摘要
丝裂原激活蛋白激酶(MAPK)酶级联反应系统参与胶质细胞中iNOS的合成.通过瞬时转染p38MAPK途径中上游激酶,MAPK激酶3(MKK3)和MAPK激酶6 (MKK6 )表达质粒,进一步了解p38MAPK级联传导信号系统调节iNOS基因在胶质细胞中的转录激活机制.MKK3或MKK6表达质粒与接有荧光素酶(luciferase ,Luc)的大鼠iNOS启动基因质粒(iNOS Luc)联合转染C6星形胶质细胞株引起iNOS Luc的激活,并且使细胞因子诱导的iNOSmRNA的表达增强.这两种效应都能够被p38MAPK抑制剂SB2 0 35 80所抑制.MKK3 6也可以诱导核因子κB(NFκB Luc)依赖的转录活性.这些分子水平的研究结果为p38MAPK信号级联传导途径在调节大鼠胶质细胞中iNOS基因转录激活中的重要作用,包括转录因子NFκB的作用提供了证据.通过阻断iNOS表达或NO的生成,抑制细胞炎症发生,为防治神经细胞炎症反应性疾病提供实验依据.
The objective of this study is to evaluate the role of p38MAPK in the induction of iNOS expression during costimulation of C-6 glial cells with lipopolysaccharide(LPS),TNF-α and IFN-γ. Measurement of cellular NO content showed that treatment of C-6 glial cells with LPS,IFN-γ and TNF-α alone did not cause significant changes of NO levels,while combination of the three agents induced markedly increase of NO production from the cells implying there was cooperation effect between cytokines and LPS. In view of SB203589, a specific inhibitor of p38MAPK markedly inhibited cytokines and LPS induced production of NO,we further found that transient transferction of C-6 glial cells with eukaryotic expression vectors harboring MAPK kinase 3(MKK3) and MAPK kinase 6(MKK6),which are both constitutively active upstream kinase in p38MAPK resulted in an activation of the p38MAPK pathway,resulted in an induction of the activation of p38MAPK,this action was further confirmed by iNOS promoter-derived luciferase assay showing that co-transfection of the cells with expression vectors of three agents gave rise to 3-fold increases in iNOS activity as compared with control group. In addition,the mechanisms for MKK3/6 to active iNOS were revealed by phosphorezation of p38MAPK as demonstrated using antiphosphorised p38MAPK antibody.These results provide evidenced for an important role of the p38MAPK pathway in transcription activation of the iNOS gene in rat glial cells.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2005年第3期315-321,共7页
Chinese Journal of Biochemistry and Molecular Biology
