1) The nuclear receptor FXR is expressed in hepatic stellate cells and its activation reduces stellate cell proliferation and the expression of fibrotic markers. 2) FXR activation induces the expression of SHP, which inhibits stellate cell activation and collagen production. 3) FXR ligands have shown antifibrotic effects in animal models of liver fibrosis by modulating stellate cell function and cross-talking with other nuclear receptors like PPARγ.

1. FXR and liver fibrosis
Stefano Fiorucci, MD
University of Perugia

2. The Superfamily of Human Nuclear Receptors
Endocrine Hormone Orphan Nuclear Receptors
Receptors 1. Chicken Ovalbumin Upsteram (COUP)
. Estrogen Receptor-β (ER-β) 2. Dosage-sensitive Sex Reversal (DAX)
. Estrogen Receptor-α (ER-α) 3. Germ Cell Nuclear Factor (GCNF)
. Glucocorticoid Receptor (GR) 4. Liver Related Homologue-1 (LRH-1)
. Mineralcorticoid Receptor (MR) 5. NGF-induced clone B (NGFI-B)
. Androgen Receptor (AR) 6. Photoreceptor Nuclear Receptor (PNR)
. Progesterone Receptor (PR) 7. Reverse ErbA (RevErbA)
. Retinoic Acid Receptor (RAR) 8. Small Heterodimer Partner
. Tyroid Hormone Receptor (TR) 9. Steroidogenic Factor-1 (SF-1)
. Vitamin-D Receptor (VDR) 10.Testis Receptor-2 (TR-2)
Adopted Orphan Receptors
Metabolic Nuclear Receptors
1. Androstan Receptor (CAR)
2. Estrogen Related Receptor-α (ERR)
3. Farnesoid X Receptor (FXR)
4. Hepatocyte Nuclear Factor-4 (HNF-4)
5. Liver X Receptor (LXR)
6. Peroxisome Proliferator-Activated Receptor (PPAR)
7. Pregnane X Receptor (PXR)
8. Retinoid X Receptor (RXR)

3. The Superfamily of Human Nuclear Receptors
Endocrine Hormone Orphan Nuclear Receptors
Receptors 1. Chicken Ovalbumin Upsteram (COUP)
. Estrogen Receptor-β (ER-β) 2. Dosage-sensitive Sex Reversal (DAX)
. Estrogen Receptor-α (ER-α) 3. Germ Cell Nuclear Factor (GCNF)
. Glucocorticoid Receptor (GR) 4. Liver Related Homologue-1 (LRH-1)
. Mineralcorticoid Receptor (MR) 5. NGF-induced clone B (NGFI-B)
. Androgen Receptor (AR) 6. Photoreceptor Nuclear Receptor (PNR)
. Progesterone Receptor (PR) 7. Reverse ErbA (RevErbA)
. Retinoic Acid Receptor (RAR) 8. Small Heterodimer Partner
. Tyroid Hormone Receptor (TR) 9. Steroidogenic Factor-1 (SF-1)
. Vitamin-D Receptor (VDR) 10.Testis Receptor-2 (TR-2)
Adopted Orphan Receptors
Metabolic Nuclear Receptors
1. Androstan Receptor (CAR)
2. Estrogen Related Receptor-α (ERR)
3. Farnesoid X Receptor (FXR)
4. Hepatocyte Nuclear Factor-4 (HNF-4)
5. Liver X Receptor (LXR)
6. Peroxisome Proliferator-Activated Receptor (PPAR)
7. Pregnane X Receptor (PXR)
8. Retinoid X Receptor (RXR)

4. FXR IN LIVER and GI

5. Clinical targets of FXR ligands
Cholestatic liver diseases
Primary biliary cirrhosis
Sclerosing cholangitis
Cholestatic disorders in pregancy
Cholestatic complications of TPN
Non cholestatic liver disorders
NASH
Liver fibrosis
Metabolic disorders
Atherosclerosis
Diabetes

