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

20

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

23

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

26

• 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

27

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?