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Functional analysis of transcriptional regulator induced by ODF in the signal transduction of formation and activation of osteoclasts

ODF诱导的转录调控因子在破骨细胞形成和激活信号转导中的功能分析

基本信息

批准号：
13671942
负责人：
KUKITA Akiko
金额：
$ 1.92万
依托单位：
Saga Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13671942/
关键词：
osteoclast differentiation macrophage histone deacetylate inhibitor 転写因子

项目摘要

(1) Analysis of role of OCZF in the signal transduction of osteoclastogenesisOCZF inhibit transcriptional activity of macrophage transcriptional factor, egr-1. Histone deacetylase inhibitor, trichostatin A (TSA) inhibits transcriptional repressive activity of OCZF. TSA and a another histone deacerylase inhibitor, sodium butyrate, NaB inhibited osteoclast differentiation. On the other hand, TSA and NaB did not have the effect on the differentiation into macrophages. In a macrophage cell line, RAW 264 which is able to differentiate into osteoclasts by the addition of RANKL, TSA and NaB inhibited the translocation of NFkappaB into nucleus and phospholylation of MAPK, whose are regulators of osteoclastogenesis. Data suggest the target genes of T5A and NaB may be regulated by OCZF and involved in the signal transduction of osteoclastogenesis.Stimulative activity of NFkB of OCZF was analyzed. OCZF did not affect the binding activity to this consensus sequence. On the other hand, we found OCZ … More F stimulated the transcription from the reporter plasmids which have promoter region of p21 and p27, cyclin-dependent kinase (CDK) inhibitors whose expression is stimulated by ODF in osteoclastogenesis. Difference of the expression of protein and mRNA between macrophage cell, RAW264 expressing OCZF and control was compared by western analysis and DNA microarray.(2) Analysis of OCZF expression by in situ hybridization of rat adjuvant arthritis.In situ hybridization analysis revealed that OCZF is strongly expressed in osteoclasts and some cells in germinal center.(3) Analysis of signaling molecules interacting with OCZFWe isolated RNA and prepared cDNA expressed in RAW 264 cells and are analyzing some genes which are possibly interacting with OCZF with yeast two-hybrid system. At same time we establish the cell line in which the expression of OCZF is regulated by tetracycline. Using the cell lines, we are going to find out the molecules which interact with OCZF by immunoprecipitation methods. Less

(1)OCZF在破骨细胞生成信号转导中的作用分析OCZF抑制巨噬细胞转录因子β 1的转录活性。组蛋白去乙酰化酶抑制剂，抑制素A（TSA）抑制OCZF的转录抑制活性。TSA和另一种组蛋白脱乙酰酶抑制剂丁酸钠NaB抑制破骨细胞的分化。TSA和NaB对巨噬细胞的分化没有影响。在巨噬细胞系中，RAW 264能够通过添加RANKL、TSA和NaB而分化成破骨细胞，其抑制NF κ B易位到细胞核中和MAPK的磷酸化，其是破骨细胞生成的调节剂。结果表明，OCZF对T5A和NaB靶基因的表达有调控作用，并参与破骨细胞生成的信号转导。OCZF不影响与该共有序列的结合活性。另一方面，我们发现OCZ ...更多信息 F刺激具有p21和p27启动子区的报告质粒的转录，p21和p27是细胞周期蛋白依赖性激酶（CDK）抑制剂，其表达在破骨细胞生成中被ODF刺激。Western分析和DNA微阵列分析比较巨噬细胞、表达OCZF的RAW264细胞和对照细胞蛋白质和mRNA表达的差异。(2)大鼠佐剂性关节炎原位杂交分析OCZF表达原位杂交分析显示OCZF在破骨细胞和生发中心的一些细胞中强烈表达。(3)与OCZF相互作用的信号分子分析我们从RAW264细胞中提取RNA，制备cDNA，并利用酵母双杂交系统对可能与OCZF相互作用的基因进行分析。同时建立了四环素调控OCZF表达的细胞系。利用这些细胞系，我们将通过免疫沉淀的方法找出与OCZF相互作用的分子。少