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Genes Dev. 2003 July 1; 17(13): 1581–1591. doi: 10.1101/gad.1083503. | PMCID: PMC196131 |
Copyright © 2003, Cold Spring Harbor Laboratory
Press Definition of a novel growth factor-dependent signal cascade for the
suppression of bile acid biosynthesis Jason A. Holt,1 Guizhen Luo,1 Andrew N. Billin,1 John Bisi,2 Y. Yvette McNeill,3 Karen F. Kozarsky,4 Mary Donahee,4 Da Yuan Wang,5 Traci A. Mansfield,6 Steven A. Kliewer,1,7 Bryan Goodwin,1 and Stacey A. Jones1,8 1Nuclear Receptor Discovery Research, High Throughput Biology, 2Gene Interference, 3Transgenics, GlaxoSmithKline, Research Triangle Park, North Carolina 27709, USA 4Protein Agents and Human Gene Therapy, 5Protein Biochemistry, GlaxoSmithKline, King of Prussia, Pennsylvania 19406, USA 6CuraGen Corporation, New Haven, Connecticut 06511, USA Received February 11, 2003; Accepted May 6, 2003. The nuclear bile acid receptor FXR has been proposed to play a central role
in the feedback repression of the gene encoding cholesterol
7α-hydroxylase (CYP7A1), the first and rate-limiting step in the
biosynthesis of bile acids. We demonstrate that FXR directly regulates
expression of fibroblast growth factor-19 (FGF-19), a secreted growth factor
that signals through the FGFR4 cell-surface receptor tyrosine kinase. In turn,
FGF-19 strongly suppresses expression of CYP7A1 in primary cultures
of human hepatocytes and mouse liver through a c-Jun N-terminal kinase
(JNK)-dependent pathway. This signaling cascade defines a novel mechanism for
feedback repression of bile acid biosynthesis and underscores the vital role
of FXR in the regulation of multiple pathways of cholesterol catabolism in the
liver. Keywords: CYP7A1, FXR, bile acid, fibroblast growth factor, JNK The catabolism of cholesterol to bile acids represents a major pathway for
the elimination of this potentially pathogenic sterol fromthe body. Bile acids
subserve a number of important physiological functions, including the
solubilization of cholesterol, fat soluble vitamins, and other lipids in the
intestine (Vlahcevic et al.
1994,
1996). In addition, bile acids
contribute to the generation of bile flow and promote the secretion of lipids,
notably phosphatidylcholine and cholesterol, fromthe canalicular membrane into
the bile canaliculus. However, because of their intrinsic toxicity,
intracellular levels of bile acids must be tightly regulated, which is largely
accomplished by transcriptional regulation of genes encoding proteins involved
in bile acid biosynthesis, transport, and metabolism. The conversion of cholesterol to the primary bile acids, cholic acid and
chenodeoxycholic acid (CDCA), involves at least 14 distinct enzymes and is
accomplished via 2 pathways ( Bjorkhem
1985; Russell and Setchell
1992). The first and rate-limiting step in the neutral (classic)
pathway of bile acid biosynthesis is catalyzed by cholesterol 7
α-hydroxylase (CYP7A1; Bjorkhem
1985; Russell and Setchell
1992; Chiang 1998).
Expression of the gene encoding CYP7A1 is known to be suppressed by a number
of factors including insulin, protein kinase C activators, cytokines such as
tumor necrosis factor α (TNF-α), steroid hormones, and,
importantly, bile acids (for review, see
Chiang 1998). The bile
acid-dependent feedback repression of CYP7A1 is important in
preventing a potentially harmful expansion of the bile acid pool. A number of
studies have focused on characterizing the molecular mechanisms by which bile
acids suppress CYP7A1 expression, and it is now apparent that
multiple, redundant pathways exist
( Stravitz et al. 1995;
Antes et al. 2000;
Goodwin et al. 2000;
Lu et al. 2000;
Miyake et al. 2000;
De Fabiani et al. 2001;
Kerr et al. 2002;
Wang et al. 2002). Notably,
these signaling cascades converge on a common bile acid responsive element
(BARE) in the CYP7A1 promoter
( Chiang and Stroup 1994;
Stroup et al. 1997). This
element is highly conserved across species and is a well-documented binding
site for members of the nuclear receptor superfamily of ligand activated
transcription factors, including liver receptor homolog-1 (LRH-1, NR5A2) and
