Alteration of nuclear factor-κB (NF-κB) expression in bone marrow stromal cells treated with etoposide11Abbreviations: VP-16, etoposide; VCAM-1, vascular cell adhesion molecule-1; VLA-4, very late antigen-4; NF-κB, nuclear factor-κB; IRF-1, interferon response factor-1; ECL, enhanced chemiluminescence; EMSA, electrophoretic mobility shift assay; GM-CSF, granulocyte macrophage-colony stimulating factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PCR, polymerase chain reaction; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; poly(dI-dC), polydeoxyinosinic-deoxycytidylic acid; and HRP, horseradish peroxidase.

2001 ◽  
Vol 61 (10) ◽  
pp. 1243-1252 ◽  
Author(s):  
Brett M. Hall ◽  
James E. Fortney ◽  
Laura F. Gibson
Keyword(s):  
Nuclear Factor Κb ◽  
Interferon Response ◽  
Nuclear Factor ◽  
Mobility Shift ◽  
Gm Csf ◽  
Enhanced Chemiluminescence ◽  
Late Antigen ◽  
Mobility Shift Assay ◽  
Macrophage Colony ◽  
Cell Adhesion Molecule 1

scholarly journals Cyclolinteinone, a sesterterpene from sponge Cacospongia linteiformis, prevents inducible nitric oxide synthase and inducible cyclo-oxygenase protein expression by blocking nuclear factor-κB activation in J774 macrophages

2000 ◽  
Vol 346 (3) ◽  
pp. 793-798 ◽  
Author(s):  
Fulvio D'ACQUISTO ◽  
Virginia LANZOTTI ◽  
Rosa CARNUCCIO
Keyword(s):  
Nitric Oxide ◽  
Protein Expression ◽  
Nuclear Translocation ◽  
Inflammatory Diseases ◽  
Nuclear Factor Κb ◽  
Nuclear Factor ◽  
Mobility Shift ◽  
J774 Cells ◽  
Cox 2 ◽  
Mobility Shift Assay

We investigated the effect of cyclolinteinone, a sesterterpene from Caribbean sponge Cacospongia linteiformis, on inducible NO synthase (iNOS) and cyclo-oxygenase-2 (COX-2) protein expression in lipopolysaccharide (LPS)-stimulated J774 macrophages. Incubation of J774 cells with LPS (1 μg/ml) caused an increase of both iNOS and COX-2 protein expression, which was prevented in a concentration-dependent fashion by cyclolinteinone (12.5, 25 and 50 μM). Electrophoretic mobility-shift assay indicated that cyclolinteinone blocked the activation of nuclear factor-ĸB (NF-ĸB), a transcription factor necessary for either iNOS or COX-2 induction. Cyclolinteinone also blocked disappearance of IĸB-α from cytosolic fraction and nuclear translocation of NF-ĸB subunits p50 and p65. These results show that cyclolinteinone down-regulates iNOS and COX-2 protein expression by inhibiting NF-ĸB activation and suggest that it may represent a novel anti-inflammatory compound capable of controlling the excessive production of prostaglandins and nitric oxide occurring in several inflammatory diseases.

Inhibition of the nuclear factor—κB activation with pyrrolidine dithiocarbamate attenuating inflammation and oxidative stress after experimental spinal cord trauma in rats

2004 ◽  
Vol 1 (3) ◽  
pp. 311-321 ◽  
Author(s):  
Giovanni La Rosa ◽  
Salvatore Cardali ◽  
Tiziana Genovese ◽  
Alfredo Conti ◽  
Rosanna Di Paola ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Nuclear Factor Κb ◽  
Pyrrolidine Dithiocarbamate ◽  
Nuclear Factor ◽  
Mobility Shift ◽  
Content Type ◽  
Cord Injury ◽  
Mobility Shift Assay ◽  
And Oxidative Stress ◽  
Fine Print

