Post on 12-Jul-2015
1
Mechanisms of Liver Fibrosis by Hepatitis C Virus
Lance D. Presser
Department of Microbiology and ImmunologyH.M. Bligh Cancer Research Laboratories
School of Graduate and Postdoctoral StudiesRosalind Franklin University of Medicine and Science
2
Outline
• Hepatitis C Virus Background• Liver Fibrosis• Mechanisms of Induction of TGF-β1 by
Hepatitis C Virus• Future Studies
3
HCV Introduction
• 9.6 Kb enveloped (+)ssRNA • Blood-Borne• Hepatotropic• Flaviviridae family
– Yellow Fever– West Nile– Dengue Fever
• Hepacivirus genus• Major cause of:
– Chronic Hepatitis– Liver Fibrosis– Cirrhosis– Hepatocellular Carcinoma
http://en.wikipedia.org/wiki/Hepatitis_C
4
HCV Infection
Nature. 2005 Aug 18;436(7053):930-2.
10-20%
80-90%
1-4%
5
Epidemiology
• HCV infects an estimated 170-200 million worldwide– 4 million in U.S. – 35,000 new infections/year in U.S.– 10-15,000 deaths/year due to HCV in
the U.S.
• 80-90% of HCV infected individuals develop chronic disease– HCV is the leading cause of liver
cancer in the U.S.– Chronic HCV is now the most common
reason for liver transplantation
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
Current HCV Entry Model
Lemon et al. 2009, Gastroenterology
Putative ReceptorsLDLRSR-B1CD-81CLDN-1Occludin
7
HCV Life Cycle
Tibotec.com
8
HCV Genome9.6 kb
Polyprotein processing
Penin et al. 2004
RNP complex
3’-NTR Replication
9
In vitro: Transient expression systems Stable transfected cell lines (constitutive/inducible expression) Replicons (subgenomic/full-length; selectable/transient) Infectious cell culture system
In vivo: Transgenic mice (PXB-mouse 95% humanized liver) Tupaia (Tree Shrew) Immunodeficient mice/hepatocellular reconstitution models Chimpanzee Clinical Samples
HCV Model Systems
10
Disease ProgressionHCV>Chronic Inflammation>Steatosis>Fibrosis>Cirrhosis>HCC
Normal Healthy Liver Hepatocellular Carcinoma
Cirrhotic Liver
11
Liver Fibrosis• Accumulation of fibrous scar tissue in the liver
• The injury or death of hepatocytes stimulates inflammatory immune cells to release cytokines and growth factors
• These chemical messengers activate hepatic stellate cells to produce collagens, glycoproteins (such as fibronectin), and proteoglycans
• These substances are deposited in the liver, causing the build-up of extracellular matrix (nonfunctional connective tissue)
12
Liver Fibrosis• Wound healing response to chronic liver injury • Accumulation of ECM molecules (collagen type I, III and IV)
• Formation of scar tissue
Normal Liver Liver Injury
Profibrogenic Factors including TGF-β1
13
Nucleus
TGF-β1 Expression
Hepatic stellate cells
Profibrogenic Gene Expression
Hepatocytes
α-SMAVimentinFibronectinCollagensCTGF
HCV
HCV infects hepatocytes
14
What Cytokines are Secreted from HCV-infected Hepatocytes?
15
Does HCV increase secreted TGF-β1?
TGF-β1 ELISA
0
200
400
600
800
1000
1200
1400
1600
Huh-7 Day 1 Day 2 Day 3
TGF-
β1 (p
g/m
l)
Huh-7
HCV
*
*
Presser et al. 2011
HCV-infection of human hepatocytes induces secretion of
TGF- β1
16
Does HCV induce TGF-β1 mRNA?Quantitative RT-PCR
0
0.5
1
1.5
2
2.5
3
3.5
4
Huh-7 Day 1 Day 2 Day 3
TGF-
β1 m
RNA/
18s r
RNA
Huh-7
HCV *
*
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- β1
mRNA
17
TGF-β1 ELISA
0100200300400500600700800900
1000
Vector Core E1/E2 NS3 NS3/4A NS4B NS5A NS5B
TGF-β
1 (pg
/ml)
*
**
*
*
Huh
-7H
CV
cor
e
Core
Huh
-7
HC
V N
S3H
CV
NS3
/4A
Huh
-7
HC
V N
S4B
Huh
-7H
CV
NS5
A
Huh
-7
HC
V N
S5B
Huh
-7E
1/E
2
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-β1?
