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Resolvin D1 Increases Mucin Secretion in Cultured Rat Conjunctival Goblet Cells via Multiple Signaling Pathways

Purpose Goblet cells in the conjunctiva secrete mucin into the tear film protecting the ocular surface. The proresolution mediator resolvin D1 (RvD1) regulates mucin secretion to maintain homeostasis during physiological conditions and in addition, actively terminates inflammation. We determined the signaling mechanisms used by RvD1 in cultured rat conjunctival goblet cells to increase intracellular [Ca2+] ([Ca2+]i) and induce glycoconjugate secretion. Methods Increase in [Ca2+]i were measured using fura 2/AM and glycoconjugate secretion determined using an enzyme-linked lectin assay with the lectin Ulex Europaeus Agglutinin 1. Signaling pathways activated by RvD1 were studied after goblet cells were pretreated with signaling pathway inhibitors before stimulation with RvD1. The results were compared with results when goblet cells were stimulated with RvD1 alone and percent inhibition calculated. Results The increase in [Ca2+]i stimulated by RvD1 was blocked by inhibitors to phospholipases (PL-) -D, -C, -A2, protein kinase C (PKC), extracellular signal-regulated kinases (ERK)1/2 and Ca2+/calmodulin-dependent kinase (Ca2+/CamK). Glycoconjugate secretion was significantly inhibited by PLD, -C, -A2, ERK1/2 and Ca2+/CamK, but not PKC. Conclusions We conclude that RvD1 increases glycoconjugate secretion from goblet cells via multiple signaling pathways including PLC, PLD, and PLA2, as well as their signaling components ERK1/2 and Ca2+/CamK to preserve the mucous layer and maintain homeostasis by protecting the eye from desiccating stress, allergens, and pathogens.

T he conjunctiva is a mucous membrane that covers the sclera and the inner surface of the eyelid. A major cell type in the conjunctival epithelium is the goblet cell, which produces mucins, especially the mucin MUC5AC. 1,2 Mucin glycoconjugates produced from the corneal epithelia and conjunctival goblet cells form the inner mucus layer of the tear film. The tear film plays a crucial role in innate immune defense of the eye creating a barrier between the environment and the ocular surface. [3][4][5][6] However, the production of mucin from conjunctival goblet cells may be altered in inflammatory diseases, such as dry eye disease, allergic conjunctivitis, and bacterial and viral conjunctivitis. [1][2][3] Inflammation can be actively terminated by proresolution mediators. 7,8 One of the proresolution mediators is resolvin D1 (RvD1), which is biosynthesized from the x-3-fatty acid, docosahexaenoic acid. Resolvins are biosynthesized during inflammation when there is a class-switch of enzymes, decreasing prostaglandin and leukotriene production, and enhancing biosynthesis of specialized proresolving mediators. 8 In experimental systems, RvD1 limits neutrophil infiltration, stimulates macrophage phagocytosis, and reduces the level of inflammatory pain and fibrosis. 9 Not only do the proresolution mediators function during disease, but they are also active during normal, noninflamed conditions. RvD1 is present in human peripheral blood, lymphoid tissue, urine, and tears under physiological conditions. [10][11][12] In addition, RvD1 maintains homeostasis in bone marrow by enhancing macrophage removal of apoptotic osteoblasts. 13 We previously found that another proresolving mediator, lipoxin A 4 (LXA 4 ), is an active mediator of mucin secretion in cultured rat conjunctival goblet cells 14 and could function to maintain homeostasis in the conjunctiva.
In the eye, RvD1 functions during inflammation by blocking glycoconjugate secretion stimulated by the proinflammatory mediators leukotriene D4 and histamine in cultured human and rat conjunctival goblet cells. 15,16 RvD1 binds to the G protein-coupled receptors ALX/FPR2 (mouse and human) and human DRV1 (identified earlier as GPR32). [17][18][19] Both receptors are present in human conjunctival goblet cells while ALX/FPR2 has been found in rat conjunctival goblet cells. 14 Under noninflamed conditions, activation of these receptors leads to an increase in intracellular [Ca 2þ ] ([Ca 2þ ] i ), extracellular regulated kinase (ERK) 1/2 activation, and stimulation of glycoconjugate secretion from cultured conjunctival goblet cells. 16 In the present study, three signaling pathways, known to be activated by ALX/FPR2, were investigated with resolvin D1 as ligand: phospholipase C (PLC), phospholipase D (PLD), and phospholipase A 2 (PLA 2 ). 20 PLC breaks down phosphatidylinositol 4,5 bisphosphate (PIP 2 ), producing inositol triphosphate (IP 3 ) and diacylglycerol (DAG). IP 3 binds to the IP 3 receptor on the intracellular calcium store, the endoplasmic reticulum, opening a ligand-gated Ca 2þ channel, increasing [Ca 2þ ] i . 21 Ca 2þ and DAG together activate protein kinase C (PKC). Using inhibitors to these different pathways, we found that all three of the phospholipases were activated by RvD1 in rat conjunctival goblet cells and that they activate the downstream signaling molecules ERK1/2 and Ca 2þ /calmodulin-dependent kinase (Ca 2þ /CamK) to increase [Ca 2þ ] i and stimulate mucin secretion.

