Studies On Transcriptional Regulation And Its Mechanism Of INOS Gene Expression By P38 MAP Kinase

Objective:NO is a free radical generated from guanido group of L-arginine catalyted by NOSs. NO plays important roles in many biological processes,such as killing tumor cells,defending cellular microorganisms,vasodilatation,neurotransmission and inhibiting platelet aggregation. NOSs are composed of constitutive NOS (cNOS) including endothelial NOS (eNOS) and neural NOS (nNOS) and inducible NOS (iNOS). NO that derived from cNOS generally acts as a kind of vasodilatation agent or neurotransmitter,while iNOS that is not present in rest cells can be induced by various stimuli,such as LPS,TNF-a,IL-1,pyridine carboxylic acid,phorbol ester and hypoxia,etc. iNOS is mainly regulated at transcriptional level. The elevated expression of iNOS is closely associated with some diseases such as infectious and hemorrhagic shock,rheumatic arthritis and pulmonary tuberculosis.Since mouse iNOS gene being cloned,scientists in the world have performed extensive studies on the transcription regulation of iNOS gene,but there are still many confusing questions. Our experiments focus mainly on the role of four p38 subtypes in transcriptional regulation on iNOS gene and the possible mechanisms underlying. Expect that we can shed some light on understanding the action of p38 MAPK hi imflammation.Methods:Using co-transfection techniques mediated by liposomes,we transfected Flag-tagged p38 isoforms of wild form or enzyme-dead mutants in pcDNA3,pcDNA3,piNOS-Luc,pCMV-B into HEK 293 cells as being indicated in the experimental design. The luciferase intensity was assayed with internal-control of P-gal for the understanding of the effects of four members of p38 MAPK on transcription regulation of iNOS. Co-transfection of Flag-tagged p38a in pcDNA3,piNOS-Luc,pCMV-p,and AP-1W ODN or AP-1M ODN into HEK 293 cells for the understanding of the function of AP-1 binding sites in the transcriptional regulation of iNOS gene. Flag-tagged p38 isoforms in pcDNA3,pcDNA3,pCMV-p,ATF2/GAL and 5XGAL4-Luc were introduced into HEK293 cells for the study of how ATF-2 acts as one of substrates of p38 to exert the effect on iNOS promoter transactivity induced by p38 MAPK.Results:1. Without the stimulation of LPS,p38