hepatocyte nuclear factor 4α (HNF-4α, NR2A1;
Crestani et al. 1998;
Nitta et al. 1999;
Lu et al. 2000;
Stroup and Chiang 2000;
De Fabiani et al. 2001;
Chiang 2002). The farnesoid X receptor (FXR; NR1H4) is a bile acid-activated
transcription factor that also belongs to the nuclear receptor family
( Makishima et al. 1999;
Parks et al. 1999;
Wang et al. 1999). FXR binds
DNA as an obligate heterodimer with the retinoid X receptors (RXRs;
Forman et al. 1995;
Seol et al. 1995). The FXR/RXR
heterodimer typically binds to an inverted repeat of the hexanucleotide motif
AG G/ TTCA separated by a single nucleotide, a so-called
IR-1 ( Forman et al. 1995;
Seol et al. 1995). FXR is
known to be expressed in tissues that are exposed to bile acids, including
liver, intestine, gallbladder (C. Housset, pers. comm.), kidney, and adrenal
gland ( Forman et al. 1995;
Seol et al. 1995). In liver,
the biological consequences of FXR activation have recently become
increasingly clear. Upon activation, FXR initiates transcription of a cohort
of genes that function to decrease the concentration of bile acids within the
hepatocyte. Specifically, FXR induces the expression of ATP-binding cassette
(ABC) transporters bile salt export pump (BSEP; ABCB11;
Sinal et al. 2000;
Ananthanarayanan et al. 2001;
Plass et al. 2002), multidrug
resistance protein 3 (MDR3, ABCB4; B. Goodwin and S.A. Jones, unpubl.), and
multidrug resistance-associated protein 2 (MRP2; ABCC2;
Kast et al. 2002). These
transporters function to transport bile acids and bile constituents fromthe
hepatocytes into the bile. In addition, activation of FXR by both naturally
occurring (CDCA) and synthetic ligands leads to the repression of two
important genes in the bile acid biosynthetic pathway, namely CYP7A1
and CYP8B1, which encodes oxysterol 12α hydroxylase
( Goodwin et al. 2000;
Lu et al. 2000;
Sinal et al. 2000;
del Castillo-Olivares and Gil
2001; Zhang and Chiang
2001). The FXR-dependent suppression of CYP7A1 is
mediated by the transcriptional repressor short heterodimer partner-1 (SHP;
NR0B2), an atypical nuclear receptor that lacks a DNA-binding domain
( Goodwin et al. 2000;
Lu et al. 2000). Thus,
activation of FXR results in increased expression of the SHP gene. In turn,
SHP interacts with LRH-1, a known positive regulator of CYP7A1
(discussed above) and represses its transcriptional activity. Elegant studies
performed in mice harboring a disrupted SHP gene confirmthe
importance of the FXR–SHP–LRH-1 cascade in suppression of
CYP7A1, however, they also demonstrate the existence of additional
SHP-independent pathways, possibly involving the c-Jun N-terminal kinase (JNK)
mitogen-activated protein kinase ( Kerr et
al. 2002; Wang et al.
2002). In this study, we describe the discovery of a novel FXR-dependent signaling
cascade for the suppression of CYP7A1. We show that FXR directly
regulates expression of FGF-19, a member of the fibroblast growth factor (FGF)
family of signaling molecules ( Nishimura
et al. 1999; Xie et al.
1999). The FGFs bind the extracellular domain of their cognate
cell surface receptor (FGFR) and induce receptor dimerization and tyrosine
kinase phosphorylation, which, in turn, leads to the activation of a number of
intracellular pathways ( Goldfarb
2001; Ornitz and Itoh
2001). For many years, it has been understood that the FGFs
regulate cell growth, differentiation, and morphogenesis, however, it is now
apparent that some of these proteins are also important components of specific
homeostatic pathways ( Yu et al.
2000; Shimada et al.
2001; Tomlinson et al.
2002). We demonstrate that FGF-19, acting as an FXR-induced
signaling molecule, represses expression of the CYP7A1 gene. Our
findings define a novel regulatory pathway for the suppression of bile acid
biosynthesis. Induction of FGF-19 by FXR agonists In experiments aimed at identifying changes in gene expression following
FXR activation in liver cells, primary human hepatocytes were treated with the
potent, selective, synthetic FXR agonist GW4064
( Maloney et al. 2000). Samples
were then examined for differential gene expression using the CuraGen
GeneCalling technology ( Shimkets et al.
