Activation of Nuclear Factor κB by Different Agents; Influence of Culture Conditions in a Cell-Based Assay

Abstract

The transcription factor nuclear factor κB (NF-κB) or other components of this pathway have been identified as possible therapeutic targets in inflammatory processes, cancer, and autoimmune diseases. In order to clarify the role of NF-κB in epithelial cells in response to different stresses, a cell-based screening assay for activation of NF-κB-dependent gene transcription in human embryonic kidney cells (HEK/293) was developed. This assay allows detection of NF-κB activation by measurement of the fluorescence of the reporter protein destabilized enhanced green fluorescent protein (d2EGFP). For characterization of the cell-based assay, activation of the pathway by several agents, for example, tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), lipopolysaccharide (LPS), camptothecin and phorbol ester (PMA), and the influence of the culture conditions on NF-κB activation by TNF-α were examined. NF-κB was activated by TNF-α, IL-1β,PMA,and camptothecin in a dose-dependent manner, but not by LPS. TNF-α results in the strongest induction of NF-κB-dependent gene expression. However, this response fluctuated from 30 to 90% of the cell population showing d2EGFP expression. This variation can be explained by differences in growth duration and cell density at the time of treatment. With increasing confluence of the cells, the activation potential decreased. In a confluent cell layer, only 20–35% of the cell population showed d2EGFP expression. The underlying mechanism of this phenomenon can be the production of soluble factors by the cells inhibiting the NF-κB activation or direct communication via gap junctions in the cell layer diminishing the TNF-α response.