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A3861 - Epidermal Growth Factor Receptor (EGFR) Signaling in the Nucleus Modulates Antiviral Immunity
Author Block: A. D. Osafo-Addo1, M. Haslip1, Y. Sun1, A. M. Mulenos1, N. Li2, J. L. Koff1; 1Internal Medicine, Yale School of Medicine, New Haven, CT, United States, 2Fluidigm Corporation, South San Francisco, CA, United States.
RATIONALE: Previously, we showed that viruses [e.g. Influenza virus, Rhinovirus, and Respiratory Syncytial Virus (RSV)] activate epidermal growth factor receptor (EGFR) to suppress Interferon Regulatory Factor (IRF)1-dependent Interferon lambda (IFN-λ) and CXCL10, which results in increased viral infection and inflammation. However, the mechanism for this effect is unknown. In cancer, EGFR traffics to the nucleus to increase tumor survival. In addition, nuclear EGFR has been shown to stimulate calcium (Ca2+) signaling in the nucleus. However, a role for nuclear EGFR to regulate Ca2+ in the nucleus that affects IRF1-induced IFN-λ and CXCL10 has not been explored. METHODS: An airway epithelial cell (BEAS-2b) line was stimulated by EGF, an EGFR ligand, and Influenza A virus (IAV; MOI=5), and EGFR nuclear localization was evaluated by collecting cytoplasmic and nuclear fractions for Western Blot (WB). Additional experiments used confocal microscopy to visualize nuclear EGFR trafficking. Subsequently, BEAS-2b cells cultured in Callisto™ were transfected with adenoviral (Adv) constructs that inhibit inositol 1,4,5-triphosphate (InsP3) selectively in the cytoplasm (Adv-InsP3-CM) and nucleus by targeting nuclear localization sequence (Adv-InsP3-NLS) to suppress Ca2+ production, as well as an Adv control (Adv-C). IRF1 expression was evaluated by WB and qPCR. Additional planned experiments will use siRNA to inhibit molecules implicated in EGFR trafficking to the nucleus [e.g. Rab5, syntaxin-6 and Sec61β]. RESULTS: EGF and IAV stimulation of BEAS-2b cells induced EGFR phosphorylation (EGFR-p) in cell lysate nuclear fractions at 15 min, and total EGFR was observed in the nucleus by confocal microscopy at 2 h. We confirmed prior experiments that EGF decreased IRF1 protein and mRNA in BEAS-2b cells. In addition, BEAS-2b cells transfected with Adv-InsP3-NLS at multiplicity of infection (MOI) 10:1 rescued EGF-induced suppression of IRF1 mRNA at 2 h, which was not observed with transfection of Adv-InsP3-CM and Adv-C. Inhibition of Rab5, syntaxin-6 and Sec61β by siRNA are currently ongoing.CONCLUSIONS: Preliminary experiments suggest that EGF and IAV stimulate total EGFR and EGFR-p in the nucleus, which provides a potential mechanism for nuclear EGFR to affect IRF1. Nuclear EGFR regulates Ca2+ signaling in the nucleus, and we found that selective inhibition of nuclear InsP3 rescued EGF-induced suppression of IRF1. Additional experiments will include stimulation with respiratory viruses and inhibition of nuclear EGFR trafficking. These experiments will better characterize this novel mechanism of EGFR and Ca2+ signaling in the nucleus to suppress IRF1, which affects antiviral immunity.