Ivana Vancurova

NF-κB Activation in Tumor Necrosis Factor α-stimulated Neutrophils Is Mediated by Protein Kinase Cδ CORRELATION TO NUCLEAR IκBα

The transcription factor NF-κB is critical for the expression of multiple genes involved in inflammatory responses and apoptosis. However, the signal transduction pathways regulating NF-κB activation in human neutrophils in response to stimulation with tumor necrosis factor-α (TNFα) are undefined. Since recent studies implicated activation of NF-κB as well as protein kinase C-δ (PKCδ) in neutrophil apoptosis, we investigated involvement of PKCδ in the activation of NF-κB in TNFα-stimulated neutrophils. Specific inhibition of PKCδ by rottlerin prevented IκBα degradation and NF-κB activation in TNFα-stimulated neutrophils. This regulation of NF-κB activation by PKCδ was specific only for TNFα signaling, since lipopolysaccharide- or interleukin-1β-induced NF-κB activation and IκBα degradation were not inhibited by rottlerin. In addition, we show that in human neutrophils, but not monocytes, IκBα localizes in significant amounts in the nucleus of unstimulated cells, and the amount of IκBα in the nucleus, as well as in the cytoplasm, correlates with the NF-κB DNA binding. These results suggest that in human neutrophils, the presence of IκBα in the nucleus may function as a safeguard against initiation of NF-κB dependent transcription of pro-inflammatory and anti-apoptotic genes, and represents a distinct and novel mechanism of NF-κB regulation.

NF-kappaB Activation in TNFalpha-Stimulated Neutrophils is Mediated by Protein Kinase C-delta: Correlation to Nuclear I-kappaB-alpha

The transcription factor NF-κB is critical for the expression of multiple genes involved in inflammatory responses and apoptosis. However, the signal transduction pathways regulating NF-κB activation in human neutrophils in response to stimulation with tumor necrosis factor-α (TNFα) are undefined. Since recent studies implicated activation of NF-κB as well as protein kinase C-δ (PKCδ) in neutrophil apoptosis, we investigated involvement of PKCδ in the activation of NF-κB in TNFα-stimulated neutrophils. Specific inhibition of PKCδ by rottlerin prevented IκBα degradation and NF-κB activation in TNFα-stimulated neutrophils. This regulation of NF-κB activation by PKCδ was specific only for TNFα signaling, since lipopolysaccharide- or interleukin-1β-induced NF-κB activation and IκBα degradation were not inhibited by rottlerin. In addition, we show that in human neutrophils, but not monocytes, IκBα localizes in significant amounts in the nucleus of unstimulated cells, and the amount of IκBα in the nucleus, as well as in the cytoplasm, correlates with the NF-κB DNA binding. These results suggest that in human neutrophils, the presence of IκBα in the nucleus may function as a safeguard against initiation of NF-κB dependent transcription of pro-inflammatory and anti-apoptotic genes, and represents a distinct and novel mechanism of NF-κB regulation.

NF-κB Regulation in Human Neutrophils by Nuclear IκBα: Correlation to Apoptosis

Neutrophils are among the first circulating leukocytes involved in acute inflammatory processes. Transcription factor NF-κB plays a key role in the inflammatory response, regulating the expression of proinflammatory and anti-apoptotic genes. Recently we have shown that human neutrophils contain a significant amount of NF-κB inhibitor, IκBα, in the nucleus of unstimulated cells. The present objective was to examine the mechanisms controlling the nuclear content of IκBα in human neutrophils and to determine whether increased accumulation of IκBα in the nucleus is associated with increased neutrophil apoptosis. We show for the first time that neutrophil stimulation with pro-inflammatory signals results in degradation of IκBα that occurs in both cytoplasm and nucleus. Prolonged (2-h) stimulation with TNF and LPS induces resynthesis of IκBα that is again translocated to the nucleus in human neutrophils, but not in monocytic cells. Leptomycin B, a specific inhibitor of nuclear export, increases nuclear accumulation of IκBα in stimulated neutrophils by blocking the IκBα nuclear export, and this is associated with inhibition of NF-κB activity, induction of caspase-3 activation, and apoptosis. Based on our data we present a new model of NF-κB regulation in human neutrophils by nuclear IκBα. Our results demonstrate that the NF-κB activity in human neutrophils is regulated by mechanisms clearly different from those in monocytes and other human cells and suggest that the increased nuclear content of IκBα in human neutrophils might represent one of the underlying mechanisms for the increased apoptosis in these cells.

