Julie Horion

Withaferin a strongly elicits IkappaB kinase beta hyperphosphorylation concomitant with potent inhibition of its kinase activity

The transcription factor NFκB plays a critical role in normal and pathophysiological immune responses. Therefore, NFκB and the signaling pathways that regulate its activation have become a major focus of drug development programs. Withania somnifera (WS) is a medicinal plant that is widely used in Palestine for the treatment of various inflammatory disorders. In this study we show that the leave extract of WS, as well as its major constituent withaferin A (WA), potently inhibits NFκB activation by preventing the tumor necrosis factor-induced activation of IκB kinase β via a thioalkylation-sensitive redox mechanism, whereas other WS-derived steroidal lactones, such as withanolide A and 12-deoxywithastramonolide, are far less effective. To our knowledge, this is the first communication of IκB kinase β inhibition by a plant-derived inhibitor, coinciding with MEK1/ERK-dependent Ser-181 hyperphosphorylation. This prevents IκB phosphorylation and degradation, which subsequently blocks NFκB translocation, NFκB/DNA binding, and gene transcription. Taken together, our results indicate that pure WA or WA-enriched WS extracts can be considered as a novel class of NFκB inhibitors, which hold promise as novel anti-inflammatory agents for treatment of various inflammatory disorders and/or cancer.

DNA methylation and cancer diagnosis: new methods and applications.

Methylation of cytosines in cytosine–guanine (CpG) dinucleotides is one of the most important epigenetic alterations in animals. The presence of methylcytosine in the promoter of specific genes has profound consequences on local chromatin structure and on the regulation of gene expression. Changes in DNA methylation play a central role in carcinogenesis. Hypermethylation and consecutive transcriptional silencing of tumor-suppressor genes has been documented in numerous cancers. The identification of target genes silenced by this modification has a great impact on diagnosis, classification, definition of risk groups and prognosis of cancer patients. Here we outline genome-wide techniques aiming at the identification of relevant methylated promoters. Methods and applications allowing clinicians to monitor the methylation of target genes will be also reviewed.

Promoter-dependent Effect of IKKα on NF-κB/p65 DNA Binding

IKKα regulates many chromatin events in the nuclear phase of the NF-κB program, including phosphorylation of histone H3 and removal of co-repressors from NF-κB-dependent promoters. However, all of the nuclear functions of IKKα are not understood. In this study, using mouse embryonic fibroblasts IKKα knock-out and reexpressing IKKα after retroviral transduction, we demonstrate that IKKα contributes to NF-κB/p65 DNA binding activity on an exogenous κB element and on some, but not all, endogenous NF-κB-target promoters. Indeed, p65 chromatin immunoprecipitation assays revealed that IKKα is crucial for p65 binding on κB sites of icam-1 and mcp-1 promoters but not on iκbα promoter. The mutation of IKKα putative nuclear localization sequence, which prevents its nuclear translocation, or of crucial serines in the IKKα activation loop completely inhibits p65 binding on icam-1 and mcp-1 promoters and rather enhances p65 binding on the iκbα promoter. Further molecular studies demonstrated that the removal of chromatin-bound HDAC3, a histone deacetylase inhibiting p65 DNA binding, is differentially regulated by IKKα in a promoter-specific manner. Indeed, whereas the absence of IKKα induces HDAC3 recruitment and repression on the icam-1 promoter, it has an opposite effect on the iκbα promoter, where a better p65 binding occurs. We conclude that nuclear IKKα is required for p65 DNA binding in a gene-specific manner.

