The Molecular Mechanism Of JWA Involved In Base Excision Repair Induced By Oxidative Stress

Oxidative stress is the most popular stress responses in the organism.Cells undergo oxidative stress when intracellular levels of reactiveoxygen species (ROS) exceed their counter-regulatory antioxidantcapacity, which may result from an elevated production and/oraccumulation of ROS or a diminution in cellular antioxidant defensecapacity. Oxidative stress, or the excessive production of oxygen-centered free radicals, has been hypothesized as one of the major sourcesresulting in DNA damage that leads to a variety of diseases, includingcancer and chronic cardiovascular diseases. Base excision repair (BER)which was the main form of DNA damage and repair induced byoxidative, copes with many of the base damages formed in DNA byattacking of reactive oxygen species.JWA (GenBank: AF070523.1, 1998), a newly identified ATRA-responsive gene, was initially cloned from primary human trachealbronchial epithelial cells by Zhou. There is an increasing amount of data to indicate that JWA is a structurally novel microtubule-associated protein(MAP), induced by various agents such as TPA, ATRA, 4HPR or As₂O₃,as well as stress conditions including heat shock and cold shock, andinitiation of cell differentiation in leukemia cells. Several JWAhomologues (e.g., ARL-6, GTRAP3-18, addicsin, and JM4) wereidentified in recent years. ARL-6 is a member of the ADP-ribosylationfactor-like family, which shows 93% homology with JWA and itsfunctions mainly referred to DNA damage and repair.In this study, series cell culture models include oxidative stressmodels, base excision repair models were employed to investigate theroles of JWA gene in regulation of cells responding to oxidative stressand the behind molecular mechanism of JWA gene involving in baseexcision repair signal pathway, especially the potential association withthe BER proteins. Hopefully, it will help to further elucidate themolecular mechanisms of oxidative stress and base excision repairpathway.1. Intracellular H₂O₂ Mediates JWA Up-regulationWe employed H₂O₂ and B[a]P to treat NIH-3T3 and HELF cells, andfound that oxidative stress exposure stimulated production of JWAprotein in a time-dependent manner. To demonstrate the dependence ofJWA induction on the presence of ROS following either H₂O₂ or B[a]Pexposure, NIH-3T3 and HELF cells were incubated with H₂O₂ or B[a]Pin the presence of either SOD or catalase. SOD treatment increased JWAprotein expression which correlated with an increase in intracellular H₂O₂levels. With concurrent scavenging of intracellular H₂O₂ by catalase,there was a diminished JWA expression. These results suggested thatintracellular ROS were also generated in cells exposed to B[a]P exposure,and that intracellular H₂O₂ mediated JWA expression.2. JWA Promoter Activation with NFI Binding to a CCAAT BoxInduced by Oxidative Stress To elucidate whether transcriptional regulation played a role in theobserved increase in JWA protein levels in response to oxidative stress,an attempt was made to determine critical sequences within the JWApromoter responsible for H₂O₂- mediated JWA expression. Promoterdeletion analysis showed that the region spanning-107 to +107 of JWApromoter mediates a potent response to H₂O₂ and B[a]P stimulation.These results indicate that the region between the -107 and +107 bp playsan important role on transcriptional activity of the JWA promoter inducedby H₂O₂ and B[a]P. EMSA assay using this fragment showed that underH₂O₂- and B[a]P-stimulation, a protein complex was bound to the-107/-28 promoter region. To characterize the nuclear factors binding tothe JWA (-107/-28) promoter region, southwestern blotting was carriedout. The results revealed a single strong band in both H₂O₂- and B[a]P-stimulated NIH-3T3 cells with a molecular weight of approximately 47kDa. To determine which molecule(s) was bind to JWA, we performedthe EMSA supershift and RNA interference assays and testified that thenuclear protein was NFI. These results indicate that NFI is essential forROS mediated JWA protein induction, suggesting that oxidative stressstimulates NFI binding and transactivation of the JWA promoter. Todetermine the manner by which NFI binds to the two closed CCAAT sitesof the JWA promoter under oxidative stress, we employed southwesternanalysis, EMSA supershift analysis, site directed mutation assay andreporter gene assays and found that, NFI may only bind to the secondCCAAT site under oxidative