Transcription Factor SP1Regulates Nox1Gene Expression In TNF Alpha Mediated Oxidative Stress

Background and ObjectionAcute respiratory distress syndrome (ARDS) is a kind of acute diffuse lung damage related to risk factors such as pneumonia, sepsis, aspiration. The main features are increased puLmonary vascuLar permeability and gas-containing lung tissue decreases due to lung inflammation. The incidence among patients exposed to risk factors is very high. As early as in1995, Hudson [1] published a study which reported that100%probability of ARDS within7days for patients got sepsis (sepsis) and trauma.Morbidity and mortality declined with the improvement of diagnosis and treatment and the Berlin Definition P1which introduce new diagnosis and treatment standards. However, ARDS sets on rapidly, deteriorates quickly,and the prognosis is poor, which is a worldwide medical problem.Inflammatory response out of control is important to the pathogenesis of ARDS. Bacterial or viral pneumonia is the most common cause of ARDS[3]. Lipopoly-saccharide of Gram-negative bacteria (LPS) initiates the inflammatory response, by combination with monocytes-macrophages, neutrophils, endothelial cells and other cell membrane receptors in vivo and leads to generation of a variety and a large number of of cytokines such as tumor necrosis factor a (TNF-a), interleukin-1β (IL-1β) and IL-6. These factors further role to other cells, causing expanded inflammation, and uLtimately lead to ARDS. TNF-a levels of ARDS patients or animal models are significantly elevated, TNF-a starts and amplifies the inflammatory response of inflammatory mediators [4,5]. Its concentration is closely related to the progression and prognosis of the disease [6]. PuLmonary oxidative stress injury is one of the characteristics of ARDS. ARDS mouse model of LPS-induced inflammation and oxidative stress levels positively correlated with LPS dose, the more high levels of ROS the more serious injury of the lung[7]. Syrkina[8] found oxidative stress imbalanced in lung of mild ARDS mice early. Treating mice with acetylcysteine inhibits glutathione decline, reduces neutrophil infiltration and inflammatory factor in bronchoalveolar lavage fluid, reduces the apoptosis level of airway cell. TNF-a is able to reduce oxidative stress injury in LPS-induced ARDS animal model[9]. Therefore, for patients with ARDS, lung protection strategy shouLd include immune intervention which keeps balance of the excessive inflammation and oxidative stress level, in order to maintain the integrity of the alveolar structure as well as to maintain the alveolar-capillary barrier function.Noxl is an important enzyme to generate ROS. The inflammation-oxidative stress research suggests that, TNF-a upreguLates expression of NADPH and celluLar ROS level [10l The knckout of Noxl gene can reduce the level of ROS induced by the TNF-a treatment within the smooth muscle cells[11]. In model of intestinal catarrh, Noxl expression raised, ROS accordingly increased, inflammatory cytokines also raised, which induced apoptosis in intestinal mucosal cells [12]. Oxidative stress injury mediated by ROS is a pathological basis of many inflammatory diseases. Therefore, to study the expression and reguLation of Noxl is a basis for ROS cause oxidative stress injury. Transcription reguLation is the most important for gene expression. Studies for transcriptional reguLation of Noxl maily focused on cardiovascuLar and intestinal diseases. Kuwano[13] analyzed the source of ROS in colon of intestinal inflammation through a pathway in which TNF-a activates Noxol(a subunits of Noxl) and therefore increases the secretion of ROS by9times. Constructing luciferase expression vector with truncated sequence of Noxol5’ end, found that a TNF-a activation promoter region is located from-585to-452bp. AP1sites in the region play an important role in TNF-a activated Noxl expression. Manea[14] further confirmed the AP1transcriptional reguLation for Noxl. NF-κB is also an important transcription factor. Inhibition of transcription factor NF-kB,Noxl can be significantly downreguLated[15]. TNF-a dependent Noxl and ROS expression levels are significantly reduced in a NF-κB blocked cell model, which is the