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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Contributes to the Antifibrotic Activity of FXR Ligands in Rodent Models of Liver CirrhosisDipartimento di Medicina Clinica e Sperimentale, Università degli Studi di Perugia, Perugia, Italy (S.F., G.R., E.A., B.R., A.Me., L.R., A.Mo.); Intercept Pharmaceuticals, New York, New York (M.P.); and Dipartimento di Chimica e Tecnologia del Farmaco, Università di Perugia, Perugia, Italy (R.P.)
Received for publication
March 15, 2005
Accepted
June 20, 2005.
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Abstract |
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exert counter-regulatory effects
on hepatic stellate cells (HSCs) and protect against liver fibrosis
development in rodents. Here, we investigated whether FXR ligands regulate
PPAR expression in HSCs and models of liver fibrosis induced in rats by
porcine serum and carbon tetrachloride administration and bile duct ligation.
Our results demonstrate that HSCs trans-differentiation associated
with suppression of PPAR mRNA expression, whereas FXR mRNA was
unchanged. Exposure of cells to natural and synthetic ligands of FXR,
including 6-ethyl chenodeoxycholic acid (6-ECDCA), a synthetic derivative of
chenodeoxycholic acid, reversed this effect and increased PPAR mRNA by
40-fold. Submaximally effective concentrations of FXR and PPAR
ligands were additive in inhibiting 
1(I) collagen mRNA accumulation
induced by transforming growth factor (TGF)β1. Administration
of 6-ECDCA in rats rendered cirrhotic by porcine serum and carbon
tetrachloride administration or bile duct ligation reverted down-regulation of
PPAR mRNA expression in HSCs. Cotreatment with 6-ECDCA potentiates the
antifibrotic activity of rosiglitazone, a PPAR ligand, in the porcine
serum model as measured by morphometric analysis of liver collagen content,
hydroxyproline, and liver expression of 1(I) collagen mRNA,
-smooth muscle actin, fibronectin, TGFβ1, and tissue
inhibitor of metalloprotease 1 and 2, whereas it enhanced the expression of
PPAR and uncoupling protein 2, a PPAR-regulated gene, by 2-fold.
In conclusion, by using an in vitro and in vivo approach, we demonstrated that
FXR ligands up-regulate PPAR mRNA in HSCs and in rodent models of liver
fibrosis. A FXR-PPAR cascade exerts counter-regulatory effects in HSCs
activation.
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Editorial Expression of Concern |
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Hepatic fibrosis is a scarring process of the liver that includes both
increased and altered deposition of extracellular matrix components
(Friedman, 2003
). In chronic
liver disease, hepatic stellate cells (HSCs) undergo a process of
trans-differentiation (Friedman,
2003) from a resting, fat-storing, phenotype toward a
myofibroblast-like phenotype characterized by expression of fibroblastic cell
markers such as 1(I) collagen and -smooth muscle actin
(-SMA). Although the mediators involved in this process are not
completely understood, a growing body of evidence suggests that members of the
nuclear receptor (NR) superfamily (Fiorucci
et al., 2004b) exert counter-regulatory effects acting as braking
signals to prevent HSCs trans-differentiation.
The farnesoid X receptor (FXR) is a ligand-activated transcription factor
that regulates cholesterol and fatty acid metabolism and functions as an
endogenous sensor for bile acids (Forman et
al., 1995; Makishima et al.,
1999; Parks et al.,
1999; Wang et al.,
1999). FXR alters the transcription of target genes by binding as
a heterodimer with the retinoid X receptor to response elements (FXR response
elements) consisting of an inverted repeat of the canonical AGGTCA
hexanucleotide core motif spaced by 1 base pair
(Forman et al., 1995). We have
recently shown that in addition to its ability to modulate bile acid synthesis
and excretion, FXR functions as a negative regulator of 1-collagen (I)
synthesis in HSCs and attenuates/reverses fibrosis in rodent models of liver
fibrosis (Fiorucci et al.,
2004b). Activation of FXR in HSCs leads to induction of the short
heterodimer partner (SHP) (Goodwin et al.,
2000) that counteracts HSCs activation induced by transforming
growth factor (TGF)β1 and thrombin
(Fiorucci et al., 2004b).
