Role of FXR and other nuclear receptors in liver fibrosis - Prof Stefano Fiorucci

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  • Role of FXR and other nuclear receptors in liver fibrosis - Prof Stefano Fiorucci

    1. 1. Role of FXRand other nucler receptors in liver fibrosis Stefano Fiorucci, MD University of Perugia Keystone symposia March 14, 2007
    2. 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. 3. Metabolic NRs expressed in the liver and gastrointestinal tract Constitutive 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. 4. Metabolic NRs that function as bile acid receptors Constitutive 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. 5. 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-1Fiorucci et al. Trends in Molecular Medicine, 2007, in press
    6. 6. Hepatic stellate cells (HSC)J Clin Invest. 2005 February 1; 115(2): 209–218
    7. 7. NRs in liver cells NR Hepatocytes 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 ‫ﻻ‬Fiorucci et al. Trends in Molecular Medicine, 2007, in press
    8. 8. PPARγ • HSC express PPARγ • PPARγ ligands inhibit profibrogenic and proinflammatory activities of HSCs• PPARγ ligands regulate adipogenic genes in HSCs • PPARγ induces a phenotypic switch from activated to quiescent HSCs • PPARγ ligands prevent hepatic steatosis, fibrosis in rodent models of liver cirrhosis
    9. 9. PPARγ Ito cells HSCs (adipocytes like) PPARγ TGFβPPARγSREPB1cLXR
    10. 10. Adipocytic characteristics of quiescent HSCsShe, H. et al. J. Biol. Chem. 2005;280:4959-4967
    11. 11. Expression of SREBP-1c induces other adipogenic factors and HSCs quiescenceShe, H. et al. J. Biol. Chem. 2005;280:4959-4967
    12. 12. PPARβ HSCs constitutively express high levels of PPARβ, which become further induced during culture activation and in vivo fibrogenesis. PPARβ activation enhances HSC proliferation. Treatment of rats with a single bolus of CCl 4 in combination with L165041 (a PPARβ ligand) enhances the expression of fibrotic markersGastroenterology 2003 Jan;124(1):184-201
    13. 13. Metabolic NRs that function as bile acid receptors Constitutive 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)
    14. 14. PXR Ligands Rifampicin in humans, PCN in rodents, phenobarbital,dexamethasone, LCA, statins, St. Johns wort, clotrimazole, possible UDCA Molecular targets MRP2/Mrp2, MRP3, Oatp1a4, MDR1, CYP3A4, SULT2A1/Sult2a1, (indirectly) CYP7A1 UGT1A1 Biological effectsInduction of canalicular and alternative basolateral bile acid secretion induction of phase I and II bile acid and bilirubin detoxification systems indirect repression of CYP7A1
    15. 15. PXR• Pregnenolone-16alpha-carbonitrile inhibits liver fibrogenesis via PXR-dependent and PXR-independent mechanisms. Biochem J. 2005 May 1;387(Pt 3):601-8• PXR activators inhibit human HSCs transdifferentiation in vitro Gastroenterology. 2006 Jul;131(1):194-209
    16. 16. PXR regulates HSCs functionGastroenterology. 2006 Jul;131(1):194-209
    17. 17. FXR
    18. 18. FXR Ligands CDCA, DCA, CA, LCA (weak ligand) synthetic: GW4064,6 -ethyl-CDCA, fexaramines Molecular targets PXR, SHP, BSEP/Bsep, I-BABP, Mrp2,OATP1B3, OSTαβ, Sult2a1, CYP3A4, UGT2B4, UGT2B7 Biological effects Induction of canalicular and alternativebasolateral bile acid excretion induction of phase I and II bile acid detoxification systems Inhibition of bile acid synthesis
    19. 19. GENES REGULATED BY FXR IN A NEGATIVE OR POSITIVE MANNERGene D/I FXRE Regulation Gene D/I FXRE Regulation
    20. 20. FXRSome of the effect of FXR are mediated by the activation of SHP, an atypical nuclear receptor that lacks ligand binding domain
    21. 21. SHP Ligands Molecular targets CYP7A1/Cyp7a1, CYP8B1/Cyp8b1, CYP27A1, Ntcp, ASBT/Asbt Biological effectsRepression of bile acid synthesis and basolateral bile acid uptake
    22. 22. NRs Regulation of CholesterolOrganic Anion andBile Salt Transport Oxysterols LRH-1 RXR LXR +c-Jun c-Fos CYP7A1 - CYP8B1 Isbt SHP - LRH-1 RXR FXR Bile Salts SHP +- HNF-1 RXR RAR + RXR FXR + RXR PXR + LRH1 Bsep Oatp2 MRP3 Ntcp Mrp2 Mrp2 Mrp2 I-Babp Trauner and Boyer Phys Rev ‘03
    23. 23. FXR 20 FXR-/- α1 collagen mRNA FXR wild type 10 0 0 3 6 9 12 Time (months) FXR deficient mice develop a pro-inflammatory and pro-fibrotic fenotypeYang F et al.,Spontaneous development of liver tumors in the absence of thebile acid receptor FXR. Cancer Res. 2007 Feb 1;67(3):863-7.
