Molecular roots of FGFR3-related skeletal dysplasia
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Molecular roots of FGFR3-related skeletal dysplasia. Pavel Krejci , PhD Institute of Experimental Biology, Masaryk University, Brno Department of Cytokinetics, Institute of Biophysics AVCR, Brno Cedars-Sinai Medical Center, Los Angeles, USA.

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Molecular roots of FGFR3-related skeletal dysplasia

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Molecular roots of fgfr3 related skeletal dysplasia

Molecular roots of FGFR3-related skeletal dysplasia

Pavel Krejci, PhD

Institute of Experimental Biology, Masaryk University, Brno

Department of Cytokinetics, Institute of Biophysics AVCR, Brno

Cedars-Sinai Medical Center, Los Angeles, USA


Molecular roots of fgfr3 related skeletal dysplasia

NavaznostiPrednaska navazuje na obecne vymezeni podstaty bunecneho signalovaniObecne zakonitosti budou demonstrovany na priklade FGF signalingu v skeletalni dysplasii


Molecular roots of fgfr3 related skeletal dysplasia

How do the limbs grow?

age

resting cartilage

proliferating cartilage

bone

proliferating cartilage

resting cartilage


Molecular roots of fgfr3 related skeletal dysplasia

FGFR3-related skeletal dysplasia

Achondroplasia


Molecular roots of fgfr3 related skeletal dysplasia

FGFR3-related skeletal dysplasia

STATURE

I

AC

Hypochondroplasia

Achondroplasia

SADDAN

Thanatophoric Dysplasia

II

FGF binds here

III

TM

TK


Molecular roots of fgfr3 related skeletal dysplasia

Thanatophoric Dysplasia

healthy

- short long bones

- brachydactyly

- macrocephaly

- low nasal bridge

- spinal stenosis

- temporal lobe malformations

TD


Molecular roots of fgfr3 related skeletal dysplasia

resting

proliferating

hypertrophic

bone

healthy

TD


Molecular roots of fgfr3 related skeletal dysplasia

Sahni et al., Genes Dev 1999, 13, 1361-66.

Sahni et al., Development 2001, 128, 2119-29.

FGFR3

?

STAT1

?

CKI

?

FGFR3-related skeletal dysplasia

Growth arrest


Molecular roots of fgfr3 related skeletal dysplasia

FGF inhibitschondrocyte proliferation by arresting their cell cycle in G1 phase

3H-thymidine (cpm x 103)

% of cells

FGF2 (h)

FGF concentration (ng/ml)


Molecular roots of fgfr3 related skeletal dysplasia

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

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


Molecular roots of fgfr3 related skeletal dysplasia

FGF alters the cartilage-like phenotype of chondrocytes

FGF (h)

control FGF

- 0.5 1 4 8 12 24 48 72

aggrecan

collagen II

collagen I

GAPDH


Molecular roots of fgfr3 related skeletal dysplasia

……via MMP-mediated degradation of extracellular matrix

control

FGF2

control

FGF2

MMP2

proMMP9

matureMMP9

MMP3

proMMP2

proMMP2

MMP9

intermediate

MMP10

matureMMP2

MMP13

pro MMP3/10

mature MMP3/10

GAPDH

proMMP13

matureMMP13

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


Molecular roots of fgfr3 related skeletal dysplasia

FGF2 activates Erk and p38 MAPK, PLCg and PKB in chondrocytes

FGF2

C1 C2 1’ 5’ 10’ 30’ 1h 2h 4h 8h

P-Erk1/2

Erk1/2

P-p38

p38

P-PLCg1

PLCg1

1’ 5’ 30’ 1h 4h 6h 12h 18h 24h 0h 4h 12h

F 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

P-PKB

PKB


Molecular roots of fgfr3 related skeletal dysplasia

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

SU5402

80

Ras

RasN17

60

40

cells/wellx103

cell colonies/100 cells (%)

20

0

- F1 F10 F100

U0126

80

RasV12

60

40

cells/wellx103

cells/wellx103

20

0

.001 .01 .1 1 10 100

Inhibitor concentration (mM)

- U0126


Molecular roots of fgfr3 related skeletal dysplasia

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.


Molecular roots of fgfr3 related skeletal dysplasia

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

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


Molecular roots of fgfr3 related skeletal dysplasia

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

50

control

40

30

cells per well [x104]

control

20

FGF2

10

FGF2

0

_ 0.1 0.2 0.5 1_ _ _ _CNP [mM]

_ _ _ _ _ 10 100 200 500pCPT-cGMP [mM]


Molecular roots of fgfr3 related skeletal dysplasia

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

unstimulated FGF2

control

CNP

pCPT-cGMP


Molecular roots of fgfr3 related skeletal dysplasia

CNP counteracts FGF2-mediated activation of Erk MAP kinase in chondrocytes

FGF2

_+ ++ + _ _ + + + + _FGF2

_ _0.1 0.2 0.5 0.1_ _ _ _ _ _CNP [mM]

__ _ _ _ _ _ _100 200 500 100pCPT-cGMP [mM]

FGFR3

P-Erk1/2

FRS2

Erk1/2

Ras

FGF2_ + + _

CNP_ _ ++_Ab

FGF2_ ++ _

CNP_ _ ++

IP:Raf-1

Raf-1

Raf-1

Ras-GST

WB

P-MEK

MEK

Ras total

P-Erk1/2

P-Erk1/2

Erk

Erk1/2

Erk1/2


Molecular roots of fgfr3 related skeletal dysplasia

CNP inhibits Erk MAP kinase module at the Raf level

FGF2

CNP

FGFR3

NP-R

FRS2

cGMP

STOP

Ras

PKG

Raf-1

Growth arrest

MEK

Erk

Matrix degradation

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


Molecular roots of fgfr3 related skeletal dysplasia

Is protein kinase C (PKC) involved in FGFR3- mediated activation of Erk in chondrocytes?

