CRP2Regulates Nox1Gene Expression In TNF-α Mediated Inflammation-oxidative Stress In A549

1BackgroudInflammation relate closely to oxidative stress, imbalanced inflammation oftenaccompanied by oxidative stress.During the biological evolution, inflammation andoxidative stress should be coordinated, which can remove antigens effectively, butalso take balance between inflammation and oxidative stress,and this balance caninduce cell damage. If breaking the balance of inflammation and oxidative stress, itwill cause great damage to cells. Many articles suggest excessive inflammation andoxidative stress are important pathogenesis of ALI.Making inflammation andoxidative stress to a rational level is a important key to reduce the ALI. ARDS is asevere ALI. In the past few decades, although it had made great progress on the ALI/ARDS, ARDS mortality remains high. Because of the feature of this disease is rapidand progessive, high mortality rate, the research of treatment strategy for ALI/ARDSis still the current hot research and urgent task of clinical medicine.Alveolar epithelial typeⅡcells is an important component of the respiratorymembrane, and it is called alveolar epithelial "stem cells",which can be metaplasia asalveolar type I alveolar epithelial cells,and synthesize and secrete surfactantsubstance to maintain normal morphology. Reducing inflammation and followingoxidative damage in acute lung injury is very important to maintain the structural ofalveolar epithelial typeⅡcells,which can reduce the occurrence and progression ofALI. ARDS mouse model of LPS-induced inflammation and oxidative stress levelspositively correlated with LPS dose, the more high levels of ROS the more seriousinjury of the lung. Syrkina found oxidative stress imbalanced in lung of mild ARDS mice early. Treating mice with acetylcysteine inhibits glutathione decline, reducesneutrophil infiltration and inflammatory factor in bronchoalveolar lavage fluid,reduces the apoptosis level of airway cell.Inflammatory cytokines TNF-α, IL-6weregreatly generated due to ROS production. TNF-α is one of the early cytokine in ALI,and TNF-α can start and amplify the inflammatory response,and induce endothelialcells and neutrophils to produce large amounts of ROS, which caused oxidativedamage to structure of the cell, eventually causing cell death. Animal model studieshave confirmed serum or lavage fluid TNF-α concentrations positively correlated withthe severity of ALI. ALI is often accompanied by lung structures cells damage. Thenwe still use TNF-α stimulate A549to construct a model of inflammation-oxidativestress damage of ALI.NADPH oxidase1(Nox1), is one of the important oxidase catalysis to generateROS. Many studies have shown that the activation of Nox can produce lots of H2O2,which cause cellular damage. Nox1mainly expressed in lung during Nox1~5. Our labalready have been Researched transcription factor NF-κB、SP1，we found thatsilencing NF-κB、SP1can reduce the expression of ROS, decreasing the rate of cellapoptosis. Nox1is an important source of ROS, the regulation of the genetranscription level is the most critical step of gene expression, therefore regulate thetranscription of Nox1to maintain a low level of Nox1activity during inflammation,which can reduces endogenous ROS, resulting low cell damage.In the early research of our lab,we use TNF-α stimulate A549cells,findingdifferences binding proteins of Nox1promoter. we use TNF-α stimulate A549cells inorder to establish inflammation-oxidative damage model, through the DNA-pulldown, two-dimensional gel electrophoresis and mass spectrometry experiments found13different binding proteins of Nox1promoter,then we select CRP2with a zincfinger structure as a target protein. CRP2is composed of193amino acids, molecularweight is about21KD, CRP2contains two LIM domain structure,distributed in C andN side, there is a signal (KKYGPK) between the two structure of LIM domains.CPR2belongs to LIM protein which is a kind of cysteine-rich proteins or zinc fingerprotein.Currently we already found more than60kinds of LIM protein,which involved in the cell transcriptional regulation, cytoskeleton regulation etc. LIMproteins are divided into four categories: LIM-HD, LIM-Only, LIM-K and one proteinwith LIM domain. CRP protein belongs to LIM-Only, which is characterizedcomposed with LIM domains, but do not have DNA binding domain.Although theCRP2protein with zinc finger protein