflin B.A. Mehta K.D.J. J. Lipid Res. 1997; 38: 2240-2248Abstract Full Text PDF PubMed Google Scholar, 25Brady L.M. Beno D.W. Davis B.H. Biochem. J. 1996; 316: 765-769Crossref PubMed Scopus (36) Google Scholar). Other studies have shown that tumor necrosis factor α (TNFα), which activates the JNK signaling pathway, can repress the mRNA and activity of CYP7A1 (26Feingold K.R. Spady D.K. Pollock A.S. Moser A.H. Grunfeld C. J. Lipid Res. 1996; 37: 223-228Abstract Full Text PDF PubMed Google Scholar, 27Miyake J.H. Wang S.L. Davis R.A. J. Biol. Chem. 2000; 275: 21805-21808Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Thus, evidence exists supporting the hypothesis that activation of signaling cascades may also be important in the regulation of CYP7A1 and other genes by bile acids. However, it is unclear which signaling pathways activated by bile acids may be important in the regulation ofCYP7A1 and how the bile acid-activated signaling pathways interact with FXR to regulate overall bile acid biosynthesis. The studies reported herein demonstrate that bile acids rapidly down-regulate CYP7A1 gene transcription via a JNK/c-Jun-dependent mechanism. We also provide evidence that the SHP-1 promoter is a direct target of activated c-Jun, suggesting “cross-talk” occurs between the bile acid-activated JNK signaling cascade and FXR in sensing bile acid levels in primary rat hepatocytes. Primary rat hepatocyte monolayer cultures were prepared from male Harlan Sprague-Dawley rats (200–300 g) using the collagenase-perfusion technique of Bissell and Guzelian (28Bissell D.M. Guzelian P.S. Ann. N. Y. Acad. Sci. 1980; 349: 85-98Crossref PubMed Scopus (282) Google Scholar). Hepatocytes were plated on collagen-coated culture dishes in serum-free William's E medium containing penicillin (100 units/ml), dexamethasone (0.1 μm), thyroxine (1 μm), and insulin (100 nm). Before plating, cells were judged to be greater than 90% viable using trypan blue exclusion. Cells were routinely incubated for 22 h at 37 °C in humidified 5% CO2, and either prewarmed bile acids or TNFα was added in the indicated concentrations. After treatments, hepatocytes (1.3 × 106 cells/35-mm dish) were washed with ice-cold phosphate-buffered saline followed by homogenization in cold lysis buffer (25 mm HEPES, pH 7.4, 5 mm EDTA, 5 mm EGTA, 50 mm NaCl, 1 mmNa3VO4, 1 mm sodium pyrophosphate, 0.05% SDS, 0.05% sodium deoxycholate, 1% Triton X-100, 5 mm NaF, 0.1% 2-mercaptoethanol, 1 mmphenylmethylsulfonyl fluoride, 1 μm Microcystin-LR, and 40 μg/ml each of pepstatin A, aprotinin, and leupeptin). Cell supernatants (500 μg) were incubated with 1 μg of anti-JNK1 antibody (Santa Cruz Biotechnology) at 4 °C for 2–3 h. The immune complexes were isolated by the addition of Protein A-agarose beads. The immunoprecipitates were recovered by centrifugation and washed (10 min) sequentially with lysis buffer, phosphate-buffered saline, and kinase assay buffer (25 mm HEPES, pH 7.4, 15 mmMgCl2, 0.1 mm Na3VO4, and 0.1% (v/v) 2-mercaptoethanol). JNK activity was determined by incubating the washed immunoprecipitates in a reaction mixture containing 40 μl of kinase assay buffer, 0.1 mm ATP, 1 μm Microcystin-LR, 10 μCi of [γ-32P]ATP, and 10 μg of recombinant GST-c-Jun (amino acids 1–169) for 20 min at 37 °C. Reactions were terminated by adding 5× SDS-polyacrylamide gel electrophoresis sample buffer and boiling for 5 min. Phosphorylated GST-c-Jun was resolved in 10% SDS-polyacrylamide gel electrophoresis, the gels were dried and autoradiographed, and the radioactivity incorporated in GST-c-Jun determined by laser scanning the autoradiograms. This assay was performed similarly to the JNK activity assay described above. After treatments, cell extracts were incubated with 1 μg each of rabbit anti-ERK1 and anti-ERK2 antibody (Santa Cruz Biotechnology), and the immunoprecipitates were washed as described above. ERK activity was determined in a reaction mixture