Work In Progress 03.17.11 Final Edit

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  • Also a cause of insulin resistance and autoimmune disease. The hepatitis C virus is the major cause of the autoimmune disorder mixed cryoglobulinemia. which is an immune complex-mediated form of systemic vasculitis that primarily affects small blood vessels.
  • HCV is a noncytopathic hepatotropic virus. When infected there is an acute hepatitis which spontaneously clears in 10-20% of the patients. In 80-90% of HCV infections Chronic Hepatitis occurs and in one third of these cases chronic cirrhosis occurs of these cases 1-4% progress to hepatocellular carcinoma
  • How HCV Binds and enters into hepatocytes is not well understood, nor are the receptors. There is currently no specific receptor identified for HCV but Based on these putative receptors the current model is… HCV associates with lipoproteins in circulation, this aides in binding to the low density lipoprotein receptor. The lipoprotein associated viron next binds to Scavenger receptor B1 and glycoprotein CD81 as well as Claudin 1 and occludin located at the tight junctions of the cell. Endocytosis occurs probably via a clathrin dependent mechanism and eventually fuses with the endosome which allows for genome release. LDLR – Low density lipoprotein receptor SR-B1 - Scavenger receptor class B type 1 CD-81 – Glycoprotein shown to complex with integrins CLDN-1 – Claudin 1 integral membrane protein and component of tight junction strands Occludin – Tight Junction protein, Are Any of these Required? Not required
  • This is the HCV genome. HCV contains a 5 and 3’ non translated region, the IRES sits at the 5’ NTR and promotes translation of the polyprotein. HCV is translated as a single polyprotein and is cleaved by both host cell and viral proteases into at least 10 proteins. Among these, are the Nonstructurals which make up the replication complex which assembles at the 3’ NTR and replicates the viral genome. NS3 is an important serine protease of which NS4a is a cofactor. NS4b is important for the alteration of the membranous web which is a key component of the replication complex of hcv as well as other + strand rna viruses. NS5a is a phosphoprotein and it also plays a role in IFN resistance. NS5b is the RNA dependent RNA polymerase Core – forms the nucleocapsid, regulation of host and viral genes affects host functions such as gene transcription, lipid metabolism and apoptosis, ox stress, induces ros thru mito E1/E2 – Envelope glycoproteins form heterodimers which form the viral envelope P7 – located at the junction between the structural and nonstructural region reported to form hexamers with ion channel activity not essential for replication NS2/3 – autoprotease consisting of NS2 and N-terminal third of NS3 NS3 – N-terminal third is a serine protease C-terminal two-thirds is an NTPase/RNA helicase has been shown to inhibit RIG-1 and TLR3 signaling required for replication, NS3 induction of ROS and oxidative stress NS4A – Required for proper serine protease activity of NS3 NS4B – shown to induce a specific membrane alteration, membranous web, serves as a scaffold for the formation of the viral replication complex NS5A – shown that NS5A directly interacted with dsRNA dependent kinase PKR and inactivate it thereby modulating the IFN stimulated antiviral response. Also shown previously by dr. waris to induce oxidative stress and activate stat-3 and nf-kb NS5B – RNA dependent RNA polymerase
  • There is currently a lack of a good model system for HCV, but these are the current tools at our disposal. Our lab focuses on the transient expression of individual HCV viral proteins, as well as the subgenomic replicon system and the infectious cell culture system Chimpanzees can be infected but they spontaneously clear the virus and don’t develop any pathogenesis thereby limiting their efficacy as a model system.
  • Mention lipid accumulation and fibrosis between cirrhosis and hcc as well as regenerative nodules for cirrhotic liver also talk about what steatosis is, fatty liver, accumulation of lipid droplets, dylan works on this. I work on fibrosis HCC - The usual lobular architecture is replaced by irregular and thickened trabeculae of malignant hepatocytes. There is mild nuclear pleomorphism
  • Kwee escent In the normal liver HSC are localized within the space of Disse and Kupffer cells are attached to the sinusoidal wall. Upon insult, Kupffer cells become activated and produce fibrogenic mediators. hepatocytes become apoptotic and release TGFB and other fibrogenic mediators these activate HSC’s which transdifferentiate into myofibroblast like cells, migrate and accumulate at the site of tissue repair and synthesize ECM components like collagen type I and III. Additionally inflammatory lymphocytes infiltrate the hepatic parenchyma. With the loss of the fenestration character of the sinusoidal endothelial cells the resistance of the blood flow increases and liver function decreases.
