Introduction Fibroblast growth factor receptor 3 (FGFR3) is one of four distinct members of the membrane-spanning tyrosine kinase family that serve as high affinity receptors (FGFRs) for at least 20 different fibroblast growth factors (FGFs). Binding of the FGF ligand, in concert with heparan sulfate, induces FGFR dimerization and transphosphorylation, followed by activation of downstream signal-transduction pathways (reviewed in Givol and Yayon, 1992; Burke et al., 1998; Klint and Claesson-Welsh, 1999; Ornitz, 2000). Signaling via the FGF-FGFR system plays a major role in regulating various cellular processes, including proliferation, differentiation and survival. In vitro, the effect of FGF on most cell types, such as endothelial cells (Gerwins et al., 1998), fibroblasts (Basilico and Moscatelli, 1992) or de- differentiated chondrocytes (Kato and Iwamoto, 1990; Hill et al., 1991; Wroblewski and Edwall-Arvidsson, 1995), is stimulation of proliferation and inhibition of terminal differentiation. However, the effect of FGF on growth-plate chondrocytes, the cells responsible for bone growth and skeletal development, and Rat Chondrosarcoma (RCS) cells, which retain mature chondrocyte markers and express high levels of FGFR3, is potent inhibition of proliferation (Sahni et al., 1999), which may ultimately result in apoptosis (Legeai-Mallet et al., 1998). The expression of FGFRs is tightly regulated during embryonal development and tissue regeneration (Basilico and Moscatelli, 1992; Yamaguchi et al., 1995; Goldfarb, 1996; Martin, 1998; Szebnyi and Fallon, 1999; Xu et al., 1999). FGFR3 is particularly highly expressed during embryonic development in the pre-cartilaginous mesenchyme (Peters et al., 1992; Peters et al., 1993) and later on in the maturation zone of the epiphyseal growth-plates, where it is involved in long bone development (Naski et al., 1998). The discovery that specific activating mutations in FGFR3 underlie a variety of human skeletal disorders, such as Achondroplasia, the most common form of human genetic dwarfism, has linked FGFR3 signaling and skeletal development (reviewed in Webster and Donoghue, 1997; Burke et al., 1998; Naski and Ornitz, 1998). Moreover, FGFR3-null mice exhibit bone overgrowth accompanied by expansion of proliferating and hypertrophic chondrocytes within the growth-plate (Colvin et al., 1996; Deng et al., 1996). Transgenic mice harboring FGFR-activating mutations (Naski et al., 1998; Chen et al., 1999; Li et al., 1999; Wang et al., 1999; Segev et al., 2000) or overexpressing FGF2 (Coffin et al., 1995) or FGF9 (Garofalo et al., 1999) display a dwarf phenotype similar to the human disorders where attenuated proliferation and 553 The effect of fibroblast growth factor (FGF) on mature chondrocytes, the cells responsible for axial skeletal development, is growth attenuation rather than stimulation. This singular response has been linked to signaling via FGF receptor 3 (FGFR3), partly because mutations causing chronic FGFR3 activation lead to various human disorders of bone growth. In order to study how FGF inhibits growth, we analyzed its effect on a rat chondrocyte-derived cell line. We show that the FGF- induced growth arrest occurs at the G1 phase, accompanied by profound changes in gene expression and cytoskeletal organization. Within minutes of binding, FGF induces tyrosine kinase activity in the focal substrate adhesions where it colocalizes with vinculin. Upon FGF stimulation, FGFR3 is selectively removed from the focal adhesions, which is followed by their disassembly and disruption of the organized cytoskeleton. Multiple genes are induced following FGF stimulation in chondrocytes, which has been shown by DNA array screening and confirmed for some by immunoblotting. These genes include regulators of cell differentiation and proliferation such as c-jun, JunD, cyclin-D1, NFκB1 and of plasma- membrane microdomain morphology, such as ezrin. The transcription factor Id1 is downregulated, consistent with the cells’ exit from the mitotic cycle. Moreover, following FGF stimulation, levels of FGFR3 mRNA and protein decline, as does downstream signaling through the MAPK pathway. The importance of this FGFR3-mediated on-off control is illustrated in transgenic mice expressing mutant, hyperactive FGFR3, where abnormally high levels of NFκB are expressed throughout their bone growth-plates. A working model is presented of the signaling network involved in regulating FGF-induced chondrocyte differentiation and receptor downregulation. Key words: Chondrocytes, FGF signaling, FGF receptor 3, Focal adhesions, G1 arrest Summary Induction of chondrocyte growth arrest by FGF: transcriptional and cytoskeletal alterations Orit Rozenblatt-Rosen 1 , Efrat Mosonego-Ornan 1,2 , Einat Sadot 1 , Liora Madar-Shapiro 2 , Yuri Sheinin 2 , Doron Ginsberg 1 and Avner Yayon 1,2, * 1 Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel 2 ProChon Biotech Ltd, Kiryat Weizmann, Rehovot 76114, Israel Author for correspondence (e-mail: yayon@prochon.co.il) Accepted 10 October 2001 Journal of Cell Science 115, 553-562 (2002) © The Company of Biologists Ltd Research Article