domain, but do not have the ablity of binding tothe DNA directly.CRP2can locate in cytoplasm and nuclear in the cells, CRPs cancombined with nuclear proteins, especially combined with transcription factors, andCRP2itself is also an important transcription factor. Early research on CSRP familyhave always focus on smooth muscle cells, then gradually spread to atery andintravenous vascular smooth muscle cells. Later, Jarvinen, PM etc.found that CRP1/CRP2can express in the idiopathic pulmonary fibrosis mice lungs.In the early in ourlab we observed CRP2under the stimulation of TNF-α one hour,CRP2from thecytoplasm moved into the nucleus by immunofluorescence experiments.CRP2maydirectly or indirectly involved in the Nox1gene regulation.So far there has no evidences suggest that CRP2can directly as transcriptionfactors acts as a role in regulating gene expression, but it can play a role oftranscriptional regulation through target protein (such as transcription factors). Kong,etc shows that CRP2locates in the nuclear can regulate muscle cells MyoD family;Yet etc. shows that there is a potential transcription start site at the5’end of CSRP2whcic can possibly combines with GATA, SP1.transient transfection of CRP2cansignificantly improve luciferase activity in arterial smooth muscle cells of rats, but theprecisely mechanism is still not clear. CRP2can form a complex with SRF andGATA–6then involved in regulation of gene regulation,in this prosess CRP2playsa role of transcription cofactors. In smooth muscle cells, CRP2could be activated bySRF, hlcalponin, SM22etc, through the c-terminal LIM structure as a bridge of SRFand GATA6molecules results inducing transdifferentiation of smooth muscle.Base onall above results, we use bioinformatics software analysis, Nox1promoter regions(1415bp) contain two SRF combining elements, we hypothesized that CRP2moveinto the nucleus after activation may be through one of SRF,resulting regulate thetranscription of Nox1. Clarifing the mechanism of CRP2regulate Nox1transcription, we use siRNA of CRP2to control Nox1gene expression, then it can control thegeneration of ROS.2objectivesTO assess CRP2whether involved in the Nox1gene transcription regulationdirectly or indirectly; Clarifying the mechanism of transcription factor CRP2regulateNox1gene expression in TNF-α mediated A549cells; Finding the possible bindingforms of CRP2in the promoter region of Nox1gene.3methods3.1Screening and identification of differential proteins binding to Nox1promoter in TNF-α-induced A549cells3.1.1preparation of biotin labeling Nox1promoter DNA probesPGL3-Basic-Nox1recombinant plasmid are saved the lab, according to themethod of molecular cloning, then amplify PGL3-Basic-Nox1recombinantplasmid, extract PGL3-Basic-Nox1recombinant plasmid as a template, preparationof biotin labeling Nox1promoter probe by PCR, The product of PCR was observedby1%agarose electrophoresism, Nox1promoter DNA probes were recycledaccording to Agarose gel DNA extraction kit manual.3.2.2DNA pull-down testUsing3~5generation of A549cells were seeded in15cm plate, when the cellsgrew to70%~80%,3%serum medium was given for12h, then treated with TNF-α at10ng/ml and incubated for1h, while the control group were incubated withequivalence3%serum medium. After the cells precipitation were collected, nuclearproteins were extracted accoding to the cytoplasmic nuclear protein extractionmanmual, then determinated the concentration accoding to the EttanTM2D Quant Kitmanmual. The proteins binding to Nox1promoter in nuclear proteins were isolatedthrough DNA pull-down following the Dynabeads kilobase BINDERTMKit manual.3.1.3Identificate differences protein CRP2by Western blotGroups: control group and experimental group. The expression of differences protein CRP2by Western blot3.1.4To construction and identify of eukaryotic expression plasmidpcDNA3-CSRP2-HA.According to the sequence of CSRP2CDS in GeneBank, a Pair of Primers wererespectively designed and Synthesized, and the total RNA was isolated from A549cells. After amplification with reverse transcription polymerase chainreaction(RT-PCR), the product was cloned into pcDNA3-HA vector, and they wereidentified by PCR and double restrictive edonuclease digestion and sequence analysis.Then the recombinant expression plasmid was transferred into A549cells, and theCSRP2protein expression was identified by Western-blot.3.1.5CRP2regulates Nox1gene transcriptional expression