  • In our model, HCV infects hepatocytes causing an increase in tgfb expression and secretion. Tgf-b can then bind to receptors on hepatocytes creating an autocrine loop or it can bind to receptors on hepatic stellate cells, activating them and causing them to increase expression of profibrogenic genes such as…..
  • Think sandwich ELISA, primary I cocktail of biotin Ab, secondary is stretavidin Densitometric analysis of the cytokine array blot was performed to determine the difference in the release of human cytokines from serum starved Huh-7 and HCV-infected cells at 72 hr. The values were normalized to identical background levels using the Ray Bio Human Cytokines antibody array V analysis tool. Vascular endothelial growth factor ( VEGF ) is a chemical signal produced by cells that stimulates the growth of new blood vessels. It is part of the system that restores the oxygen supply to tissues when blood circulation is inadequate. Epidermal growth factor or EGF is a growth factor that plays an important role in the regulation of cell growth , proliferation , and differentiation by binding to its receptor EGFR . Tumor necrosis factor ( TNF , cachexin or cachectin is a cytokine involved in systemic inflammation and is a member of a group of cytokines that stimulate the acute phase reaction . The primary role of TNF is in the regulation of immune cells . TNF is also able to induce apoptotic cell death, to induce inflammation , and to inhibit tumorigenesis and viral replication . Angiogenin (Ang) is a small polypeptide that is implicated in angiogenesis (formation of new blood vessels) in tumor growth . However, angiogenin is unique among the many proteins that are involved in angiogenesis in that it is also an enzyme with an amino acid sequence 33% identical to that of bovine pancreatic ribonuclease (RNase) A).
  • Mock-infected and HCV-infected cells (day 4) were grown to ~70% confluence and washed twice with cold 1x PBS, followed by culture in DMEM containing 0.2% FBS for 24 hr. Cell culture conditioned medium was collected, centrifuged at 1,000 rpm for 10 min to remove cell debris. Total secreted TGF-β1 protein in cell culture conditioned medium at day 4 after HCV infection was determined by enzyme-linked immunosorbent assay (ELISA) according to manufacturer’s protocol (Promega). For activation 10 μl of 1N HCl was added to 500 μl of cell culture supernatant, mixed, and incubated for 15 min at room temperature. The samples were neutralized by adding 10 μl of 1N NaOH, mixed, and used for the sandwich TGF-β1 ELISA. A standard curve was constructed by using serial dilutions of human recombinant TGF-β1. TGF-β1 levels were measured in triplicate determinants.
  • Take the top one, #4 wt and crop the others out
  • NS3 cannot do it alone due to lack of 4a cofactor but NS3/4a together has an active protease domain can activate the arginine deletion has little effect due to it being outside the protease domain, but the point mutation S to A has an effect cause it causes a dead protease region NS3 can partially cleave substrates alone, but the mutants cannot Think of this in terms of viral infection, NS3/4a can prevent the innate immune response (IFN-B) but NS3, NS4A, and NS3/4a mutants cannot prevent the IFN-B response
  • Add IFA data
  • Er signaling retention N terminus When N terminus is deleted from the NS5A construct it does not signal to the ER but instead the nucleus (Shown in PNAS paper by GW).
