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Christoph Ullmer, Elisabeth Anna Zirwes, Anja Osterwald; Lysophosphatidic acid (LPA) elicits pro-inflammatory responses in ARPE-19 cells via activation of LPA2 receptors. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1746.
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© ARVO (1962-2015); The Authors (2016-present)
The consequences of increased levels of the bioactive phospholipid LPA in the vitreous fluid from patients with proliferative diabetic retinopathy (PDR) compared to non-diabetic patients remain unknown. Due to the high inflammatory pathophysiology of PDR, we hypothesized that LPA acts on retinal pigment epithelial (RPE) cells to increase the production of inflammatory mediators by activating LPA receptors and respective signaling pathways.
Expression of LPA receptors was examined in ARPE-19 cells by real-time PCR. The induction of cytokine levels was determined by ELISA and real-time PCR. Cells were transfected with a NF-kappa B (NFkB) responsive reporter gene using a lentivirus and LPA mediated NFkB activation was measured as luciferase activity. LPA receptor activity was investigated by transfection of LPA receptor siRNA, and by pre-incubation with LPA receptor antagonists, pertussis toxin or inhibitors of signal transduction proteins.
ARPE-19 cells predominantly express LPA2 receptor mRNA; LPA3, LPA4, LPA5 and LPA6 receptors were below level of detection. Exogenously applied LPA (2.5 μM for 24h) induces cells to secrete interleukin-6 (IL-6), and interleukin-8 (IL-8). LPA-mediated interleukin secretion and mRNA induction could be fully inhibited by co-incubation with a LPA2 receptor antagonist (cpd 35) but not or partially by a LPA1 receptor antagonist (AM095). Furthermore, LPA activated the NFkB reporter gene by 17-fold, which was fully prevented by transfection of a LPA2 selective siRNA. The EC50 of LPA to activate NFkB (304 nM) matched the induction of IL-6 (275 nM) and IL-8 (294 nM) mRNA. The LPA response was fully antagonized by cpd 35, but not by AM095. LPA failed to modulate cAMP or intracellular Ca2+ levels. Full inhibition of the pro-inflammatory LPA2 pathway was achieved by inhibition of JAK3, PI3K, AKT, and IKK.
This study demonstrates that LPA promotes ARPE-19 cells to secrete inflammatory mediators such as IL-6 and IL-8 via activation of LPA2 receptors. These data suggest that activation of LPA2 receptors may potentially contribute to the inflammatory pathophysiology in PDR that displays elevated vitreous LPA as well as IL-6 and IL-8 levels. Activation of JAK3, PI3K, AKT, and NFkB are important signaling components in LPA2 receptor-mediated cytokine secretion, which is unprecedented and will be further analyzed.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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