Triiodothyronine and L-Triiodothyronine and L-thyroxine (T3/T4) , their thyroxine (T3/T4) , their

receptors and liver fibrosisreceptors and liver fibrosis

Effect of T3 on liver fibrosisEffect of T3 on liver fibrosis

Treatment Time Pathological score (0-3)

TAA6 weeks0

12 weeks2.7+/- 0.1

TAA+PTU12 weeks0.85+/-0.15

TAA+MMI12 weeks0.55+/-0.12

TAA+surgical thyroid removal

12 weeks0.10+/-0.06

TAA+T3 (Eltroxin)6 weeks1.75+/-0.20

TAA+T3 (Eltroxin)12 weeks2.75+/-0.10

TR beta 1

TR alpha 1

461

410

86

DNA

82

ligand

A/B C D E

Affinity of T3: TR alpha1 >TR beta 1

Classic thyroid hormone receptors

Two genes, 4 isoforms

DNA binding

corepressorAF-1 Corepressor RXR

Ligand

DNA binding

AF-1 RXRT3

Ligand

Coactivator

NCoR SMRT SUN-CoR

Thyroid hormone receptor alpha 1

Trip1 TIF1 TRAP CBP ACTR

Histone acetylase, active chromatin

Histone deacetylase (HDAC), inactive chromatin

AF-2

Phosphorylation

Collagen I (cardiac fibroblasts),

TSH, c-myc, EGFR, prolactin, CD44

PEPCK, growth hormone,

Myosin , fatty acid synthetase

Phenotypic features of hepatic Phenotypic features of hepatic stellate cell activationstellate cell activation

Rat HSC-T6

Human LX-2

TRα1 TRβ1 GAPDH

Rat hepatic stellate cells expressRat hepatic stellate cells express

TRα1, but not TRβ1TRα1, but not TRβ1

The second T4/T3 receptor:Integrin The second T4/T3 receptor:Integrin vv3 and hepatic stellate cells3 and hepatic stellate cells

vv3 is a 3 is a receptorreceptor for L-thyroxine - for L-thyroxine -T4T4

• Receptor for vitronectin and fibronectin

• Vitronectin induced in liver injury; vv33 present only on activated stellate cells

• SiRNA of vv3 3 apoptosisapoptosis via induction of via induction of MMP-9MMP-9

3βα v

vitronectin

fibronectin

T4

T3 TRα1

T3

T3

T4

deiodination

Collagen I

Fibrosis

pMAPK

pSTAT3

proliferation

PDGFR

PDGFR

TGFBRI

III

pSTAT3 pMAPK

General aimsGeneral aims

• Verify our working hypothesisVerify our working hypothesis

• Role of Role of T4/T3T4/T3 and their receptors nuclear and their receptors nuclear receptors receptors TRTR11 and integrin and integrin vv33, in inducing , in inducing hepatic fibrosishepatic fibrosis

• Resolution of fibrosis by blocking Resolution of fibrosis by blocking TRTR11 and and integrin integrin vv33 signaling signaling

Specific aimsT3/TR1 • Effect and mechanisms of T3 effect on cell

proliferation (cyclin D, c-myc, p27)• Regulation of transcription of collagen I gene by T3

in rat and human HSC• Phenotype of TR1 and TR1 overexpressing HSC

and of siRNA of TR1

T4/Integrin v3• Regulation of HSC proliferation by T4• Effect of T4 and ECM on growth factor receptors• Effect of T4 on HSC activation

Does T3 affect HSC proliferationDoes T3 affect HSC proliferation? ?

Average of 5 exp

0.00

20.00

40.00

60.00

80.00

100.00

120.00

con T3 10-7M T3 10-9M T3 10-11M

* *

0.00

20.00

40.00

60.00

80.00

100.00

120.00

con T3 10-7M T3 10-9M T3 10-11M

*

*

T3 inhibits HSC proliferation-24h T3 inhibits HSC proliferation-48h

Average of 6 exp

T3 in low doses inhibits proliferation of rat HSC at 24 and 48h

What about humansWhat about humans??

Low dose of T3 inhibits LX-2 proliferation

0

20

40

60

80

100

120

con-24h T3 10-11M con-48h T3 10-11M

(%)

How does T3 inhibits proliferation

Cell cycle arrest Apoptosis

Cell CycleCell Cycle

In neuroblastoma cellsIn neuroblastoma cells

Gracia-Silva,JBC 1999/Toxicology 2002

CDK2

Cyclin D1

RbE2F

PP

Rb-PPP

E2FE2F

DP

G1 SCell proliferation

CKI-p27

C-MycnTRE

5’UTR of c-Myc

T3+TR

Cyclin D1 Promoter

Transcription of Cyclin D1 T3+TR

Incubation of Na2-b with T3 leads to:

1.Rapid down regulation of c-myc gene and protein.

2.Decrease of cyclin D1 levels of mRNA protein.

