Growth factors in experimental liver fibrogenesisRalf Weiskirchen

Institute of Clinical Chemistry and Pathobiochemistry, UKA

Falk Symposium 162: Leberzirrhose: Von der Pathophysiologie zur Klinik13. Oktober 2007

Inflammation Fibrosis

Cytokines in Liver Fibrogenesis

PRO-• IL-1β• IL-12• TNF-α• TNF-β• IFN-γ• IL-18• IL1-Ra

ANTI-• IL-4• IL-10• IL-13• IL1-Ra• sTNFα-R

PRO-• TGF-β• TNF-α• PDGF• bFGF• ET-1• IL-1• IGF-1

ANTI-• HGF• IFN-α• IL-10• BMP-7

Gen

eral

quiescent HSC activated HSC

Activation

MFB

Transdifferentiation

• Transcriptional events• Paracrine stimulation• Early ECM changes

Initiation phase

• Increased cytokine secretion• Receptor tyrosine phosphorylation• ECM synthesis and remodeling

Perpetuation phase

adapted from Friedman (2000) J. Biol. Chem. 275, 2247-50

Overview of TGF-β1 effects duringactivation/transdifferentiation of HSC

α-SMA expression

TGF-β

collagen expression

TGF-β BMP-7

Gen

eral

SRF

GATA

SRF function in smooth muscle cell(SMC) differentiation and α-SMA expression

Chang et al., Dev Cell 2003

CRP2(Weiskirchen et al., Oncogene 1993)

pluripotent fibroblast(10T1/2)

SMC

CRP2BP

(Weiskirchen & Gressner, BBRC 2000)

Gen

eral

1d 2d 3d 4d 5d 6d 7d 2d 4d

HSC MFB

SRF (67 kDa)

α-SMA (42 kDa)

Ponceau S

1d 2d 3d 4d 5d 6d 7d 2d 4d

HSC MFB

SRF (67 kDa)

α-SMA (42 kDa)

Ponceau S

Herrmann et al., in revision

Serum response factor (SRF) isupregulated during transdifferentiation

Western blot

Immunocytochemistry

SRF

Herrmann et al., in revision

1d 3d 5d 7d 4d

HSC MFB

1d 3d 5d 7d 4d

HSC MFBconsensus SRE Xmutant SRE Xanti-SRF Xcontrol IgG X

consensus SRE Xmutant SRE Xanti-SRF Xcontrol IgG X

consensus SRE Xmutant SRE Xanti-SRF Xcontrol IgG X

Activity of SRF during transdifferentiation of HSC

Electrophoretic mobility shift assay Supershift

5’-GGA TGT CCA TAT TAG GAC ATC T-3’

SRF

CC (A/T)6 GGSR

F

co-factors

5’-GGA TGT CCA TAT TAT TAC ATC T-3’

SMC-gene expression

consensus SRE

Serum response element (SRE)

mutant SRE

SRF

Myocardin(102 kDa)

1d 3d 5d 7d 4d

HSC MFB

Ponceau S

Myocardin(102 kDa)

1d 3d 5d 7d 4d

HSC MFB

Ponceau S

Serum response factor (SRF) targetgene expression during transdifferentiation

”reporter gene assay“Myocardin expression

GFPCMV

GFPSM22α

GFPTIMP-1

Ad5-CMV-GFP

Ad5-SM22α-GFP

Ad5-TIMP-1-GFP

A

B

SRF

Herrmann et al., in revision

The expression of SRF is TGF-β1-dependent

TGF-β1 (ng/ml) --- --- --- --- 1FCS (%) 0.2 10 0.2 0.2 10 0.2

5d HSC24 h starved

6d HSC48 h starved

SRF

Ponceau S

TGF-β1 (ng/ml) --- --- 1 --- ---FCS 0.2

-β1 --- --- ---

B

C

Herrmann et al., in revision

SB-431542(ALK5 inhibitor)

4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide

A

SRF (67 kDa)

Ponceau S

--- + --- + STR

Ι ΙΙ Preparation

STR = soluble TGF-β type II receptor

SRF (67 kDa)