identical to that described for JNK, except for the substrate (0.5 mg/ml myelin basic protein). Reactions were terminated by spotting 30 μl of supernatant reaction mixture onto phosphocellulose discs and washing the discs four times (10 min each) in 180 mmphosphoric acid. The radioactivity incorporated in the substrate was determined by scintillation counting. Expression vectors encoding for dominant-negative JNK1 (pCMV-dnJNK1), dominant-negative MKK4 (pCMV-dnMKK4), or wild-type c-Jun (pCMV-c-Junwt) (29Auer K.L. Contessa J. Brenz-Verca S. Pirola L. Rusconi S. Cooper G. Abo A. Wymann M.P. Davis R.J. Birrer M. Dent P. Mol. Biol. Cell. 1998; 9: 561-573Crossref PubMed Scopus (114) Google Scholar) were introduced using conjugated DNA-adenovirus complexes (29Auer K.L. Contessa J. Brenz-Verca S. Pirola L. Rusconi S. Cooper G. Abo A. Wymann M.P. Davis R.J. Birrer M. Dent P. Mol. Biol. Cell. 1998; 9: 561-573Crossref PubMed Scopus (114) Google Scholar, 30Park J.S. Boyer S. Mitchell K. Gilfor D. Birrer M. Darlington G. El Deiry W. Firestone G.L. Munger K. Band V. Fisher P.B. Dent P. J. Biol. Chem. 2000; 275: 18-28Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 31Park J.S. Qiao L. Gilfor D. Yang M.Y. Hylemon P.B. Benz C. Darlington G. Firestone G. Fisher P.B. Dent P. Mol. Biol. Cell. 2000; 11: 2915-2932Crossref PubMed Scopus (68) Google Scholar, 32Cristiano R.J. Smith L.C. Kay M.A. Brinkley B.R. Woo S.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11548-11552Crossref PubMed Scopus (152) Google Scholar). Freshly isolated hepatocytes at 50% confluency were cultured for 4 h prior to transfections. Using polystyrene tubes, 1 μg (35-mm dish) or 7 μg (100-mm dish) of either control plasmid (pCMV) or the plasmid encoding for the gene of interest was incubated with poly-l-lysine-coupled virus in HEPES-buffered saline (HBS; 20 mm HEPES, pH 7.3, 150 mm NaCl) solution in the dark for 30 min at room temperature. After 30 min, additional poly-l-lysine (Sigma Chemical Co.,Mr 29,300) (1.3 μg of poly-l-lysine/μg of DNA) in HBS was added, and the complexes were incubated for another 30 min. The DNA-conjugated virus was added to the hepatocytes at a multiplicity of infection of 300. The cells were incubated for 22–24 h to allow for the expression of the gene products and experimental additions were made thereafter. Primary rat hepatocytes were plated on 150-mm culture dishes for 24 h under culture conditions as described above. After 24 h, the cultures were infected with unpurified recombinant adenovirus encoding for dominant-negative c-Jun mutant (TAM67) (33Brown P.H. Alani R. Preis L.H. Szabo E. Birrer M.J. Oncogene. 1993; 8: 877-886PubMed Google Scholar) at a multiplicity of 1–10 plaque-forming units/cell. All experiments were compared with control (null) virus infection. After 3 h of infection, the virus was removed and replaced with fresh medium, and the cells were allowed to incubate for an additional 24 h. Experimental additions were made 24 h after the media change. Total RNA was prepared from cultured hepatocytes using the guanidinium thiocyanate CsCl centrifugation. CYP7A1 mRNA levels were determined by RNase protection assay as described previously (34Hylemon P.B. Gurley E.C. Stravitz R.T. Litz J.S. Pandak W.M. Chiang J.Y. Vlahcevic Z.R. J. Biol. Chem. 1992; 267: 16866-16871Abstract Full Text PDF PubMed Google Scholar). Rat cyclophilin mRNA was used as an internal control. The rat SHP promoter firefly luciferase construct (bases −441 to +19) was kindly provided by Dr. Steven A. Kliewer (Glaxo Wellcome, NC). FXR expression plasmid (pCMV-rFXR) was a gift from Dr. Gregorio Gil (Department of Biochemistry, Medical College of Virginia, Virginia Commonwealth University). Site-directed mutagenesis of the putative AP-1 binding site in the rat SHP promoter luciferase construct was performed using the Stratagene QuikChange site-directed mutagenesis kit with the ΔratAP1 (bases −298 to −256, 