Mutations of fibroblast growth factor receptor 3 (FGFR3) are responsible for achondroplasia (ACH) and related dwarfing conditions in humans. The pathogenesis involves constitutive activation of FGFR3, which inhibits proliferation and differentiation of growth plate chondrocytes. Here we report that activating mutations in FGFR3 increase the stability of the receptor. Our results suggest that the mutations disrupt c-Cbl-mediated ubiquitination that serves as a targeting signal for lysosomal degradation and termination of receptor signaling. The defect allows diversion of actively signaling receptors from lysosomes to a recycling pathway where their survival is prolonged, and, as a result, their signaling capacity is increased. The lysosomal targeting defect is additive to other mechanisms proposed to explain the pathogenesis of ACH.

Chondrogenesis results from a complex equilibrium between chondrocyte proliferation and differentiation. Insulin-like growth factors (IGFs) have a crucial role in chondrogenesis, but their mechanisms of action are not well defined. IGF-binding protein-3 (IGFBP-3) is the major carrier for circulating IGFs in postnatal life, and has been shown to have IGF-independent effects on proliferation of several cancer cell lines. In this study, we have evaluated the IGF-independent and -dependent effects of IGFBP-3 on chondrocyte proliferation and the relationship of these effects with chondrocyte differentiation stage. We used the RCJ3.1C5.18 nontransformed mesenchymal chondrogenic cell line, which, over 2 weeks of culture, progresses through the differentiation pathway exhibited by chondrocytes in the growth plate. We demonstrated that IGFBP-3 inhibited, in a dose-dependent manner (1-30 nM), the proliferation of chondroprogenitors and early differentiated chondrocytes, stimulated by des-(1-3)-IGF-I and longR 3 -IGF-I (IGF-I analogs with reduced affinity for IGFBP-3), and by insulin and IGF-I. In terminally differentiated chondrocytes, IGFBP-3 retained the ability to inhibit cell proliferation stimulated by IGF-I, but had no effect on cell growth stimulated by insulin, or des-(1-3)-IGF-I or longR 3 IGF-I. By monolayer affinity cross-linking, we demonstrated a specific IGFBP-3-associated cell-membrane protein of ϳ20 kDa. We determined that IGFBP-3 has an antiproliferative effect on chondrocytes and, that this effect is related to the differentiation process. In chondroprogenitors and early differentiated chondrocytes, antiproliferative effect of IGFBP-3 is mainly IGF-independent, whereas, following terminal differentiation this effect is IGF-dependent.

The chondrogenesis process requires the ordered proliferation and differentiation of chondrocytes. Insulinlike growth factor-binding protein (IGFBP)-3, well characterized as the carrier of insulin-like growth factor (IGF), has been reported to have intrinsic bioactivity that is independent of IGF binding. The mechanisms involved in this IGF-independent action are still unclear. Using the RCJ3.1C5.18 chondrogenic cells, which in culture progresses from undifferentiated to terminally differentiated chondrocytes, we have shown previously that IGFBP-3 has an IGF-independent, antiproliferative effect in undifferentiated and early differentiated but not in terminally differentiated chondrocytes. In the present study, cDNA microarray analysis was used to screen for genes: 1) that were regulated by IGFBP-3 in early but not in terminally differentiated chondrocytes; 2) that were regulated specifically by IGFBP-