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Control of liver fibrosis by nuclear receptors - Prof Stefano Fiorucci

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Control of liver fibrosis by nuclear receptors - Prof Stefano Fiorucci

  1. 1. Control of liver fibrosis by nuclear receptor Stefano Fiorucci, MD University of Perugia EASL BASIC SCHOOL OF HEPATOLOGY
  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 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 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. 5. NRs general structure AF1 AF2 A/B C D E FNH2 DBD hinge LBD COOH AF1 AF2 DIMERIZATION
  6. 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 acidsAF1 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. 7. Hepatic stellate cellsJ Clin Invest. 2005 February 1; 115(2): 209–218
  8. 8. Nuclear receptors and HSCsReceptor Hepatic cells Stellate cellsPPARα ‫ﻻ‬ not foundPPARγ ‫ﻻ‬ ‫ﻻ‬PPARβ ‫ﻻ‬ ‫ﻻ‬FXR ‫ﻻ‬ ‫ﻻ‬PXR ‫ﻻ‬ ‫ﻻ‬ERR ‫ﻻ‬ ‫ﻻ‬CAR ‫ﻻ‬ -LXR ‫ﻻ‬ ‫-/ﻻ‬RXR ‫ﻻ‬ ‫ﻻ‬SHP ‫ﻻ‬ ‫ﻻ‬
  9. 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. 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. 11. Adipocytic characteristics of quiescent HSCShe, H. et al. J. Biol. Chem. 2005;280:4959-4967
  12. 12. PPARγ transduction induces other adipogenic transcription factorsShe, H. et al. J. Biol. Chem. 2005;280:4959-4967
  13. 13. Expression of SREBP-1c induces other adipogenic factors And HSCs quiescenceShe, H. et al. J. Biol. Chem. 2005;280:4959-4967
  14. 14. PXRLigandsRifampicin in humans, PCN in rodents, phenobarbital,dexamethasone, LCA, statins, St. Johns wort, clotrimazole,possible UDCAMolecular targetsMRP2/Mrp2, MRP3, Oatp1a4, MDR1, CYP3A4,SULT2A1/Sult2a1, (indirectly) CYP7A1 UGT1A1Biological effectsInduction of canalicular and alternative basolateralbile acid excretion induction of phase I and II bileacid and bilirubin detoxification systems indirectrepression of CYP7A1
  15. 15. 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.
  16. 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. 17. PXR PXR is expressed in HSCsGastroenterology. 2006 Jul;131(1):194-209
  18. 18. PXR regulates HSCs functionGastroenterology. 2006 Jul;131(1):194-209
  19. 19. FXR IN LIVER and GI
  20. 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. 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. 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. 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 IICYP7A cholesterol 7alpha-hydroxylaseCYP8B sterol 12 alpha-hydroxylase P450
  24. 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. 25. 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
  26. 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 GW4064Fiorucci et al., Gastroenterology 2004
  27. 27. FXR Co-Crystal Structure COOH . HO OH 6ECDCAMol. Cell, 2003, 11, 1093-1100
  28. 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. 29. 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
  30. 30. Control (TAA + PBS) Treated (TAA + INT-747) Group 1 Group 2 Group 3Albanis et al. Hepatology 2005, Abs
  31. 31. 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
  32. 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 TGFβ1Fiorucci et al., GASTROENTEROLOGY 2004;127:1497–1512
  33. 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. 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 UDCAFiorucci et al. JEPT 2005
  35. 35. 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 receptor31. Mol Endocrinol. 2003 Feb;17(2):259-722. JPET 315:58–68, 2005 3. J Biol Chem. 2006 Jul 14;281(28):19081-91
  36. 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 FXROstα/β
  37. 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 1µMFiorucci S., et al. JPET 315:58–68, 2005
  38. 38. FXR, PXR and PPARγCross-talk between FXR, PXR and PPARγ might contribute to the antifibrotic activity of FXR ligands in rodent models of liver cirrhosisFiorucci S., et al. JPET 315:58–68, 2005
  39. 39. 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
  40. 40. FXR Regulation of Hepatic Stellate Cell Phenotype CO2H INITIATION Hepatic Stellate Cells HO . OHQuiescent ActivatedPhenotype 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. 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 PLTPbile 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

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