regulation of limb development by fibroblast growth factor (fgf) system pavel krejci
TRANSCRIPT
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REGULATION OF LIMB DEVELOPMENT BY FIBROBLAST GROWTH FACTOR (FGF) SYSTEM
Pavel Krejci
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4 receptors: FGFR1-422 ligands: FGF1-23
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wt Fgf10-/-
AER
wt Fgf10-/-
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Fgf8/Fgf4-/-wt Fgf8/Fgf4-/-wt
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How do the limbs grow?
resting cartilage
bone
proliferating cartilage
proliferating cartilage
age
resting cartilage
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FGFR3
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Fgfr3+/-
Fgfr3+/-
Fgfr3-/-
Tail
Fgfr3-/-
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FGFR3-related skeletal dysplasia
HypochondroplasiaAchondroplasiaSADDANThanatophoric Dysplasia
STATURE AC
TM
TK
I
II
III
FGF binds here
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FGFR3-related skeletal dysplasia
Achondroplasia
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Thanatophoric Dysplasia
- short long bones
- brachydactyly
- macrocephaly
- low nasal bridge
- spinal stenosis
- temporal lobe malformations
healthy
TD
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Fgfr3+/-
Loss-of-function vs. Gain-of-function
Fgfr3-/-
Fgfr3+/+
Fgfr3TD/+
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healthy TD
resting
proliferating
hypertrophic
bone
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0.01 0.1 1 10 1000
5e+4
1e+5
2e+5
2e+5FGF1 FGF1 + HeparinFGF2FGF9ControlHeparin
3 H-th
ymid
ine
( cpm
x 1
03 )
0 10 20 30 400
20
40
60
80
100
G0-G1SG2-M
FGF2 (h)
% o
f cel
ls
FGF concentration (ng/ml)
FGFR3 inhibits chondrocyte proliferation by arresting
their cell cycle in G1 phase
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….via inhibition of cdk activity necessary for progression through the G1 phase of a cell cycle
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control FGF
FGF alters the cartilage-like phenotype of chondrocytes
FGF (h)
- 0.5 1 4 8 12 24 48 72
aggrecan
collagen II
collagen I
GAPDH
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proMMP2
……via MMP-mediated degradation of extracellular matrix
MMP2
MMP3
MMP9
MMP10
MMP13
GAPDH
con
trol
FG
F2
proMMP9 matureMMP9
intermediate
matureMMP2
proMMP13 matureMMP13
con
trol
FG
F2
proMMP2
mature MMP3/10
pro MMP3/10
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FGF2 activates Erk and p38 MAPK, PLC and PKB in chondrocytes
C1 C2 1’ 5’ 10’ 30’ 1h 2h 4h 8h
FGF2
P-Erk1/2
Erk1/2
P-p38
p38
P-PLC1 PLC1
P-PKB
PKB
1’ 5’ 30’ 1h 4h 6h 12h 18h 24h 0h 4h 12hF F/H F F/H F F/H F F/H F F/H F F/H F F/H F F/H F F/H - - H - H
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0.001 0.01 0.1 1 10 1000
20
40
60
80
.001 .01 .1 1 10 100Inhibitor concentration (M)
U0126
cells
/wel
lx10
3
0
2
4
6
8
10
12
1 2
- U0126
RasV12
cells
/wel
lx10
3
cells
/wel
lx10
3
0
20
40
60
80
control f1 f10 f100
Series1Series2
cel
l col
onie
s/10
0 ce
lls (%
)
RasRasN17
0.001 0.01 0.1 1 10 1000
20
40
60
80
FGF2control
SU5402
……but only Ras/Erk activity is involved in FGF-induced growth arrest
80
60
40
20
0
80
60
40
20
0
- F1 F10 F100
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Erk MAP kinase activity is necessary for FGFR3 phenotype in cartilage
FGFR3
Ras
Raf-1
MEK
Erk
FRS2
FGF2
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C-type Natriuretic Peptide (CNP) over-expression results in skeleton overgrowth in mice
CNP
wild-type
CNP over-expression??? CNPwild-type
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wild-type
Fgfr3Ach
Fgfr3Ach/CNP
CNP rescues dwarfism caused by ACH mutation in FGFR3
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Series2Series3
control
FGF2
FGF2 control
_ 0.1 0.2 0.5 1 _ _ _ _ CNP [M] _ _ _ _ _ 10 100 200 500 pCPT-cGMP [M]
50
40
30
20
10
0
cells
per
wel
l [x1
04 ]CNP counteracts FGF2-mediated chondrocyte growth arrest
through cGMP-dependent pathway
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CNP antagonizes FGF2-mediated loss of cartilage extracellular matrix in chondrocytes
control
CNP
pCPT-cGMP
unstimulated FGF2
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CNP counteracts FGF2-mediated activation of Erk MAP kinase in chondrocytes
_ + + + + _ _ + + + + _ FGF2 _ _ 0.1 0.2 0.5 0.1 _ _ _ _ _ _ CNP [M] _ _ _ _ _ _ _ _ 100 200 500 100 pCPT-cGMP [M]
Erk1/2
P-Erk1/2
Ras total
Ras-GST
FGF2 _ + + _
CNP _ _ + +
Erk1/2
P-Erk1/2
FGF2 _ + + _
CNP _ _ + + _Ab
Erk1/2
Raf-1 IP: Raf-1
WB P-MEK
P-Erk1/2
FGFR3
Ras
Raf-1
MEK
Erk
FRS2
FGF2
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CNP inhibits Erk MAP kinase module at the Raf level
FGFR3
Ras
CNP
cGMP
PKG Raf-1
MEK
Erk
NP-R FRS2
STOP
FGF2
Growth arrest
Matrix degradation
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Is protein kinase C (PKC) involved in FGFR3- mediated activation of Erk in chondrocytes?