Promoter-dependent effect of IKK alpha on NF-kappa B/p65 DNA binding

IKKα regulates many chromatin events in the nuclear phase of the NF-κB program, including phosphorylation of histone H3 and removal of co-repressors from NF-κB-dependent promoters. However, all of the nuclear functions of IKKα are not understood. In this study, using mouse embryonic fibroblasts IKKα knock-out and reexpressing IKKα after retroviral transduction, we demonstrate that IKKα contributes to NF-κB/p65 DNA binding activity on an exogenous κB element and on some, but not all, endogenous NF-κB-target promoters. Indeed, p65 chromatin immunoprecipitation assays revealed that IKKα is crucial for p65 binding on κB sites of icam-1 and mcp-1 promoters but not on iκbα promoter. The mutation of IKKα putative nuclear localization sequence, which prevents its nuclear translocation, or of crucial serines in the IKKα activation loop completely inhibits p65 binding on icam-1 and mcp-1 promoters and rather enhances p65 binding on the iκbα promoter. Further molecular studies demonstrated that the removal of chromatin-bound HDAC3, a histone deacetylase inhibiting p65 DNA binding, is differentially regulated by IKKα in a promoter-specific manner. Indeed, whereas the absence of IKKα induces HDAC3 recruitment and repression on the icam-1 promoter, it has an opposite effect on the iκbα promoter, where a better p65 binding occurs. We conclude that nuclear IKKα is required for p65 DNA binding in a gene-specific manner.

Actin cytoskeleton differentially modulates NF-κB-mediated IL-8 expression in myelomonocytic cells

Many physiopathological events such as phagocytosis, pathogen invasion, cellular adhesion and chemotaxis governed by actin-based cytoskeleton are often accompanied by nuclear factor kappaB (NF-kappaB) activation and expression of pro-inflammatory genes. In the present study, we demonstrated that reorganization of actin cytoskeleton induced by Cytochalasin D (CytD), an actin-polymerization inhibitor, enhanced il-8 gene expression induced by TNFalpha and LPS in HL-60 monocyte-like cells. Both transcriptional and post-transcriptional mechanisms were involved. CytD potentiated NF-kappaB-mediated transcription induced by both TNFalpha and LPS but via different mechanisms. In the case of LPS, the perturbation of actin dynamics increased the TLR4 levels at the cell membrane and consequently enhanced the IKK complex activation and NF-kappaB nuclear translocation. However, the canonical pathway involving the IKK complex and leading to the NF-kappaB translocation into the nucleus was not affected by actin remodelling in the case of TNFalpha. Interestingly, actin disruption primed p65 phosphorylation induced by TNFalpha and LPS, on Ser(276) and Ser(536), respectively, which suggested actin cytoskeleton could also modulate p65 transactivating activity.

Histone Deacetylase Inhibitor Trichostatin A Sustains Sodium Pervanadate-induced NF-κB Activation by Delaying IκBα mRNA Resynthesis COMPARISON WITH TUMOR NECROSIS FACTOR α

NF-κB is a crucial transcription factor tightly regulated by protein interactions and post-translational modifications, like phosphorylation and acetylation. A previous study has shown that trichostatin A (TSA), a histone deacetylase inhibitor, potentiates tumor necrosis factor (TNF) α-elicited NF-κB activation and delays IκBα cytoplasmic reappearance. Here, we demonstrated that TSA also prolongs NF-κB activation when induced by the insulino-mimetic pervanadate (PV), a tyrosine phosphatase inhibitor that initiates an atypical NF-κB signaling. This extension is similarly correlated with delayed IκBα cytoplasmic reappearance. However, whereas TSA causes a prolonged IKK activity when added to TNFα, it does not when added to PV. Instead, quantitative reverse transcriptase-PCR revealed a decrease of iκbα mRNA level after TSA addition to PV stimulation. This synthesis deficit of the inhibitor could explain the sustained NF-κB residence in the nucleus. In vivo analysis by chromatin immunoprecipitation assays uncovered that, for PV induction but not for TNFα, the presence of TSA provokes several impairments on the iκbα promoter: (i) diminution of RNA Pol II recruitment; (ii) reduced acetylation and phosphorylation of histone H3-Lys14 and -Ser10, respectively; (iii) decreased presence of phosphorylated p65-Ser536; and (iv) reduction of IKKα binding. The recruitment of these proteins on the icam-1 promoter, another NF-κB-regulated gene, is not equally affected, suggesting a promoter specificity of PV with TSA stimulation. Taken together, these data suggest that TSA acts differently depending on the NF-κB pathway and the targeted promoter in question. This indicates that one overall histone deacetylase role is to inhibit NF-κB activation by molecular mechanisms specific of the stimulus and the promoter.