Peroxisome proliferator-activated receptors (PPARs) are a family of
ligand-activated NR that, similarly to FXR, bind regulatory element in
responsive genes after the formation of a heterodimeric complex with retinoid
X receptor (Berger and Moller,
2002). Three mammalian PPAR subtypes have been identified:
PPAR, β (or 
), and
(Berger and Moller, 2002).
PPARβ and have been found in rat and human HSCs
(Galli et al., 2000;
Marra et al., 2000;
Miyahara et al., 2000;
Hellemans et al., 2003).
However, although PPAR ligands
(Galli et al., 2002;
Kon et al., 2002) inhibit
proliferation, migration, and chemokine expression of HSCs and protect against
development of liver fibrosis, induction of PPARβ in HSCs favors the
development of an activated phenotype
(Hellemans et al., 2003).
Previous studies have provided evidence that FXR ligands increase
PPAR mRNA expression in human hepatocytes
(Pineda Torra et al., 2003).
Whether FXR interacts with PPAR, however, is unknown. In the present
study, we demonstrate that natural and synthetic FXR ligands induce
PPAR expression in HSCs and provide evidence that a FXR ligand protects
against PPAR down-regulation caused by liver diseases and enhances the
antifibrotic activity of PPAR ligands. These results provide the first
molecular evidence for a cross-talk between the FXR and PPAR and
suggest that NRs provide a network of counter-regulatory signals that limit
HSCs activation.
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Materials and Methods |
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TopAbstractEditorial Expression of ConcernMaterials and MethodsResultsDiscussionReferences |
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-Ethyl-chenodeoxycholic
acid (6-ECDCA) was synthesized as described previously
(Pellicciari et al., 2002;
Mi et al., 2003). GW4064
(Maloney et al., 2000) was
kindly donated by Tim Willson (GlaxoSmithKline, Research Triangle Park, NC).
GW9662 was from Alexis Biochemicals (Florence, Italy).
In Vitro Studies
Isolation and Culture of HSCs. In vitro studies were performed on
primary cultures of rat HSCs and HSC-T6, a rat immortalized HSC line. Primary
rat HSCs were isolated from control and cirrhotic rats according to techniques
described previously (Fiorucci et al.,
2004a,b).
The HSCs were more than 90% viable as assessed by trypan blue exclusion and
>95% pure. Cells were cultured at 37°C in an atmosphere of 5%
CO2 in Dulbecco's modified minimal essential medium (Invitrogen,
Carlsbad, CA) containing 10% fetal calf serum (FCS), 2 mM
L-glutamine, and 5000 IU/ml penicillin/5000 g/ml streptomycin.
To investigate the expression of FXR and PPAR, β, and
in HSCs, and the effect of FXR and PPAR ligands on HSCs activation,
primary culture of rat HSCs (days 0 and 7) and 24-h starved HSC-T6 cells were
incubated for 18 h with medium alone or increasing concentrations of 6-ECDCA,
a semisynthetic derivative of CDCA (0.1–10 µM); GW406, a nonsteroidal
FXR ligand (0.01–1 µM); and rosiglitazone, a PPAR ligand
(0.1–10 µM) and mRNA expression for FXR, PPARs, 1(I) collagen,
SHP, TIMP-1, TIMP-2, MMP-2, and TGFβ1 investigated by
quantitative (q)RT-PCR (Fiorucci et al.,
2004b).
qRT-PCR. Quantization of the expression level of selected genes was
performed by real-time PCR (qRT-PCR) as described previously
(Fiorucci et al., 2004b). All
PCR primers (Table 1) were
designed using the software PRIMER3-OUTPUT using published sequence data
obtained from the National Center for Biotechnology Information database.