FGF2

FGF2_+ ++ + _ ++ + __ + ++ + _

Gö6983 (mM)__ 1 5 10 5 _____ _ _ _ _ _

Bis I (mM)______ 1 5 10 5 _ _ _ _ _ _

Gö6976 (mM)____________ 1 5 10 5

FGFR3

FRS2

Ras

P-Erk1/2

Raf-1

Erk2

MEK

FGF2_+ ++ + + ++ + _

GFX (mM)__ 0.5 1 5 10 20 50 _ 10

vehicle (DMSO)__ + ++ + + ++ +

kinase

Erk

P-Elk


Molecular roots of fgfr3 related skeletal dysplasia

FGF2_+ + _

Bis I__ + +

FGF2_+ + _

Bis I__ + +

IP:Raf-1

FGF2

Raf-1

WB

Ras-GST

FGFR3

MEK1

Ras total

FRS2

-MEK1

-MEK1

-MEK1

P

P

P

MEK1

Ras

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

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

Raf inhibitor(mM)____ _ _ __ 1_ _ 1

vehicle (DMSO)__++ + + + + + + + +

Raf-1

kinase

MEK

Raf-1

Erk

MEK1


Molecular roots of fgfr3 related skeletal dysplasia

FGF2_ ++ ++ ++ +++ +

Bis I(mM)__ 1 5 10 20 50_ _ _ _

SU5402(mM)_ _ _ _ _ _ _1 10 20_

vehicle(DMSO)__ + + + + + + + + +

FGF2

kinase

FGFR3

FRS2

Gab1

IP:FRS2

SHP2

SHP2

GRB2

SOS

WB

FRS2

-Y-FRS2

P

FGF2_5’ 10’ 30’ 1h_30’ 30’_

Bis I_ __ __ _ _+ +

IP:Gab1

SHP2

GRB2

SOS

WB

Ras

Gab1

FGFR3

Raf-1

FRS2

MEK

Erk


Molecular roots of fgfr3 related skeletal dysplasia

Bis I

Bis I

Shp2-Grb2-SOS

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

FGF2

FGFR3

?

FRS2

Grb2-SOS

Gab1

SHC

Ras

Raf-1

MEK

Erk

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


Molecular roots of fgfr3 related skeletal dysplasia

FGFR3-K508M

FGFR3-wt

FGFR3-K650M

GFP

FLAG

actin

IP: FLAG

FLAG

WB

STAT1

FGFR3-K650E

FGFR3-wt

substrate: STAT1++ ++ ++

SU5402(mM)_ +_ _ +_

ATP++_++_

FGFR3 associates with STAT1 and

acts as STAT1-kinase in chondrocytes

FGFR3

?

STAT1

?

?

CKI

P-Y701-STAT1

Growth arrest

STAT1

FGFR3


Molecular roots of fgfr3 related skeletal dysplasia

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

FGFR3

STAT1

?

?

CKI

Growth arrest

Krejci et al., J Cell Sci. In revision


Molecular roots of fgfr3 related skeletal dysplasia

150kDa

STAT3-YFP

100

STAT3

75

STAT3-YFP DAPI merge DIC/merge

control

FGF2 2h

FGF2 48h

IL6 30m

FGF2 48h

IL6 30m

Chronic FGF stimulus inhibits cytokine/STAT signaling in chondrocytes


Molecular roots of fgfr3 related skeletal dysplasia

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

IL6__++______

IL11____++__ _ _

LIF______ ++_ _

IFNg________++

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

IL6 (minutes) __5 5 10 10 30 30 60 60

P-Y705-STAT3

1.2

STAT3

0.9

actin

STAT3 activation (OD450-650nm)

0.6

0.3

0

Chronic FGF stimulus inhibits cytokine/STAT signaling in chondrocytes


Molecular roots of fgfr3 related skeletal dysplasia

FGF2

IL6

IL11, LIF

FGFR3

IL6RA

Shp2

Frs2

gp130

Cish

Socs1

Socs3

STAT3

nucleus

STAT3

Chronic FGF stimulus inhibits cytokine/STAT signaling in chondrocytes

Krejci et al., manuscript in preparation


Molecular roots of fgfr3 related skeletal dysplasia

control FGF2

FGF2 [ng/ml] - 5 10 20

48

36

24

12

0

SA-b-gal

FGF2 causes premature senescence in chondrocytes

SA-b-gal positive cells

per 50 cells [n=10]

Krejci et al., manuscript in preparation


Molecular roots of fgfr3 related skeletal dysplasia

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

Krejci et al., manuscript in preparation


Molecular roots of fgfr3 related skeletal dysplasia

FGF2 signals towards the cytoskeleton in chondrocytes

Phalloidin DAPI merge

control

FGF2


Molecular roots of fgfr3 related skeletal dysplasia

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

b-catenin

actin

FGF2 accumulates b-catenin in chondrocytes


Molecular roots of fgfr3 related skeletal dysplasia

20012007

FGFR3

IL6, LIF, IL11, IFNg

FGFR3

CNP

?

PKC

NPR-B

STAT1

Frs2, Gab1, SHC

STAT1/3

STAT1

?

cGMP

CKI

?

Ras/Raf/MEK/Erk

PKG

Growth arrest

CKIMMP

Growth arrest

Matrix degradation


Molecular roots of fgfr3 related skeletal dysplasia

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 FGFR3

Prochon Biotech Ltd.


Molecular roots of fgfr3 related skeletal dysplasia

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

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