Regulation of the expression of interleukin-8 induced by 25-hydroxycholesterol in retinal pigment epithelium cells

Authors

  • Steve Catarino,

    1. Centre of Ophthalmology and Vision Sciences (COCV), IBILI, Faculty of Medicine, University of Coimbra, Coimbra, Portugal
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  • Carla F. Bento,

    1. Centre of Ophthalmology and Vision Sciences (COCV), IBILI, Faculty of Medicine, University of Coimbra, Coimbra, Portugal
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  • Ana Brito,

    1. Centre of Ophthalmology and Vision Sciences (COCV), IBILI, Faculty of Medicine, University of Coimbra, Coimbra, Portugal
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  • Eliana Murteira,

    1. Centre of Ophthalmology and Vision Sciences (COCV), IBILI, Faculty of Medicine, University of Coimbra, Coimbra, Portugal
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  • Alexandre F. Fernandes,

    1. Centre of Ophthalmology and Vision Sciences (COCV), IBILI, Faculty of Medicine, University of Coimbra, Coimbra, Portugal
    2. Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA
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  • Paulo Pereira

    1. Centre of Ophthalmology and Vision Sciences (COCV), IBILI, Faculty of Medicine, University of Coimbra, Coimbra, Portugal
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  • These authors contributed equally to this work.

Paulo Pereira
COCV – IBILI, Faculty of Medicine
University of Coimbra
Azinhaga de Santa Comba
Celas, 3000-548 Coimbra
Portugal
Tel: + 351 239480220
Fax: + 351 239480280
Email: ppereira@ibili.uc.pt

Abstract.

Purpose:  This study aimed at elucidating the molecular mechanisms involved in the regulation of IL-8 production by several oxysterols in retinal pigment epithelium (RPE) cells.

Methods:  A human cell line from RPE (ARPE-19) was used to test the role of cholesterol and several oxysterols (25-OH, 7-KC and 7β-OH) in the expression and secretion of IL-8. Expression of IL-8 was assessed by real-time PCR, while IL-8 secretion was evaluated by ELISA. PI3K-, MEK1/2-, ERK1/2- and NF-κB-specific inhibitors were used to assess the specific role of the several players on the regulation of IL-8 production by oxysterols. A gene-reporter assay for AP-1 activity was also conducted to evaluate the putative role of this transcription factor on IL-8 expression induced by oxysterols.

Results:  Here, we demonstrate that 25-OH specifically increases transcription and secretion of the cytokine IL-8 in ARPE-19 cells. Indeed, treatment of ARPE-19 with 25-OH, but not with 7-KC, 7β-OH or cholesterol, induced the secretion of IL-8 from cells. 25-OH also induced the activation/phosphorylation of ERK1/2 through a mechanism dependent on MEK, ERK1/2 and PI3K kinase activity. Real-time PCR and ELISA experiments demonstrated that 25-OH increased transcription and secretion of IL-8 through a mechanism that is dependent on ERK1/2 and PI3K activity. Furthermore, 25-OH triggered the activation/phosphorylation of the AP-1 component c-Jun and, consistently, increased the transcriptional activity of AP-1. Additionally, we also found that 25-OH decreases the levels of IκB and increases the nuclear levels of NF-κB p65 subunit and that inhibition of NF-κB activity partially prevents the increased secretion of IL-8 induced by 25-OH.

Conclusions:  The results presented in this study suggest a role for 25-OH in inducing IL-8 production through pathways that are likely to involve AP-1 and NF-κB in ARPE-19 cells. Our data may also provide new molecular targets for the treatment of AMD.

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