Transactivation Of Nuclear factor-kappaB Research Articles

Regulation of cells of the arterial wall by hypoxia and its role in the development of atherosclerosis.

The cell's response to hypoxia depends on stabilization of the hypoxia-inducible factor 1 complex and transactivation of nuclear factor kappa-B (NF-κB). HIF target gene transcription in cells resident to atherosclerotic lesions adjoins a complex interplay of cytokines and mediators of inflammation affecting cholesterol uptake, migration, and inflammation. Maladaptive activation of the HIF-pathway and transactivation of nuclear factor kappa-B causes monocytes to invade early atherosclerotic lesions, maintaining inflammation and aggravating a low-oxygen environment. Meanwhile HIF-dependent upregulation of the ATP-binding cassette transporter ABCA1 causes attenuation of cholesterol efflux and ultimately macrophages becoming foam cells. Hypoxia facilitates neovascularization by upregulation of vascular endothelial growth factor (VEGF) secreted by endothelial cells and vascular smooth muscle cells lining the arterial wall destabilizing the plaque. HIF-knockout animal models and inhibitor studies were able to show beneficial effects on atherogenesis by counteracting the HIF-pathway in the cell wall. In this review the authors elaborate on the up-to-date literature on regulation of cells of the arterial wall through activation of HIF-1α and its effect on atherosclerotic plaque formation.

The Bradykinin-BDKRB1 Axis Regulates Aquaporin 4 Gene Expression and Consequential Migration and Invasion of Malignant Glioblastoma Cells via a Ca2+-MEK1-ERK1/2-NF-κB Mechanism.

Glioblastoma multiforme (GBM) is the most common form of brain tumor and is very aggressive. Rapid migration and invasion of glioblastoma cells are two typical features driving malignance of GBM. Bradykinin functionally prompts calcium influx via activation of bradykinin receptor B1/B2 (BDKRB1/2). In this study, we evaluated the roles of bradykinin in migration and invasion of glioblastoma cells and the possible mechanisms. Expressions of aquaporin 4 (AQP4) mRNA and protein were upregulated in human glioblastomas. Furthermore, exposure of human U87 MG glioblastoma cells to bradykinin specifically increased levels of BDKRB1. Successively, bradykinin stimulated influx of calcium, phosphorylation of MEK1 and extracellular signal-regulated kinase (ERK)1/2, translocation and transactivation of nuclear factor-kappaB (NF-κB), and expressions of AQP4 mRNA and protein. Concomitantly, migration and invasion of human glioblastoma cells were elevated by bradykinin. Knocking-down BDKRB1 concurrently decreased AQP4 mRNA expression and cell migration and invasion. The bradykinin-induced effects were further confirmed in murine GL261 glioblastoma cells. Therefore, bradykinin can induce AQP4 expression and subsequent migration and invasion through BDKRB1-mediated calcium influx and subsequent activation of a MEK1-ERK1/2-NF-κB pathway. The bradykinin-BDKRB1 axis and AQP4 could be precise targets for treating GBM patients.

Abstract 481: Dualistic regulation of NF-kB signaling by CD99 and EWS-FLI1 in Ewing sarcoma: impact on cell differentiation

