Sustained phosphorylation of mutated FGFR3 is a crucial feature of genetic dwarfism and induces apoptosis in the ATDC5 chondrogenic cell line via PLCγ-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.

Analysis of STAT1 Activation by Six FGFR3 Mutants Associated with Skeletal Dysplasia Undermines Dominant Role of STAT1 in FGFR3 Signaling in Cartilage

“…As determined by densitometry, the activation of STAT1 by wild-type FGFR3 in HeLa cells was ∼5.5-fold lower than in K650M (Fig. 2), similar to Harada et al [10] or Su et al [5], who found the wild-type FGFR3 activating STAT1 to the levels 4.8-fold or 20-fold lower than K650M.…”

Disruption of Phosphoinositide-Specific Phospholipases Cγ1 Contributes to Extracellular Matrix Synthesis of Human Osteoarthritis Chondrocytes

“…Several lines of evidence supported that PLCγ is widely distributed, activated by receptor or non-receptor tyrosine kinases, and involved in cell proliferation, differentiation, apoptosis, and metastasis of mammal cells, including chondrocytes [4,5,6,8]. For example, Harada et al [8] reported that PLCγ mediated FGFR3-induced STAT1 activation and this signaling cascade involved in the induction of apoptosis in the chondrogenic cell line ATDC5.…”

“…For example, Harada et al [8] reported that PLCγ mediated FGFR3-induced STAT1 activation and this signaling cascade involved in the induction of apoptosis in the chondrogenic cell line ATDC5. Here, we demonstrated that PLCγ1 was expressed in normal articular and OA chondrocytes, and PLCγ1 was highly expressed in OA chondrocytes.…”

FGFR3 signaling induces a reversible senescence phenotype in chondrocytes similar to oncogene-induced premature senescence

“…Increased apoptosis was found in the cartilage of mice overexpressing FGF2 [38], however other studies failed to confirm this effect [39, 40] (P. Krejci and W. R. Wilcox, unpublished). Ectopic expression of activating FGFR3 mutants induces apoptosis in ATDC5 chondrogenic cells and RCS chondrocytes [41] (Figs 5, S2), however this effect may represent an artifact of FGFR3 overexpression rather then a true physiological response.…”