METHODS AND MATERIALS
RPMI-1640 cell culture medium, penicillin/streptomycin, and L-glutamine were purchased from Lonza (Walkerville, IL, USA). Fetal bovine serum (FBS) was from Atlanta Biologicals (Norcross, GA, USA). Fura-2/AM and Amplex Red were from ThermoFisher (Waltham, MA, USA). Inhibitors and suppliers are listed in the Table. Resolvin

Cell Culture
Goblet cells from rats were grown in organ culture as described previously. 6,15,[22][23][24] The conjunctival explants were grown in 6-well plates containing RPMI 1640 medium supplemented with 10% FBS, 2-mM glutamine, and 100-lg/ mL penicillin-streptomycin. The explant was removed before trypsinization of the cells for first passage. First passage goblet cells were seeded in either glass bottom petri dishes (for Ca 2þ measurements) or 24 well plates (for mucin secretion measurements) were used in all experiments. Cultured cells were identified by evaluating staining with the lectin UEA-1, which detects goblet cell secretory product, and the antibody to cytokeratin 7, which detects goblet cell bodies, to ensure that goblet cells predominated. 6,15,16,[22][23][24][25]

Measurement of [Ca 2þ ] i
Cultured rat conjunctival goblet cells were plated onto 35-mm glass bottom culture dish and incubated overnight. Cells were then incubated for 1 hour at 378C with KRB-HEPES plus 0.5% bovine serum albumin containing 0.5-lM fura-2/AM (Invitrogen, Grand Island, NY, USA), 8-lM pluronic acid F127 (Sigma-Aldrich, St. Louis, MO, USA) and 250 lM sulfinpyrazone (Sigma-Aldrich) followed by washing in KRB-HEPES containing sulfinpyrazone. Calcium measurements were made with a ratio imaging system (In Cyt Im2; Intracellular Imaging, Cincinnati, OH, USA) using wavelengths of 340 and 380 nm and an emission wavelength of 505 nm. 26 At least five cells were selected in each experimental condition, and the experiments were repeated in at least three separate animals. The goblet cells were incubated with inhibitors before RvD1 (10 À8 M) was added. The cholinergic agonist carbachol (Cch, 10 À4 M) was used as a positive control. The inhibitors and the pathways they affect are listed in the Table. BOC-2, BAPTA, autocamtide-2-related inhibitory peptide (AIP), and U0126 were added 30 minutes prior to RvD1-or Cch-stimulation. U73122, the negative control U73343, 1-butanol (1-but), and its negative control t-butanol (t-but) were added 15 minutes before RvD1or Cch-stimulation. 2-Aminoethyl diphenylborate (2-APB) and Ro 31-8220 were added 10 minutes in advance of RvD1-or Cch-stimulation. Aristolochic acid (aris) was added 10 minutes prior to stimulation. Inhibitors were initially dissolved in DMSO and diluted to working concentrations in KRB-HEPES. We have