Abstract Introduction: Ewing sarcoma (ES) is a high-grade rare malignant bone tumor in children and adolescents. This disease is primarily characterized by: i) Chromosomal translocations that fuse EWS gene and a gene of the ETS family of transcription factors (around 85% EWS-FLI1 and 10% EWS-ERG, respectively) which are the oncogenic drivers of this tumor; ii) High expression of CD99, a cell surface glycoprotein virtually present in all of ES cells. EWS-FLI1 and CD99 were reported to have divergent roles in modulating ES malignancy and neural differentiation In this study we investigated the possible role of nuclear factor-kappa B (NF-kB) signaling pathway on the neural differentiation of ES cells and its relationship with EWS-FLI1 and CD99. Materials and Methods: Transcriptional activity of several transcription factors (AP1, SRE, CREB, E2F and NF-kB) were evaluated by gene reporter assay. Using a knockdown approach in stable and transient conditions we silenced CD99, EWS-FLI1 and NF-kB itself, alone or in combination, in three ES cell lines (TC-71, IOR/CAR and ASP14). NF-kBp65 was also transiently overexpressed in TCshCD99 and CARshCD99 clones with pCMVp65 construct. Neural differentiation was detected evaluating β-III tubulin by immunofluorescence microscopy. Results: NF-kB was found heterogeneously expressed in a panel of ES cell lines. Stable CD99 silencing was found to decrease NF-kB activity (pValue <0.05) without any differences at RNA and protein levels. On the contrary, EWS-FLI1 knockdown increased NF-kB transactivation (pValue <0.01). CD99 affects NF-kBp65 activity and its effect are dominant with respect to EWS-FLI1. Accordingly, EWS-FLI1 silencing was not able to revert the reduction of NF-kB activity observed after CD99 deprivation. In addition cells silenced for CD99 but not for EWS-FLI1 (TCshCD99 and CARshCD99 clones) showed a neural differentiated phenotype, which was lost when cells were induced to re-express NF-kB. Conclusion: Our findings support the concept that inhibition of NF-kB is able to induce terminal neural differentiation of ES cells. This identifying NF-kB as a potential therapeutic target against Ewing sarcoma progression. (Supported by AIRC_2013_IG 14049 and Liddy Shriver Foundation) Citation Format: Selena Ventura, Chiara Manzalini, Dave Aryee, Piero Picci, Heinrich Kovar, Katia Scotlandi. Dualistic regulation of NF-kB signaling by CD99 and EWS-FLI1 in Ewing sarcoma: impact on cell differentiation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 481. doi:10.1158/1538-7445.AM2014-481

Sphingosine kinase regulates oxidized low density lipoprotein-mediated calcium oscillations and macrophage survival

We recently reported that oxidized LDL (oxLDL) induces an oscillatory increase in intracellular calcium ([Ca(2+)](i)) levels in macrophages. Furthermore, we have shown that these [Ca(2+)](i) oscillations mediate oxLDL's ability to inhibit macrophage apoptosis in response to growth factor deprivation. However, the signal transduction pathways by which oxLDL induces [Ca(2+)](i) oscillations have not been elucidated. In this study, we show that these oscillations are mediated in part by intracellular mechanisms, as depleting extracellular Ca(2+) did not completely abolish the effect. Inhibiting sarco-endoplasmic reticulum ATPase (SERCA) completely blocked [Ca(2+)](i) oscillations, suggesting a role for Ca(2+) reuptake by the ER. The addition of oxLDL resulted in an almost immediate activation of sphingosine kinase (SK), which can increase sphingosine-1-phosphate (S1P) levels by phosphorylating sphingosine. Moreover, S1P was shown to be as effective as oxLDL in blocking macrophage apoptosis and producing [Ca(2+)](i) oscillations. This suggests that the mechanism in which oxLDL generates [Ca(2+)](i) oscillations may be 1) activation of SK, 2) SK-mediated increase in S1P levels, 3) S1P-mediated Ca(2+) release from intracellular stores, and 4) SERCA-mediated Ca(2+) reuptake back into the ER.

Effects of mushroom‐derived β‐glucan‐rich polysaccharide extracts on nitric oxide production by bone marrow‐derived macrophages and nuclear factor‐κB transactivation in Caco‐2 reporter cells: Can effects be explained by structure?

