title: Functional TRPV1 expression in human corneal fibroblasts.
abstract: Corneal wound healing in mice subsequent to an alkali burn results in dysregulated inflammation and opacification. Transient receptor potential vanilloid subtype 1 (TRPV1) channel activation in all tissue layers by endogenous ligands contributes to this sight compromising outcome since in TRPV1 knockout mice wound healing results instead in tissue transparency restoration. However, it is not known if primary human stromal fibroblasts exhibit such expression even though functional TRPV1 expression is evident in an immortalized human corneal epithelial cell line. In primary human corneal fibroblasts (HCF), TRPV1 gene expression and localization were identified based on the results of quantitative RT-PCR and immunocytochemistry, respectively. Western blot analysis identified a 100 kD protein corresponding to TRPV1 protein expression in a positive control. Single-cell fluorescence imaging detected in fura2-AM loaded cells Ca(2+) transients that rose 1.8-fold above the baseline induced by a selective TRPV1 agonist, capsaicin (CAP), which were blocked by a TRPV1 antagonist, capsazepine (CPZ) or exposure to a Ca(2+) free medium. The whole-cell mode of the planar patch-clamp technique identified TRPV1-induced currents that rose 1.76-fold between -60 and +130 mV. CAP-induced time dependent changes in the phosphorylation status of mitogen activated protein kinase (MAPK) signaling mediators that led to a 2.5-fold increase in IL-6 release after 24 h. This rise did not occur either in TRPV1 siRNA gene silenced cells or during exposure to SB203580 (10 μM), a selective p38 MAPK inhibitor. Taken together, identification of functional TRPV1 expression in HCF suggests that in vivo its activation by injury contributes to corneal opacification and inflammation during wound healing. These undesirable effects may result in part from increases in IL-6 expression mediated by p-p38 MAPK signaling.
authors.affiliation: Department of Biological Sciences, State University of New York, State College of Optometry, New York, NY 10036, USA.
authors.forename: Yuanquan
authors.initials: Y
authors.lastname: Yang
authors.forename: Hua
authors.initials: H
authors.lastname: Yang
authors.forename: Zheng
authors.initials: Z
authors.lastname: Wang
authors.forename: Stefan
authors.initials: S
authors.lastname: Mergler
authors.forename: J Mario
authors.initials: JM
authors.lastname: Wolosin
authors.forename: Peter S
authors.initials: PS
authors.lastname: Reinach
mesh_terms: D015153:Blotting, Western; D002211:Capsaicin / Q000494:pharmacology; D002478:Cells, Cultured; D060527:Corneal Keratocytes / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D007150:Immunohistochemistry; D015850:Interleukin-6 / Q000378:metabolism; D018408:Patch-Clamp Techniques; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050916:TRPV Cation Channels / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D015850:Interleukin-6; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D050916:TRPV Cation Channels; C494482:TRPV1 protein, human; D048051:p38 Mitogen-Activated Protein Kinases; D002211:Capsaicin