1999). The most highly induced gene following GW4064 treatment was
a member of the FGF family of secreted signaling molecules, FGF-19. To further
examine the FXR-dependent regulation of FGF-19, primary human
hepatocytes were cultured in the presence of various concentrations of GW4064
or the naturally occurring FXR agonist chenodeoxycholic acid (CDCA). FGF-19
mRNA was undetectable in control cultures; however, treatment with either
GW4064 or CDCA caused a robust, dose-dependent, increase in FGF-19 mRNA levels
(). Such increases were
not seen for other members of the FGF family, including FGF-1, FGF-2, FGF-9,
FGF-21, or FGF-22. In parallel, expression of the FXR target gene SHP
( Goodwin et al. 2000;
Lu et al. 2000;
Sinal et al. 2000) was also
induced. As expected, activation of FXR by either GW4064 or CDCA resulted in a
profound suppression of CYP7A1 expression
(). Pretreatment of
primary human hepatocytes with the protein synthesis inhibitor cycloheximide
did not block the induction of FGF-19 by GW4064, indicating that the
induction is likely to be a direct effect (data not shown). Identification of an FXRE in the FGF-19 gene The ability of GW4064 and CDCA to strongly induce expression of
FGF-19 suggested that this gene is directly regulated by FXR. To
delineate potential FXR-responsive elements (FXRE) in the FGF-19
promoter, multiple fragments, up to 6.3 kb in size, of the FGF-19
5′-flanking region were isolated. Chimeric FGF-19-luciferase
reporter gene constructs were prepared, and the ability of FXR to
transactivate luciferase expression was examined by transient transfection
into a human liver-derived cell line, HuH7. Despite extensive analysis, we
were unable to identify an FXRE in this region of the FGF-19 gene
(data not shown). To investigate the possibility that FXR-responsive regions
lay downstreamof the transcription initiation site, within the FGF-19
structural gene, three BamHI fragments encompassing the entire
FGF-19 gene were isolated (). These fragments, corresponding to bases –2031 to 494,
494 to 848, and 848 to 7904 (relative to the FGF-19 transcription
initiation site, ) were
inserted into the downstreamenhancer region of the pGL3-tk-LUC vector, which
contains a minimal thymidine kinase (tk) promoter linked to a luciferase
reporter gene. Upon transient transfection into HuH7 cells, only constructs
harboring the 7051-bp BamHI fragment [pGL3-tk-FGF19(848–7899)
and pGL3-tk-FGF19(7899–848)] exhibited FXR-responsiveness
(). Regardless of
orientation, >10-fold induction was observed when these constructs were
cotransfected into HuH7 cells with an FXR expression vector and exposed to
GW4064, indicating that this region contained an FXR-responsive element
(). This 7051-bp
BamHI fragment contains a portion of intron 1, all of exons 2 and 3
including intron 2, and ~2.8 kb of the 3′-flanking region of the
FGF-19 gene ().
Typically, FXR-response elements are comprised of inverted repeats of the
AG( G/ T)TCA hexad with a single nucleotide spacer (IR-1
element; Forman et al. 1995;
Seol et al. 1995). Inspection
of the 7051-bp BamHI region revealed the presence of a perfect IR-1
motif located in intron 2 of the FGF-19 gene (bases 3322–3334
relative to the transcription initiation site,
). To investigate
whether this site mediated the FXR response, mutations were introduced into
the IR-1 of the 7051-bp fragment. Mutation of the putative FXRE completely
abolished FXR-responsiveness of this construct, demonstrating that this site
mediated the FXR response. Thus, the FGF-19 gene contains a
functional FXR responsive element within the second intron. To determine whether FXR–RXRα heterodimers could directly bind
the FXRE identified in the second intron of FGF-19, electrophoretic
mobility-shift assays were performed. Radiolabeled probes corresponding to the
putative FXRE fromthe FGF-19 gene (FGF-19 IR-1) and a previously
characterized FXRE fromthe human I-BABP gene (hI-BABP IR-1;
Grober et al. 1999) were
prepared and examined for their ability to bind FXR or RXRα alone or in
combination (). Neither
FXR nor RXRα were capable of binding to these elements when added to the
binding reaction alone (). However, when both proteins were present, a robust complex
formed on the control h I-BABP IR-1 and the IR-1 from the
FGF-19 gene ().
Competition binding analyses showed that these interactions were specific; no
competition was seen when a mutated derivative of the IR-1 motif derived from
the FGF-19 gene was added to the binding reaction
(). Thus,
FXR–RXRα heterodimers bind and activate an FXRE within intron 2 of
the FGF-19 gene. Repression of CYP7A1 by FGF-19 FGFs signal by activating transmembrane tyrosine kinase receptors. Four FGF
receptors (FGFR1–FGFR4) have been identified
( Goldfarb 2001;
Ornitz and Itoh 2001). FGF-19
is a high-affinity ligand for FGFR4, and unlike other FGF family members,
exhibits exclusive binding to FGFR4 ( Xie
et al. 1999). Interestingly, mice harboring a disrupted
FGFR4 gene exhibit elevated expression of CYP7A1 and an
expanded bile acid pool ( Yu et al.