    24. 24. FXR• HSCs constitutively express high levels of FXR, which become slightly induced during cell activation and in vivo in rodent models of liver injury • FXR activation by FXR ligands reduces HSC transdifferention • Treatment of rats with FXR ligands reduces the expression of fibrotic markers in rodent models of liver cirrhosis.Fiorucci, et al. Gastroenterology 2004
    25. 25. FXR Co-Crystal Structure COOH . HO OH 6ECDCAMol. Cell, 2003, 11, 1093-1100
    26. 26. FXR 3 (fold of increase versus d1) * FXR expression WB: anti-FXR 2 * HSC HSC-T6 1 D1 D7 FXR 0 HSC HSC-T6α-SMA D1 D7 2 HSC HSC-T6 D1 D7 * FXR mRNA (qRT-PCR) FXR 1β-actin 0 HSC HSC-T6 D1 D7
    27. 27. FXR HSC-T6 Bp – 1 2 3 4 Agent alone α1(I) 6-ECDCA 30 CDCA α1 (I) collagen mRNA GW4064 * β-actin 20 * ** ** 10 1.5 ** ** ** ** α1 (I) collagen mRNA 0 1.0 Control Thrombin TGFβ 1 * 0.5 * * 0.0 Control CDCA 6-ECDCA GW4064Fiorucci et al., Gastroenterology 2004
    28. 28. FXR 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. 29. Control (TAA + PBS) Treated (TAA + INT-747) Group 1 Group 2 Group 3Albanis et al. Hepatology 2005, Abs
    30. 30. FXR INT-747 Decreases Portal Pressure 25 20 15 cm H20 Control 10 INT-747 5 0 Group 1 Group 2 Group 3Albanis et al. Hepatology 2005, Abs
    31. 31. FXR acts via SHP 1.5 Collagen α1(I) 1.0 mRNA WT HA-SHP 0.5 * HA-SHP 0.0 WB: anti-HA (28 KDa) WT SHP * 3 WT SHP+ * Collagen α1 (I) 2 mRNA 1 ** ** 0 Control Thrombin TGFβ1Fiorucci et al., GASTROENTEROLOGY 2004;127:1497–1512
    32. 32. FXR 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
    33. 33. FXREffect of FXR ligands on rodent models of liver cirrhosisModel Antifibrotic effectCCL4 ++BDL +Porcine serum +++TAA ++
    34. 34. Nuclear receptors cross talk FXR ligands upregulates PPARα and PPARγ expression/function1,2Some of the metabolic effects of FXR ligands are negatively modulated by PPARγ antagonists1,2 PXR is a target of farnesoid X receptor 31. Mol Endocrinol. 2003 Feb;17(2):259-722. JPET 315:58–68, 20053. J Biol Chem. 2006 Jul 14;281(28):19081-91
    35. 35. 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 1µMFiorucci S., et al. JPET 315:58–68, 2005
    36. 36. FXR, PXR and PPARγ NRs (PPARγ, FXR and PXR) are pharmacological targets in the treatment of liver fibrosis Cross-talk between FXR, PXR and PPARγ suggests a synergistic effects between selective ligandsFiorucci S., et al. JPET 315:58–68, 2005
    37. 37. FXR ligands: clinical trialsIntercept Pharmaceuticals is now advancing 6-ECDCA (INT-747) in to clinical trials Phase I, was completed early this yearPhase II studies for NASH and PBC will start in II-III qt 2007
    38. 38. AcknowledgementsDipartimento di MedicinaClinica e Sperimentale(Università di Perugia) Giovanni Rizzo Barbara Renga Piero Vavassori Andrea Mencarelli GSK (NC, USA) Moses di Sante Timothy M. Willson Bryan GoodwinDipartimento di Chimica e Intercept Pharmaceuticals (New York)Tossicologia del farmaco Mark Pruzanski(Universià di Perugia)Roberto PellicciariEmidio CamaioniGabriele CostantinoAntonio MacchiaruloAntimo GioielloBahman SadeghpourUdo Mayer
    39. 39. FXR ligands & bile acid metabolism Cholesterol NTCP CYP7A CYP7A NTCP Portal Blood CYP8B MR P2 Bile SHP bile acids BSEPcanaliculus 3 MDR MRP3 MRP4 FXR RXR CYP3A4 Phase IICYP7A cholesterol 7alpha-hydroxylaseCYP8B sterol 12 alpha-hydroxylase P450
    40. 40. FXR 1 2 3 4 5 6 α-SMA * 8 10.0 4 * * ralpha-SMA mRNA 7rCOL1A1 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
    41. 41. Protection against bile duct ligation-induced necrosis, mitosis, and bile duct proliferation by GW4064 Liu, Y. et al. J. Clin. Invest. 2003;112:1678-1687Copyright ©2003 American Society for Clinical Investigation
    42. 42. PXR PXR is expressed in HSCsGastroenterology. 2006 Jul;131(1):194-209
    43. 43. FXR 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 UDCAFiorucci et al. JEPT 2005
    44. 44. PPARγ Ito cells HSCs (adipocytes like) PPARγ TGFβPPARγ CollagenSREPB1c SMALXR TIMP1
    45. 45. PXR LIGANDS PNAS | March 13, 2001 | vol. 98 | no. 6 | 3369-3374Chemical structures of xenobiotics that bind to and activate PXR. Hyperforin is a constituent of St. Johns wort.
    46. 46. NRs general structure AF1 AF2 A/B C D E F NH2 DBD hinge LBD COOH AF1 AF2 DIMERIZATIONFiorucci et al. Trends in Molecular Medicine, 2007, in press
    47. 47. PPARγ transduction induces adipogenic transcription factorsShe, H. et al. J. Biol. Chem. 2005;280:4959-4967
    48. 48. 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

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