same as the study resuLts of our laboratory.From the foregoing, current researches about the function and transcription reguLation of Noxl maily focuse on cardiovascuLar disease and colitis model. However, as lung is the first targeting organ of excessive oxidative stress, mechanisms for respiratory inflammatory diseases induced by oxidative stress injury shouLd be studied.In our previous study, alveolar type II epithelial cells A549treated by TNF-a couLd mimic inflammation-oxidative damage and apoptosis in ALI of lung structural cells in vitro. However, we found that disruption of NF-κB only partially down reguLated Noxl expression. Although gene expression is also influenced by non-transcriptional level, we still hypothesize that there are other transcription factors which might be involved in the reguLation of Nox1expression.Our previous study found a remarkable upreguLation of NF-κB in TNF-a treated A549cells. SP1is a potent transcription factor which functions widely. SP1promotes cell growth, embryonic development and the generation and development of tumors by combining with the promoter sequence in the target gene [16]. A study on human glioma cells points out that TNF-a can upreguLate SPlmRNA expression by5-fold [17]. Bioinformatics analysis found that muLtiple SP1transcription factor binding sites is located in the upstream of translation initiation site of Noxl gene. Therefore, we further assumed:The SP1reguLates the Noxl expression by combination with transcription factor binding sites in Noxl promoter.In summary, the objectives of this subject are as follows:1Use our established cell model to assess whether the TNF-a representative inflammation level had function in expression of oxidative stress representative Noxl and its catalytic ROS;2Investigate whether the transcription factor SP1participates in the reguLation of TNF-a-mediated Noxl expression;3Constructe luciferase expression vector of Noxl promoter in order to analyze core region of Noxl promoter and to clarify the role of TNF-a on Noxl promoter activity;4Analyse the promoter activity and binding ability of SP1binding sites in the Noxl promoter core region.MethodsThe A549cells were cuLtured in RPMI-1640medium withlO%fetal calf serum, and incubated at37℃with5%CO2. The cells were grown to about80%confluence when passaged. Select3-5generations of the cells used for experiments,with cells seeded in six-well plates at the density of5×105cells/mL. The cells used for transfection were not incubated with antibiotics, the other experiments cells cuLture in medium plus l00U/mL penicillin and100U/ml streptomycin. 1TNF-a stimuLated Noxl expression and increased the level of its catalytic ROS in alveolar epithelial type II cellsMTT assay divided into Ong/mL (control group), lOng/mL,25ng/mL,50ng/mL,100ng/mL group. According to MTT resuLts, follow-up experiments canceled the100ng/mL group. Noxl expression and ROS levels were categorized according to the TNF-a concentrations:Ong/mL,10ng/mL,25ng/mL,50ng/mL4groups. Noxl silencing identification and ROS levels assay are grouped into:the silence, the silence negative control, silence+TNF-a, silencing the negative control+of TNF-a4groups. In no concentration gradient set experiments of this topic, use the TNF-a concentration with50ng/mL.MTT assay was used for apoptosis. Noxl expression of TNF-a stimuLated A549cells detected by RT-PCR and Western-blotReactive oxygen fluorescent probe DCFH-DA detect TNF-a-stimuLated A549cells ROS level. Transiently transfected with siRNA of eukaryotic cells then use RT-PCR for silence efficiency, DCFH-DA detect ROS level of Noxl knockout A549cells.2the change of Noxl expression when transcription factor SP1is disrupted.The SP1levels grouped by the TNF-a concentration used to stimuLate the A549cells:Ong/mL,10ng/mL,25ng/mL,50ng/mL4groups.Nox1expression after SP1knockout and SP1silence verification divided into silence, silent negative control, Silence+of TNF-a, silent negative control+of TNF-a4groups. SP1expression of TNF-a stimuLated A549cells was detected by RT-PCR and Western-blot. Transiently transfected the eukaryotic cells with siRNA, RT-PCR for Noxl expression when knockout SP1and SP1silence efficiency.3Construct luciferin enzyme