9. MECHANISMS OF DEVELOPMENT I – REGULATION OF LIMB DEVELOPMENT BY FIBROBLAST GROWTH

FACTORS (FGF)

4 receptors: FGFR1-422 ligands: FGF1-23

wt Fgf10-/-

AER

wt Fgf10-/-

Fgf8/Fgf4-/-wt Fgf8/Fgf4-/-wt

How do the limbs grow?

resting cartilage

bone

proliferating cartilage

proliferating cartilage

age

resting cartilage

FGFR3

Fgfr3+/-

Fgfr3+/-

Fgfr3-/-

Tail

Fgfr3-/-

FGFR3-related skeletal dysplasia

HypochondroplasiaAchondroplasiaSADDANThanatophoric Dysplasia

STATURE AC

TM

TK

I

II

III

FGF binds here

FGFR3-related skeletal dysplasia

Achondroplasia

Thanatophoric Dysplasia

- short long bones

- brachydactyly

- macrocephaly

- low nasal bridge

- spinal stenosis

- temporal lobe malformations

healthy

TD

Fgfr3+/-

Loss-of-function vs. Gain-of-function

Fgfr3-/-

Fgfr3+/+

Fgfr3TD/+

healthy TD

resting

proliferating

hypertrophic

bone

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)

FGFR3 inhibits chondrocyte proliferation by arresting

their cell cycle in G1 phase

….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

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

0.001 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

80

60

40

20

0

80

60

40

20

0

- F1 F10 F100

Erk MAP kinase activity is necessary for

FGFR3 phenotype in cartilage

FGFR3

Ras

Raf-1

MEK

Erk

FRS2

FGF2

C-type Natriuretic Peptide (CNP) over-expression results in skeleton overgrowth in mice

CNP

wild-type

CNP over-expression??? CNPwild-type

wild-type

Fgfr3Ach

Fgfr3Ach/CNP

CNP rescues dwarfism caused by ACH mutation in FGFR3

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

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

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

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

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

IL11, LIF

CishSocs1Socs3

FGF2

IL6

IL6RA Shp2

gp130

STAT3

STAT3

nucleus

FGFR3

Frs2

Chronic FGF stimulus inhibits cytokine/STAT signaling in

chondrocytes

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]

FGF2 signals towards the cytoskeleton in chondrocytes

Phalloidin DAPI merge

control

FGF2

actin

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

Wnt

b-catenin

Where is Wnt?

Frs2, Gab1, SHC

CKI

?

STAT1

Ras/Raf/MEK/Erk

STAT1

CKI MMP

PKC

CNP

NPR-B

cGMP

PKG

2001 2008 IL6, LIF, IL11, IFN

STAT1/3

growth arrest

growth arrest

matrix degradation

?

?

?

proliferation

SOCS1/3

FGFR3

FGFR3