HCV proteins NS3, NS3/4A and NS5A cause the highest amount of
secreted TGF-β1
18
What Effect do HCV Proteins have on the TGF-β1 Promoter?
TGF-β1 luciferase assay
0
1
2
3
4
5
Vector Core E1/E2 NS3 NS3/4A NS4B NS5A NS5B
Rel
ativ
e lu
cife
rase
act
ivit
y
* *
*
phTG1 + + + + + + + +
*
-1362 (phTG1)
+11
Luc
USF1/2 AP-1 Egr-1Sp-1
Presser et al. 2011
19
NS3/4A Mutational Studies
Actin
NS3
wt
NS3
/4A
NS
3/4A
(∆
Arg
)
NS
3/4A
S11
65A
1 2 3 4 5 6
Mar
ker
Huh
7
76 kDa
52 kDa
NS3/4A
Presser et al. 2011
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TGF-β1 ELISA
0
100
200
300
400
500
600
700
800
Vector NS3 Wt NS3/4A NS3/4A (Δ1487-1501)
NS3/4A(S1156A)
TGF-
β1 (p
g/m
l)
****
*
*
What effect do wild-type NS3/4A and mutants have on the secretion
of TGF-β1?
Wild-type NS3/4A with its active protease domain induces the significantly higher amounts of TGF-β1 than mutant NS3/4A
Presser et al. 2011
21
What Effect do the NS3/4A Mutants have on the TGF-β1 Promoter?
TGF-β1 luciferase assay
0
1
2
3
4
5
Vector NS3 Wt NS3/4A NS3/4A(Δ1487-1501)
NS3/4A(S1156A)
Rel
ativ
e lu
cife
rase
act
ivity
*
*
**
**
phTG1 + + + + +
-1362 (phTG1)
+11
Luc
USF1/2 AP-1 Egr-1Sp-1
Presser et al. 2011
22
NS5A Mutational Studies
1 2 3 4
NS5A (58 kDa)
Actin (42 kDa)
Hu
h-7
NS
5A W
t
pC
NS
M1
pC
NS
M3
76 kDa
31 kDa
Presser et al. 2011
Wild-type NS5A
NS5A mutant-1
NS5Amutant-3
1973 2419Mut-1
2135 2419Mut-3
1973 2135Wt
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TGF-β1 ELISA
0
100
200
300
400
500
600
700
Vector NS5A Wt pCNSM1 pCNSM3
TG
F-β
1 (p
g/m
l)
Presser et al. 2011
What effect do wild-type NS5A and mutants have on the secretion of
TGF-β1?
Wild-type NS5A requires its N-terminus to efficiently induce secretion of TGF-β1
*
**
24
What Effect do NS5A Mutants have on the wild-type TGF-β1 Promoter
TGF-β1 luciferase assay
0
1
2
3
4
5
Vector NS5A Wt pCNSM1 pCNSM3
Relat
ive lu
cifer
ase a
ssay
**
*
TGF-β1 Luc + + + +
Presser et al. 2011
-1362 (phTG1)
+11
Luc
USF1/2 AP-1 Egr-1Sp-1
Wild-type NS5A requires its N-
terminus to efficiently induce wild-type TGF-
β1 promoter
25
Summary
• HCV proteins activate the wild-type TGF-β1 promoter and increase secretion of TGF-β1 – Core, NS3, NS3/4A, NS5A
• Mutants of NS3/4A and NS5A– NS3/4A requires an active protease domain to fully
induce wild-type TGF-β1 promoter and secretion of TGF-β1
– NS5A requires N-terminus region for efficient wild-type TGF-β1 promoter activation and secretion of TGF-β1
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• G Waris, G Gong et al. PNAS 2001– Human Hepatitis C Virus NS5A Protein Alters Intracellular Calcium Levels,
Induces Oxidative Stress, and Activates STAT-3 and NF-κB
• G Waris and Aleem Siddiqui J. Virol 2005– Hepatitis C Virus Stimulates the Expression of Cyclooxygenase-2 via Oxidative
Stress: Role of Prostaglandin E2 in RNA Replication
• G Waris, et al. J. Virol 2005– Hepatitis C Virus Constitutively Activates STAT-3 via Oxidative Stress: Role of
STAT-3 in HCV Replication
• Masaaki Korenaga, et al. JBC 2005– Hepatitis C Virus Core Protein Inhibits Mitochondrial Electron Transport and
Increases Reactive Oxygen Species Production
• C Piccoli, et al. Hepatology 2007– Hepatitis C Virus Protein Expression Causes Calcium-Mediated Mitochondrial
