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Kupffer cells mediate_leptin-induced_liver_fibrosis

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Kupffer Cells Mediate Leptin-Induced Liver Fibrosis.

GASTROENTEROLOGY 2009;137:713–723

JIANHUA WANG,* ISABELLE LECLERCQ,‡ JOANNE M. BRYMORA,* NING XU,* MEHDI RAMEZANI–MOGHADAM,* ROSLYN M. LONDON,* DAVID BRIGSTOCK,§ and JACOB GEORGE*

*Storr Liver Unit, Westmead Millennium Institute, University of Sydney and Westmead Hospital, Westmead, Australia; ‡Laboratory of Gastroenterology, Faculty of
Medicine, Université Catholique de Louvain, Brussels, Belgium; and §Center for Cell and Vascular Biology, Children’s Research Institute, Columbus, Ohio

瘦素(Leptin)是一由脂肪細胞(Adipocyte)所分泌之荷爾蒙,是調控體重及新陳代謝之重要因子。過去研究發現病態肥胖(Obese)、脂肪肝(Nonalcoholic steatohepatitis)及酒精性肝炎(Alcoholic liver disease)等病患之血液循環中,Leptin量有明顯增加。而近期研究報告指出leptin具有促進肝臟纖維化(Liver fibrosis)之能力,當中分子機理並未明確。
在肝纖維化過程中,肝臟星狀細胞(HSC)會被活化增生及促進胞外基質(ECM)產生,而鄰近之Kupffer細胞(KC)則已知可透過促發炎因子(Proinflammatory factor)和促纖維化因子(Profibrogenic factors)例如TGF-β1和ROS影響HSC表現。雖然HSC是肝纖維化過程中重要角色,前人研究卻發現leptin似對HSC無任何調控作用。故本篇作者針對Leptin是否透過間接作用於HSC鄰近之KC,刺激其產生促纖維化因子,以活化HSC。
為探討leptin直接或間接影響HSC之分子機理,本篇作者透過RT-PCR、Immunoblot等分子生物學方法,分別測定leptin刺激後HSC及KC中Collagen I、TIMP1等促纖維化因子基因及蛋白表現,發現leptin雖可促使HSC增生,但對其纖維化能力之影響甚微。而leptin可刺激KC中TGF-β1及CTGF/CCN2等肝纖維化中重要之cytokines表現。另發現Leptin-treated KC-conditioned培養液可刺激HSC增生及增加其中Collagen I、TIMP1等表現,得出了leptin是透過刺激KC來活化HSC之推論。作者亦於後續實驗中,透過磷酸化測定、EMSA等方法探討leptin訊號傳遞作用,發現leptin可活化KC中STAT3、ERK1/2、AKT等路徑,及下游因子AP-1、NF-κB,而此兩種蛋白具有增強TGF-β1及CTGF/CCN2基因表現之能力。

Veröffentlicht in: Gesundheit & Medizin
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Kupffer cells mediate_leptin-induced_liver_fibrosis

  1. 1. Kupffer Cells Mediate Leptin-Induced Liver Fibrosis GASTROENTEROLOGY 2009;137:713–723 JIANHUA WANG,* ISABELLE LECLERCQ,‡ JOANNE M. BRYMORA,* NING XU,* MEHDI RAMEZANI–MOGHADAM,* ROSLYN M. LONDON,* DAVID BRIGSTOCK,§ and JACOB GEORGE* *Storr Liver Unit, Westmead Millennium Institute, University of Sydney and Westmead Hospital, Westmead, Australia; ‡Laboratory of Gastroenterology, Faculty of Medicine, Université Catholique de Louvain, Brussels, Belgium; and §Center for Cell and Vascular Biology, Children’s Research Institute, Columbus, Ohio IF: 12.591 Design by ChauChanLao 2010.03
  2. 2. Liver Fibrosis <ul><li>KCs and SECs play important roles in modulating stellate cell behavior </li></ul>J Clin Invest. 2005 February 1; 115(2): 209–218.
