Molecular roots of FGFR3-related skeletal dysplasia

Pavel Krejci, PhD

Institute of Experimental Biology, Masaryk University, BrnoDepartment of Cytokinetics, Institute of Biophysics AVCR, Brno

Cedars-Sinai Medical Center, Los Angeles, USA

Navaznosti

Prednaska navazuje na obecne vymezeni podstaty bunecneho signalovani

Obecne zakonitosti budou demonstrovany na priklade FGF signalingu v skeletalni dysplasii

How do the limbs grow?

resting cartilage

bone

proliferating cartilage

proliferating cartilage

age

resting cartilage

FGFR3-related skeletal dysplasia

Achondroplasia

FGFR3-related skeletal dysplasia

HypochondroplasiaAchondroplasiaSADDANThanatophoric Dysplasia

STATURE AC

TM

TK

I

II

III

FGF binds here

Thanatophoric Dysplasia

- short long bones

- brachydactyly

- macrocephaly

- low nasal bridge

- spinal stenosis

- temporal lobe malformations

healthy

TD

healthy TD

resting

proliferating

hypertrophic

bone

CKI

?

FGFR3

STAT1

Growth arrest

?

?

FGFR3-related skeletal dysplasia

Sahni et al., Genes Dev 1999, 13, 1361-66. Sahni et al., Development 2001, 128, 2119-29.

0.01 0.1 1 10 1000

5e+4

1e+5

2e+5

2e+5FGF1 FGF1 + HeparinFGF2FGF9ControlHeparin

3H-thymidine (cpm x 103)

0 10 20 30 400

20

40

60

80

100

G0-G1SG2-M

FGF2 (h)

% of cells

FGF concentration (ng/ml)

FGF inhibits chondrocyte proliferation by arresting their

cell cycle in G1 phase

Krejci et al., Exp Cell Res 2004, 295, 152-64

….via inhibition of cdk activity necessary for progression through the G1 phase of a cell cycle

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

proMMP2

……via MMP-mediated degradation of extracellular matrix

Krejci et al., J Cell Sci 2005, 118, 5089-100

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

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

0.001 0.01 0.1 1 10 100

0

20

40

60

80

.001 .01 .1 1 10 100Inhibitor concentration (M)

U0126

cells/wellx103

0

2

4

6

8

10

12

1 2

- U0126

RasV12

cells/wellx103

cells/wellx103

0

20

40

60

80

control f1 f10 f100

Series1Series2

cell colonies/100 cells (%)

RasRasN17

- F1 F10 F1000.001 0.01 0.1 1 10 100

0

20

40

60

80

FGF2control

SU5402

……but only Ras/Erk activity is involved in FGF-induced growth arrest (Krejci et al., Exp Cell Res 2004, 295, 152-64)

80

60

40

20

0

80

60

40

20

0

Erk MAP kinase activity is necessary for

FGFR3 phenotype in cartilage

Murakami et al., Genes Dev 2004, 18, 290-305.Raucci et al., J Biol Chem 2004, 279, 1747-1756.Krejci et al., Exp Cell Res 2004, 297, 152-164.

Murakami et al., Genes Dev 2004, 18, 290-305.

Raucci et al., J Biol Chem 2004, 279, 1747-1756.

Krejci et al., Exp Cell Res 2004, 297, 152-164.

C-type Natriuretic Peptide (CNP) rescues achondroplastic phenotype in FGFR3-ACH mice.

