From the 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
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ABSTRACT |
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Cholesterol 7 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 7 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 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 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
TNF 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
[ 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
Statistical Analyses--
Data were analyzed by Student's
t-test. Level of significance was set at p < 0.05.
Taurine-conjugated Bile Acids and TNF
Treatment of hepatocytes with TNF 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.
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 TNF
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.
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.
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 TNF 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).
-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-
,
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
-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).
(TNF
), 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.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was added in the indicated concentrations.
-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.
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
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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Stimulate JNK Activity in
Primary Cultures of Rat Hepatocytes--
Bile acids have been reported
to induce the expression of inflammatory cytokines such as TNF
and
interleukin-1 in macrophages (27). TNF
has been reported to
down-regulate CYP7A1 in the liver (26, 27). In numerous cell
lines, TNF
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.
(2 ng/ml) also caused a rapid
increase (~2-fold) in JNK activity (Fig.
2A). Moreover, TNF
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 TNF
did not induce apoptosis under our culture
conditions.
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Fig. 2.
Effect of TNF on
JNK1 activity and CYP7A1 mRNA levels in primary
rat hepatocytes. A, time course of TNF
(2 ng/ml)
activation of JNK. B, effect of TNF
(2 ng/ml) on
CYP7A1 mRNA levels. TNF
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.
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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.
. 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.
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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.
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[in a new window]
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 (
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
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 TNF
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).
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[in a new window]
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.
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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
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ABBREVIATIONS |
---|
The abbreviations used are:
CYP7A1, cholesterol 7-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,
1-fetoprotein transcription factor;
PKC, protein kinase C;
ERK, extracellular signal-regulated kinase;
JNK, c-Jun N-terminal kinase;
TNF
, tumor necrosis factor
;
TCA, taurocholate;
DCA, deoxycholate;
MKK4, mitogen-activated protein kinase kinase 4;
AP-1, activator protein-1;
CYP8B1, sterol 12
-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.
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