FGF2 _ + + + + _ + + + _ _ + + + + _
Gö6983 (M) _ _ 1 5 10 5 _ _ _ _ _ _ _ _ _ _
Bis I (M) _ _ _ _ _ _ 1 5 10 5 _ _ _ _ _ _
Gö6976 (M) _ _ _ _ _ _ _ _ _ _ _ _ 1 5 10 5
Erk2
P-Erk1/2
FGF2 _ + + + + + + + + _
GFX (M) _ _ 0.5 1 5 10 20 50 _ 10
vehicle (DMSO) _ _ + + + + + + + +
kinase
P-Elk
FGFR3
Ras
Raf-1
MEK
Erk
FRS2
FGF2
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FRS2
FGFR3
-Y-FRS2P
FGF2 _ + + + + + + + + + +
Bis I (M) _ _ 1 5 10 20 50 _ _ _ _
SU5402(M) _ _ _ _ _ _ _ 1 10 20 _
vehicle(DMSO) _ _ + + + + + + + + +
kinase
Ras
Raf-1
MEK
Erk
FGF2
Gab1
SHP2GRB2SOS
GRB2SOS
FRS2
FGFR3
FGF2 _ 5’ 10’ 30’ 1h _ 30’ 30’ _
Bis I _ _ _ _ _ _ _ + + IP: FRS2
WB FRS2
SHP2
IP: Gab1
WB Gab1
SHP2
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Protein kinase C inhibitor Bisindolylmaleimide I (Bis I)suppresses the FGF2-mediated activation of Erk MAP kinase pathway in
chondrocytes by preventing the SHP2 association with FRS2 and Gab1 adaptor proteins
FGFR3
Ras
Raf-1
MEK
Erk
FRS2
FGF2
Gab1 SHC
Grb2-SOSShp2-Grb2-SOS
Bis I Bis I
?
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FGFR3 associates with STAT1 and acts as STAT1-kinase in chondrocytes
CKI
FGFR3
STAT1
Growth arrest
?
?
?
FLAG
actin
FLAG IP: FLAG WB
STAT1
FGFR
3-w
t
FGFR
3-K
650M
FGFR
3-K
508M
GFP
FGFR3-wt
substrate: STAT1 + + + + + +
SU5402(M) _ + _ _ + _
ATP + + _ + + _
FGFR3-K650E
P-Y701-STAT1
STAT1
FGFR3
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Chronic FGF stimulus inhibits cytokine/STAT signaling in chondrocytes
STAT3-YFP DAPI merge DIC/merge
control
IL6 30mFGF2 48h
FGF2 2h
IL6 30m
FGF2 48h
STAT3
STAT3-YFP
75
150kDa
100
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IL11, LIF
CishSocs1Socs3
FGF2
IL6
IL6RA Shp2
gp130
STAT3
STAT3
nucleus
FGFR3
Frs2
Chronic FGF stimulus inhibits cytokine/STAT signaling in chondrocytes
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FGF2 causes premature senescence in chondrocytes
SA--gal
control FGF2 FGF2 [ng/ml] - 5 10 20
0
12
24
36
48
SA-
-gal
pos
itiv
e ce
lls
per
50 c
ells
[n=
10]
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FGF2 signals towards the cytoskeleton in chondrocytes
Phalloidin DAPI merge
control
FGF2
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actin
FGF2: C1 15‘ 1h 3h 6h 12h 24h 48h 72h C2 C3
Wnt
b-catenin
Where is Wnt?
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Frs2, Gab1, SHC
CKI
?
STAT1
Ras/Raf/MEK/Erk
STAT1
CKI MMP
PKC
CNP
NPR-B
cGMP
PKG
2001 NOW IL6, LIF, IL11, IFN
STAT1/3
growth arrest
growth arrest
matrix degradation
?
?
? proliferation
SOCS1/3
FGFR3
FGFR3