title: TRPV1 activation is required for hypertonicity-stimulated inflammatory cytokine release in human corneal epithelial cells.
abstract: OBJECTIVE
  To determine whether hypertonic stress promotes increases in inflammatory cytokine release through transient receptor potential vanilloid channel type 1 (TRPV1) signaling pathway activation in human corneal epithelial cells (HCECs).
  
  
  METHODS
  Hyperosmotic medium was prepared by supplementing isotonic Ringers solution with sucrose. Ca2+ signaling was measured in fura2-AM-loaded HCECs using a single-cell fluorescence imaging system. Western blot analysis evaluated the phosphorylation status of EGFR, ERK, p38 MAPK, and nuclear factor (NF)-κB. ELISA assessed the effect of TRPV1 activation on the release of IL-6 and IL-8.
  
  
  RESULTS
  A 450 mOsm hypertonic stress elicited 2-fold Ca2+ transients that were suppressed by the TRPV1-selective antagonists capsazepine and JYL 1421. Such transients were enhanced by PGE2. Hypertonicity-induced EGF receptor (EGFR) transactivation was suppressed by preincubating HCECs with capsazepine, matrix metalloproteinase 1 (MMP1) inhibitor TIMP-1, broad-spectrum MMP inhibitor GM 6001, heparin-bound (HB)-EGF inhibitor CRM 197, or EGFR inhibitor AG 1478. ERK and p38 MAPK and NF-κB activation after EGFR transactivation occurred in tonicity and in a time-dependent manner. Hypertonicity-induced increases in IL-6 and IL-8 releases were suppressed by exposure to capsazepine, AG 1478, ERK inhibitor PD 98059, p38 inhibitor SB 203580, or NF-κB inhibitor PDTC.
  
  
  CONCLUSIONS
  Hypertonic stress-elicited TRPV1 channel stimulation mediates increases in a proinflammatory cytokine IL-6 and a chemoattractant IL-8 by eliciting EGFR transactivation, MAPK, and NF-κB activation. Selective drug modulation of either TRPV1 activity or its signaling mediators may yield a novel approach to suppressing inflammatory responses occurring in dry eye syndrome.
authors.affiliation: Department of Biological Sciences, College of Optometry, State University of New York, New York, New York 10065, USA. zap2001@med.cornell.edu
authors.forename: Zan
authors.initials: Z
authors.lastname: Pan
authors.forename: Zheng
authors.initials: Z
authors.lastname: Wang
authors.forename: Hua
authors.initials: H
authors.lastname: Yang
authors.forename: Fan
authors.initials: F
authors.lastname: Zhang
authors.forename: Peter S
authors.initials: PS
authors.lastname: Reinach
mesh_terms: D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002211:Capsaicin / Q000031:analogs & derivatives / Q000494:pharmacology; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D019573:Epithelium, Corneal / Q000187:drug effects* / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D016257:Fura-2 / Q000031:analogs & derivatives; D006801:Humans; D006982:Hypertonic Solutions / Q000494:pharmacology*; D015850:Interleukin-6 / Q000378:metabolism*; D016209:Interleukin-8 / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism; D010766:Phosphorylation; D015398:Signal Transduction; D013312:Stress, Physiological; D013449:Sulfonamides / Q000494:pharmacology; D050916:TRPV Cation Channels / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013890:Thiourea / Q000031:analogs & derivatives / Q000494:pharmacology; D013997:Time Factors; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D006982:Hypertonic Solutions; D015850:Interleukin-6; D016209:Interleukin-8; C466314:JYL 1421; D016328:NF-kappa B; D013449:Sulfonamides; D050916:TRPV Cation Channels; C494482:TRPV1 protein, human; C049925:fura-2-am; C512478:EGFR protein, human; D066246:ErbB Receptors; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D013890:Thiourea; C071423:capsazepine; D002211:Capsaicin; D002118:Calcium; D016257:Fura-2