Abstract
Purpose::
TRPV1 is a vanilloid subtype of the transient receptor potential protein superfamily. This isoform is a subunit of a non-selective cation channel mediating downstream responses to heat, low pH, or noxious stimuli. TRPV1 expression has been recently described in some epithelial tissues and induces proinflammatory cytokine release through mitogen-activated protein kinase (MAPK) superfamily stimulation. We describe here in human corneal epithelial cells (HCEC) the signaling pathways mediating TRPV1-induced increases in proinflammatory cytokine release.
Methods::
Immunohistology, immunocytochemistry and Western Blot analysis identified TRPV1 expression in intact human corneal epithelium, primary and SV40-immortalized human corneal epithelial cells (pHCEC and HCEC, respectively). The effects of the TRPV1 agonist, capsaicin (CAP), on MAPK superfamily activation were evaluated by Western Blot analysis. ELISA quantified CAP-induced increases in proinflammatory cytokine (i.e.IL-8 and IL-6) release. The involvement of p38 JNK and ERK MAPK branches were evaluated based on the relative inhibitory effects of specific MAPK inhibitors: 10 µM SB203580, 10 µM SP600125 and 10 µM U0126, respectively on these responses.
Results::
TRPV1 expression was detected in pHCEC, HCEC and the intact human corneal epithelium. In HCEC, TRPV1 staining was delimited to the cell margins. CAP(1 µM) induced after 5 min maximal global p38 MAPK, JNK and Erk1/2 activation. Over the next 90 min, such stimulation gradually waned towards their baseline values. p38 MAPK was maximally activated by all CAP concentrations (i.e.0.5-100 µM). On the other hand, maximal JNK and Erk1/2 activation occurred with 10 µM and 0.1 µM CAP, respectively. Pretreatment with the TRPV1 receptor antagonist capsazepine (CPZ) blocked its activation. CAP (1 µM) maximally increased IL-8 and IL-6 8- and 4- fold release, respectively. Such rises were completely suppressed by CPZ (10 µM) whereas they were only partially suppressed by either SB203580, SP600125 or U0126.
Conclusions::
TRPV1 receptor stimulation in HCEC induces through three MAPK limbs increases in proinflammatory cytokine release. These results suggest that epithelial TRPV1 expression contributes in-vivo to mounting proinflammatory reactions to noxious stimuli.
Keywords: cornea: epithelium • ion channels • signal transduction