--- + --- + SB-431542

2d HSC 7d HSC

Ponceau S

SRF

61 171

COOHGRR

10 120 194

NH2

1

GRRLIM1 LIM2

C-X2-C-X17-H-X2-C-X2-C-X2-C-X17-C-X23-C-

C

C

H

CZn

C

C

C

CZn

NH2 COOH

CRP2 is a molecular adaptor protein

J Biol Chem 272, 12001-7 (1997)Biochemistry 37, 7127-34 (1998)J Am Chem Soc 120, 7127-8 (1998)J Biol Chem 273, 23233-40 (1998)J Mol Biol 292, 893-908 (1999)

Weiskirchen et al., (2001), Biochem. J. 359, 485-96

CR

P2

CRP2

no BDL 2d BDL 7d BDL 14d BDL

α-SMA

β-actin

CRP2 expression during transdifferentiation and liver injury

Weiskirchen et al. (2001) Biochem J 359:485-96; Neyzen et al. (2006) J Hepatol 44:910-7; Herrmann et al. (2006) BBRC 345:1526-35

A

C

Normal Liver BDL LiverDTransdifferentiation

B

CR

P2

STR w/o +

CRP2

β-actin

00.20.40.60.8

11.21.41.61.8

β-actin CRP2

STR

vs.

Unt

reat

ed(n

orm

alis

edto

β-a

ctin

)

p = 0.038

CRP2

β-actin

--- --- + TGF-β1--- + + Starvation

CRP2

--- + --- + SB-431542

5d HSC 7d HSC

β-actin

The expression of CRP2 is TGF-β1 dependent

A

B

C

CSRP2

GAPDH

CFSC

Western blot

--- --- + TGF-β1--- + + Starvation

Primary HSC

Northern blot

untreated 7d BDL

D

E

CR

P2

LIM1 LIM2NH2 COOH hCRP2

qCRP2

LIM1NH2

LIM2 COOHNH2

hCRP2(LIM1)

hCRP2(LIM2)

1

1

193

193

81

82

LDB

LIM1 LIM2NH2 COOH

1 194

LDB

LDB

LDB

1 3 4kD

20

26

38

50

61

ALIM1 LIM22

LIM12 COOH

LIM22

LDB

LIM1 LIM22

1

LDB

LDB

BLDB

2 5C

CRP2BP is a binding partner of CRP2 (Y2H)

F

D E

Weiskirchen and Gressner (2000) BBRC 274:655-63

G

CR

P2

CRP2BP is a Histone-Acetyltransferase (HAT)

Paged structuresB

C

Recognition fold

A

For “Recognition fold“ methodology see:Hepatology 2001; 34:230-3

CR

P2B

P

cont

rol

siSR

F

SRF (67 kDa)

α-SMA (42 kDa)

SM22α (22 kDa)

S6 (32 kDa)

• Induction of SRF expression

• Induction of SRF cofactors (CRP2, myocardin)

• Forming of SRF • cofactor complexes

• Expression of specific SM genes (α-SMA; SM22α)

CFSC0 8 24 32 48 72 hrs starvation

CRP2

β-actin

HSC

Li et al., in revision

SM geneexpression

SRF

HATAcetylation

SRFC

RP2

CR

P2

quiescent HSC activated HSCTGF-β1

Early activation phase

SRF-

Net

wor

k

TGF-β signal transduction in HSC

C

Meurer et al., J. Biol. Chem. 2005;280:3078-87

A

B B

C

A

D

TGF-β/

HSC

sc-3

98 (A

LK-5

)

sc-4

00 (R

II)AF

242 (

RIII)P4

A4 (En

dogl

in)

Cross-linking andImmunoprecipitation

Endoglin binds TGF-β and isassociated with other TGF-β receptors

B

D

Endoglin(NFκB AB)

HSP-70

- + 48 72 96 120 144 168 48 72 96 120 144 168 hrs

24h siRNA Fluo siRNA Eng

HSC

2dHS

C 7d

MFB

4d

kD

A

C

HSC have highercontent of p-Endoglin(possibly preventingsurface exposition)

TGF-β/

HSC

ALK5

type III receptors

type I receptors

type II receptor

Smads

Analysis of the Smad1/5signalling pathway in transdifferentiating HSC

A B

Northern Blot

TGF-β/

HSC

Activation of artificial Smad reporters(CAGA)12-MLP-Luc and (BRE)2-Luc

Adenoviral expression (4 d HSC, 3 x 108 pfu/ml)

(CAGA)12- MLP-Luc reporter (“TGF-β reporter”)

LuciferaseMLP(minimal latepromoter)

PAI(Plasminogen activatorInhibitor promoter element)

Transcription

0

5

10

15

20

25

control TGF-β BMP-7

rel.