Relative efficiency of the primer used for qRT-PCR was calculated through the
determination of standard curves for every gene. Standard curves were
performed using standard concentration of cDNA template and estimating the
unit of relative fluorescence. Optimization experiments were performed to
obtain a primers efficiency value of 100% for every gene.
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Western Blot Analysis of FXR and PPAR Expression on HSCs. Day
0 and day 7 HSCs were incubated with or without 1 µM 6-ECDCA for 24 h at
37°C in Dulbecco's modified Eagle's medium. Cell lysates were prepared by
solubilization of cells in sample buffer (62.5 mM Tris-HCl, pH 6.8, 10%
glycerol 2% SDS, and 0.015% bromphenol blue) and separated by polyacrylamide
gel electrophoresis. The proteins were then transferred to nitrocellulose
membranes (Bio-Rad, Hercules, CA) and probed with primary antibodies to FXR
and PPAR (Santa Cruz Biotechnology Inc., Santa Cruz, CA). The
anti-immunoglobulin G horseradish peroxidase conjugate (Bio-Rad) was used as
the secondary antibody and specific protein bands were visualized using
enhanced chemiluminescence (GE Healthcare, Little Chalfont, Buckinghamshire,
UK) following the manufacturer's suggested protocol.
In Vivo Studies
All studies were approved by the Animal Study Committee of the University
of Perugia. Male Wistar rats (200–250 g) were obtained from Charles
River Breeding Laboratories (Portage, MI) and maintained on standard
laboratory rat chow on a 12-h light/dark cycle. Three different models of
liver fibrosis were used to examine the effect of FXR ligands on PPAR
expression. In the first model, liver fibrosis was induced by repeated
intraperitoneal (i.p.) administrations of 0.5 ml of porcine serum twice a week
for 8 weeks (Fiorucci et al.,
2004b). To investigate whether 6-ECDCA was effective in regulating
PPAR expression, porcine serum-administered rats (6–8 each group)
were randomized to receive 1 and 3 mg/kg 6-ECDCA via gavage 5 times a week.
Control rats were administered 3% carboxymethyl cellulose (CMC) by gavage. At
the end of the study, rats were sacrificed under anesthesia with sodium
pentobarbital (50 mg/kg i.p.) and terminally bled via cardiac puncture; the
liver was removed for examination and blood samples were taken. In the second
model, hepatic fibrosis was induced by bile duct ligation (BDL) of 8- to
9-week old male Wistar rats as reported previously (Fiorucci et al.,
2004a,b).
Sham-operated rats (n = 6) received the same laparoscopic procedure,
except that the bile duct was manipulated, but not ligated and sectioned. In
total, 24 animals were operated. Two weeks after surgery, surviving rats were
randomized to receive placebo, i.e., 3% CMC (six rats) or 6-ECDCA, 3 mg/kg
(eight rats) by gavage. Animals were then treated for 14 days. In the third
model, liver fibrosis was induced in rats by i.p. injection of
CCl4, 100 µl/100 g body weight, in an equal volume of paraffin
oil two times a week for 4 weeks. Control rats were injected i.p. with 100
µl/100 g body weight of paraffin oil alone. Rats (six per group) were then
treated by oral administration of 3 mg/kg 6-ECDCA in CMC five times a week or
3% CMC alone (control) for 8 weeks.
In another set of experiments, we investigated whether 6-ECDCA interacts with rosiglitazone on liver fibrosis induced by porcine serum administration (Table 1). Groups and duration of treatment are described in Table 2. Animals were followed for 8 weeks.
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Liver Histology and Hydroxyproline Determination. For histological
examination, portions of the right and left liver lobes (10–15 mg/each)
from each animal were fixed in 10% formalin, embedded in paraffin, sectioned,
and stained with Sirius red (Lopez-De Leon
and Rojkind, 1985; Fiorucci et al.,
2004a,b).
