Epidermal growth factor receptor transactivation by the cannabinoid receptor (CB1) and transient receptor potential vanilloid 1 (TRPV1) induces differential responses in corneal epithelial cells.

Corneal transparency maintenance depends on continuous renewal of its outermost epithelial layer. This process replaces the uppermost terminally differentiated layers that are being sloughed off into the tears. Their replacement assures preservation of epithelial integrity and its smooth optical properties. In addition, epithelial renewal preserves other needed functions for visual clarity that include: 1) tight junction barrier intactness, which protects the cornea from becoming translucent due to tissue swelling caused by exposure to environmental stresses such as pathogens and anisosmotic challenges (Lu et al., 2001); 2) innate immune responsiveness, which detects the presence of pathogens and provides signals that activate the corneal defense system (Zhang et al., 2005); 3) aldehyde dehydrogenase expression, which protects this layer against UV- and 4-hydroxynonenal-induced cellular damage (Pappa et al., 2005). As epithelial turnover is modulated by a host of cytokines, extensive effort is devoted to identifying cognate receptor-linked cell signaling drug targets for hastening this process subsequent to corneal injury (Reinach and Pokorny, 2008). These studies employ injury-induced in vitro epithelial and in vivo wound healing models to determine which receptor-linked cell signaling pathways mediate control of cell proliferation, migration, differentiation, inflammation and apoptosis. In addition to improving our understanding of corneal biology, these studies have identified potential novel drug targets to lessen corneal scarring and inflammation that can persist subsequent to wound closure. These complications may be severe enough to prevent restoration of corneal transparency and visual function (Saika et al., 2007). In human corneal epithelial cells (HCEC), epidermal growth factor receptors (EGFRs) contribute to mediating corneal epithelial renewal. EGFR activation by EGF results in stimulation of cell proliferation and migration through activation of: a) the three member pathways of the mitogen-activated protein kinase (MAPK) signaling cassette (i.e., ERK, p38 and JNK); b) the phosphoinositide 3-kinase (PI3-K)/Akt/GSK-3 pathway; c) adenylate cyclase and phospholipase C (PLC)-induced Ca2+ signaling as well as phospholipase D (PLD)-mediated phosphatidic acid formation (Islam and Akhtar, 2000; Kang et al., 2000; Kang et al., 2001; Mazie et al., 2006; Wang et al., 2006; Wang et al., 2009; Yang et al., 2005; Yang et al., 2001; Yin and Yu, 2009; Zhang and Akhtar, 1998). EGFR activation also can occur through transactivation by other receptors and mediators (Block and Klarlund, 2008; Lyu et al., 2006; Spix et al., 2007; Xu et al., 2006; Xu et al., 2007). In this process, agonists other than EGF activate their cognate receptors, which leads to matrix metalloproteinase activation and scission of EGF from membrane bound heparin. Therefore, the EGFR-linked cell signaling pathways serve as a conduit for eliciting tissue responses to a number of different mediators besides EGF. Members of the transient receptor potential (TRP) protein superfamily are polymodal in that they are activated by numerous different stimuli. In the corneal epithelium, some members of the vanilloid (V) TRP subfamily were identified. In HCEC, there is functional expression of TRPV1, 3 and 4 (Pan et al., 2008; Yamada et al., 2010; Zhang et al., 2007). TRPV1 is a nonselective ion channel which is activated by injury-induced endogenous mediators such as endocannabinoids, endovanilloids, declines in pH, elevated temperature and hypertonicity as well as capsaicin, which is present in red pepper extracts. Capsaicin (CAP) is a selective TRPV1 agonist and in HCEC induces increases in the release of proinflammatory cytokine mediators, such as interleukin (IL)-6 and the chemoattractant, IL-8. MAPK activation is a contributor to their increases (Zhang et al., 2007). These rises induced by CAP have physiological relevance since TRPV1 activation by injury in a mouse corneal wound healing model contributes to the development of severe inflammation that persists subsequent to wound closure. Evidence of its role stems from our finding that in homozygous TRPV1(−/−) knockout mice the wound healing response to injury is more favorable. This is apparent since inflammation and scarring are less severe at the time of wound closure (Okada et al., 2008). Even though EGFR-linked pathways are activated by CAP, it is not known if EGFR transactivation contributes to the development of inflammation and scarring. The cannabinoid receptor subtype 1 (CB1) modulates, through the GTP binding protein (Gi), a number of important physiological processes in different tissues including neurotransmitter release, pain and analgesia, energy homeostasis regulation, and control of immune cell function (Graham et al., 2009; Howlett, 2005; Kress and Kuner, 2009; Pertwee, 2006; Stephens, 2009). CB1 activation by cannabinoids has immunosuppressive effects, which have beneficial effects in the treatment of autoimmune disorders. These results suggest that the cannabinoid system has various roles in disease pathologies and provides potential therapeutic targets. A functional role for CB1 in the human corneal epithelium has not yet been described even though CB1 expression was detected in the corneas of isolated human eyes (Straiker et al., 1999). In some other tissues, TRPV1 and CB1 are coexpressed and functionally interact with one another. Such is the case in the colonic epithelium, in neuronal enriched mesencephalic cultures, primary sensory neurons and myometrial smooth muscle cells (Brighton et al., 2009; Kim et al., 2008; Mahmud et al., 2009; Sibaev et al., 2006). The coexpression of TRPV1 and CB1 in the corneal epithelium prompted us to probe for a functional interaction between them in HCEC. We show in HCEC that there is a functional interaction between TRPV1 and CB1. Together they mediate increases in cell proliferation and migration through EGFR transactivation and MAPK/Akt-linked signaling. On the other hand, other EGFR independent TRPV1-linked pathway(s) contribute to mediating TRPV1 stimulation of IL-6 and IL-8 release. In contrast, CB1 activation counters TRPV1-induced increases in IL-8. It is conceivable in a clinical setting that drugs targeted to activate CB1 receptors may be effective in reducing TRPV1-induced inflammation caused by corneal injury.