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

in Journal of Biological Chemistry (2007), 282(29), 21308-21318

IKK alpha regulates many chromatin events in the nuclear phase of the NF-kappa B program, including phosphorylation of histone H3 and removal of co-repressors from NF-kappa B-dependent promoters. However ... [more ▼]

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

Stat5 Is an Ambivalent Regulator of Neutrophil Homeostasis

in PLoS ONE (2007), 2(1), 727

Although STAT5 promotes survival of hematopoietic progenitors, STAT5-/- mice develop mild neutrophilia. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that in STAT5-/- mice, liver endothelial cells (LECs ... [more ▼]

Although STAT5 promotes survival of hematopoietic progenitors, STAT5-/- mice develop mild neutrophilia. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that in STAT5-/- mice, liver endothelial cells (LECs) autonomously secrete high amounts of G-CSF, allowing myeloid progenitors to overcompensate for their intrinsic survival defect. However, when injected with pro-inflammatory cytokines, mutant mice cannot further increase neutrophil production, display a severe deficiency in peripheral neutrophil survival, and are therefore unable to maintain neutrophil homeostasis. In wild-type mice, inflammatory stimulation induces rapid STAT5 degradation in LECs, G-CSF production by LECs and other cell types, and then sustained mobilization and expansion of long-lived neutrophils. CONCLUSION: We conclude that STAT5 is an ambivalent factor. In cells of the granulocytic lineage, it exerts an antiapoptotic function that is required for maintenance of neutrophil homeostasis, especially during the inflammatory response. In LECs, STAT5 negatively regulates granulopoiesis by directly or indirectly repressing G-CSF expression. Removal of this STAT5-imposed brake contributes to induction of emergency granulopoiesis. [less ▲]

Potentiation of tumor necrosis factor-induced NF-kappa B activation by deacetylase inhibitors is associated with a delayed cytoplasmic reappearance of I kappa B alpha

in Molecular and Cellular Biology (2003), 23(17), 6200-6209

Previous studies have implicated acetylases and deacetylases in regulating the transcriptional activity of NF-kappaB. Here, we show that inhibitors of deacetylases such as trichostatin A (TSA) and sodium ... [more ▼]

Previous studies have implicated acetylases and deacetylases in regulating the transcriptional activity of NF-kappaB. Here, we show that inhibitors of deacetylases such as trichostatin A (TSA) and sodium butyrate (NaBut) potentiated TNF-induced expression of several natural NF-kappaB-driven promoters. This transcriptional synergism observed between TNF and TSA (or NaBut) required intact kappaB sites in all promoters tested and was biologically relevant as demonstrated by RNase protection on two instances of endogenous NF-kappaB-regulated gene transcription. Importantly, TSA prolonged both TNF-induced DNA-binding activity and the presence of NF-kappaKB in the nucleus. We showed that the p65 subunit of NF-kappaB was acetylated in vivo. However, this acetylation was weak, suggesting that other mechanisms could be implicated in the potentiated binding and transactivation activities of NF-kappaB after TNF plus TSA versus TNF treatment. Western blot and immunofluorescence confocal microscopy experiments revealed a delay in the cytoplasmic reappearance of the IkappaBalpha inhibitor that correlated temporally with the prolonged intranuclear binding and presence of NF-kappaB. This delay was due neither to a defect in IkappaBalpha mRNA production nor to a nuclear retention of IkappaBalpha but was rather due to a persistent proteasome-mediated degradation of IkappaBalpha. A prolongation of IkappaB kinase activity could explain, at least partially, the delayed IkappaBalpha cytoplasmic reappearance observed in presence of TNF plus TSA. [less ▲]

Purification of bovine leukemia virus gp70 and bovine leukemia virus p24. Detection by radioimmunoassay of antibodies directed against these antigens.

in Burny, Arsène (Ed.) Bovine leukosis: various methods of molecular virology (1977)