Bortezomib Induces Nuclear Translocation of IκBα Resulting in Gene-Specific Suppression of NF-κB–Dependent Transcription and Induction of Apoptosis in CTCL

Cutaneous T-cell lymphoma (CTCL) is characterized by constitutive activation of nuclear factor κB (NF-κB), which plays a crucial role in the survival of CTCL cells and their resistance to apoptosis. NF-κB activity in CTCL is inhibited by the proteasome inhibitor bortezomib; however, the mechanisms remained unknown. In this study, we investigated mechanisms by which bortezomib suppresses NF-κB activity in CTCL Hut-78 cells. We demonstrate that bortezomib and MG132 suppress NF-κB activity in Hut-78 cells by a novel mechanism that consists of inducing nuclear translocation and accumulation of IκBα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha), which then associates with NF-κB p65 and p50 in the nucleus and inhibits NF-κB DNA binding activity. Surprisingly, however, while expression of NF-κB–dependent antiapoptotic genes cIAP1 and cIAP2 is inhibited by bortezomib, expression of Bcl-2 is not suppressed. Chromatin immunoprecipitation indicated that cIAP1 and cIAP2 promoters are occupied by NF-κB p65/50 heterodimers, whereas Bcl-2 promoter is occupied predominantly by p50/50 homodimers. Collectively, our data reveal a novel mechanism of bortezomib function in CTCL and suggest that the inhibition of NF-κB–dependent gene expression by bortezomib is gene specific and depends on the subunit composition of NF-κB dimers recruited to NF-κB–responsive promoters. Mol Cancer Res; 9(2); 183–94. ©2011 AACR.

Proapoptotic effect of curcumin on human neutrophils: activation of the p38 mitogen-activated protein kinase pathway.

Objective:Despite advances in the management of sepsis and acute respiratory distress syndrome, the mortality rate remains high. Delayed apoptosis of neutrophils is associated with multiple organ failure under those conditions. Thus, development of nontoxic neutrophil apoptosis regulating molecules may provide a novel therapeutic strategy. Curcumin is a promising dietary supplement for cancer prevention. However, the effect of curcumin on human neutrophil apoptosis remains unknown. We therefore hypothesized that curcumin would produce a proapoptotic effect on neutrophils. Design:Prospective, controlled, and randomized in vitro study. Setting:Research institute laboratory. Subjects:Human peripheral neutrophils obtained from normal subjects. Interventions:None. Measurements and Main Results:In the presence or absence of curcumin, both spontaneous neutrophil apoptosis and apoptosis of neutrophils following transmigration across a human lung endothelium-epithelium bilayer were studied by morphology and terminal dUTP nucleotide end labeling analyses, respectively. Myeloperoxidase activity and migration assays were performed to determine the impact of curcumin on neutrophil function. To elucidate the potential mechanism, the p38 mitogen-activated protein kinase pathway and caspase-3 activity were examined by Western blotting and enzymatic analyses. The data demonstrate that curcumin increased constitutive neutrophil apoptosis and abrogated the transbilayer migration-induced delay in neutrophil apoptosis. Neutrophil activation was reduced by curcumin treatment as evidenced by a decrease in migration and myeloperoxidase release. A marked increase in p38 phosphorylation and caspase-3 activity was observed following curcumin exposure. In addition, inhibition of p38 mitogen-activated protein kinase with SB203580 suppressed apoptosis and caspase-3 activation induced by curcumin. Thus, activation of p38 mitogen-activated protein kinase or an increase in caspase-3 activity appears to contribute to the proapoptotic effect of human neutrophil apoptosis by curcumin. Conclusion:The characteristics of curcumin, including its proapoptotic effect and antidegranulation effect, make it a potential candidate for the therapy of neutrophil-induced lung injury and sepsis.