Bioenergetic Dysfunction and Nitro-Oxidative Stress
HCV Gene Expression Induces ROS via Ca2+ Signaling
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Core (21 kDa)
Huh7 HCV
Actin (42 kDa)
1 2
ROS levels
0
2
4
6
8
10
12
Mock-InfectedHuh-7
HCV-InfectedHuh-7
BAPTA-AM TMB-8 Ruthenium RedCel
l cou
nts (
HC
V/M
ock-
infe
cted
)ROS levels
0
20
40
60
80
100
120
Mock-Infected Huh-7 Huh-7+ H202
Cel
l cou
nts
(Pos
itive
co
ntro
l/Moc
k-in
fect
ed)
Mock-infection
HCV-infection
BAPTA-AMtreatment
TMB-8treatment
Ruthenium Redtreatment
Mock-infection+ H2O2 treatment
Presser et al. 2011
Dye = CM-H2DCFDA
HCV-infection increases ROS levels, which can be abrogated by treatment with intracellular Ca2+ chelators
and an inhibitor of mitochondrial Ca2+ uptake
28
TGF-β1 Luciferase assay
0
1
2
3
4
5
6
Huh7 HCV BAPTA TMB-8 EGTA RR PDTC DPI
Rela
tive L
ucife
rase
Act
ivity
*
****
****
HCV infection
TGF-β1 Luc + + + + + + + +
Presser et al. 2011
-1362 (phTG1)
+11
Luc
USF1/2 AP-1 Egr-1Sp-1
Does Ca2+ and ROS play a role in the induction of the wild-type
TGF-β1 promoter?
29
Quantitative RT-PCR
0
1
2
3
4
5
Huh-7 HCV BAPTA TMB-8 EGTA RR PDTC DPI NAC
TG
F-β
1 m
RN
A/1
8s m
RN
A
**
**
**
****
HCV infection
*
Does Ca2+ and ROS play a role in the induction of the
TGF-β1 mRNA?
Presser et al. 2011
30
Summary
• HCV infection induces ROS• Intracellular Ca2+ chelators and an
inhibitor of Ca2+ uptake by the mitochondria decrease TGF-β1 induction– Extracellular chelators does not have an
effect
• Antioxidants (NAC and PDTC) decrease induction of TGF-β1 – DPI has no effect
31
TGF-β1 Promoter Transcriptional Activators
• Published Binding Sites– Sp1, AP-1, Egr-1, ARE, USF1/2
• Hypothetical Binding Sites (TFSEARCH Threshold = >90%)– C/EBP, MZF1, GATA-1-3, p300, AML-1a, USF
• Hypothetical Binding Sites (AliBaba2.1 Threshold = >90%)– C/EBP, MZF1, GATA-1, Ttx, USF, Krox-20, Elf-1
Wei Q. J. Biochem. 2008Birchenall-Roberts MC. Mol. Cell Bio.1990Yongseok K. JBC. 1998Seong-Jin K. JBC 1989Peralta-Zaragoza O. V. Immuno. 2006
32
Transcription Factor Background
• S Lee et al. Virology 2001– HCV Core protein transactivates Insulin-like Growth Factor II gene transcription
through acting concurrently on Egr1 and Sp1 sites
• T Tsutsumi et al. Virology 2002– Alteration of intrahepatic cytokine expression and AP-1 activation in transgenic
mice expressing HCV core protein
• W Lin, et al. Gastroentrol. 2010– HCV regulates TGF-β1 production through the generation of reactive oxygen
species in an NFκB-dependent manner
• G Waris, G Gong et al. PNAS 2001– Human Hepatitis C Virus NS5A Protein Alters Intracellular Calcium Levels,
Induces Oxidative Stress, and Activates STAT-3 and NF-κB
• V Christen et al. Hepatology 2007– Activation of ER stress response by HCV up-regulates protein phosphatase 2A
• Z Xiang et al. Biochem & Biophys Res. Comm.– HCV NS5A activates SREBP-1c through transcription factor Sp1
33
-1362 (phTG1)
-453 (phTG5)
-323 (phTG6)
-175 (phTG7)
-60 (phTG7-4)
+11
Luc
Luc
Luc
Luc
Luc
USF1/2 AP-1 Egr-1Sp-1
Design of TGF-β1 Promoter Region
USF1/2-1013 to -1002Egr-1 -119 to -111 and -82 to -74AP-1 -418 to -412 and -371 to -364Sp1 -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
What effect does HCV infection have on the TGF-β1 promoter region?
