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

Seema GuptaDagger , R. Todd StravitzDagger , Paul Dent§, and Phillip B. Hylemon||

From the Dagger  Department of Medicine-Gastroenterology, § Department of Radiation Oncology, and  Department of Microbiology and Immunology, Virginia Commonwealth University, Medical College of Virginia Campus, Richmond, Virginia 23298

Received for publication, December 1, 2000, and in revised form, February 12, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cholesterol 7alpha -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 represses CYP7A1 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-alpha , 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-regulate CYP7A1 mRNA. In contrast, overexpression of wild-type c-Jun (c-Junwt) enhanced the repression of CYP7A1 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 regulating CYP7A1 levels in primary rat hepatocytes.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (1). 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 (2). 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 7alpha -hydroxylase (CYP7A1),1 is the most highly regulated step in this feedback inhibitory loop (3); however, other genes encoding bile acid biosynthetic enzymes are also under dynamic control by the bile acid pool (4-8). 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 (9-11) and to primary rat hepatocyte cultures (12).

Bile acids have been shown to bind and activate the orphan nuclear receptor, farnesoid X receptor (FXR) (13-15). 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) (16). Although the activated FXR/RXR heterodimer repressed CYP7A1 promoter activity (14, 17, 18), 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 (19, 20). 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 maximal CYP7A1 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 (7). 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 (21, 22). Moreover, activation of PKC by phorbol esters repressed CYP7A1 transcriptional activity (22). 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 (23). 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 (24, 25). Other studies have shown that tumor necrosis factor alpha  (TNFalpha ), which activates the JNK signaling pathway, can repress the mRNA and activity of CYP7A1 (26, 27). 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 of CYP7A1 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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Primary Rat Hepatocyte Cultures-- 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 (28). 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 TNFalpha was added in the indicated concentrations.

JNK Activity Assay-- 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 mM Na3VO4, 1 mM sodium pyrophosphate, 0.05% SDS, 0.05% sodium deoxycholate, 1% Triton X-100, 5 mM NaF, 0.1% 2-mercaptoethanol, 1 mM phenylmethylsulfonyl 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 mM MgCl2, 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 [gamma -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.

ERK Activity Assay-- 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 mM phosphoric acid. The radioactivity incorporated in the substrate was determined by scintillation counting.

Poly-L-lysine-conjugated Adenovirus Infection/Transfection of Hepatocytes-- Expression vectors encoding for dominant-negative JNK1 (pCMV-dnJNK1), dominant-negative MKK4 (pCMV-dnMKK4), or wild-type c-Jun (pCMV-c-Junwt) (29) were introduced using conjugated DNA-adenovirus complexes (29-32). 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.

Infection of Primary Hepatocytes with Adenovirus Encoding TAM67-- 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) (33) 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.

Quantitation of CYP7A1 mRNA-- 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 (34). Rat cyclophilin mRNA was used as an internal control.

Transient Transfections and Dual Luciferase Reporter Assay-- 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 Delta ratAP1 (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 µM dexamethasone, 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.

Statistical Analyses-- Data were analyzed by Student's t-test. Level of significance was set at p < 0.05.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Taurine-conjugated Bile Acids and TNFalpha Stimulate JNK Activity in Primary Cultures of Rat Hepatocytes-- Bile acids have been reported to induce the expression of inflammatory cytokines such as TNFalpha and interleukin-1 in macrophages (27). TNFalpha has been reported to down-regulate CYP7A1 in the liver (26, 27). In numerous cell lines, TNFalpha has been shown to potently activate the JNK cascade (35). 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. 1A 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. 1B). 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. 1C). 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 (36).


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Fig. 1.   Effect of different taurine-conjugated bile acids on JNK1 activity in primary rat hepatocytes. A, JNK activity in response to taurine-conjugated bile acids (50 µM for 90 min) (NA, no addition). B, time course of TCA (50 µM) activation of JNK. C, effect of TCA concentration (60-min incubations) on JNK activity. All values are means ± S.E.; n = 3-6.

Treatment of hepatocytes with TNFalpha (2 ng/ml) also caused a rapid increase (~2-fold) in JNK activity (Fig. 2A). Moreover, TNFalpha also rapidly (2 h) down-regulated (70-80%) CYP7A1 mRNA levels in primary rat hepatocytes (Fig. 2B). CYP7A1 mRNA levels rebounded with longer incubations, suggesting that TNFalpha did not induce apoptosis under our culture conditions.


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Fig. 2.   Effect of TNFalpha on JNK1 activity and CYP7A1 mRNA levels in primary rat hepatocytes. A, time course of TNFalpha (2 ng/ml) activation of JNK. B, effect of TNFalpha (2 ng/ml) on CYP7A1 mRNA levels. TNFalpha was added to hepatocyte cultures at 48 h, total RNA was isolated at time points indicated, and CYP7A1 mRNA was quantitated as described under "Experimental Procedures." All values are means ± S.E.; n = 3.