  • It is well documented that HCV gene expression in the context of subgenomic replicon induces ROS via Calcium signaling and with this previous work as shown by Dr. Waris and others, we wanted to look at how HCV Infection induces ROS via Ca2+ signaling and its effect of the regulation of TGF-B To initiate this study…
  • What ROS species does CM-H2DCFDA dye stain? Hydrogen peroxide, hydroxyl radical, peroxyl radical, peroxynitrite, Not, singlet oxygen, superoxide anion
  • 3B. (1,2- b is(o- a mino p henoxy ) ethane -N,N,N',N'- t etra a cetic acid ) DPI is a NADPH oxidase/NOS inhibitor (this is the pentose phosphate pathway) It reduces glutathione via glutathione reductase , which converts reactive H2O2 into H2O by glutathione peroxidase . If absent, the H2O2 would be converted to hydroxyl free radicals by Fenton chemistry , which can attack the cell Pyrrolidine dithiocarbamate (PDTC) is a chemical compound with the molecular formula C5H9NS2. PDTC and its salts , such as ammonium pyrrolidinecarbodithioate, are used for a variety of biochemical applications including metal chelation , induction of G1 phase cell cycle arrest, [1] and preventing induction of
  • DPI is a NADPH oxidase/NOS inhibitor (this is the pentose phosphate pathway) It reduces glutathione via glutathione reductase, which converts reactive H2O2 into H2O by glutathione peroxidase. If absent, the H2O2 would be converted to hydroxyl free radicals by Fenton chemistry, which can attack the cell Pyrrolidine dithiocarbamate (PDTC) is a chemical compound with the molecular formula C5H9NS2. PDTC and its salts, such as ammonium pyrrolidinecarbodithioate, are used for a variety of biochemical applications including metal chelation, induction of G1 phase cell cycle arrest,[1] and preventing induction of
  • V Christen paper shows induction of CREB
  • SB - A inhibitor of p38 MAP kinase. Does not significantly inhibit the JNK and p42 MAP kinase. SP - A inhibitor of c -Jun N-terminal kinase (JNK). Exhibits over 300-fold greater selectivity for JNK as compared to ERK1 and p38-2 MAP kinases. Inhibits the phosphorylation of c -Jun and blocks cellular expression of IL-2, IFN-γ, TNF-α, and COX-2. Blocks IL-1-induced accumulation of phospho-Jun and induction of c -Jun transcription. LY – A phosphatidylinositol 3-kinase inhibitor that acts on the ATP-binding site of the enzyme. SU - A Src family kinase inhibitor. Inhibits Src as well as closely related kinases such as Fyn and Yes and Lyn . TAN - Tanshinone IIA is a diterpenoid naphthoquinone found in the traditional Chinese medicine Tanshen. It inhibits AP-1 activity by suppressing jun-fos-DNA complex formation. AG - A inhibitor of epidermal growth factor receptor kinase autophosphorylation. Inhibition of JAK2 by AG 490. Inhibits the constitutive activation of STAT-3 DNA binding. AG 490 has also been shown to inhibit the autokinase activity of JAK3. BAY - inhibits the TNF-a-inducible phosphorylation of IkBa resulting in a decreased expression of NF-kB and of adhesion molecules. Does not affect constitutive IkBa autophosphorylation. NFkB – potent inhibitor of NFkB transcriptional activation UO - A inhibitor of MEK1 and MEK2.
  • Model illustrating The HCV-induced activation of TGF-b via Ca2+ signaling and oxidative stress. ER overload response causes the release of Ca2+ from the ER. The uptake of Ca2+ in the mitochondria induces oxidative stress which generates ROS. ROS then activate TGF-b and regulate the expression of profibrogenic genes.
  • Figure 4A
  • enzyme which belongs to the subtilisin -like proprotein convertase family. calcium-dependent serine endoprotease that can efficiently cleave precursor proteins at their paired basic amino acid processing sites. Some of its substrates are: pro parathyroid hormone , transforming growth factor beta 1 precursor, pro albumin , pro- beta-secretase , membrane type-1 matrix metalloproteinase , beta subunit of pro- nerve growth factor and von Willebrand factor .
