Smith Simone M-L, West Leigh A, Hassell John R
Department of Molecular Medicine, University of South Florida College of Medicine, 12901 Bruce B Downs Boulevard, Tampa, FL 33612, USA.
Arch Biochem Biophys. 2007 Dec 15;468(2):244-51. doi: 10.1016/j.abb.2007.10.006. Epub 2007 Oct 22.
Fibroblast growth factor-18 (FGF-18) has been shown to regulate the growth plate chondrocyte proliferation, hypertrophy and cartilage vascularization necessary for endochondral ossification. The heparan sulfate proteoglycan perlecan is also critical for growth plate chondrocyte proliferation. FGF-18 null mice exhibit a skeletal dwarfism similar to that of perlecan null mice. Growth plate perlecan contains chondroitin sulfate (CS) and heparan sulfate (HS) chains and FGF-18 is known to bind to heparin and to heparan sulfate from some sources. We used cationic filtration and immunoprecipitation assays to investigate the binding of FGF-18 to perlecan purified from the growth plate and to recombinant perlecan domains expressed in COS-7 cells. FGF-18 bound to perlecan with a K(d) of 145 nM. Near saturation, approximately 103 molecules of FGF-18 bound per molecule of perlecan. At the lower concentrations used, FGF-18 bound with a K(d) of 27.8 nM. This binding was not significantly altered by chondroitinase nor heparitinase digestion of perlecan, but was substantially and significantly reduced by reduction and alkylation of the perlecan core protein. This indicates that the perlecan core protein (and not the CS nor HS chains) is involved in FGF-18 binding. FGF-18 bound equally to full-length perlecan purified from the growth plate and to recombinant domains I-III and III of perlecan. These data indicate that low affinity binding sites for FGF-18 are present in cysteine-rich regions of domain III of perlecan. FGF-18 stimulated 3H-thymidine incorporation in growth plate chondrocyte cultures derived from the lower and upper proliferating zones by 9- and 14-fold, respectively. The addition of perlecan reversed this increased incorporation in the lower proliferating chondrocytes by 74% and in the upper proliferating cells by 37%. These results suggest that perlecan can bind FGF-18 and alter the mitogenic effect of FGF-18 on growth plate chondrocytes.
成纤维细胞生长因子18（FGF - 18）已被证明可调节生长板软骨细胞增殖、肥大以及软骨内骨化所必需的软骨血管生成。硫酸乙酰肝素蛋白聚糖基底膜聚糖对于生长板软骨细胞增殖也至关重要。FGF - 18基因敲除小鼠表现出与基底膜聚糖基因敲除小鼠相似的骨骼侏儒症。生长板基底膜聚糖含有硫酸软骨素（CS）和硫酸乙酰肝素（HS）链，并且已知FGF - 18可与来自某些来源的肝素和硫酸乙酰肝素结合。我们使用阳离子过滤和免疫沉淀测定法来研究FGF - 18与从生长板纯化的基底膜聚糖以及在COS - 7细胞中表达的重组基底膜聚糖结构域的结合。FGF - 18与基底膜聚糖结合，解离常数（K(d)）为145 nM。接近饱和时，每分子基底膜聚糖约结合103个FGF - 18分子。在较低浓度下，FGF - 18以27.8 nM的K(d)结合。基底膜聚糖经软骨素酶或硫酸乙酰肝素酶消化后，这种结合没有明显改变，但基底膜聚糖核心蛋白经还原和烷基化后，结合显著减少。这表明基底膜聚糖核心蛋白（而非CS或HS链）参与FGF - 18的结合。FGF - 18与从生长板纯化的全长基底膜聚糖以及基底膜聚糖的重组结构域I - III和III的结合程度相同。这些数据表明，FGF - 18的低亲和力结合位点存在于基底膜聚糖结构域III的富含半胱氨酸区域。FGF - 18分别使来自生长板下部和上部增殖区的软骨细胞培养物中的3H - 胸腺嘧啶核苷掺入增加了9倍和14倍。添加基底膜聚糖可使下部增殖软骨细胞中的这种增加的掺入逆转74%，在上部增殖细胞中逆转37%。这些结果表明，基底膜聚糖可以结合FGF - 18并改变FGF - 18对生长板软骨细胞的促有丝分裂作用。
