Resolvin D2 uses multiple Ca2+ -dependent signaling pathways to stimulate mucin secretion in rat and human conjunctival goblet cells.

The mucin layer of the tear film is produced by goblet cells in the conjunctiva to protect the ocular surface and maintain homeostasis. The pro-resolving lipid mediator resolvin D2 (RvD2) biosynthesized from an omega 3 fatty acid actively terminates inflammation and regulates mucin secretion from conjunctival goblet cells. Our objective was to determine which Ca2+ -dependent signaling pathways RvD2 uses to stimulate conjunctival goblet cell function (CGC). We hypothesize that RvD2 activates multiple intracellular Ca2+ signaling pathways to stimulate CGC secretion. Rat and human CGCs were cultured from conjunctival explants. The amount of RvD2 receptor GPR18/DRV2 message and protein were determined. The intracellular concentration of Ca2+ ([Ca2+ ]i ) was measured in CGCs using a fluorescent Ca2+ dye and mucin secretion was determined by measuring protein secretion enzymatically with a lectin. Goblet cells were incubated with signaling pathway inhibitors before stimulation with RvD2 and [Ca2+ ]i or secretion was measured. In rat and human CGCs RvD2 receptor and in rat CGCs IP3 (a molecule that releases Ca2+ from intracellular organelles) receptors 1-3 were detected. In both species of CGC RvD2 increased [Ca2+ ]i similarly to RvD1. In rat CGCs, the increase in [Ca2+ ]i and secretion stimulated by RvD2 was significantly blocked by inhibitors to phospholipase (PL-) C and IP3 -receptor, but not protein kinase C. Increase in [Ca2+ ]i was blocked by the PLD inhibitor, but not the PLA2 inhibitor. Secretion was blocked by PLA2 inhibitor, but not the PLD inhibitor. An inhibitor of the epidermal growth factor receptor blocked the increase in [Ca2+ ]i by RvD2 in both species of CGCs. In CGCs RvD2 activates multiple intracellular signaling pathways that are Ca2+ -dependent, along with one Ca2+ -independent and one cAMP/protein kinase A-dependent pathway. Activation of these pathways stimulate mucin secretion from rat and human CGCs into the tear film contributing to ocular surface homeostasis and health.

Resolvin D2 and Resolvin D1 Differentially Activate Protein Kinases to Counter-Regulate Histamine-Induced [Ca2+]i Increase and Mucin Secretion in Conjunctival Goblet Cells.

Resolvin (Rv) D2 and RvD1 are biosynthesized from docosahexaenoic acid (DHA) and promote resolution of inflammation in multiple organs and tissues, including the conjunctiva. Histamine is a mediator produced by mast cells in the conjunctiva during the allergic response. We determined the interaction of RvD2 with histamine and its receptor subtypes in cultured conjunctival goblet cells and compared them with RvD1 by measuring intracellular [Ca2+] and mucous secretion. Treatment with RvD2 significantly blocked the histamine-induced [Ca2+]i increase as well as secretion. RvD2 and RvD1 counter-regulate different histamine receptor subtypes. RvD2 inhibited the increase in [Ca2+]i induced by the activation of H1, H3, or H4 receptors, whereas RvD1 inhibited H1 and H3 receptors. RvD2 and RvD1 also activate distinct receptor-specific protein kinases to counter-regulate the histamine receptors, probably by phosphorylation. Thus, our data suggest that the counter-regulation of H receptor subtypes by RvD2 and RvD1 to inhibit mucin secretion are separately regulated.

Resolvin D2 elevates cAMP to increase intracellular [Ca2+] and stimulate secretion from conjunctival goblet cells.

