4 receptors: FGFR1-4 22 ligands: FGF1-23

1. 9. MECHANISMS OF DEVELOPMENT I – REGULATION OF LIMB DEVELOPMENT BY FIBROBLAST GROWTH FACTORS (FGF)

2. 4 receptors: FGFR1-4 22 ligands: FGF1-23

4. Fgf10-/- Fgf10-/- wt wt AER

5. Fgf8/Fgf4-/- Fgf8/Fgf4-/- wt wt

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

8. FGFR3

9. Fgfr3+/- Fgfr3-/- Fgfr3+/- Fgfr3-/- Tail

10. FGFR3-related skeletal dysplasia STATURE I AC Hypochondroplasia Achondroplasia SADDAN Thanatophoric Dysplasia II FGF binds here III TM TK

11. FGFR3-related skeletal dysplasia Achondroplasia

12. Thanatophoric Dysplasia healthy - short long bones - brachydactyly - macrocephaly - low nasal bridge - spinal stenosis - temporal lobe malformations TD

13. Fgfr3+/- Fgfr3+/+ Fgfr3-/- Fgfr3TD/+ Loss-of-function vs. Gain-of-function

15. resting proliferating hypertrophic bone healthy TD

16. FGFR3 inhibitschondrocyte proliferation by arresting their cell cycle in G1 phase 3H-thymidine (cpm x 103) % of cells FGF2 (h) FGF concentration (ng/ml)

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

18. 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

19. ……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

20. 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

21. - U0126 ……but only Ras/Erk activity is involved in FGF-induced growth arrest 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)

22. FGF2 FGFR3 FRS2 Ras Raf-1 MEK Erk Erk MAP kinase activity is necessary for FGFR3 phenotype in cartilage

23. C-type Natriuretic Peptide (CNP) over-expression results in skeleton overgrowth in mice wild-type CNP CNP over-expression??? wild-type CNP

24. CNP rescues dwarfism caused by ACH mutation in FGFR3 wild-type Fgfr3Ach Fgfr3Ach/CNP

25. 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]

26. CNP antagonizes FGF2-mediated loss of cartilage extracellular matrix in chondrocytes unstimulated FGF2 control CNP pCPT-cGMP

27. FGF2 FGFR3 FRS2 Ras Raf-1 MEK Erk 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 100pCPT-cGMP [mM] P-Erk1/2 Erk1/2 FGF2_ + + _ CNP_ _ ++_Ab FGF2_ ++ _ CNP_ _ ++ IP:Raf-1 Raf-1 Ras-GST WB P-MEK Ras total P-Erk1/2 P-Erk1/2 Erk1/2 Erk1/2

28. 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

29. 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

30. 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

31. 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

32. 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

33. 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

34. FGF2 IL6 IL11, LIF FGFR3 IL6RA Shp2 Frs2 gp130 Cish Socs1 Socs3 STAT3 nucleus STAT3 Chronic FGF stimulus inhibits cytokine/STAT signaling in chondrocytes

35. 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]

36. FGF2 signals towards the cytoskeleton in chondrocytes Phalloidin DAPI merge control FGF2

37. Where is Wnt? FGF2: C1 15‘ 1h 3h 6h 12h 24h 48h 72h C2 C3 b-catenin actin Wnt

38. 20012008 FGFR3 IL6, LIF, IL11, IFNg FGFR3 CNP SOCS1/3 ? PKC NPR-B STAT1 Frs2, Gab1, SHC STAT1/3 STAT1 ? cGMP ? CKI ? Ras/Raf/MEK/Erk PKG proliferation growth arrest CKIMMP growth arrest matrix degradation