Collagen surface density was quantified using a computerized image analysis
system (Image Acquisition System Version 005; Delta Sistemi, Rome, Italy)
(Fiorucci et al., 2004b).
Hepatic and urinary content of hydroxyproline were determined by
high-performance liquid chromatography (LC Varian Prostar HPLC; Varian, Inc.,
Palo Alto, CA) as described previously (Fiorucci et al.,
2004a,b).
Statistical Analysis. Analysis of variance followed by Dunnett or Bonferroni correction for multiple comparison was applied when appropriate. EC50 were calculated using Prism III (GraphPad Software Inc., San Diego, CA).
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Results |
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TopAbstractEditorial Expression of ConcernMaterials and MethodsResultsDiscussionReferences |
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mRNA in Human and Rat HSCs. As shown
in Fig. 1, A–C, primary
cultures of rat HSCs express PPAR, β, and . Using HSCs
cultured for 1 day, we found that expression of PPARβ was unchanged by
exposure to FXR ligands, whereas exposure to 1 µM 6-ECDCA, 20 µM CDCA,
and 100 nM GW4064 increased PPAR and mRNA by 2- to 3-fold
(Fig. 1, A–C; n
= 6; P < 0.05). Exposure to FXR ligands increased FXR mRNA
expression by 1.5-fold and SHP mRNA by 3-fold
(Fig. 1, D and E; n =
6; P < 0.05), whereas decreased 1(I) collagen mRNA by 60 to
80% (Fig. 1F; n = 6;
P < 0.05). Similarly, FXR activation decreased -SMA and
TIMP-1 mRNA by 60 to 80% (data not shown).
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expression decreases during
HSCs trans-differentiation. To investigate whether FXR ligands could
revert this pattern, primary cultures of HSCs were grown in plastic dishes for
7 days with or without 6-ECDCA, and expression of NRs was assessed. As shown
in Fig. 2, although acquisition
of an activated phenotype associates with a slight increase of FXR expression,
mRNA, and protein (Fig. 2, A, C, and
E; P < 0.05 versus day 1), HSCs
trans-differentiation associated with reduced expression of
PPAR mRNA (Fig. 2, B, D, and
F; n = 4; P < 0.001). Culturing the cells
with 1 µM 6-ECDCA increased PPAR, mRNA, and protein by 40-fold
(Fig. 2, B, D, and F;
n = 4; P < 0.01). Similar to 6-ECDCA, both natural and
synthetic FXR ligands, CDCA and GW4064 prevented the down-regulation of
PPAR- caused by HSCs activation (data not shown).
Functional Cooperation between FXR and PPAR Ligand in HSCs.
As shown in Fig. 3A, exposure
of HSC-T6 to 1 ng/ml TGFβ1 induced a 5-fold increase of
1(I) collagen mRNA expression (n = 4; P < 0.01
versus control cells). Treating cells with increasing concentrations of
6-ECDCA and rosiglitazone caused a concentration-dependent inhibition of
1(I) collagen mRNA expression. At 1 and 10 µM, respectively, 6-ECDCA
and rosiglitazone reduced 1(I) collagen mRNA expression by 90%
(Fig. 3A; n = 4;
P < 0.05 versus TGFβ1). However, the
IC50, i.e., the concentration that caused a 50% inhibition of
response to TGFβ1, was 0.08 ± 0.01 µM for the
6-ECDCA and 3.3 ± 0.2 µM for rosiglitazone (n = 4;
P < 0.01), indicating that the FXR ligand was 40-fold more
potent than the PPAR ligand in repressing 1(I) collagen mRNA
up-regulation induced by TGFβ1. Since the concentration of
rosiglitazone required to inhibit 1(I) collagen was higher than the
EC50 (Jarvinen,
2004) of this agent for the PPAR, we wondered whether the
effect of rosiglitazone was PPAR-independent. However, exposure to
GW9662, a selective PPAR antagonist fully reversed the effect of
rosiglitazone, but not 6-ECDCA, on 1(I) collagen
(Fig. 3B; n = 4;
P < 0.05 versus rosiglitazone alone).
