FLIP (Flice-like inhibitory protein) suppresses cytoplasmic double-stranded-RNA-induced apoptosis and NF-κB and IRF3-mediated signaling

<p>Abstract</p> <p>Background</p> <p>Cytoplasmic viral double-stranded RNA (dsRNA) is detected by a class of ubiquitous cytoplasmic RNA helicases, retinoic acid inducible gene-I (RIG-I) and melanoma differentiation antigen-5 (MDA5), which initiate a signaling cascade via their common adaptor called interferon-β (IFN-β) promoter stimulator-1 (IPS-1). This leads to the production of proinflammatory and antiviral cytokines, the type I Interferons, via mainly nuclear factor kappa B (NF-κB) and interferon response factor-3 (IRF3) transcription factors. Fas-associated death domain (FADD) protein, receptor-interacting protein (RIP1), caspase-8 and tumor necrosis factor receptor (TNFR)-associated death domain (TRADD) protein, all traditionally associated with death receptor signaling, are also involved in RIG-I/MDA5 signaling pathway. We previously showed that FLIP (Flice-like inhibitory protein), also designated as <it>cflar </it>(CASP8 and FADD-like apoptosis regulator), negatively regulates lipopolysaccharide (LPS)-induced toll-like receptor 4 (TLR4) signaling in endothelial cells and mouse embryonic fibroblasts (MEFs) and protected against TLR4-mediated apoptosis.</p> <p>Results</p> <p>In this study, we investigated the role of FLIP in cellular response to cytoplasmic polyinosinic:polycytidylic acid, poly(I:C), a synthetic analog of dsRNA. <it>C</it>onsistent with the previously described role of FADD in RIG-I/MDA5-mediated apoptosis, we found that FLIP^-/-MEFs were more susceptible to killing by cytoplasmic poly(I:C). However, FLIP^-/-MEFs also exhibited markedly increased expression of NF-κB-and IRF3- dependent genes in response to cytoplasmic poly(I:C). Importantly, reconstitution of FLIP in FLIP^-/-MEFs reversed the hyper-activation of IRF3- and NF-κB-mediated gene expression. Further, we found that caspase-8 catalytic activity was not required for cytoplasmic poly(I:C)-mediated NF-κB and IRF3 signaling.</p> <p>Conclusions</p> <p>These results provide evidence for a crucial dual role for FLIP in antiviral responses to cytoplasmic dsRNA: it protects from cytoplasmic dsRNA-mediated cell death while down-regulating IRF3-and NF-κB-mediated gene expression. Since the pathogenesis of several viral infections involves a heightened and dysregulated cytokine response, a possible therapy could involve modulating FLIP levels.</p

Extracellular Administration of BCL2 Protein Reduces Apoptosis and Improves Survival in a Murine Model of Sepsis

Severe sepsis and septic shock are major causes of morbidity and mortality worldwide. In experimental sepsis there is prominent apoptosis of various cell types, and genetic manipulation of death and survival pathways has been shown to modulate organ injury and survival.We investigated the effect of extracellular administration of two anti-apoptotic members of the BCL2 (B-cell lymphoma 2) family of intracellular regulators of cell death in a murine model of sepsis induced by cecal ligation and puncture (CLP). We show that intraperitoneal injection of picomole range doses of recombinant human (rh) BCL2 or rhBCL2A1 protein markedly improved survival as assessed by surrogate markers of death. Treatment with rhBCL2 or rhBCL2A1 protein significantly reduced the number of apoptotic cells in the intestine and heart following CLP, and this was accompanied by increased expression of endogenous mouse BCL2 protein. Further, mice treated with rhBCL2A1 protein showed an increase in the total number of neutrophils in the peritoneum following CLP with reduced neutrophil apoptosis. Finally, although neither BCL2 nor BCL2A1 are a direct TLR2 ligand, TLR2-null mice were not protected by rhBCL2A1 protein, indicating that TLR2 signaling was required for the protective activity of extracellularly adminsitered BCL2A1 protein in vivo.Treatment with rhBCL2A1 or rhBCL2 protein protects mice from sepsis by reducing apoptosis in multiple target tissues, demonstrating an unexpected, potent activity of extracellularly administered BCL2 BH4-domain proteins

