Fibroblast Growth Factor Inhibits Chondrocytic Growth through Induction of p21 and Subsequent Inactivation of Cyclin E-Cdk2

Abstract

Fibroblast growth factor (FGF) and its receptor (FGFR) are thought to be negative regulators of chondrocytic growth, as exemplified by achondroplasia and related chondrodysplasias, which are caused by constitutively active mutations in FGFR3. To understand the growth-inhibitory mechanisms of FGF, we analyzed the effects of FGF2 on cell cycle-regulating molecules in chondrocytes. FGF2 dramatically inhibited proliferation of rat chondrosarcoma (RCS) cells and arrested their cell cycle at the G 1 phase. FGF2 increased p21 expression in RCS cells, which assembled with the cyclin E-Cdk2 complexes, although the expression of neither cyclin E nor Cdk2 increased. In addition, the kinase activity of immunoprecipitated cyclin E or Cdk2, assessed with retinoblastoma protein (pRb) as substrate, was dramatically reduced by FGF-2. Moreover, FGF2 shifted pRb to its underphosphorylated, active form in RCS cells. FGF2 not only induced p21 protein expression in proliferating chondrocytes in mouse fetal limbs cultured in vitro but also decreased their proliferation as assessed by the expression of histone H4 mRNA, a marker for cells in S phase. Furthermore, inhibitory effects of FGF2 on chondrocytic proliferation were partially reduced in p21-null limbs, compared with those in wild-type limbs in vitro. Taken together, FGF's growth inhibitory effects of chondrocytes appear to be mediated at least partially through p21 induction and the subsequent inactivation of cyclin E-Cdk2 and activation of pRb. FGFs1 are a large family of at least 23 related polypeptides that bind to and activate a family of four tyrosine kinase receptors, FGFRs. They play important roles in regulating proliferation and differentiation of various types of cells, including those involved in limb development and long bone formation (1, 2). Long bones form by endochondral ossification, which is characterized by mesenchymal condensation, chondrogenic differentiation, chondrocytic proliferation, synthesis of cartilage matrix, hypertrophic differentiation, and replacement by bone. These sequential growth and differentiation processes are regulated by numerous growth factors and their receptors, such as parathyroid hormone-related protein, Indian hedgehog, and insulin-like growth factor-I (3, 4). Recently, achondroplasia, thanatophoric dysplasia, and hypochondroplasia have been shown to be caused by constitutively active mutations in the FGFR3 gene (5). Also, FGFR3-deficient mice displayed overgrowth of long bones (6, 7), and mice carrying dominant active FGFR3 genes exhibited dwarfism similar to that in patients with achondroplasia and thanatophoric dysplasia (8 -13). Moreover, targeted overexpression of FGF9 in cartilage results in dwarfism in mice similar to the dwarfism in achondroplasia (14). Although these findings support the hypothesis that FGF and its receptors are negative regulators of endochondral bone development, little is known about the mechanisms by which FGF inhibits chondrocytic growth. Sahni et al. recently reported that FGF inhibited chondrocytic ...

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