Yasoda et al., Nature Medicine 2004, 10, 80-86

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]

CNP counteracts FGF2-mediated chondrocyte growth arrest through cGMP-dependent pathway

CNP antagonizes FGF2-mediated loss of cartilage extracellular matrix in chondrocytes

control

CNP

pCPT-cGMP

unstimulated FGF2

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

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

Krejci et al., J Cell Sci 2005, 118, 5089-100

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

MEK1 WB

-MEK1P

MEK1

-MEK1P

Raf-1

FGF2 _ + + _

Bis I _ _ + + IP: Raf-1

Raf-1

-MEK1P

MEK1

FGF2 _ + + + + + + + + + _ _

Bis I (M) _ _ 0.5 1 5 10 20 50 _ _ 10 _

Raf inhibitor (M) _ _ _ _ _ _ _ _ 1 _ _ 1

vehicle (DMSO) _ _ + + + + + + + + + +

kinase

FGFR3

Ras

Raf-1

MEK

Erk

FRS2

FGF2 FGF2 _ + + _

Bis I _ _ + +Ras-GST

Ras total

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

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

Krejci et al., J Biol Chem 2007, 282, 2929-36

FGFR3

Ras

Raf-1

MEK

Erk

FRS2

FGF2

Gab1

SHC

Grb2-SOSShp2-Grb2-SOS

Bis I Bis I

?

FGFR3 associates with STAT1 and acts as STAT1-kinase in chondrocytes

CKI

FGFR3

STAT1

Growth arrest

?

?

?

FLAG

actin

FLAG IP: FLAG

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

STAT1 and STAT3 are not involved in FGFR3-mediated growth arrest in chondrocytes

CKI

?

FGFR3

STAT1

Growth arrest

?

Krejci et al., J Cell Sci. In revision

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

Chronic FGF stimulus inhibits cytokine/STAT signaling in

chondrocytes

2

ST

AT

3 ac

tiva

tio

n (

OD

45

0-6

50

nm)

0

0.3

0.6

0.9

1.2

FGF2 (72h) _ + _ + _ + _ + _ +

IL6 _ _ + + _ _ _ _ _ _

IL11 _ _ _ _ + + _ _ _ _

LIF _ _ _ _ _ _ + + _ _

IFN _ _ _ _ _ _ _ _ + +

STAT3

actin

P-Y705-STAT3

FGF2 (48h) _ + _ + _ + _ + _ +

IL6 (minutes) _ _ 5 5 10 10 30 30 60 60

IL11, LIF

CishSocs1Socs3

FGF2

IL6

IL6RA Shp2

gp130

STAT3

STAT3

nucleus

FGFR3

Frs2

Chronic FGF stimulus inhibits cytokine/STAT signaling in

chondrocytes

Krejci et al., manuscript in preparation

FGF2 causes premature senescence in chondrocytes

SA--gal

control FGF2 FGF2 [ng/ml] - 5 10 20

0

12

24

36

48

SA

--g

al p

osit

ive c

ells

per

50 c

ells [

n=

10]

Krejci et al., manuscript in preparation

Krejci et al., manuscript in preparation

FGFR3 recruits multiple adapter proteins to activate Ras/Erk signaling pathway

FGF2 signals towards the cytoskeleton in chondrocytes

Phalloidin DAPI merge

control

FGF2

FGF2 accumulates -catenin in chondrocytes

-catenin

actin

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

FGFR3

Frs2, Gab1, SHC

CKI

?

FGFR3

STAT1

Ras/Raf/MEK/Erk

STAT1

CKI MMP

PKC

CNP

NPR-B

cGMP

PKG

2001 2007

IL6, LIF, IL11, IFN

STAT1/3

Growth arrest

Growth arrest

Matrix degradation

?

?

From bench to bedside:

Strategies to treat achondroplasia

1. Stable CNP analog – Biomarin Pharmaceutical Inc.

2. Neutralizing antibody to FGFR3

3. Small chemical inhibitor of FGFR3Prochon Biotech Ltd.

Cedars-Sinai Medical Center

Los Angeles, California

William Wilcox

Katerina Pejchalova

Betty Mekikian

Patricia Lin

Matthew Rock

Claire Rock

UCI, Irvine

California

Leslie Thompson

Tamara Kashiwada

Lisa Salazar

UCLA, Los Angeles

California

Robert Pogue

Matthew Schibler

Laboratory of Molecular Embryology

MZLU Brno, Czech republic

Vita Bryja

Jiri Pachernik

INSERM U589, Toulouse, France

Herve Prats

Bernard Masri

Vincent Fontaine