  3. 3. Kupffer cells(KCs) <ul><li>Specialized macrophages located in the liver </li></ul><ul><li>Kupffer cells activation are responsible for early ethanol-induced liver injury, common in chronic alcoholics </li></ul>http://cindymarie95.glogster.com/
  4. 4. Pathogenesis of liver fibrosis Comparative Hepatology 2007 6 :7
  5. 5. J Clin Invest. 2005 February 1; 115(2): 209–218.
  6. 6. Circulating levels of leptin are known to be increased in overweight and obese persons, in individuals with nonalcoholic steatohepatitis, and in those with alcoholic liver disease and chronic viral hepatitis N Engl J Med 1996;334:292–295. The American Journal of Gastroenterology (2003) 98 , 1135–1141
  7. 7. Leptin( 瘦素 ) <ul><li>16kD adipocyte-derived hormone </li></ul><ul><li>Ob(Lep) gene (Ob for obese, Lep for leptin) is located on chromosome 7 in humans </li></ul><ul><li>Plays a key role in regulating appetite and metabolism </li></ul>Wikipedia.org Recently, leptin has been shown to possess direct profibrogenic activity in the liver, and the absence of leptin is associated with a marked attenuation of the hepatic response to a diverse range of fibrotic stimuli
  8. 8. <ul><li>Leptin-deficient ob/ob mice failed to develop hepatic fibrosis in a rodent nutritional model of steatohepatitis and in response to chronic CCl4-induced liver injury </li></ul><ul><li>Restitution of physiologic levels of circulating leptin restored the liver’s “fibrogenic” capacity </li></ul>www.informatics.jax.org The cellular and molecular mechanisms for this effect, however, have not been fully elucidated
  9. 9. The Conflicting Theory <ul><li>One view holds that leptin acts directly on HSCs to trigger downstream response pathways that ultimately lead to ECM deposition </li></ul><ul><li>Others suggest that Kupffer cells (KCs) and/or sinusoidal endothelial cells (SECs) contain a functional leptin receptor, which can stimulate the release of profibrogenic mediators such as transforming growth factor (TGF)-1 that in turn drives HSC activation </li></ul>
  10. 10. <ul><li>KC dysfunction as evidenced by decreased TNF- production and down-regulation of TGF-1 gene expression occurs in leptin receptor-deficient Zucker rats </li></ul>Wikipedia.org
  11. 11. The aim in this article <ul><li>To undertook detailed in vitro experiments to clarify the cellular and molecular mechanisms whereby leptin exerts profibrogenic effects on the liver. </li></ul>
  12. 12. Experimental Design <ul><li>To investigate whether leptin have direct effects on profibrotic gene and αSMA protein expression in HSCs </li></ul>Profibrotic gene(Collagen I, TIMP1, TGFβ1, CTGF/CCN2) To investigate the direct and indirect effects of leptin on the proliferation of HSCs in culture KC-conditioned medium, SEC-conditioned medium To investigate whether leptin have indirect profibrogenic effects on HSCs(via soluble mediators released from KCs)
  13. 13. <ul><li>To investigate g ene and protein expression of TGF- β 1 and CTGF/CCN2 in leptin - induced KCs </li></ul>To investigate whether leptin activates JAK/STAT, MAPK or PI3K/AKT pathways in KCs to target downstream components leading to profibrotic gene transcription
  14. 14. Nonparenchymal Cell (HSC, KC, SEC) Isolation <ul><li>Two-step collagenase perfusion technique (Collagenase B & E) </li></ul>currentprotocols.com Liver Density gradient centrifugation Nylon mesh filters washing Supernatant HSC 500g 7 mins Homogenization KC, SEC nature.com
