Cannabinoid-induced chemotaxis in bovine corneal epithelial cells

Abstract

Purpose: Cannabinoid CB1 receptors are found in abundance in the vertebrate eye, with most tissue types expressing this receptor. However, the function of CB1 receptors in corneal epithelial cells (CECs) is poorly understood. Interestingly, the corneas of CB1 knockout mice heal more slowly after injury via a mechanism proposed to involve protein kinase B (Akt) activation, chemokinesis, and cell proliferation. The current study examined the role of cannabinoids in CEC migration in greater detail.

Methods: We determined the role of CB1 receptors in corneal healing. We examined the consequences of their activation on migration and proliferation in bovine CECs (bCECs). We additionally examined the mRNA profile of cannabinoid-related genes and CB1 protein expression as well as CB1 signaling in bovine CECs.

Results: We now report that activation of CB1 with physiologically relevant concentrations of the synthetic agonist WIN55212-2 (WIN) induces bCEC migration via chemotaxis, an effect fully blocked by the CB1 receptor antagonist SR141716. The endogenous agonist 2-arachidonoylglycerol (2-AG) also enhances migration. Separately, mRNA for most cannabinoid-related proteins are present in bovine corneal epithelium and cultured bCECs. Notably absent are CB2 receptors and the 2-AG synthesizing enzyme diglycerol lipase-α (DAGLα). The signaling profile of CB1 activation is complex, with inactivation of mitogen-activated protein kinase (MAPK). Lastly, CB1 activation does not induce bCEC proliferation, but may instead antagonize EGF-induced proliferation.

Conclusions: In summary, we find that CB1-based signaling machinery is present in bovine cornea and that activation of this system induces chemotaxis.

Figure 1

Activation of CB₁ induces chemotaxis in bovine CECs in a Boyden chamber assay. (A) The CB₁ agonist WIN55212-2 enhances bCEC migration in a concentration-dependent manner during a 3-hour incubation. This enhancement is fully blocked by the CB₁ antagonist SR141716 (SR1, 1 μM). (B) The endocannabinoid 2-AG induces a similar migration at the relatively low concentration of 100 nM. This migration is partly blocked by SR141716 (1 μM). *P < 0.05, unpaired t-test versus corresponding 100 nM drug.

Figure 2

WIN55212-2 induces chemotaxis in bovine CECs in an in vitro gradient assay. (A) Sample diagram showing tracking of individual cells in response to WIN55212-2 (WIN) placed in upper end of dish. (B) Summary data from (A), showing migration before (left panel) and after (right panel) drug toward the target (direction denoted by arrow), with start-points normalized to origin (0,0). (C) Time course showing distance traveled toward the WIN target under baseline, WIN, and WIN/SR141716 conditions. *Distance was returned zero to facilitate comparison. (D) Summary of velocity toward target during final five minutes of each condition. *P < 0.01, **P < 0.001 1-way ANOVA with Bonferroni post hoc test.

Figure 3

Messenger RNA expression for various cannabinoid-related proteins in the eye of the cow. (A) We tested for mRNA expression in four bovine ocular tissues, corneal epithelium (Co Ep), and endothelium (Co End), retina, and trabecular meshwork (TM). Genes and their hypothesized roles are described in the text. (B) We also examined expression of a subset of these mRNAs in cultured bovine epithelial cells.

Figure 4

CB₁ protein expression in cultured bCECs. (A) Overview shows CB₁ expression (red, arrows) in a subset of cultured bCECs, outlined by phalloidin (green). (B) Higher magnification image shows ring-like CB₁ expression (red). (C) CB₁/phalloidin overlaid on DIC image (left); CB₁ only (right). (D) CB₁/DAPI (left). Same with DIC (middle). Flat Z stack of CB₁ in same cell (right). Scale bars: 20 μm (A), 10 μm (B, C), 5 μm (D).

Figure 5

Cannabinoids have an unusual intracellular signaling activation profile in bovine corneal epithelial cells. (A) Instead of activating MAPK, WIN, and 2-AG dephosphorylate MAPK in bCECs. (B) Mitogen-activated protein kinase inhibition occurs rapidly and persists to 30 minutes. (C) 2-arachidonoylglycerol, but not WIN, reduces cAMP levels in forskolin-treated cells. (D) 2-arachidonoylglycerol activates Akt, but WIN does so only modestly at higher concentrations.

Figure 6

Activation of CB₁ does not enhance proliferation of bCECs. (A) In a proliferation assay, CP55940 (100 nM) and WIN55212 (100 nM) do not enhance bCEC proliferation. (B) A concentration-response profile for CP55940 again shows no effect on proliferation, while EGF (50 ng/mL) enhances proliferation. (C) Epidermal growth factor enhances proliferation in a concentration-dependent manner in our preparation. (D) CP55940 (100 nM) in combination with half-maximal (5 ng/mL) or low (1 ng/mL) EGF does not enhance proliferation. (B) *P < 0.05 1-way ANOVA/Dunnett's post hoc versus control. (D) Not significant (NS), 1-way ANOVA/Bonferroni post hoc between EGF 5 ng/mL and EGF 5 ng/mL + CP (100 nM).