Glycoconjugate Secretion
Cultured rat conjunctival goblet cells were trypsinized, passaged in 24-well plates, and grown to approximately 75% confluence. The cells were serum starved in serum free RPMI 1640 containing 0.5% BSA for 2 hours before they were preincubated for 30 minutes with the inhibitors. Inhibitors were initially dissolved in DMSO and diluted to working concentrations in RPMI 1640 medium. We have previously shown that DMSO, at the concentrations used, did not have any effect on basal secretion. 23 Thereafter, the cells were incubated with RvD1 (10 À8 M) for 2 hours. To determine the amount of goblet cell glycoconjugate secretion, the lectin horseradish peroxidase-conjugated Ulex Europaeus Agglutinin I (UEA-1), which binds to glycoproteins secreted from goblet cells was used in an enzyme-linked lectin assay. The standards and supernatant were spotted onto Nunc microplates and dried overnight at 608C. The UEA-I was then detected using Amplex Red, which when oxidized by peroxidase in the presence of hydrogen peroxide produces a highly fluorescent molecule. Quantification of fluorescence was done using a fluorescence ELISA reader (model FL600; Bio-Tek, Winooski, VT, USA) with an excitation wavelength of 530 nm and an emission wavelength of 590 nm. The cells were scraped, and the cell homogenate analyzed for the total amount of protein using the Bradford protein assay. Secretion was normalized to the amount of protein in each well and expressed as fold increase above basal. Please note that experiments with Ro31-8220, 1-butanol (1-but), tertiary butanol (t-butanol), and aris were performed in the same experiments.

Statistical Analysis
The data are presented as fold-increase above basal as average 6 SEM. Student's t-test was used to perform statistical analysis and P less than 0.05 was set as statistically significant.

ALX/FPR2 Receptors Are Activated by RvD1 in Rat Conjunctival Goblet Cells
RvD1 can activate two receptors in human cells, ALX/FPR2 and DRV1. An earlier study, where the ALX/FPR2 receptor was knocked down with siRNA in cultured rat conjunctival goblet cells, showed that RvD1 uses the ALX/FPR2 receptor to increase [Ca 2þ ] i . 16 To support these findings and further investigate RvD1 intracellular signals, first passage goblet cells cultured from rat conjunctiva were preincubated with the ALX/FPR2 receptor antagonist BOC-2 (10 À4 M) for 30 minutes prior to addition of RvD1 (10 À8 M) and [Ca 2þ ] i levels over time were measured (Figs. 1A, 1B). RvD1 by itself increased [Ca 2þ ] i by maximum 425.3 6 77.4 nM (P ¼ 0.003). Inhibition of the ALX/FPR2 receptor gave a significant decrease in RvD1stimulated [Ca 2þ ] i increase, giving a 71.4 6 7.2% inhibition (P ¼ 0.2 3 10 À6 ) (Fig. 1C). Thus, RvD1 exerts its actions primarily via the ALX/FPR2.
Our studies indicate that RvD1 leads to phosphorylation and activation of ERK1/2 that increases [Ca 2þ ] i in rat conjunctival goblet cells and stimulates secretion of glycoconjugates into the tear film.

