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Identification of an NF-kappaB-dependent gene network in cells infected by mammalian reovirus.

ABSTRACT: Reovirus infection activates NF-kappaB, which leads to programmed cell death in cultured cells and in the murine central nervous system. However, little is known about how NF-kappaB elicits this cellular response. To identify host genes activated by NF-kappaB following reovirus infection, we used HeLa cells engineered to express a degradation-resistant mutant of IkappaBalpha (mIkappaBalpha) under the control of an inducible promoter. Induction of mIkappaBalpha inhibited the activation of NF-kappaB and blocked the expression of NF-kappaB-responsive genes. RNA extracted from infected and uninfected cells was used in high-density oligonucleotide microarrays to examine the expression of constitutively activated genes and reovirus-stimulated genes in the presence and absence of an intact NF-kappaB signaling axis. Comparison of the microarray profiles revealed that the expression of 176 genes was significantly altered in the presence of mIkappaBalpha. Of these genes, 64 were constitutive and not regulated by reovirus, and 112 were induced in response to reovirus infection. NF-kappaB-regulated genes could be grouped into four distinct gene clusters that were temporally regulated. Gene ontology analysis identified biological processes that were significantly overrepresented in the reovirus-induced genes under NF-kappaB control. These processes include the antiviral innate immune response, cell proliferation, response to DNA damage, and taxis. Comparison with previously identified NF-kappaB-dependent gene networks induced by other stimuli, including respiratory syncytial virus, Epstein-Barr virus, tumor necrosis factor alpha, and heart disease, revealed a number of common components, including CCL5/RANTES, CXCL1/GRO-alpha, TNFAIP3/A20, and interleukin-6. Together, these results suggest a genetic program for reovirus-induced apoptosis involving NF-kappaB-directed expression of cellular genes that activate death signaling pathways in infected cells.

SUBMITTER: O'Donnell SM

PROVIDER: S-EPMC1346919 | biostudies-literature | 2006 Feb

REPOSITORIES: biostudies-literature

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Identification of an NF-kappaB-dependent gene network in cells infected by mammalian reovirus.

O'Donnell Sean M SM Holm Geoffrey H GH Pierce Janene M JM Tian Bing B Watson Melissa J MJ Chari Ravi S RS Ballard Dean W DW Brasier Allan R AR Dermody Terence S TS

Journal of virology 20060201 3

Reovirus infection activates NF-kappaB, which leads to programmed cell death in cultured cells and in the murine central nervous system. However, little is known about how NF-kappaB elicits this cellular response. To identify host genes activated by NF-kappaB following reovirus infection, we used HeLa cells engineered to express a degradation-resistant mutant of IkappaBalpha (mIkappaBalpha) under the control of an inducible promoter. Induction of mIkappaBalpha inhibited the activation of NF-kapp ...[more]

PMID: 16414985

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Project description:Respiratory syncytial virus (RSV) is a mucosa-restricted virus that is a leading cause of epidemic respiratory tract infections in children. In epithelial cells, RSV replication activates nuclear translocation of the inducible transcription factor nuclear factor kappaB (NF-kappaB) through proteolysis of its cytoplasmic inhibitor, IkappaB. In spite of a putative role in mediating virus-inducible gene expression, the spectrum of NF-kappaB-dependent genes induced by RSV infection has not yet been determined. To address this, we developed a tightly regulated cell system expressing a nondegradable, epitope-tagged IkappaBalpha isoform (Flag-IkappaBalpha Mut) whose expression could be controlled by exogenous addition of nontoxic concentrations of doxycycline. Flag-IkappaBalpha Mut expression potently inhibited IkappaBalpha proteolysis, NF-kappaB binding, and NF-kappaB-dependent gene transcription in cells stimulated with the prototypical NF-kappaB-activating cytokine tumor necrosis factor alpha (TNF-alpha) and in response to RSV infection. High-density oligonucleotide microarrays were then used to profile constitutive and RSV-induced gene expression in the absence or presence of Flag-IkappaBalpha Mut. Comparison of these profiles revealed 380 genes whose expression was significantly changed by the dominant-negative NF-kappaB. Of these, 236 genes were constitutive (not RSV regulated), and surprisingly, only 144 genes were RSV regulated, representing numerically approximately 10% of the total population of RSV-inducible genes at this time point. Hierarchical clustering of the 144 RSV- and Flag-IkappaBalpha Mut-regulated genes identified two discrete gene clusters. The first group had high constitutive expression, and its expression levels fell in response to RSV infection. In this group, constitutive mRNA expression was increased by Flag-IkappaBalpha Mut expression, and the RSV-induced decrease in expression was partly inhibited. In the second group, constitutive expression was very low (or undetectable) and, after RSV infection, expression levels strongly increased. In this group, NF-kappaB was required for RSV-inducible expression because Flag-IkappaBalpha Mut expression blocked their induction by RSV. This latter cluster includes chemokines, transcriptional regulators, intracellular proteins regulating translation and proteolysis, and secreted proteins (complement components and growth factor regulators). These data suggest that NF-kappaB action induces global cellular responses after viral infection.