TGF-β1 Luciferase Assay
1 0 052 49
121 1 2
626
276
25
0
100
200
300
400
500
600
700
Vector phTG7-4 phTG7 phTG5 phTG1 phTG6
RL
A Huh-7
HCV
HCV infection induces the TGF-β1 promoter
12-fold
6-fold
2-fold
35
Effect of HCV proteins on TGF-β1 Promoter
TGF-β1 Luciferase Assay
0
20
40
60
80
100
120
140
Vector phTG7-4 phTG7 phTG5 phTG1 phTG6
RL
A
Huh-7
NS3
NS3/4A
NS5A
36
Does HCV infection induce AP-1?
AP-1 Luciferase Assay
0
0.5
1
1.5
2
2.5
3
3.5
4
Vector AP-1 4x
Rel
ativ
e L
ucif
eras
e A
ctiv
ity
Huh-7
HCV
Luc-35
+1
TATA
4x GCN4 Consensus
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-κB (Bay11-7085 & AI)
38
TGF-β1 Luciferase Assay
024
68
1012
1416
Vecto
r
phTG
1 unt
reat SB SP LY SU
TAN AGBAY
NFkB UO
RL
A
Huh-7.5
HCV
Ratio
What kinase pathways play a role in HCV infection-induced TGF-β1 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-β1 promoter
region activation
39Nucleus
Mitochondria
Endoplasmic Endoplasmic reticulumreticulum
EOR
CaCa 2+ 2+
releaserelease
Oxidative Stress
TGF-βactivation
Hepatic stellate cells
ROS
Ca2+
Ca2+
HCV geneHCV geneexpressionexpression
Ca2+
Profibrogenic Gene Expression
Hepatocytes
FurinTSP-1
α-SMA Vimentin Fibronectin Collagens CTGF
Kinases
Transcription Factors
40
TGF-β1 Proteolysis
• TGF-β1 is synthesized as a larger in active precursor that must undergo proteolytic processing before releasing the bioactive product
• Potential Candidates– Furin– TSP-1– Calpain
• Calpains are Ca2+-dependent cysteine proteases that have been shown by our lab to be induced by HCV subgenomic replicon
– MMP-9• Involved in the breakdown of ECM
41
Quantitative RT-PCR
0
1
2
3
4
5
6
7
Furin TSP-1 Calpain MMP-9
Relat
ive fo
ld ch
ange
Huh-7
HCV *
*
Presser et al. 2011
What potential TGF-β1 proteases show mRNA
upregulation in HCV infection?
HCV-infection increases mRNA expression of Furin and TSP-1 but not Calpain or MMP-9
42
Role of Furin• Ubiquitously expressed
– Ca2+-dependent serine protease– In our model Ca2+ signaling is increased by
HCV infection– Consensus sequence R-X-K/R-R– Processes precursors either at the trans-Golgi
network or cell surface
43
Role of TSP-1
• Has been shown to be an important activator of TGF-β1– K Breitkopf et al. Gut 2005
• Increased expression of TSP-1 has been observed in liver specimens of patients with HCC– H Kondou et al. J. Hepatol. 2003
44
Furin (60 kDa)
HCV NS3 (70 kDa)
Moc
k-in
fect
ion
TSP-1 (75 kDa)
Albumin (66 kDa)
1 2
Precursor TGF-β1 (50 kDa)
Mature TGF-β1 (25 kDa)
Actin (42 kDa)
HC
V-in
fect
ion
Presser et al. 2011
HCV-infection increases Furin, TSP-1
and TGF-β1 protein expression as well as cleavage of TGF-β1
Does HCV infection
increase protein expression of
Furin, TSP-1, or have an effect on the cleavage of
TGF-β1?
