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John Hulleman, Khiem Vu; Conditional, genetically-encoded, small molecule-regulated inhibition of NF-κB signaling in RPE cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5736.
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Nuclear factor-κB (NF-κB) is a proinflammatory transcription factor that controls the expression of hundreds of genes involved in metastasis, angiogenesis and inflammation. Classical activation of NF-κB ultimately causes upregulation of pro-forms of inflammatory cytokines such as interleukin-1β (IL-1β) and interleukin-6 (IL-6). A number of studies have implicated IL-1β/IL-6 as being detrimentally involved in age-related macular degeneration (AMD) and retinal dystrophies, thus making inhibition of NF-κB signaling a potential therapeutic target. While a plethora of pharmacologic NF-κB inhibitors exist, the complex intraocular delivery of these drugs, combined with their potential for off-target effects, limits their utility in the eye.
We developed a genetic-based, trimethoprim (TMP)-regulated approach that allows for conditional inhibition of NF-κB by fusing a destabilized domain (DD) of E. coli. dihydrofolate reductase (DHFR) to IkBα (DHFR-IkBα). In the absence of TMP, DHFR fusion proteins are degraded and not present in the cell at appreciable levels. However, after TMP addition, DHFR is stabilized, and the fusion protein can function (in this case as an inhibitor of NF-κB). We used lentivirus to generate stable ARPE-19 cells expressing dox-inducible DHFR-YFP (as a control) or DHFR-IkBα. We then used western blotting, qPCR, EMSA, ELISA and reporter assays to assess whether DHFR-IkBα could be utilized to prevent NF-κB signaling and inflammasome priming.
In the absence of dox/TMP, DHFR-YFP and DHFR-IkBα protein levels were tightly regulated in ARPE-19 cells. Addition of dox/TMP led to induction and stabilization of the fusion proteins in a concentration-dependent manner, and this strategy could be turned off by removal of dox/TMP for 48-72 h. We were able to cycle this strategy ‘on’ and ‘off’ at least three times. Without dox/TMP, DHFR-IkBα cells demonstrated identical NF-κB-related responses after stimulation with IL-1α (i.e., NF-κB nuclear translocation, IL-1β/IL-6 upregulation and secretion, etc.). Addition of dox/TMP to DHFR-IkBα cells significantly prevented NF-κB-mediated signaling, reducing these same responses by as much as 90% (p<0.05).
This DD strategy is effective at conditionally preventing NF-κB-mediated signaling and inflammasome priming. We believe that it has the potential to be a unique strategy to prevent diseases such as AMD.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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