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

Fgfr3+/-

Fgfr3+/-

Fgfr3-/-

Tail

Fgfr3-/-

Cell 1996, 84(6):911-21.

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

FGFR3-related skeletal dysplasia

HypochondroplasiaAchondroplasiaSADDANThanatophoric Dysplasia

STATURE

AC

TM

TK

I

II

III

FGF binds here

Skeleton: hypochondroplasia, achondroplasia, thanatophoric dysplasia, SADDAN, Muenke syndromeSkin: epidermal nevi, seborrhaeic keratosis, acanthosis nigricans Cancer: multiple myeloma, bladder cancer, seminoma

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

healthy TD

resting

proliferating

hypertrophic

bone

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.

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.

FGFR3 causes premature senescence in chondrocytes

SA--gal

control FGF2 FGF2 control

Bone 2010; 47:102-10.

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.

Ras

Raf-1

MEK

Erk

FGF2

FRS2

SHP2GRB2SOS

GRB2SOS

GAB1 SHC

FGFR3

J Biol Chem 2007; 282:2929-36.

CKI

FGFR3

STAT1

Growth arrest

Skeletal dysplasia

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

J Cell Sci 2008;121:272-81.

PLoS One 2008;3:e3961.

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.

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

actin

FGF2: C1 15‘ 1h 3h 6h 12h 24h 48h 72h C2 C3

-catenin

Wnt

submitted

Fgfr3Ach

Kazuwa Nakao

Nature Medicine 10, 80-86 (2004).

CNP

wild-type

Fgfr3Ach/CNP

CNPwild-type

wild-type

C-natriuretic peptide (CNP)

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

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.

A collection of 1120 biologically active compounds supplied as DMSO solutions.

Tocriscreen™ Mini Library

J Biol Chem 2010; 285:20644-53.

NF449

Leukemia 2011. 25:538-50.

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