Kupffer Cells Mediate Leptin-Induced Liver Fibrosis GASTROENTEROLOGY 2009;137:713–723 JIANHUA WANG,* ISABELLE LECLERCQ,‡ J...
Liver Fibrosis <ul><li>KCs and SECs play important roles in modulating stellate cell behavior </li></ul>J Clin Invest.  20...
Kupffer cells(KCs) <ul><li>Specialized macrophages located in the liver </li></ul><ul><li>Kupffer cells activation are res...
Pathogenesis of liver fibrosis Comparative Hepatology  2007  6 :7
J Clin Invest.  2005 February 1; 115(2): 209–218.
Circulating levels of leptin are known to be increased in overweight and obese persons, in individuals with nonalcoholic s...
Leptin( 瘦素 ) <ul><li>16kD adipocyte-derived hormone </li></ul><ul><li>Ob(Lep)  gene (Ob for obese, Lep for leptin) is loca...
<ul><li>Leptin-deficient ob/ob mice failed to develop hepatic fibrosis in a rodent nutritional model of steatohepatitis an...
The Conflicting Theory <ul><li>One view holds that leptin acts directly on HSCs to trigger downstream response pathways th...
<ul><li>KC dysfunction as evidenced by decreased TNF- production and down-regulation of TGF-1 gene expression occurs in le...
The aim in this article <ul><li>To undertook detailed in vitro experiments to clarify the cellular and molecular mechanism...
Experimental Design <ul><li>To investigate whether leptin have direct effects on profibrotic gene and αSMA protein express...
<ul><li>To investigate g ene and protein expression   of TGF- β 1 and CTGF/CCN2 in leptin - induced   KCs </li></ul>To inv...
Nonparenchymal Cell (HSC, KC, SEC) Isolation <ul><li>Two-step collagenase perfusion technique (Collagenase B & E) </li></u...
HSC Comparative Hepatology  2007,  6: 7 Journal of Young Investigators. 2008. Volume 16
SEC wikimedia.org Chinese Medical Journal, 2010, Vol. 123 No. 1 : 68-73
KC Comparative Hepatology  2003,  2: 1 www.siumed.edu
To  investigate  whether leptin have direct effects on profibrotic gene and αSMA protein expression in HSCs <ul><li>Male S...
Profibrotic gene <ul><li>Type-I collagen The most abundant collagen of the human body </li></ul><ul><li>TIMP metallopeptid...
<ul><li>Male Sprague-Dawley rats </li></ul>Fig1B HSCs(48h) Immunoblot Assays Microcon YM-10 Centrifugal Filters Leptin(10 ...
<ul><li>Male Sprague-Dawley rats </li></ul>Fig1C, 1D HSCs (48h or 6 days) Immunoblot Assays Lysis buffer Leptin(10 or 100n...
α SMA protein <ul><li>Alpha-Smooth Muscle Actin Actins are highly conserved proteins that are involved in cell motility, s...
Fig1E Leptin(10 or 100nmol/L) And/or TGFβ1(10ng/mL), 24h HSCs(48h) Total Cellular RNA TRI REAGENT RT-PCR
<ul><li>Leptin Has Minimal Direct Effects on Profibrotic Gene Expression in HSCs In Vitro </li></ul>
To investigate the direct and indirect effects of leptin on the proliferation of HSCs in culture <ul><li>Leptin Enhances S...
WST-1 assay Cell WST-1, 0.5~4h ELISA Reader www.ub.edu/web/ub/ca/
Whether leptin indirectly affects HSC proliferation Leptin(10 or 100nmol/L) And/or LPS(25ng/mL), 24h KCs, SECs (48h) WST-1...
Fig2C
Fig2D Fig2E Leptin(100nmol/L), 24h KCs(48h) ELISA HSCs, 24h Total Cellular RNA TRI REAGENT RT-PCR Coditioned medium PDGF a...
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 Diff...
To investigate whether leptin have indirect profibrogenic effects on HSCs(via soluble mediators released from KCs) Fig3A L...
Leptin(100nmol/L), 24h KCs, SECs(48h) 3 days HSCs, 24h Fig3B Coditioned medium Sirius Red Staining
Sirius Red Staining ep.physoc.org www.biocolor.co.uk Cells Fixing, 1h Sirius red solution, 1h   Washing OD, 450nm Dissolve...