expression vector contain Noxl promoter truncated segments and identify its activity Experimental groups for plasmid transfected divided into stimuLation and unstimuLated two groups, each group divided into pGL3-Bask、pGL3-143/ATG、 pGL3-192/ATG、pGL3-401/ATG、pGL3-534/ATG、 pGL3-947/ATG、 pGL3-1415/ATG7groups, according to the length of the promoter (the entire experiment the length of DNA fragment was not calculated protective base and the length of the restriction sites).Upstream1415bp of the Noxl translation initiation sites were amplified by PCR, and by design of primers, amplification gradually extended promoter fragment upstream of the translation start site. The promoter fragment was ligated into the pGL3-Basic vector used molecuLar cloning method.Transiently transfected with the pGL3-Noxl, dual luciferase reporter system detects the relative luciferase activity.4The influence of SP1binding sites to Noxl promoter activityPromoter deletion SP1binding sites named Noxl-1377/ATG, the recombinant plasmid was named pGL3-1377/ATG. The luciferase activity is divided into TNF-a stimuLation group、unstimuLated group and TNF-a stimuLation+SPlsiRNA group three major groups.Each major group is divided into the the pGL3-1377/ATG and pGL3-1415/ATG two groups. EMSA experiments to detect the activity of spl binding sites grouped into1:negative control group,2:Sample response group, the3: probe cold competition reaction group,4:mutation probe cold com petition reaction group5:spl-1group:6:spl-2,7:GATA-1group,8:Oct-1group,1-4group EMSA reaction was the deletion fragments pro-be,5-8group of EMSA reaction probe were transcription factor binding sites from deletion fragments.The overlapping PCR amplification Noxl promoter missing SP1sites in the core area fragment,which named Noxl-1377/ATG, digestion and sequencing to verify its accuracy. pGL3-Basic.In accordance with the method of MolecuLar Cloning, Noxl-1377/ATG is connected to the pGL3-Basic. Detect luciferase activity in order to determine the missing38bp exists promoter activity. According to the Alibaba2.1,binding sites of the38bp divided into a few EMS A probes, to detect whether SP1couLd bind to it.5. Statistical methodsUse spss13.0package for statistical analysis, measurement datas were expressed as mean number±standard deviation (X±s) MuLtiple comparisons use One-Way-ANOVA. First test of homogeneity of variance and the overall difference between the groups, the muLtiple comparisons with LSD when the data are equal variances, Tamhane test for unequal variances. Detection level α=0.05, P<0.05significant difference. The experiment was repeated three times.ResuLts:1Levels of Noxl expression and its catalytic product ROS in TNF-a-stimuLated alveolar epithelial type Ⅱ cells A549.1.1TNF-a couLd upreguLate Noxl expression, and increase ROS level catalytic products of Noxl, and is positively correlated with the concentration of TNF-a.1.2Silence of Noxl can down-reguLate Noxl expression by about70%, and can reduce the level of ROS.2Relationship between Noxl expression and silence of transcription factor SP1.2.1Silencing SP1can down-reguLate the the SP1expression by about70%, and reduce Noxl expression mildly.3Construction of Noxl sub-truncated fragments expression vector and viability assessment.3.1Truncated promoter fragment luciferase expression vector pGL3-Noxl is successfuLly constructed.3.2Core promoter region is between-534and-401bp.3.3TNF-a can increase the the Noxl promoter activity. 4Activity of SP1binding sites in Noxl promoter4.1The recombinant plasmid pGL3-1377/ATG missing38bp including SP1binding sites is successfuLly constructed.4.2The decrease of relative luciferase activity of pGL3-1377/ATG is statistical significantly. TNF-a can increase the luciferase activity of pGL3-1377/ATG.4.3SP1binding sites in the deletion38bp couLd bind to SP1in vitro.Conclusion:1Inflammatory cytokines TNF-a increased oxidative stress by upreguLating Noxl expression level in alveolar type II epithelial cell line A549, which is positively related to the concentration of TNF-a;2The transcription factor SP1is involved in TNF-a-mediated activation of Noxl;3SP1upreguLates the expression of Noxl through a combination to the site-427--418bp in the Noxl promoter.