2000). These data suggested that FGFR4 plays a key role in the
regulation of CYP7A1, and as a result, bile acid biosynthesis
( Yu et al. 2000). Because
FGFR4 is the FGF-19 receptor, we hypothesized that FGF-19 might play a role in
the bile acid-mediated repression of CYP7A1. Accordingly, primary
human hepatocytes were treated with purified recombinant human FGF-19 and
analyzed for CYP7A1 expression. In preliminary studies,
CYP7A1 expression was strongly suppressed after 3–6 h of
treatment with FGF-19 (data not shown). Thus, all subsequent experiments were
performed within this time frame. Treatment of human hepatocytes with
recombinant FGF-19 resulted in a profound dose-dependent suppression of
CYP7A1 expression (). In parallel, expression of the gene encoding SHP was not
altered by FGF-19 treatment (). This observation suggests that FGF-19 represses expression of
CYP7A1 through a novel mechanismthat does not involve induction of
SHP expression. Thus, SHP induction is not a prerequisite
for CYP7A1 repression and FXR can mediate bile-acid feedback
repression of CYP7A1 via two distinct mechanisms. To determine whether bile acid biosynthesis can be regulated by FGF-19 in
vivo, it was necessary to express FGF-19 without induction of SHP.
Accordingly, mice were infected with an adenovirus expressing human FGF-19 and
the levels of CYP7A1 expression determined. Three days after
infection, livers were collected for mRNA analysis
(). Northern blot
analysis showed the expected appearance of FGF-19 message in the livers of the
exposed mice. In agreement with experiments performed in primary hepatocytes,
overexpression of FGF-19 resulted in a marked suppression of CYP7A1 mRNA
levels compared with mice infected with null virus. Importantly, expression of
SHP was unchanged in these animals. These data demonstrate that
FGF-19 is capable of down-regulating the expression of the CYP7A1
gene in vivo. Suppression of CYP7A1 by FGF-19 is mediated by the
JNK-signaling cascade Upon ligand binding, FGF receptors activate a number of pathways, including
the extracellular signal-regulated kinases (ERK) and JNK
phosphoprotein-signaling cascades
( Goldfarb 2001;
Sheikh et al. 2001). In
addition, several reports have implicated the JNK pathways in the bile
acid-dependent suppression of CYP7A1
( De Fabiani et al. 2001;
Gupta et al. 2001;
Wang et al. 2002). Therefore,
we examined the ability of a specific JNK inhibitor to block the effects of
FGF-19 treatment on CYP7A1 expression. Hepatocytes were cotreated
with either recombinant FGF-19 alone or in combination with the JNK inhibitor
SP600125 ( Bennett et al. 2001).
As expected, FGF-19 treatment completely repressed CYP7A1 expression.
Pretreatment of hepatocytes with the JNK-specific inhibitor SP600125 resulted
in inhibition of the FGF-19-dependent suppression of CYP7A1.
Consistent with the negative effects of JNK activation on CYP7A1
expression, cells treated with SP600125 exhibited an approximately threefold
induction in CYP7A1 mRNA levels (). A similar observation has been reported recently by Moore and
coworkers ( Wang et al. 2002).
SP600125-treatment did not completely prevent the suppression of
CYP7A1 by FGF-19, suggesting the existence of additional
JNK-independent CYP7A1 regulatory pathways. To determine whether JNK activity is elevated by exposure of primary human
hepatocytes to FGF-19, a JNK activity assay was performed. Cell extracts were
prepared fromprimary hepatocytes and incubated with recombinant c-Jun. An
antibody that specifically detects Ser 63-phosphorylated c-Jun was used to
visualize the activated protein prior to quantitation. Exposure of hepatocytes
to FGF-19 resulted in a 25–30-fold increase in the level of
phosphorylated c-Jun, indicating activation of the JNK pathway
(). Increased JNK
activity was also seen following GW4064 treatment (data not shown).
Cotreatment with the JNK inhibitor, SP600125, strongly inhibited the FGF-19
induced c-Jun phosphorylation. Thus, FGF-19 treatment of primary human
hepatocytes is able to activate the JNK pathway. This indicates that the JNK
pathway is involved in the suppression of CYP7A1 by FGF-19 and is
consistent with previous observations that activation of the JNK pathway by
bile acids and cellular stress can repress CYP7A1 expression. Bile acids are physiologically important amphipathic molecules that
subserve a number of functions. To prevent their accumulation to potentially
harmful levels, bile acids coordinately regulate genes involved in their
biosynthesis, transport, and metabolism. Notably, bile acids suppress
expression of the CYP7A1 gene, which encodes the first and
rate-limiting step in the neutral (classic) pathway of bile acid biosynthesis.
