Hypertonicity-Induced TRPV1 Stimulation Leads to Increases in Proinflammatory Cytokine Release Through EGFR Transactivation in Human Corneal Epithelial Cells

Abstract

Purpose: : Tear film hyperosmolarity is associated with pathogenesis of keratoconjunctivitis. Transient receptor potential vanilloid channel 1 (TRPV1) activation induces increases in proinflammatory cytokine release in human corneal epithelial cells (HCEC). We hypothesized that hypertonicity-induced TRPV1 activation mediates increases in proinflammatory cytokine release through EGF receptor (EGFR) transactivation in HCEC.

Methods: : Hypertonic medium was prepared by supplementing basic medium with either NaCl or sucrose. TRPV1 mediated Ca²⁺ signaling was measured in fura2-AM loaded HCEC using a single-cell fluorescence imaging system. Western Blot analysis evaluated activation of EGFR, Erk1/2, p38 MAPK, and the NFΚB inhibitory component, IΚB. ELISA assessed hypertonicity induced IL-6 and IL-8 release.

Results: : A 450 mOsm hypertonic stress induced 3.5-fold Ca²⁺transients ([Ca²⁺]_i). These increases were enhanced by PGE₂ (1 µM) but suppressed by either capsazepine (CPZ, 10 µM) or exposure to Ca²⁺ free medium. This challenge also activated EGF receptor (EGFR) phosphorylation. Such activation was inhibited by either CPZ or AG 1478 (10 µM). EGF reversed CPZ induced inhibition, but had no effect on AG 1478’s inhibition of EGFR phosphorylation. Erk1/2 and p38 MAPK were phosphorylated by this stress. Their phosphorylations were suppressed during concomitant exposure to CPZ or AG 1478. NFΚB phosphorylation by the hypertonic stress was inhibited by CPZ, AG, PD 98059 (10 µM), SB 203580 (10 µM) or PDTC (50 µM). IL-6 and IL-8 release increased by 5- and 8-fold, respectively, following 24 hr exposure to this hypertonic medium. Inhibition of either TRPV1, EGFR, Erk1/2, p38 or NFΚB activation suppressed in turn these IL-6 and IL-8 increases. Addition of EGF in the hypertonic medium alleviated CPZ inhibition of NFΚB activation and its suppression of IL-6 and IL-8 rises.

Conclusions: : TRPV1 activation by hypertonic stress in HCEC leads to increases in IL-6, IL-8 release through EGFR transactivation followed by stimulation of Erk1/2, p38 and NFΚB signaling.

Keywords: cornea: epithelium • stress response • cornea: basic science