5′-CCCTGTTTATACACTTGtcagATCCGATAAAGGGCATCCAGGC-3′) primer. The mutated construct was sequenced prior to use to verify DNA sequence fidelity. Primary rat hepatocytes were seeded into 24-well plates (8 × 104 cells/well) in growth medium (William's E supplemented with 10% fetal calf serum, 0.1 μmdexamethasone, and 1 μm thyroxine) and incubated for 6 h. Hepatocytes were transfected using the Effectene transfection reagent (Qiagen) according to the manufacturer's instructions. Each well received 100 ng of the rSHP-luciferase construct, 2 ng of pRL-TK construct (a plasmid containing the herpes simplex thymidine kinase promoter upstream from the Renilla luciferase gene), to normalize for transfection efficiencies, and 30 ng of either pCMV-lacZ (control promoter plasmid), pCMV-rFXR, or pCMV-c-Junwt. After 16 h, the medium was changed to serum-free medium, and the cells were treated with taurocholate (TCA) (50 μm) for 24 h prior to harvesting. Dual luciferase output (Dual Luciferase Reporter Assay System, Promega) was quantified with a luminometer (Lumat LB9501, Berthold), and the results are expressed as an index of relative light units. Data were analyzed by Student'st-test. Level of significance was set at p< 0.05. Bile acids have been reported to induce the expression of inflammatory cytokines such as TNFα and interleukin-1 in macrophages (27Miyake J.H. Wang S.L. Davis R.A. J. Biol. Chem. 2000; 275: 21805-21808Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). TNFα has been reported to down-regulate CYP7A1 in the liver (26Feingold K.R. Spady D.K. Pollock A.S. Moser A.H. Grunfeld C. J. Lipid Res. 1996; 37: 223-228Abstract Full Text PDF PubMed Google Scholar, 27Miyake J.H. Wang S.L. Davis R.A. J. Biol. Chem. 2000; 275: 21805-21808Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). In numerous cell lines, TNFα has been shown to potently activate the JNK cascade (35Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3646) Google Scholar). In this context, we wanted to determine whether bile acids might also activate the JNK cascade in primary cultures of rat hepatocytes and down-regulate CYP7A1. To achieve this, primary rat hepatocytes were incubated with taurine-conjugated bile acids (50 μm) for 90 min prior to cell harvest. The two isoforms of JNK (JNK1 and JNK2) were then immunoprecipitated, and the JNK kinase activity was determined using GST-c-Jun as the substrate as described under “Experimental Procedures.” Fig.1 A shows that the addition of TCA to hepatocytes increased JNK activity by ∼4-fold. TCA caused a time-dependent increase in JNK activity with peak activation occurring at 90 min followed by a rapid decline in activity (Fig. 1 B). A dose-response curve indicated that concentrations of as low as 12.5 μm TCA resulted in detectable JNK activation and that maximal stimulation of JNK activity occurred between concentrations of 100 and 200 μm (Fig.1 C). Taurodeoxycholate and taurochenodeoxycholate, the two other relatively hydrophobic bile acids, also stimulated JNK activity by ∼1.5- to 2-fold, whereas tauroursodeoxycholate, a hydrophilic bile acid, had virtually no effect. It should be noted that the degree of JNK activation by different bile acids paralleled their ability to repress CYP7A1 mRNA levels in primary rat hepatocytes (36Stravitz R.T. Hylemon P.B. Heuman D.M. Hagey L.R. Schteingart C.D. Ton-Nu H.T. Hofmann A.F. Vlahcevic Z.R. J. Biol. Chem. 1993; 268: 13987-13993Abstract Full Text PDF PubMed Google Scholar). Treatment of hepatocytes with TNFα (2 ng/ml) also caused a rapid increase (∼2-fold) in JNK activity (Fig.2 A). Moreover, TNFα also rapidly (2 h) down-regulated (70–80%) CYP7A1 mRNA levels in primary rat hepatocytes (Fig. 2 B).CYP7A1 mRNA levels rebounded with longer incubations, suggesting that TNFα did not induce apoptosis under our culture conditions. Previous reports in the literature have shown that bile acids can sequentially activate the ERK1/2 cascade (Raf-1/MEK1,2/ERK1,2) (24Kumar A. Chambers T.C. Cloud-Heflin B.A. Mehta K.D.J. J. Lipid Res. 1997; 38: 2240-2248Abstract Full Text PDF PubMed Google Scholar, 25Brady L.M. Beno D.W. Davis B.H. Biochem. J. 1996; 316: 765-769Crossref PubMed Scopus (36) Google Scholar). In addition, data from our laboratory showed that both hydrophobic and hydrophilic bile acids could rapidly activate the ERK cascade in primary rat hepatocytes (37Stravitz R.T Vlahcevic Z.R Hylemon P.B Paumgartner G. Stiehl A. Gerok W. Keppler D. Leuschner U. in Falk Symposium 108: Bile Acids and Cholestasis. 