Regulation of cell function by FGFR3 receptor tyrosine kinase Pavel Krejci Medical Genetics Institute, Cedars-Sinai Medical Center, Los Angeles, CA Institute of Experimental Biology, Masaryk University, Brno, Czech Republic Fgfr3+/- Fgfr3+/- Fgfr3-/- Tail Fgfr3-/- Cell 1996, 84(6):911-21. Achondroplasia Thanatophoric Dysplasia FGFR3-related skeletal dysplasias Nat Genet 1995, 9:321-8.. - short long bones - brachydactyly - macrocephaly - low nasal bridge - spinal stenosis - temporal lobe malformations FGFR3-related skeletal dysplasia Hypochondroplasia Achondroplasia SADDAN Thanatophoric Dysplasia STATURE AC TM TK I II III FGF binds here Skeleton: hypochondroplasia, achondroplasia, thanatophoric dysplasia, SADDAN, Muenke syndrome Skin: epidermal nevi, seborrhaeic keratosis, acanthosis nigricans Cancer: multiple myeloma, bladder cancer, seminoma Exp Cell Res. 2004;297:152-64. J Cell Sci. 2005; 118: 5089-100. J Biol Chem. 2007 ;282:2929-36. Pediatr Res. 2007; 61(3):267-72. Invest New Drugs 2007; 25:391-95. PLoS One 2008; 3:e3961. J Cell Sci 2008; 121:272-81. Cell Signal 2009; 21:151-60. Hum Mol Genet. 2009; 18:227-40. J Biol Chem 2010; 285:20644-53. Bone 2010; 47:102-10. Leukemia 2011; 25:538-50. Human Mutation 2012; 33:29-41 healthy TD resting proliferating hypertrophic bone FGF2 (h) FGF2 concentration (ng/ml) FGFR3 inhibits chondrocyte proliferation by arresting their cell cycle in G1 phase Exp Cell Res 2004, 297:152-64. Lower expression levels of FGFR3 than FGFR1 but all zones express same amounts. control FGF2 FGFR3 alters the cartilage-like phenotype of chondrocytes FGF2 (h) M C 0.5 1 4 8 12 24 48 72 aggrecan collagen II collagen I GAPDH proMMP9 matureMMP9 intermediate matureMMP2 proMMP13 matureMMP13 proMMP2 mature MMP3/10 pro MMP3/10 J Cell Sci. 2005; 118: 5089-100. Lower expression levels of FGFR3 than FGFR1 but all zones express same amounts. FGFR3 causes premature senescence in chondrocytes SA-b-gal control FGF2 FGF2 control Bone 2010; 47:102-10. C1 C2 1’ 5’ 10’ 30’ 1h 2h 4h 8h FGF2 P-Erk1/2 Erk1/2 P-p38 p38 P-PLCg1 PLCg1 .001 .01 .1 1 10 100 Inhibitor concentration (mM) U0126 80 60 40 20 0 Ras RasN17 - F1 F10 F100 FGFR3 arrests chondrocyte growth via RAS-ERK MAP kinase pathway Exp Cell Res 2004, 297:152-64. Lower expression levels of FGFR3 than FGFR1 but all zones express same amounts. Ras Raf-1 MEK Erk FGF2 FRS2 SHP2 GRB2 SOS GRB2 SOS GAB1 SHC FGFR3 J Biol Chem 2007; 282:2929-36. CKI FGFR3 STAT1 Growth arrest Skeletal dysplasia FGFR3 associates with STAT1 and acts as STAT1-kinase in chondrocytes CKI FGFR3 STAT1 Growth arrest ? ? ? FGFR3 actin FGFR3 IP: STAT1 WB STAT1 FGFR3-wt substrate: STAT1 + + + + + + SU5402(mM) _ + _ _ + _ ATP + + _ + + _ FGFR3-K650E P-Y701-STAT1 STAT1 FGFR3 Skeletal dysplasia J Cell Sci 2008;121:272-81. PLoS One 2008;3:e3961. STAT3-YFP DAPI merge DIC/merge control IL6 30m FGF2 48h FGF2 2h IL6 30m FGF2 48h IL11, LIF Cish Socs1 Socs3 FGF2 IL6 IL6RA Shp2 gp130 STAT3 STAT3 nucleus FGFR3 Frs2 Chronic FGF stimulus inhibits cytokine/STAT signaling in chondrocytes Cell Signal 2009;21:151-60. Frs2, Gab1, SHC CKI ? STAT1 Ras/Raf/MEK/Erk STAT1 CKI MMP PKC CNP NPR-B cGMP PKG 2001 2012 IL6, LIF, IL11, IFNg STAT1/3 growth arrest matrix degradation ? ? ? proliferation SOCS1/3 FGFR3 growth arrest FGFR3 actin FGF2: C1 15‘ 1h 3h 6h 12h 24h 48h 72h C2 C3 b-catenin Wnt submitted Fgfr3Ach Kazuwa Nakao Nature Medicine 10, 80-86 (2004). CNP wild-type Fgfr3Ach/CNP CNP wild-type wild-type C-natriuretic peptide (CNP) 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 control CNP control FGF2 FGFR3 Ras CNP cGMP PKG Raf-1 MEK Erk NP-R FRS2 STOP FGF2 Growth arrest Matrix degradation J Cell Sci. 2005; 118: 5089-100. A collection of 1120 biologically active compounds supplied as DMSO solutions. Tocriscreen™ Mini Library J Biol Chem 2010; 285:20644-53. NF449 Leukemia 2011. 25:538-50. Cedars-Sinai Medical Center Los Angeles, California Betty Mekikian Anie Aklian UCI, Irvine, California Leslie Thompson Tamara Kashiwada Lisa Salazar UCLA, Los Angeles, California Matthew Schibler Masaryk University, Brno, Czech Republic Jirina Prochazkova Vita Bryja Lukas Balek Tereza Spoustova Tereza Pospisilova INSERM U589, Toulouse, France Bernard Masri Vincent Fontaine