Mushrooms are known for their immune-modulating and anti-tumour properties. The polysaccharide fraction, mainly beta-glucans, is responsible for the immune-modulating effects. Fungal beta-glucans have been shown to activate leukocytes, which depend on structural characteristics of beta-glucans. As edible mushrooms come in contact with the intestinal immune system, effects on enterocytes are also interesting. Our aim was to evaluate the effect of mushroom polysaccharide extracts varying in beta-glucan structure on nitric oxide production by bone marrow-derived macrophages (BMMs) from mice and on nuclear factor-kappaB transactivation in human intestinal Caco-2 cells. We demonstrated that extracts from Agaricus bisporus stimulated nitric oxide production by BMM, whereas extracts from Coprinus comatus and spores of Ganoderma lucidum had only minor effects. Furthermore, extracts of A. blazei Murill and Phellinus linteus had no effect at all. Almost all mushroom extracts lowered nuclear factor-kappaB transactivation in Caco-2 cells. Structural analysis of A. bisporus compared with A. blazei Murill suggests that branching of the beta-glucan chain is essential for immune-stimulating activity. In conclusion, extracts from A. bisporus activate BMM, without activating enterocytes. These characteristics make A. bisporus an attractive candidate as a nutritional compound to stimulate the immune response in depressed states of immunity.

N-acetyl-L-cysteine inhibits adenoviral E1A-involved transactivation of nuclear factor-kappaB in rat alveolar epithelial cells.

the relationship between latent adenovirus infection and airway inflammation had not been well documented. The aim of this study was to illustrate the roles of adenovirus E1A protein on the transactivation of NF-kappaB, AP-1 in response to inflammatory stimuli and the effect of N-Acetylcysteine (NAC) upon the transactivation of NF-kappaB and AP-1 in cells stably expressing E1A protein. rat alveolar epithelial cells stably expressing adenoviral E1A or control plasmid were developed. For isolation of nuclear extracts, 5 x 10(5) cells were plated and grown overnight in 60 mm dishes. Experiments were repeated 3 times. The cell model of stably expressing adenoviral E1A was stimulated by LPS or TNF-alpha and treated with NAC, a precursor for cysteine. The NF-kappaB and AP-1 transcriptional activity were measured by LUC report system. The expression of NF-kappaB and AP-1 were measured by Western blot. Differences between groups were assessed for significance by Student' t test, and multiple comparisons were made by one-way ANOVA. the luciferase activity derived by NF-kappaB element was (9 698 +/- 98) RLU in untreated E1A-positive clones and (101 195 +/- 234), and (170 385 +/- 443) RLU in LPS and TNF-alpha-stimulated cells, which were significantly higher than that of the control group 2 077 +/- 107, 67 846 +/- 332, 95 743 +/- 211 respectively. The luciferase activity derived by AP-1 element was 9 034 +/- 78 RLU in untreated E1A-positive clones and 26 343 +/- 398 and 31 731 +/- 332 RLU in LPS and TNF-alpha-stimulated cells, which were significantly higher than that of the control group 2 845 +/- 93, 10 772 +/- 432, 11 005 +/- 556 respectively. The densitometry of the NF-kappaB expression in E1A-positive clones were 79.3 +/- 4.6 and 80.3 +/- 3.8 respectively without treatment and were 81.8 +/- 3.9 - 89.9 +/- 1.6 and 94.1 +/- 1.9 to 99.8 +/- 1.6 respectively under LPS or TNF-alpha stimulation, which were significantly higher than that of the control group (68.3 +/- 3.8, 69.4 +/- 4.3 respectively) without stimulation and 70.1 +/- 2.8 to 80.8 +/- 3.6, 73.4 +/- 4.9 to 83.2 +/- 6.7 respectively under stimulation. The level of AP-1 expression did not show difference upon treatment with LPS or TNF-alpha in either cell clones. The densitometry of the NF-kappaB expression in E1A-positive clones were 3.2 +/- 0.1 and 3.3 +/- 0.1 respectively under LPS and TNF-alpha-stimulation and 1.98 +/- 0.2 and 1.9 +/- 0.2 respectively upon treatment for LPS and TNF-alpha with NAC pre-incubation. these results indicate that E1A protein upregulated NF-kappaB transcription activity induced by LPS and TNF-alpha in rat alveolar epithelial cells and this effect could be repressed by NAC. The mechanisms underlying transactivation of NF-kappaB involved by E1A may be related to oxidative stress.

