Deficiency Of JWA Induces Premature Aging Via Activating NF-?B Signaling In Mice

As people live longer,the question arises of how malleable aging is and whether it can be slowed or postponed..DNA damage is thought to contribute to aging,but the mechanisms involved remain obscure.JWA was recently demonstrated to be involved in cellular responses to environmental stress including oxidative stress.JWA works as a novel DNA base excision repair protein and plays an important role in reactive oxygen species induced DNA damage.JWA deficiency leads to the development of premature aging in mice and an over activation of NF-?B signal pathway might be the primary cause.Objective: We focuse on how JWA negatively activates the NF-?B signal pathway and whether blocking of over activated NF-?B could reverse premature aging in mice? Methods: Both the JWA+/(35)2 and Rel A+/-single heterozygous mice were crossed to create JWA+/(35)2Rel A+/-dual heterozygous progeny.Polymerized chain reaction followed DNA sequencing and immunoblotting were used to genotype the mice at the DNA and protein level,respectively;an cohort study on mice growth and development was conducted to investigate the appearance,body weight and life span of the JWA+/(35)2Rel A+/-mice;open field test was used to investigate the locomotor activity and micro-CT and Hematoxylin-Eosin staining were used to investigate the pathological alterations in bone of mice;ELISA analysis were used to evaluate the level of growth factors in serum;SA-?-gal staining was used to evaluate the senescence cells in the liver;high throughput gene microarray assay was used to evaluate the changed gene expression profile in spleen of JWA+/(35)2Rel A+/-mice;series molecular determinatins by immunoblotting or immunoprecipitation were used to further investigate the mechanisms of how JWA negatively activated NF-?B signaling pathway contributed to premature aging in mice.Results: 1.after several generations of cross breeding,we received JWA+/(35)2Rel A+/-progeny,which were confirmed at both DNA and protein levels.The blockage of NF-?B reversed the premature aging phenotypes in all 51 JWA+/(35)2Rel A+/-dual heterozygous progeny.However,it was shown that 16 out of 210(7.62%)single heterozygous JWA+/(35)2Rel A+/+ mice with premature aging phenotype(AP)such as kyphosis and tachypnea phenotype at about 4.5 month of age.The subcutaneous fat cell layers were significantly thicker in 6-month old JWA+/(35)2Rel A+/-and JWA+/(35)2Rel A+/+(NAP)mice than in JWA+/(35)2Rel A+/+(AP)mice.Histological analysis also revealed a normal appearance in JWA+/(35)2Rel A+/-and JWA+/(35)2Rel A+/+(NAP)mice whereas a significant increase of premature enlarged nuclei of hepatocytes and decrease of lymphoid follicle organization and germinal center reaction of spleen were seen in JWA+/(35)2Rel A+/+(AP)mice,whose aging phenotype was similar with JWA(35)2/(35)2Rel A+/+.Moreover,micro-CT analysis indicated that the density(P < 0.05)and trabecular thickness(P < 0.05)of bones were increased in 7-month old JWA+/(35)2Rel A+/-and JWA+/(35)2Rel A+/+(NAP)mice compared with JWA+/(35)2Rel A+/+(AP)mice.The open field test revealed that JWA+/(35)2Rel A+/-and JWA+/(35)2Rel A+/+(NAP)mice have much more locomotor activity than JWA+/(35)2Rel A+/+(AP)mice(P < 0.05).And the serum level of insulin-like growth factor IGF-1 was increased in JWA+/(35)2Rel A+/-and JWA+/(35)2Rel A+/+(NAP)mice(P < 0.05).In addition,the altered SASP factors profile in spleen of JWA+/(35)2Rel A+/+(AP)mice was reversed in JWA+/(35)2Rel A+/-mice.2.Data in the present study also provided molecular evidences how JWA negativelyregulating NF-?B.Overexpression of JWA in cells inhibited the translocation of p65 into nucleus upon VP16 treatment.Similarly,the expression of IKK? was negative regulated by JWA with or without VP16 treatment.JWA promoted IKK? ubiquitination for degradation by the proteasome.Further investigation indicated that JWA enhanced the ubiquitination of IKK? by influenced the interaction between IKK? and Hsp90.JWA,through the interaction between its carboxyl end 91-188 amino acids domain and the whole IKK?,Hsp90-IKK? complex might be shifted its physical structure and therefore exposed or released IKK? easily be ubiquitinated and degradated.Without binding of JWA,the ubiquitination and degradation of IKK? were inhibited.This process further promoted phosphorylation of IkB and released p65 for translocated into nucleus,and therefore activated downstream target genes.Finally,the altered essential physiological and biochemical homeostasis resulted in the premature aging in mice.Conclusions: In the present study,we provided evidences to show that the premature aging produced by loss of single JWA could be almost completely reversed in JWA-Rel A dual knockout mice.We also elucidated the molecular mechanisms of how JWA deficiency activated NF-?B signaling pathways.We believe that the present data are not only with obvious significane academically,but potentally applicable in clinic.