  • subunit of a disulfide-linked homotrimeric protein. This protein is an adhesive glycoprotein that mediates cell-to-cell and cell-to-matrix interactions. This protein can bind to fibrinogen , fibronectin , laminin , type V collagen and integrins alpha-V/beta-1. This protein has been shown to play roles in platelet aggregation, angiogenesis , and tumorigenesis . [3
  • Remove percent inhibiton…write relative mrna levels
  • Explain system
  • Work In Progress 03.17.11 Final Edit

    1. 1. Mechanisms of Liver Fibrosis by Hepatitis C Virus Lance D. Presser Department of Microbiology and Immunology H.M. Bligh Cancer Research Laboratories School of Graduate and Postdoctoral Studies Rosalind Franklin University of Medicine and Science
    2. 2. Outline <ul><li>Hepatitis C Virus Background </li></ul><ul><li>Liver Fibrosis </li></ul><ul><li>Mechanisms of Induction of TGF- β 1 by Hepatitis C Virus </li></ul><ul><li>Future Studies </li></ul>
    3. 3. HCV Introduction <ul><li>9.6 Kb enveloped (+)ssRNA </li></ul><ul><li>Blood-Borne </li></ul><ul><li>Hepatotropic </li></ul><ul><li>Flaviviridae family </li></ul><ul><ul><li>Yellow Fever </li></ul></ul><ul><ul><li>West Nile </li></ul></ul><ul><ul><li>Dengue Fever </li></ul></ul><ul><li>Hepacivirus genus </li></ul><ul><li>Major cause of: </li></ul><ul><ul><li>Chronic Hepatitis </li></ul></ul><ul><ul><li>Liver Fibrosis </li></ul></ul><ul><ul><li>Cirrhosis </li></ul></ul><ul><ul><li>Hepatocellular Carcinoma </li></ul></ul>http://en.wikipedia.org/wiki/Hepatitis_C
    4. 4. HCV Infection Nature. 2005 Aug 18;436(7053):930-2. 10-20% 80-90% 1-4%
    5. 5. Epidemiology <ul><li>HCV infects an estimated 170-200 million worldwide </li></ul><ul><ul><li>4 million in U.S. </li></ul></ul><ul><ul><li>35,000 new infections/year in U.S. </li></ul></ul><ul><ul><li>10-15,000 deaths/year due to HCV in the U.S. </li></ul></ul><ul><li>80-90% of HCV infected individuals develop chronic disease </li></ul><ul><ul><li>HCV is the leading cause of liver cancer in the U.S. </li></ul></ul><ul><ul><li>Chronic HCV is now the most common reason for liver transplantation </li></ul></ul>Rank City Pop. (2009) 1 New York 8.4 Million 2 Los Angeles 3.8 Million 3 Chicago 2.9 Million 4 Houston 2.3 Million 5 Phoenix 1.6 Million Rank City Pop. (2009) 1 Shanghai 13.8 Million 2 Mumbai 13.8 Million 3 Karachi 12.9 Million 4 Delhi 12.6 Million 5 Istanbul 12.5 Million
    6. 6. Current HCV Entry Model Lemon et al. 2009, Gastroenterology Putative Receptors LDLR SR-B1 CD-81 CLDN-1 Occludin
    7. 7. HCV Life Cycle Tibotec.com
    8. 8. HCV Genome Penin et al. 2004 RNP complex 3’-NTR Replication 9.6 kb Polyprotein processing
    9. 9. <ul><li>In vitro : </li></ul><ul><li>Transient expression systems </li></ul><ul><li>Stable transfected cell lines (constitutive/inducible expression) </li></ul><ul><li>Replicons (subgenomic/full-length; selectable/transient) </li></ul><ul><li>Infectious cell culture system </li></ul><ul><li>In vivo: </li></ul><ul><li>Transgenic mice (PXB-mouse 95% humanized liver) </li></ul><ul><li>Tupaia (Tree Shrew) </li></ul><ul><li>Immunodeficient mice/hepatocellular reconstitution models </li></ul><ul><li>Chimpanzee </li></ul><ul><li>Clinical Samples </li></ul>HCV Model Systems
    10. 10. Disease Progression HCV>Chronic Inflammation>Steatosis> Fibrosis >Cirrhosis>HCC Normal Healthy Liver Hepatocellular Carcinoma Cirrhotic Liver
    11. 11. Liver Fibrosis <ul><li>Accumulation of fibrous scar tissue in the liver </li></ul><ul><li>The injury or death of hepatocytes stimulates inflammatory immune cells to release cytokines and growth factors </li></ul><ul><li>These chemical messengers activate hepatic stellate cells to produce collagens , glycoproteins (such as fibronectin ), and proteoglycans </li></ul><ul><li>These substances are deposited in the liver, causing the build-up of extracellular matrix (nonfunctional connective tissue) </li></ul>
    12. 12. Liver Fibrosis <ul><li>Wound healing response to chronic liver injury </li></ul><ul><li>Accumulation of ECM molecules (collagen type I, III and IV) </li></ul><ul><li>Formation of scar tissue </li></ul>Normal Liver Liver Injury Profibrogenic Factors including TGF- β 1
    13. 13. Nucleus TGF- β 1 Expression Hepatic stellate cells Profibrogenic Gene Expression Hepatocytes α -SMA Vimentin Fibronectin Collagens CTGF HCV HCV infects hepatocytes
    14. 14. What Cytokines are Secreted from HCV-infected Hepatocytes?
