Abstract
Once activated, the intracellular receptor NLRP3 assembles an inflammasome protein complex that facilitates the caspase-1-mediated maturation of IL-1β and IL-18. Inactive NLRP3 is guarded by a protein complex containing Hsp90. In response to stress stimuli, Hsp90 is released, and NLRP3 can be activated to promote inflammation. In this study, we blocked Hsp90 with geldanamycin and studied the fate of NLRP3 in human retinal pigment epithelial (RPE) cells. RPE cells play a central role in the development of age-related macular degeneration (AMD), a progressive eye disease causing severe vision loss in the elderly. IL-1α-primed ARPE-19 cells, human embryonal stem cell (hESC)-derived RPE cells, and primary human RPE cells were exposed to MG-132 and bafilomycin A to activate NLRP3 via the inhibition of proteasomes and autophagy, respectively. Additionally, RPE cells were treated with geldanamycin at different time points and the levels of NLRP3 and IL-1β were determined. Caspase-1 activity was measured using a commercial assay. Geldanamycin prevented the activation of the inflammasome in human RPE cells. NLRP3 released from its protective complex became degraded by autophagy or secreted from the cells. Controlled destruction of NLRP3 is a potential way to regulate the inflammation associated with chronic diseases, such as AMD.
Highlights
The nucleotide-binding domain and Leucine-rich repeat Receptor containing a Pyrin domain 3 (NLRP3) inflammasome is an intracellular signaling complex involved in the induction of inflammation[1]
We have previously shown that inhibition of the intracellular clearance systems with MG-132 and bafilomycin A1 (BafA) activates NLRP3 inflammasomes, leading to the secretion of IL-1β from ARPE-19 cells[18]
The secreted IL-1β levels induced by MG-132 + BafA remained below 0.2 pg/ml but in the present study, the same activators on primed ARPE-19 cells raised the levels of released cytokine up to 3.4 ± 0.18 pg/ml
Summary
The nucleotide-binding domain and Leucine-rich repeat Receptor containing a Pyrin domain 3 (NLRP3) inflammasome is an intracellular signaling complex involved in the induction of inflammation[1]. Protein suppressor of the G2 allele of SKP1 (SGT1), Hsp[90] forms a complex with NLRP3 and retains the receptor protein in an inactive but competent form for activation after reception of the priming signal[14,15,16]. If this complex is not formed, the NLRP3 protein will be degraded, but it is far from clear whether the degradation takes place in proteasomes or via the lysosomal (autophagy) pathway[14,17]. The effects of the Hsp[90] inhibitor geldanamycin (GA) on the fate of NLRP3 were explored in human RPE cells
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