Conditional, Genetically Encoded, Small Molecule-Regulated Inhibition of NF?B Signaling in RPE Cells.

ABSTRACT:

Purpose

Nuclear factor ?B (NF?B) 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 NF?B-dependent cytokines or complement-related factors as being detrimentally involved in retinal diseases, thus making inhibition of NF?B signaling a potential therapeutic target. We sought to develop a conditional and reversible method that could regulate pathogenic NF?B signaling by the addition of a small molecule.

Methods

We developed a genetically based, trimethoprim (TMP)-regulated approach that conditionally inhibits NF?B signaling by fusing a destabilized dihydrofolate reductase (DHFR) domain to an inhibitor of NF?B, I?B?, in ARPE-19 cells. We then challenged ARPE-19 cells with a number of stimuli that have been demonstrated to trigger NF?B signaling, including LPS, TNF?, IL-1?, and A2E. Western blotting, electrophoretic mobility shift assay, quantitative PCR, ELISA, and NF?B reporter assays were used to evaluate the effectiveness of this DHFR-I?B? approach.

Results

This destabilized domain approach, coupled with doxycycline-inducibility, allowed for accurate control over the abundance of DHFR-I?B?. Stabilization of DHFR-I?B? with TMP prevented IL-1?-, A2E-, LPS-, and TNF?-induced NF?B-mediated upregulation and release of the proinflammatory cytokines IL-1? 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.

SUBMITTER: Vu KT 

PROVIDER: S-EPMC5566385 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature