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Khiem T. Vu, Fang Zhang, John D. Hulleman; Conditional, Genetically Encoded, Small Molecule–Regulated Inhibition of NFκB Signaling in RPE Cells. Invest. Ophthalmol. Vis. Sci. 2017;58(10):4126-4137. doi: https://doi.org/10.1167/iovs.17-22133.
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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.
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.
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.
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.
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