    15. 15. Does HCV increase secreted TGF- β 1? Presser et al. 2011 HCV-infection of human hepatocytes induces secretion of TGF- β 1 * *
    16. 16. Does HCV induce TGF- β 1 mRNA? 1 2 3 4 HCV core (21 kDa) Actin (42 kDa) Huh-7 D1 D2 D3 HCV-infection Presser et al. 2011 HCV-infection of human hepatocytes induces TGF- β 1 mRNA * *
    17. 17. * * * * * Huh-7 HCV core Core Huh-7 HCV NS3 HCV NS3/4A Huh-7 HCV NS4B Huh-7 HCV NS5A Huh-7 HCV NS5B Huh-7 E1/E2 1 2 E1/E2 Actin Actin Actin Core (21 kDa) NS3 (70 kDa) E1/E2 (27 kDa) NS4B (27 kDa) NS5A (58 kDa) NS5B (66 kDa) 1 2 1 2 1 2 1 2 3 1 2 NS3/4A NS4B NS5A NS5B Presser et al . 2011 What effect do HCV proteins have on the secretion of TGF- β 1? HCV proteins NS3, NS3/4A and NS5A cause the highest amount of secreted TGF- β 1
    18. 18. What Effect do HCV Proteins have on the TGF- β 1 Promoter? * * * phTG1 + + + + + + + + * -1362 ( phTG1) +11 Luc USF1/2 AP-1 Egr-1 Sp-1 Presser et al. 2011
    19. 19. NS3/4A Mutational Studies Presser et al. 2011 Actin NS3 wt NS3/4A NS3/4A (  Arg) NS3/4A S1165A 1 2 3 4 5 6 Marker Huh7 76 kDa 52 kDa NS3/4A
    20. 20. ** ** * * What effect do wild-type NS3/4A and mutants have on the secretion of TGF- β 1? Wild-type NS3/4A with its active protease domain induces the significantly higher amounts of TGF- β 1 than mutant NS3/4A Presser et al. 2011
    21. 21. What Effect do the NS3/4A Mutants have on the TGF- β 1 Promoter? * * ** ** phTG1 + + + + + -1362 ( phTG1) +11 Luc USF1/2 AP-1 Egr-1 Sp-1 Presser et al. 2011
    22. 22. NS5A Mutational Studies 1 2 3 4 NS5A (58 kDa) Actin (42 kDa) Huh-7 NS5A Wt pCNSM1 pCNSM3 76 kDa 31 kDa Presser et al. 2011 Wild-type NS5A NS5A mutant-1 NS5A mutant-3 1973 2419 Mut-1 2135 2419 Mut-3 1973 2135 Wt
    23. 23. Presser et al. 2011 What effect do wild-type NS5A and mutants have on the secretion of TGF- β 1? Wild-type NS5A requires its N-terminus to efficiently induce secretion of TGF- β 1 * **
    24. 24. What Effect do NS5A Mutants have on the wild-type TGF- β 1 Promoter ** * TGF- β 1 Luc + + + + Presser et al. 2011 -1362 ( phTG1) +11 Luc USF1/2 AP-1 Egr-1 Sp-1 Wild-type NS5A requires its N-terminus to efficiently induce wild-type TGF- β 1 promoter
    25. 25. Summary <ul><li>HCV proteins activate the wild-type TGF- β 1 promoter and increase secretion of TGF- β 1 </li></ul><ul><ul><li>Core, NS3, NS3/4A, NS5A </li></ul></ul><ul><li>Mutants of NS3/4A and NS5A </li></ul><ul><ul><li>NS3/4A requires an active protease domain to fully induce wild-type TGF- β 1 promoter and secretion of TGF- β 1 </li></ul></ul><ul><ul><li>NS5A requires N-terminus region for efficient wild-type TGF- β 1 promoter activation and secretion of TGF- β 1 </li></ul></ul>
    26. 26. <ul><li>G Waris, G Gong et al. PNAS 2001 </li></ul><ul><ul><li>Human Hepatitis C Virus NS5A Protein Alters Intracellular Calcium Levels, Induces Oxidative Stress, and Activates STAT-3 and NF- κ B </li></ul></ul><ul><li>G Waris and Aleem Siddiqui J. Virol 2005 </li></ul><ul><ul><li>Hepatitis C Virus Stimulates the Expression of Cyclooxygenase-2 via Oxidative Stress: Role of Prostaglandin E 2 in RNA Replication </li></ul></ul><ul><li>G Waris, et al. J. Virol 2005 </li></ul><ul><ul><li>Hepatitis C Virus Constitutively Activates STAT-3 via Oxidative Stress: Role of STAT-3 in HCV Replication </li></ul></ul><ul><li>Masaaki Korenaga, et al. JBC 2005 </li></ul><ul><ul><li>Hepatitis C Virus Core Protein Inhibits Mitochondrial Electron Transport and Increases Reactive Oxygen Species Production </li></ul></ul><ul><li>C Piccoli, et al. Hepatology 2007 </li></ul><ul><ul><li>Hepatitis C Virus Protein Expression Causes Calcium-Mediated Mitochondrial Bioenergetic Dysfunction and Nitro-Oxidative Stress </li></ul></ul>HCV Gene Expression Induces ROS via Ca 2+ Signaling
