Screening Of Differential Proteins Binding To Nox1 Promoter In TNF-α-induced A549 Cells And Preliminary Functional Study Of CSRP2

Background and objectivesAcute respiratory distress syndrome (ARDS) is a group of clinical syndrome with progressive dyspnea and refractory hypoxemia caused by refractory hypoxemia, such as severe infection, trauma, aspiration. It is a common severe acute disease in respiratory department. Based on the NIH Heart,60-day mortality in patients with ARDS maintained more than 25% during the last 10 years. The present studies show that the mainly pathophysiological process is vascular endothelial cells and alveolar epithelial cells out-of-control inflammatory response, which leads to cell dysfunction, permeability increased, and then appear diffuse pulmonary interstitial and alveolar edema.In the process of cell or tissue injury, inflammation and oxidative stress are both important parts of the defense responses, and they intract each other. The inflammatory response is often accompanied by the generation of oxygen free radicals to clears the pathogenic microorganism, and oxygen free radicals can also act as a second messenger to modulate the inflammatory response. Alveolar type Ⅱ cells, "stem cells" of alveolar cells with the proliferation and secretion function, play an important role in inflammation and oxidative stress in the process of lung infection, produce proinflammatory factors, such as IL-8, TGF-β and TNF-α, to promote a "cascade of inflammatory cascade reaction". TNF-α is a main pro-inflammatory during inflammation and oxidative stress, it produces reactive oxygen species possess with the function of destructing pathogenic microorganisms and second messenger, then trigger the reaction of the downstream series inflammation oxidative stress, and mediated apoptosis and necrosis. Previous studies in our laboratory have shown that, treatment with TNFR-Fc significantly reduced LPS-induced ALI The transcription level of Nox and XO was down-regulated by anti-TNF-a therapy, followed by oxidative stress attenuation.In alveolar epithelial cells with inflammatory oxidative stress, NADPH oxidase (Nox) family is an important source of ROS. Nox family is consisting of the Nox1-5 and Duoxl-2 isoforms, among them, the alveolar epithelial cells mainly express Nox1. Liu proved that Noxl gene expression was up-regulated in A549 cells stimulated with TNF-α. Previous studies in our laboratory have shown that, TNF-a-induced A549 cells can be successfully simulated inflammation and oxidative stress model in alveolar epithelial cell, and NF-κ B/p65 silencing could down-regulate the over inflammation and cell apoptosis induced by TNF-α, and NF-κ B p65 can bind to Noxl promoter to regulate the express of Noxl. But there is no clear conclusion about other proteins participate the regulation of Noxl except NF-κ B. In the view of this, we focus on the regulatory proteins involved in Noxl protomer to study the express of Noxl.In genetics, a promoter is a region of DNA with the function of RNA enzyme recognition, initiate transcription of a particular gene. In eukaryotes, the promoter itself does not directly regulate gene transcription and translation unless combined with the transcriptional regulation of protein. So the study on protein binding to promoter DNA is an important point to understand the target gene transcription regulatory mechanism. Lambeth’s research shows that the upstream sequence of more than 4000 base of Noxl gene transcription start site contains IRSE, GAS, kappa B, GATA, C/ERB transcription factor binding sites, which can regulate Noxl transcription. Therefore, study on differential proteins binding to Noxl promoter in the oxidative stress model is of great significance to study the activation of Noxl pathway.At present, to study proteins binding to promoter includs intracellular and extracellular methods. Intracellular method contains the yeast one-hybrid technique, phage surface display technology, and so on, the known promoter sequences are used to screen the protein combined with the coding genes, then to determine the protein by bioinformatics. This method has the characteristics of high throughput