REGULATION OF LIMB DEVELOPMENT BY FIBROBLAST GROWTH FACTOR (FGF) SYSTEM Pavel Krejci 4 receptors: FGFR1-4 22 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 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+/+ Fgfr3TD/+ healthy TD resting proliferating hypertrophic bone FGF2 (h) FGF concentration (ng/ml) FGFR3 inhibits chondrocyte proliferation by arresting their cell cycle in G1 phase Lower expression levels of FGFR3 than FGFR1 but all zones express same amounts. ….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 Lower expression levels of FGFR3 than FGFR1 but all zones express same amounts. proMMP2 ……via MMP-mediated degradation of extracellular matrix MMP2 MMP3 MMP9 MMP10 MMP13 GAPDH proMMP9 matureMMP9 intermediate matureMMP2 proMMP13 matureMMP13 proMMP2 mature MMP3/10 pro MMP3/10 FGF2 activates Erk and p38 MAPK, PLCg and PKB in chondrocytes C1 C2 1’ 5’ 10’ 30’ 1h 2h 4h 8h FGF2 P-Erk1/2 Erk1/2 P-p38 p38 P-PLCg1 PLCg1 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 Lower expression levels of FGFR3 than FGFR1 but all zones express same amounts. .001 .01 .1 1 10 100 Inhibitor concentration (mM) U0126 - U0126 RasV12 Ras RasN17 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??? CNP wild-type wild-type Fgfr3Ach Fgfr3Ach/CNP CNP rescues dwarfism caused by ACH mutation in FGFR3 control FGF2 FGF2 control _ 0.1 0.2 0.5 1 _ _ _ _ CNP [mM] _ _ _ _ _ 10 100 200 500 pCPT-cGMP [mM] 50 40 30 20 10 0 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 [mM] _ _ _ _ _ _ _ _ 100 200 500 100 pCPT-cGMP [mM] 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 (mM) _ _ 1 5 10 5 _ _ _ _ _ _ _ _ _ _ Bis I (mM) _ _ _ _ _ _ 1 5 10 5 _ _ _ _ _ _ Gö6976 (mM) _ _ _ _ _ _ _ _ _ _ _ _ 1 5 10 5 Erk2 P-Erk1/2 FGF2 _ + + + + + + + + _ GFX (mM) _ _ 0.5 1 5 10 20 50 _ 10 vehicle (DMSO) _ _ + + + + + + + + kinase P-Elk FGFR3 Ras Raf-1 MEK Erk FRS2 FGF2 FRS2 FGFR3 -Y-FRS2 P FGF2 _ + + + + + + + + + + Bis I (mM) _ _ 1 5 10 20 50 _ _ _ _ SU5402(mM) _ _ _ _ _ _ _ 1 10 20 _ 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 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 substrate: STAT1 + + + + + + SU5402(mM) _ + _ _ + _ 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 75 150kDa 100 IL11, LIF Cish Socs1 Socs3 FGF2 IL6 IL6RA Shp2 gp130 STAT3 STAT3 nucleus FGFR3 Frs2 Chronic FGF stimulus inhibits cytokine/STAT signaling in chondrocytes Resting -- some membranous staining detected, mostly non-specific nuclear staining Proliferative and Hypertrophic -- membranous staining mostly seen in these two zones. FGF2 causes premature senescence in chondrocytes SA-b-gal control FGF2 FGF2 [ng/ml] - 5 10 20 0 12 24 36 48 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, IFNg STAT1/3 growth arrest growth arrest matrix degradation ? ? ? proliferation SOCS1/3 FGFR3 FGFR3