A novel histone deacetylase inhibitor prevents IL-1β induced metabolic dysfunction in pancreatic β-cells

The histone deacetylase (HDAC) inhibitor trichostatin A (TSA) has recently been shown to inhibit deleterious effects of cytokines on beta-cells, but it is unable to protect beta-cells from death due to its own cytotoxicity. Herein, we investigated novel HDAC inhibitors for their cytoprotective effects against IL-1beta-induced damage to isolated beta-cells. We report that three novel compounds (THS-73-44, THS-72-5 and THS-78-5) significantly inhibited HDAC activity and increased the acetylation of histone H4 in isolated beta-cells. Further, these compounds exerted no toxic effects on metabolic cell viability in these cells. However, among the three compounds tested, only THS-78-5 protected against IL-1beta-mediated loss in beta-cell viability. THS-78-5 was also able to attenuate IL-1beta-induced inducible nitric oxide synthase expression and subsequent NO release. Our data also indicate that the cytoprotective properties of THS-78-5 against IL-1beta-mediated effects may, in part, be due to inhibition of IL-1beta-induced transactivation of nuclear factor kappaB (NF-kappaB) in these cells. Together, we provide evidence for a novel HDAC inhibitor with a significant potential to prevent IL-1beta-mediated effects on isolated beta-cells. Potential implications of these findings in the development of novel therapeutics to prevent deleterious effects of cytokines and the onset of autoimmune diabetes are discussed.

PPARα and PPARγ effectively protect against HIV‐induced inflammatory responses in brain endothelial cells

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors which down-regulate inflammatory signaling pathways. Therefore, we hypothesized that alterations of PPAR functions can contribute to human immunodeficiency virus-1 (HIV-1)-induced dysfunction of brain endothelial cells. Indeed, treatment with HIV-1 transactivator of transcription (Tat) protein decreased PPAR transactivation in brain endothelial cells. We next stably over-expressed PPARalpha and PPARgamma in a newly developed cell line of human brain endothelial cells (hCMEC/D3 cells). Tat-induced up-regulation of inflammatory mediators, such as interleukin (IL)-1beta, tumor necrosis factor-alpha, CCL2, and E-selectin were markedly attenuated in hCMEC/D3 over-expressing PPARalpha or PPARgamma. These results were confirmed in CCL2 and E-selectin promoter activity studies. Similar protective effects were observed in hCMEC/D3 after activation of PPARgamma by exogenous PPAR agonists (dPGJ(2) and rosiglitazone). PPAR over-expression also prevented Tat-induced binding activity and transactivation of nuclear factor-kappaB. Importantly, increased PPAR activity attenuated induction of IL-1beta, tumor necrosis factor-alpha, CCL2, and E-selectin in hCMEC/D3 cells co-cultured with HIV-1-infected Jurkat cells. The protective effects of PPAR over-expression were reversed by the antagonists of PPARalpha (MK886) or PPARgamma (GW9662). The present data suggest that targeting PPAR signaling may provide a novel therapeutic approach to attenuate HIV-1-induced local inflammatory responses in brain endothelial cells.

SUMOylation Attenuates Sensitivity toward Hypoxia- or Desferroxamine-Induced Injury by Modulating Adaptive Responses in Salivary Epithelial Cells

Hypoxic stress activates various signal transduction pathways including posttranslational modification with the ubiquitin-like SUMO protein (SUMOylation). However, the molecular mechanisms by which SUMOylation regulates hypoxic responses remain unclear. Here, we investigated the ability of rat salivary Pa-4 epithelial cells to resist cell injury elicited by 1% O(2)- or hypoxia-mimetic desferroxamine (DFO)-stimulated SUMOylation processes. By using Pa-4 cells stably transduced with lenti-SUMO-1 and a cell-permeant peptide harboring SUMO-binding motif to interfere with SUMO-dependent protein-protein interactions, we demonstrate that SUMOylation augments cell survival against DFO treatment. This appeared to be partly mediated through attenuation of Protein Kinase C (PKC)-delta activation and caspase-3 cleavage, hallmarks of pro-apoptotic signaling. Intriguingly, DFO-induced phosphorylation of DNA damage marker ataxia-telangiectasia-mutated protein S1981 preceded activation of PKCdelta and caspase-3. Constitutive SUMOylation facilitated 1% O(2)- or DFO-induced nuclear factor kappaB transactivation, possibly via activation of genotoxic signaling cascade. In addition, we observed transient preservation of transepithelial electrical resistance during the early stage of hypoxia (1% O(2)) as well as enhanced transepithelial electrical resistance recovery after prolonged hypoxia in SUMO-1-expressing cell monolayers. In conclusion, our results unveil a previously unrecognized mechanism by which SUMOylation and activation of ataxia-telangiectasia-mutated protein, PKCdelta, caspase-3, and nuclear factor kappaB signaling pathways modulate salivary adaptive responses to stress in cells exposed to either 1% O(2) or DFO.

