May 2004
Volume 45, Issue 13
ARVO Annual Meeting Abstract  |   May 2004
Persistence and termination of cytokine–mediated pro–inflammatory signaling in retinal pigment epithelial cells (RPE)
Author Affiliations & Notes
  • Z. Faghiri
    Neuroscience Center/Ophthalmology, LSU Health Sciences Center, New Orleans, LA
  • N.G. Bazan
    Neuroscience Center/Ophthalmology, LSU Health Sciences Center, New Orleans, LA
  • Footnotes
    Commercial Relationships  Z. Faghiri, None; N.G. Bazan, None.
  • Footnotes
    Support  NIH EY05121
Investigative Ophthalmology & Visual Science May 2004, Vol.45, 1308. doi:
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      Z. Faghiri, N.G. Bazan; Persistence and termination of cytokine–mediated pro–inflammatory signaling in retinal pigment epithelial cells (RPE) . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1308.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract: : Purpose: Oxidative stress and pro–inflammatory cytokines activate signalings that include transcription factors AP–1 and NF–ΚB and result in gene induction. ECSIT (evolutionarily conserved signaling intermediate in Toll pathways), an adaptor protein linking TRAF6 to MEKK–1, is specific for Toll/IL–1 pathways. Here we investigated how ECSIT interacts with IL–1ß actions on TRAF6, IΚB–α and IΚB kinases IKKα, ß, and γ in ARPE–19 cells. Methods: ARPE–19 cells were grown in 6–well plates up to 100% confluence, serum–starved for 18 hr, then stimulated with IL–1ß, or TNF–α/H2O2 to trigger oxidative stress. Cells at 80% confluence were transfected with ECSIT. TRAF6, IΚB–α and IΚB kinase expression was examined in cytosolic and membrane fractions by Western blot analysis. Results:Oxidative stress caused translocation of IΚB–α to the membrane followed by a sustained down–regulation and induced a limited down–regulation of cytosolic IΚB–α . Phosphorylation of membrane TRAF6 was observed but did not affect phosphorylation state in cytosol under these conditions. There was no phosphorylation of IKKα, ß, or γ. IL–1ß–induced IΚB–α translocation from cytosol to the membrane 1 min after stimulation was followed by its down–regulation in both cytosol and membrane for 30 min. IΚB–α returned to basal level within 4 hr in the presence of IL–1ß. IL–1ß induced phosphorylation of membrane TRAF6 and cytosolic IKKß without affecting cytosolic TRAF6, IKKα and γ. TNF–α induced down–regulation of IΚB–α in both cytosol and membrane without recovery to the normal level. TNF–α did not induce TRAF6 or IKKα, ß, or γ phosphorylation. ECSIT–transfected cells also had IL–1ß–induced IΚB–α down–regulation and TRAF6 phosphorylation that started later (15 and 5 min, respectively) compared to non–transfected cells (5 and 1 min, respectively). Conclusions: Our data show that IL–1ß–induced IΚB–α degradation is via activation of membrane TRAF6 and cytosolic IKKß but not IKKα or γ. However, TNF–α alone causes activation and degradation of IΚB–α without affecting TRAF6 or IKKα, ß, and γ. Oxidative stress has stimulatory effects on IΚB–α that could be mediated through activation of TRAF6 but not IKKα, ß, or γ. Furthermore, ECSIT participates in IL–1ß signaling by phosphorylating TRAF6 and down–regulating IΚB–α and is, therefore, an intermediate in the activation of both NF–ΚB and AP–1. Altogether, our data highlight signaling modulated by the adaptor protein ECSIT relevant for sustained activation of pro–inflammatory responses as well as for its termination in human ARPE–19 cells. Supported by NIH EY05121.

Keywords: oxidation/oxidative or free radical damage • inflammation • signal transduction 

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