MEK1 Inhibitor Does Not Block the Bile Acid-mediated Down-regulation of CYP7A1 Expression in Primary Hepatocytes-- Previous reports in the literature have shown that bile acids can sequentially activate the ERK1/2 cascade (Raf-1/MEK1,2/ERK1,2) (24, 25). In addition, data from our laboratory showed that both hydrophobic and hydrophilic bile acids could rapidly activate the ERK cascade in primary rat hepatocytes (37). Studies were therefore carried out to determine if the ERK cascade was involved in the bile acid-dependent down-regulation of CYP7A1. 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. 3A, 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. 3B). 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.


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Fig. 3.   Effect of inhibition of the ERK pathway on ERK activity and DCA-mediated repression of CYP7A1 mRNA levels. A, cultured hepatocytes were treated as indicated with a vehicle control of dimethyl sulfoxide (DMSO), the MEK1 inhibitor, PD98059 in DMSO (50 µM for a 30-min pretreatment), and/or DCA (50 µM for 20 min), or left untreated. The cells were then harvested and assayed for ERK activity. Values are mean ± S.E.; n = 3. *, p < 0.001. Statistical significance was calculated between DCA-treated samples. B, cultured hepatocytes were pretreated with PD98059 as described above, followed by treatment with DCA (50 µM) for 6 h. Total RNA was then isolated and subjected to RNase protection assay for CYP7A1 and cyclophilin (CYC) mRNAs.

Bile Acid-dependent Down-regulation of CYP7A1 mRNA Is Mediated by the JNK Pathway-- The data presented above suggest that the JNK pathway may be important in the down-regulation of CYP7A1 by bile acids and TNFalpha . 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. 4A). 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. 4B, 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.


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Fig. 4.   Effect of dominant-negative mutants of MKK4 and JNK1 on JNK activity and effect of dnJNK1 on CYP7A1 mRNA levels. A, hepatocytes were transfected with either control plasmid (pCMV), dominant-negative MKK4 plasmid (pCMV-dnMKK4), or dominant-negative JNK1 plasmid (pCMV-dnJNK1) for 24 h to allow for expression of dominant-negative gene products. After 24 h, the cells were treated with TCA (50 µM, 90 min) or left untreated. The cells were then harvested and assayed for JNK activity. Top panel, representative autoradiogram depicting JNK activity in transfected hepatocytes in the absence (-) and in the presence (+) of TCA as assayed by incorporation of labeled ATP in GST-c-Jun. Lower panel, quantitative results of JNK activity assay. Values are mean ± S.E.; n = 3. **, p < 0.025; *, p < 0.001. Statistical significance was calculated between TCA-treated control (pCMV) versus TCA-treated dnMKK4 or dnJNK1. B, cultured hepatocytes were transfected as described above, followed by treatment with TCA (50 µM) for 18 h. Total RNA was then isolated and subjected to RNase protection assay for CYP7A1 and cyclophilin mRNAs. Values are mean ± S.E.; n = 4. *, p < 0.025. Statistical significance was calculated between TCA-treated pCMV and dnJNK1.

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 CYP7A1 transcription, 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. 5A). 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. 5B). Moreover, expressing a dominant-negative c-Jun also significantly blocked the ability of TCA to down-regulate CYP7A1 mRNA (Fig. 5C). These experiments strongly support a role for c-Jun as the rapid mediator of CYP7A1 regulation by bile acids.


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Fig. 5.   Effect of overexpression of wild-type c-Jun and TAM67 on CYP7A1 mRNA levels. A, hepatocytes were transfected with either control plasmid (pCMV) or a plasmid encoding for wild-type c-Jun (pCMV-c-Junwt) for 24 h to allow for overexpression of c-Jun. After 24 h, the cells were treated with TCA (50 µM, 18 h) or left untreated. Total RNA was then isolated and subjected to RNase protection assay for CYP7A1 and cyclophilin mRNAs. Values are mean ± S.E.; n = 3. *, p < 0.05. Statistical significance was calculated between TCA-treated samples. B, recombinant adenovirus encoding a dominant-negative c-Jun mutant (TAM67) or adenovirus without expression vector (NULL) in the indicated multiplicity of infection (MOI) were added to cultured hepatocytes for 24 h. Total RNA was isolated and subjected to RNase protection assay for CYP7A1 and cyclophilin (CYC) mRNAs. C, hepatocytes were treated with either the null virus or the TAM67 adenovirus as described above. Following infection, TCA (50 µM) was added to the hepatocytes for 6 h, and total RNA were isolated and subjected to RNase protection assay for CYP7A1 and cyclophilin (CYC) mRNAs. Values are mean ± S.E.; n = 4.