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To investigate whether ligands of FXR and PPAR might cooperate in
repressing 1(I) collagen gene expression, we exposed HSC-T6 to
submaximally effective concentrations of the two ligands. As shown in
Fig. 3, C and D, although 0.1
µM 6-ECDCA and 1 µM rosiglitazone individually decreased 1(I)
collagen and -SMA mRNA by 30 to 40%, the combination of the two leads
to significant increase in this effect, resulting in 3-fold induction of
PPAR and 80% reduction of 1(I) collagen and -SMA mRNA
(n = 4; P < 0.05 versus 6-ECDCA or rosiglitazone alone). Similarly
to rosiglitazone, coincubation of HSCs with 6-ECDCA in combination with
pioglitazone and 15-deoxy-
12,14-prostaglandin J2
(PGJ2) (Kliewer et
al.,1995) i.e., the putative natural ligand of PPAR,
resulted in a significant additive effect in repressing
TGFβ1-regulated genes (Fig.
3D; n = 5; P < 0.05 versus pioglitazone or
15-deoxy-12,14-prostaglandin J2 alone).
In Vivo Activation of FXR Increases Liver PPAR mRNA. We then
investigated whether in vivo administration of FXR ligands modulates the
expression of PPAR in HSCs. Three different models of liver
fibrosis—porcine serum administration, BDL, and CCL4
intoxication—were used for this experiment. As shown in
Fig. 4, development of liver
fibrosis associates with a significant reduction in the expression of
PPAR that became almost undetectable in HSCs prepared from rats treated
with porcine serum or CCL4 for 8 weeks (n = 4 rat/group; P
< 0.05 versus control rats). Similarly, expression of PPAR was
nearly undetectable in HSCs obtained 4 weeks after BDL. In contrast to
PPAR, development of liver cirrhosis had no effect on the expression of
FXR and SHP mRNA expression. Administration of rats with the FXR ligand
resulted in a robust induction of PPAR expression in all three models.
Thus, although treatment with 3 mg/kg 6-ECDCA increased FXR and SHP mRNA by
1.8- to 4-fold, the FXR ligand increased PPAR mRNA expression by 30- to
50-fold (Fig. 4; P
< 0.01 versus cirrhotic rats).
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ligands on
the deposition of liver collagen were additive. Rats administered porcine
serum for 8 weeks developed extensive liver fibrosis as demonstrated by
morphometric analysis of Sirius red-stained livers, with minimal inflammation
and necrosis (Fig. 5B).
Accordingly, although no differences in biochemical parameters were observed
compared with controls (Table
1). Histological evaluation of liver specimens obtained from rats
administered with porcine serum for 8 weeks showed extensive perilobular
fibrosis resulting in a 10-fold increase of the surface area of hepatic
collagen in comparison with control rats (Figs.
5B and
6A; n = 8–12;
P < 0.001). The number of -SMA-positive HSCs in the fibrous
septa increased significantly in cirrhotic rats compared with control rats
(data not shown; P < 0.001 versus controls).
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-SMA, 1(I) collagen, fibronectin,
TGFβ1, TIMP-1, and TIMP-2 mRNAs (Fig.