  15. 15. HSC Comparative Hepatology 2007, 6: 7 Journal of Young Investigators. 2008. Volume 16
  16. 16. SEC wikimedia.org Chinese Medical Journal, 2010, Vol. 123 No. 1 : 68-73
  17. 17. KC Comparative Hepatology 2003, 2: 1 www.siumed.edu
  18. 18. To investigate whether leptin have direct effects on profibrotic gene and αSMA protein expression in HSCs <ul><li>Male Sprague-Dawley rats </li></ul>Fig1A HSCs(48h or 6 days) Total cellular RNA TRI REAGENT RT-PCR Leptin(10 or 100nmol/L, 24h)
  19. 19. Profibrotic gene <ul><li>Type-I collagen The most abundant collagen of the human body </li></ul><ul><li>TIMP metallopeptidase inhibitor 1 The glycoprotein is a natural inhibitor of the matrix metalloproteinases (MMPs), a group of peptidases involved in degradation of the extracellular matrix </li></ul><ul><li>TGFβ-1 A polypeptide member of the transforming growth factor beta superfamily of cytokines. It is a secreted protein that performs many cellular functions, including the control of cell growth, cell proliferation, cell differentiation and apoptosis </li></ul><ul><li>CTGF/CCN2 CTGF mirrors some of the effects of TGF beta on skin fibroblasts, such as stimulation of extracellular matrix production, chemotaxis, proliferation and integrin expression </li></ul>Wikipedia.org
  20. 20. <ul><li>Male Sprague-Dawley rats </li></ul>Fig1B HSCs(48h) Immunoblot Assays Microcon YM-10 Centrifugal Filters Leptin(10 or 100nmol/L, 24h) Culture medium
  21. 21. <ul><li>Male Sprague-Dawley rats </li></ul>Fig1C, 1D HSCs (48h or 6 days) Immunoblot Assays Lysis buffer Leptin(10 or 100nmol/L) And/or TGFβ1(1 or 10ng/mL), 24h Cell lysates
  22. 22. α SMA protein <ul><li>Alpha-Smooth Muscle Actin Actins are highly conserved proteins that are involved in cell motility, structure and integrity. Alpha actins are a major constituent of the contractile apparatus </li></ul>Wikipedia.org images1.clinicaltools.com/
  23. 23. Fig1E Leptin(10 or 100nmol/L) And/or TGFβ1(10ng/mL), 24h HSCs(48h) Total Cellular RNA TRI REAGENT RT-PCR
  24. 24. <ul><li>Leptin Has Minimal Direct Effects on Profibrotic Gene Expression in HSCs In Vitro </li></ul>
  25. 25. To investigate the direct and indirect effects of leptin on the proliferation of HSCs in culture <ul><li>Leptin Enhances Stellate Cell Proliferation </li></ul>Leptin(10 or 100nmol/L) And/or PDGF BB(25ng/mL), 24h HSCs (48h or 6 days) WST-1 Assay Fig2A, 2C
  26. 26. WST-1 assay Cell WST-1, 0.5~4h ELISA Reader www.ub.edu/web/ub/ca/
  27. 27. Whether leptin indirectly affects HSC proliferation Leptin(10 or 100nmol/L) And/or LPS(25ng/mL), 24h KCs, SECs (48h) WST-1 Assay 3 days HSCs 24h Fig2B Coditioned medium 2.3X 3.1X
  28. 28. Fig2C
  29. 29. Fig2D Fig2E Leptin(100nmol/L), 24h KCs(48h) ELISA HSCs, 24h Total Cellular RNA TRI REAGENT RT-PCR Coditioned medium PDGF and its receptors are not the principal mediators of the HSC proliferative effects of KC-conditioned medium
  30. 30. Fig2F HSCs(1,3,7 days) KCs(1,3 days) Total Cellular RNA TRI REAGENT RT-PCR HSCs and KCs Express OB-Rb but Demonstrate Differential Expression Patterns in Culture
  31. 31. To investigate whether leptin have indirect profibrogenic effects on HSCs(via soluble mediators released from KCs) Fig3A Leptin(100nmol/L), 24h KCs, SECs(48h) 3 days HSCs, 24h Total Cellular RNA TRI REAGENT RT-PCR Coditioned medium