Luc-

activ

ity

(BRE)2- Luc reporter (“BMP reporter”)

LuciferaseMLP(minimal latepromoter)

Id-1(Inhibitor of differentiation-1promoter element)

Transcription

0

1

2

3

4

5

6

7

8

rel.

Luc-

activ

ity

control TGF-β BMP-7

A

B

*

*

TGF-β/

HSC

Endoglin: Impact on TGF-β1 and BMP-7 pathways

25

20

15

10

5

0

Blockade of (CAGA)12-Signals Stimulation of (BRE)2-Signals

Reporter-assay in L6E9 myoblasts (lacking endogenous endoglin)

Scherner et al., J Biol Chem 2007;282:13934-43

TGF-β/

HSC

TGF-β, BMP-7and Endoglin in activated HSC

Fibrosis

TGF-β

Smad2

Smad3

pSmad2

pSmad3

+

Resolution

BMP-7

Smad1

Smad5

pSmad1

pSmad5

Endoglin

-

TGF-β/

HSC

rel.

Luc.

-act

ivity

7

6

5

4

3

2

1

0

siCon siEngcontrol controlTGF-β TGF-β

(CAGA)12- Luciferase assay

Specificity and duration of Endoglin „knock down“

A BTG

F-β

- TGF-

β

-Endoglin

β-actin

19197

41

siCon siEng

kD

D

4d 7d 10d 13d

siCon siEng

Endoglin

HSP-70

4d 7d 10d 13d191

97

70

kD

C

*

*

TGF-β/

HSC

Analysis of BMP-7 and BMP-7 receptor expression in HSC

A

RT-PCRBM

P-7

Rec

epto

rs

B Western blot

C

HSC/MFB don‘t expressbut are responsive towards BMP-7

TGF-β/

HSC

Antagonisms ofTGF-β1 and BMP-7 in HSC

Cytosol Nucleus

BMP-7 [ng/ml]BMP-7 [ng/ml]

BMP-7 [ng/ml]

A

B

C

D

TGF-β/

HSC

SUMMARY

• SRF expression and activation

• expression of SRF cofactors (e.g. CRP2, myocardin)

• generation of SRF-cofactor complexes

• activation of SMC specific genes (e.g. SMA)

• Smad phosphorylation

• ECM synthesis expression (e.g. collagen type I)

quiescent HSC activated HSC

Activation

MFB

Transdifferentiation

TGF-β1

BMP-7

Endoglin

+

Sum

mar

y

AcknowledgementInstitute of Clinical Chemistryand Pathobiochemistry, UKA

Steffen K. MeurerOlav SchernerWanda N. VredenLidia TihaaErawan Borkham-KamphorstSabine WeiskirchenSenait MengsteabEddy van de LeurCarmen G. TagSimone MohrenOlav A. GressnerJens HerrmannFalko Drews

Axel M. Gressner

Martin Roderfeld, Elke RoebDepartment of Internal Medicine II,University Hospital Giessen

Reinhard BüttnerInstitute of Pathology,University Hospital Bonn

Florian Winau, Stefan KaufmannDepartment of Immunology,Max-Planck-Institute for Infection Biology (Berlin)

Sandip M. Kanse, Klaus T. PreissnerInstitute of Biochemistry,University Hospital Giessen

SFB 542SFB 542

INNOVATIONSPROGRAMMF O R S C H U N G

Ankang Li, Robert J. SchwartzBaylor College of Medicine, Center for MolecularDevelopment and Disease, Houston, TX, USAJoachim GrötzingerChristian Albrecht University Kiel, Institute of Bio-chemistry, Med. Faculty, Structural Biology Group