title: Sustained phosphorylation of mutated FGFR3 is a crucial feature of genetic dwarfism and induces apoptosis in the ATDC5 chondrogenic cell line via PLCgamma-activated STAT1.
abstract: The most frequent type of rhizomelic dwarfism, achondroplasia (ACH), is caused by mutations in the fibroblast growth factor receptor 3 (FGFR3) gene. Mutations in FGFR3 result in skeletal dysplasias of variable severity, including mild phenotypic effects in hypochondroplasia (HCH), severe phenotypic effects in thanatophoric dysplasia types I (TDI) and II (TDII), and severe but survivable phenotypic effects in severe achondroplasia with developmental delay and acanthosis nigricans (SADDAN). To explore the molecular mechanisms that result in the different phenotypes, we investigated the kinetics of mutated versions of FGFR3. First, we assayed the phosphorylation states of the mutated FGFR3s and found that the level of phosphorylation in TDI-FGFR3 was lower than in ACH-FGFR3, although the other mutants were phosphorylated according to phenotypic severity. Second, we analyzed the duration of the phosphorylation. TDI-FGFR3 was not highly phosphorylated under ligand-free conditions, but the peak phosphorylation levels of TDI-FGFR3 and ACH-FGFR3 were maintained for 30 min after stimulation with FGF-1. Moreover, ligand-dependent phosphorylation of TDI-FGFR3, but not ACH-FGFR3, lasted for more than 8 h after FGF-1 administration. The other mutant proteins showed sustained phosphorylation independent of ligand presence. Third, we investigated the intracellular localization of the mutant proteins. Immunofluorescence analysis showed accumulations of TDII-FGFR3, SADDAN-FGFR3, and a portion of TDI-FGFR3 in the endoplasmic reticulum (ER). Based on these data, we concluded that sustained phosphorylation of FGFR3 causes chondrodysplasia, and the phenotypic severity depends on the proportion of ER-localized mutant FGFR3. In FGFR3 signaling, the transcription factor, signal transducer and activator of transcription 1 (STAT1) inhibit proliferation and induce apoptosis of chondrocytes. Here we reveal that phospholipase C gamma (PLCgamma) mediates FGFR3-induced STAT1 activation. Both PLCgamma and STAT1 were activated by FGFR3 signaling, but a dominant-negative form of PLCgamma (DN-PLCgamma) remarkably reduced STAT1 phosphorylation. Apoptosis assays revealed that the constitutively active forms of FGFR3 (TDII-FGFR3) and STAT1 (STAT1-C) induce apoptosis of chondrogenic ATDC5 cells via caspase activity. DN-PLCgamma reduced the apoptosis of ATDC5 cells expressing TDII-FGFR3, but over-expression of both DN-PLCgamma and STAT1-C induced apoptosis. Therefore, we conclude that a PLCgamma-STAT1 pathway mediates apoptotic signaling by FGFR3.
authors.affiliation: Department of Pediatrics, Okayama University, Graduate School of Medicine and Dentistry, 2-5-1 Shikata-Cho, Okayama 700-8558, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Harada
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Yamanaka
authors.forename: Koso
authors.initials: K
authors.lastname: Ueda
authors.forename: Riko
authors.initials: R
authors.lastname: Nishimura
authors.forename: Tsuneo
authors.initials: T
authors.lastname: Morishima
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Seino
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Tanaka
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002460:Cell Line; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism; D004392:Dwarfism / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed; D051966:Phospholipase C gamma / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051498:Receptor, Fibroblast Growth Factor, Type 3 / Q000235:genetics / Q000378:metabolism*; D050794:STAT1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology
chemical_list: D034741:RNA, Small Interfering; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; C496365:FGFR3 protein, human; D051498:Receptor, Fibroblast Growth Factor, Type 3; D051966:Phospholipase C gamma