Conditional, Genetically Encoded, Small Molecule-Regulated Inhibition of NFκB Signaling in RPE Cells.

Purpose: Nuclear factor κB (NFκB) is a ubiquitously expressed, proinflammatory transcription factor that controls the expression of genes involved in cell survival, angiogenesis, complement activation, and inflammation. Studies have implicated NFκB-dependent cytokines or complement-related factors as being detrimentally involved in retinal diseases, thus making inhibition of NFκB signaling a potential therapeutic target. We sought to develop a conditional and reversible method that could regulate pathogenic NFκB signaling by the addition of a small molecule. Methods: We developed a genetically based, trimethoprim (TMP)-regulated approach that conditionally inhibits NFκB signaling by fusing a destabilized dihydrofolate reductase (DHFR) domain to an inhibitor of NFκB, IκBα, in ARPE-19 cells. We then challenged ARPE-19 cells with a number of stimuli that have been demonstrated to trigger NFκB signaling, including LPS, TNFα, IL-1α, and A2E. Western blotting, electrophoretic mobility shift assay, quantitative PCR, ELISA, and NFκB reporter assays were used to evaluate the effectiveness of this DHFR-IκBα approach. Results: This destabilized domain approach, coupled with doxycycline-inducibility, allowed for accurate control over the abundance of DHFR-IκBα. Stabilization of DHFR-IκBα with TMP prevented IL-1α-, A2E-, LPS-, and TNFα-induced NFκB-mediated upregulation and release of the proinflammatory cytokines IL-1β and IL-6 from ARPE-19 cells (by as much as 93%). This strategy is dosable, completely reversible, and can be cycled "on" or "off" within the same cell population repeatedly to confer protection at desired time points. Conclusions: These studies lay the groundwork for the use of destabilized domains in retinal pigment epithelium (RPE) cells in vivo and in this context, demonstrate their utility for preventing inflammatory signaling.