title: NF-κB feedback control of JNK1 activation modulates TRPV1-induced increases in IL-6 and IL-8 release by human corneal epithelial cells.
abstract: OBJECTIVE
  The corneal wound healing response to an alkali burn results in dysregulated inflammation and opacity. Transient receptor 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-terminal kinase1 (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 status of cell signaling mediators. Enzyme-linked immunosorbent assay (ELISA) evaluated interleukin-6 (IL-6) and interleukin-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 increased nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) phosphorylation, rises in both PKCδ 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-κB1 subline, these IL-6/8 responses were absent, JNK1 activation was attenuated and there was a concomitant increase in DUSP1 expression compared to the control. In the DUSP1 subline, JNK1 phosphorylation was enhanced and prolonged and 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 and JNK1-independent signaling pathways. Their joint activation is required for NF-κB to elicit sufficient positive feedback control of JNK1/2 phosphorylation to elicit increases in IL-6/8 release. Such regulation depends on NF-κB modulation of DUSP1 expression levels and associated changes in PKCδ protein levels.
authors.affiliation: Department of Biological Sciences, State University of New York, State College of Optometry, New York, NY 10036, USA.
authors.forename: Z
authors.initials: Z
authors.lastname: Wang
authors.forename: Y
authors.initials: Y
authors.lastname: Yang
authors.forename: H
authors.initials: H
authors.lastname: Yang
authors.forename: J E
authors.initials: JE
authors.lastname: Capó-Aponte
authors.forename: S D
authors.initials: SD
authors.lastname: Tachado
authors.forename: J M
authors.initials: JM
authors.lastname: Wolosin
authors.forename: P S
authors.initials: PS
authors.lastname: Reinach
mesh_terms: D002211:Capsaicin / Q000494:pharmacology; D054638:Dual Specificity Phosphatase 1 / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology*; D019573:Epithelium, Corneal / Q000166:cytology; D025461:Feedback, Physiological / Q000187:drug effects; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism*; D016209:Interleukin-8 / Q000378:metabolism*; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects; D048055:Mitogen-Activated Protein Kinase 8 / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D051745:Protein Kinase C-delta / Q000378:metabolism; D050916:TRPV Cation Channels / Q000378:metabolism*; D000099151:MAP Kinase Kinase Kinase 7
chemical_list: D002211:Capsaicin; D054638:Dual Specificity Phosphatase 1; D015850:Interleukin-6; D016209:Interleukin-8; D020930:MAP Kinase Kinase Kinases; D048055:Mitogen-Activated Protein Kinase 8; D016328:NF-kappa B; D051745:Protein Kinase C-delta; D050916:TRPV Cation Channels; D000099151:MAP Kinase Kinase Kinase 7; C508606:CXCL8 protein, human; C508600:IL6 protein, human; C494482:TRPV1 protein, human; C517019:DUSP1 protein, human