45
46
Summary
• Furin and TSP-1 mRNA and protein are upregulated by HCV-infection
• MMP-9 and Calpain mRNA expression does not appear to increase upon HCV-infection of Huh-7 cells
47
TGF-β1 ELISA
0200400600800
100012001400160018002000
Huh-7 GFP siRNA HCV GFP siRNA TGF-β1siRNA
TSP-1siRNA
FurinsiRNA
TG
F-β1
(pg/
ml)
Percent inhibition by siRNA
0
20
40
60
80
100
120
140
GFP siRNA Furin siRNA TGF-B1 siRNA TSP-1 siRNA
Per
cen
t In
hib
itio
n o
f T
arg
et G
enes What effect does
knock down of TSP-1 and furin
using siRNA have on amount
of secreted TGF-β1?
Presser et al. 2011
Rel
ativ
e m
RN
A L
evel
s
siRNA against furin
and TSP-1 reduces the amount of secreted TGF-β1
48
Mink Lung Epithelial Cells Growth Inhibition Assay
0
500
1000
1500
Huh-7 HCV GFP siRNA TGF-β1 siRNA TSP-1 siRNA Furin siRNARela
tive L
ucife
rase
Uni
tsIs the secreted TGF-β1 induced by
HCV infection bioactive? Do TSP-1 and furin siRNA affect amount of bioactive TGF-β1
HCV-infection increases amount of secreted bioactive TGF-β1 and knock down with TSP-1 or furin siRNA results in a
decrease of bioactive TGF-β1
Presser et al. 2011
49
HCV Replication: Quantitative RT-PCR
0
5
10
15
20
Huh-7 GFPsiRNA
HCV GFPsiRNA
TGF-β1siRNA
TSP-1siRNA
FurinsiRNA
Relat
ive F
old C
hang
e
Huh-7
HCV Infection
* *
**
****
What effect does knock down of TGF-β1, TSP-1 and furin using siRNA have on HCV replication?
Presser et al. 2011
Knock down of furin, TSP-1, and TGF-β1 significantly decreases HCV replication
50
Hepatocyte
Endoplasmic reticulum
ER stress
Ca2+
release
Oxidative Stress
TGF-β1precursor
ROS
Ca2+
Ca2+
HCV Ca2+
BioactiveTGF-β1
Furin and/orTSP-1Ca2+
Ca2+
BioactiveTGF-β1
HCV replication
Intracellular signaling
Mitochondria
Ca2+
51
Future Directions
• Continue to characterize the effect of HCV infection on the TGF-β1 promoter
– Unknown transcription factors• Identify unknown profibrogenic factors
secreted from HCV infected cells by proteomics
• Define the effect of profibrogenic factors on Hepatic Stellate Cells
– Migration/Invasion– Proliferation
• Determine what role TGF-β1 plays in the HCV life cycle
52
AcknowledgementsCommittee Members
David EverlyNeelam Sharma-Walia
Gulam WarisCarl White
Chao-Lan Yu
Laboratory MembersAdam HaskettDylan Burdette
Matthew OlivarezJawed Iqbal
Thi Mai
Acknowledgements
FundingRFUMS H.M Bligh Cancer Research FundACS IL DivisionNIH R21 AI078532-01
CollaboratorsSeong-Jin Kim – National Cancer Institute, NIHCraig Cameron – Pennsylvania State University
Takaji Wakita – Tokyo Metropolitan Institute for NeuroscienceDaniel B. Rifkin - NYU Langone Medical CenterMichael Gale – University of Washington, SeattleScott Friedman - Mount Sinai School of MedicineNancy Colburn – National Cancer Institute, NIH
Charles Rice – Rockefeller University, NY
Thank youFlow Cytometry Core - RFUMS
Microscopy Core - RFUMS
53
• Questions?