DISCUSSION
Herein, we report that RvD1 activates ALX/FPR2 in rat conjunctival goblet cells by stimulating PLC, PLD, and PLA 2 (Fig. 10) . PLC via IP 3 production activates the IP 3 receptor to release intracellular Ca 2þ . In turn activation of PLC also produces diacylglycerol (DAG) that activates PKC and PKC increases [Ca 2þ ] i . Stimulation of PLD and PLA 2 likewise increases [Ca 2þ ] i . As activation of ERK1/2 and Ca 2þ /CamK elevate [Ca 2þ ] i ERK1/2 and Ca 2þ /CamK could be in the signaling pathways activated PLC, PLD, or PLA 2 . Glycoconjugate secretion is dependent upon Ca 2þ as chelation of Ca 2þ with BAPTA blocked RvD1-stimulated glycoconjugate secretion. This is similar to LXA 4 -stimulated secretion, which was also blocked by BAPTA. Use of these pathways to regulate [Ca 2þ ] i , and stimulate secretion strengthens our previous findings that resolvins and the proresolution mediator LXA 4 preserve ocular surface homeostasis during noninflamed, normal physiological processes in conjunctival goblet cells by stimulating mucin secretion. 15,16,23 In human tissues including goblet cells, RvD1 is known to bind to two receptors, DRV1 and ALX/FPR2. 17 In the present study, inhibition of ALX/FPR2 blocked RvD1-stimulated increase in [Ca 2þ ] i by over 70%. This indicates that the majority of actions stimulated by RvD1 are mediated thorough ALX/ FPR2. In contrast to rat goblet cells, in goblet cells cultured from human conjunctiva, RvD1-stimulated increase in [Ca 2þ ] i was not altered by inhibition of ALX/FPR2. 24 Additionally, RvD1 and LXA 4 , another proresolution mediator that also binds to the ALX/FPR2 receptor, desensitize each other when added sequentially in rat conjunctival goblet cells whereas in human goblet cells, RvD1 does not desensitize the LXA 4 response. 14,24 These data support the hypothesis that in human cells RvD1 preferentially binds to the DRV1 receptor, while in rats RvD1 preferentially acts via ALX/FPR2 receptor to increase [Ca 2þ ] i and stimulate glycoconjugate secretion. Given the important role of ALX/FPR2 in rat compared human goblet cells stimulated by RvD1, the role of rat DRV1 in goblet cells is likely limited.
In the conjunctiva, glycoconjugate secretion was measured 2 hours after addition of RvD1 while Ca 2þ is measured within minutes after addition. Glycoconjugate secretion is the end result of the activation of numerous signal transduction pathways, movement of granules, and fusion of membranes, 31 whereas the release of Ca 2þ is one of the earliest events that occur after agonist addition. Glycoconjugate release is directly linked to the increase in [Ca 2þ ] i as chelation of Ca 2þ blocks it. While it is well-established that MUC5AC, the primary glycoconjugate present in conjunctival goblet cells is stored in granules and secreted upon appropriate stimuli, [32][33][34][35] it is possible that the release of glycoconjugates could be due to protein synthesis and discharged in an unregulated manner. The rate of MUC5AC synthesis in conjunctival goblet cells has not been determined, the amount of MUC5AC mRNA in response to hypoxia in nasal epithelial cells was not significantly increased for 6 hours, 36 and in mouse stomach, newly synthesized MUC5AC did not appear at the cell surface for 5 hours. 37 In addition, in previous studies release of gylcoconjugates stimulated by RvD1 and other stimuli occurs in a time-dependent manner, which were blocked by receptor antagonists. 15,16,23,38 Therefore, it is unlikely that in the 2hour time point at which release of glycoconjugates was measured in this study, new protein synthesis plays a significant role. The time frame required to detect the glycoconjugates most likely reflect the sensitivity of this method versus that of the high sensitivity of the Ca 2þ fluorescent dye Fura 2.