GEA 3162, a peroxynitrite donor, induces Bcl-2-sensitive, p53-independent apoptosis in murine bone marrow cells

AbstractApoptosis may be regulated by oxidants such as peroxynitrite (ONOO−). The tumour suppressor, p53, has been reported to play a crucial role in apoptosis induced by oxidants, therefore we assessed the ability of a ONOO− donor, GEA 3162, to activate caspases and induce mitochondrial permeability in a p53-deficient murine bone marrow cell line, Jaws II. Furthermore, these cells were stably transfected with Bcl-2, in order to investigate the impact of this survival protein on ONOO−-induced apoptosis. GEA 3162 activated caspases and induced loss of mitochondrial membrane potential in Jaws II cells. In particular, caspases 3 and 2 were activated, alongside minor activation of caspases 8 and 9, and apoptosis was partially dependent upon p38 MAP kinase activation, with little or no role for JNK. Overexpression of Bcl-2 abolished activation of all caspases and reduced the change in mitochondrial membrane potential. Thus, we have demonstrated that the ONOO− donor, GEA 3162, induces apoptosis in Jaws II murine myeloid cells despite lacking functional p53, via a pathway that principally involves caspases 2 and 3 and mitochondrial changes. This is blocked by overexpression of Bcl-2 via a mechanism that does not appear to merely reflect stabilisation of the mitochondrial membrane

The Fas-associated death domain protein suppresses activation of NF-κB by LPS and IL-1β

Activation of NF-κB by bacterial LPS promotes the upregulation of proinflammatory cytokines that contribute to the pathogenesis of Gram-negative septic shock. LPS activation of NF-κB is dependent upon the interaction of two death domain–containing (DD-containing) proteins, MyD88 and IL-1 receptor–associated kinase IRAK. Another DD-containing protein, Fas-associated death domain (FADD), also binds MyD88 through respective DD-DD interactions. Although FADD has been classically described as a proapoptotic signaling molecule, several reports have implicated a role for FADD in mediating NF-κB activation. In the present report, we investigated whether FADD could mediate LPS activation of NF-κB. Overexpression of FADD blocked LPS-induced NF-κB activation, whereas absence of FADD enhanced activation of NF-κB by LPS. Further, LPS-induced expression of two NF-κB–dependent gene products, IL-6 and KC, was enhanced in FADD(–/–) mouse embryo fibroblasts (MEFs) compared with wild-type. This increase in NF-κB activity correlated with enhanced IκB degradation. FADD(–/–) MEFs were also resistant to NF-κB activation induced by IL-1β. Finally, reconstitution of full-length FADD in the FADD(–/–) MEFs completely reversed the enhanced activation of NF-κB elicited by either LPS or IL-1β. Together, these data indicate that FADD negatively regulates LPS- and IL-1β–induced NF-κB activation and that this regulation occurs upstream of IκB degradation

Phosphoinositide 3 Kinase Mediates Toll-Like Receptor 4-Induced Activation of NF-κB in Endothelial Cells