Okadaic acid induces sustained activation of NFκB and degradation of the nuclear IκBα in human neutrophils

Abstract Human neutrophils differ from other cells by containing high amount of IκBα in the nucleus, and this increased nuclear IκBα accumulation is associated with the inhibition of NFκB activity and increased apoptosis. However, the mechanisms regulating NFκB activation and IκBα degradation in human neutrophils are little understood. The objective of this study was to provide a further insight into the mechanisms regulating NFκB activity and IκBα degradation in human neutrophils. We show that okadaic acid (OA), an inhibitor of protein phosphatases PP1 and PP2A, induces sustained activation of NFκB and degradation of the nuclear IκBα, and increases interleukin-8 expression in the neutrophils. Furthermore, inhibitors of protein kinase C-δ (PKCδ) and IκB kinase (IKK) inhibit the OA-induced activation of NFκB. Collectively, our results indicate that in human neutrophils, the sustained activation of NFκB is regulated by a continuous phosphorylation and degradation of the nuclear IκBα.

Activation of Nuclear Factor-κB and Its Suppression by Dexamethasone in Polymorphonuclear Leukocytes: Newborn Versus Adult

Polymorphonuclear leukocytes (PMN) of the newborn, to a greater extent than those of the adult, have the ability to amplify PMN recruitment to an inflammatory site by their own release of IL-8, and this process is inhibited by dexamethasone. The aim of the present study was to determine whether the regulation of nuclear factor-κB (NF-κB) could explain the previous observations. NF-κB is a transcription factor pivotal for expression of genes encoding inflammatory cytokines such as IL-8, but NF-κB has not been previously studied in the PMN of the newborn. NF-κB activation was measured by an electrophoretic mobility shift assay in nuclear extracts prepared from PMN isolated from adults and cord blood from newborns. Two distinct molecular forms of NF-κB were identified after tumor necrosis factor-α stimulation; this included the previously characterized p50/65 heterodimer and a newly identified p50/50 homodimer. Both NF-κB dimers were activated by tumor necrosis factor-α to significantly higher levels in the neutrophil of the newborn versus adult. An additional new finding was that pretreatment of PMN with dexamethasone (10-7 M, therapeutic range) inhibited activation of both NF-κB complexes in both the newborn and the adult PMN. We conclude that the increased activation of NF-κB by the PMN of the newborn may play an important role in neonatal inflammatory reactions. Eventually, specific targeting of NF-κB activation in the neutrophil may be an effective molecular approach for the treatment of neutrophil-mediated disorders in the newborn.

Gene-Specific Repression of Proinflammatory Cytokines in Stimulated Human Macrophages by Nuclear IκBα

