Abstract
Previous studies revealed that phenylmethimazole (C10) inhibits IRF3 signaling, preventing dsRNA-induction of type 1 interferon gene expression, production, and downstream signaling. In the present study, we investigated the molecular basis for C10 inhibition of dsRNA-stimulated IRF3 signaling. IRF-3 Trans-AM assays were used to measure C10 effects on dsRNA induction of IRF3 DNA binding. Green fluorescent protein-labeled IRF3 was used to measure C10 effects on dsRNA-induced IRF3 nuclear translocation. Native PAGE, SDS PAGE, and western blotting were used to identify effects of C10 on IRF3 homodimer formation and phosphorylation, respectively. There was a significant impairment of dsRNA-induced IRF3 DNA binding activity in human embryonic kidney and pancreatic cancer cells with C10 treatment. C10 also blocked dsRNA-induced IRF3 nuclear translocation and homodimer formation without blocking serine 396 phosphorylation of IRF3. Together, these results indicate that C10 interferes with IRF3 signaling by blocking dsRNA-induced IRF3 homodimer formation, a prerequisite for nuclear translocation and DNA binding activities.
Highlights
Interferon regulatory factor 3 (IRF3) is a critical mediator of innate immune signaling
Identified pattern recognition receptors (PRRs) which recognize cytoplasmic dsRNA and activate IRF3 include toll-like receptor 3 (TLR3), retinoic acid-inducible gene 1 (RIG1), and melanoma differentiation associated factor 5 (MDA5). dsRNA-activated TLR3 interacts with Toll/IL-1 receptor (TIR)-domain-containing adaptor inducing IFN-beta (TRIF), which will subsequently interact with tumor necrosis factor (TNF)R-associated factor 3 (TRAF3) [15]
In order to study the effect of C10 on IRF3 DNA binding activity in human cells, human embryonic kidney 293 (HEK293) cells which stably overexpress human TLR3 tagged with hemagglutinin (HA), HEK293-hTLR3-HA cells, and human pancreatic cancer cells (PANC-1) were transfected with pUNO-hIRF3 as described in the Experimental section
Summary
Interferon regulatory factor 3 (IRF3) is a critical mediator of innate immune signaling. In the nucleus the activated IRF3 homodimer complexes with the chaperone proteins CBP and p300, binds to its transcriptional regulatory DNA sequences, and initiates the activation of target gene transcription [i.e., type 1 interferons The innate immune response to pathogenic infectious agents is initiated by pattern recognition receptors (PRRs) which recognize pathogen-associated molecular patterns (PAMPs) such as viral dsRNA [10,11,12,13,14]. Identified PRRs which recognize cytoplasmic dsRNA and activate IRF3 include toll-like receptor 3 (TLR3), retinoic acid-inducible gene 1 (RIG1), and melanoma differentiation associated factor 5 (MDA5). DsRNA-activated TLR3 interacts with Toll/IL-1 receptor (TIR)-domain-containing adaptor inducing IFN-beta (TRIF), which will subsequently interact with tumor necrosis factor (TNF)R-associated factor 3 (TRAF3) [15]. The studies described were designed to explore the molecular basis for C10 inhibition of dsRNA-mediated IRF3 signaling
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