Moraxella catarrhalisstimulates the release of proinflammatory cytokines and prostaglandin E2from human respiratory epithelial cells and monocyte-derived macrophages

The outer membrane proteins of Moraxella catarrhalis, a bacterial pathogen which causes disease in both children and adults, play an important role in its phenotypic properties. However, their proinflammatory potential with regard to respiratory epithelium and macrophages is unclear. To this end, we examined the cytokine- and mediator-inducing capacity of a heat-killed wild-type M. catarrhalis strain and a nonautoagglutinating mutant as well as their outer membrane proteins and secretory/excretory products using the A549 respiratory epithelial cell line. The outer membrane proteins and secretory/excretory products from both isolates as well as the heat-killed bacteria all induced interleukin (IL)-6, IL-8 and prostaglandin E2, but not IL-1beta, from the A549 cell line in a dose- and time-dependent manner. Heat-killed bacteria and secretory/excretory products stimulated the release of IL-1beta, IL-6, IL-8 and prostaglandin E2 from human monocyte-derived macrophages. Both heat-killed isolates also stimulated nuclear translocation and transactivation of nuclear factor-kappaB. The heat-killed wild-type autoagglutinating isolate induced significantly greater amounts of IL-6 and IL-8 from A549 cells than the nonautoagglutinating mutant compared with the monocyte-derived macrophages but no significant differences in the amounts induced by the two strains were observed. These differences were also evident when the respiratory cell line was stimulated with outer membrane proteins as well as in the degree of nuclear factor-kappaB transactivation. There was little difference in the stimulatory activity of the secretory/excretory products. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analyses revealed some differences in the outer membrane proteins and secretory excretory products between the two isolates. Combined, these data show that M. catarrhalis secretory excretory products and outer membrane proteins are associated with the induction of inflammatory responses in both respiratory epithelium and macrophages.

HTLV-I Tax induces and associates with Crk-associated substrate lymphocyte type (Cas-L)

Crk-associated substrate lymphocyte type (Cas-L) is a docking protein that is heavily tyrosine phosphorylated by the engagement of beta1 integrins in T cells. In the present study, we attempted to evaluate the role of Cas-L in the pathophysiology of adult T-cell leukemia (ATL). Examination of peripheral blood mononuclear cells from ATL patients as well as ATL-derived T cell lines showed an elevation of Cas-L in these cells. We showed that tyrosine phosphorylation as well as expression of Cas-L was markedly elevated through the induction of human T-lymphotropic virus type I (HTLV-I) Tax in JPX-9 cells, with these cells showing marked motile behavior on the ligands for integrins. We next performed yeast two-hybrid screening of cDNA library from an HTLV-I-transformed T cell line, which resulted in the identification of Tax as a putative binding partner for Cas-L. Co-precipitation experiments revealed that the serine-rich region of Cas-L might serve as the binding site with the highest affinity for Tax. Co-localization study showed that Tax and Cas-L partly merged in the cytoplasm. Finally, we showed that exogenous Cas-L inhibited Tax-mediated transactivation of nuclear factor kappaB (NF-kappaB), while Tax-independent activation of NF-kappaB remained intact, hence indicating that Cas-L might specifically regulate Tax-NF-kappaB pathway.

