Cannabinoid Receptor Induces EGFR Transactivation Through Transient Receptor Potential Vanilloid 1 Channel Stimulation in Human Corneal Epithelial Cells

Abstract

Purpose: : Cannabinoid receptor 1 (CB1) protein expression was described in the human cornea, but its functional role is not known. In brain, CB1 receptor stimulation induces activation of a specific transient receptor potential (TRP) isoform in the vanilloid subfamily (TRPV1). We probed for such an interaction in human corneal epithelial cells (HCEC). In addition, we evaluated whether or not TRPV1 activation induces increases in proinflammatory cytokine release and proliferation through epidermal growth factor receptor (EGFR) transactivation.

Methods: : Immunostaining and Western blot analysis probed for CB1 and TRPV1 expression. A single cell fluorescence imaging system monitored Ca²⁺ transients. The mixed CB1 and TRPV1 endocannabinoid agonist: anandamide (AEA) was used. The respective selective CB1 agonist/antagonist pair: WIN55, 212-2 (WIN) and AM251 (AM) and the TRPV1 agonist/antagonist pair: capsaicin (CAP), and capsazepine (CPZ) were also individually used. A relatively selective EGFR inhibitor, AG1478 (AG) was used to assess for crosstalk between these different receptors. Changes in EGFR phosphorylation status elicited by these different agonists and antagonists monitored its activation. [³H] thymidine incorporation measured cell proliferation. ELISA determined TRPV1-induced increases in proinflammatory cytokine release.

Results: : WIN (10 µM) induced a 3-fold [Ca²⁺]_i transient, whereas preincubation with 10 µM AM blocked CB1 activation by 83 %. AEA induced a 2.1-fold [Ca²⁺]_i transient, which was suppressed by 79% with 10 µM CPZ. Preincubation with AG (100 nM) blocked CAP and WIN -induced [Ca²⁺]_i transients by 42% and 81%, respectively. CAP (10 µM) or WIN (3 µM) increased the EGFR phosphorylation status by 4 and 2.2-fold, respectively. Preincubation with either 10 µM CPZ, 3 µM AM or 5 µM AG abolished CB1 or TRPV1-induced EGFR phosphorylation. Both EGF and CAP increased proliferation by 1.7-fold. Similarly, WIN (3 µM) increased this response by 1.4 -fold. Each of these increases were blocked by 5 µM AG, 10 µM CPZ, or 3 µM AM. TRPV1 -induced increases in IL-6 and IL-8 expression were suppressed during exposure to AG (10 µM) or CPZ (10 µM) to nearly baseline levels.

Conclusions: : Taken together, CB1 receptor activation mediates through TRPV1 and EGFR stimulation downstream signaling events leading to increases in proinflammatory cytokine release and cell proliferation. Ca²⁺ signaling contributes to the stimulation of these responses by mediating CB1-induced EGFR transactivation.

Keywords: cornea: epithelium • receptors: pharmacology/physiology • ion channels