<ul><li>Zucker rats </li></ul>Fig3C KCs Leptin (100nmol/L, 24h) HSCs, 24h Total Cellular RNA TRI REAGENT RT-PCR This confi...
Zucker rats <ul><li>There are two types of Zucker rat:  </li></ul><ul><li>Lean Zucker rat denoted as the dominant trait (F...
Whether leptin indirectly affects HSC αSMA protein expression Fig3D Supp.Fig2 HSCs Immunoblot Assays Lysis buffer Leptin(1...
<ul><li>Leptin activates KCs to release soluble profibrogenic mediators that are transferred in the conditioned medium to ...
To investigate g ene and protein expression   of TGF- β 1 and CTGF/CCN2 in leptin -  induced   KCs Leptin (10 or 100nmol/L...
Fig4C Leptin(100nmol/L) sTGFβR fusion protein(50μg/mL) Human IgG( 50μg/mL ), 24h KCs antibodies against CTGF  Quantitative...
Schematic diagram of the hierarchy and interplay between ET-1, TGF-β and CTGF Arthritis Research & Therapy  2007,  9 (Supp...
<ul><li>Leptin Up-Regulates TGF-β1 and CTGF/CCN2 Expression in KCs </li></ul>
<ul><li>Zucker </li></ul><ul><li>rats </li></ul>Fig4D Leptin (100nmol/L) 24h KCs Total Cellular RNA RT-PCR TRI REAGENT The...
Fig4E Leptin (100nmol/L) 24h KCs These data suggest that, at least in vitro, leptin’s profibrogenic effects that are media...
H 2 O 2  Assay www.ricercaitaliana.it Fluorescence spectrophotometer
To clarify further whether TGF β -1 is indeed one of the soluble mediators of the profibrogenic effects of leptin on KC Fi...
To investigate whether leptin activates JAK/STAT, MAPK or PI3K/AKT pathways in KCs to target downstream components leading...
Fig5C Leptin did not increase JNK or p38 phosphorylation
Leptin-related pathway Biochem J. 2006 Jan 1;393(Pt 1):7-20.
Fig5D Leptin(10 or 100nmol/L) 60mins KCs Nuclear Protein Extraction EMSA
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...
To clarify which of the activated signaling pathways contributes to the observed increase in TGF-β1 gene expression Fig5E ...
Summary
Conclusion <ul><li>Leptin mediates HSC activation and liver fibrosis through indirect effects on KC, these effects are par...
TGF-1 treatment of HSCs did not increase SMA protein expression, whereas leptin-treated KC-conditioned media did <ul><li>T...
It has been previously reported that leptin facilitates HSC proliferation by increased PDGF receptor expression Biochem Bi...
<ul><li>These data suggest that HSC proliferation by leptin-treated KC medium may be a complex phenomenon and must involve...
Thanks for your attentions
Mechanisms of JAK/STAT activation through OB-Rb   Biochem J. 2006 Jan 1;393(Pt 1):7-20.
The MAPK pathway in leptin signalling Biochem J. 2006 Jan 1;393(Pt 1):7-20.
The  PI3K/PDE3B/cAMP  cascade   Biochem J. 2006 Jan 1;393(Pt 1):7-20.
Role of phosphotyrosines of OB-Rb in leptin signalling Biochem J. 2006 Jan 1;393(Pt 1):7-20.  SOCS: Suppressor of cytokine...
Leptin-induced HSC profibrogenic mechanisms induced by Jak activation and OB-Rb phosphorylation FASEB J 2004;18:1612–1614....
Wikipedia.org
END
Elutriation nature.com
To exclude the possibility that effect above was due to endotoxin contamination of the recombinant leptin protein Supp.Fig...
Proposed model for the participation of SOCS3 in leptin resistance Biochem J. 2006 Jan 1;393(Pt 1):7-20.
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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。

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  • Changes in the hepatic architecture ( A ) associated with advanced hepatic fibrosis ( B ). Following chronic liver injury, inflammatory lymphocytes infiltrate the hepatic parenchyma. Some hepatocytes undergo apoptosis, and Kupffer cells activate, releasing fibrogenic mediators. HSCs proliferate and undergo a dramatic phenotypical activation, secreting large amounts of extracellular matrix proteins. Sinusoidal endothelial cells lose their fenestrations, and the tonic contraction of HSCs causes increased resistance to blood flow in the hepatic sinusoid.