AUTOCRINE OVER EXPRESSION OF FIBRONECTIN BY HUMAN TRANSITIONAL CARCINOMA CELLS IMPAIRS BACILLUS CALMETTE-GUERIN ADHERENCE AND SIGNALING

Bacillus Calmette-Guerin (BCG) binds to the tumor cell as a result of mycobacterial receptors for fibronectin (FN). Cell surface bound FN serves as a bridge through which BCG attaches to the tumor cell. Despite the importance of FN studies have demonstrated an idiosyncratic decrease in BCG adherence in response to exogenous FN. We evaluated the effect of exogenous and autocrine FN on the ability of BCG to adhere to the tumor cell surface and initiate cellular signaling. BCG adherence to parental 253J and FN over expressing 253JTGFbeta1-8 cells as well as to the intrinsic FN expressing cell line 647V was quantified using green fluorescent protein-BCG. Experiments were performed to assess the effect of FN on BCG initiated signal transduction through nuclear factor kappaB and AP1. Finally, the integrity of the BCG activated signaling pathway in transforming growth factor-beta1/FN over expressors was assessed using antibody mediated cross-linking of the FN receptor. BCG adherence was decreased in cell lines with high autocrine expression of FN. Exogenous FN prevented BCG induced transactivation of nuclear factor kappaB and AP1 reporter constructs. No BCG stimulated signaling to these reporters could be detected in FN over expressing 253J cells. NonFN dependent alpha5beta1 cross-linking initiated signal transduction in FN over expressing cells. We propose that by saturating cellular and BCG receptors excess FN expression decreases the ability of cellular or mycobacterial bound FN to bind vacant receptors on BCG or on the cell. Excess FN inhibits BCG adherence and BCG initiated signal transduction.

Enhanced survival effect of pyruvate correlates MAPK and NF-kappaB activation in hydrogen peroxide-treated human endothelial cells.

We recently reported that pyruvate inhibited translocation and activation of p53 caused by DNA damage due to oxidant injury (Lee YJ, Kang IJ, Bünger R, and Kang YH. Microvasc Res 66: 91-101, 2003); this was associated with increased expression of apoptosis-related bcl-2 and decreased expression of bax gene. This study attempted to delineate possible regulatory sites and mechanisms of antiapoptotic pyruvate, focusing on reactive oxygen species-mediated signaling in a human umbilical vein endothelial cell model. We compared the effects of the cytosolic reductant l-lactate and malate-aspartate shuttle blocker aminooxyacetate, both of which increase cytosolic NADH, on the downstream signaling pathway. Hydrogen peroxide (0.5 mM H2O2) depleted intracellular total glutathione that was prevented by pyruvate but not by l-lactate or aminooxyacetate. Activation of caspase-3 and the cleavage of procaspase-6 and procaspase-7 were strongly inhibited by pyruvate but markedly enhanced by l-lactate and aminooxyacetate, implicating redox-related antiapoptotic mechanisms of pyruvate. Western blot analysis and immunochemical data revealed that H2O2-induced transactivation of nuclear factor-kappaB (NF-kappaB) was also inhibited by pyruvate but not by l-lactate or aminooxyacetate. In addition, H2O2 downregulated extracellular signal-regulated kinase (ERK1/2) and phosphorylated p38 mitogen-activated protein kinase (MAPK), effects that were fully reversed by pyruvate within 2 h. Collectively, these findings indicate that pyruvate can protect cellular glutathione, thus enhancing cellular antioxidant potential, and that enhanced antioxidant potential can desensitize NF-kappaB transactivation due to reactive oxygen species, suggesting possible metabolic redox relations to NF-kappaB. Furthermore, pyruvate blocked the p38 MAPK pathway and activated the ERK pathway in an apparently redox-sensitive manner, which may regulate expression of genes believed to prevent apoptosis and promote cell survival. Thus pyruvate may have therapeutic potential for reducing endothelial dysfunction and improving survival during oxidative stress.