How Do Both c-Jun and FXR Down-regulate CYP7A1 Gene Expression?-- Previous work has demonstrated that bile acids down-regulate CYP7A1 gene expression by activating the nuclear hormone receptor FXR (14, 17, 18). It was recently shown that the mechanism by which activated FXR repressed CYP7A1 transcription is through induction of the gene encoding SHP-1 protein (19, 20). The data presented above suggests that bile acids regulate CYP7A1 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. 6A). 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. 6B, 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.


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Fig. 6.   Wild-type c-Jun and FXR activate the rat SHP-1 promoter. A, alignment of the proximal regions of the rat, mouse, and human SHP-1 promoters. The conserved FXR binding site (IR-1) and the putative AP-1 binding site are boxed. Shown below the figure are the AP-1 site consensus sequence and the sequence of the mutated AP-1 site in the rat SHP-1 promoter. B, hepatocytes were transfected with the luciferase reporter plasmid containing the proximal promoter of the rat (-441 to +19) SHP-1 gene or with the corresponding reporter plasmid in which the AP-1 site had been mutated (Delta AP1) in combination with expression plasmids for either FXR, c-Junwt, or lacz (-) (control promoter plasmid). Following transfection, cells were treated with 50 µM TCA for 24 h and harvested for measurement of luciferase activity. Values are mean ± S.E. of at least two experiments performed in triplicate.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 of CYP7A1 transcription in response to increasing concentrations of bile acids (9-12, 36). Moreover, it was shown that hydrophobic bile acids are more potent repressors of CYP7A1 mRNA than hydrophilic bile acids (11, 36). 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 (13-15). 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 transporter NTCP (7). 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 (16). In contrast, the bile acid-responsive elements (BAREs) in the promoters of the CYP7A1 and CYP8B1 genes lack a functional FXR binding site (17, 18). Recent studies reveal that FXR induces the expression of the SHP-1 protein (19, 20). SHP-1 represses CYP7A1 transcription by inhibiting the activity of LRH-1, a positive transcription factor that binds to the BARE region in the CYP7A1 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 of CYP7A1 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 (36). Interestingly, hydrophobic bile acids have been recently shown to induce the expression of cytokines TNFalpha and interleukin-1 in macrophages (27). Induction of these cytokines has been correlated with the down-regulation of CYP7A1 mRNA and activity in intact animals (26, 27). In the current investigation, we demonstrate that activation of the JNK signaling pathway by TNFalpha 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-regulate CYP7A1 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 (38, 39). 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 of CYP7A1 by TCA (Fig. 5A). 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. 5B). Together, these observations indicate that activated c-Jun acts as a repressor of CYP7A1 mRNA 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. 6B). 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 for CYP7A1 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 (40).


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Fig. 7.   Model for regulation of CYP7A1 gene expression by FXR and JNK signaling cascade. This model predicts that bile acids entering the hepatocyte activate both the FXR and the JNK signaling cascade. Activated FXR and c-Jun in turn enhance SHP-1 transcription by binding to the IR-1 and AP-1 elements in the SHP-1 promoter, respectively. Elevated SHP-1 protein levels repress CYP7A1 transcription by interacting with LRH-1, a positive transcription factor that binds to the BARE-II region in the CYP7A1 promoter. We speculate that c-Jun might also directly interact with LRH-1 to repress CYP7A1 transcription. See text for details.

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 of CYP7A1 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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants PO1-DK38030 (to P. B. H.) and RO1-DK52825 (to P. D.) and by Department of Defense Career Development Award BC98-0148 (to P. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Medical College of Virginia Campus, Virginia Commonwealth University, P. O. Box 980678, Richmond, VA 23298-0678. Tel.: 804-828-2331; Fax: 804-828-0676; E-mail: hylemon@hsc.vcu.edu.

Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M010878200

    ABBREVIATIONS

The abbreviations used are: CYP7A1, cholesterol 7alpha -hydroxylase; FXR, farnesoid X receptor; RXR, retinoid X receptor; SHP-1, small heterodimer partner 1; LRH-1, liver receptor homolog-1; CPF, CYP7A1 promoter binding factor; FTF, alpha 1-fetoprotein transcription factor; PKC, protein kinase C; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; TNFalpha , tumor necrosis factor alpha ; TCA, taurocholate; DCA, deoxycholate; MKK4, mitogen-activated protein kinase kinase 4; AP-1, activator protein-1; CYP8B1, sterol 12alpha -hydroxylase; GST, glutathione S-transferase; CMV, cytomegalovirus; MEK, mitogen-activated protein kinase/ERK kinase; dn, dominant-negative; bp, base pair(s); BARE, bile acid-responsive element; IR-1, inverted repeat-1.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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