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

(1) OCZF在破骨细胞发生信号转导中的作用分析OCZF抑制巨噬细胞转录因子egr-1的转录活性。组蛋白去乙酰化酶抑制剂trichostatin A （TSA）抑制OCZF的转录抑制活性。TSA和另一种组蛋白去乙酰化酶抑制剂丁酸钠（NaB）抑制破骨细胞分化。另一方面，TSA和NaB对巨噬细胞的分化没有影响。在巨噬细胞细胞系中，通过添加RANKL、TSA和NaB能够分化为破骨细胞的RAW 264抑制了作为破骨细胞发生调节因子的NFkappaB向细胞核的易位和MAPK的磷酸化。数据提示，T5A和NaB的靶基因可能受OCZF调控，参与破骨细胞发生的信号转导。分析了OCZF对NFkB的刺激活性。OCZF对该一致序列的结合活性没有影响。另一方面，我们发现OCZ . More F刺激具有p21和p27启动子区域的报告质粒的转录，细胞周期蛋白依赖性激酶（CDK）抑制剂的表达在破骨细胞发生中被ODF刺激。采用western分析和DNA芯片技术比较巨噬细胞、表达OCZF的RAW264与对照组蛋白和mRNA的表达差异。(2)原位杂交法分析大鼠佐剂性关节炎OCZF的表达。原位杂交分析显示OCZF在破骨细胞和生发中心部分细胞中强烈表达。(3)与OCZF相互作用的信号分子分析我们分离RNA，制备在RAW 264细胞中表达的cDNA，并对酵母双杂交系统中可能与OCZF相互作用的部分基因进行分析。同时建立了四环素调控OCZF表达的细胞系。利用这些细胞系，我们将用免疫沉淀法找出与OCZF相互作用的分子。少

(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