screening, but unsatisfactory specificity. Extracellular methods contains gel retardation assay, DNase I footprinting experiments, and so on, recombination proteins are used to combine the promoter to validate the proteins in vitro. This method has the characteristics of high specificity, but low efficiency and complex operation.So, a method with high-throughput and high specificity should be used to screen the differential proteins binding to Noxl promoter. We use the biotin-streptavidin system, two-dimensional fluorescence difference protein gel electrophoresis and protein mass spectrometry to screen and identify differential proteins binding to Noxl promoter inTNF-a-induced A549 cells. We hope to provide an experimental basis for the follow-up study of cell signal transduction pathways in Nox1 transcription regulatory mechanism.Methods1 Screening of differential proteins binding to Nox1 promoter in TNF-α-induced A549 cells1.1 Preparation of biotin-labeled Nox1 promoter DNA probesE. coli bacteria transfected with recombinant plasmid PGL3-Basic-Nox1 retained in our aboratory were intermediate cultured, and recombinant plasmid PGL3-Basic-Nox1 (as template) was extracted according to the manual. Nox1 promoter upstream primer was biotinylated in 5’end. Biotin-labeled Nox1 promoter DNA probes were amplificated theough PCR. The product of PCR was observed by 1% agarose electrophoresism, Nox1 promoter DNA probes were recycled according to Agarose gel DNA extraction kit manual.1.2 DNA pull-down research1.5 × 106 A549 cells were seeded in 15cm plate, serum-free medium was given for 12h when the cells grew to 80%, then treated with TNF-α at 10 ng/ml and incubated for 1h, while the control group were incubated with equivalence serum-free medium. After the cells precipitation were collected, nuclear proteins were extracted accoding to the cytoplasmic nuclear protein extraction manmual, then determinated the concentration accoding to the EttanTM 2D Quant Kit manmual. The proteins binding to Nox1 promoter in nuclear proteins were isolated through DNA pull-down following the Dynabeads(?) kilobase BINDERTM Kit manual.1.3 Two-dimensional fluorescence difference gel electrophoresis (2D DIGE)Nuclear proteins were labeled with fluorescent dye CyDye, added to the 24cm pH 3-10 NL IPG strips to finish the IEF in EttanTM IPGPhor. After that, the IPG strip were removed and placed on the top of the 12.5% polyacrylamide gel after equilibrium liquid balance, and then the second dimension electrophoresis was finished in EttanTM DALT Six electrophoresis instrument in the condition of light avoiding.The gel labeled with CyDye is used to be analyzed. After electrophoresis, the plastic sheet image was scanned and imaged by Typhoon Imager 9400, Cy2. Cy3 and Cy5 images can be seen through the ImageQuant TL software respectively blue, green and red, represent internal standard proteins, proteins binding to Nox1 promoter in control group and treatment group. Overlay image was overlaid with both Cy3 and Cy5 channels.1.4 Preparation of gel, imaging, analysis of differential proteinsSamples containing same amount of every group proteins were added to gel, and then finished the first and second dimension electrophoresis with the same parameter as the analysis gel. After electrophoresis, the gel was stained with fixation and coomassie brilliant blue G250 stain solution. The imaging of the gel was acquired through scanned by Image Scanner, anlalyses of differential proteins in or between gels was done by DeCyder V6.5 analysis software, and then the average volume of protein spots greater than 1.5 times between the two groups in analysis gel were filtered. The protein spots were dug in the gel by EttanTM Spot picker after picked.2. Mass spectrometry and bioinformatics analysis in differences binding proteins to Noxl promoter in TNF-a-induced A549 cells2.1 Mass SpectrometryDifferential proteins were identified by mass spectrometry after we finished sample processing multistep such as differential protein spots digesting by trypsin. Samples were input to ABI 4800 MALDI-TOF/TOF mass spectrometer to get peptide mass fingerprint (PMF). Mode MS and MS/MS were used to search