Mechanism of Bone Growth : Research on proliferation and apoptosis of growth plate chondrocyte.

本课题旨在探讨生长板软骨细胞增殖、分化和细胞死亡(凋亡)的分子基础。为此，我们选择软骨发育不全作为生长板软骨细胞的模型疾病。这种疾病的致病基因是编码成纤维细胞生长因子受体3(FGFR3)的基因。主要的突变是G380R，它结构性地激活受体。而与之相关的严重形式，死亡性发育不良是由于突变K650E，它更深刻地诱导受体激活。我们首先将这些突变导入成软骨细胞系ATDC5，并研究了这些细胞的表型变化。表达突变的FGFR3的软骨细胞生长恶化，细胞凋亡率增加。抑制细胞生长是通过激活p21介导的，而细胞凋亡率的增加是由于bcl2表达的降低。此外，K650E突变还诱导了STAT1的激活和核转位，以及L…更倾向于位于细胞生长恶化和凋亡增加的上游。生长激素诱导的IGF-I可能激活PI3K，进而增加bcl2的表达。这一机制可能与挽救软骨细胞的凋亡和生长激素刺激软骨生长有关。从软骨发育不全的类比到PTHrP KO小鼠，我们接下来研究了PTHrP在这个细胞模型中的表达。表达突变的FGFR3的细胞PTHrP基因表达降低。此外，这种下降在K650E突变中更为严重。接下来，我们通过多种途径将PTHrP作用于细胞，证实PTHrP信号可能阻断FGFR3结构性激活的致病途径，使细胞免于凋亡。软骨特异性突变的FGFR3转基因小鼠骨器官培养系统显示骨长度增加，这主要是由于增殖的软骨细胞区的扩张所致。体内实验同样表明，甲状旁腺素对软骨发育不全的骨生长有良好的效果。较少

We aimed to explore molecular basis of the cell proliferation, differentiation, and cell death(apoptosis) of the growth plate chondrocyte in this project. To this aid, we selected achondroplasia as a model disorder of growth plate chondrocyte. The responsible gene to this disorder is the gene coding fibroblast growth factor receptor 3 (FGFR3). The major mutation is G380R, which activate receptor constitutively. And related severe form, thanatophoric dysplasia is due to the mutation, K650E, which induce receptor activation more profoundly. We at first introduced these mutations into chondrogenic cell line, ATDC5, and studied phenotypic change of these cells. The chondrocytes expressing mutated FGFR3 showed deteriorated cell growth and increased apoptosis. The inhibition of the cell growth was mediated by the activation of p21 and increased apoptosis was due to the decreased bcl-2 expression. Moreover K650E mutation induced stat 1 activation and its nuclear translocation and this event l … More ooked to located upstream of deteriorated cell growth and increased apoptosis. IGF-I induced by the Growth hormone treatment may activate PI3K and then increase bcl-2 expression. This mechanism may be responsible to the rescue chondrocyte from apoptosis and to GH stimulated bone growth in achondroplasia.From thee analogy of achondroplasia to PTHrP KO mice, we next studied PTHrP expression in this cellular model. The cells expressing mutated FGFR3 showed decreased expression of PTHrP mRNA. Moreover, this decrease was more severe in K650E mutation. Next we administered PTHrP to the cells by several methods and confirmed that PTHrP signal may blockk pathogenic pathway induced by constitutively activated FGFR3 and rescue the cells from apoptosis. Bone organ culture system using cartilage specific mutated FGFR3 transgenic mice showed increased bone length, which was mainly due to the expansion of proliferative chondrocyte zone. In vivo experiment similarly indicates promising effect of PTH on the bone growth of achondroplasia. Less