The effects of IKK-beta inhibition on early NF-kappa-B activation and transcription of downstream genes

Abstract

Small molecule approaches targeting the nuclear factor kappa B (NF-kB) pathway, a regulator of inflammation, have thus far proven unsuccessful in the clinic in part due to the complex pleiotropic nature of this network. Downstream effects depend on multiple factors including stimulus-specific temporal patterns of NF-kB activity. Despite considerable advances, genome-level impact of changes in temporal NF-kB activity caused by inhibitors and their stimulus dependency remains unexplored. This study evaluates the effects of pathway inhibitors on early NF-κB activity and downstream gene transcription. 3T3 fibroblasts were treated with SC-514, an inhibitor targeted to the NF-kB pathway, prior to stimulation with interleukin 1 beta (IL-1β) or tumor necrosis factor alpha (TNF-α). Stimulus induced NF-κB activation was quantified using immunofluorescence imaging over 90-minutes and gene expression tracked over 6-hours using mRNA TagSeq. When stimulated with IL-1β or TNF-α, significant differences (P < 0.05, two-way ANOVA), were observed in the temporal profiles of NF-κB activation between treated and untreated cells. Increasing numbers of differentially expressed genes (P < 0.01) were observed at higher inhibitor concentrations. Individual gene expression profiles varied in an inhibitor concentration and stimulus-dependent manner. The results in this study demonstrate small molecule inhibitors acting on pleiotropic pathway components can alter signal dynamics in a stimulus-dependent manner and affect gene response in complex ways.

Keywords: IKK; Inflammation; Pathway analysis; Signaling dynamics; Temporal control.