Object. The nuclear factor—κB (NF-κB) is a transcription factor that plays a pivotal role in the induction of genes involved in physiological processes and in the response to inflammation. The authors of recent studies have demonstrated that NF-κB and oxidative stress contribute to secondary injury after impact-induced spinal cord injury (SCI) in the rat. Dithiocarbamates are antioxidants that are potent inhibitors of NF-κB. The authors postulated that pyrrolidine dithiocarbamate (PDTC) would attenuate NF-κB—related inflammatory and oxidative events that occur after SCI. Methods. Spinal cord injury was induced by the application of vascular clips (force of 50 g) to the dura mater after a four-level T5–8 laminectomy. The authors investigated the effects of PDTC (30 mg/kg administered 30 minutes before SCI and 6 hours after SCI) on the development of the inflammatory response associated with SCI in rats. Levels of myeloperoxidase activity were measured as an indicator of polymorphonuclear infiltration; malondialdehyde levels in the spinal cord tissue were determined as an indicator of lipid peroxidation. The following studies were performed: immunohistochemical analysis to assess levels of inducible nitric oxide synthase (iNOS), nitrotyrosine formation, poly([adenosine diphosphate]-ribose) polymerase (PARP) activity; Western blot analysis to determine cytoplasmic levels of inhibitory—κB-α (IκB-α); and electrophoretic mobility-shift assay to measure the level of DNA/NF-κB binding. The PDTC treatment exerted potent antiinflammatory effects with significant reduction of polymorphonuclear cell infiltration, lipid peroxidation, and iNOS activity. Furthermore, administration of PDTC reduced immunohistochemical evidence of formation of nitrotyrosine and PARP activation in the spinal cord section obtained in the SCI-treated rats. Additionally, PDTC treatment significantly prevented the activation of NF-κB (electrophoretic mobility-shift assay and immunoblot analysis). Conclusions. Overall, the results clearly demonstrate that PDTC-related prevention of the activation of NF-κB reduces the development of some secondary injury events after SCI. Therefore, inhibition of NF-κB may represent a novel approach in the treatment of SCIs.

Nuclear factor-κB is constitutively activated in primitive human acute myelogenous leukemia cells

Blood ◽  
2001 ◽  
Vol 98 (8) ◽  
pp. 2301-2307 ◽  
Author(s):  
Monica L. Guzman ◽  
Sarah J. Neering ◽  
Donna Upchurch ◽  
Barry Grimes ◽  
Dianna S. Howard ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myelogenous Leukemia ◽  
Nuclear Factor Κb ◽  
Myelogenous Leukemia ◽  
Nuclear Factor ◽  
Mobility Shift ◽  
Human Cd34 ◽  
Acute Myelogenous ◽  
Mobility Shift Assay ◽  
Gene Expression Studies

Abstract Human acute myelogenous leukemia (AML) is thought to arise from a rare population of malignant stem cells. Cells of this nature, herein referred to as leukemic stem cells (LSCs), have been documented for nearly all AML subtypes and appear to fulfill the criteria for stem cells in that they are self-renewing and give rise to the cells found in many leukemic populations. Because these cells are likely to be critical for the genesis and perpetuation of leukemic disease, the present studies sought to characterize unique molecular properties of the LSC population, with particular emphasis on the transcription factor, nuclear factor-κB (NF-κB). Previous experiments have shown that unstimulated human CD34+ progenitor cells do not express NF-κB. In contrast, primary AML CD34+ cells display readily detectable NF-κB activity as assessed by electrophoretic mobility shift assay and gene expression studies. Furthermore, detailed analyses of enriched AML stem cells (CD34+/CD38−/CD123+) indicate that NF-κB is also active in the LSC population. Given the expression of NF-κB in leukemic, but not normal primitive cells, the hypothesis that inhibition of NF-κB might induce leukemia-specific apoptosis was tested by treating primary cells with the proteasome inhibitor MG-132, a well-known inhibitor of NF-κB. Leukemic CD34+/CD38− cells displayed a rapid induction of cell death in response to MG-132, whereas normal CD34+/CD38− cells showed little if any effect. Taken together, these data indicate that primitive AML cells aberrantly express NF-κB and that the presence of this factor may provide unique opportunities to preferentially ablate LSCs.

scholarly journals Enhanced survival of lung granulocytes in an animal model of asthma: evidence for a role of GM-CSF activated STAT5 signalling pathway

Thorax ◽  
2001 ◽  
Vol 56 (9) ◽  
pp. 696-702
Author(s):  
R K Turlej ◽  
L Fiévez ◽  
C F Sandersen ◽  
S Dogné ◽  
N Kirschvink ◽  
...  
Keyword(s):  
Allergic Inflammation ◽  
Annexin V ◽  
Inflammatory Cells ◽  
Binding Activity ◽  
Mobility Shift ◽  
Gm Csf ◽  
Mobility Shift Assay ◽  
Macrophage Colony ◽  
Receptor Antibodies

