in-vitro-method-to-study-sex-based-differences-conjunctival-goblet

<em>In Vitro</em> Method to Study Sex-Based Differences in Conjunctival Goblet Cells

Dry eye is a multi-factorial disease affecting ocular surface health, with a profoundly higher prevalence in women. Disruption of the gel-forming mucin ...

Harvard Medical School

The authors determined the role of the protein kinase C (PKC) isoforms cPKCalpha and nPKCepsilon in EGF-stimulated proliferation of cultured rat and human conjunctival goblet cells.

Role of cPKCalpha and nPKCepsilon in EGF-stimulated goblet cell proliferation.

To determine whether a constitutively active protein kinase C (PKC)-alpha stimulates rat and human conjunctival goblet cell proliferation through activation of ERK 1/2.

Stimulatory role of PKCalpha in extracellular regulated kinase 1/2 pathway in conjunctival goblet cell proliferation.

The purpose of this study was to identify the signaling pathways that epidermal growth factor (EGF) uses to stimulate mucin secretion from cultured rat conjunctival goblet cells and to compare the pathways used by EGF with those used by the known secretagogue muscarinic, cholinergic agonists. To this end, goblet cells from rat conjunctiva were grown in culture using RPMI media. For immunofluorescence experiments, antibodies against EGF receptor (EGFR) and ERK 2 as well as muscarinic receptors (M(1)AchR, M(2)AchR, and M(3)AchR) were used, and the cells viewed by fluorescence microscopy. Intracellular [Ca(2+)] ([Ca(2+)](i)) was measured using fura 2/AM. Glycoconjugate secretion was determined after cultured goblet cells were preincubated with inhibitors, and then stimulated with EGF or the cholinergic agonist carbachol (Cch). Goblet cell secretion was measured using an enzyme-linked lectin assay with UEA-I or ELISA for MUC5AC. In cultured goblet cells EGF stimulated an increase in [Ca(2+)](i) in a concentration-dependent manner. EGF-stimulated increase in [Ca(2+)](i) was blocked by inhibitors of the EGF receptor and removal of extracellular Ca(2+). Inhibitors against the EGFR and ERK 1/2 blocked EGF-stimulated mucin secretion. In addition, cultured goblet cells expressed M(1)AchR, M(2)AchR, and M(3)AchRs. Cch-stimulated increase in [Ca(2+)](i) was blocked by inhibitors for the M(1)AchRs, matrix metalloproteinases, and EGF receptors. Inhibitors against the EGF receptor and ERK 1/2 also blocked Cch-stimulated mucin secretion. We conclude that in conjunctival goblet cells, EGF itself increases [Ca(2+)](i) and activates ERK 1/2 to stimulate mucin secretion. EGF-stimulated secretion is dependent on extracellular Ca(2+). This mechanism of action is similar to cholinergic agonists that use muscarinic receptors to transactivate the EGF receptor, increase [Ca(2+)](i), and activate ERK 1/2 leading to an increase in mucin secretion.

Signaling pathways used by EGF to stimulate conjunctival goblet cell secretion.

Conjunctival goblet cells play a major role in maintaining the mucus layer of the tear film under physiological conditions as well as in inflammatory diseases like dry eye and allergic conjunctivitis. Resolution of inflammation is mediated by proresolution agonists such as lipoxin A (LXA) that can also function under physiological conditions. The purpose of this study was to determine the actions of LXA on cultured rat conjunctival goblet cell mucin secretion, intracellular [Ca] ([Ca]), and identify signaling pathways activated by LXA. ALX/FPR2 (formyl peptide receptor2) was localized to goblet cells in rat conjunctiva and in cultured goblet cells. LXA significantly increased mucin secretion, [Ca], and extracellular regulated kinase 1/2 (ERK 1/2) activation. These functions were inhibited by ALX/FPR2 inhibitors. Stable analogs of LXA increased [Ca] to the same extent as LXA. Sequential addition of either LXA or resolvin D1 followed by the second compound decreased [Ca] of the second compound compared with its initial response. LXA activated phospholipases C, D, and A and downstream molecules protein kinase C, ERK 1/2, and Ca/calmodulin-dependent kinase to increase mucin secretion and [Ca]. We conclude that conjunctival goblet cells respond to LXA to maintain the homeostasis of the ocular surface and could be a novel treatment for dry eye diseases.

Lipoxin A activates ALX/FPR2 receptor to regulate conjunctival goblet cell secretion.

