Characterization of Aging-Associated Up-Regulation of Constitutive Nuclear Factor-κB Binding Activity

Abstract

Changes occur in gene expression during aging in vivo and in replicative senescence in vitro, suggesting that aging can affect gene regulation. We have recently observed age-related changes in ubiquitously expressed, oxidative stress-responsive nuclear factor-kappa B (NF-kappa B) pathway during aging. Here we report a significant age-related increase in nuclear NF-kappa B binding activity together with increased protein levels of p52 and p65 components in rat liver. An additional, higher molecular weight protein band seen in their western blots suggests that their post-translational modification (but not phosphorylation) occurs in liver, which might affect their nuclear localization and binding activity during aging. However, aging did not affect the protein levels of the main I kappa B inhibitors (I kappa B alpha and I kappa B beta) or I kappa B kinase (IKK)-complex subunits (IKK alpha, -beta, and -gamma) involved in NF-kappa B activation. In addition, the level of Ser32-phosphorylated I kappa B alpha was unaffected by age, suggesting that neither the IKK complex nor altered level of the main inhibitors is involved in the observed up-regulation of NF-kappa B binding activity. Furthermore, the expression of NF-kappa B mRNAs (p50, p52, p65, and c-rel) and the mRNAs of their inhibitors (I kappa B alpha and I kappa B beta) did not show any statistically significant age-related changes. These results indicate that the expression level of NF-kappa B genes is not significantly affected by aging. The up-regulation of constitutive nuclear NF-kappa B binding activity and increased levels of nuclear p52 and p65 proteins might affect the expression of some NF-kappa B target genes in the aging liver.

Inhibition of NF-κB by ZAS3, a zinc-finger protein that also binds to the κB motif

“…However, incubation of the samples with shrimp alkaline phosphatases had no effect on the gel mobility of p65 (data not shown), suggesting the difference might not be due to protein phosphorylation. Previously, two such closely migrating p65-antibody reacting species were observed in immunoblot analysis of rat livers, and treatment with calf intestine alkaline phosphatases also did not resolve the difference in their gel mobility (26).…”

Regulation of microglial inflammatory response by sodium butyrate and short‐chain fatty acids

“…EMSA assays were performed as described earlier in detail (Helenius et al, 2001). Nuclear proteins were isolated according to the modified protocol of Dignam et al (1983).…”

“…Probes were labelled with T4 polynucleotide kinase (Promega). Unspecific binding was blocked by 2 mg of poly(dI-dC) : poly (dI-dC) (Roche Applied Science) in an assay volume of 20 ml (Helenius et al, 2001). The binding assays were performed as described earlier (Helenius et al, 1996).…”

Roles of Reactive Oxygen Species in Rheumatoid Arthritis Pathogenesis

“…NF-κB signaling is activated during aging [52] and is a potent inducer of the expression of several NADPH oxidase components including gp91phox and p22phox [53]. SIRT1 has also been suggested as a potent inhibitor of NF-κB signaling by suppressing oxidative stress and inflammatory responses [54].…”