NF-κB feedback control of JNK1 activation modulates TRPV1-induced increases in IL-6 and IL-8 release by human corneal epithelial cells

Department of Medicine, Beth Israel Deaconess Medical Centerand Harvard Medical School, Boston, MAPurpose: The corneal wound healing response to an alkali burn results in dysregulated inflammation and opacity. Transientreceptor potential vanilloid type1 (TRPV1) ion channel activation by such a stress contributes to this unfavorable outcome.Accordingly, we sought to identify potential drug targets for mitigating this response, in human corneal epithelial cells(HCEC).Methods: SV40-immmortalized HCEC were transduced with lentiviral vectors to establish stable c-Jun N-terminalkinase1 (JNK1), nuclear factor- κB1 (NF- κB1), and dual specificity phsophatase1 (DUSP1) shRNAmir sublines.Immunoblotting evaluated the expression of NF-κB1, DUSP1, protein kinase Cδ (PKCδ), and the phosphorylation statusof cell signaling mediators. Enzyme-linked immunosorbent assay (ELISA) evaluated interleukin-6 (IL-6) andinterleukin-8 (IL-8) release.Results: Capsaicin (CAP; a selective TRPV1 agonist), induced time-dependent activation of transforming growth factor-activated kinase 1 (TAK1) and mitogen- activated protein kinase (MAPK) cascades temporally followed by increasednuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (I κBα) phosphorylation, rises in bothPKCδ protein levels and IL-6 and IL-8 release. All of these responses were blocked by the TAK1 inhibitor 5z-7-oxozeaenol(5z-OX). In the JNK1 subline, CAP failed to increase IL-6/8 release, but still stimulated NF- κB by 50%. In the NF- κB1subline, these IL-6/8 responses were absent, JNK1 activation was attenuated and there was a concomitant increase inDUSP1 expression compared to the control. In the DUSP1 subline, JNK1 phosphorylation was enhanced and prolongedand accompanied by larger increases in IL-6/8 release.Conclusions: TRPV1 induced increases in IL-6/IL-8 release occur through TAK1 activation of JNK1-dependent andJNK1-independent signaling pathways. Their joint activation is required for NF- κB to elicit sufficient positive feedbackcontrol of JNK1/2 phosphorylation to elicit increases in IL-6/8 release. Such regulation depends on NF- κB modulationof DUSP1 expression levels and associated changes in PKCδ protein levels.