Transcription factor nuclear factor-kappa B is activated in neurons after focal cerebral ischemia.

Nuclear factor-kappa B (NF-kappaB) is a multisubunit transcription factor that when activated induces the expression of genes encoding acute-phase proteins, cell adhesion molecules, cell surface receptors, and cytokines. NF-kappaB is composed of a variety of protein subunits of which p50-and p65-kDa (RelA) are the most widely studied. Under resting conditions, these subunits reside in the cytoplasm as an inactive complex bound by inhibitor proteins, IkappaB alpha and IkappaB beta. On activation, IkappaB is phosphorylated by IkappaB kinase and ubiquitinated and degraded by the proteasome; simultaneously, the active heterodimer translocates to the nucleus where it can initiate gene transcription. In the periphery, NF-kappaB is involved in inflammation through stimulation of the production of inflammatory mediators. The role of NF-kappaB in the brain is unclear. In vitro, NF-kappaB activation can be either protective or deleterious. The role of NF-kappaB in ischemic neuronal cell death in vivo was investigated. Adult male rats were subjected to 2 hours of focal ischemia induced by middle cerebral artery occlusion (MCAO). At 2, 6, and 12 hours after reperfusion, the expression and transactivation of NF-kappaB in ischemic versus nonischemic cortex and striatum were determined by immunocytochemistry and by electrophoretic mobility gel-shift analysis. At all time points studied, p50 and p65 immunoreactivity was found exclusively in the nuclei of cortical and striatal neurons in the ischemic hemisphere. The contralateral nonischemic hemisphere showed no evidence of nuclear NF-kappaB immunoreactivity. Double immunofluorescence confirmed expression of p50 in nuclei of neurons. Increased NF-kappaB DNA-binding activity in nuclear extracts prepared from the ischemic hemisphere was further substantiated by electrophoretic mobility gel-shift analysis. Because the activation of NF-kappaB by many stimuli can be blocked by antioxidants in vitro, the effect of the antioxidant, LY341122, previously shown to be neuroprotective, on NF-kappaB activation in the MCAO model was evaluated. No significant activation of NF-kappaB was found by electrophoretic mobility gel-shift analysis in animals treated with LY341122. These results demonstrate that transient focal cerebral ischemia results in activation of NF-kappaB in neurons and supports previous observations that neuroprotective antioxidants may inhibit neuronal death by preventing the activation of NF-kappaB.

Induction of High Mobility Group-I(Y) Protein by Endotoxin and Interleukin-1β in Vascular Smooth Muscle Cells: ROLE IN ACTIVATION OF INDUCIBLE NITRIC OXIDE SYNTHASE

Nonhistone chromosomal proteins of the high mobility group (HMG) affect the transcriptional regulation of certain mammalian genes. For example, HMG-I(Y) controls cytokine-mediated promoters that require transcription factors, such as nuclear factor-kappaB, for maximal expression. Even though a great deal is known about how HMG-I(Y) facilitates expression of other genes, less is known about the regulation of HMG-I(Y) itself, especially in cells in primary culture. Therefore we investigated the effect of endotoxin and the cytokine interleukin-1beta on HMG-I(Y) expression in vascular smooth muscle cells. Induction of HMG-I(Y) peaked after 48 h of interleukin-1beta stimulation (6.2-fold) in cells in primary culture, and this increase in mRNA corresponded to an increase in HMG-I(Y) protein. Moreover, immunohistochemical staining revealed a dramatic increase in HMG-I(Y) protein expression in vascular smooth muscle cells after endotoxin stimulation in vivo. This increase in HMG-I(Y) expression (both in vitro and in vivo) mirrored an up-regulation of inducible nitric oxide synthase, a cytokine-responsive gene. The functional significance of this coinduction is underscored by our finding that HMG-I(Y) potentiated the response of inducible nitric oxide synthase to nuclear factor-kappaB transactivation. Taken together, these studies suggest that induction of HMG-I(Y), and subsequent transactivation of iNOS, may contribute to a reduction in vascular tone during endotoxemia and other systemic inflammatory processes.