stress, suggesting that oxidative stressresponsiveness of the JWA promoter region (-107/+107) requires thefunctional integrity of the second CCAAT site.3. JWA Protects Cells from DNA Damage Induced by OxidativeStressWe have constructed a JWA knock-down vector which was stablytransfected into NIH-3T3 cell lines, and was further confirmed bywestern blot analysis. JWA NIH-3T3 vector control and knock-down cells were treated with 100μM of B[a]P for 3 h and then allowed torecover for 3 h prior to screening for DNA repair. The DNA damage inJWA knock-down cells was greater than that seen in vector controlNIH-3T3 cells, as determined by comet assay. We also found that the rateof DNA repair in JWA knock-down cells was considerably weaker thanthat found in vector control cells following B[a]P exposure. It wassuggested that JWA is likely to be involved in protecting cells from DNAdamage induced by oxidative stress.4. JWA Involves in Intracellular Base Excision Signal PathwayIt is possible that JWA may function as a DNA repair gene responsiveto oxidative stress produced DNA damage. Since the DNA damageinduced by intracellular H₂O₂ is believed to be repaired through baseexcision repair (BER) pathway. We employed western blot to determinethe expression of JWA, and BER pathway associate proteins, includingXRCC1 and PARP1 in vector-control and JWA knock-down NIH-3T3cells, and found that both JWA and XRCC1 expression levels wereenhanced by H₂O₂ or B[a]P exposure in vector control cells butdown-regulated in JWA knock-down NIH-3T3 cells. PARP1 expression,however, showed an opposite pattern. In order to further understandwhether JWA participates in BER associated signal pathways, wetransiently transfected JWA cDNA pEGFP vector (sense vector) into JWAknock-down NIH-3T3 cells and to rescue the expression of JWA protein.As a result, following exposure to H₂O₂ and B[a]P, JWA and XRCC1expressions were up-regulated, whereas PARP1 expression wasdown-regulated. These results suggest that functional links were existedbetween JWA and XRCC1, as well as between JWA and PARP1 underthese exposure conditions. To determine whether JWA interacts withXRCC1 in NIH-3T3 cells, co-immunoprecipitation analysis wasemployed. JWA or XRCC1 were immunoprecipitated from lysatesfollowing treatment of NIH-3T3 cells, with anti-XRCC1 or anti-JWAantibodies, respectively. Co-precipitating proteins were detected by probing western blots of the immunoprecipitates with anti-JWA andanti-XRCC1 antibodies. The results confirmed that JWA interacts withXRCC1.5. The Localization of JWA in Base Excision Repair Signal PathwayTo elucidate the localization of JWA gene in the base excision repairsignal pathway, we employed western blot to determine the expression ofJWA, and BER pathway associate proteins APE1 and Ligâ…¢invector-control and JWA knock-down NIH-3T3 cells, and found that bothJWA and Ligâ…¢expression levels were enhanced by H₂O₂ or B[a]Pexposure in vector control cells but down-regulated in JWA knock-downNIH-3T3 cells. APE1 expression, however, showed no relation with theJWA expression. These results suggest that JWA regulated Ligâ…¢(gapfilling protein) expression and not regulated APE1 (damage site detectionprotein) expression. It is further suggested that JWA involved in thedamage site repair and gap filling.Taken together, we found that JWA is up-regulated followingexposure to oxidative stress produced by the treatment of H₂O₂ or B[a]P.Our studies demonstrated that in oxidative stress cell culture models,JWA was up- regulated and H₂O₂ is the primary oxidative productresponsible for B[a]P-induced JWA expression. Furthermore, we foundthat both H₂O₂ and B[a]P exposure activated nuclear transcription factor I(NFI) in NIH-3T3 cells which specifically bound to the CCAAT elementin the JWA proximal promoter (-58/-28 bp) and thereby induced JWAexpression. Consistently siRNA mediated knockdown of NFI whichprevented JWA induction. Knockdown of JWA enhanced H₂O₂ (100μM)- and B[a]P (100μM) -induced DNA damage. Under oxidative stress,the expression of JWA was regulated and paralleled to the rise of the baseexcision repair (BER) protein XRCC1 but reduction of PARP1. Finally,we demonstrated that JWA involved in the damage site repair and gapfilling, but not responds to the damage recognition in base excision repairsignal pathway. These findings indicate that JWA may serve as a novel and effective base excision repair protein to protect cells against reactiveoxygen species (ROS)-triggered DNA damage.