1. Role of TLR4 Ligand EDA-Fn in mediating
signal transduction of Skin Fibrosis
Sofia Hossain
8/12/11

2. •All ten TLRs encode transmembrane glycoproteins with a large extracellular
domain consisting of leucine-rich repeats (LRR), a transmembrane portion
• and a cytoplasmic tail with a conserved ‘Toll-IL-1 receptor’ (TIR) domain
that is essential for mediating signal transduction of both TLRs and the IL-1
receptor (IL-1R)
•The first characterized member of the mammalian tolls, referred to as TLR4
Introduction

3. Introduction
• Fibronectin (Fn) is a glycoprotein found in the extracellular
matrix of the cells and plays a key role in cell adhesive and
migratory behavior
• EDA-Fn serves as a prime example of proteins generated by
alternative splicing
EDAEDBEDA

4. • EDA-FN embodies a very unique embryonic splicing pattern,
garnering interest among researchers
Introduction
Embryonic
development •Tissue repair
(e.g. skin wound
healing)
•Tissue fibrosis
•angiogenesis
Development
FrequencyofSplice
patternDisplay

5. Introduction
• Tissue repair situations are characterized by differentiation
and activation of myofibroblasts
• TGFβ promotes EDA exon into the mature pre-mRNA through
investigated mechanisms
Pre-mRNA
TGFβ
EDA Exon
EDAFn
Pre- mRNA
?

6. NonSmad
PSmad
SB40
TGFβ
MEK1/2 ERK 1/2
CABL
PI3K
U0126 IM Ly20
Receptor Level
Pre-
mRN
A
TGFβ
EDA Exon
?
EDAFn
Pre- mRNA

7. 0
5
10
15
20
25
30
35
40
EDAFnandtotalFnmRNA
TGF-b - + + + +
1d 3d
SBh 68a: Effect of TGF-b on EDA-Fn and total Fn
in human foreskin fibroblasts
6.20.11
Human foreskin fibroblasts were
starved (24h, 0.1%BSA) and
treated with TGF-b (1,5 ng/ml)
After 1 day and 3days RNAs were
isolated and were subjected to
realtime qPCR
Pre-
mRN
A
EDAFn
TGFβ

8. 0
0.5
1
1.5
2
2.5
3
3.5
4
COL1A1mRNA
0
0.5
1
1.5
2
2.5
3
aSMAmRNA
Fn-EDA - + + 3d - + + 3d
SBh 68b: Effect of EDA-Fn on COL1A1 and aSMA
in human foreskin fibroblasts
6.21.11

9. Human foreskin fibroblasts were
starved (24h, 0.1%BSA) and
treated with TGF-b2 (1,5 ng/ml)
After 1 day and 3days cells were
harvested and cell lysates were
subjected to realtime qPCR
EDA-Fn Lys
EDA-Fn secreted
Tubulin
pSmad2
TGF-b - 1 5 1 5 ng/ml
1d 3d
↓
SBh 69: Effect of TGF-b on EDA-Fn
in human foreskin fibroblasts
6.23.11
Pre-
mRN
A
TGFβ
EDAFn

10. 0
2
4
6
8
10
12
14
16
18
Fn-EDAmRNA
DMSO SB431542 U0126 IM Ly 10mM
TGF-b - + - + - + - + - +
Human foreskin fibroblasts
were starved (24h) (0.1%BSA)
↓24h
Pretreated SB431542, U0126,
IM and LY204002 followed
by TGF-b2 (10ng/ml) treatment
↓24h
RNA isolated and subjected to qPCR
SBh 72: Effect of inhibitors on Fn-EDA mRNA
in human foreskin fibroblasts
6.21.11