6, C–H; P < 0.01 versus control rats), whereas
MMP-2 mRNA was unchanged. Administration of 1 and 3 mg/kg 6-ECDCA and
rosiglitazone, respectively, did not affect liver function as measured by
plasma alanine aminotransferase, alkaline phosphatase, and bilirubin
(P > 0.05 versus control and porcine serum-treated rats). However,
both drugs effectively protected rats against development of liver fibrosis at
the dose of 3 mg/kg (Figs. 6
and 7). Although expression of
selected profibrogenetic markers was reduced in animals treated with 1 mg/kg
6-ECDCA (-SMA, fibronectin, and TGFβ1), this dose
slightly reduced the histological score and the liver hydroxyproline content
in comparison with animals treated with porcine serum alone. No significant
effects were observed in any biochemical or molecular marker of liver fibrosis
in animals treated with 1 mg/kg rosiglitazone. In contrast, administration of
rats with 3 mg/kg 6-ECDCA or rosiglitazone decreased the area of liver
parenchyma occupied by fibrotic tissue and hepatic levels of hydroxyproline as
well as expression of -SMA, 1(I) collagen, fibronectin,
TGFβ1, TIMP-1, and TIMP-2 mRNAs by 50 to 60% in comparison with rats
administered porcine serum alone (n = 8–12; P <
0.01 versus porcine serum alone; Figs.
6 and
7).
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Coadministration of 1 mg/kg 6-ECDCA together with 1 mg/kg rosiglitazone resulted in higher antifibrotic activity with respect to that observed with either of the two drugs alone and reduced the extent of liver fibrosis as measured by morphometric analysis by 50 to 60% in comparison with porcine serum alone (Figs. 5, 6, 7; n = 8–12; P < 0.01 versus porcine serum alone).
Similarly, coadministration of 6-ECDCA and rosiglitazone at the dose 3
mg/kg each resulted in a significant potentiation of the antifibrotic effect
exerted separately by the two drugs. Indeed, this combination ameliorated the
histological score, reduced the liver hydroxyproline content and decreased the
expression of -SMA, 1(I) collagen, fibronectin, TGFβ1,
TIMP-1 and TIMP-2 mRNAs by 90% (P < 0.05 versus 6-ECDCA and
rosiglitazone). The beneficial effect observed in rats treated with the
combination of FXR and PPAR ligands correlated with a significant
induction of PPAR mRNA expression in the liver. Thus, whereas
administering rats with 1 mg/kg 6-ECDCA or 1 and 3 mg/kg roglitazione alone
increased PPAR mRNA by 1- to 2-fold, administration of 3 mg/kg 6-ECDCA
resulted in 3- to 4-fold induction. Furthermore, the FXR ligand, but not the
PPAR ligand, increased the expression of SHP (3–4-fold) and
reduced Cyp7A1 by 70 to 80% (Fig.
7D). Furthermore, similarly to rosiglitazone 6-ECDCA (3 mg/kg)
increased the hepatic expression of UCP-2, a PPAR regulated gene, by 2-
to 4-fold (P < 0.05 versus control and porcine serum alone).
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Discussion |
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TopAbstractEditorial Expression of ConcernMaterials and MethodsResultsDiscussionReferences |
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expression, suggesting that an FXR-PPAR cascade exerts
counter-regulatory effects in HSCs. The demonstration that FXR ligands
increase PPAR and expression is consistent with the finding
that PPAR and , similar to FXR, function as a braking signal for
activation of HSCs and indicates that regulated expression of these NRs has
the potential to prevent HSCs trans-differentiation
(Friedman, 2003). Indeed, the
decrease in PPAR expression observed at the early stages of HSCs
activation and the fact that PPAR ligands inhibit at least some of the
characteristics associated with the activated phenotype of HSCs suggest that
PPAR may be involved in the maintenance of a quiescent phenotype (Galli
et al., 2000,
2002;
Marra et al., 2000;
Kon et al., 2002). In contrast
to the effect on PPAR and , FXR ligands failed to modulate
PPARβ expression. The increased expression of PPARβ, together with
the reduced expression of PPAR observed upon HSCs activation, suggests
that a balance between these transcription factors might be responsible for
some of the key phenotypic changes responsible for the profibrogenic role of
HSCs (Hellemans et al., 2003).
In this regard, PPARβ has been shown to act as a potent inhibitor of
PPAR activated transcription by the recruitment of corepressors and
histone deacetylases to PPAR-responsive elements
(Shi et al., 2002). Thus, the
opposite effects that FXR exerts on PPARβ and PPAR and
suggest that inhibition of HSCs activity caused by its ligands is due, at
least in part, to modulation of the ratio of PPARβ versus PPAR or
.