Histone deacetylase inhibitor trichostatin a sustains sodium pervanadate-induced NF-κB activation by delaying IκBα mRNA resynthesis - comparison with TNFα

NF-κB is a crucial transcription factor tightly regulated by protein interactions and post-translational modifications, like phosphorylation and acetylation. A previous study has shown that trichostatin A (TSA), a histone deacetylase inhibitor, potentiates tumor necrosis factor (TNF) α-elicited NF-κB activation and delays IκBα cytoplasmic reappearance. Here, we demonstrated that TSA also prolongs NF-κB activation when induced by the insulino-mimetic pervanadate (PV), a tyrosine phosphatase inhibitor that initiates an atypical NF-κB signaling. This extension is similarly correlated with delayed IκBα cytoplasmic reappearance. However, whereas TSA causes a prolonged IKK activity when added to TNFα, it does not when added to PV. Instead, quantitative reverse transcriptase-PCR revealed a decrease of iκbα mRNA level after TSA addition to PV stimulation. This synthesis deficit of the inhibitor could explain the sustained NF-κB residence in the nucleus. In vivo analysis by chromatin immunoprecipitation assays uncovered that, for PV induction but not for TNFα, the presence of TSA provokes several impairments on the iκbα promoter: (i) diminution of RNA Pol II recruitment; (ii) reduced acetylation and phosphorylation of histone H3-Lys14 and -Ser10, respectively; (iii) decreased presence of phosphorylated p65-Ser536; and (iv) reduction of IKKα binding. The recruitment of these proteins on the icam-1 promoter, another NF-κB-regulated gene, is not equally affected, suggesting a promoter specificity of PV with TSA stimulation. Taken together, these data suggest that TSA acts differently depending on the NF-κB pathway and the targeted promoter in question. This indicates that one overall histone deacetylase role is to inhibit NF-κB activation by molecular mechanisms specific of the stimulus and the promoter.

Varicella-Zoster Virus Modulates NF-κB Recruitment on Selected Cellular Promoters

ABSTRACT Intercellular adhesion molecule 1 (ICAM-1) expression is down-regulated in the center of cutaneous varicella lesions despite the expression of proinflammatory cytokines such as gamma interferon and tumor necrosis factor alpha (TNF-α). To study the molecular basis of this down-regulation, the ICAM-1 induction of TNF-α was analyzed in varicella-zoster virus (VZV)-infected melanoma cells (MeWo), leading to the following observations: (i) VZV inhibits the stimulation of icam-1 mRNA synthesis; (ii) despite VZV-induced nuclear translocation of p65, p52, and c-Rel, p50 does not translocate in response to TNF-α; (iii) the nuclear p65 present in VZV-infected cells is no longer associated with p50 and is unable to bind the proximal NF-κB site of the icam-1 promoter, despite an increased acetylation and accessibility of the promoter in response to TNF-α; and (iv) VZV induces the nuclear accumulation of the NF-κB inhibitor p100. VZV also inhibits icam-1 stimulation of TNF-α by strongly reducing NF-κB nuclear translocation in MRC5 fibroblasts. Taken together, these data show that VZV interferes with several aspects of the immune response by inhibiting NF-κB binding and the expression of target genes. Targeting NF-κB activation, which plays a central role in innate and adaptive immune responses, leads to obvious advantages for the virus, particularly in melanocytes, which are a site of viral replication in the skin.