11. 0
2
4
6
8
10
12
14
16
18
Fn-EDAmRNA
Human foreskin fibroblasts
were starved (24h) (0.1%BSA)
↓24h
Pretreated SB431542, U0126,
IM and LY204002 followed
by TGF-b2 (10ng/ml) treatment
↓24h
RNA isolated and subjected to qPCR
SBh 72: Effect of inhibitors on Fn-EDA mRNA
in human foreskin fibroblasts
6.21.11
TGFβ
MEK1/2 ERK 1/2 CABL PI3K
U0126 IM Ly20
Receptor Level
PSmad
SB40
NonSmad
DMSO SB431542 U0126 IM Ly 10mM
TGF-b - + - + - + - + - +

12. 0
2
4
6
8
10
12
14
16
18
Fn-EDAmRNA
Human foreskin fibroblasts
were starved (24h) (0.1%BSA)
↓24h
Pretreated SB431542, U0126,
IM and LY204002 followed
by TGF-b2 (10ng/ml) treatment
↓24h
RNA isolated and subjected to qPCR
SBh 72: Effect of inhibitors on Fn-EDA mRNA
in human foreskin fibroblasts
6.21.11
TGFβ
MEK1/2 ERK 1/2 CABL PI3K
U0126 IM Ly20
Receptor Level
PSmad
SB40
NonSmad
DMSO SB431542 U0126 IM Ly 10mM
TGF-b - + - + - + - + - +

13. 0
2
4
6
8
10
12
14
16
18
Fn-EDAmRNA
Human foreskin fibroblasts
were starved (24h) (0.1%BSA)
↓24h
Pretreated SB431542, U0126,
IM and LY204002 followed
by TGF-b2 (10ng/ml) treatment
↓24h
RNA isolated and subjected to qPCR
SBh 72: Effect of inhibitors on Fn-EDA mRNA
in human foreskin fibroblasts
6.21.11
TGFβ
MEK1/2 ERK 1/2 CABL PI3K
U0126 IM Ly20
Receptor Level
PSmad
SB40
NonSmad
DMSO SB431542 U0126 IM Ly 10mM
TGF-b - + - + - + - + - +

14. 0
2
4
6
8
10
12
14
16
18
Fn-EDAmRNA
Human foreskin fibroblasts
were starved (24h) (0.1%BSA)
↓24h
Pretreated SB431542, U0126,
IM and LY204002 followed
by TGF-b2 (10ng/ml) treatment
↓24h
RNA isolated and subjected to qPCR
SBh 72: Effect of inhibitors on Fn-EDA mRNA
in human foreskin fibroblasts
6.21.11
TGFβ
MEK1/2 ERK 1/2 CABL PI3K
U0126 IM Ly20
Receptor Level
PSmad
SB40
NonSmad
DMSO SB431542 U0126 IM Ly 10mM
TGF-b - + - + - + - + - +

15. 0
1
2
3
4
5
6
FnmRNA
DMSO SB431542 U0126 IM Ly 10mM
TGF-b - + - + - + - + - +
SBh 72: Effect of inhibitors on EDA-Fn mRNA
in human foreskin fibroblasts
7.11.11

16. Human foreskin fibroblasts were starved (24h) (0.1%BSA)
↓24h
Pretreated SB431542, U0126, IM and LY204002 (10 mM) followed
by TGF-b2 (10ng/ml) treatment
↓24h
Cell lysates subjected to Western blot
EDA-Fn
Tubulin
DMSO SB43 IM U0126 Ly20 10mM
TGF-b - + - + - + + - +
SBh 74: Effect of inhibitors on Fn-EDA protein
in foreskin fibroblasts
7.19.11

17. Purified EDA-Fn

18. TGFBR
EDA-Fn
Smad
TLR4 sensitizes fibroblasts to TGF-b
TGF-ß
TLR4
EDA-Fn

19. ECM
production Migration Proliferation Contraction
Adhesion
EMT
Pericyte
Endo MT
TGF-β

20. TGF-β
FnEDA
TβR
Smad2/3
Fn Myofibroblast
TLR4 α4β7 β1
?
ERK
GSKβ3
FnEDA