Previous studies have shown that bile acids regulate PPAR expression
in hepatoma cell lines and primary hepatocytes and that a functional FXR
response elements is expressed within the human PPAR promoter, which
confers responsiveness to FXR ligands
(Pineda Torra et al., 2003).
Consistent with this finding, we have now shown that 6-ECDCA, a potent FXR
ligand, increases rat PPAR mRNA expression in HSCs. Although the exact
role of PPAR in regulating HSCs is still undefined, administration of
the PPAR agonist Wy-14,643 to mice ameliorates established fibrosis and
reduces hepatic levels of 1(I) collagen, TIMP-1, TIMP-2, and MMP-13
(Ip et al., 2004), suggesting
that similarly to PPAR, PPAR might function as a negative
regulator of trans-differentiation in HSCs.
Despite the fact FXR and PPAR are expressed by HSCs and ligands for
these receptors decrease the expression of myofibroblastic markers in HSCs
(Galli et al., 2000;
Marra et al., 2000;
Hazra et al., 2004), their
expression shows opposite regulation during the process of
trans-differentiation. In fact, although the levels of PPAR
mRNA decrease to an almost undetectable level during the process of activation
in vitro and in vivo, the expression of FXR remains unchanged
(Fig. 2). This differential
regulation suggests that although an early reduction in PPAR plays a
permissive role, allowing HSCs to trans-differentiate, the same does
not apply to FXR, which regulates the profibrogenetic phenotype at a later
stages of activation. Thus, although molecular cross-talk exists between the
FXR and PPAR signaling pathways with FXR ligands inducing PPAR
expression, they seem to affect different regulatory mechanisms.
The ability of FXR ligands to increase PPAR expression in HSCs
raises the question of whether the antifibrotic activity of FXR ligands is
mediated through the activation of this nuclear receptor. Experiments carried
out with a selective PPAR antagonist suggest that although a certain
overlap between the two signaling pathways exists, inhibition of the
profibrogenetic activity of HSCs by FXR ligands seems to be largely
independent of the effect on PPAR. Support for this concept comes from
the demonstration that although treatment of HSCs with GW9662, a potent and
selective PPAR antagonist
(Leesnitzer et al., 2002),
fully reversed the inhibition of 1(I) collagen mRNA caused by
rosiglitazone, it was only marginally effective in blocking the inhibitory
effect of 6-ECDCA. Together with the fact that 6-ECDCA is 40-fold more potent
than rosiglitazone in blocking the up-regulation of 1(I) collagen
induced by TGFβ1, these data indicate that FXR ligands
modulate HSCs activity by both PPAR-dependent and -independent
mechanisms.
One important finding of the present study was the demonstration that in
vivo treatment with a FXR ligand reverses the down-regulation of PPAR
in HSCs obtained from rats administered porcine serum and CCL4 as
well as in BDL rats. Administering rats with 6-ECDCA caused an 2-fold of
increase in FXR and SHP mRNA, indicating that FXR ligands have the potential
to prevent PPAR down-regulation in these models.
Because thiazolidinediones, rosiglitazone, and pioglitazone effectively
prevent HSCs trans-differentiation in vitro and reduce fibrogenesis
in rodent model of liver cirrhosis (Galli
et al., 2002), it has been suggested that these glucose-lowering
agents might be beneficial in treating fibrosis in human disease
(Tsukamoto, 2002). Indeed,
although clinical studies in human fibrotic diseases are lacking,
thiazolidinediones have been used to treat insulin resistance in patients with
nonalcoholic steatohepatitis, resulting in a significant improvement of
fibrosis as judged by liver histology
(Neuschwander-Tetri et al.,
2003). However, in contrast to animal models of liver fibrosis,
where PPAR expression is abrogated, it is noteworthy that PPAR
is expressed at elevated levels in the liver of a number of murine models of
diabetes or obesity (Bedoucha et al.,
2001) suggesting that these conditions (i.e., liver fibrosis and
liver steatosis) involve different pathogenetic mechanisms.
