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Trivendra Tripathi, Mahshid Abdi, Hassan Alizadeh; Protease-Activated Receptor (PAR)2, But not PAR1, is Upregulated by Acanthamoeba Plasminogen Activator (aPA) and Induces Proinflammatory Cytokines in Human Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):6278.
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© ARVO (1962-2015); The Authors (2016-present)
Acanthamoeba plasminogen activator (aPA), is a serine protease elaborated by Acanthamoeba trophozoites, facilitates invasion of trophozoites to the host and contributes to the pathogenesis of Acanthamoeba keratitis (AK). The goal of the present study was to determine if aPA induces proinflammatory cytokines in human corneal epithelial (HCE) cells via the protease-activated receptor PAR1 and PAR2 pathways.
A. castellanii trophozoites were grown in peptone-yeast extract glucose for 7 days and the supernatants were collected and centrifuged. The aPA was purified using the fast protein liquid chromatography system and aPA activity was determined by zymography assays. Human corneal epithelial (HCE) cells were pre-incubated with or without aPA (100 µg/ml), PAR1 agonists (Thrombin, 10 µM; TRAP-6, 10 µM), and PAR2 agonists (SLIGRL-NH2, 100 µM; AC 55541, 10 µM) for 1 hour. Inhibition of PAR1 and PAR2 involved pre-incubating the HCE cells for 1 hour with the antagonist of PAR1 (SCH 79797, 60 μM) and PAR2 (FSLLRY-NH2, 100 μM) with or without aPA. Expression of PAR1 and PAR2 was examined by qRT-PCR, flow cytometry, and immunocytochemistry. IL-8 and IL-6 expression was quantified by qRT-PCR and secretion of these cytokines was determined by ELISA assays 24 hours after treatments.
Both PAR1 and PAR2 transcript were expressed in HCE cells. aPA and PAR2 agonists significantly upregulate PAR2 expression while PAR2 antagonist significantly inhibits aPA-induced PAR2 expression in HCE cells. aPA and PAR1 agonists did not significantly affect PAR1 expression in HCE cells. aPA and PAR2 agonists triggered upregulation of proinflammatory cytokines IL-8 and IL-6 mRNA expression. Both IL-6 and IL-8 protein secretion were significantly (P<0.05) upregulated in HCE cells stimulated with aPA and PAR2 agonists and the level of these cytokines were significantly attenuated by PAR2 antagonist. In contrast, PAR1 agonists failed to induce IL-8 and IL-6 expression and PAR1 antagonist did not inhibit aPA-induced expression of IL-8 and IL-6 in HCE cells.
aPA specifically induces expression and production of proinflammatory cytokines in HCE cells via PAR2 pathway and PAR2 antagonists may be a therapeutic target in AK.
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