6. Clinical targets of FXR ligands
Cholestatic liver diseases
Primary biliary cirrhosis
Sclerosing cholangitis
Cholestatic disorders in pregancy
Cholestatic complications of TPN
Non cholestatic liver disorders
NASH
Liver fibrosis
Metabolic disorders
Atherosclerosis
Diabetes

7. J Clin Invest. 2005 February 1; 115(2): 209–218

8. Nuclear receptors and HSCs
Receptor Hepatic cells Stellate cells
FXR ‫ﻻ‬ ‫ﻻ‬
PXR ‫ﻻ‬ ‫ﻻ‬
PPARα ‫ﻻ‬ not found
PPARγ ‫ﻻ‬ ‫ﻻ‬
PPARβ ‫ﻻ‬ ‫ﻻ‬
ERR ‫ﻻ‬ ‫ﻻ‬
CAR ‫ﻻ‬ -
LXR ‫ﻻ‬ ‫-/ﻻ‬
RXR ‫ﻻ‬ ‫ﻻ‬
SHP ‫ﻻ‬ ‫ﻻ‬

9. PPARβ
PPARβ signaling contributes to enhanced
proliferation of HSC
• HSCs constitutively express high levels of PPARβ, which
become further induced during culture activation and in vivo
fibrogenesis.
• PPARβ activation by L165041 enhanced HSC proliferation.
• Treatment of rats with a single bolus of CCl4 in combination
with L165041 enhanced the expression of fibrotic markers.
Gastroenterology 2003 Jan;124(1):184-201

10. PPARγ
• Ligands of PPARγ modulate profibrogenic and
proinflammatory actions in HSCs.
• PPARγ ligands regulate adipogenic genes in
HSC.
• PPARγ induces a phenotypic switch from
activated to quiescent HSC.
• PPARγ ligands prevent hepatic steatosis,
fibrosis in rodent models of liver cirrhosis.

11. Adipocytic characteristics of quiescent HSC
She, H. et al. J. Biol. Chem. 2005;280:4959-4967

12. PPARγ transduction induces other adipogenic
transcription factors
She, H. et al. J. Biol. Chem. 2005;280:4959-4967

13. PXR
• Pregnenolone-16alpha-carbonitrile inhibits
rodent liver fibrogenesis via PXR
-dependent and PXR-independent
mechanisms.
Biochem J. 2005 May 1;387(Pt 3):601-8
• PXR activators inhibit human hepatic
stellate cell transdifferentiation in vitro
Gastroenterology. 2006 Jul;131(1):194-209

15. FXR
• HSCs constitutively express high levels of FXR, which
become slightly induced during culture activation and in
vivo fibrogenesis.
• FXR activation by FXR ligands reduces HSC
proliferation.
• Treatment of rats with FXR ligands reduces the
expression of fibrotic markers in rodent models of
cirrhosis.
• Fiorucci, et al. Gastroenterology 2004

16. 3
(fold of increase versus d1)
*
FXR expression
WB: anti-FXR 2 *
HSC HSC-T6
1
D1 D7
FXR
0
HSC HSC-T6
α-SMA
D1 D7
2
HSC HSC-T6
D1 D7 *
FXR mRNA
(qRT-PCR)
FXR
1
β-actin
0
HSC HSC-T6
D1 D7

17. HSC-T6
Bp – 1 2 3 4
α1(I)
Agent alone
6-ECDCA
β-actin 30 CDCA
α1 (I) collagen mRNA
GW4064 *
20
*
** **
**
10
1.5 ** ** **
**
α1 (I) collagen mRNA
0
1.0
Control Thrombin TGFβ 1
*
0.5 * *
0.0
Control CDCA 6-ECDCA GW4064
Fiorucci et al., Gastroenterology 2004

