Cannabinoid CB2R receptors are upregulated with corneal injury and regulate the course of corneal wound healing
Introduction
Cannabinoid receptors were once chiefly known as the endogenous target for the psychoactive ingredients of marijuana (Gaoni and Mechoulam, 1964). However, it is now appreciated that the endogenous cannabinoid signaling system is important in its own right, playing physiological roles in pain, neurodegeneration, appetite and energy regulation, learning and memory, addiction, bone homeostasis and remodeling, cancer, immune function, cardiovascular function, and reproduction (Howlett et al., 2004; Kano et al., 2009). Cannabinoids are also active in the eye, where CB1R receptors are widely expressed (Straiker et al., 1999a, 1999b). CB1R activation alters intraocular pressure, iridial contraction and perhaps vision (reviewed in (Cairns et al., 2015)). Deleterious mutations of the endocannabinoid-metabolizing enzyme ABHD12 in humans result in the development of cataracts and retinal degeneration (Fiskerstrand et al., 2010). CB1R receptors are abundant in human corneal epithelial cells (CECs) (Straiker et al., 1999b) and mediate chemotaxis in cultured bovine CECs (Murataeva et al., 2016). Importantly, CB1R deletion slows wound healing (Yang et al., 2013), suggesting that cannabinoids, via CB1R, regulate the rate of wound closure via a chemotaxic mechanism, using an endocannabinoid gradient to provide directional cues to CECs during the healing process (Murataeva et al., 2015).
CB2R receptors were identified shortly after CB1R (Munro et al., 1993) but the data on ocular CB2R has been mixed: most receptor expression studies have not detected CB2R in the anterior eye (Buckley et al., 1998; Lu et al., 2000; Porcella et al., 1998, 2000), but a few pharmacological studies provide evidence for functional CB2R in the eye (He and Song, 2007; Zhong et al., 2005). Cannabinoid pharmacology can however be problematic; we have shown for instance that CB2R agonist JWH015 (used in (He and Song, 2007; Zhong et al., 2005)) is also a potent and efficacious CB1R agonist (Murataeva et al., 2012).
CB2R receptors have demonstrated beneficial effects in wound healing in several models (Li et al., 2016; Wang et al., 2016; Wright et al., 2005; Zheng et al., 2012) and been shown to regulate chemotaxis in immune-related cells such as lymphocytes (e.g. (Ghosh et al., 2006)). The current study investigates CB2R upregulation upon corneal injury and its role in corneal wound healing.
Section snippets
Animals
Experiments were conducted at the Indiana University campus. All mice used for experiments were handled according to the guidelines of the Indiana University animal care committee and in accordance with the ARVO animal statement. Mice (age 3ā8 months) were kept on a 12āÆh (06:00ā18:00) light dark cycle, and fed ad libitum. Male and female C57BL/6āÆJ (C57) and CD1 strain mice were kindly provided by Dr. Ken Mackie (Indiana University, Bloomington IN). Mice were allowed to acclimatize to the animal
CB2R ligands induce chemorepulsion in cultured bovine corneal epithelial cells
The CB2R agonist JWH133 (300āÆnM) was embedded in a cube of agar and placed in a 60āÆmm petri dish containing recently plated bCECs. As depicted schematically in Fig. 1A, the JWH133-containing agar block was positioned at the edge of the dish while the migration of epithelial cells at the center of the dish was monitored. The sample migration tracks from one experiment are shown in Fig. 1B. Migration of cells was arrested after subsequent bath application (i.e. no gradient) of CB2 antagonist
Discussion
Our chief findings are 1) a CB2R signaling system is upregulated by injury in murine cornea; 2) CB1R deletion in mouse slows healing, consistent with a published finding; 3) CB2R deletion in mouse also slows wound closure; 4) in experiments using a different species, the cow, we find that levels of N-acyl ethanolamines including the eCB anandamide are increased upon injury in bovine cornea; 5) in epithelial cells cultured from cow eyes we find that CB2R activation mediates repulsive chemotaxis.
Acknowledgements
This work was supported in part by a grant from the National Institutes of Health [RO1-EY24625, AS].
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