database Swiss-Prot, Humon was used as search Species. Mascot score, numbers of fragments matching and t coverage judgment were estimated to get the result.2.2 Bioinformatic analysisWoLF PSORT predicted software and Swiss-Prot NCBI were used to analysis the subcellular localization of the differential proteins binding to Nox1 promoter in TNF-α-induced A549 cells screened by DIGE. Interaction between proteins was predicted by String database.3. Preliminary functional study of CSRP23.1 Construction of plasmid pcDNA3-CSRP2-HACultured A549 cells in vitro, total RNA was extracted, full-length fragment of the CDS of CSRP2 mRNA was amplificated by RT-PCR. Plasmid pcDNA3-CSRP2-HA was constructed by molecular cloning.3.2 Localization of CSRP2 detected by immunofluorescence1.0 X 105 A549 cells were seeded in 24-well, plasmid pcDNA3-CSRP2-HA and pcDNA3-HA were respectivly transiently transfected when the cells grew to 80% to keep on cultured for 24h, then treated with TNF-a at 10 ng/ml and incubated for 1h, while the control group were incubated with equivalence medium. Then the localization of CSRP2 was dected by immunofluorescence.3.3 Effects of CSRP2 on transcriptional expression of Noxl dectecd by luciferase reporter gene assayCells culture was the same as step 3.2. Plasmid pcDNA3-CSRP2-HA and Plasmid PGL3-Basic-Noxl were transiently co-transfected when the cells grew to 80% to keep on cultured for 24h, then treated with TNF-α at 10 ng/ml and incubated for 1h, while the control group were incubated with equivalence medium. Plasmid pcDNA3-HA was used to act as negative control. Then the relative luciferase activity was detected.Result1. Screening of differential proteins binding to Noxl promoter in TNF-α-induced A54924 protein points (differential expression of greater than 1.5 times) was screened by software analysis after gels picked, among them,23 proteins were up-regulated while the lasted was down-regulated.2. Mass spectrometry and bioinformatics analysis in differences binding proteins to Noxl promoter in TNF-a-induced A549 cells2.1 Mass SpectrometryWe identified 13 proteins among the 24 proteins picked by 2D DIGE, they are GLE1, ALB, EXD2, KRT9 TTC34 of KRT10, KRT1, DDX19A GSTM5 GATC SSX11, H4, CSRP2, KRT1 and KRT10 were the same kind of protein (keratin), point 724,725,727,742 were the same protein (KRT9), point 728,741,745,754 were the same protein (KRT10), point 1556,1591 were the same protein GSTM5,that meant the proteins had modified change during TNF-a stimulation.2.2 Subcellular localization of the differential proteinsAmong the 13 proteins identified by mass spectrometry, GLE1、EXD2、KRT9、 TTC34、KRT10、KRT1、DDX19A、GATC、Histone H4、CSRP2 can locate in nuclei.2.3 Interaction between proteinsKRT1, KRT9, ALB and Histone H4 interacted reciprocally. KRT10 and GLE1 interacted reciprocally. 3. Preliminary functional study of CSRP2 3.1 Construction of plasmid pcDNA3-CSRP2-HAPlasmid pcDNA3-CSRP2-HA was proved to be constructed successfully by sequencing.3.2 Localization of CSRP2 detected by immunofluorescenceCSRP2 were distributed in cytoplasm and nuclei, especially in nuclei at rest. After stimulus of TNF-α for 1h, CSRP2 uptaked in nuclear membrane, partly translocated into nuclei.3.3 Effects of CSRP2 on transcriptional expression of Noxl dectecd by luciferase reporter gene assayAssayed by One-way ANOVA, the relative luciferase activity in A549 cells transfected with plasmid pcDNA3-CSRP2-HA was significantly higher than plasmid pcDNA3-HA (F=21.114, P=0.006) at rest. After stimulus of TNF-α for 1h, the relative luciferase activity was significantly increased(F=46.189, P=0.000); A549 cells transfected with plasmid pcDNA3-CSRP2-HA was significantly higher than plasmid pcDNA3-HA(F=26.868, P=0.000). There was an interaction between the factors of TNF-α and plasmid transfected(F=5.447, P=0.042).Conclusion1. Proteins GLE1, ALB, EXD2, KRT9, TTC34, KRT10, KRT1, DDX19A, GSTM5, GATC, SSX11, H4 and CSRP2 may involve in the activation of transcription of Nox1.2. Preliminaried to prove that CSRP2 is a regulatory protein with the translocation from cytoplasm to nuclei after stimulus of TNF-α for 1h, and involved in the regulation of Noxl promoter.