Thrombospondin-1-deficient (TSP-1) mice are used as an animal model of Sjögren's Syndrome because they exhibit many of the symptoms associated with the autoimmune type of dry eye found in primary Sjögren's Syndrome. This type of dry eye is linked to the inflammation of the lacrimal gland, conjunctiva, and cornea, and is thought to involve dysfunction of the complex neuronal reflex arc that mediates tear production in response to noxious stimuli on the ocular surface. This study characterizes the structural and functional changes to the corneal nerves that are the afferent arm of this arc in young and older TSP-1 and wild type (WT) mice. The structure and subtype of nerves were characterized by immunohistochemistry, in vivo confocal microscopy, and confocal microscopy. Cytokine expression analysis was determined by Q-PCR and the number of monocytes was measured by immunohistochemistry. We found that only the pro-inflammatory cytokine MIP-2 increased in young corneas of TSP-1 compared to WT mice, but tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-2 (MIP-2) all increased in older TSP-1 mouse corneas. In contrast, CD11b+ pro-inflammatory monocytes did not increase even in older mouse corneas. Calcitonin gene-related peptide (CGRP)-, but not Substance P (SubP)-containing corneal nerves decreased in older, but not younger TSP-1 compared to WT mouse corneas. We conclude that CGRP-containing corneal sensory nerves exhibit distinct structural deficiencies as disease progresses in TSP-1 mice, suggesting that: (1) TSP-1 is needed for the development or repair of these nerves and (2) impaired afferent corneal nerve structure and hence function may contribute to ocular surface dysfunction that develops as TSP-1 mice age.

Thrombospondin-1 Is Necessary for the Development and Repair of Corneal Nerves.

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 [Ca] ([Ca]) using Fura-2, and cellular cAMP levels by ELISA. RvD2 (10-10 M) stimulated secretion, increased cellular cAMP levels and the [Ca]. RvD2-stimulated increase in [Ca] and secretion was blocked by Ca chelator 1,2-bis(2-aminophenoxy)ethane-,,','-tetraacetic acid tetrakis and the PKA inhibitor -[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 [Ca]. Increasing cAMP with 8-Br-cAMP inhibited the increase in [Ca] stimulated by the cAMP-independent agonist cholinergic agonist carbachol. In conclusion, RvD2 uses both cellular cAMP and [Ca] to stimulate glycoconjugate secretion from CGCs, but the interaction of cAMP and [Ca] 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 [Ca] and stimulate secretion from conjunctival goblet cells.

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

Mucin secretion from conjunctival goblet cells forms the tear film mucin layer and requires regulation to function properly. Maresin 1 (MaR1) is a specialized proresolving mediator produced during the resolution of inflammation. We determined if MaR1 stimulates mucin secretion and signaling pathways used. Cultured rat conjunctival goblet cells were used to measure the increase in intracellular Ca ([Ca ] ) concentration and mucin secretion. MaR1-increased [Ca ] and secretion were blocked by inhibitors of phospholipase C, protein kinase C, Ca /calmodulin-dependent protein kinase II, and extracellular-regulated kinase 1/2. MaR1 added before addition of histamine counterregulated histamine-stimulated increase in [Ca ] and secretion. We conclude that MaR1 likely has two actions in conjunctival goblet cells: first, maintaining optimal tear film mucin levels by increasing [Ca ] and stimulating mucin secretion in health and, second, attenuating the increase in [Ca ] and overproduction of mucin secretion by counterregulating the effect of histamine as occurs in ocular allergy.

Maresin 1, a specialized proresolving mediator, stimulates intracellular [Ca ] and secretion in conjunctival goblet cells.

Leukotriene B4 (LTB4) is a major proinflammatory mediator important in host defense, whereas resolvins (Rvs) are produced during the resolution phase of inflammation. The authors determined the actions of both RvE1 and RvD1 on LTB4-induced responses of goblet cells cultured from rat conjunctiva. The responses measured were an increase in the intracellular [Ca] ([Ca]) and high-molecular-weight glycoprotein secretion. Treatment with RvE1 or RvD1 for 30 minutes significantly blocked the LTB4-induced [Ca] increase. The actions of RvE1 on LTB4-induced [Ca] increase were reversed by siRNA for the RvE1 receptor, and the actions of RvD1 were reversed by an RvD1 receptor inhibitor. The RvE1 and RvD1 block of LTB4-stimulated increase in [Ca] was also reversed by an inhibitory peptide to β-adrenergic receptor kinase. LTB4 and block of the LTB4-stimulated increase in [Ca] by RvE1 and RvD1 were partially mediated by the depletion of intracellular Ca stores. RvE1, but not RvD1, counterregulated the LTB4-induced high-molecular-weight glycoprotein secretion. Thus, both RvE1 and RvD1 receptors directly inhibit LTB4 by phosphorylating the LTB4 receptor using β adrenergic receptor kinase. RvE1 receptor counterregulates the LTB4-induced increase in [Ca] and secretion, whereas RvD1 receptor only counterregulates LTB4-induced [Ca] increase.

Resolvin E1 Reduces Leukotriene B4-Induced Intracellular Calcium Increase and Mucin Secretion in Rat Conjunctival Goblet Cells.

To determine the effect of povidone iodine (PI), an antiseptic commonly used prior to ocular surgery, on viability of mixed populations of conjunctival stratified squamous and goblet cells, purified conjunctival goblet cells and purified conjunctival stromal fibroblasts in primary culture.

Povidone iodine treatment is deleterious to human ocular surface conjunctival cells in culture.

