| Background and issues: It was reported that endothelial nitric oxide synthase (eNOS) not only participates in regulating blood pressure and vascular permeability , preventing white blood cell adhesion and platelet aggregation, but also concerns some pathological processes such as shock, hypertension, atherosclerotic heart disease, diabetes, ischemia strock, vascular stenosis and occlusion. Unless flow shear stress it is also influenced by a variety of stimuli, at least including hypoxia, lysophosphatidycholine (LPC), transforming growth factor-p (TGFp),tumor necrosis factor-a (TNFa), oxidized low density lipoprotein, physical exercise and estrogen, which may up-regulate eNOS mRNA level. eNOS, therefore, plays a key role in maintaining blood pressure homeostasis and vascular integrity.lt is very significant to elucidate signal transduction pathways by which expressions of eNOS gene are increased. eNOS activity, at present, are observed mainly by a luciferase reporter gene, but it is not easy to control influencing factors, experimental cells have to be splitted, and it is impossible to make quality and quantity for the same sample at the same time. Hence, real time observation can not be reached. According to a few reports, different regions of human eNOS promoter may exert different functions, with SP1 and GATA-2 elements playing a critical role in regulating eNOS promoter activity. Although eNOS promoter contains many cis- elements such as API that appear different functions in other genes, characteristics and functional regions of it remain unclear. Strong evidence supports a role for LPC in the processes of atherogenesis, inflammation, and vascular wound healing, and ithas used as a model system to elucidate the mechanism by which eNOS gene transcription is regulated. A preliminary report has been obtained that eNOS promoter is activated by a signaling pathway including PI-3gamma (phosphatidylinositol-3 kinase r) -Jak2 (Janus kinase 2) -MEK1-ERK1/2. Therefore, mechanisms concerning signal transduction remain to be clarified in detail.Purposes and methods: In this study, a red fluorescent protein reporter gene vector containing human eNOS promoter was constructed with molecular cloning techniques, and expression of it in different mammalian cells observed. Then four different regions of eNOS promoter- driving red fluorescent protein reporter gene vectors were constructed by DNA sequence deletion, and also ten mutants of SPK API and SSRE cis elements were generated through DNA site-directed mutagenesis, by which transcriptional activity of different regions of human eNOS promoter and functions of SP1, API and SSRE cis elements were investigated. This report is to shed light on the MAPK signal regulation of human eNOS gene expression by LPC stimulus, checking from MAPKK, MAPK, transcriptional factor and cis element levels respectively, utilizing red fluorescent protein reporter gene vectors containing human eNOS promoter, applying wild MAPK and MAPKK vectors or active and negative mutants of them, using different signal molecules-selective inhibitors, employing co-transfecting MAPK or MAPKK signal molecules into cells, depending on decoy ODN techniques, .adopting electrophoretic mobility shift assay, and making use of Western blot. Results and conclusions: Our main findings showed as follows: (1) The four red fluorescent protein reporter gene vectors containing human eNOS promoter, which were testified to be right via qualification of PCR, double restriction ribonuclease digestion and DNA sequencing, were first successfully constructed and highly expressed in NIH3T3, HEK293, ECV304 cell lines. This reporter gene system is characteristics of real time observation, quality and quantity analysis for the same sample at the same time. So, we offer an efficient and practical tool to investigate correspondingmechanisms about transcriptional regulation of eNOS.(2) It was found that 32 percent of basal eNOS promoter activity was located at-1034-1600bp region, and 68 percent at l~-166bp region, which is...
|
PDF Full Text Request