  • 肝竇為一特化的血管性構造,位於肝動脈與門靜脈末端、匯流入肝靜脈之前,所以腸道血液流入肝臟後,必須經過肝竇才能進到中央靜脈。此系統為單層的竇狀內皮細胞 (sinusoidal endothelial cell) 所覆蓋,形成狹小的管道空間(約 6~15 微米),和淋巴球大小相當( 7~12 微米),所以克布霍細胞 (Kupffer cell) 、 B 淋巴球細胞及 T 淋巴球細胞等免疫細胞就可以在此進行把關工作,把細菌、內毒素等加以攔截與排除。
  • The &amp;quot;canonical principle&amp;quot; of fibrogenesis starts with necrosis or apoptosis of hepatocytes and inflammation-connected activation of hepatic stellate cells (HSC triggering), their transdifferentiation to myofibroblasts with enhanced expression and secretion of extracellular matrix and matrix deposition (fibrosis). The latter is a precondition for cirrhosis. New pathogenetic mechanisms concern the influx of bone marrow-derived cells (fibrocytes) and of circulating monocytes and their TGF-β driven differentiation to fibroblasts in the damaged liver tissue. A further new mechanism is epithelial-mesenchymal transition (EMT) of bile duct epithelial cells and potentially of hepatocytes. All three complementary mechanisms enlarge the pool of matrix-synthesizing (myo-)fibroblasts in the damaged liver. The most important fibrogenic mediators are transforming growth factor (TGF)-β, platelet-derived growth factor (PDGF), insulin-like growth factor 1 (IGF-1), endothelin-1 (ET-1), and reactive oxygen species (ROS including hydroxyl radicals, superoxid anions). Abbreviations: ASH – alcoholic steatohepatitis; NAFLD – non-alcoholic fatty liver disease. Inset shows an electron micrograph of HSC with numerous lipid droplets indenting the nucleus.
  • Cellular mechanisms of liver fibrosis. Different types of hepatotoxic agents produce mediators that induce inflammatory actions in hepatic cell types. Damaged hepatocytes and biliary cells release inflammatory cytokines and soluble factors that activate Kupffer cells and stimulate the recruitment of activated T cells. This inflammatory milieu stimulates the activation of resident HSCs into fibrogenic myofibroblasts. Activated HSCs also secrete cytokines that perpetuate their activated state. If the liver injury persists, accumulation of activated HSCs and portal myofibroblasts occurs, synthesizing large amounts of ECM proteins and leading to tissue fibrosis. ECM degradation is inhibited by the actions of cytokines such as TIMPs. Apoptosis of damaged hepatocytes stimulates the fibrogenic actions of HSCs. If the cause of the liver injury is removed, fibrosis is resolved. This phase includes apoptosis of activated HSCs and regeneration of hepatocytes. Collagen is degraded by increased activity of MMPs induced by decreased TIMP expression. CCL21, C-C chemokine ligand 21; MCP-1, monocyte chemoattractant protein–1; MIP-2, macrophage inflammatory protein–2; NS3, HCV nonstructural protein 3; NS5, HCV nonstructural protein 5; PAF, platelet-activating factor.
  • There was a strong positive correlation (r = 0.85, P&lt;0.001) between the serum leptin concentration and the percentage of body fat Correlation between serum leptin concentrations and percentage hepatocyte steatosis( 脂肪變性 ) in chronic hepatitis C patients segregated with respect to genotype
  • There are two types of Zucker rat: a lean Zucker rat, denoted as the dominant trait (Fa/Fa) or (Fa/fa); and the characteristically obese (or fatty) Zucker rat, which is 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
  • Animal preparation and operation procedure. (A) Orientation and securing of the animal to the workstation. Recommended incision lines and intraperitoneal injection points are indicated. (B) Opened abdomen after median and horizontal incisions. (C) Loosely tied threads around the inferior vena cava 下腔靜脈 (IVC) and portal vein. (D) Perfused liver after cutting the IVC and the abdominal artery and opening the thorax. The catheter is placed in the portal vein, fixed with a thread, and the IVC is ligated.
  • A hepatic stellate cell activated by the p75 neurotrophin receptor promotes repair in the liver. Image courtesy of USCD.