3. Academic Publishers, Boston1999: 39-50Google Scholar). Studies were therefore carried out to determine if the ERK cascade was involved in the bile acid-dependent down-regulation ofCYP7A1. Primary rat hepatocytes were pretreated (30 min) with PD98059 (50 μm), a specific inhibitor of MEK1 activation, which prevents activation of downstream ERK1 and ERK2. Next, DCA (50 μm) was added to the cells for 20 min, and ERK1/ERK2 activity was determined. As shown in Fig.3 A, pretreatment with PD98059 prevented activation of the ERK pathway by DCA. Furthermore, PD98059 failed to block the down-regulation of CYP7A1 mRNA by DCA (50 μm, 6 h) (Fig. 3 B). PD98059 alone had no effect on CYP7A1 mRNA levels. This result indicates that activation of the ERK pathway does not result in the feedback repression of CYP7A1 by bile acids in primary rat hepatocytes. The data presented above suggest that the JNK pathway may be important in the down-regulation ofCYP7A1 by bile acids and TNFα. To test this hypothesis, we employed dominant-negative mutants of MKK4 (kinase upstream of JNK1,2) and JNK1 to block the ability of TCA to activate the endogenous JNKs. A plasmid expressing either dominant-negative MKK4 (pCMV-dnMKK4), dominant-negative JNK1 (pCMV-dnJNK1), or the control plasmid (pCMV) was transfected into hepatocytes using a poly-l-lysine-conjugated adenovirus system. After 24-h incubation, hepatocytes were treated with 50 μm TCA for 90 min, and the cells were harvested. As expected, in control plasmid-infected cells, TCA treatment stimulated JNK1 activity (Fig.4 A). However, stimulation of JNK1 activity by TCA was almost completely blocked in cells overexpressing the dominant-negative mutants of MKK4 or JNK1. We next tested the ability of TCA to down-regulate CYP7A1 mRNA in hepatocytes expressing the dominant-negative JNK1 protein. As shown in Fig. 4 B, the dominant-negative JNK1 significantly blocked the ability of TCA to down-regulate CYP7A1 mRNA, suggesting a role for the JNK pathway in the rapid bile acid-mediated repression of CYP7A1. Stimulation of the JNK pathway by extracellular stimuli results in the activation of the immediate-early transcription factor c-Jun. To assess the role of c-Jun in bile acid-mediated repression of CYP7A1transcription, we transfected hepatocytes with a wild-type c-Jun expression construct (pCMV-c-Junwt) using a poly-l-lysine-conjugated adenovirus system. In parallel experiments, hepatocytes were also infected with a recombinant adenovirus to express dominant-negative c-Jun (TAM67). TAM67 is deleted in the N-terminal domain (amino acids 3–122) of c-Jun and is incapable of being transactivated by JNK. Hepatocytes were treated 24 h after infection with either media control or 50 μm TCA, and the effect of TCA on CYP7A1 mRNA levels was determined 18 h later. TCA treatment of control-infected hepatocytes repressed CYP7A1 mRNA levels by ∼60%. This TCA-mediated down-regulation of CYP7A1 mRNA levels was further enhanced in cells overexpressing wild-type c-Jun (Fig.5 A). In contrast, overexpression of TAM67 resulted in an increase of ∼2- to 3-fold in the basal levels of CYP7A1 mRNA as compared with cells infected with a control (null) virus (Fig. 5 B). Moreover, expressing a dominant-negative c-Jun also significantly blocked the ability of TCA to down-regulate CYP7A1 mRNA (Fig.5 C). These experiments strongly support a role for c-Jun as the rapid mediator of CYP7A1 regulation by bile acids. Previous work has demonstrated that bile acids down-regulate CYP7A1 gene expression by activating the nuclear hormone receptor FXR (14Makishima M. Okamoto A.Y. Repa J.J. Tu H. Learned R.M. Luk A. Hull M.V. Lustig K.D. Mangelsdorf D.J. Shan B. Science. 