Studies performed in a number of laboratories have demonstrated the existence
of multiple, redundant pathways for the bile acid-dependent repression of this
gene. Previously, we and others described a pathway in which induction of the
repressor protein SHP by bile acid-activated FXR leads to suppression of
CYP7A1 promoter activity through a direct interaction with LRH-1
( Goodwin et al. 2000;
Lu et al. 2000). This pathway
relies on a network of nuclear receptor interactions that presumably all occur
within the nucleus of an individual hepatocyte. Thus, within a single cell,
when bile acid concentrations are high enough to activate FXR, that individual
cell can suppress bile acid biosynthesis through the FXR–SHP–LRH-1
mechanism. The pathway described in this report is distinct in that it
provides a mechanism for cell-to-cell signaling and coordinate down-regulation
of CYP7A1 among neighboring hepatocytes and for a potential paracrine
pathway to reinforce the activity of the FXR–SHP–LRH-1 mechanism.
Here, we report that bile acid activation of FXR results in the induction of
FGF-19 gene expression and that the FGF-19-signaling pathway
represses CYP7A1 gene expression without elevating SHP expression,
via a JNK-dependent pathway. FGF-19 is a member of the FGF family of secreted signaling molecules. In
the adult, FGF-19 mRNA has been reported to be localized in the brain as well
as adult liver, gallbladder, kidney, spleen, heart, and leukocytes
( Nishimura et al. 1999;
Xie et al. 1999). Unlike other
members of the FGF family, FGF-19 displays little mitogenic activity toward
fibroblasts and binds exclusively to FGFR4, one of the four known FGFRs
( Xie et al. 1999). Thus, bile
acid activation of FXR would lead to secretion of FGF-19, which could then
bind to FGFR4 and initiate a signaling pathway that results in suppression of
CYP7A1 in neighboring cells (). Importantly, FGFR4 is known to be expressed on the cell
surface of hepatocytes and bile duct epithelium
( Partanen et al. 1991;
Hughes 1997). Moreover,
McKeehan and coworkers demonstrated that mice harboring a disrupted
FGFR4 gene exhibit elevated levels of CYP7A1 compared with
their wild-type littermates, resulting in an enlarged bile acid pool
( Yu et al. 2000). However,
cholic acid feeding resulted in suppression of CYP7A1 in both the
wild-type and FGFR4-deficient mice ( Yu et
al. 2000). Taken together, these observations suggest that FGFR4
plays a role in the suppression of CYP7A1 expression, but other
FGF-independent pathways are also active in this process. The importance of kinase-signaling pathways in the regulation of
CYP7A1 gene expression has been recognized for several years. Phorbol
ester activation of protein kinase C (PKC), specifically PKCδ, results
in repression of CYP7A1 expression
( Stravitz et al. 1996;
De Fabiani et al. 2001).
Additionally, TNF-α has been shown to repress CYP7A1 gene
expression by activating the JNK pathway
( Feingold et al. 1996;
Miyake et al. 2000;
De Fabiani et al. 2001;
Gupta et al. 2001). More
recently, two groups have demonstrated that bile acid activation of the JNK
pathway is able to mediate repression of CYP7A1 gene expression
( De Fabiani et al. 2001;
Gupta et al. 2001). Although
it is possible that FGF-19 regulates CYP7A1 mRNA levels through
post-transcriptional mechanisms, JNK signaling is thought to exert its effects
at the level of the CYP7A1 promoter, and we suggest that a similar
mechanism is responsible for mediating the effects of FGF-19 on
CYP7A1 expression. DeFabiani et al.
( 2001) reported that the
HNF-4α-binding site in the BARE of the CYP7A1 promoter was
necessary for TNF-α and JNK-mediated repression of CYP7A1.
Further, they demonstrated that inactivation of this signaling pathway by a
dominant negative JNK kinase prevented the repression of CYP7A1 by
bile acids ( De Fabiani et al.
2001). The precise mechanism of HNF-4α inactivation by the
JNK pathway is not yet clear, but it could be analogous to the negative
regulation of RXR via MKK4 and JNK phosphorylation of RXR that inhibits
retinoid signaling ( Lee et al.
2000). Importantly, studies by Wang et al.
( 2002) in mice lacking a
functional SHP gene suggest that SHP itself may be a component of the
JNK signaling cascade. It is also noteworthy that whereas the bile
acid-dependent suppression of CYP7A1 expression is largely intact in
the SHP-knockout mice, repression of this gene by the synthetic FXR agonist
GW4064 is completely lost ( Kerr et al.
2002; Wang et al.
2002). In addition, it is intriguing that DeFabiani et al.
