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The effects of IKK-beta inhibition on early NF-kappa-B activation and transcription of downstream genes.

ABSTRACT: Small molecule approaches targeting the nuclear factor kappa B (NF-kB) pathway, a regulator of inflammation, have thus far proven unsuccessful in the clinic in part due to the complex pleiotropic nature of this network. Downstream effects depend on multiple factors including stimulus-specific temporal patterns of NF-kB activity. Despite considerable advances, genome-level impact of changes in temporal NF-kB activity caused by inhibitors and their stimulus dependency remains unexplored. This study evaluates the effects of pathway inhibitors on early NF-κB activity and downstream gene transcription. 3T3 fibroblasts were treated with SC-514, an inhibitor targeted to the NF-kB pathway, prior to stimulation with interleukin 1 beta (IL-1β) or tumor necrosis factor alpha (TNF-α). Stimulus induced NF-κB activation was quantified using immunofluorescence imaging over 90-minutes and gene expression tracked over 6-hours using mRNA TagSeq. When stimulated with IL-1β or TNF-α, significant differences (P < 0.05, two-way ANOVA), were observed in the temporal profiles of NF-κB activation between treated and untreated cells. Increasing numbers of differentially expressed genes (P < 0.01) were observed at higher inhibitor concentrations. Individual gene expression profiles varied in an inhibitor concentration and stimulus-dependent manner. The results in this study demonstrate small molecule inhibitors acting on pleiotropic pathway components can alter signal dynamics in a stimulus-dependent manner and affect gene response in complex ways.

SUBMITTER: Bloom MJ

PROVIDER: S-EPMC6348059 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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The effects of IKK-beta inhibition on early NF-kappa-B activation and transcription of downstream genes.

Bloom Meghan J MJ Saksena Sachit D SD Swain George P GP Behar Marcelo S MS Yankeelov Thomas E TE Sorace Anna G AG

Cellular signalling 20181210

Small molecule approaches targeting the nuclear factor kappa B (NF-kB) pathway, a regulator of inflammation, have thus far proven unsuccessful in the clinic in part due to the complex pleiotropic nature of this network. Downstream effects depend on multiple factors including stimulus-specific temporal patterns of NF-kB activity. Despite considerable advances, genome-level impact of changes in temporal NF-kB activity caused by inhibitors and their stimulus dependency remains unexplored. This stud ...[more]

PMID: 30543861

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Project description:Proinflammatory factors play important roles in the pathogenesis of African swine fever virus (ASFV), which is the causative agent of African swine fever (ASF), a highly contagious and severe hemorrhagic disease. Efforts in the prevention and treatment of ASF have been severely hindered by knowledge gaps in viral proteins responsible for modulating host antiviral responses. In this study, we identified the I10L protein (pI10L) of ASFV as a potential inhibitor of the TNF-α- and IL-1β-triggered NF-κB signaling pathway, the most canonical and important part of host inflammatory responses. The ectopically expressed pI10L remarkably suppressed the activation of NF-κB signaling in HEK293T and PK-15 cells. The ASFV mutant lacking the I10L gene (ASFVΔI10L) induced higher levels of proinflammatory cytokines production in primary porcine alveolar macrophages (PAMs) compared with its parental ASFV HLJ/2018 strain (ASFVWT). Mechanistic studies suggest that pI10L inhibits IKKβ phosphorylation by reducing the K63-linked ubiquitination of NEMO, which is necessary for the activation of IKKβ. Morever, pI10L interacts with the kinase domain of IKKβ through its N-terminus, and consequently blocks the association of IKKβ with its substrates IκBα and p65, leading to reduced phosphorylation. In addition, the nuclear translocation efficiency of p65 was also altered by pI10L. Further biochemical evidence supported that the amino acids 1-102 on pI10L were essential for the pI10L-mediated suppression of the NF-κB signaling pathway. The present study clarifies the immunosuppressive activity of pI10L, and provides novel insights into the understanding of ASFV pathobiology and the development of vaccines against ASF. IMPORTANCE African swine fever (ASF), caused by the African swine fever virus (ASFV), is now widespread in many countries and severely affects the commercial rearing of swine. To date, few safe and effective vaccines or antiviral strategies have been marketed due to large gaps in knowledge regarding ASFV pathobiology and immune evasion mechanisms. In this study, we deciphered the important role of the ASFV-encoded I10L protein in the TNF-α-/IL-1β-triggered NF-κB signaling pathway. This study provides novel insights into the pathogenesis of ASFV and thus contributes to the development of vaccines against ASF.

Dataset Information

The effects of IKK-beta inhibition on early NF-kappa-B activation and transcription of downstream genes.

Publications

The effects of IKK-beta inhibition on early NF-kappa-B activation and transcription of downstream genes.

Similar Datasets

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