Transcript (T3/T4) , their receptors and liver fibrosis

Triiodothyronine and Lthyroxine (T3/T4) , their
receptors and liver fibrosis
Effect of T3 on liver fibrosis
Treatment
Time
TAA
6 weeks
Pathological score
(0-3)
0
12 weeks
2.7+/- 0.1
TAA+PTU
12 weeks
0.85+/-0.15
TAA+MMI
12 weeks
0.55+/-0.12
TAA+surgical
thyroid removal
12 weeks
0.10+/-0.06
TAA+T3 (Eltroxin)
6 weeks
1.75+/-0.20
TAA+T3 (Eltroxin)
12 weeks
2.75+/-0.10
Classic thyroid hormone
receptors
A/B
TR beta 1
C
DNA
D
E
461
ligand
410
TR alpha 1
86
Affinity of T3: TR alpha1 >TR beta 1
Two genes, 4 isoforms
82
Thyroid hormone
receptor alpha 1
Histone deacetylase (HDAC),
inactive chromatin
NCoR SMRT SUN-CoR
corepressor
Corepressor
AF-1
RXR
DNA binding
Ligand
Phosphorylation
Trip1 TIF1 TRAP
CBP ACTR
T3
AF-1
RXR
DNA binding
Coactivator
Ligand
AF-2
Histone acetylase,
active chromatin
PEPCK, growth hormone,
Collagen I (cardiac fibroblasts),
Myosin a , fatty acid synthetase
TSH, c-myc, EGFR, prolactin, CD44
Phenotypic features of hepatic
stellate cell activation
Rat hepatic stellate cells express
TRα1, but not TRβ1
TRα1
TRβ1
GAPDH
Rat HSC-T6
Human LX-2
The second T4/T3 receptor:Integrin
avb3 and hepatic stellate cells
 avb3 is a receptor for L-thyroxine -T4
• Receptor for vitronectin and fibronectin
• Vitronectin induced in liver injury;
avb3 present only on activated stellate cells
• SiRNA of avb3
MMP-9
apoptosis via induction of
PDGFR
I
TGFBR
I
proliferation
II
Collagen I
pSTAT3 pMAPK
T3
pSTAT3
pMAPK
Fibrosis
TRα1
deiodination T3
T4
T3
PDGFR
vitronectin
T4
αv
β3
fibronectin
General aims
• Verify our working hypothesis
• Role of T4/T3 and their receptors nuclear
receptors TRa1 and integrin avb3, in inducing
hepatic fibrosis
• Resolution of fibrosis by blocking TRa1 and
integrin avb3 signaling
Specific aims
T3/TRa1
• 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 TRa1 and TRb1 overexpressing HSC
and of siRNA of TRa1
T4/Integrin avb3
• 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 proliferation ?
T3 inhibits HSC proliferation-24h
T3 inhibits HSC proliferation-48h
120.00
120.00
100.00
100.00
*
*
80.00
60.00
60.00
40.00
40.00
20.00
20.00
0.00
*
80.00
*
0.00
con
T3 10-7M
Average of 5 exp
T3 10-9M
T3 10-11M
con
T3 10-7M
Average of 6 exp
T3 in low doses inhibits proliferation of rat
HSC at 24 and 48h
T3 10-9M
T3 10-11M
What about humans?
Low dose of T3 inhibits LX-2 proliferation
120
100
(%)
80
60
40
20
0
con-24h
T3 10-11M
con-48h
T3 10-11M
How does T3 inhibits proliferation
Cell cycle arrest
Apoptosis
Cell Cycle
In neuroblastoma cells
Transcription of Cyclin D1
T3+TRb
Cyclin D1 Promoter
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.
Cyclin D1
CKI-p27
CDK2
E2F
Rb
PP
Rb-PPP
Growth arrest and differentiation
E2F
E2F
T3+TRb
nTRE
C-Myc
5’UTR of c-Myc
DP
G1 S
Cell proliferation
Gracia-Silva,JBC 1999/Toxicology 2002
Low dose of T3 inhibits Cyclin D1
in HSC-T6
T3 effect on CycD1 in HSC
120.00
100.00
*
(%)
80.00
60.00
40.00
20.00
0.00
con
Average of 6 exp
T3 10-7M
T3 10-9M
T3 10-11M
Low dose of T3 induces p27 expression in
HSC-T6
p27
Tub
1
2
3
4
*
Average of 5 exp
T3 inhibits CycD1 in LX-2 after 48h
CyclinD1
Tubulin
*
Average of 3 exp
T3 induces p27 expression in LX-2 after
48h starvation in STD
P27
Tubulin
*
Average of 3 exp
How does T3 affect collagen I
expression ?
TGF-b
Collagen I
GAPDH
1
2.7
2.4
7
The Collagen promoter
Collagen Promoter
Luciferase gene
Reporter Vector
LUC
LUC
LUC
LUC
LUC
Collagen 1A1 Gene
Promoter of Collagen 1A1
AP-1 Sp1
AP-1
TRE
Sp1
TRE
TRE AP-1
5’ UTR
ATG
Transfection procedure
Col1A1 promoter-Luc
Transfection Reagent
Cell
Transfection procedure
Cell
Cell
Incubation for 24-48h
then lysis and results
reading
LUC
Col1A1-Luc
LUC
LUC
LUC
T3 affects collagen promoter activity
T3 effect promoter activity in HSC
140
120
*
*
T3 10-9
T3 1011
80
60
40
20
0
T3 effects collagen promoter in LX-2
140
120
100
80
60
40
20
T3
-9
+
TR
T3
-7
+
TR
co
n
-T
R
T3
10
-1
1M
T3
10
-9
M
0
T3
10
-7
M
T3 10-7
co
n
con
(%)
(%)
100
Summary
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.
Aims
T3/TRa1
• 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 TRa1 and TRb1 overexpressing HSC
and of siRNA of TRa1
T4/Integrin αvβ3
•Regulation of HSC proliferation by T4
•Effect of T4 and ECM on growth factor receptors
•Effect of T4 on HSC activation
Fibronectin
(ECM)
T4/T3
T4
αV
Vitronectin
(ECM)
β3
CYTOPLASM
p
Deiodinase
Cyclin D
MAPK
p
Stat3
T3
RXR TR
Proliferation
Genes activation
Col. I transcription
Fibrosis Activation
Extracellular receptors like
PDGFR, TGFbR1
and integrin αVβ3.
NUCLEUS
The integrins are the fastest sense of the cell!
T4 induces proliferation of human
hepatic stellate cells LX-2 and
expression of cyclin D
Cyclin D
LX-2 Proliferation
200
150
100
50
0
TCP
* TCP - Plastic
T410-7
T4 increases phosphorylation of MAPK
and decreases phosphorylation of STAT3
pMAPK 42/44, pSTAT3
1.8
1.6
1.4
1.2
1
c
0.8
0.6
0.4
T4
0.2
0
pMAPK - 44
pMAPK - 42
pSTAT 3
T4 increases expression of integrin αv in LX-2 on
TCP and Fibronectin
PDGFR
TCP
±T4
-
+
Fibronectin Vitronectin
-
+
-
+
PDGFR
TGFbR1
TGFbR1
αv
* TCP - Plastic
αv
The future
T3/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 TRa1 specific antagonist
T4/ avb3 antagonists
• Vitaxin- antibody against avb3, clinical trials