( 2001) only observed the
JNK-dependent repression of CYP7A1 in HepG2 cells, which express high
constitutive levels of SHP (B. Goodwin, unpubl.). Here, we
demonstrate that GW4064 is a strong inducer of FGF-19 expression and
that FGF-19 is capable of acting directly on the hepatocyte to suppress
CYP7A1 expression. Taken together, these observations suggest that
the FXR–FGF-19–JNK signal cascade may converge on the nuclear
receptor SHP (). However,
despite extensive analysis, we have been unable to demonstrate modulatory
activity of FGF-19 on the CYP7A1 promoter or on proteins known to
regulate CYP7A1 expression, including SHP, LRH1, and HNF-4α.
Nuclear extracts prepared from primary cultures of human hepatocytes treated
with vehicle or FGF-19 show no difference in the binding of LRH-1 or
HNF-4α to the BARE in the CYP7A1 promoter (data not shown).
Additionally, protein–protein interaction studies failed to show an
increased affinity of SHP for LRH-1 or HNF-4α in nuclear extracts from
FGF-19-treated human hepatocytes, and chromatin immunoprecipitation
experiments did not detect any changes in the occupancy of the CYP7A1
promoter by these nuclear receptors (data not shown). Thus, the mechanism by
which FGF-19 suppresses CYP7A1 expression remains obscure and is the
focus of ongoing studies. Recently, transgenic mice expressing FGF-19 under the control of
the myosin light-chain promoter have been reported to have increased metabolic
rate, decreased adiposity, and increased insulin sensitivity compared with
control mice ( Tomlinson et al.
2002). These mice also develop hepatocellular carcinomas as they
age ( Nicholes et al. 2002). On
the basis of their observations with the FGF-19 transgenic mouse, Tomlinson et
al. propose the liver as the primary site of action of FGF-19. Our studies
further support this hypothesis by showing that FGF-19 is under the
transcriptional control of FXR, a liver-enriched nuclear receptor, and by
showing that it is a key regulator of cholesterol and bile acid homeostasis in
primary human hepatocytes. Tomlinson et al.
( 2002) suggest increased brown
adipose tissue mass and decreased liver expression of acetyl-CoA carboxylase 2
(ACC2) as a mechanism whereby FGF-19 modulates metabolismand insulin
sensitivity in the transgenic mouse. ACC2 catalyzes the conversion of
acetyl-CoA to malonyl-CoA. Decreased expression of ACC2 relieves malonyl CoA
repression of carnitine palmitoyl transferase 1 (CPT1) initiation of fatty
acid oxidation in the mitochondria. It will be interesting to see whether
physiological concentrations of FGF-19, such as would be expected following
activation of FXR, elicit similar changes in metabolic rate and insulin
sensitivity. It may not be straightforward to ascertain this in rodent models,
as a rodent homolog of FGF-19 has not been identified. In summary, the observations reported here connect, for the first time, the
activity of the bile acid receptor, FXR, with the JNK pathway and the negative
regulation of CYP7A1 gene expression. Bile acid activation of FXR
initiates two distinct pathways that lead to repression of bile acid
biosynthesis. One is strictly intracellular and consists of the regulatory
cascade of the nuclear receptors SHP and LRH-1. The second pathway provides
for extracellular signaling and regulation of bile acid homeostasis across
colocalized cells. This work illustrates an alternate pathway for bile acid
feedback regulation of CYP7A1. Materials Primary cultures of human hepatocytes were obtained from Dr. Steve
Strom(University of Pittsburgh, PA), BioWhittaker, or In Vitro Technologies.
CDCA, dexamethasone, and charcoal-stripped, delipidated calf serumwere
acquired fromthe Sigma Chemical Co. Charcoal/dextran-treated FBS was from
Hyclone Laboratories Inc. Trizol, insulin-transferrin-selenium(ITS-G) and all
other tissue culture reagents were from Invitrogen. Hybond N+,
[α 32P]dCTP, poly(dI-dC) · poly(dI-dC), and the
Mega-prime DNA Labeling Systemwere purchased from Amersham Pharmacia Biotech.,
Inc. Recombinant human FGF-19 was provided by R&D Systems. Restriction
endonucleases were provided by Roche Molecular Biochemicals. The BAC clone
RP11-300I6 (GenBank accession no. AP001888) was obtained from BACPAC Resource
Center (Children's Hospital Oakland Research Institute). GW4064 was prepared
at GlaxoSmithKline as described elsewhere
( Maloney et al. 2000). Primary culture of human hepatocytes Primary human hepatocytes were cultured on Matrigel-coated 6-well plates at
a density of 1.5 × 10 6 cells per well. Culture medium
consisted of serum-free Williams' E medium supplemented with 100 nM
dexamethasone, 100 U/mL penicillin G, 100 μg/mL streptomycin and ITS-G.