    27. 27. Core (21 kDa) Huh7 HCV Actin (42 kDa) 1 2 Mock-infection HCV-infection BAPTA-AM treatment TMB-8 treatment Ruthenium Red treatment Mock-infection + H 2 O 2 treatment Presser et al. 2011 Dye = CM-H 2 DCFDA HCV-infection increases ROS levels, which can be abrogated by treatment with intracellular Ca2+ chelators and an inhibitor of mitochondrial Ca2+ uptake
    28. 28. * ** ** ** ** HCV infection TGF-  1 Luc + + + + + + + + Presser et al. 2011 -1362 ( phTG1) +11 Luc USF1/2 AP-1 Egr-1 Sp-1 Does Ca2+ and ROS play a role in the induction of the wild-type TGF- β 1 promoter?
    29. 29. ** ** ** ** ** HCV infection * Does Ca2+ and ROS play a role in the induction of the TGF- β 1 mRNA? Presser et al. 2011
    30. 30. Summary <ul><li>HCV infection induces ROS </li></ul><ul><li>Intracellular Ca2+ chelators and an inhibitor of Ca2+ uptake by the mitochondria decrease TGF- β 1 induction </li></ul><ul><ul><li>Extracellular chelators does not have an effect </li></ul></ul><ul><li>Antioxidants (NAC and PDTC) decrease induction of TGF- β 1 </li></ul><ul><ul><li>DPI has no effect </li></ul></ul>
    31. 31. TGF- β 1 Promoter Transcriptional Activators <ul><li>Published Binding Sites </li></ul><ul><ul><li>Sp1, AP-1, Egr-1, ARE, USF1/2 </li></ul></ul><ul><li>Hypothetical Binding Sites (TFSEARCH Threshold = > 90%) </li></ul><ul><ul><li>C/EBP, MZF1, GATA-1-3, p300, AML-1a, USF </li></ul></ul><ul><li>Hypothetical Binding Sites (AliBaba2.1 Threshold = > 90%) </li></ul><ul><ul><li>C/EBP, MZF1, GATA-1, Ttx, USF, Krox-20, Elf-1 </li></ul></ul>Wei Q. J. Biochem. 2008 Birchenall-Roberts MC. Mol. Cell Bio.1990 Yongseok K. JBC. 1998 Seong-Jin K. JBC 1989 Peralta-Zaragoza O. V. Immuno. 2006
    32. 32. Transcription Factor Background <ul><li>S Lee et al. Virology 2001 </li></ul><ul><ul><li>HCV Core protein transactivates Insulin-like Growth Factor II gene transcription through acting concurrently on Egr1 and Sp1 sites </li></ul></ul><ul><li>T Tsutsumi et al. Virology 2002 </li></ul><ul><ul><li>Alteration of intrahepatic cytokine expression and AP-1 activation in transgenic mice expressing HCV core protein </li></ul></ul><ul><li>W Lin, et al. Gastroentrol. 2010 </li></ul><ul><ul><li>HCV regulates TGF- β 1 production through the generation of reactive oxygen species in an NF κ B-dependent manner </li></ul></ul><ul><li>G Waris, G Gong et al. PNAS 2001 </li></ul><ul><ul><li>Human Hepatitis C Virus NS5A Protein Alters Intracellular Calcium Levels, Induces Oxidative Stress, and Activates STAT-3 and NF- κ B </li></ul></ul><ul><li>V Christen et al. Hepatology 2007 </li></ul><ul><ul><li>Activation of ER stress response by HCV up-regulates protein phosphatase 2A </li></ul></ul><ul><li>Z Xiang et al. Biochem & Biophys Res. Comm . </li></ul><ul><ul><li>HCV NS5A activates SREBP-1c through transcription factor Sp1 </li></ul></ul>