by luciferase reportsystem1x105/ml A549cells were seeded in24plates grew to to80%~90%, accordingto instructions of LipofectamineTM2000separately transfect pcDNA3-CSRP2-HA plasmid and PGL3-Basic-Nox1plasmid t, meanwhile given a control group.Give4h grew after6h Transfection, the experimental group was given10ng/mlTNF-α stimulation500ul, control group was given the equivalent medium, continueto grew after24h,then test the luciferase activity.3.2CRP2participated in the TNF-α mediated Nox1gene regulation in A549cells3.2.1CSRP2deletion affect Nox1gene mRNA and protein expressionGroups: the blank group, TNF-α group, CSRP2siRNA group+TNF-α group,siRNA control+TNF-α group. CSRP2silence efficiency and Nox1mRNA expressionlevel by RT-PCR; Nox1protein, CRP2cytoplasm、 nucleus protein expression byWestern blot.3.2.2CSRP2deletion affect ROS expressionGroups: the blank group, TNF-α group, CSRP2siRNA group+TNF-α group,siRNA control+TNF-α group. ROS expression by DCFH-DA, at the same timegiven fluorescence microscope to save images. 3.3The study of CRP2combined forms in Nox1promoter regions3.3.1Construction of different SRF combination site deletionment of Nox1promoterluciferate enzyme vectorUse PGL3-Basic-Nox1(1415bp) as the template, design two different SRFdeletion sites primers, and two SRF deletion primer at the same time, amplified byPCR, then the PCR products connected to PGL3-Basic expression vector, veriftiedby sequence test.3.3.2Identification of luciferate activity of different SRF deletion sites of Nox1promoterGroups: blank group, TNF-α group, transfection of pcDNA3-HA+PGL3-Basic-Nox1+TNF-α group, transfection of pcDNA3-CSRP2-HA+PGL3-Basic-Nox1+TNF-α group, transfection of pcDNA3-CSRP2-HA+PGL3-Basic-Nox1-SRF1deletion+TNF-α group, transfection of pcDNA3-CSRP2-HA+PGL3-Basic-Nox1-SRF2deletion+TNF-α group, transfection of pcDNA3-CSRP2-HA+PGL3-Basic-Nox1-SRF (1,2) deletion+TNF-α group. luciferateactivity by luciferase reporter gene assay.3.4Statistical methodsUse spss13.0package for statistical analysis, measurement datas were expressedas mean number±standard deviation （X s） Multiple comparisons useOne-Way-ANOVA. First test of homogeneity of variance and the overall differencebetween the groups, the multiple comparisons with LSD when the data are equalvariances, Dunnett test for unequal variances. Detection level α=0.05, P <0.05significant difference. The experiment was repeated three times.4Results4.1Screening and identification of differential proteins binding to Nox1promoter in TNF-α-induced A549cells4.1.1compared with blank group,CRP2protein expression of TNF-α group was obviously up-regulated, which showed that CRP2protein expression higher under thestimulation of TNF-α, and shift from the cytoplasm into nucleus.4.1.2The pcDNA3-CSRP2-HA eukaryotic expression plasmid was successfullyestablished, and the expression of pcDNA3-CSRP2-HA could be detected in A549byWestern-blot.4.1.3Assayed by One-way ANOVA,Luciferate activity in A549cells was statisticallysignificant between groups.Pretransfection of pcDNA3-CSRP2-HA+PGL3-Basic-Nox1group，luciferase activity was significantly lower than Pretransfection ofpcDNA3-HA+PGL3-Basic-Nox1group，but significantly higher than controlgroup (F=260.753, P=0.000). Under the stimulation of TNF-α, Pretransfection ofpcDNA3-CSRP2-HA+PGL3-Basic-Nox1+TNF-α group，luciferase activitywas significantly lower than Pretransfection of pcDNA3-HA+PGL3-Basic-Nox1+TNF-α group, but significantly higher than TNF-α group (F=446.061, P=0.000).Assayed by One-way ANOVA, under the TNF-α stimulation，TNF-α group luciferaseactivity was significantly higher than control group (F=54.753, P=0.002).Pretransfection of pcDNA3-HA+PGL3-Basic-Nox1+TNF-α group, luciferaseactivity was higher than Pretransfection of pcDNA3-HA+PGL3-Basic-Nox1group (F=360.488, P=0.000). Pretransfection of pcDNA3-CSRP2-HA+PGL3-Basic-Nox1group+TNF-α group, luciferase activity was higher thanPretransfection of pcDNA3-CSRP2-HA+PGL3-Basic-Nox1group (F=117.988,P=0.000). There was an interaction between the factors of TNF-α and plasmidtransfected Assayed by factors analysis (F=15.000, P=0.010).4.2CRP2participated in the TNF-α mediated Nox1gene regulation in A549cells4.2.1CSRP2mRNA expression in A549cells was statistically significant betweengroups(F=800.366, P=0.000). After TNF-α stimulation, CSRP2mRNA expressionwas significantly higher than blank control group (P <0.05). Pretransfection