DNA Repair Protein JWA Involves In Aging Regulation In Mice

With improved life style and health care, the aging population is rapidly increasing. This phenomenon is currently receiveing worldwide attention due to increased incidence of aging related diseases, such as late life cancer and neurodegenerative disease. Aging is, at the cellular level, the outcome of a fine balance between damage and repair. Damage is accumulated but the repair efficiency is decreased with increasing age, finally, resulting in organ malfunction and disease. Many mechanisms are required to repair various types of DNA damage, such as base excision repair (BER), nucleotide excision repair (NER), non-homologous end joining (NHEJ) and homologous recombination (HR). Mutation or deficiency of genes involved in these repair pathways often causes aging and/or aging related disease.JWA, a gene known for its role in environmental response, had been found to be upregulated under different stress conditions. Recently, it was found to be involved in oxidative stress induced DNA repair. After exposure to oxidative stress, JWA was translocated into the nucleus and colocalized with XRCC1 and LIG3. Moreover, the expression of XRCC1 and LIG3 was positively regulated by JWA. It is still unclear whether JWA, as a potential DNA repair protein, is involved in DNA double strand break (DSB) repair and whether JWA plays a role in aging.Objective:To investigate the role and the underlying mechanisms of JWA repair function in vivo and to provide the new evidence of the significance of JWA in aging.Methods:VP16 and CPT were selected for inducing DNA DSBs and immunoblotting and immufluorescense were used to detect the expression and localization of JWA; Cre-Loxp mediated recombination was used to establish conditional conventional JWA knockout mice; PCR, RT-PCR, sequencing techniques and immunoblotting were used to genotype the mice at the DNA, RNA and protein level; naked eye observation, cohort study, and body weight follow up study were used to investigate the appearance, body weight and life span of JWA knockout mice; X-ray, Micro-CT and Hematoxylin-Eosin staining were used to investigate the pathological change of JWA knockout mice. FACS and ELISA analysis were used to evaluate the immune function and level of growth factors; open field test was used to investigate the locomotor activity of JWA knockout mice; Microarray was used to evaluate the changed gene expression profile in JWA knockout mice; OATFA analysis was used to analyze the activity of transcriptional factors in JWA knockout liver tissues; GSEA, DAVID and oPOSSUM were used to investigate the gene enrichment, GO changing in JWA knockout mice and at last, EMSA, Q-PCR, reporter gene array, immunoblotting and immunoprecipitation were used to investigate the mechanisms of JWA via NF-κB signaling pathway induces aging.Results:1. JWA expression was induced to 2.4 and 2.3 fold (P<0.05) and partially translocated into nucleus upon VP16 and CPT treatment respectively. We also found that JWA colocalized withγH2AX and p-ATM in nucleus, moreover, the more severe the damage, much more JWA entered into the nucleus.2.With a Cre-Loxp recombination strategy, we constructed the conditional conventional JWA knockout mice, which were verified by DNA, RNA genotyping and protein analysis.3. JWA knockout mice showed advanced decreased body weight (P<0.05), vertebral malformation (lateral Cobb’s were 6.5±1.2 and 25±2.5, P<0.001 and cyrphosis Cobb’s were 32.7±1.6 and 80.7±5.8, P<0.001 for JWA+/+ and JWA-/- mice respectively), osteoporosis (the bone density were 635.56±25.5 mgHA/ccm and 662.43±15.67 mgHA/ccm for JWA-/- mice and JWA+/+ mice respectively), subcutaneous fat layers and spleen atrophy, decreased locomotor activity (running distance in 10 min were 29.5±23.99m and 62.3±23.99m for JWA-/-and JWA++ mice respectively), low level of IGF-1 and shorten life span (one year survival rate for JWA-/-mice was 22.7%).4. SA-β-gal positive staining cells in JWA-1-mice liver was 10.6±2.6% and 0.29±0.49% in JWA+/+ mice, P<0.01; SA-β-gal positive staining cells in JWA-/-1P6 MEFs was 27.4±2.7% and 17±2.4% in JWA+/+ P6 MEFs, P<0.01; G2 phase cells in JWA-1- MEFs was 22.4±2.8% and 12.8±0.7% in JWA+/+ MEFs, P<0.05; Apoptotic cells in JWA-/- MEFs were 2.3±0.67% and 6.27±1.12% in JWA+/+ MEFs, P<0.01; The expression of p53, p16 and p21 were increased in JWA-/- mice livers and MEFs; Microarray found that part of SASPs were increased in JWA-/- spleen, liver and MEFs.5. Comet assay found the DNA damaged cells were increased in JWA-/- primary spleen cells and MEFs; TRAP analysis found the telomerase activity decreased in JWA-/- mice, although JWA knockout has no effect on the ROS levels in tissues and MEFs.6. GSEA analysis found the target genes of E2Fs and NF-κB were enriched in JWA-/ spleen. Leading edge analysis further found that most target genes of E2Fs were decreased, in constract, the most target genes of NF-κB were increased in JWA-/- mice spleen, respectively.7. OATFA analysis found some transcriptional factor activities was changed in JWA-/ mice liver. The oPOSSUM analysis further found the activities of Rela and c-Rel was overrepresented.8. NF-κB activity increased significantly in JWA-/- MEFs and JWA knock-down cells, indicated by increased reporter gene activity and increased expression of NF-κB target gene Bcl-xL, Icaml, ILla, Nfkbl, Nfkbl and Nfkbia. Moreover, immunoprecipitation analysis found JWA interacting with IKKβand this interaction increased upon DNA damage stimuli. Interfere the expression of p65 with specific shRNA reverse the senescence state of JWA-/- MEFs induced by oxidative stress. The PAR was increased in JWA-/- MEFs and JWA knockdown cell upon DNA damage. Inhibit the PAR with 3-AB reversed the activity of NF-κB in JWA-/- cells.Conclusions: Based on the understanding of JWA widely involved in several types of DNA repair proecesses, we constructed the Cre-Loxp mediated conditional conventional JWA knockout mice and followed by series phenotype analysis, we found at first time that JWA knockout induced aging phenotypes in about 84.2% mice, which may be caused by the accumulated DNA damage, loss activity of telomerase, increased NF-κB activity and increased cellular senescence. Thus, the present work provided novel evidence for an important role of JWA in the aging process in vivo and this could be a potential intervention target for aging and aging related diseases.