18. COOH
.
HO OH 250
200
Rluc/β Gal
150
100
150
Relative Luciferase/βGal
50
GW4064
100
6-ECDCA 0
LX α
l
b
ER
PP R
VD
C R
PP α
CDCA
δ
t ro
PP γ
R
R
AR
AR
AR
G
FX
LX
on
50
0
-10 -9 -8 -7 -6 -5 -4
Agonist concentration (Log M)

19. 8 *
7
(percent of total)
6
Fibrotic area
5
4
3 **
2 ** **
1
0
1500
Liver HP content ( µg/g
1250 *
1000
Normal Porcine serum
tissue)
750
**
500 **
**
250
0
150
HP/creatinine ( µg/mg)
*
100
**
** **
50
PS + INT- 747 5 mg/kg 0
PS
RL
g
g
g
g
3m
1m
5m
m
CT
10
A-
A-
A-
A-
DC
DC
DC
DC
EC
EC
EC
EC
6-
6-
6-
6-
Fiorucci et al., GASTROENTEROLOGY 2004;127:1497–1512

20. 1 2 3 4 5 6
α-SMA
*
8 10.0 4
* *
ralpha-SMA mRNA
7
rCOL1A1 mRNA
rTGFb mRNA
6 7.5 3
5
**
4 5.0 2 **
3
** **
** ** **
**
2 ** 2.5 ** 1
**
1
0 0.0 0
Control Porcine serum Control Porcine serum Control Porcine serum
Alone 1 3 5 10 Alone 1 3 5 10 Alone 1 3 5 10
6-ECDCA (mg/kg/day) 6-ECDCA (mg/kg/day) 6-ECDCA (mg/kg/day)
Fiorucci et al., GASTROENTEROLOGY 2004;127:1497–1512

21. Control (TAA + PBS) Treated (TAA + INT-747)
Group 1
Group 2
Group 3
Albanis et al. Hepatology 2005, Abs

22. INT-747 Decreases Portal Pressure
25
20
15
cm H20
Control
10 INT-747
5
0
Group 1 Group 2 Group 3
Albanis et al. Hepatology 2005, Abs

23. SHP
FXR ligands
+
CDCA
FXR RXR
SHP

24. 1.5
Collagen α1(I)
1.0
mRNA
0.5 *
WT HA-SHP
0.0
HA-SHP WT SHP
WB: anti-HA
(28 KDa)
*
3 WT
SHP+ *
Collagen α1 (I)
2
mRNA 1
**
**
0
Control Thrombin TGFβ1
Fiorucci et al., GASTROENTEROLOGY 2004;127:1497–1512

25. siRNA - siRNA +
1.5
2.0
1.8
1.6 **
Collagen α1(I)
SHP mRNA
QRT-PCR
1.0 1.4 **
QRT-PCR
**
1.2
* **
1.0
* 0.8
0.5 *
0.6
* *
0.4
* 0.2
0.0 0.0
0 1 5 10 15 Control CDCA 6-ECDCA GW4064
SHP-siRNA (nM)
Fiorucci et al., GASTROENTEROLOGY 2004;127:1497–1512

26. Nuclear receptors cross talk
FXR ligands upregulates PPARα and PPARγ
expression/function1,2
Some of the metabolic effects of FXR ligands are
negatively modulated by PPARγ antagonists1,2
PXR is a target of FXR3
1. Mol Endocrinol. 2003;17:259-72
2. JPET 315:58–68, 2005
3. J Biol Chem. 2006 14;281:19081-91

27. 6-ECDCA
7.0 4
Rosiglitazone *
6.5
α (I) collagen mRNA
6.0 **
*
PPAR-γ mRNA
5.5 3
Fold of basall 5.0
qRT-PCR
qRT-PCR
4.5
4.0 2
3.5 *
3.0 *
2.5 *
1
2.0
1.5 * *
1
1.0 * * *
e
*
on
0.5 0
.z
l
tro
os
0.0
µM
µM
µM
A M
on
R
C µ
C
+
D 1
0
5
.1
1
0.01 0.1 0.5 1.0 5.0 10.0
e
E C ne
A
0
on
C
A
6- o
D
C
az
az
FXR or PPAR- γ ligand
EC
D
lit
lit
EC
ig
ig
6-
os
os
(µ
6-
M)
R
R
7.5 * α 1 (I) collagen
α-SMA
α (I) collagen mRNA
*
qRT-PCR
5.0
**
** **
**
2.5
1
*** ***
0.0
Medium TGF β 1 ng/ml
1
Alone 6-ECDCA RGT 6-ECDCA
+RGT
0.1 µM 1µM
Fiorucci S., et al. JPET 315:58–68, 2005

