Repression of hedgehog signaling and BMP4 expression in growth plate cartilage by fibroblast growth factor receptor 3

Michael C. Naski 1 Jennifer S. Colvin 1 J. Douglas Coffin 0 David M. Ornitz ) 1 0 Department of Pharmaceutical Sciences, School of Pharmacy and Allied Health Sciences, University of Montana , Missoula, MT 59812 , USA 1 Department of Molecular Biology and Pharmacology, Washington University School of Medicine , Campus Box 8103, 660 S. Euclid Ave, St. Louis, MO 63110 , USA SUMMARY Fibroblast growth factor receptor 3 (FGFR3) is a key regulator of skeletal growth and activating mutations in Fgfr3 cause achondroplasia, the most common genetic form of dwarfism in humans. Little is known about the mechanism by which FGFR3 inhibits bone growth and how FGFR3 signaling interacts with other signaling pathways that regulate endochondral ossification. To understand these mechanisms, we targeted the expression of an activated FGFR3 to growth plate cartilage in mice using regulatory elements from the collagen II gene. As with humans carrying the achondroplasia mutation, the resulting transgenic mice are dwarfed, with axial, appendicular and craniofacial skeletal hypoplasia. We found that FGFR3 inhibited endochondral bone growth by markedly inhibiting chondrocyte proliferation and by Skeletal growth is regulated by a hierarchy of genetic, endocrine and mechanical regulatory programs. These programs ensure coordinated growth of both the cartilaginous and bony portions of the skeleton. Recently, fibroblast growth factor (FGF) receptor 3 (FGFR3) has been identified as a critical regulator of endochondral bone growth. Autosomal dominant mutations in Fgfr3 cause the dwarfing chondrodysplasias, achondroplasia (Rousseau et al., 1994; Shiang et al., 1994), hypochondroplasia (Bellus et al., 1995), and thanatophoric dysplasia (Tavormina et al., 1995a,b). Additionally, mice homozygous for null alleles of Fgfr3 exhibit skeletal overgrowth (Colvin et al., 1996; Deng et al., 1996). The contrasting phenotypes between the Fgfr3- /mice and the human dwarfing conditions resulting from mutations in Fgfrs suggest that the mutations causing dwarfism are gain of function alleles (this has recently been proved biochemically; Naski et al., 1996; Webster et al., 1996; Webster and Donoghue, 1996), and that Fgfr3 negatively regulates bone growth. Whether FGFR3 achieves this regulation directly or indirectly through interactions with regulatory signaling pathways is not known. slowing chondrocyte differentiation. Significantly, FGFR3 downregulated the Indian hedgehog (Ihh) signaling pathway and Bmp4 expression in both growth plate chondrocytes and in the perichondrium. Conversely, Bmp4 expression is upregulated in the perichondrium of Fgfr3- /mice. These data support a model in which Fgfr3 is an upstream negative regulator of the hedgehog (Hh) signaling pathway. Additionally, Fgfr3 may coordinate the growth and differentiation of chondrocytes with the growth and differentiation of osteoprogenitor cells by simultaneously modulating Bmp4 and patched expression in both growth plate cartilage and in the perichondrium. Endochondral bone growth is a tightly regulated developmental process that occurs in the epiphyseal growth plate, a specialized cartilaginous tissue found at the ends of growing long bones (Caplan and Pechak, 1987). Growth plate chondrocytes are arranged in columns that sequentially and synchronously progress through proliferative, prehypertrophic and hypertrophic stages (Caplan and Pechak, 1987; Caplan, 1988). The hypertrophic chondrocytes die and are replaced by trabecular bone and bone marrow through a process that includes apoptosis of hypertrophic chondrocytes, vascular invasion of the growth plate, resorption of the cartilaginous matrix and recruitment of osteoblasts that deposit the trabecular bone matrix. Fgfr3 is expressed in the epiphyseal growth plate and is most highly expressed in a histomorphological domain that encompasses proliferating and prehypertrophic chondrocytes. This expression pattern suggests a direct role for FGFR3 in regulating chondrocyte proliferation and possibly differentiation. Trabecular bone is formed by endochondral ossification in the growth plate. In a separate process, osteoblasts derived from osteoprogenitor cells in the perichondrium generate cortical bone. Longitudinal bone growth requires synchronous cortical and endochondral bone formation. This implies that endochondral bone formation, the process of chondrocyte growth and differentiation, must be coordinated with osteoblast differentiation and the synthesis of cortical bone derived from osteoprogenitor cells in the perichondrium. The mechanisms coordinating these two processes are poorly understood. FGFR3 profoundly regulates longitudinal bone growth but is only expressed in the cartilaginous growth plate (Shiang et al., 1994; Tavormina et al., 1995a,b; Colvin et al., 1996; Deng et al., 1996). This suggests that signals downstream of FGFR3 must regulate bone formation adjacent to the epiphyseal growth plate. Studies of Fgfr3 null mice show prolonged expression of markers for cell proliferation (Deng et al., 1996) and overexpression of FGFR3 in a chondrocytic cell line results in diminished cell proliferation (J. Henderson, M. C. Naski and D. M. Ornitz, unpublished data). In addition to affecting chondrocyte proliferation, evidence also suggests that FGFR3 may regulate chondrocyte differentiation. Histological studies of biopsies from individuals with achondroplasia show either extensive or focal disorganization of the growth plate (Ponseti, 1970; Rimoin et al., 1970; Briner et al., 1991). Furthermore, Fgfr3- /- mice have an expanded zone of hypertrophy in the epiphyseal growth plate (Colvin et al., 1996; Deng et al., 1996) and in vitro experiments demonstrate that the addition of FGF to cultured chondrocytes inhibits chondrocyte differentiation (Kato and Iwamoto, 1990). These observations suggest that FGFR3 signaling may affect chondrocyte differentiation in vivo. Along with FGFs, endochondral bone growth is regulated by many signaling molecules including growth hormone, insulinlike growth factor-1 (IGF-1), parathyroid hormone related protein (PTHrP), Indian hedgehog (Ihh) and bone morphogenetic proteins (BMPs) (Reddi, 1994; Erlebacher et al., 1995). Recently, a feedback loop was described in which Ihh and PTHrP interact to coordinate chondrocyte differentiation (Lanske et al., 1996; Vortkamp et al., 1996). However, the relationship between the Ihh/PTHrP and FGF signaling pathways has not been determined. In this study we have created a mouse model for the human genetic disease, achondroplasia. We show that expressing an activated FGFR3 in the growth plate downregulates the expression of Ihh, the Ihh receptor, patched and Bmp4, whereas patched and Bmp4 expression are upregulated in Fgfr3- /mice. Significantly, Bmp4 expression is modulated in both growth plate chondrocytes and in the perichondrium. These data suggest that Fgfr3 is genetically upstream of the Ihh signaling pathway and is a global coord (...truncated)