    33. 33. -1362 ( phTG1) -453 (phTG5) -323 (phTG6) -175 (phTG7) -60 (phTG7-4) +11 Luc Luc Luc Luc Luc USF1/2 AP-1 Egr-1 Sp-1 Design of TGF- β 1 Promoter Region USF1/2 -1013 to -1002 Egr-1 -119 to -111 and -82 to -74 AP-1 -418 to -412 and -371 to -364 Sp1 -236 to -227, -217 to -209, -121 to -113, -109 to -100, -81 to -71 Negative Regulatory Region -1362 to -1132 and -731 to -453 Constructs were a generous gift from Dr. Seong-Jin Kim
    34. 34. What effect does HCV infection have on the TGF- β 1 promoter region? HCV infection induces the TGF- β 1 promoter 12-fold 6-fold 2-fold
    35. 35. Effect of HCV proteins on TGF- β 1 Promoter
    36. 36. Does HCV infection induce AP-1? Luc -35 +1 TATA 4x GCN4 Consensus
    37. 37. AP-1 ( Tanshinone II ) p38 ( SB203580 ) JNK ( SP600125 ) PI3K ( LY294002 ) Src (Fyn, Yes, Lyn) ( SU6656 ) HCV Infection MEK1/2 ( UO126 ) JAK2/3/STAT3 ( AG490 ) NF- κ B ( Bay11-7085 & AI )
    38. 38. What kinase pathways play a role in HCV infection-induced TGF- β 1 promoter region activation? p38, JNK, MEK1/2 as well as AP-1 and NFkB appear to be playing a role in HCV infection-induced TGF- β 1 promoter region activation
    39. 39. Nucleus Mitochondria Endoplasmic reticulum EOR Ca 2+ release Oxidative Stress TGF-  activation Hepatic stellate cells ROS Ca 2+ Ca 2 + HCV gene expression Ca 2+ Profibrogenic Gene Expression Hepatocytes Furin TSP-1 α -SMA Vimentin Fibronectin Collagens CTGF Kinases Transcription Factors
    40. 40. TGF- β 1 Proteolysis <ul><li>TGF- β 1 is synthesized as a larger in active precursor that must undergo proteolytic processing before releasing the bioactive product </li></ul><ul><li>Potential Candidates </li></ul><ul><ul><li>Furin </li></ul></ul><ul><ul><li>TSP-1 </li></ul></ul><ul><ul><li>Calpain </li></ul></ul><ul><ul><ul><li>Calpains are Ca 2+ -dependent cysteine proteases that have been shown by our lab to be induced by HCV subgenomic replicon </li></ul></ul></ul><ul><ul><li>MMP-9 </li></ul></ul><ul><ul><ul><li>Involved in the breakdown of ECM </li></ul></ul></ul>
    41. 41. * * Presser et al. 2011 What potential TGF- β 1 proteases show mRNA upregulation in HCV infection? HCV-infection increases mRNA expression of Furin and TSP-1 but not Calpain or MMP-9
    42. 42. Role of Furin <ul><li>Ubiquitously expressed </li></ul><ul><ul><li>Ca 2+ -dependent serine protease </li></ul></ul><ul><ul><li>In our model Ca 2+ signaling is increased by HCV infection </li></ul></ul><ul><ul><li>Consensus sequence R-X-K/R-R </li></ul></ul><ul><ul><li>Processes precursors either at the trans-Golgi network or cell surface </li></ul></ul>
    43. 43. Role of TSP-1 <ul><li>Has been shown to be an important activator of TGF- β 1 </li></ul><ul><ul><li>K Breitkopf et al. Gut 2005 </li></ul></ul><ul><li>Increased expression of TSP-1 has been observed in liver specimens of patients with HCC </li></ul><ul><ul><li>H Kondou et al. J. Hepatol. 2003 </li></ul></ul>
    44. 44. Furin (60 kDa) HCV NS3 (70 kDa) Mock-infection TSP-1 (75 kDa) Albumin (66 kDa) 1 2 Precursor TGF-  1 (50 kDa) Mature TGF-  1 (25 kDa) Actin (42 kDa) HCV-infection Presser et al. 2011 HCV-infection increases Furin, TSP-1 and TGF- β 1 protein expression as well as cleavage of TGF- β 1 Does HCV infection increase protein expression of Furin, TSP-1, or have an effect on the cleavage of TGF- β 1?