BACKGROUNDAs granulocyte/macrophage colony stimulating factor (GM-CSF) mediated delay of granulocyte apoptosis contributes to the accumulation of inflammatory cells at the site of inflammation in many diseases, we sought to determine whether asthma is also associated with a GM-CSF dependent increase in lung granulocyte survival. Moreover, because GM-CSF mediates its effects through activation of signal transducer and activator of transcription 5 (STAT5), we also investigated the potential role of STAT5 in allergic inflammation.METHODSBlood granulocytes were recovered from six healthy and six heaves affected horses, a model of asthma. Lung granulocytes were obtained by bronchoalveolar lavage (BAL) from the same horses. Granulocytes were cultured in the presence or absence of anti-GM-CSF receptor antibodies for different times and apoptosis was determined using the Annexin-V/propidium iodide detection method. Nuclear protein extracts from cultured granulocytes were analysed for STAT5 binding activity by electrophoretic mobility shift assay.RESULTSBAL fluid granulocytes from heaves affected horses demonstrated a significant delay in apoptosis compared with blood granulocytes from the same horses and blood and BAL fluid granulocytes from healthy horses. Conversely, the rate of apoptosis in blood granulocytes from healthy and heaves affected horses was comparable. The enhanced survival of BAL fluid granulocytes from affected horses was suppressed in the presence of antibodies directed against GM-CSF receptors. Increased levels of active STAT5 were found in BAL fluid granulocytes from heaves affected horses and were markedly reduced after treatment with anti-GM-CSF receptor antibodies.CONCLUSIONSThese data indicate that granulocyte survival is enhanced in the lung of heaves affected horses and suggest a role for a GM-CSF activated STAT5 pathway in delaying apoptosis of lung granulocytes in this model of asthma.

scholarly journals Characterization of nuclear factors that bind to a critical positive regulatory element of the human granulocyte-macrophage colony- stimulating factor promoter

Blood ◽  
1994 ◽  
Vol 84 (8) ◽  
pp. 2523-2530 ◽  
Author(s):  
JK Fraser ◽  
S Tran ◽  
SD Nimer ◽  
JC Gasson
Keyword(s):  
Regulatory Element ◽  
Point Mutations ◽  
Regulatory Sequence ◽  
Colony Stimulating Factor ◽  
Mobility Shift ◽  
Gm Csf ◽  
Nuclear Factors ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor that stimulates the proliferation, maturation, and functional activity of myeloid cells in peripheral blood and bone marrow. Expression of GM-CSF is tightly regulated and is limited to cells stimulated directly (T cells, macrophages) or indirectly (fibroblasts, endothelial cells) by immune challenge. Several studies of the transcriptional control of GM-CSF expression have elucidated a region of the GM-CSF promoter that mediates positive regulatory activity in a number of cell types. This region contains a direct repeat of the sequence CATTA/T that extends from nucleotides -37 to -48 upstream of the start of mRNA synthesis. Although specific DNA:protein interactions have been shown within this region, neither the nature nor the number of nuclear factors responsible for these interactions have been characterized. In this study, we use DNase I footprinting analysis to demonstrate that point mutations, which inactivate the GM-CSF promoter, disrupt DNA:protein interactions within this region. By combined electrophoretic mobility shift and ultraviolet cross-linking analysis, we have detected several protein species that bind specifically to the positive regulatory sequence.

PDGF Up-regulates CSF-1 Gene Transcription in Ameloblast-like Cells

2008 ◽  
Vol 87 (1) ◽  
pp. 33-38 ◽  
Author(s):  
Y. Wittrant ◽  
B. Sriniketan Bhandari ◽  
H. Abboud ◽  
N. Benson ◽  
K. Woodruff ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Binding Motif ◽  
Rt Pcr ◽  
Mobility Shift ◽  
Protein Levels ◽  
Macrophage Colony Stimulating Factor ◽  
Cellular Pathways ◽  
Mobility Shift Assay ◽  
Macrophage Colony ◽  
Stimulating Factor

Macrophage colony-stimulating factor (CSF-1) is a key regulatory cytokine for amelogenesis, and ameloblasts synthesize CSF-1. We hypothesized that PDGF stimulates DNA synthesis and regulates CSF-1 in these cells. We determined the effect of PDGF on CSF-1 expression using MEOE-3M ameloblasts as a model. By RT-PCR, MEOE-3M expressed PDGFRs and PDGF A- and B-chain mRNAs. PDGF-BB increased DNA synthesis and up-regulated CSF-1 mRNA and protein in MEOE-3M. Cells transfected with CSF-1 promoter deletion constructs were analyzed. A PDGF-responsive region between −1.7 and −0.795 kb, containing a consensus Pea3 binding motif, was identified. Electrophoretic mobility shift assay (EMSA) showed that PDGF-BB stimulated protein binding to this motif that was inhibited in the presence of anti-Pea3 antibody. Analysis of these data provides the first evidence that PDGF-BB is a mitogen for MEOE-3M and increases CSF-1 protein levels, predominantly by transcription. Elucidation of the cellular pathways that control CSF-1 expression may provide novel strategies for the regulation of enamel matrix formation.

