regulation of cell function by fgfr3 receptor tyrosine kinase pavel krejci medical genetics...
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Regulation of cell function by FGFR3 receptor tyrosine kinase
Pavel Krejci
Medical Genetics Institute, Cedars-Sinai Medical Center, Los Angeles, CA Institute of Experimental Biology, Masaryk University, Brno, Czech Republic
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Fgfr3+/-
Fgfr3+/-
Fgfr3-/-
Tail
Fgfr3-/-
Cell 1996, 84(6):911-21.
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Achondroplasia
Thanatophoric Dysplasia
FGFR3-related skeletal dysplasias
Nat Genet 1995, 9:321-8..
- short long bones- brachydactyly- macrocephaly- low nasal bridge- spinal stenosis- temporal lobe malformations
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FGFR3-related skeletal dysplasia
HypochondroplasiaAchondroplasiaSADDANThanatophoric Dysplasia
STATURE
AC
TM
TK
I
II
III
FGF binds here
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Skeleton: hypochondroplasia, achondroplasia, thanatophoric dysplasia, SADDAN, Muenke syndromeSkin: epidermal nevi, seborrhaeic keratosis, acanthosis nigricans Cancer: multiple myeloma, bladder cancer, seminoma
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Exp Cell Res. 2004;297:152-64.J Cell Sci. 2005; 118: 5089-100. J Biol Chem. 2007 ;282:2929-36. Pediatr Res. 2007; 61(3):267-72.Invest New Drugs 2007; 25:391-95. PLoS One 2008; 3:e3961.J Cell Sci 2008; 121:272-81. Cell Signal 2009; 21:151-60.Hum Mol Genet. 2009; 18:227-40. J Biol Chem 2010; 285:20644-53.Bone 2010; 47:102-10. Leukemia 2011; 25:538-50.Human Mutation 2012; 33:29-41
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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-t
hym
idin
e (
cpm
x 1
03 )
0 10 20 30 400
20
40
60
80
100
G0-G1SG2-M
FGF2 (h)
% o
f ce
lls
FGF2 concentration (ng/ml)
FGFR3 inhibits chondrocyte proliferation by arresting their cell cycle in G1 phase
Exp Cell Res 2004, 297:152-64.
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control FGF2
FGFR3 alters the cartilage-like phenotype of chondrocytes
FGF2 (h)
M C 0.5 1 4 8 12 24 48 72
aggrecan
collagen II
collagen I
GAPDH
proMMP9 matureMMP9
intermediate matureMMP2
proMMP13 matureMMP13
con
trol
FG
F2
proMMP2
mature MMP3/10
pro MMP3/10
J Cell Sci. 2005; 118: 5089-100.
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FGFR3 causes premature senescence in chondrocytes
SA--gal
control FGF2 FGF2 control
Bone 2010; 47:102-10.
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C1 C2 1’ 5’ 10’ 30’ 1h 2h 4h 8h
FGF2
P-Erk1/2
Erk1/2
P-p38
p38
P-PLC1
PLC1
0.001 0.01 0.1 1 10 100
0
20
40
60
80
.001 .01 .1 1 10 100Inhibitor concentration (M)
U0126
cel
ls/w
ellx
103
80
60
40
20
0
0
20
40
60
80
control f1 f10 f100
Series1Series2
cel
l co
lon
ies/
100
cells
(%
)
RasRasN17
- F1 F10 F100
FGFR3 arrests chondrocyte growth via RAS-ERK MAP kinase pathway
Exp Cell Res 2004, 297:152-64.
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Ras
Raf-1
MEK
Erk
FGF2
FRS2
SHP2GRB2SOS
GRB2SOS
GAB1 SHC
FGFR3
J Biol Chem 2007; 282:2929-36.
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CKI
FGFR3
STAT1
Growth arrest
Skeletal dysplasia
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FGFR3 associates with STAT1 and acts as STAT1-kinase in chondrocytes
CKI
FGFR3
STAT1
Growth arrest
?
?
?
FGFR3
actin
FGFR3 IP: STAT1
WB STAT1
FG
FR
3-w
t
FG
FR
3-K
650M
FG
FR
3-K
508M
GF
P
FGFR3-wt
substrate: STAT1 + + + + + +
SU5402(M) _ + _ _ + _
ATP + + _ + +
_
FGFR3-K650E
P-Y701-STAT1
STAT1
FGFR3
Skeletal dysplasia
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J Cell Sci 2008;121:272-81.
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PLoS One 2008;3:e3961.
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STAT3-YFP DAPI merge DIC/merge control
IL6 30m FGF2 48h
FGF2 2h
IL6 30m
FGF2 48h
IL11, LIF
CishSocs1Socs3
FGF2
IL6
IL6RA Shp2
gp130
STAT3
STAT3
nucleus
FGFR3
Frs2
Chronic FGF stimulus inhibits cytokine/STAT signaling in chondrocytes
Cell Signal 2009;21:151-60.
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Frs2, Gab1, SHC
CKI
?
STAT1
Ras/Raf/MEK/Erk
STAT1
CKI MMP
PKC
CNP
NPR-B
cGMP
PKG
2001 2012 IL6, LIF, IL11, IFN
STAT1/3
growth arrest
matrix degradation
?
?
?
proliferation
SOCS1/3
FGFR3
growth arrest
FGFR3
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actin
FGF2: C1 15‘ 1h 3h 6h 12h 24h 48h 72h C2 C3
-catenin
Wnt
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submitted
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Fgfr3Ach
Kazuwa Nakao
Nature Medicine 10, 80-86 (2004).
CNP
wild-type
Fgfr3Ach/CNP
CNPwild-type
wild-type
C-natriuretic peptide (CNP)
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Series2
Series3
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 ]
control
CNP
control FGF2
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FGFR3
Ras
CNP
cGMP
PKG Raf-1
MEK
Erk
NP-R FRS2
STOP
FGF2
Growth arrest
Matrix degradation
J Cell Sci. 2005; 118: 5089-100.
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A collection of 1120 biologically active compounds supplied as DMSO solutions.
Tocriscreen™ Mini Library
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J Biol Chem 2010; 285:20644-53.
NF449
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Leukemia 2011. 25:538-50.
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Cedars-Sinai Medical Center
Los Angeles, California
Betty Mekikian
Anie Aklian
UCI, Irvine, California
Leslie Thompson
Tamara Kashiwada
Lisa Salazar
UCLA, Los Angeles, California
Matthew Schibler
Masaryk University, Brno, Czech Republic
Jirina Prochazkova
Vita Bryja
Lukas Balek
Tereza Spoustova
Tereza Pospisilova
INSERM U589, Toulouse, France
Bernard Masri
Vincent Fontaine