1999; 284: 1362-1365Crossref PubMed Scopus (2164) Google Scholar, 17Chiang J.Y. Kimmel R. Weinberger C. Stroup D.J. J. Biol. Chem. 2000; 275: 10918-10924Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 18del Castillo-Olivares A. Gil G. Nucleic Acids Res. 2000; 28: 3587-3593Crossref PubMed Google Scholar). It was recently shown that the mechanism by which activated FXR repressed CYP7A1transcription is through induction of the gene encoding SHP-1 protein (19Lu T.T. Makishima M. Repa J.J. Schoonjans K. Kerr T.A. Auwerx J. Mangelsdorf D.J. Mol. Cell. 2000; 6: 507-515Abstract Full Text Full Text PDF PubMed Scopus (1229) Google Scholar, 20Goodwin B. Jones S.A. Price R.R. Watson M.A. McKee D.D. Moore L.B. Galardi C. Wilson J.G. Lewis M.C. Roth M.E. Maloney P.R. Willson T.M. Kliewer S.A. Mol. Cell. 2000; 6: 517-526Abstract Full Text Full Text PDF PubMed Scopus (1515) Google Scholar). The data presented above suggests that bile acids regulateCYP7A1 transcription by activating the transcription factor c-Jun. This observation raised the question whether in addition to FXR, SHP-1 expression was also up-regulated by activated c-Jun. To test this hypothesis, the proximal 500–600 bp of the mouse, rat, and human SHP-1 promoters were examined for potential c-Jun (AP-1) binding sites. A highly conserved AP-1 binding site was identified ∼250–300 bp upstream of the transcription initiation site in the SHP-1 promoter of all three species (Fig. 6 A). To test if the SHP-1 promoter was regulated by c-Jun, primary rat hepatocytes were co-transfected with an expression plasmid for wild-type c-Jun (pCMV-c-Junwt) and a luciferase reporter construct under the control of the rat SHP-1 promoter. In a parallel experiment, an FXR expression plasmid (pCMV-rFXR) was co-transfected with the SHP-1 luciferase promoter construct as a positive control for these studies. Following transfection, cells were incubated with TCA (50 μm) for 24 h and harvested, and luciferase activity was measured. As shown in Fig. 6 B, overexpression of FXR induced SHP-1 promoter activity ∼ 2-fold in the presence of TCA. Similarly, TCA treatment of cells transfected with the c-Jun expression plasmid also resulted in an increase of ∼2-fold in SHP-1 reporter activity. Mutating the AP-1 site eliminated the c-Jun responsiveness of the SHP promoter. These data provide strong evidence that bile acids stimulate SHP-1 transcription in an AP-1-dependent manner in primary rat hepatocytes. The conversion of cholesterol to bile acids via the neutral pathway is regulated at the level of CYP7A1 gene expression. Several groups have demonstrated the feedback inhibition ofCYP7A1 transcription in response to increasing concentrations of bile acids (9Horton J.D. Cuthbert J.A. Spady D.K. J. Biol. Chem. 1995; 270: 5381-5387Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 10Jelinek D.F. Andersson S. Slaughter C.A. Russell D.W. J. Biol. 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In total, these data suggest that bile acids can regulate their own biosynthesis and that they may do so by either binding to a specific nuclear hormone receptor and/or by activating one or more signaling cascades in the liver. A major breakthrough in understanding the regulation of gene expression by bile acids came with the discovery that FXR is activated by bile acids (13Wang H. Chen J. Hollister K. Sowers L.C. Forman B.M. Mol. Cell. 1999; 3: 543-553Abstract Full Text Full Text PDF PubMed Scopus (1298) Google Scholar, 14Makishima M. Okamoto A.Y. Repa J.J. Tu H. Learned R.M. Luk A. Hull M.V. Lustig K.D. Mangelsdorf D.J. Shan B. Science. 