Molecular roots of FGFR3-related skeletal dysplasia
Download
1 / 36

Molecular roots of FGFR3-related skeletal dysplasia - PowerPoint PPT Presentation


  • 176 Views
  • Uploaded on

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.

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about ' Molecular roots of FGFR3-related skeletal dysplasia' - posy


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript

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


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


How do the limbs grow?

age

resting cartilage

proliferating cartilage

bone

proliferating cartilage

resting cartilage



FGFR3-related skeletal dysplasia

STATURE

I

AC

Hypochondroplasia

Achondroplasia

SADDAN

Thanatophoric Dysplasia

II

FGF binds here

III

TM

TK


Thanatophoric Dysplasia

healthy

- short long bones

- brachydactyly

- macrocephaly

- low nasal bridge

- spinal stenosis

- temporal lobe malformations

TD


resting

proliferating

hypertrophic

bone

healthy

TD


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


FGF inhibitschondrocyte proliferation by arresting their cell cycle in G1 phase

3H-thymidine (cpm x 103)

% of cells

FGF2 (h)

FGF concentration (ng/ml)


….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


FGF alters the cartilage-like phenotype of through the G1 phase of a cell cyclechondrocytes

FGF (h)

control FGF

- 0.5 1 4 8 12 24 48 72

aggrecan

collagen II

collagen I

GAPDH


……via MMP-mediated degradation of extracellular matrix through the G1 phase of a cell cycle

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


FGF2 activates Erk and p38 MAPK, PLC through the G1 phase of a cell cycleg 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


……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


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


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]


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

unstimulated FGF2

control

CNP

pCPT-cGMP


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


CNP inhibits Erk MAP kinase module at the Raf level in chondrocytes

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


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


FGF activation of Erk in chondrocytes2_+ + _

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


FGF2 activation of Erk in chondrocytes_ ++ ++ ++ +++ +

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


Bis I activation of Erk in chondrocytes

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


FGFR3-K508M activation of Erk in chondrocytes

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


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


150kDa arrest in chondrocytes

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


FGF2 (72h) arrest in chondrocytes_ +_+_ +_ +_+

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


FGF2 arrest in chondrocytes

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


control FGF2 arrest in chondrocytes

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


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

Krejci et al., manuscript in preparation


FGF2 signals towards the cytoskeleton in chondrocytes signaling pathway

Phalloidin DAPI merge

control

FGF2


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

b-catenin

actin

FGF2 accumulates b-catenin in chondrocytes


2001 signaling pathway2007

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


From bench to bedside: signaling pathway

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.


UCLA, Los Angeles signaling pathway

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

  • 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


ad