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

(1)OCZF在破骨细胞形成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。组蛋白去乙酰酶抑制剂曲古抑素A(TSA)可抑制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。而TSA和NAB对其向巨噬细胞的分化没有影响。在巨噬细胞系中，RAW 264可通过加入RANKL、TSA和NAB而分化为破骨细胞，并抑制NFkappaB的核内转位和MAPK的磷酸化，而MAPK是破骨细胞生成的调节因子。结果提示，OCZF可能调控T5A和NAB的靶基因，并参与破骨细胞的信号转导。OCZF不影响与该共有序列的结合活性。另一方面，我们发现了OCZ…在破骨细胞形成过程中，含p21和p27启动子区域的报告质粒、细胞周期蛋白依赖性激酶(CDK)抑制剂的转录被更多的F刺激。(2)大鼠佐剂性关节炎OCZF表达的原位杂交分析：OCZF在破骨细胞和生发中心部分细胞中高表达。(3)与OCZF相互作用的信号分子分析。我们分离了OCZF在原始264细胞中表达的RNA，并制备了在原始264细胞中表达的cDNA，并正在利用酵母双杂交系统分析一些可能与OCZF相互作用的基因。同时建立了四环素调控OCZF表达的细胞系。利用细胞系，我们将通过免疫沉淀的方法找出与OCZF相互作用的分子。较少

(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