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Prof Stefano Fiorucci -  FXR and liver fibrosis
 

Prof Stefano Fiorucci - FXR and liver fibrosis

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    Prof Stefano Fiorucci -  FXR and liver fibrosis Prof Stefano Fiorucci - FXR and liver fibrosis Presentation Transcript

    • FXR and liver fibrosis Stefano Fiorucci, MD University of Perugia
    • 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)
    • 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)
    • FXR IN LIVER and GI
    • 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
    • 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
    • J Clin Invest. 2005 February 1; 115(2): 209–218
    • Nuclear receptors and HSCsReceptor Hepatic cells Stellate cellsFXR ‫ﻻ‬ ‫ﻻ‬PXR ‫ﻻ‬ ‫ﻻ‬PPARα ‫ﻻ‬ not foundPPARγ ‫ﻻ‬ ‫ﻻ‬PPARβ ‫ﻻ‬ ‫ﻻ‬ERR ‫ﻻ‬ ‫ﻻ‬CAR ‫ﻻ‬ -LXR ‫ﻻ‬ ‫-/ﻻ‬RXR ‫ﻻ‬ ‫ﻻ‬SHP ‫ﻻ‬ ‫ﻻ‬
    • 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
    • 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.
    • Adipocytic characteristics of quiescent HSC She, H. et al. J. Biol. Chem. 2005;280:4959-4967
    • PPARγ transduction induces other adipogenic transcription factors She, H. et al. J. Biol. Chem. 2005;280:4959-4967
    • 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
    • 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
    • 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
    • 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
    • COOH . HO OH 250 200 Rluc/β Gal 150 100 150Relative 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)
    • 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
    • 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
    • Control (TAA + PBS) Treated (TAA + INT-747) Group 1 Group 2 Group 3Albanis et al. Hepatology 2005, Abs
    • 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
    • SHPFXR ligands + CDCA FXR RXR SHP
    • 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
    • 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
    • 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 FXR31. Mol Endocrinol. 2003;17:259-722. JPET 315:58–68, 20053. J Biol Chem. 2006 14;281:19081-91
    • 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
    • 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.
    • AcknowledgementsDipartimento 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
    • 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
    • 13:10 Partenza Roma (FCO)Arrivo Basilea (BSL) 17:05 ven 6-ottDurata: 3h 55m Lufthansa 3859 / 3824Collegamento Monaco(Munich F. J. Strauss International Airport)14:40 Partenza Basilea (BSL)Arrivo Roma (FCO) 18:15 sab 7-ottDurata: 3h 35m Lufthansa 3813 / 3848Collegamento Francoforte (Frankfurt Intl.)
    • GENE EXPRESSION PROFILING FXR-Regulated Genes 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
    • 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 UDCAFiorucci et al. JEPT 2005
    • PPARγ transduction induces other adipogenic transcription factors She, H. et al. J. Biol. Chem. 2005;280:4959-4967
    • Expression of SREBP-1c induces other adipogenic factors and HSCs quiescenceShe, H. et al. J. Biol. Chem. 2005;280:4959-4967