Regulation of NF-[kappa]B in response to age-related energy stress in human skin fibroblasts

Nirupama Yalamanchili

doi:10.17918/etd-4003

Aging is associated with cumulative damage to nuclear and mitochondrial genomes, misfolding and oxidization of proteins, and organelle dysfunction. It encompasses not only the accumulation of damage over time, but also protective/adaptive responses regulated by complex signaling processes. Whereas the loss of molecular fidelity and cellular damage has been, in large part, deciphered and quantified, no clear picture has emerged on the relative importance, interconnectivity and sequence of events that characterize the adaptive response to cellular aging. Our previous work has implicated activation of inflammatory pathways as an adaptive mechanism to cellular aging. Specifically, we observed that fibroblasts from donors at advanced age exhibit a gene expression signature in vitro reflective of an inflammatory cell stress response. We found that these age-associated changes in transcriptional patterns were cell autonomous and accompanied by an enhanced NF-[kappa]B DNA binding activity (Kriete, Mayo et al. 2008). The central objective of this thesis project was to identify pathways and mechanisms involved in NF-[kappa]B activation of aging fibroblasts. Since mitochondrial dysfunction has been implicated in aging we first investigated the basal levels of ROS and intracellular ATP in pre-senescent fibroblasts from young and old donors (22 to 92 years old). Though there was no noticeable change in the intracellular ROS levels, we found an overall decrease in the total basal ATP levels with aging. Based on this observation, we explored a potential link between cellular energy levels and increased NF-[kappa]B p65 DNA binding activity in fibroblasts. In our approach we subjected 'young' fibroblasts to experimental conditions (glycolysis reduction, disruption of oxidative phosphorylation) that mimicked reduced basal ATP levels in 'old' fibroblasts. These treatments triggered increased NF-[kappa]B p65 DNA binding activity. Activation of NF-[kappa]B under these conditions occurred through the canonical NF-[kappa]B pathway and was independent of PI3K/Akt and p53 pathways. Further analysis revealed autophagy induction in parallel to energy deprivation-triggered NF-[kappa]B. Autophagy, a fundamental cellular 'housekeeping' process and usually pro-survival in response to metabolic stress, might be induced presumably as an adaptive response. Pharmacological inhibition of autophagy reduced energy deprivation-triggered NF-[kappa]B indicating its crucial role as a mediator of NF-[kappa]B activation in response to reduced cellular energy levels in 'young' fibroblasts. These findings motivated us to probe for autophagy in pre-senescent fibroblasts aged in vivo. In doing so, we found evidence that supports the involvement of autophagy. However, further studies are required to elucidate additional intracellular signal transduction pathways that may contribute to NF-[kappa]B regulation in aging. While the aging process is inherently driven by damage accumulation and multiple pathways in response to various cell stressors over time, our studies establish a new mechanism of whereby 'chronic inflammation' as evidenced by enhanced NF-[kappa]B activity may be induced in response to energy stress increasing during the normal aging process.

Regulation of NF-[kappa]B in response to age-related energy stress in human skin fibroblasts

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Regulation of NF-[kappa]B in response to age-related energy stress in human skin fibroblasts

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