28. FXR Regulation of Hepatic Stellate Cell Phenotype
CO2H
INITIATION
Hepatic Stellate Cells
HO
. OH
Quiescent Activated
Phenotype Phenotype 6-ECDCA
+ + +
(myofibroblast-like) +
CDCA
α -SMA Collα -1 TIMP-1 FXR RXR
Up-Regulation
Procollagen Genes +
DNA Binding +
- SHP
+
PPARγ RXR
AP-1 Small Heterodimer Partner
DNA Binding Inhibition Transcriptional
Fiorucci S, Pellicciari R., Enhancement
DNA Binding Gastroenterology.2004 submitted Nishizawa, H.,
Inhibition - J Biol Chem. 2000, 277, 1586-1592.

29. Acknowledgements
Dipartimento di Medicina Clinica e Sperimentale (Università di Perugia)
Giovanni Rizzo
Barbara Renga
Piero Vavassori
Andrea Mencarelli
Moses di Sante
Dipartimento di Chimica e Tossicologia del farmaco (Università di
Perugia)
Roberto Pellicciari
Emidio Camaioni
Gabriele Costantino
Antonio Macchiarulo
Antimo Gioiello
Bahman Sadeghpour
Udo Mayer
GSK (NC, USA)
Timothy M. Willson
Bryan Goodwin
Intercept Pharmaceuticals (New York)
Mark Pruzanski

31. FXR, PXR and PPARγ
Cross-talk between FXR, PXR and
PPARγ might contribute to the
antifibrotic activity of FXR ligands
in rodent models of liver cirrhosis
Fiorucci S., et al. JPET 315:58–68, 2005

32. 13:10 Partenza Roma (FCO)
Arrivo Basilea (BSL) 17:05 ven 6-ott
Durata: 3h 55m Lufthansa 3859 / 3824Collegamento Monaco
(Munich F. J. Strauss International Airport)
14:40 Partenza Basilea (BSL)
Arrivo Roma (FCO) 18:15 sab 7-ott
Durata: 3h 35m Lufthansa 3813 / 3848Collegamento
Francoforte (Frankfurt Intl.)

34. GENE EXPRESSION PROFILING
FXR-Regulated Genes
Primary human
hepatocytes
CYP7A CYP8B I-BABP MDR3 BSEP SHP MRP2 MRP3 PLTP
bile acid synthesis HDL -> VLDL
bile acid synthesis organic anion transporter
bile acid binding proteinbile acid transporter conversion
phospholipid transporter sulfate conjugate transporter
small heterodimer partner

35. 12
11
10 *
*
(percent of total)
9
Fibrotic area
8
7
6
5 **
4
3
2
1
Naive 0
25
*
αSMA positive cells
20
*
15
10 **
5
CCL4 0
1500 *
1250
*
Liver HP content
(µg/g tissue)
1000
750
500
CCL4 + INT747 250
**
0
150
(µg/mg creatinine)
* *
Urinary HP
100
50 **
CCL4 + UDCA
0
Control CCL4
Alone 6-ECDCA UDCA
Fiorucci et al. JEPT 2005

36. PPARγ transduction induces other adipogenic
transcription factors
She, H. et al. J. Biol. Chem. 2005;280:4959-4967

37. Expression of SREBP-1c induces other adipogenic factors and
HSCs quiescence
She, H. et al. J. Biol. Chem. 2005;280:4959-4967