We have previously shown that increased nuclear accumulation of IκBα inhibits NF-κB activity and induces apoptosis in human leukocytes. In this study, we wanted to explore the possibility that the nucleocytoplasmic distribution of IκBα can be used as a therapeutic target for the regulation of NF-κB–dependent cytokine synthesis. Treatment of LPS-stimulated human U937 macrophages with an inhibitor of chromosome region maintenance 1-dependent nuclear export, leptomycin B, resulted in the increased nuclear accumulation of IκBα and inhibition of NF-κB DNA binding activity, caused by the nuclear IκBα-p65 NF-κB interaction. Surprisingly, however, whereas mRNA expression and cellular release of TNF-α, the β form of pro-IL-1 (IL-1β), and IL-6 were inhibited by the leptomycin B-induced nuclear IκBα, IL-8 mRNA expression and cellular release were not significantly affected. Analysis of in vivo recruitment of p65 NF-κB to NF-κB–regulated promoters by chromatin immunoprecipitation in U937 cells and human PBMCs indicated that although the p65 recruitment to TNF-α, IL-1β, and IL-6 promoters was inhibited by the nuclear IκBα, p65 recruitment to IL-8 promoter was not repressed. Chromatin immunoprecipitation analyses using IκBα and S536 phosphospecific p65 NF-κB Abs demonstrated that although the newly synthesized IκBα induced by postinduction repression is recruited to TNF-α, IL-1β, and IL-6 promoters but not to the IL-8 promoter, S536-phosphorylated p65 is recruited to IL-8 promoter, but not to TNF-α, IL-1β, or IL-6 promoters. Together, these data indicate that the inhibition of NF-κB–dependent transcription by nuclear IκBα in LPS-stimulated macrophages is gene specific and depends on the S536 phosphorylation status of the recruited p65 NF-κB.

Increased p50/p50 NF-κB Activation in Human Papillomavirus Type 6- or Type 11-Induced Laryngeal Papilloma Tissue

ABSTRACT We have observed elevated NF-κB DNA-binding activity in nuclear extracts from human papillomavirus type 6- and 11-infected laryngeal papilloma tissues. The predominant DNA-binding species is the p50/p50 homodimer. The elevated NF-κB activity could be correlated with a reduced level of cytoplasmic IκBβ and could be associated with the overexpression of p21 CIP1/WAF1 in papilloma cells. Increased NF-κB activity and cytoplasmic accumulation of p21 CIP1/WAF1 might counteract death-promoting effects elicited by overexpressed PTEN and reduced activation of Akt and STAT3 previously noted in these tissues.

Proteasome inhibition increases recruitment of IκB kinase β (IKKβ), S536P-p65, and transcription factor EGR1 to interleukin-8 (IL-8) promoter, resulting in increased IL-8 production in ovarian cancer cells.

Background: IL-8 promotes angiogenesis and metastasis in ovarian cancer. Results: Proteasome inhibition induces specific recruitment of IKKβ, EGR-1, and S536P-p65 to the IL-8 promoter. Conclusion: The increased IKKβ, EGR-1, and S536P-p65 recruitment results in the increased IL-8 expression and release in ovarian cancer cells. Significance: The BZ-increased IL-8 release may be responsible for the BZ-limited effectiveness in ovarian cancer treatment. Proinflammatory and pro-angiogenic chemokine interleukin-8 (IL-8, CXCL8) contributes to ovarian cancer progression through its induction of tumor cell proliferation, survival, angiogenesis, and metastasis. Proteasome inhibition by bortezomib, which has been used as a frontline therapy in multiple myeloma, has shown only limited effectiveness in ovarian cancer and other solid tumors. However, the responsible mechanisms remain elusive. Here, we show that proteasome inhibition dramatically increases the IL-8 expression and release in ovarian cancer cells. The responsible mechanism involves an increased nuclear accumulation of IκB kinase β (IKKβ) and an increased recruitment of the nuclear IKKβ, p65-phosphorylated at Ser-536, and the transcription factor early growth response-1 (EGR-1) to the endogenous IL-8 promoter. Coimmunoprecipitation studies identified the nuclear EGR-1 associated with IKKβ and with p65, with preferential binding to S536P-p65. Both IKKβ activity and EGR-1 expression are required for the increased IL-8 expression induced by proteasome inhibition in ovarian cancer cells. Interestingly, in multiple myeloma cells the IL-8 release is not increased by bortezomib. Together, these data indicate that the increased IL-8 release may represent one of the underlying mechanisms responsible for the decreased effectiveness of proteasome inhibition in ovarian cancer treatment and identify IKKβ and EGR-1 as potential new targets in ovarian cancer combination therapies.