1. Control of liver fibrosis
by nuclear receptor
Stefano Fiorucci, MD
University of Perugia
EASL BASIC SCHOOL
OF HEPATOLOGY

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. Metabolic NRs expressed in the
liver and gastrointestinal tract
 Androstan Receptor (CAR)
 Estrogen Related Receptor-α (ERR)
 Farnesoid X Receptor (FXR)
 Hepatocyte Nuclear Factor-4 (HNF-4)
 Liver X Receptor (LXR)
 Peroxisome Proliferator-Activated Receptor (PPAR)
 Pregnane X Receptor (PXR)
 Retinoid X Receptor (RXR)
 Vitamin D Receptor (VDR)

4. Metabolic NRs expressed in the
liver and gastrointestinal tract
 Androstan Receptor (CAR)
 Estrogen Related Receptor-α (ERR)
 Farnesoid X Receptor (FXR)
 Hepatocyte Nuclear Factor-4 (HNF-4)
 Liver X Receptor (LXR)
 Peroxisome Proliferator-Activated Receptor (PPAR)
 Pregnane X Receptor (PXR)
 Retinoid X Receptor (RXR)
 Vitamin D Receptor (VDR)

5. NRs general structure
AF1 AF2
A/B C D E F
NH2 DBD hinge LBD COOH
AF1 AF2
DIMERIZATION

6. NRs mode of action
HAT
HDAC
+ligands LX
N-CoR LL XL
Histones LXX L
Histones
AF2 AF2 De-Acetylation AF2 AF2 Acetylation
Bile acids
AF1 AF1 CDCA or INT-747 AF1 AF1 Ac Ac
Ac Ac
Ac Ac
DBD DBD DBD DBD
FXR RXR FXR RXR
9-cis RA Ac Ac
Ac Ac
Ac Ac
CDCA
INT-747
FXR RXR FXR RXR
9-cis RA
9-cis RA
IR-1 IR-1

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

8. Nuclear receptors and HSCs
Receptor Hepatic cells Stellate cells
PPARα ‫ﻻ‬ not found
PPARγ ‫ﻻ‬ ‫ﻻ‬
PPARβ ‫ﻻ‬ ‫ﻻ‬
FXR ‫ﻻ‬ ‫ﻻ‬
PXR ‫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. Expression of SREBP-1c induces other adipogenic
factors And HSCs quiescence
She, H. et al. J. Biol. Chem. 2005;280:4959-4967

14. PXR
Ligands
Rifampicin in humans, PCN in rodents, phenobarbital,
dexamethasone, LCA, statins, St. John's wort, clotrimazole,
possible UDCA
Molecular targets
MRP2/Mrp2, MRP3, Oatp1a4, MDR1, CYP3A4,
SULT2A1/Sult2a1, (indirectly) CYP7A1 UGT1A1
Biological effects
Induction of canalicular and alternative basolateral
bile acid excretion induction of phase I and II bile
acid and bilirubin detoxification systems indirect
repression of CYP7A1

15. PXR LIGANDS
PNAS | March 13, 2001 | vol. 98 | no. 6 | 3369-3374
Chemical structures of xenobiotics that bind to and activate PXR.
Hyperforin is a constituent of St. John's wort.

16. 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

17. PXR
PXR is expressed in HSCs
Gastroenterology. 2006 Jul;131(1):194-209

18. PXR regulates HSCs function
Gastroenterology. 2006 Jul;131(1):194-209

19. FXR IN LIVER and GI

20. Ntcp Bsep Mdr2
Na+
BS- X BS- PC
OA-
Ostαβ
X
BS-
X
OA-
OC+ Mrp3 & 4 Proximal Renal Tubule
BS -
BS-
Oatps Mrp2 Mdr1
Asbt
Cholangiocyte
X
Na+
Hepatocyte
BS-
Asbt Ostαβ ? OA-
Na+ Na+ BS-
Enterocyte
Bile Duct BS- BS- Mrp2 Ostαβ
Mrp4
OA-
+
? Mrp3 OC
BS-
OA- Mdr1 ? Mrp3
BS-
Na+ BS- Kidney
Mdr1
?
Asbt
X Mrp2 Adaptive changes in
transporter expression in
Intestine
cholestasis
Trauner & Boyer.
Phys. Rev 83:’03

21. FXR
 Ligands
CDCA, DCA, CA, LCA possibly UDCA (weak ligand)
synthetic: GW4064,6 -ethyl-CDCA, fexaramines
 Molecular targets
SHP, BSEP/Bsep, I-BABP, Mrp2, OATP1B3,
OSTαβ, Sult2a1, CYP3A4, UGT2B4, UGT2B7
 Biological effects
Induction of canalicular and alternative basolateral
bile acid excretion induction of phase I and II bile
acid detoxification systems

22. SHP
 Ligands
 Molecular targets
CYP7A1/Cyp7a1, CYP8B1/Cyp8b1, CYP27A1, Ntcp, ASBT/Asbt
 Biological effects
Repression of bile acid synthesis and basolateral bile acid uptake

23. FXR ligands & bile acid metabolism
Cholesterol
NTCP
CYP7A
NTCP
Portal Blood
CYP8B MR
P2
Bile
SHP bile acids BSEP canaliculus
3
MDR
MRP3
FXR RXR
MRP4
CYP3A4 Phase II
CYP7A cholesterol 7alpha-hydroxylase
CYP8B sterol 12 alpha-hydroxylase P450

24. 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

25. 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

26. 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

27. FXR Co-Crystal Structure
COOH
.
HO OH
6ECDCA
Mol. Cell, 2003, 11, 1093-1100

28. 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

29. 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

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

31. 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

32. 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

33. 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

34. 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 1 2 3 4
10 **
5 α-SMA
CCL4 0
TIMP-1
1500 *
1250 *
MMP-2
Liver HP content
(µg/g tissue)
1000
FXR
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

35. 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 farnesoid X receptor3
1. Mol Endocrinol. 2003 Feb;17(2):259-72
2. JPET 315:58–68, 2005
3. J Biol Chem. 2006 Jul 14;281(28):19081-91

36. Regulation of Transporter Expression
by NRs
SHP
RXRα RARα HNF-1
Ntcp
FXR PXR CAR
RARα RXRα
Mrp2
SP-1 SP-3 LRH-1
Mrp3
RXRα FXR
Bsep
RXRα FXR
Ostα/β

37. 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

38. 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

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

40. 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.

41. GENE EXPRESSION PROFILING
FXR-Regulated Genes
+ 6α-Ethyl-CDCA Liver
Intestine Gene Array
Experiments
DGE
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