The amount of mucin secreted by conjunctival goblet cells is regulated to ensure the optimal level for protection of the ocular surface. Under physiological conditions lipid specialized pro-resolving mediators (SPM) are essential for maintaining tissue homeostasis including the conjunctiva. The protein Annexin A1 (AnxA1) can act as an SPM. We used cultured rat conjunctival goblet cells to determine if AnxA1 stimulates an increase in intracellular [Ca] ([Ca]) and mucin secretion and to identify the signaling pathways. The increase in [Ca] was determined using fura2/AM and mucin secretion was measured using an enzyme-linked lectin assay. AnxA1 stimulated an increase in [Ca] and mucin secretion that was blocked by the cell-permeant Ca chelator BAPTA/AM and the ALX/FPR2 receptor inhibitor BOC2. AnxA1 increased [Ca] to a similar extent as the SPMs lipoxin A and Resolvin (Rv) D1 and histamine. The AnxA1 increase in [Ca] and mucin secretion were inhibited by blocking the phospholipase C (PLC) pathway including PLC, the IP receptor, the Ca/ATPase that causes the intracellular Ca stores to empty, and blockade of Ca influx. Inhibition of protein kinase C (PKC) and Ca/calmodulin-dependent protein kinase also decreased the AnxA1-stimulated increase in [Ca] and mucin secretion. In contrast inhibitors of ERK 1/2, phospholipase A (PLA), and phospholipase D (PLD) did not alter AnxA1-stimulated increase in [Ca], but did inhibit mucin secretion. Activation of protein kinase A did not decrease either the AnxA1-stimulated rise in [Ca] or secretion. We conclude that in health, AnxA1 contributes to the mucin layer of the tear film and ocular surface homeostasis by activating the PLC signaling pathway to increase [Ca] and stimulate mucin secretion and ERK1/2, PLA, and PLD to stimulate mucin secretion from conjunctival goblet cells.

Pro-Resolving Mediator Annexin A1 Regulates Intracellular Ca and Mucin Secretion in Cultured Goblet Cells Suggesting a New Use in Inflammatory Conjunctival Diseases.

Specialized pro-resolving mediators (SPMs), including Maresins (MaR)-1 and 2, contribute to tear film homeostasis and resolve conjunctival inflammation. We investigated MaR2's signaling pathways in goblet cells (GC) from rat conjunctiva. Agonist-induced [Ca] and high-molecular weight glycoconjugate secretion were measured. MaR2 increased [Ca] and stimulated secretion. MaR2 and MaR1 stimulate conjunctival goblet cell function, especially secretion, by activating different but overlapping GPCR and signaling pathways, and furthermore counter-regulate histamine stimulated increase in [Ca]. Thus, MaR2 and MaR1 play a role in maintaining the ocular surface and tear film homeostasis in health and disease. As MaR2 and MaR1 modulate conjunctival goblet cell function, they each may have potential as novel, but differing, options for the treatment of ocular surface inflammatory diseases including allergic conjunctivitis and dry eye disease. We conclude that in conjunctival GC MaR2 and MaR1, both increase the [Ca] and stimulate secretion to maintain homeostasis by using one set of different, but overlapping, signaling pathways to increase [Ca] and another set to stimulate secretion. MaR2 also resolves ocular allergy.

Article	Year
Role of cPKCalpha and nPKCepsilon in EGF-stimulated goblet cell proliferation. Investigative ophthalmology & visual science\| PubMed ID: 18824739	2009
Stimulatory role of PKCalpha in extracellular regulated kinase 1/2 pathway in conjunctival goblet cell proliferation. Investigative ophthalmology & visual science\| PubMed ID: 19074803	2009
Signaling pathways used by EGF to stimulate conjunctival goblet cell secretion. Experimental eye research\| PubMed ID: 22975404	2012
Lipoxin A activates ALX/FPR2 receptor to regulate conjunctival goblet cell secretion. Mucosal immunology\| PubMed ID: 27072607	2017
Thrombospondin-1 Is Necessary for the Development and Repair of Corneal Nerves. International journal of molecular sciences\| PubMed ID: 30332778	2018
Resolvin D2 elevates cAMP to increase intracellular [Ca] and stimulate secretion from conjunctival goblet cells. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 31013438	2019
Maresin 1, a specialized proresolving mediator, stimulates intracellular [Ca ] and secretion in conjunctival goblet cells. Journal of cellular physiology\| PubMed ID: 32510663	2021
Resolvin E1 Reduces Leukotriene B4-Induced Intracellular Calcium Increase and Mucin Secretion in Rat Conjunctival Goblet Cells. The American journal of pathology\| PubMed ID: 32561135	2020
Povidone iodine treatment is deleterious to human ocular surface conjunctival cells in culture. BMJ open ophthalmology\| PubMed ID: 32995498	2020
Pro-Resolving Mediator Annexin A1 Regulates Intracellular Ca and Mucin Secretion in Cultured Goblet Cells Suggesting a New Use in Inflammatory Conjunctival Diseases. Frontiers in immunology\| PubMed ID: 33968020	2021
Signaling Pathways Used by the Specialized Pro-Resolving Mediator Maresin 2 Regulate Goblet Cell Function: Comparison with Maresin 1. International journal of molecular sciences\| PubMed ID: 35682912	2022

Jeffrey A. Bair

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