Since both our in vivo and in vitro data indicate that FXR ligands can
reverse down-regulation of PPAR, a study was designed to investigate
whether a combination of submaximally effective doses of FXR and PPAR
ligands exert an additive effect in protecting against liver fibrosis induced
by porcine serum administration (Fiorucci
et al., 2004b). Using this model, we demonstrated that although
treatment of rats with 1 mg/kg 6-ECDCA and rosiglitazone, i.e., submaximally
effective doses of these agents caused a 20 to 30% reduction of markers of
hepatic fibrosis, coadministration of the two ligands reduces liver fibrosis
as assessed by liver morphometry by 60%. Furthermore, although a
significant reduction in liver fibrosis was observed in rats treated with 3
mg/kg of both agents, administration of the combination of 6-ECDCA and
rosiglitazone at 3 mg/kg for 8 weeks resulted in a 90% reduction in liver
collagen content. The reduction in collagen deposition obtained by the
combination of FXR and PPAR ligands was associated with a reduction in
the parenchymal area occupied by -SMA-positive cells, suggesting a
causal relationship between the decreased number of activated HSCs and the
reduced accumulation of extracellular matrix components. Protection against
development of liver fibrosis induced by 6-ECDCA associated with a significant
induction of PPAR and SHP gene expression.
Although we have shown that the effect of FXR ligands might be additive to
the effects of PPAR ligands in reducing liver fibrosis, these results
extend to other diseases. Indeed, FXR ligands increase the expression of
UCP-2, a PPAR regulated gene involved in regulation of energy
metabolism (Matsusue et al.,
2003), and suggest that FXR ligands might enhance the
glucose-lowering effects of PPAR ligands.
The synergistic activity of FXR ligand and PPAR ligand could also
contribute to limit the incidence of side effects associated with the use of
PPAR agonist. In fact 6 to 15% of diabetic patient taking rosiglitazone
or pioglitazone develop a diuretic-resistant edema
(Nesto et al., 2003;
Jarvinen, 2004). Since the
incidence of side effects caused by these two drugs is dose-dependent, it
seems likely that a combination of FXR and PPAR ligand could contribute
to limit the dose of PPAR ligand reducing the burden of side effects
associated with their use.
In conclusion, the present study demonstrates that FXR ligands regulate
PPAR gene expression and that FXR and PPAR ligands synergize in
regulating profibrogentic activities of HSCs.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: HSC, hepatic stellate cell; -SMA,
-smooth muscle actin; NR, nuclear receptor; FXR, farnesoid X receptor;
SHP, small heterodimer partner; TGF, transforming growth factor; PPAR,
proliferator-activated receptor; 6-ECDCA, 6-ethyl chenodoexyxholic acid;
GW4064,
3-(2,6-dichlorophenyl)-4-(3'-carboxy-2-chloro-stilben-4-yl)-oxymethyl-5-isopropyl-isoxazole;
GW9662, 2-chloro-5-nitrobenbanilide; CDCA, chenodeoxycholic acid; TIMP, tissue
inhibitor of matrix metalloproteinase; MMP, matrix metalloproteinase; qRT-PCR,
quantitative reverse transcription-polymerase chain reaction; CMC,
carboxymethyl cellulose; BDL, bile duct ligation; Wy-14,643,
(4-chloro-6-[(2,3-dimethylphenyl)amino]-2-pyrimidinyl)thioacetic acid
(pirinixic acid).
Address correspondence to: Dr. Stefano Fiorucci, Gastroenterologia ed Epatologia, Policlinico Monteluce, Via E dal Pozzo, 06111 Perugia, Italy. E-mail: fiorucci{at}unipg.it
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