Many of the proinflammatory effects of gram-negative bacteria are elicited by the interaction of bacterial lipopolysaccharide (LPS) with Toll-like receptor 4 (TLR4) expressed on host cells. TLR4 signaling leads to activation of NF-κB and transcription of many genes involved in the inflammatory response. In this study, we examined the signaling pathways involved in NF-κB activation by TLR4 signaling in human microvascular endothelial cells. Akt is a major downstream target of phosphoinositide 3 kinase (PI3-kinase), and PI3-kinase activation is necessary and sufficient for Akt phosphorylation. Consequently, Akt kinase activation was used as a measure of PI3-kinase activity. In a stable transfection system, dominant-negative mutants of myeloid differentiation factor 88 (MyD88) and interleukin-1 (IL-1) receptor-associated kinase 1 (IRAK-1) (MyD88-TIR and IRAK-DD, respectively) blocked Akt kinase activity in response to LPS and IL-1β. A dominant-negative mutant (Mal-P/H) of MyD88 adapter-like protein (Mal), a protein with homology to MyD88, failed to inhibit LPS- or IL-1β-induced Akt activity. Moreover, a dominant-negative mutant of p85 (p85-DN) inhibited the NF-κB luciferase activity, IL-6 production, and IκBα degradation elicited by LPS and IL-1β but not that stimulated by tumor necrosis factor alpha. The dominant-negative mutant of Akt partially inhibited the NF-κB luciferase activity evoked by LPS and IL-1β. However, expression of a constitutively activated Akt failed to induce NF-κB luciferase activity. These findings indicate that TLR4- and IL-1R-induced PI3-kinase activity is mediated by the adapter proteins MyD88 and IRAK-1 but not Mal. Further, these studies suggest that PI3-kinase is an important mediator of LPS and IL-1β signaling leading to NF-κB activation in endothelial cells and that Akt is necessary but not sufficient for NF-κB activation by TLR4

Bcl-2 Overexpression Leads to Increases in Suppressor of Cytokine Signaling-3 Expression in B Cells and De Novo Follicular Lymphoma

The t(14;18)(q32;q21), resulting in deregulated expression of B-cell-leukemia/lymphoma-2 (Bcl-2), represents the genetic hallmark in human follicular lymphomas. Substantial evidence supports the hypothesis that the t(14;18) and Bcl-2 overexpression are necessary but not solely responsible for neoplastic transformation and require cooperating genetic derangements for neoplastic transformation to occur. To investigate genes that cooperate with Bcl-2 to influence cellular signaling pathways important for neoplastic transformation, we used oligonucleotide microarrays to determine differential gene expression patterns in CD19+ B cells isolated from Eμ-Bcl-2 transgenic mice and wild-type littermate control mice. Fifty-seven genes were induced and 94 genes were repressed by ≥2-fold in Eμ-Bcl-2 transgenic mice (P \u3c 0.05). The suppressor of cytokine signaling-3 (SOCS3) gene was found to be overexpressed 5-fold in B cells from Eμ-Bcl-2 transgenic mice. Overexpression of Bcl-2 in both mouse embryo fibroblast-1 and hematopoietic cell lines resulted in induction of SOCS3 protein, suggesting a Bcl-2-associated mechanism underlying SOCS3 induction. Immunohistochemistry with SOCS3 antisera on tissue from a cohort of patients with de novo follicular lymphoma revealed marked overexpression of SOCS3 protein that, within the follicular center cell region, was limited to neoplastic follicular lymphoma cells and colocalized with Bcl-2 expression in 9 of 12 de novo follicular lymphoma cases examined. In contrast, SOCS3 protein expression was not detected in the follicular center cell region of benign hyperplastic tonsil tissue. These data suggest that Bcl-2 overexpression leads to the induction of activated signal transducer and activator of transcription 3 (STAT3) and to the induction of SOCS3, which may contribute to the pathogenesis of follicular lymphoma

The role of institutional quality in the international trade of a Latin American country: evidence from Colombian export performance

Abstract This paper analyses the relevance of Colombian institutional quality in recent years in terms of the performance of its exports within a framework of trade openness. Based on the trade gravity model, we examine the effect of governance on the evolution of Colombian exports through an econometric approach that identifies, on the one hand, the influence of institutional quality, and on the other hand, the influence of the institutional distance between Colombia and its trading partners. We use a panel data set for 2005–2018, through which the export flows from Colombia to 136 of its trading partners are considered. The findings indicate that Colombian institutional quality and the institutional distance between the country and its partners are statistically significant and affect its foreign sales. Similarly, there is a prominent influence of regulatory quality and the rule of law variables in the performance of Colombian exports in relation to other variables included in the model. We conclude that the Colombian government must improve its institutional quality considerably as a fundamental step towards boosting its overseas sales, not least because the country’s institutional distance from the world average is notable, which also affects its exports