3.Increase levels of p27 half-life.

Growth arrest and differentiation

Low dose of T3 inhibits Cyclin D1 Low dose of T3 inhibits Cyclin D1 in HSC-T6in HSC-T6

Average of 6 exp

T3 effect on CycD1 in HSC

0.00

20.00

40.00

60.00

80.00

100.00

120.00

con T3 10-7M T3 10-9M T3 10-11M

(%)

*

Low dose of T3 induces p27 expression in Low dose of T3 induces p27 expression in

HSC-T6HSC-T6

p27

Tub

1 2 3 4

p27 expression

0

20

40

60

80

100

120

140

con T3-7 T3-9 T3-11

Average of 5 exp

*

T3 inhibits CycD1 in LX-2 after 48hT3 inhibits CycD1 in LX-2 after 48h

Cyclin D1 in LX-2

0.0

20.0

40.0

60.0

80.0

100.0

120.0

con-HDM T3-7M T3-9M T3-11M

*

Average of 3 exp

CyclinD1

Tubulin

T3 induces p27 expression in LX-2 after T3 induces p27 expression in LX-2 after 48h starvation in STD48h starvation in STD

P27

Tubulin

p27 in LX-2

0.0

50.0

100.0

150.0

200.0

con-HDM T3-7M T3-9M T3-11M

Average of 3 exp

*

How does T3 affect collagen I How does T3 affect collagen I expressionexpression? ?

C

TGF-

T3

Collagen I

C T3

GAPDH

1 2.7 2.4 7

The Collagen promoterThe Collagen promoter

Promoter of Collagen 1A1Collagen 1A1 Gene

5’ UTR

TRETREAP-1 TREAP-1 AP-1

ATG

Sp1 Sp1

Collagen Promoter Luciferase gene

Reporter Vector

LUCLUC

LUC

LUCLUC

Transfection procedureTransfection procedure

Cell

Col1A1 promoter-Luc

Transfection Reagent

Transfection procedureTransfection procedure

Incubation for 24-48h then lysis and resultsreading

Col1A1-Luc

Cell

Cell

LUC LUC

LUC

LUC

T3T3 affects collagen promoter activityaffects collagen promoter activity

T3 effect promoter activity in HSC

0

20

40

60

80

100

120

140

con T3 10-7 T3 10-9 T3 10-11

(%)

* *

T3 effects collagen promoter in LX-2

0

20

40

60

80

100

120

140

con

T310

-7M

T310

-9M

T310

-11M

con

-TRT3

-7+ TR

T3-9

+ TR

(%)

SummarySummary

Low dose of T3 (10 –11M):

• Inhibits proliferation of rat and human hepatic stellate cell lines.

• Down regulates cyclin D1 and up regulates cell cycle inhibitor p27.

• Inhibits the promoter activity of collagen I.

AimsT3/TR1 • Mechanisms of T3 inhibition of cell proliferation

(cyclin D, c-myc, p27)• Regulation of transcription of collagen I gene by T3

in rat and human HSC• Phenotype of TR1 and TR1 overexpressing HSC

and of siRNA of TR1

T4/Integrin αvβ3

•Regulation of HSC proliferation by T4•Effect of T4 and ECM on growth factor receptors•Effect of T4 on HSC activation

CYTOPLASM

NUCLEUS

RXR TRT3 Col. I transcriptionFibrosis Activation

αV β3

Extracellular receptors likePDGFR, TGFbR1and integrin αVβ3.

Proliferation Genes activation

T4/T3Vitronectin

(ECM)

Fibronectin(ECM)T4

DeiodinaseStat3

MAPKCyclin D

p

p

The integrins are the fastest sense of the cell!

T4 induces proliferation of human T4 induces proliferation of human hepatic stellate cells LX-2 and hepatic stellate cells LX-2 and

expression of cyclin Dexpression of cyclin D

LX-2 Proliferation

0

50

100

150

200

TCP T410-7

Cyclin D

*TCP - Plastic

T4 increases phosphorylation of MAPK T4 increases phosphorylation of MAPK and decreases phosphorylation of STAT3and decreases phosphorylation of STAT3

pMAPK 42/44, pSTAT3

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

pMAPK - 44 pMAPK - 42 pSTAT 3

c

T4

T4 increases expression of integrin T4 increases expression of integrin αv in LX-2 on in LX-2 on TCP and FibronectinTCP and Fibronectin

PDGFR

TGFbR1

- - -+ + +

TCP Fibronectin Vitronectin

α v

PDGFR

TGFbR1

αv

±T4

*TCP - Plastic

The futureT3/TR antagonists

• TR antagonists : 250,000 compounds screened using a virtual program, the most potent used to design new compounds that were synthesized and tested

• Dronerarone- new TR1 specific antagonist

T4/ v3 antagonists

• Vitaxin- antibody against v3, clinical trials