Anti-Inflammatory and Pro-resolving Actions of the N-Terminal Peptides Ac2-26, Ac2-12 and Ac9-25 of Annexin A1 on Conjunctival Goblet Cell Function

Abstract

Purpose : Annexin A1 (AnxA1) is the main mediator of the anti-inflammatory actions of glucocorticoids. AnxA1 functions as a pro-resolving mediator in cultured rat conjunctival goblet cells to ensure tissue homeostasis through stimulation of intracellular [Ca²⁺] ([Ca²⁺]_i) and mucin secretion. AnxA1 has several N-terminal peptides with anti-inflammatory properties of their own, including Ac2-26, Ac2-12 and Ac9-25. We measured the increase in [Ca²⁺]_i caused by AnxA1 and its N-terminal peptides in goblet cells to determine which formyl peptide receptors (FPRs) the compounds use and the action of the peptides on histamine stimulation.

Methods : Goblet cells were cultured from pieces dissected from the bulbar and forniceal conjunctiva in male 8-week old rats. Changes in [Ca²⁺]_i were determined using a fluorescent Ca²⁺ indicator fura-2 after no additions, antagoinists, or agonists of annexin A1 and its N-terminal peptides Ac2-26, Ac2-12 and Ac9-25. The action of agonists on stimulation with the allergic mediator histamine was also determined.

Results : AnxA1 and its peptides each activated FPRs in goblet cells. AnxA1 and Ac2-26 at 10^-12 M and Ac2-12 at 10^-9 M inhibited the histamine-stimulated increase in [Ca²⁺]_i as did RvD1 and LXA₄ at 10^-12 M, while Ac9-25 did not. AnxA1 and Ac2-26 counter-regulated the H1 receptor through the p42p44MAPK/ERK1/2, β-adrenergic receptor kinase (βARK) and protein kinase C (PKC) pathways, whereas Ac2-12 counter-regulated only through βARK.

Conclusions : We conclude that the N-terminal peptides Ac2-26 and Ac2-12, but not Ac9-25, share multiple functions with the full-length AnxA1 in goblet cells, including inhibition of histamine-stimulated increase in [Ca²⁺]_i and counter-regulation of the H1 receptor. These actions suggest a potential pharmaceutical application of the AnxA1 N-terminal peptides Ac2-26 and Ac2-12 in homeostasis and ocular inflammatory diseases.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.