REGULATION OF LIMB DEVELOPMENT BY FIBROBLAST GROWTH FACTOR (FGF) SYSTEM Pavel Krejci

4 receptors: FGFR ligands: FGF1-23

wt Fgf10 -/- AER wt Fgf10 -/-

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

How do the limbs grow? resting cartilage bone proliferating cartilage age resting cartilage

FGFR3

Fgfr3 +/- Fgfr3 -/- Tail Fgfr3 -/-

FGFR3-related skeletal dysplasia Hypochondroplasia Achondroplasia SADDAN Thanatophoric 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 +/+ Fgfr3 TD/+

healthy TD resting proliferating hypertrophic bone

3 H-thymidine (cpm x 10 3 ) 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) aggrecan collagen II collagen I GAPDH

proMMP2 ……via MMP-mediated degradation of extracellular matrix MMP2 MMP3 MMP9 MMP10 MMP13 GAPDH control FGF2 proMMP9 matureMMP9 intermediate matureMMP2 proMMP13 matureMMP13 control FGF2 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-PLC  1 PLC  1 P-PKB PKB 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

Inhibitor concentration (  M) U0126 cells/wellx U0126 RasV12 cells/wellx10 3 cell colonies/100 cells (%) Ras RasN17 SU5402 ……but only Ras/Erk activity is involved in FGF-induced growth arrest 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??? CNP wild-type

Fgfr3 Ach Fgfr3 Ach/CNP CNP rescues dwarfism caused by ACH mutation in FGFR3

control FGF2 control _ _ _ _ _ CNP [  M] _ _ _ _ _ pCPT-cGMP [  M] cells per well [x10 4 ] 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 _ _ _ _ _ _ _ _ CNP [  M] _ _ _ _ _ _ _ _ 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) _ _ _ _ _ _ _ _ _ _ _ _ Bis I (  M) _ _ _ _ _ _ _ _ _ _ _ _ Gö6976 (  M) _ _ _ _ _ _ _ _ _ _ _ _ Erk2 P - Erk1/2 FGF2 _ _ GFX (  M) _ _ _ 10 vehicle (DMSO) _ _ kinase P - Elk FGFR3 Ras Raf-1 MEK Erk FRS2 FGF2

FRS2 FGFR3 - Y-FRS2 P FGF2 _ Bis I (  M ) _ _ _ _ _ _ SU5402(  M) _ _ _ _ _ _ _ _ vehicle(DMSO) _ _ kinase Ras Raf-1 MEK Erk FGF2 Gab1 SHP2 GRB2 SOS GRB2 SOS 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-SOS Shp2-Grb2-SOS 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 FGFR3-wt FGFR3-K650M FGFR3-K508M GFP FGFR3-wt substrate: STAT 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 30m FGF2 48h FGF2 2h IL6 30m FGF2 48h STAT3 STAT3-YFP kDa 100

IL11, LIF Cish Socs1 Socs3 FGF2 IL6 IL6RA Shp2 gp130 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] SA-  -gal positive cells per 50 cells [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 NOW IL6, LIF, IL11, IFN  STAT1/3 growth arrest matrix degradation ? ? ? proliferation SOCS1/3 FGFR3