Twenty-four hours after isolation, cells were treated with the indicated
concentration of either GW4064 or CDCA, which were added to the culture
mediumas 1000× stocks in DMSO. Control cultures received vehicle (0.1%
DMSO) alone. Total RNA was isolated using Trizol reagent (Invitrogen)
according to the manufacturer's instructions. Differentially regulated genes
were identified using CuraGen GeneCalling Technology
( Shimkets et al. 1999).
Recombinant human FGF-19 was reconstituted in PBS containing 0.1% BSA, and
added directly to the cell culture medium to produce the indicated final
concentration. The phosphorylation inhibitor SP600125, a selective JNK
inhibitor (BioMol), was added to the medium 30 min prior to treatment with
recombinant FGF-19 as a 1000× stock in DMSO (final concentration 10
μM). RNA was isolated as described above and resolved on a 1% agarose/2.2 M
formaldehyde denaturing gel, then transferred to a Hybond N+ membrane, and a
Northern Blot analysis was performed. Blots were hybridized with
32P-labeled cDNA probes corresponding to human CYP7A1, SHP
( Goodwin et al. 2000), FGF-19
(bases 464–1114, GenBank accession no. NM_005117),
glyceraldehyde-3-phosphate (G3PDH), or β-actin (BD Biosciences
Clon-tech). Plasmid constructs Chimeric FGF-19-reporter gene constructs were prepared by subcloning
fragments of genomic DNA from the BAC clone RP11-300I6 into pGL3-tk-LUC, which
contains the minimal thymidine kinase promoter (bases –105 to +51)
linked to a luciferase reporter gene. Three BamHI fragments of
genomic DNA corresponding to bases –2031 to 499, 494 to 853, and 848 to
7904 (relative to the FGF-19 transcription initiation site) were subcloned in
both orientations into the BamHI site of pGL3-tk-Luc, which lies
downstreamof the luciferase reporter gene. These constructs, designated
pGL3-tk-FGF19(–2031–499), pGL3-tk-FGF19[499–(–2031)],
pGL3-tk-FGF19(494–853), pGL3-tk-FGF19(853–494),
pGL3-tk-FGF19(848–7904), and pGL3-tk-FGF19(7904–848), encompass
the entire FGF-19 structural gene. Mutations were introduced in the FXRE in
the pGL3-tk-FGF19(7904–848) using the Quick Change XL Site-Directed
Mutagenesis Kit (Stratagene) and the oligonucleotide 5′-CAC
CCACCTGCAGaaCAGTcttCTTTGCAGACTTAG-3′.
Mutant constructs were sequenced and verified to be free of nonspecific base
changes. Transient transfection assay A human liver-derived cell line, HuH7, was used to delineate FXR-responsive
regions of the FGF-19 gene. Cells were maintained in Dulbecco's Modified
Eagle's medium (DMEM) supplemented with 10% FBS, 100 U/mL penicillin G, 100
μg/mL streptomycin. Plasmid DNA was transfected into HuH7 cells using the
Lipofectamine Plus Reagent (Invitrogen) according to the manufacturer's
instructions. Thus, 96-well plates were inoculated with 20,000 cells per well
in phenol red-free DMEM/F-12 nutrient mixture containing 15 mM HEPES, 2 mM
glutamine, and 10% charcoal/dextran-treated FBS 24 h prior to transfection.
Cells were transfected for 4 h in OptiMEM with 2 ng of human FXR expression
vector (pSG5-hFXR), 8 ng of the control plasmid pβ-Actin-SPAP, 8 ng of
luciferase reporter gene construct. All transfection mixes were complemented
with the pBluescript plasmid to an identical total amount of DNA (65 ng).
