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Pro-Resolving Mediator Annexin A1 Regulates Intracellular Ca²⁺ and Mucin Secretion in Cultured Goblet Cells Suggesting a New Use in Inflammatory Conjunctival Diseases.

ABSTRACT: 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 [Ca²⁺] ([Ca²⁺]_i) and mucin secretion and to identify the signaling pathways. The increase in [Ca²⁺]_i was determined using fura2/AM and mucin secretion was measured using an enzyme-linked lectin assay. AnxA1 stimulated an increase in [Ca²⁺]_i and mucin secretion that was blocked by the cell-permeant Ca²⁺ chelator BAPTA/AM and the ALX/FPR2 receptor inhibitor BOC2. AnxA1 increased [Ca²⁺]_i to a similar extent as the SPMs lipoxin A₄ and Resolvin (Rv) D1 and histamine. The AnxA1 increase in [Ca²⁺]_i and mucin secretion were inhibited by blocking the phospholipase C (PLC) pathway including PLC, the IP₃ receptor, the Ca²⁺/ATPase that causes the intracellular Ca²⁺ stores to empty, and blockade of Ca²⁺ influx. Inhibition of protein kinase C (PKC) and Ca²⁺/calmodulin-dependent protein kinase also decreased the AnxA1-stimulated increase in [Ca²⁺]_i and mucin secretion. In contrast inhibitors of ERK 1/2, phospholipase A₂ (PLA₂), and phospholipase D (PLD) did not alter AnxA1-stimulated increase in [Ca²⁺]_i, but did inhibit mucin secretion. Activation of protein kinase A did not decrease either the AnxA1-stimulated rise in [Ca²⁺]_i 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 [Ca²⁺]_i and stimulate mucin secretion and ERK1/2, PLA₂, and PLD to stimulate mucin secretion from conjunctival goblet cells.

SUBMITTER: Lyngstadaas AV

PROVIDER: S-EPMC8100605 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The amount of mucin on the ocular surface is regulated by the rate of mucin synthesis, mucin secretion, and the number of goblet cells. We have previously shown that cholinergic agonists are potent stimuli of mucin secretion. In contrast, there have been no studies on the control of goblet cell proliferation. In this study we investigate the presence of the EGF family of growth factors and their receptors in rat conjunctiva and cultured rat conjunctival goblet cells as well as their effects on activation of signaling intermediates and goblet cell proliferation. Rat conjunctival goblet cells were grown in organ culture and identified as goblet cells by their morphology and positive staining for the lectin UEA-1 and cytokeratin 7. In the rat conjunctiva, the presence of the EGF family members epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), heparin binding EGF (HB-EGF), and heregulin was determined by RT-PCR. The receptors for these ligands, EGF receptor (EGFR), erbB2, erbB3, and erbB4 were detected in both rat conjunctiva and goblet cells by Western blot analysis. Immunofluorescence microscopy of conjunctival tissue determined that EGFR was present as punctate staining in the cytoplasm of conjunctival goblet cells while ErbB2 was present in the basolateral and lateral membranes of goblet cells. ErbB3 was localized to the cytosol of rat conjunctival goblet cells. In cultured goblet cells, EGFR and ErbB2 were present in the perinuclear area of the cells. ErbB3 was widely distributed throughout the cytoplasm of the cells. ErbB4 was not detected in either the conjunctiva or goblet cells by immunofluorescence microscopy. Using a multiplex assay system we measured phosphorylation (activation) of p44/p42 mitogen-activated protein kinase (MAPK), also known as ERK, Jun N-terminal kinase (JNK), p38 MAPK and AKT (also known as protein kinase B), molecules known to be activated by EGF receptor members. EGF, TGF-alpha and HB-EGF activated the signaling intermediate proteins whereas heregulin did not. No EGF family member significantly activated AKT. Consistent with these findings, EGF, TGF-alpha and HB-EGF each stimulated goblet cell proliferation as measured by WST-1 assay or immunofluorescence microscopy using an antibody against Ki-67, a protein expressed in dividing cells. Heregulin did not cause goblet cell proliferation. We conclude that multiple members of the EGF family, EGF, TGF-alpha and HB-EGF, and heregulin are present with three of the four erbB receptor subtypes. EGF, TGF-alpha and HB-EGF all stimulated the activation of signaling intermediates and caused goblet cell proliferation.

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Pro-Resolving Mediator Annexin A1 Regulates Intracellular Ca²⁺ and Mucin Secretion in Cultured Goblet Cells Suggesting a New Use in Inflammatory Conjunctival Diseases.

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Pro-Resolving Mediator Annexin A1 Regulates Intracellular Ca2+ and Mucin Secretion in Cultured Goblet Cells Suggesting a New Use in Inflammatory Conjunctival Diseases.

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OmicsDI is part of the ELIXIR infrastructure

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Pro-Resolving Mediator Annexin A1 Regulates Intracellular Ca²⁺ and Mucin Secretion in Cultured Goblet Cells Suggesting a New Use in Inflammatory Conjunctival Diseases.