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N. C. Sears, S. Chattopadhyay, G. Sen, G. Stark; Caspase-8, Deficient in Retinal Pigment Epithelial Cells, is Necessary for Degradation of IRF-3. Invest. Ophthalmol. Vis. Sci. 2010;51(13):504.
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© ARVO (1962-2015); The Authors (2016-present)
Intracellular dsRNA is a chief sign of replication for many viruses. dsRNA binds endosomal toll-like receptor (TLR)-3 or cytoplasmic helicase RIG-I, to activate interferon response factor 3 (IRF3), leading to the induction of many antiviral genes, including interferon-beta. Within 12 hours of activation, IRF3 is proteasomally degraded to attenuate the inflammatory gene induction program. The purpose of this investigation is to determine the molecular pathways that underlie IRF3 degradation.
RIG-I dependent N terminal IRF-3 degradation was analyzed by western blot in P2.1 cells, a mutagenized HT1080 derived cell line low in IRF3, using lentivral directed expression of IRF3-Flag. We measured the time dependent degradation of IRF-3 in HT1800 cells after RIG-I or TLR3 activation with and without selective caspase inhibition. We next compared IRF3 degradation in ARPE-19 cells after stable transfection of caspase-8. Cells were cultured in DMEM with 5% FBS and treated with transfected dsRNA (4 ug/ml) to selectively activate the RIG-I pathway.
A 40 kD C-terminal fragment of IRF-3 was observed after RIG-I activation. Broad inhibition of caspases by Z-VAD-FMK as well as specific inhibition of caspase 8 by Z-IETD-FMK prevented IRF3 degradation. ARPE-19 cells, which express 100x less caspase-8 than HT1080 cells, showed no signal dependent IRF3 degradation. Ectopic expression of caspase-8 in ARPE-19 cells allowed IRF-3 degradation.
We have defined a necessary role for caspase-8 in the degradation of IRF-3. We think it possible that viral dsRNA can initiate two different antiviral responses, depending on the level of caspase 8 expression. First, if caspase-8 is present, an immediate type-I interferon response is initiated which is then attenuated by IRF3 degradation, followed by programmed cell death. However, if caspase-8 is not present, cells initiate a sustained type-I interferon response, not followed by IRF3 degradation and cell death. This new finding has significant implications in the response of the RPE to inflammatory stimulus from viral dsRNA.
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