Regulation of NF-κB in response to age-related energy stress in human skin fibroblasts

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-κB DNA binding activity (Kriete, Mayo et al. 2008). The central objective of this thesis project was to identify pathways and mechanisms involved in NF-κB 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-κB 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-κB p65 DNA binding activity. Activation of NF-κB under these conditions occurred through the canonical NF-κB pathway and was independent of PI3K/Akt and p53 pathways. Further analysis revealed autophagy induction in parallel to energy deprivation-triggered NF-κB. 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-κB indicating its crucial role as a mediator of NF-κB 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-κB 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-κB activity may be induced in response to energy stress increasing during the normal aging process.Ph.D., Biomedical Engineering -- Drexel University, 201

Combinatorial biomarkers for aging research

Poster presented at Biomedical Technology Showcase 2006, Philadelphia, PA. Retrieved 18 Aug 2006 from http://www.biomed.drexel.edu/new04/Content/Biomed_Tech_Showcase/Poster_Presentations/Kriete.pdf.The biology of aging has been recognized as our biggest risk factor in developing a range of chronic and costly diseases like cancer, diabetes or Alzheimer’s. An increasingly aging population demands investigation of mid-life decline and development of individualized treatment strategies. Key in this view is to identify biomarkers reflecting biological age. Previous attempts to find such biomarkers have failed; one reason might be that chronological age as a primary classification parameter does not well reflect biological age. Further, most bioinformatics search strategies are tuned towards identification of the most up- or down-regulated genes in age groups, without consideration of individual responses. Inspired by a systems biology view, we have developed a novel method to define correlative biomarkers across scales. Hereby we use a bioimaging based hyperquantification of cells and tissues to enrich the data mining process of related gene expression profiles. Dissection of data heterogeneity and consideration of individual responses is likely relevant to identify early onset markers of age related diseases

Quantifying gene network connectivity in silico: Scalability and accuracy of a modular approach

Large, complex data sets that are generated from microarray experiments, create a need for systematic analysis techniques to unravel the underlying connectivity of gene regulatory networks. A modular approach, previously proposed by Kholodenko and co-workers, helps to scale down the network complexity into more computationally manageable entities called modules. A functional module includes a gene\u27s mRNA, promoter and resulting products, thus encompassing a large set of interacting states. The essential elements of this approach are described in detail for a three-gene model network and later extended to a ten-gene model network, demonstrating scalability. The network architecture is identified by analysing in silico steady-state changes in the activities of only the module outputs, communicating intermediates, that result from specific perturbations applied to the network modules one at a time. These steady-state changes form the system response matrix, which is used to compute the network connectivity or network interaction map. By employing a known biochemical network, the accuracy of the modular approach and its sensitivity to key assumptions are evaluated

Cell autonomous expression of inflammatory genes in biologically aged fibroblasts associated with elevated NF-kappaB activity

<p>Abstract</p> <p>Background</p> <p>Chronic inflammation is a well-known corollary of the aging process and is believed to significantly contribute to morbidity and mortality of many age-associated chronic diseases. However, the mechanisms that cause age-associated inflammatory changes are not well understood. Particularly, the contribution of cell stress responses to age-associated inflammation in 'non-inflammatory' cells remains poorly defined. The present cross-sectional study focused on differences in molecular signatures indicative of inflammatory states associated with biological aging of human fibroblasts from donors aged 22 to 92 years.</p> <p>Results</p> <p>Gene expression profiling revealed elevated steady-state transcript levels consistent with a chronic inflammatory state in fibroblast cell-strains obtained from older donors. We also observed enhanced NF-κB DNA binding activity in a subset of strains, and the NF-κB profile correlated with mRNA expression levels characteristic of inflammatory processes, which include transcripts coding for cytokines, chemokines, components of the complement cascade and MHC molecules. This intrinsic low-grade inflammatory state, as it relates to aging, occurs in cultured cells irrespective of the presence of other cell types or the <it>in vivo </it>context.</p> <p>Conclusion</p> <p>Our results are consistent with the view that constitutive activation of inflammatory pathways is a phenomenon prevalent in aged fibroblasts. It is possibly part of a cellular survival process in response to compromised mitochondrial function. Importantly, the inflammatory gene expression signature described here is cell autonomous, i.e. occurs in the absence of prototypical immune or pro-inflammatory cells, growth factors, or other inflammatory mediators.</p

Distinct Cell Stress Responses Induced by ATP Restriction in Quiescent Human Fibroblasts

Quiescence is the prevailing state of many cell types under homeostatic conditions. Yet, surprisingly little is known about how quiescent cells respond to energetic and metabolic challenges. To better understand compensatory responses of quiescent cells to metabolic stress, we established, in human primary dermal fibroblasts, an experimental ‘energy restriction’ model. Quiescence was achieved by short-term culture in serum-deprived media and ATP supply restricted using a combination of glucose transport inhibitors and mitochondrial uncouplers. In aggregate, these measures led to markedly reduced intracellular ATP levels while not compromising cell viability over the observation period of 48 h. Analysis of the transcription factor landscape induced by this treatment revealed alterations in several signal transduction nodes beyond the expected biosynthetic adaptations. These included increased abundance of NF-κB regulated transcription factors and altered transcription factor subsets regulated by Akt and p53. The observed changes in gene regulation and corresponding alterations in key signaling nodes are likely to contribute to cell survival at intracellular ATP concentrations substantially below those achieved by growth factor deprivation alone. This experimental model provides a benchmark for the investigation of cell survival pathways and related molecular targets that are associated with restricted energy supply associated with biological aging and metabolic diseases