Project description:BackgroundPancreatic beta-cells are the target of an autoimmune attack in type 1 diabetes mellitus (T1DM). This is mediated in part by cytokines, such as interleukin (IL)-1beta and interferon (IFN)-gamma. These cytokines modify the expression of hundreds of genes, leading to beta-cell dysfunction and death by apoptosis. Several of these cytokine-induced genes are potentially regulated by the IL-1beta-activated transcription factor (TF) nuclear factor (NF)-kappaB, and previous studies by our group have shown that cytokine-induced NF-kappaB activation is pro-apoptotic in beta-cells. To identify NF-kappaB-regulated gene networks in beta-cells we presently used a discriminant analysis-based approach to predict NF-kappaB responding genes on the basis of putative regulatory elements.ResultsThe performance of linear and quadratic discriminant analysis (LDA, QDA) in identifying NF-kappaB-responding genes was examined on a dataset of 240 positive and negative examples of NF-kappaB regulation, using stratified cross-validation with an internal leave-one-out cross-validation (LOOCV) loop for automated feature selection and noise reduction. LDA performed slightly better than QDA, achieving 61% sensitivity, 91% specificity and 87% positive predictive value, and allowing the identification of 231, 251 and 580 NF-kappaB putative target genes in insulin-producing INS-1E cells, primary rat beta-cells and human pancreatic islets, respectively. Predicted NF-kappaB targets had a significant enrichment in genes regulated by cytokines (IL-1beta or IL-1beta + IFN-gamma) and double stranded RNA (dsRNA), as compared to genes not regulated by these NF-kappaB-dependent stimuli. We increased the confidence of the predictions by selecting only evolutionary stable genes, i.e. genes with homologs predicted as NF-kappaB targets in rat, mouse, human and chimpanzee.ConclusionThe present in silico analysis allowed us to identify novel regulatory targets of NF-kappaB using a supervised classification method based on putative binding motifs. This provides new insights into the gene networks regulating cytokine-induced beta-cell dysfunction and death.

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Project description:Picroliv, an iridoid glycoside derived from the plant Picrorhiza kurroa, is used traditionally to treat fever, asthma, hepatitis, and other inflammatory conditions. However, the exact mechanism of its therapeutic action is still unknown. Because nuclear factor-kappaB (NF-kappaB) activation plays a major role in inflammation and carcinogenesis, we postulated that picroliv must interfere with this pathway by inhibiting the activation of NF-kappaB-mediated signal cascade. Electrophoretic mobility shift assay showed that pretreatment with picroliv abrogated tumor necrosis factor (TNF)-induced activation of NF-kappaB. The glycoside also inhibited NF-kappaB activated by carcinogenic and inflammatory agents, such as cigarette smoke condensate, phorbol 12-myristate 13-acetate, okadaic acid, hydrogen peroxide, lipopolysaccharide, and epidermal growth factor. When examined for the mechanism of action, we found that picroliv inhibited activation of IkappaBalpha kinase, leading to inhibition of phosphorylation and degradation of IkappaBalpha. It also inhibited phosphorylation and nuclear translocation of p65. Further studies revealed that picroliv directly inhibits the binding of p65 to DNA, which was reversed by the treatment with reducing agents, suggesting a role for a cysteine residue in interaction with picroliv. Mutation of Cys(38) in p65 to serine abolished this effect of picroliv. NF-kappaB inhibition by picroliv leads to suppression of NF-kappaB-regulated proteins, including those linked with cell survival (inhibitor of apoptosis protein 1, Bcl-2, Bcl-xL, survivin, and TNF receptor-associated factor 2), proliferation (cyclin D1 and cyclooxygenase-2), angiogenesis (vascular endothelial growth factor), and invasion (intercellular adhesion molecule-1 and matrix metalloproteinase-9). Suppression of these proteins enhanced apoptosis induced by TNF. Overall, our results show that picroliv inhibits the NF-kappaB activation pathway, which may explain its anti-inflammatory and anticarcinogenic effects.

Dataset Information

Identification of an NF-kappaB-dependent gene network in cells infected by mammalian reovirus.

Publications

Identification of an NF-kappaB-dependent gene network in cells infected by mammalian reovirus.

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