  • Immunohistochemistry staining shows the expression and localization of Bcl-2 and Bax. Both Bcl-2 and Bax protein were expressed in SECs (arrows) at the periphery of the sinusoidal space. The expression of Bcl-2 was higher in group 2 ( B ) after 1 hour of reperfusion compared with the same time point in group 1 ( A ). The expression of Bax was lower in group 2 ( D ) after 1 hour of reperfusion compared with the same time point in group 1 ( C ) (original magnification ×400).
  • This specimen comes from an animal which was injected intravenously with a suspension of carbon particles.  These particles are scavenged by macrophages , most notably by those in the liver which are called Kupffer cells ., whose cytoplasm becomes packed with black carbon particles. In the absence of such experimental demonstration, Kupffer cells can be recognized by their oval nuclei closely associated with sinusoidal spaces. Endothelial cells appear similar, but with thinner (flatter) and denser nuclei and with less conspicuous cytoplasm. In contrast, the hepatocytes which comprise the hepatic cords have round nuclei surrounded by abundant cytoplasm.   The space of Disse is visible here and there on this image, appearing as a thin bright band between hepatocyte cytoplasm and the thinner, darker band which represents the endothelium.
  • 在肝纖維化過程中,肝星狀細胞 (hepatic stellate cell HSC ) 被活化常是重要關鍵;而轉化生長因子 -β1 (transforming growth factor-β1 TGF-β1 ) 的分泌會促使 HSC 轉型為肌纖維母細胞 (myofibroblast) ,並刺激細胞外間質 (extracellular matrix ECM) 的合成和抑制其降解,如此可使肝臟內增加 ? 多纖維組織。 connective tissue growth factor is a cysteine -rich, matrix -associated, heparin -binding protein . In vitro , CTGF mirrors some of the effects of TGF beta on skin fibroblasts , such as stimulation of extracellular matrix production, chemotaxis , proliferation and integrin expression. CTGF can promote endothelial cell growth, migration, adhesion and survival and is thus implicated in endothelial cell function and angiogenesis [1] .
  • αSMA protein was unaltered following leptin treatment Leptin and TGFβ-1 alone or the combination of leptin and TGFβ-1 did not enhance αSMA protein expression
  • 一個主要組成部分的收縮設備
  • TGFβ-1 treatment was associated with up-regulation of collagen I and TIMP1 mRNA expression Coadministration of TGF-β1 with leptin in HSCs failed to have any synergistic effects on profibrotic gene expression
  • platelet-derived growth factor ( PDGF ) is one of the numerous growth factors , or proteins that regulate cell growth and division . In particular, it plays a significant role in blood vessel formation (angiogenesis), the growth of blood vessels from already existing blood vessel tissue. Uncontrolled angiogenesis is a characteristic of cancer. Chemically, platelet-derived growth factor is dimeric glycoprotein composed of two A (-AA) or two B (-BB) chains or a combination of the two (-AB).
  • Fig. 1: Cleavage of the tetrazolium salt WST-1 (4-[3-(4-Iodophenyl)- 2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) to formazan. (EC = electron coupling reagent! RS = mitochondrial succinate-tetrazolium-reductase system)
  • KC-conditioned medium increased HSC proliferation by 2.3-fold, whereas pretreatment of KC with leptin enhanced HSC proliferation 3.1-fold SEC-conditioned medium did not affect HSC proliferation Low-dose lipopolysaccharide (LPS) to activate KCs enhanced the proliferative effects of KC-conditioned medium on HSCs but, together with leptin, had no additional effect on proliferation Lipopolysaccharides ( LPS ), also known as lipoglycans, are large molecules consisting of a lipid and a polysaccharide joined by a covalent bond ; they are found in the outer membrane of Gram-negative bacteria, act as endotoxins and elicit strong immune responses in animals
  • Similar results were obtained when KC-conditioned medium was applied on activated HSCs
  • PDGF is known to be the most potent mitogen for HSCs, we assessed whether leptin facilitates PDGF-induced HSC proliferation
  • The expression of all profibrogenic genes was elevated at least 2-fold in HSCs incubated with KC- but not with SEC-conditioned medium
  • Consistent with Fig3A, collagen protein was also augmented
  • Cell layers were fixed for 1 h in Bouin’s fixative followed by addition of Sirius red F3BA solution (0.1% in saturated picric acid). After 1-h staining, cell layers were washed in running tap water and again in 0.01 N HCl to remove the non-bound dye. For quantification of collagen content, the dye was dissolved in 0.1 N NaOH. The absorbance was determined at 550 nm.
