Regulation of the expression of interleukin-8 induced by 25-hydroxycholesterol in retinal pigment epithelium cells.
OBJECTIVE
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.
Centre of Ophthalmology and Vision Sciences (COCV), IBILI, Faculty of Medicine, University of Coimbra, Coimbra, Portugal.
Steve
S
Catarino
Carla F
CF
Bento
Ana
A
Brito
Eliana
E
Murteira
Alexandre F
AF
Fernandes
Paulo
P
Pereira
D015153:Blotting, Western; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology*; D017930:Genes, Reporter; D006801:Humans; D006888:Hydroxycholesterols / Q000494:pharmacology*; D016209:Interleukin-8 / Q000235:genetics* / Q000378:metabolism*; D007653:Ketocholesterols / Q000494:pharmacology; D016328:NF-kappa B / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D055213:Retinal Pigment Epithelium / Q000187:drug effects* / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism
D004791:Enzyme Inhibitors; D006888:Hydroxycholesterols; D016209:Interleukin-8; D007653:Ketocholesterols; D016328:NF-kappa B; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; C011724:cholest-5-en-3 beta,7 alpha-diol; C007997:25-hydroxycholesterol; D019869:Phosphatidylinositol 3-Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; C003001:7-ketocholesterol