Under physiologic conditions, conjunctival goblet cells (CGCs) secrete mucins into the tear film to preserve ocular surface homeostasis. Specialized proresolving mediators (SPMs), like resolvins (Rvs), regulate secretion from CGCs and actively terminate inflammation. The purpose of this study was to determine if RvD2 stimulated mucin secretion and to investigate the cellular signaling components. Goblet cells were cultured from rat conjunctiva. Secretion was measured by an enzyme-linked lectin assay, change in intracellular [Ca2+] ([Ca2+]i) using Fura-2, and cellular cAMP levels by ELISA. RvD2 (10-11-10-8 M) stimulated secretion, increased cellular cAMP levels and the [Ca2+]i. RvD2-stimulated increase in [Ca2+]i and secretion was blocked by Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis and the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride but not by the cAMP exchange protein inhibitor α-[2-(3-chlorophenyl)hydrazinylidene]-5-(1,1-dimethylethyl)-b-oxo-3-isoxazolepropanenitrile. Forskolin, 3-isobutyl-1-methylxanthine, and 8-bromo-cAMP (8-Br-cAMP) increased [Ca2+]i. Increasing cAMP with 8-Br-cAMP inhibited the increase in [Ca2+]i stimulated by the cAMP-independent agonist cholinergic agonist carbachol. In conclusion, RvD2 uses both cellular cAMP and [Ca2+]i to stimulate glycoconjugate secretion from CGCs, but the interaction of cAMP and [Ca2+]i is context dependent. Thus RvD2 likely assists in the maintenance of the mucous layer of the tear film to sustain ocular surface homeostasis and has potential as a novel treatment for dry eye disease.-Botten, N., Hodges, R. R., Li, D., Bair, J. A., Shatos, M. A., Utheim, T. P., Serhan, C. N., Dartt, D. A. Resolvin D2 elevates cAMP to increase intracellular [Ca2+] and stimulate secretion from conjunctival goblet cells.

Resolvin D1 treatment on goblet cell mucin and immune responses in the chronic allergic eye disease (AED) model.

Severe, chronic eye allergy is an understudied, vision-threatening condition. Treatments remain limited. We used a mouse model of severe allergic eye disease (AED) to determine whether topical application of the pro-resolution mediator Resolvin D1 (RvD1) terminates the response. AED was induced by injection of ovalbumin (OVA) followed by topical challenge of OVA daily. RvD1 was applied topically prior to OVA. Clinical symptoms were scored. Eye washes were assayed for MUC5AC. After 7 days, eyes were removed and the number of goblet cells, T helper cell responses and presence of immune cells in draining lymph nodes and conjunctiva determined. Topical RvD1 treatment significantly reduced symptoms of AED. RvD1 did not alter the systemic type 2 immune response in the lymph nodes. AED increased the total amount of goblet cell mucin secretion, but not the number of goblet cells. RvD1 prevented this increase, but did not alter goblet cell number. Absolute numbers of CD4 + T cells, total CD11b + myeloid cells, eosinophils, neutrophils, and monocytes, but not macrophages increased in AED versus RvD1-treated mice. We conclude that topical application of RvD1 reduced the ocular allergic response by local actions in conjunctival immune response and a decrease in goblet cell mucin secretion.

Resolvin D1, but not resolvin E1, transactivates the epidermal growth factor receptor to increase intracellular calcium and glycoconjugate secretion in rat and human conjunctival goblet cells.

PURPOSE: To identify interactions of the epidermal growth factor receptor (EGFR) with the pro-resolving mediator receptors for RvD1 and RvE1 to stimulate an increase in intracellular [Ca2+] ([Ca2+]i) and mucin secretion from cultured human and rat conjunctival goblet cells. METHODS: Goblet cells from human and rat conjunctiva were grown in culture using RPMI media. Cultured goblet cells were pre-incubated with inhibitors, and then stimulated with RvD1, RvE1, EGF or the cholinergic agonist carbachol (Cch). Increase in [Ca2+]i was measured using fura-2/AM. Goblet cell secretion was measured using an enzyme-linked lectin assay with UEA-1. Western blot analysis was performed with antibodies against AKT and ERK 1/2. RESULTS: In cultured human conjunctival goblet cells RvE1 -stimulated an increase in [Ca2+]i. RvD1-, but not the RvE1-, stimulated increase in [Ca2+]i and mucin secretion was blocked by the EGFR inhibitor AG1478 and siRNA for the EGFR. RvD1-, but not RvE1-stimulated an increase in [Ca2+]i that was also inhibited by TAPI-1, an inhibitor of the matrix metalloprotease ADAM 17. Inhibition of the EGFR also blocked RvD1-stimulated increase in AKT activity and both RvD1-and RvE1-stimulated increase in ERK 1/2 activity. Pretreatment with either RvD1 or RvE1 did not block the EGFR-stimulated increase in [Ca2+]i. CONCLUSIONS: We conclude that in cultured rat and human conjunctival goblet cells, RvD1 activates the EGFR, increases [Ca2+]i, activates AKT and ERK1/2 to stimulate mucin secretion. RvE1 does not transactivate the EGFR to increase [Ca2+]I and stimulate mucin secretion, but does interact with the receptor to increase ERK 1/2 activity.