ofCSRP2siRNA,CSRP2mRNA expression was significantly lower compared withblank control group, TNF-α group, negative control group, the difference was statistically significant (P <0.05).4.2.2CRP2cytoplasm protein expression in each A549cell level was statisticallysignificant (F=62.081, P=0.000). After TNF-α stimulation, cytoplasm CRP2proteinexpression was significantly lower than blank control group (P <0.05).Pretransfection of CSRP2siRNA,CRP2cytoplasm protein expression wassignificantly lower compared with TNF-α group, negative control group, thedifference was statistically significant (P <0.05).4.2.3CRP2nuclear expression in each A549cell level was statistically significant (F=45.609, P=0.000). After TNF-α stimulation, nuclear CRP2protein expression wassignificantly lower than blank control group (P <0.05). Pretransfection of CSRP2siRNA,CRP2nuclear protein expression was significantly lower compared TNF-αgroup, negative control group, the difference was statistically significant (P <0.05).4.2.4Nox1mRNA expression in A549cells was statistically significant betweengroups(F=298.868, P=0.000). After TNF-α stimulation, Nox1mRNA expressionwas significantly higher than blank control group (P <0.05). Pretransfection ofCSRP2siRNA, Nox1mRNA expression was significantly higher compared withblank control group, TNF-α group, negative control group, the difference wasstatistically significant (P <0.05).4.2.5Nox1protein expression in A549cells was statistically significant betweengroups(F=39.094, P=0.000). After TNF-α stimulation, Nox1mRNA expressionwas significantly higher than blank control group (P <0.05). Pretransfection ofCSRP2siRNA, Nox1protein expression was significantly higher compared withblank control group, TNF-α group, negative control group, the difference wasstatistically significant (P <0.05).4.2.6ROS expression in A549cells was statistically significant between groups(F=29.121, P=0.000). After TNF-α stimulation, ROS expression was significantlyhigher than blank control group (P <0.05). Pretransfection of CSRP2siRNA, ROSexpression was significantly higher compared with blank control group, TNF-α group,negative control group, the difference was statistically significant (P <0.05).ROS positive percentage expression in A549cells was statistically significant between groups(F=148.167, P=0.000). After TNF-α stimulation, ROS positivepercentage expression was significantly higher than blank control group (P <0.05).Pretransfection of CSRP2siRNA, ROS positive percentage expression wassignificantly higher compared with blank control group, TNF-α group, negativecontrol group, the difference was statistically significant (P <0.05).4.3The study of CRP2combined forms in Nox1promoter regions4.3.1The luciferin enzyme expression vector of different SRF binding site deletionwere successfully established.4.3.2Luciferate activity in A549cells was statistically significant between groups(F=31.788, P=0.000). After TNF-α stimulation, Luciferate activity expression wassignificantly higher than blank control group (P <0.05). Pretransfection ofpcDNA3-HA+PGL3-Basic-Nox1+TNF-α group, Luciferate activity expression wassignificantly higher compared with blank control group, TNF-α group, the differencewas statistically significant (P <0.05).Pretransfection of pcDNA3-CSRP2-HA+PGL3-Basic-Nox1+TNF-α group, Luciferate activity expression was significantlylower compared with pcDNA3-HA+PGL3-Basic-Nox1+TNF-α group, the differencewas statistically significant(P<0.05). Pretransfection of pcDNA3-CSRP2-HA+PGL3-Basic-Nox1-SRF1deletion+TNF-α group, Luciferate activity expressioncompared with pcDNA3-CSRP2-HA+PGL3-Basic-Nox1+TNF-α group was notstatistically significant (P＞0.05).Pretransfection of pcDNA3-CSRP2-HA+PGL3-Basic-Nox1-SRF2deletion+TNF-α group, Luciferate activity expressioncompared with pcDNA3-CSRP2-HA+PGL3-Basic-Nox1+TNF-α group、pcDNA3-CSRP2-HA+PGL3-Basic-Nox1-SRF1deletion+TNF-α group wassignificantly lower, the difference was statistically significant (P＜0.05).Pretransfection of pcDNA3-CSRP2-HA+PGL3-Basic-Nox1-SRF （1、2）deletion+TNF-α group, Luciferate activity expression compared withpcDNA3-CSRP2-HA+PGL3-Basic-Nox1-SRF2deletion+TNF-α group was notstatistically significant (P＞0.05). 5conlusionsCSRP2deletion can increase Nox1expression, and ROS expression; CRP2actsas a transcription factor involved in TNF-α mediated oxidative damage regulation ofNox1gene expression indirectly,which related closed to SRF.