    45. 45.
    46. 46. Summary <ul><li>Furin and TSP-1 mRNA and protein are upregulated by HCV-infection </li></ul><ul><li>MMP-9 and Calpain mRNA expression does not appear to increase upon HCV-infection of Huh-7 cells </li></ul>
    47. 47. What effect does knock down of TSP-1 and furin using siRNA have on amount of secreted TGF- β 1? Presser et al. 2011 Relative mRNA Levels siRNA against furin and TSP-1 reduces the amount of secreted TGF- β 1
    48. 48. Is the secreted TGF- β 1 induced by HCV infection bioactive ? Do TSP-1 and furin siRNA affect amount of bioactive TGF- β 1 HCV-infection increases amount of secreted bioactive TGF- β 1 and knock down with TSP-1 or furin siRNA results in a decrease of bioactive TGF- β 1 Presser et al. 2011
    49. 49. Huh-7 HCV Infection * * ** ** ** What effect does knock down of TGF- β 1, TSP-1 and furin using siRNA have on HCV replication? Presser et al. 2011 Knock down of furin, TSP-1, and TGF- β 1 significantly decreases HCV replication
    50. 50. Hepatocyte Endoplasmic reticulum ER stress Ca 2+ release Oxidative Stress TGF-  1 precursor ROS Ca 2+ Ca 2+ HCV Ca 2 + BioactiveTGF-  1 Furin and/or TSP-1 Ca 2+ Ca 2+ BioactiveTGF-  1 HCV replication Intracellular signaling Mitochondria Ca 2+
    51. 51. Future Directions <ul><li>Continue to characterize the effect of HCV infection on the TGF- β 1 promoter </li></ul><ul><ul><li>Unknown transcription factors </li></ul></ul><ul><li>Identify unknown profibrogenic factors secreted from HCV infected cells by proteomics </li></ul><ul><li>Define the effect of profibrogenic factors on Hepatic Stellate Cells </li></ul><ul><ul><li>Migration/Invasion </li></ul></ul><ul><ul><li>Proliferation </li></ul></ul><ul><li>Determine what role TGF- β 1 plays in the HCV life cycle </li></ul>
    52. 52. Acknowledgements <ul><li>Committee Members </li></ul><ul><li>David Everly </li></ul><ul><li>Neelam Sharma-Walia </li></ul><ul><li>Gulam Waris </li></ul><ul><li>Carl White </li></ul><ul><li>Chao-Lan Yu </li></ul><ul><li>Laboratory Members </li></ul><ul><li>Adam Haskett </li></ul><ul><li>Dylan Burdette </li></ul><ul><li>Matthew Olivarez </li></ul><ul><li>Jawed Iqbal </li></ul><ul><li>Thi Mai </li></ul>Acknowledgements Funding RFUMS H.M Bligh Cancer Research Fund ACS IL Division NIH R21 AI078532-01 Collaborators Seong-Jin Kim – National Cancer Institute, NIH Craig Cameron – Pennsylvania State University Takaji Wakita – Tokyo Metropolitan Institute for Neuroscience Daniel B. Rifkin - NYU Langone Medical Center Michael Gale – University of Washington, Seattle Scott Friedman - Mount Sinai School of Medicine Nancy Colburn – National Cancer Institute, NIH Charles Rice – Rockefeller University, NY Thank you Flow Cytometry Core - RFUMS Microscopy Core - RFUMS
    53. 53. <ul><li>Questions? </li></ul>

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