1999; 284: 1362-1365Crossref PubMed Scopus (2164) Google Scholar, 15Parks D.J. Blanchard S.G. Bledsoe R.K. Chandra G. Consler T.G. Kliewer S.A. Stimmel J.B. Willson T.M. Zavacki A.M. Moore D.D. Lehmann J.M. Science. 1999; 284: 1365-1368Crossref PubMed Scopus (1843) Google Scholar). It was shown that both conjugated and unconjugated bile acids could bind to and activate FXR at physiological concentrations (10–100 μm). Studies with FXR “knock-out” mice revealed that a number of genes involved in cholesterol homeostasis are regulated by FXR, namely, CYP7A1, CYP8B1, intestinal bile acid binding protein (I-BABP), canalicular bile salt excretory pump (BSEP), phospholipid transfer protein (PLTP), and hepatic basolateral transporterNTCP (7Sinal C.J. Tohkin M. Miyata M. Ward J.M. Lambert G. Gonzalez F.J. Cell. 2000; 102: 731-744Abstract Full Text Full Text PDF PubMed Scopus (1426) Google Scholar). FXR can regulate gene expression by either a direct or an indirect mechanism. FXR induces gene expression by binding to an FXR-response element (IR1-motif) in the promoters of target genes (16Forman B.M. Goode E. Chen J. Oro A.E. Bradley D.J. Perlmann T. Noonan D.J. Burka L.T. McMorris T. Lamph W.W. Cell. 1995; 81: 687-693Abstract Full Text PDF PubMed Scopus (973) Google Scholar). In contrast, the bile acid-responsive elements (BAREs) in the promoters of the CYP7A1 and CYP8B1 genes lack a functional FXR binding site (17Chiang J.Y. Kimmel R. Weinberger C. Stroup D.J. J. Biol. Chem. 2000; 275: 10918-10924Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 18del Castillo-Olivares A. Gil G. Nucleic Acids Res. 2000; 28: 3587-3593Crossref PubMed Google Scholar). Recent studies reveal that FXR induces the expression of the SHP-1 protein (19Lu T.T. Makishima M. Repa J.J. Schoonjans K. Kerr T.A. Auwerx J. Mangelsdorf D.J. Mol. Cell. 2000; 6: 507-515Abstract Full Text Full Text PDF PubMed Scopus (1229) Google Scholar, 20Goodwin B. Jones S.A. Price R.R. Watson M.A. McKee D.D. Moore L.B. Galardi C. Wilson J.G. Lewis M.C. Roth M.E. Maloney P.R. Willson T.M. Kliewer S.A. Mol. Cell. 2000; 6: 517-526Abstract Full Text Full Text PDF PubMed Scopus (1515) Google Scholar). SHP-1 repressesCYP7A1 transcription by inhibiting the activity of LRH-1, a positive transcription factor that binds to the BARE region in theCYP7A1 promoter. Consistent with this model are the findings that FXR null mice fail to feedback repress CYP7A1 and are defective in the bile acid induction of SHP-1. In the current study, we provide evidence that feedback repression ofCYP7A1 by bile acids is also mediated by activation of the JNK signaling cascade. We show that the JNK cascade is rapidly activated by bile acids, specifically TCA, in a time- and concentration-dependent manner. Additionally, the rank order of potency for bile acid induction of the JNK pathway mirrors their ability to repress CYP7A1 mRNA levels in primary hepatocytes (36Stravitz R.T. Hylemon P.B. Heuman D.M. Hagey L.R. Schteingart C.D. Ton-Nu H.T. Hofmann A.F. Vlahcevic Z.R. J. Biol. Chem. 1993; 268: 13987-13993Abstract Full Text PDF PubMed Google Scholar). Interestingly, hydrophobic bile acids have been recently shown to induce the expression of cytokines TNFα and interleukin-1 in macrophages (27Miyake J.H. Wang S.L. Davis R.A. J. Biol. Chem. 2000; 275: 21805-21808Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Induction of these cytokines has been correlated with the down-regulation of CYP7A1 mRNA and activity in intact animals (26Feingold K.R. Spady D.K. Pollock A.S. Moser A.H. Grunfeld C. J. Lipid Res. 1996; 37: 223-228Abstract Full Text PDF PubMed Google Scholar, 27Miyake J.H. Wang S.L. Davis R.A. J. Biol. Chem. 2000; 275: 21805-21808Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). In the current investigation, we demonstrate that activation of the JNK signaling pathway by TNFα rapidly down-regulates CYP7A1 mRNA levels in primary rat hepatocytes. The role of