Function analysis of Na^+ dependent ion transporters in bone metabolism

Na^依赖性离子转运蛋白在骨代谢中的功能分析

基本信息

批准号：
12671815
负责人：
AMANO Hitoshi
金额：
$ 2.37万
依托单位：
Showa university
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12671815/
关键词：
mechanical stress osteoblast RT-PCR osteoclast resorption pit co-culture Na^+Ca^<2+> exchanger NPT2 CSF-1 KB-R7943

项目摘要

Na^+/Ca^<2+> exchanger (NCX) catalyzes the electrogenic exchange of 3 Na^+ for 1 Ca2^+ across the plasma membrane in many mammalian cells. We have previously reported that the Na^+/Ca^<2+> exchange activity had an important role for CSF-1-induced osteoclast differentiation. In the present study, we sought the effect of mechanical stress to confirm the role of NCX1, in osteoblastic bone formation and osteoclastic bone resorption formation.Osteoblast were obtained from calvalia of new bone rat. Mechanical stress induced NCX1 mRNA and F-actin polyme ralization in osteoblast. The application of Gadlinium ion, a blocker of SAchannel, inhibited stretch-activated cell responces.Bone marrow cells were obtained from tibiae and femuor of 5- to 8-week-old male ddy mice. We used Sephadex G-10 column (G10) in order to remove macrophages and stroma cells. Non adherent G10-passed cells were cultured in a-MEM containing the final concentration of 15 % FBS, 100 ng/ml RANKL, 20 ng/ml CSF-1. KB-R7943 inhibited the osteoclast differentiation and bone resorption in vitro dose-dependently. In addition, NCX1 antisense oligo deoxy nucleotide (ODN) reduced the number of tartrate-resistant acid phosphatase-positive multinucleated cells and decreased the amount of NCX1 in protein level. Eight week-old female ddY mice received sham-operation or ovar iectomy (OVX) and were fed a laboratory chow for 4 weeks after the operation. Animals were given subcutaneously 337.5 mg/kg KB-R 7943 or DMSO every other day for 4 weeks. After 4 weeks treatment with KB-R7943, the BMD in the femur of OVX group was significantly decreased compared with sham group. However the BMD in KB-R7943 treated OVX group was quantitatively similar to the sham group. These results suggest that bone resorption was also blocked by treatment with KB-R7943 in vivo.In conclusion, the activation of NCX1 is essential for osteoclast differentiation and activation.

Na^+/ca^<2+>交换器（NCX）在许多哺乳动物细胞中催化跨质膜的3 na^+的电源交换为1 Ca2^+。我们先前已经报道说，Na^+/Ca^<2+>交换活性对于CSF-1诱导的破骨细胞分化具有重要作用。在本研究中，我们寻求机械应力的作用来确认NCX1的作用，在成骨细胞骨形成和整骨骨吸收形成中。机械应力诱导的NCX1 mRNA和成骨细胞中的F-肌动蛋白多矩阵。 gadlinium离子的应用是s频道的阻断剂，抑制了拉伸激活的细胞呼吸。骨髓细胞是从胫骨和5周至8周大的雄性DDY小鼠的肌肉中获得的。我们使用Sephadex G-10柱（G10）来去除巨噬细胞和基质细胞。在含有15％FBS，100 ng/ml RANKL，20 ng/ml CSF-1的最终浓度的A-MEM中培养非粘附的G10细胞。 KB-R7943抑制了破骨细胞分化和体外剂量依赖性的骨吸收。此外，NCX1反义寡核苷酸（ODN）减少了耐锈蚀酸酸磷酸酶阳性多核细胞的数量，并减少了蛋白质水平的NCX1量。八个星期大的雌性DDY小鼠接受了假手术或卵形体化学切除术（OVX），并在手术后4周接受了实验室CHOW。在4周内，每隔一天给动物皮下注射337.5 mg/kg KB-R 7943或DMSO。在用KB-R7943治疗4周后，与假模组相比，OVX组股骨的BMD显着降低。但是，在KB-R7943处理过的OVX组中的BMD在定量上与假模组相似。这些结果表明，在体内用KB-R7943处理也阻塞了骨吸收。总之，NCX1的激活对于破骨细胞分化和激活至关重要。