  • Leptin-treated WT KC-conditioned medium significantly increased αSMA protein expression in HSC SEC-conditioned medium, however, did not result in increased αSMA expression
  • TGF-1 and CTGF/CCN2 are major profibrogenic cytokines in the development of liver fibrosis Leptin treatment significantly up-regulated TGF-β1 and CTGF/CCN2 mRNA expression in KCs, also TGF-β1 protein expression
  • CTGF/CCN2 protein was also expressed at higher levels in leptin-treated KCs than in control Coexposure of KCs to leptin and sTGFβR significantly attenuated CTGF/CCN2 protein expression induced by leptin
  • Schematic diagram of the hierarchy and interplay between ET-1, TGF-β and CTGF. CTGF, connective tissue growth factor; ET, endothelin; NF-κB, nuclear factor-κB; TGF, transforming growth factor.
  • H2O2 is a well-recognized profibrogenic factor
  • Production of cellular ROS was analyzed by measuring the intracellular deacylation and oxidation of 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) to the fluorescent compound 2,7-dichloroflurescein (DCF). DCFH-DA is highly reactive with hydrogen peroxide.
  • Exposure of KCs to leptin resulted in increased activation of ERK1/2 and AKT after 5 and 10 minutes incubation, respectively, and in a time-dependent manner
  • Leptin, in a dose-dependent fashion, increased AP-1 and NF-κB DNA binding abilities in KCs We speculated that leptin may also activate downstream transcription factors such as AP-1 and/or NF-B because these transcription factors can be activated by MAPK/ERK1/2 and PI3K/AKT signaling,28,29 and AP-1 and/or NF-B binding motifs are known to be present in the promoter regions of TGF-1 and CTGF/CCN2
  • Schematic illustration of exon 1 of the human TGF- 1 gene, in which the TGF- 1 promoter and the 5&apos; end of the CDS are included. The locations of the B3 and AP-1-3 sites and the two major transcription initiation sites (P1 and P2) described by Kim et al. (15 ) are indicated. The DNA segments amplified by PCR primer pairs TP1 (between P1 and P2) and TP2 (in CDS) are also shown. The numbers indicate nucleotide positions in relation to the translation start site
  • STAT3 inhibitor but not the MEK(PD98059) or PI3K (LY294002) inhibitors resulted in a significant reduction of TGF-β1 mRNA expression in KCs
  • Fig 1.Effect of leptin on BrdU labeling in isolated HSCs. Isolated rat HSCs were cultured for 3 days as described detail in Materials and methods. Cells were incubated with PDGF-BB (5 ng/ml) and leptin (10–100 nM) in the presence of 10% FBS for 6–8 h. BrdU was added to the culture 1 h prior to fixation, and incorporation of BrdU into nuclei was detected by immunocytochemical staining. Percentages of BrdU-positive cells in two different time-points (A; black bars: controls, light gray bars: PDGF alone, dark gray bars: PDGF and 100 nM leptin), and different doses of leptin at 8 h (B) are plotted ( n  = 7, * P  &lt; 0.05 vs. controls, # P  &lt; 0.05 vs. PDGF alone). Fig. 2. Effect of leptin on expression of PDGF-R subunits in isolated HSCs. Total RNA was prepared from 3-day cultured HSCs after incubation with leptin (100 nM) for 3–6 h, and expression of PDGF-Rα and β subunit mRNA was detected by RT-PCR. PCR products were separated on 1.5% agarose gels, and specific bands were visualized by ethidium bromide staining. Representative photographs of specific bands (A, PDGF-Rα: 398 bp, PDGF-Rβ: 352 bp, β-actin: 281 bp) and densitometrical data (B,C) are shown ( n  = 4, * P  &lt; 0.05 vs. controls). Similarly, whole cell protein extracts were prepared from leptin-treated HSCs, and PDGF-Rα and β protein levels were analyzed by Western blotting. Representative photographs of specific bands from 4 separate experiments are shown (D).