the JNK pathway in mediating the inhibitory effects of bile acids on CYP7A1 expression is supported by the observation that overexpression of dominant-negative JNK1 blocked the ability of TCA to activate JNK and to down-regulateCYP7A1 mRNA (Fig. 4). In human colon carcinoma cell line HCT116 and in human adenocarcinoma cells, treatment with bile acids enhanced the phosphorylation of c-Jun, a transcription factor substrate of JNK and a component of the classic AP-1 heterodimer (38Qiao D. Chen W. Stratagoules E.D. Martinez J.D. J. Biol. Chem. 2000; 275: 15090-15098Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 39Zhang F. Subbaramaiah K. Altorki N. Dannenberg A.J. J. Biol. Chem. 1998; 273: 2424-2428Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). In these cells, bile acids stimulated gene transcription by enhancing the AP-1 transcriptional activity and DNA binding to promoter constructs. In contrast, in the present study, overexpression of wild-type c-Jun enhanced the repression ofCYP7A1 by TCA (Fig. 5 A). Moreover, overexpression of TAM67, a non-transactivable mutant of c-Jun, significantly blocked the ability of TCA to repress CYP7A1 mRNA. Interestingly, the basal levels of CYP7A1 mRNA were 2- to 3-fold higher in TAM67-overexpressing cells compared with null virus-infected cells (Fig. 5 B). Together, these observations indicate that activated c-Jun acts as a repressor of CYP7A1mRNA in primary rat hepatocyte cultures. Previous reports in the literature and the data presented in this study suggest that bile acids entering the hepatocyte activate both FXR and the cell signaling cascades. It is of interest to note that both FXR and the JNK pathway are activated by similar concentrations of bile acids (10–100 μm). Therefore, how does bile acid-activated FXR and -activated c-Jun both interact to coordinately regulate CYP7A1 gene expression in the hepatocyte? Our data indicate that activated c-Jun induces SHP-1 promoter activity (Fig.6 B). In this regard, a consensus AP-1 binding site was identified in close proximity to the FXR binding site in the mouse, rat, and human SHP-1 promoters. Mutations in the AP-1 binding site abolished bile acid responsiveness of the rat SHP-1 promoter. Thus, we believe that activation of the JNK/c-Jun pathway by bile acids results in the induction of SHP-1 expression. SHP-1, in turn, interacts with LRH-1 and indirectly represses CYP7A1 transcription (Fig.7). However, we do not exclude additional mechanisms of c-Jun-mediated bile acid repression of CYP7A1. It is possible that activated c-Jun might also directly interact with LRH-1 or another positive transcription factor required forCYP7A1 gene expression. Finally, it is possible that LRH-1 might be a substrate of activated JNK. In this regard, it has been recently reported that both mitogen-activated protein kinase kinase 4 (MKK4) and JNK phosphorylate RXR, altering its functional properties (40Lee H.Y. Suh Y.A. Robinson M.J. Clifford J.L. Hong W.K. Woodgett J.R. Cobb M.H. Mangelsdorf D.J. Kurie J.M. J. Biol. Chem. 2000; 275: 32193-32199Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). In conclusion, our combined results suggest that bile acids activate the JNK/c-Jun cascade in primary rat hepatocytes. Activation of this protein kinase cascade is involved in the down-regulation ofCYP7A1 by bile acids. We hypothesize that activation of this pathway provides the hepatocytes with another “sensing mechanism” for detecting bile acid levels as well as stress signals. Together, FXR and the JNK/c-Jun signaling pathway allow the hepatocytes great flexibility in responding to both intra- and extracellular signals for maintaining bile acid homeostasis in the liver. We thank Emily Gurley, Pat Bohdan, and Elaine Studer for their invaluable technical help. We also thank Drs. D. J. Mangelsdorf and Gregorio Gil for critical review of the manuscript.

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