Following transfection, cells were incubated for 24 h in DMEM/F-12 medium
containing 10% heat-inactivated, charcoal-stripped, delipidated bovine calf
serum. Cells were incubated for a further 24 h in the same medium supplemented
with either GW4064 (1 μM) or vehicle alone (0.1% DMSO). An aliquot of
medium was assayed for SPAP activity and the cells were lysed prior to
determination of luciferase expression. Luciferase values were normalized to
SPAP. Electrophorectic mobility-shift assay Human FXR and RXRα proteins were synthesized in vitro using the TNT
rabbit reticulocyte lysate coupled in vitro transcription/translation system
(Promega) according to the manufacturer's instructions. Double-stranded
oligonucleotides were end labeled with [α-32P]dCTP using
Klenow enzyme (Promega). Reactions contained 10 mM HEPES (pH 7.8), 60 mM KCl,
0.2% Nonidet P-40, 6% glycerol, 2 mM dithiothreitol, 1 μg poly(dI-dC)
· poly(dI-dC), and recombinant FXR and/or RXRα proteins (2.5
μL each) in a final volume of 20 μL. Double-stranded, unlabeled,
competitor oligonucleotides, corresponding to the wild-type FXRE (FGF-19 IR-1;
5′-GATCCCTGCAGTTCAGTGACCTTTGCA-3′), mutated wild-type element (Mut
IR-1;
5′-GATCCCTGCAGaaCAGTGttCTTTGCA-3′),
and a previously characterized FXRE from the human I-BABP promoter (I-BABP
IR-1; 5′-GATCGGCCAGGGTGAATAACCTCGGGG-3′) were included at 25-,
100-, or 500-fold excess. After a 10-min incubation on ice, 25 nM of probe was
added, and the incubation continued for an additional 20 min on ice, followed
by 3 min at room temperature. DNA–protein complexes were resolved on a
4% polyacrylamide gel in 0.5× TBE. Gels were dried and subjected to
autoradiography at –70°C. FGF-19 adenovirus Recombinant adenovirus expressing FGF-19 was made using standard protocols.
Briefly, FGF-19 cDNA was subcloned into pAd.CMVlink and recombinant adenovirus
was generated by cotransfection of 293 cells as described previously
( Kozarsky et al. 1997). Plaque
purification, expansion, and purification of the recombinant adenovirus
yielded sufficient virus for in vivo studies. Transgene expression was
confirmed by Western blotting of medium from Ad-FGF-19-infected HeLa cells.
Six-week-old male FVB mice were injected with 200 μL saline, null virus (2
× 10 11 particles in saline) or Ad-FGF-19 expressing virus (2
× 10 11 particles in saline) via tail vein. Mice were housed
in the AAALAC-accredited GlaxoSmithKline Research Triangle Park facility under
a 12-h light/dark cycle at 72°F ± 2°F, 50% humidity, and
allowed food and water ad libitum. Experimental protocols were approved by the
GlaxoSmithKline Institutional Animal Care and Use Committee. Mice were
sacrificed 72 h after injection, and livers were removed for analysis of gene
expression. RNA isolation and Northern Blot analysis are described above.
Blots were hybridized with 32P-labeled cDNA probes corresponding to
mouse CYP7A1 (bases 772–2256 of GenBank accession no. L23754), mouse SHP
( Goodwin et al. 2000), FGF-19
(bases 464–1114, GenBank accession no. NM_005117), and
glyceraldehyde-3-phosphate (G3PDH). JNK activity assay JNK activity in primary human hepatocytes was determined using a
commercially available assay system (Cell Signaling Technology). Briefly,
mediumwas aspirated fromhuman hepatocytes cultured on Matrigel, and the cells
were overlaid with 2 mL of ice-cold PBS supplemented with 5 mM EDTA. Following
a 45-min incubation on ice to dissolve the Matrigel, the cells were pelleted
by centrifugation at 2000 rpmfor 5 min. The supernatant was aspirated, and the
cells were lysed in 20 mM Tris buffer (pH 7.4) supplemented with 150 mM NaCl,
1 mM EDTA, 1 mM EGTA, 1% Nonidet P-40, 2.5 mM sodium pyrophosphate, 1 mM
β-Glycerolphosphate, 1 mM sodium orthovanadate, 1 μg/mL leupeptin, and
Complete, Mini, EDTA-free protease inhibitor cocktail tablets (Roche Molecular
Biochemicals). Samples were sonicated and centrifuged to remove cellular
debris. The supernatant was transferred to a fresh tube and the protein
concentration determined. Samples were processed as directed by the
manufacturer (Cell Signaling Technology) and electrophoresed on a 4%–20%
Tris-Glycine gel (Invitrogen) and transferred to a nitrocellulose membrane
filter (Invitrogen). The level of phosphorylated c-Jun was determined by
standard techniques using a primary antibody that specifically recognizes Ser
63-phosphorylated c-Jun (Cell Signaling Technology). Blots were probed with an
HRP-conjugated secondary antibody (Santa Cruz Biotechnology), and the bands
visualized using a chemiluminescence reagent (Western Blotting
Chemiluminescence Luminol, Santa Cruz Biotechnology). The signal was
quantitated using a Molecular Dynamics Densitometer SI (Molecular
Dynamics). We thank Steven Haneline for assistance in obtaining FGF-19-containing BAC
clones and John Moore for his input throughout this project. We also thank
Kelvin Nurse, Michael McQueney, and Karen Kabnick for helpful discussions on
FGF biology. The publication costs of this article were defrayed in part by payment of
page charges. This article must therefore be hereby marked
“advertisement” in accordance with 18 USC section 1734 solely to
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