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