Differential regulation of endochondral bone growth and joint development by FGFR1 and FGFR3 tyrosine kinase domains

Qing Wang 0 Rebecca P. Green 0 Guoyan Zhao 0 David M. Ornitz ) 0 0 Department of Molecular Biology and Pharmacology, Washington University Medical School , Campus Box 8103, 660 S. Euclid Avenue, St Louis, MO 63110 , USA SUMMARY Fibroblast growth factor receptors (FGFR) 1 and 3 have distinct mitogenic activities in vitro. In several cultured cell lines, FGFR1 transmits a potent mitogenic signal, whereas FGFR3 has little or no mitogenic activity. However, in other in vitro assays the FGFR3 intracellular domain is comparable with that of FGFR1. In vivo, FGFR3 negatively regulates chondrocyte proliferation and differentiation, and activating mutations are the molecular etiology of achondroplasia. By contrast, FGFR1 transmits a proliferative signal in various cell types in vivo. These observations suggest that inhibition of the proliferating chondrocyte could be a unique property of FGFR3 or, alternatively, a unique property of the proliferating chondrocyte. To test this hypothesis, FGFR1 signaling was activated in the growth plate in cells that normally express FGFR3. Comparison of transgenic mice with an activated FGFR1 signaling pathway with an achondroplasia-like mouse that expresses a similarly activated FGFR3 signaling pathway demonstrated that both transgenes result in Fibroblast growth factors (FGFs) and FGF receptors (FGFRs) have essential roles in organogenesis and morphogenesis (Szebenyi and Fallon, 1999; Yamaguchi and Rossant, 1995). Autosomal dominant missense mutations in FGFR1-FGFR3 account for a large number of human skeletal dysplasia and craniosynostosis syndromes (Burke et al., 1998; Naski and Ornitz, 1998; Wilke et al., 1997). Biochemical and genetic studies indicate that most of the point mutations in FGFRs result in increased or ectopic FGFR signaling (Naski et al., 1996; Neilson and Friesel, 1996; Webster and Donoghue, 1996). Molecular mechanisms by which mutations in FGFRs activate signaling include constitutive ligand-independent receptor dimerization (Galvin et al., 1996; Naski et al., 1996; Robertson et al., 1998), increased ligand-binding affinity (Anderson et al., 1998), altered ligand-binding specificity (Yu et al., 2000) and decreased ligand-mediated receptor downregulation (Monsonego-Ornan et al., 2000). Gain-of-function mutations in FGFR3 inhibit endochondral a similar achondroplasia-like dwarfism. These data demonstrate that suppression of mitogenic activity by FGFR signaling is a property that is unique to growth plate chondrocytes. Surprisingly, we observed that in transgenic mice expressing an activated FGFR, some synovial joints failed to develop and were replaced by cartilage. The defects in the digit joints phenocopied the symphalangism that occurs in Apert syndrome and the number of affected joints was dependent on transgene dose. In contrast to the phenotype in the growth plate, the joint phenotype was more severe in transgenic mice with an activated FGFR1 signaling pathway. The failure of joint development resulted from expanded chondrification in the presumptive joint space, suggesting a crucial role for FGF signaling in regulating the transition of condensed mesenchyme to cartilage and in defining the boundary of skeletal elements. bone growth and cause the diseases hypochondroplasia, achondroplasia and thanatophoric dysplasia. By contrast, mutations in FGFR2, and a single mutation in FGFR1, are associated with the craniosynostosis syndromes, some of which also include phenotypes affecting the appendicular skeleton (Burke et al., 1998; Naski and Ornitz, 1998; Wilke et al., 1997). The phenotype of each syndrome correlates with a specific FGFR mutation, and with the spatial expression pattern of FGFRs in mesenchymal condensations and in developing endochondral and membranous bone (Delezoide et al., 1998; Iseki et al., 1999; Johnson et al., 2000; Orr-Urtreger et al., 1991; Peters et al., 1992). In growing long bones with established growth plates, FGFR3 is highly expressed in proliferating chondrocytes and acts to inhibit proliferation (Colvin et al., 1996; Deng et al., 1996; Naski et al., 1998; Naski and Ornitz, 1998; Naski et al., 1996; Peters et al., 1992; Webster and Donoghue, 1996; Webster and Donoghue, 1997b). This activity of FGFR3 is remarkable considering the classical view that FGFs and their receptors transmit mitogenic signals. This raises the question of whether the inhibition of chondrocyte proliferation is a unique property of FGFR3 or a unique property of the chondrocyte. In vitro, activated FGFR3 inhibits proliferation of several cell types. In 293T cells, constitutively active FGFR3 (containing the activation loop mutation K650E) specifically activated the transcription factor STAT1, which upregulates p21 expression, a known cell cycle inhibitor (Su et al., 1997). This observation was supported by the study of Stat1- /bone explants, in which treatment with FGF was unable to inhibit longitudinal growth (Sahni et al., 1999). In CFK2 chondrocytes, FGFR3 (containing the weakly activating transmembrane domain mutation G380R), inhibited cell growth (Henderson et al., 2000). In contrast to these data, intracellular domains of FGFR1, FGFR3 or FGFR4 containing the constitutive activation loop mutation K650E and a plasma membrane targeting myristylation signal, all induced a transformed phenotype in NIH3T3 cells (Hart et al., 2000; Webster and Donoghue, 1997a). Furthermore, chromosomal translocations involving FGFR3 and constitutive activating mutations have been implicated as the etiological agent of some bladder carcinomas and some forms of myeloma (Cappellen et al., 1999; Chesi et al., 1997; Plowright et al., 2000; Richelda et al., 1997). These data demonstrate that constitutively activated FGFR3 can be mitogenic for some cell types. In contrast to Fgfr3, which is expressed in proliferating chondrocytes, Fgfr1 is expressed in the adjacent hypertrophic chondrocytes and in articular chondrocytes. Fgfr1 and Fgfr2 are expressed in the perichondrium (Delezoide et al., 1998; Orr-Urtreger et al., 1991; Peters et al., 1993; Peters et al., 1992). The function of FGFR1 and FGFR2 in endochondral bone growth is not known; however, the non-overlapping expression patterns of FGFR1-FGFR3 suggest that these receptors have unique functions, mediated by differences in their ligand-binding specificity and/or downstream signaling. The FGFR intracellular region contains a juxta-membrane domain, a bipartite tyrosine kinase domain and a kinase insert sequence, and is responsible for signal transduction. The intracellular regions of FGFR1 and FGFR3 share 73% amino acid sequence identity. Several studies have demonstrated differences in FGFR signaling potency in a variety of in vitro assays. In BaF3 cells, FGFR1 and FGFR2 elicit a strong mitogenic response whereas FGFR3 and FGFR4 fail to maintain cell proliferation, even in the presence of saturating ligand concentrations (Naski et al., 1996; Ornitz et al., 1996; Wang et al., 1994). Simil (...truncated)