The signaling pathways activated upon binding of RvD1 to the ALX/FPR2 receptor are similar to the pathways stimulated by LXA 4 when it interacts with the ALX/FPR2 receptor. 17,39 Both proresolution mediators activate the same signaling pathways to induce glycoconjugate secretion. RvD1 and LXA 4 have also shown the same effects in disease. 40,41 For example, in a mouse animal model, RvD1 and the LXA 4 analog, aspirin-triggered LXA 4 , both significantly reduced neovascularization in inflamed cornea. 41 In the present study, conjunctival goblet cells from rats were used for all the experiments. As RvD1 appears to bind to DRV1 preferentially over ALX/FPR2 in human cells, a comparison between signaling pathways activated by RvD1 in human compared with rat goblet cells could be warranted. 17,18 On the other hand, it is plausible that RvD1 activates similar pathways in rat and human goblet cells, as the same receptors are found in both species and RvD1 stimulates an increase in [Ca 2þ ] i and glycoconjugate secretion in both human and rat conjunctival goblet cells. 16 In addition, RvD1 blocks LTD 4 -and histamine-stimulated mucin secretion in goblet cells of both species. 15,16 In order to examine whether RvD1 activates a PLC pathway in goblet cells, we used the PLC inhibitor U73122, which gave a significant blockage of RvD1-stimulated [Ca 2þ ] i increase and mucin secretion. However, the inactive analog of U73144, also decreased RvD1-stimulated [Ca 2þ ] i increase. This could indicate that the decrease in [Ca 2þ ] i levels with U73122 was not FIGURE 10. Schematic diagram of signaling pathways of RvD1 in conjunctival goblet cells. RvD1 binds to the ALX/FPR2 receptor and activates the signaling pathways of PLA 2 , PLC, and PLD. PLA 2 and PLD are believed to activate ERK 1/2 to increase [Ca 2þ ] i and Ca 2þ /CamK. PLC increases IP 3 and DAG. IP 3 releases Ca 2þ while DAG activates PKC. Activation of these pathways leads to mucin secretion. due to PLC inhibition. However, U73144 did not affect either the RvD1-stimulated mucin secretin, Cch-stimulated increase in [Ca 2þ ] i or Cch-stimulated glycoconjugate secretion. In addition, 2-APB, the IP 3 receptor inhibitor, significantly decreased RvD1stimulated [Ca 2þ ] i increase and glycoconjugate secretion, implying that RvD1 does indeed activate a PLC pathway in rat conjunctival goblet cells.
We found that RvD1 stimulated mucin secretion through an increase in intracellular Ca 2þ via multiple signaling pathways. This illustrates the importance of RvD1 in stimulating mucin secretion, including MUC5AC. MUC5AC is a gel-forming protein and is believed to be involved in the clearance of allergens, pathogens, and debris to protect the ocular surface and maintain a clear vision. Lack of sufficient MUC5AC secretion, as seen in atopic keratoconjunctivitis, can lead to corneal ulcers and decreased lubrication. 42 Decreased levels of MUC5AC is also seen in dry eye disease. [43][44][45][46] Treatment with RvD1 has been studied in several animal models, and shows promising results regarding resolution of inflammation. 41,[47][48][49][50][51][52][53][54] In addition, resolvins decrease the level of pain, [55][56][57][58] and lower the need for antibiotics, 59 which may be a significant benefit, considering the spread of antibiotic resistance. In contrast to glucocorticoids, used as topical treatment in ocular inflammatory diseases, resolvins are not immunosuppressive.
In the eye, ALX/FPR2 receptors are synthesized by uninjured corneal epithelium 60 and play a role in corneal wound healing in mice. 61 We recently demonstrated that several proresolving mediators including RvD1 are present in human emotional tears from males but not females. 12 Interestingly, Gao et al. 62 demonstrated that the ALX/FPR2 receptors play a role in autoimmune dry eye that occurs more prominently in females than males.
In conclusion, RvD1 functions under noninflamed conditions to maintain homeostasis of the ocular surface by regulating mucin secretion through activation of ALX/FPR2 and perhaps the DRV1 receptors. Activation of these receptors leads to an increase in [Ca 2þ ] i and stimulation of secretion through stimulation of phospholipases -D, -C, and -A 2 , as well as the signaling intermediates ERK1/2 and Ca 2þ /CamK; thus, maintaining the mucin layer of the tear film that is critical for ocular surface health and disease prevention.