Epoxomicin, a Selective Proteasome Inhibitor, Activates AIM2 Inflammasome in Human Retinal Pigment Epithelium Cells

Abstract

Emerging evidence suggests that the intracellular clearance system plays a vital role in maintaining homeostasis and in regulating oxidative stress and inflammation in retinal pigment epithelium (RPE) cells. Dysfunctional proteasomes and autophagy in RPE cells have been associated with the pathogenesis of age-related macular degeneration. We have previously shown that the inhibition of proteasomes using MG-132 activates the NLR family pyrin domain containing 3 (NLRP3) inflammasome in human RPE cells. However, MG-132 is a non-selective proteasome inhibitor. In this study, we used the selective proteasome inhibitor epoxomicin to study the effect of non-functional intracellular clearance systems on inflammasome activation. Our data show that epoxomicin-induced proteasome inhibition promoted both nicotinamide adenine dinucleotide phosphate oxidase and mitochondria-mediated oxidative stress and release of mitochondrial DNA to the cytosol, which resulted in potassium efflux-dependent absence in melanoma 2 (AIM2) inflammasome activation and subsequent interleukin-1β secretion in ARPE-19 cells. The non-specific proteasome inhibitor MG-132 activated both NLRP3 and AIM2 inflammasomes and oxidative stress predominated as the activation mechanism, but modest potassium efflux was also detected. Collectively, our data suggest that a selective proteasome inhibitor is a potent inflammasome activator in human RPE cells and emphasize the role of the AIM2 inflammasome in addition to the more commonly known NLRP3 inflammasome.

Inflammasome activation in response to aberrations of cellular homeostasis in epithelial cells from human cornea and retina

“…This result is in line with the fact that poly(dA:dT) was reported to induce AIM2 inflammasome activation instead of NLRP3 (Hornung et al., 2009). In comparison, MCC950 decreased the production of IL‐1β in MG‐132 and BafA‐treated ARPE‐19 cells as this pathway is known to be dependent on NLRP3 in addition to AIM2 (Piippo et al., 2014; Piippo, Korhonen, Hytti, Skottman, et al., 2018; Sridevi Gurubaran et al., 2022). In line with the finding obtained with MCC950, NLRP3 knockdown significantly reduced the production of IL‐1β in D407 RPE cells (Figure 22b).…”

“…Impaired intracellular protein clearance induced by autophagy and proteasome inhibitors has been demonstrated to be capable of activating AIM2 and NLRP3 inflammasomes in human RPE cells (Piippo et al., 2014; Piippo, Korhonen, Hytti, Skottman, et al., 2018; Sridevi Gurubaran et al., 2022). As p62/SQSTM1 is an indicator of dysfunctional intracellular protein clearance (Bjørkøy et al., 2009), its response to inflammation was studied in Publication V. For this purpose, ARPE‐19 cells were transfected with EGFP‐p62/SQSTM1 or p62/SQSTM1 siRNA with or without the IL‐1β exposure.…”

“…K + efflux is a well‐studied inflammasome activation mechanism in myeloid cells but in RPE cells, it has been less extensively examined. Our recent study found that the regulation of inflammasome activation could be dependent on both ROS generation and K + efflux in human RPE cells in conditions of declined protein clearance, but the predominant upstream pathway was dependent on the selectivity of the proteasomal inhibitor used in the in vitro experiments (Piippo, Korhonen, Hytti, Kinnunen, et al., 2018; Sridevi Gurubaran et al., 2022). Another less studied mechanism in RPE cells involves mitochondrial damage‐induced release of mtDNA and the subsequent activation of the inflammasome.…”

“…Another less studied mechanism in RPE cells involves mitochondrial damage‐induced release of mtDNA and the subsequent activation of the inflammasome. Recently, we have reported that a selective proteasomal inhibitor, epoxomicin, is able to induce mtDNA‐mediated AIM2 inflammasome activation and the production of IL‐1β in ARPE‐19 cells (Sridevi Gurubaran et al., 2022). In line with that transfected mtDNA was previously shown to induce caspase‐1 activation followed by the maturation of IL‐1β but not IL‐18 in ARPE‐19 cells (Dib et al., 2015).…”

“…Therefore, it was deemed beneficial to examine inflammasome activation and its mechanisms in human RPE cells with damaged mitochondria. Moreover, it is recognized that impaired intracellular protein clearance is capable of inducing inflammasome activation and the production of other inflammatory cytokines in human RPE cells and iPS‐derived RPE cells from AMD patients (Hytti et al., 2021; Karema‐Jokinen et al., 2023; Piippo et al., 2014; Sridevi Gurubaran et al., 2022). p62/SQSTM1 is a key adaptor protein in the regulation of protein aggregate clearance via autophagy and the proteosomal pathways (Dikic, 2017).…”