Hyperosmotic Stress–Induced TRPM2 Channel Activation Stimulates NLRP3 Inflammasome Activity in Primary Human Corneal Epithelial Cells

Abstract

PURPOSE. The purpose of this study was to determine whether either a hyperosmotic or oxidative stress induces NLRP3 inflammasome activation and increases in bioactive IL-1b secretion through transient receptor potential melastatin 2 (TRPM2) activation in primary human corneal epithelial cells (PHCECs). METHODS.Real-time PCR, Western blots, and immunofluorescent staining were used to evaluate TRPM2 and NLRP3, ASC, caspase-1, and IL-1b mRNA and protein expression levels, respectively. A CCK-8 assay evaluated cell viability. Hyperosmotic 500 mOsm and oxidative 0.5 mM H 2 O 2 stresses were imposed. TRPM2 expression was inhibited with a TRPM2 inhibitor, 20 lM N-(p-amylcinnamoyl) anthranilic acid (ACA), or TRPM2 siRNA knockdown.RESULTS. In the hypertonic medium, TRPM2, NLRP3, ASC, caspase-1, and IL-1b gene and protein expression levels rose after 4 hours (P 0.043), whereas ACA preincubation suppressed these rises (P 0.044). Similarly, H 2 O 2 upregulated TRPM2 protein expression by 80%, and induced both NLRP3 inflammasome activation and increased bioactive IL-1b secretion (P 0.036), whereas ACA pretreatment suppressed these effects (P 0.029). TRPM2 siRNA transfection reduced TRPM2 gene expression by 70% (P ¼ 0.018) in this hyperosmotic medium and inhibited the increases in NLRP3, caspase-1, and IL-1b gene (P 0.028) and protein expression (P 0.037).CONCLUSIONS. TRPM2 activation by either a hyperosmotic or oxidative stress contributes to mediating increases in NLRP3 inflammasome activity and bioactive IL-1b expression because inhibiting TRPM2 activation or its expression blunted both of these responses in PHCECs. This association points to the possibility that TRPM2 is a viable target to suppress hyperosmotic-induced corneal epithelial inflammation.

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