  • Upon leptin (L) binding, a conformational change takes place ( A ) that allows juxtaposition of JAKs, which then become activated and are able to tyrosine-phosphorylate other JAKs and tyrosine residues on the receptor ( B ). Activation of JAK2 occurs by transphosphorylation and subsequent phosphorylation of tyrosine residues in the cytoplasmic region of the receptor. Phosphorylation of Tyr1138 allows association of STATs, which then become substrates of receptor-associated JAKs. Phosphorylation of STATs leads to their dissociation from the receptor and the formation of active dimers ( C ), which translocate to the nucleus to regulate gene expression, binding to the promoter regions of target genes ( D ).
  • The ERK members of the MAPK family are components of the well-defined Ras/Raf/MAPK signalling cascade and have become activated by leptin (L). For more detailed information, see the text. DAG, diacylglycerol; Grb-2, growth factor receptor binding-2; PI3, PtdIns(3,4,5) P 3; PLC, phospholipase C; SOS, son of sevenless. In many cell types, activation of this pathway promotes cell division
  • Stimulation of the PI3K pathway by leptin (L) represents a key cascade to exert several different effects of the hormone at multiple sites. For more detailed information, see the text. C/EBP, CCAAT/enhancer-binding protein; eNOS, endothelial nitric oxide synthase; GSK3, glycogen synthase kinase 3.
  • JAK2 associates with the receptor via the box1 motif. The long isoform leptin (L) receptor (OB-Rb) contains four important tyrosine residues (Tyr974, Tyr985, Tyr1077 and Tyr1138). These phosphorylated tyrosine residues provide docking sites for signalling proteins with SH2 domains. Most importantly, Tyr1138 recruits the transcription factor STAT3, which is subsequently phosphorylated by JAK2, dimerizes and translocates to the nucleus, where it induces SOCS3 and POMC (pro-opiomelanocortin) expression, while repressing AgRP (agouti-related peptide). SOCS proteins inhibit signalling by binding to phosphorylated JAK proteins or interacting directly with tyrosine-phosphorylated receptors. The ability of SOCS3 to inhibit leptin-stimulated phosphorylation of JAK2 and ERK provides a negative-feedback mechanism on the leptin signalling system. Grb-2, growth factor receptor binding-2.
  • Leptin signals as any gp130 cytokine primarily by Jak2 phosphorylation of several key tyrosine residues. OB-Rb phosphorylation promotes HSC proliferation and survival by Erk and Akt activation. SOCS-3, a feedback inhibitor of OB-Rb signaling, and AG490, a chemical inhibitor of Jak2 kinase activity, both block HSC proliferation and survival by blocking phosphorylation of Erk or Akt. HSC survival was inhibited by PI3-kinase inhibitor LY294002. HSC proliferation was blocked by MAPK inhibitor PD98059, PI3-kinase inhibitor LY294002, as well as pharmacologic and biologic OB-Rb signal blockade. Leptin could not suppress apoptosis in the presence of LY, indicating that Akt is highly protective against HSC apoptosis.
  • The centrifugal and the drag forces are acting in opposite directions. ( a ) Unfractionated cells enter the elutriation chamber. ( b ) Size gradient balanced by centrifugal force and counterflow (drag force) of elutriation fluid keeps cells inside the chamber. ( c ) Increasing flow rate elutes smaller cells first followed by larger ones. The centrifuge is run at 20 °C and constant speed (2,200 r.p.m., 683 g ). The pump is turned on and the system is washed first with 100 ml of 70% ethanol, then with 200 ml of physiological buffer (saline or PBS) to remove traces of alcohol. The physiological buffer is then replaced by freshly made elutriation fluid at an initial flow rate used for the introduction of cells into the chamber. Approximately 100 ml of the elutriation fluid is collected separately and discarded. The further, but still preliminary, run serves to remove bubbles from the system. Bubbles tend to be left behind in the elutriation chamber, in the sample-mixing chamber, in the tubing and inside the manometer. The Beckman loading chamber, which also serves as sample mixer, is used in the bypass position to trap bubbles and to compensate for pump pulsation. The bypass valve helps to remove bubbles from the sample-mixer chamber.
  • During prolonged receptor stimulation by leptin (L), the inhibition of JAK2 and ERK phosphorylation is mediated by SOCS3 independently of Tyr985 of OB-Rb.
  • 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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