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The role of cathepsin E in osteoclast and the research for substrate molecule binding with cathepsin E

组织蛋白酶E在破骨细胞中的作用及其与组织蛋白酶E结合的底物分子的研究

基本信息

批准号：
21592369
负责人：
OKAMOTO Kuniaki
金额：
$ 3万
依托单位：
Nagasaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2009
资助国家：
日本
起止时间：
2009 至 2011
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-21592369/
关键词：
破骨細胞骨粗鬆症プロテアーゼ aspartic proteinase cathepsin E osteoclast RANK RANKL

项目摘要

An axis of RANK(Receptor Activator of NF-kB)/ RANKL(RANK Ligand), OPG(Osteoprotegerin) and Macrophage colony stimulating factor(M-CSF)/ c-fms is one of the most important signal pathways in differentiation and activation of osteoclast. In previous study, we reported that number of osteoclast significantly decreased in cathepsin E(CE) knockout mice. In this time, real time PCR analyses showed that mRNA expression levels of M-CSF, c-fms, RANK, c-fos and osteoclast specific marker, cathepsin K and TRAP tended to decrease in CE KO mice, although no statistical differences were recognized. Moreover, mRNA of fusion-related protein such as DC-STAMP and OC-STAMP also decreased. Therefore, we examined mRNA profile with wild(C57BL/ 6) and CE KO mice using DNA microarray. Then, lots of factors related with oxidative stress significantly increased in CE KO mice compared with wild type mice. Furthermore, hydrogen peroxide also increased in CE KO mice after adding RANKL. Studies of autophagy related molecules such as p62, LC3 and ubiquitin showed that they migrated toward non soluble fraction from soluble fraction. These results suggest that CE deficiency causes impairment of autophagy, a degradation system for aggregated proteins and damaged organelles, and increase of oxidative stress. Finally, number of osteoclast significantly decrease in CE KO mice.

RANK(核因子-kB受体激活剂)/RANKL(RANK配体)、OPG(骨保护素)和巨噬细胞集落刺激因子(M-CSF)/c-FMS轴是破骨细胞分化和激活过程中最重要的信号通路之一。在之前的研究中，我们报道了组织蛋白酶E(CE)基因敲除小鼠的破骨细胞数量显著减少。实时荧光定量聚合酶链式反应分析显示，CEKO小鼠M-CSF、c-FMS、RANK、c-fos和破骨细胞特异性标志物组织蛋白酶K、TRAP的mRNA表达水平有下降趋势，但差异无统计学意义。此外，DC-STAMP和OC-STAMP等融合相关蛋白的mRNA也有所下降。因此，我们用基因芯片检测了野生型(C57BL/6)和CEKO小鼠的基因表达谱。与野生型小鼠相比，CEKO小鼠体内许多与氧化应激相关的因子显著增加。此外，加入RANKL后，CEKO小鼠体内的过氧化氢含量也增加。对自噬相关分子如p62、Lc3和泛素的研究表明，它们从可溶组分向非可溶性组分迁移。这些结果表明，CE缺乏导致自噬功能受损，自噬是聚集蛋白和受损细胞器的降解系统，并导致氧化应激增加。最后，CEKO小鼠破骨细胞数量明显减少。