Inhibition of NF-kB signaling modulates epithelial to mesenchymal transition in human stem cell-derived retinal pigment epithelial cells

Abstract

Purpose : RPE injury can induce epithelial to mesenchymal transition (EMT), and the RPE dysfunction associated with EMT has been implicated in retinal diseases such as age-related macular degeneration (AMD). Based on the hypothesis that inhibition of RPE EMT could be therapeutically beneficial, we have been testing various small molecules for their ability to modulate RPE EMT. Several major EMT transcription factors are direct targets of NF-kappaB (NF-kB). Using transforming growth factor β (TGF-β) induced RPE EMT as a model, we tested the ability of the NF-kB signaling inhibitor BAY 65-1942 (kinase of IkappaB (IkB, inhibitor of NF-kB) to inhibit EMT of human stem cell-derived RPE cells. Further, we performed transcriptomic analysis to dissect the molecular pathways altered by inhibition of NF-kB signaling in the context of EMT.

Methods : Human RPE monolayers were differentiated from iPS cells. To induce EMT, RPE monolayer cultures were dissociated or co-treated with TGF-β and the inflammatory cytokine TNF-α, and the cultures were treated with BAY 65-1942. Expression of endogenous RPE and EMT genes was measured by RT-qPCR. For global transcriptomic analysis, RNA-Seq libraries were constructed, sequenced and analyzed using Tophat, cuffquant and Cuffnorm. Biological pathway analysis was performed using Ingenuity Pathway Analysis (IPA) and Gene Set Enrichment Analysis (GSEA).

Results : Expression of a core set of down-regulated visual cycle (RPE65, RLBP1, RDH5) and other RPE-related genes (BEST1, PMEL17, CRX) was significantly restored by BAY 65-1942 treatment. Elevated EMT regulators (SNAIL, JAG1, HMGA2 and CDH2) and NF-kB target genes (ICAM1, IRF1, FAS and FTH) were significantly suppressed with IKKβ inhibition. Ingenuity pathway analysis showed that alterations of gene expression related to EMT, axon guidance, actin cytoskeleton, BMP, retinoic acid receptor (RAR) activation, matrix metalloproteinases, and ILK/IL-8 signaling were significantly attenuated.

Conclusions : Targeting the NF-kB pathway through inhibition of IKKβ attenuates TGF-β/TNF-α induced RPE EMT and promotes RPE identity. These results suggest that targeted inhibition of NF-kB signaling might be a promising therapeutic approach for AMD and other retinal disease. Additionally, exploring altered transcriptional networks in human RPE cells undergoing EMT may identify novel therapeutic targets for AMD.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.