  32. 32. Leptin(100nmol/L), 24h KCs, SECs(48h) 3 days HSCs, 24h Fig3B Coditioned medium Sirius Red Staining
  33. 33. Sirius Red Staining ep.physoc.org www.biocolor.co.uk Cells Fixing, 1h Sirius red solution, 1h Washing OD, 450nm Dissolved in 0.1 N NaOH Collagen stains strongly with acid red dyes due to the affinity of the cationic groups of the proteins for the anionic reactive groups of the acid dyes
  34. 34. <ul><li>Zucker rats </li></ul>Fig3C KCs Leptin (100nmol/L, 24h) HSCs, 24h Total Cellular RNA TRI REAGENT RT-PCR This confirmed that the observed effects were specifically because of leptin acting through its receptor Coditioned medium
  35. 35. Zucker rats <ul><li>There are two types of Zucker rat: </li></ul><ul><li>Lean Zucker rat denoted as the dominant trait (Fa/Fa) or (Fa/fa) </li></ul><ul><li>Obese (or fatty) Zucker rat actually a recessive trait (fa/fa) of the leptin receptor, capable of weighing up to 1 kilogram (2.2 lb)—more than twice the average weight </li></ul>www.med.howard.edu
  36. 36. Whether leptin indirectly affects HSC αSMA protein expression Fig3D Supp.Fig2 HSCs Immunoblot Assays Lysis buffer Leptin(100nmol/L) 24h Cell lysates KCs, SECs Coditioned medium
  37. 37. <ul><li>Leptin activates KCs to release soluble profibrogenic mediators that are transferred in the conditioned medium to HSCs. </li></ul>These factors promote the activation, proliferation, and profibrogenic activities of HSCs
  38. 38. To investigate g ene and protein expression of TGF- β 1 and CTGF/CCN2 in leptin - induced KCs Leptin (10 or 100nmol/L) 24h KCs Total Cellular RNA TRI REAGENT RT-PCR Fig4A, 4B Immunoblot Assays Microcon YM-10 Centrifugal Filters Culture medium
  39. 39. Fig4C Leptin(100nmol/L) sTGFβR fusion protein(50μg/mL) Human IgG( 50μg/mL ), 24h KCs antibodies against CTGF Quantitative analysis
  40. 40. Schematic diagram of the hierarchy and interplay between ET-1, TGF-β and CTGF Arthritis Research & Therapy 2007, 9 (Suppl 2) : S4
  41. 41. <ul><li>Leptin Up-Regulates TGF-β1 and CTGF/CCN2 Expression in KCs </li></ul>
  42. 42. <ul><li>Zucker </li></ul><ul><li>rats </li></ul>Fig4D Leptin (100nmol/L) 24h KCs Total Cellular RNA RT-PCR TRI REAGENT These results further corroborate our data that the effects of leptin in inducing TGF- β 1 and CTGF/CCN2 are indeed leptin receptor dependent
  43. 43. Fig4E Leptin (100nmol/L) 24h KCs These data suggest that, at least in vitro, leptin’s profibrogenic effects that are mediated via KCs are not due to the production of H 2 O 2 H 2 O 2 Assay
  44. 44. H 2 O 2 Assay www.ricercaitaliana.it Fluorescence spectrophotometer
  45. 45. To clarify further whether TGF β -1 is indeed one of the soluble mediators of the profibrogenic effects of leptin on KC Fig4F These data confirm that TGF β -1 is likely to be the principal profibrogenic mediator that is released on leptin treatment of KCs Leptin(100nmol/L) TGFβ antibody(10 μ g/mL) KCs Total Cellular RNA RT-PCR TRI REAGENT
  46. 46. To investigate whether leptin activates JAK/STAT, MAPK or PI3K/AKT pathways in KCs to target downstream components leading to profibrotic gene transcription Fig5A, 5B Leptin(100nmol/L) 0, 5, 10, 30, 60mins KCs Immunoblot Assays Lysis buffer Cell lysates
  47. 47. Fig5C Leptin did not increase JNK or p38 phosphorylation
  48. 48. Leptin-related pathway Biochem J. 2006 Jan 1;393(Pt 1):7-20.
  49. 49. Fig5D Leptin(10 or 100nmol/L) 60mins KCs Nuclear Protein Extraction EMSA
  50. 50. AP-1 & NF-κB <ul><li>AP-1 and NF- κ B can be activated by MAPK/ERK1/2 and PI3K/AKT signaling </li></ul><ul><li>AP-1 and/or NF- κ B binding motifs are known to be present in the promoter regions of TGF-β1 and CTGF/CCN2 </li></ul>The Journal of Immunology , 2006, 176: 603-615.
  51. 51. To clarify which of the activated signaling pathways contributes to the observed increase in TGF-β1 gene expression Fig5E Leptin(100nmol/L) PI3K inhibitor(25 μ mol/L) MEK inhibitor(50 μ mol/L) STAT3 inhibitor peptide(50 μ mol/L) KCs Total Cellular RNA RT-PCR TRI REAGENT
  52. 52. Summary
  53. 53. Conclusion <ul><li>Leptin mediates HSC activation and liver fibrosis through indirect effects on KC, these effects are partly mediated by TGF-1 </li></ul>biology.about.com Laboratory Investigation (2008) 88, 96–97
  54. 54. TGF-1 treatment of HSCs did not increase SMA protein expression, whereas leptin-treated KC-conditioned media did <ul><li>This suggests that collagen I, TIMP1, and CTGF/CCN2 are at least, in part, regulated by TGF-1 </li></ul><ul><li>In contrast, SMA may be regulated by another soluble factor, possibly including CTGF/CCN2 </li></ul>Fig3D Fig1C, 1D
  55. 55. It has been previously reported that leptin facilitates HSC proliferation by increased PDGF receptor expression Biochem Biophys Res Commun 2004;323:1091–1095
  56. 56. <ul><li>These data suggest that HSC proliferation by leptin-treated KC medium may be a complex phenomenon and must involve other pro-proliferative factors, the identity of which is unclear </li></ul>Fig2D Fig2E
  57. 57. Thanks for your attentions
  58. 58. Mechanisms of JAK/STAT activation through OB-Rb Biochem J. 2006 Jan 1;393(Pt 1):7-20.
  59. 59. The MAPK pathway in leptin signalling Biochem J. 2006 Jan 1;393(Pt 1):7-20.
  60. 60. The PI3K/PDE3B/cAMP cascade Biochem J. 2006 Jan 1;393(Pt 1):7-20.
  61. 61. Role of phosphotyrosines of OB-Rb in leptin signalling Biochem J. 2006 Jan 1;393(Pt 1):7-20. SOCS: Suppressor of cytokine signaling proteins
  62. 62. Leptin-induced HSC profibrogenic mechanisms induced by Jak activation and OB-Rb phosphorylation FASEB J 2004;18:1612–1614. Wikipedia.org SOCS3
  63. 63. Wikipedia.org
  64. 64. END
  65. 65. Elutriation nature.com
  66. 66. To exclude the possibility that effect above was due to endotoxin contamination of the recombinant leptin protein Supp.Fig1 LPS(0.16ng/mL), 24h KCs(48h) 3 days HSCs, 24h Total Cellular RNA TRI REAGENT RT-PCR Coditioned medium
  67. 67. Proposed model for the participation of SOCS3 in leptin resistance Biochem J. 2006 Jan 1;393(Pt 1):7-20.

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