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Expression of an IKKgamma splice variant determines IRF3 and canonical NF-kappaB pathway utilization in ssRNA virus infection.

ABSTRACT: Single stranded RNA (ssRNA) virus infection activates the retinoic acid inducible gene I (RIG-I)- mitochondrial antiviral signaling (MAVS) complex, a complex that coordinates the host innate immune response via the NF-kappaB and IRF3 pathways. Recent work has shown that the IkappaB kinase (IKK)gamma scaffolding protein is the final common adapter protein required by RIG-I.MAVS to activate divergent rate-limiting kinases downstream controlling the NF-kappaB and IRF3 pathways. Previously we discovered a ubiquitous IKKgamma splice-variant, IKKgammaDelta, that exhibits distinct signaling properties.We examined the regulation and function of IKKgamma splice forms in response to ssRNA virus infection, a condition that preferentially induces full length IKKgamma-WT mRNA expression. In IKKgammaDelta-expressing cells, we found increased viral translation and cytopathic effect compared to those expressing full length IKKgamma-WT. IKKgammaDelta fails to support viral-induced IRF3 activation in response to ssRNA infections; consequently type I IFN production and the induction of anti-viral interferon stimulated genes (ISGs) are significantly attenuated. By contrast, ectopic RIG-I.MAVS or TNFalpha-induced canonical NF-kappaB activation is preserved in IKKgammaDelta expressing cells. Increasing relative levels of IKKgamma-WT to IKKgammaDelta (while keeping total IKKgamma constant) results in increased type I IFN expression. Conversely, overexpressing IKKgammaDelta (in a background of constant IKKgamma-WT expression) shows IKKgammaDelta functions as a dominant-negative IRF3 signaling inhibitor. IKKgammaDelta binds both IKK-alpha and beta, but not TANK and IKKepsilon, indicating that exon 5 encodes an essential TANK binding domain. Finally, IKKgammaDelta displaces IKKgammaWT from MAVS explaining its domainant negative effect.Relative endogenous IKKgammaDelta expression affects cellular selection of inflammatory/anti-viral pathway responses to ssRNA viral infection.

SUBMITTER: Liu P

PROVIDER: S-EPMC2778955 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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Expression of an IKKgamma splice variant determines IRF3 and canonical NF-kappaB pathway utilization in ssRNA virus infection.

Liu Ping P Lu Muping M Tian Bing B Li Kui K Garofalo Roberto P RP Prusak Deborah D Wood Thomas G TG Brasier Allan R AR

PloS one 20091126 11

<h4>Unlabelled</h4>Single stranded RNA (ssRNA) virus infection activates the retinoic acid inducible gene I (RIG-I)- mitochondrial antiviral signaling (MAVS) complex, a complex that coordinates the host innate immune response via the NF-kappaB and IRF3 pathways. Recent work has shown that the IkappaB kinase (IKK)gamma scaffolding protein is the final common adapter protein required by RIG-I.MAVS to activate divergent rate-limiting kinases downstream controlling the NF-kappaB and IRF3 pathways. P ...[more]

PMID: 19956647

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Project description:NF-kappaB is an inducible transcription factor mediating innate immune responses whose activity is controlled by the multiprotein IkappaB kinase (IKK) "signalsome". The core IKK consists of two catalytic serine kinases, IKKalpha and IKKbeta, and a noncatalytic subunit, IKKgamma. IKKgamma is required for IKK activity by mediating kinase oligomerization and serving to couple the core catalytic subunits to upstream mitogen-activated protein 3-kinase cascades. We have discovered an alternatively spliced IKKgamma mRNA isoform, encoding an in-frame deletion of exon 5, termed IKKgamma-delta. Using a specific reverse transcription-PCR assay, we find that IKKgamma-delta is widely expressed in cultured human cells and normal human tissues. Because IKKgamma-Delta protein is lacking a critical coiled-coil domain important in protein-protein interactions, we sought to determine its signaling properties by examining its ability to self associate, couple to activators of the canonical pathway, and mediate human T-cell leukemia virus type 1 (HTLV-1) Tax-induced NF-kappaB activity. Coimmunoprecipitation and confocal colocalization assays indicate IKKgamma-delta has strong homo- and heterotypic association with wild-type (WT) IKKgamma and, like IKKgamma WT, associates with the IKKbeta kinase. Similarly, IKKgamma-delta mediates IKK kinase activity and downstream NF-kappaB-dependent transcription in response to tumor necrosis factor (TNF) and the NF-kappaB-inducing kinase-IKKalpha signaling pathway. Surprisingly, however, in contrast to IKKgamma WT, IKKgamma-delta is not able to mediate HTLV-1 Tax-induced NF-kappaB-dependent transcription, even though IKKgamma-delta binds and colocalizes with Tax. These observations suggest that IKKgamma-delta is a functionally distinct alternatively spliced mRNA product differentially mediating TNF-induced, but not Tax-induced, signals converging on the IKK signalsome. Differing levels of IKKgamma-delta expression, therefore, may affect signal transduction cascades coupling to IKK.

Project description:The ability of the transcription factor NF-kappaB to upregulate anti-apoptotic proteins has been linked to the chemoresistance of solid tumors to standard chemotherapy. In contrast, recent studies have proposed that, in response to doxorubicin, NF-kappaB can be pro-apoptotic through repression of anti-apoptotic target genes. However, there is little evidence analyzing the outcome of NF-kappaB inhibition on the cytotoxicity of doxorubicin in studies describing pro-apoptotic NF-kappaB activity. In this study, we further characterize the activation of NF-kappaB in response to doxorubicin and evaluate its role in chemotherapy-induced cell death in sarcoma cells where NF-kappaB is reported to be pro-apoptotic. Doxorubicin treatment in U2OS cells induced canonical NF-kappaB activity as evidenced by increased nuclear accumulation of phosphorylated p65 at serine 536 and increased DNA-binding activity. Co-treatment with a small molecule IKKbeta inhibitor, Compound A, abrogated this response. RT-PCR evaluation of anti-apoptotic gene expression revealed that doxorubicin-induced transcription of cIAP2 was inhibited by Compound A, while doxorubicin-induced repression of other anti-apoptotic genes was unaffected by Compound A or siRNA to p65. Furthermore, the combination of doxorubicin and canonical NF-kappaB inhibition with Compound A or siRNA to p65 resulted in decreased cell viability measured by trypan blue staining and MTS assay and increased apoptosis measured by cleaved poly (ADP-ribose) polymerase and cleaved caspase 3 when compared to doxorubicin alone. Our results demonstrate that doxorubicin-induced canonical NF-kappaB activity associated with phosphorylated p65 is anti-apoptotic in its function and that doxorubicin-induced repression of anti-apoptotic genes occurs independent of p65. Therefore, combination therapies incorporating NF-kappaB inhibitors together with standard chemotherapies remains a viable method to improve the clinical outcomes in patients with advanced stage malignancies.

Dataset Information

Expression of an IKKgamma splice variant determines IRF3 and canonical NF-kappaB pathway utilization in ssRNA virus infection.

Publications

Expression of an IKKgamma splice variant determines IRF3 and canonical NF-kappaB pathway utilization in ssRNA virus infection.

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