scholarly journals Thrombin Stimulation of the Vascular Cell Adhesion Molecule-1 Promoter in Endothelial Cells Is Mediated by Tandem Nuclear Factor-κB and GATA Motifs

2001 ◽  
Vol 276 (50) ◽  
pp. 47632-47641 ◽  
Author(s):  
Takashi Minami ◽  
William C. Aird
Keyword(s):  
Endothelial Cells ◽  
Adhesion Molecule ◽  
Umbilical Vein ◽  
Fold Increase ◽  
Nuclear Factor Κb ◽  
Nuclear Factor ◽  
Mobility Shift ◽  
Response Element ◽  
Vascular Adhesion ◽  
Stimulation Of

The goal of this study was to delineate the transcriptional mechanisms underlying thrombin-mediated induction of vascular adhesion molecule-1 (VCAM-1). Treatment of human umbilical vein endothelial cells with thrombin resulted in a 3.3-fold increase in VCAM-1 promoter activity. The upstream promoter region of VCAM-1 contains a thrombin response element, two nuclear factor κB (NF-κB) motifs, and a tandem GATA motif. In transient transfection assays, mutation of the thrombin response element had no effect on thrombin induction. In contrast, mutation of either NF-κB site resulted in a complete loss of induction, whereas a mutation of the two GATA motifs resulted in a significant reduction in thrombin stimulation. In electrophoretic mobility shift assays, nuclear extracts from thrombin-treated endothelial cells displayed markedly increased binding to the tandem NF-κB and GATA motifs. The NF-κB complex was supershifted with anti-p65 antibodies, but not with antibodies to RelB, c-Rel, p50, or p52. The GATA complex was supershifted with antibodies to GATA-2, but not GATA-3 or GATA-6. A construct containing tandem copies of the VCAM-1 GATA motifs linked to a minimal thymidine kinase promoter was induced 2.4-fold by thrombin. Taken together, these results suggest that thrombin stimulation of VCAM-1 in endothelial cells is mediated by the coordinate action of NF-κB and GATA transcription factors.

scholarly journals Consistent loss of the D5S89 locus mapping telomeric to the interleukin gene cluster and centromeric to EGR-1 in patients with 5q- chromosome

Blood ◽  
1994 ◽  
Vol 83 (1) ◽  
pp. 199-208 ◽  
Author(s):  
L Nagarajan ◽  
J Zavadil ◽  
D Claxton ◽  
X Lu ◽  
J Fairman ◽  
...  
Keyword(s):  
Cpg Islands ◽  
Early Response ◽  
Interferon Response ◽  
Artificial Chromosomes ◽  
Gm Csf ◽  
Chromosome 5Q ◽  
Macrophage Colony ◽  
Locus Mapping ◽  
Principal Region ◽  
Yeast Artificial Chromosomes

Abstract Interstitial deletions of the long arm of chromosome 5 are common in a number of disorders of leukemic and preleukemic myeloid disorders. Although the limits of these deletions vary among patients, a region of cytogenetic overlap that includes band 5q31 is deleted consistently, suggesting loss of 5q31 loci critical for normal myeloid differentiation and leukemogenesis. An anonymous genomic DNA segment D5S89, previously mapped to 5q21–31, detects consistent loss of alleles in cases showing the 5q- chromosome at presentation or relapse. Analysis of a panel of natural-deletion somatic-cell hybrids in conjunction with irradiation hybrids containing fragments of human chromosome 5q shows that the D5S89 locus is telomeric to the interleukin (IL) genes (IL-3, IL-4, IL-5, IL-9, and granulocyte- macrophage colony-stimulating factor [GM-CSF]) and interferon response factor-1 (IRF-1) gene and centromeric to the early response transcription factor (early growth response gene-1 [EGR-1]) on 5q31. To further define the principal region of loss, we have isolated and characterized yeast artificial chromosomes (YACs) spanning D5S89. The presence of several CpG islands within the 300-kb YAC is suggestive of multiple transcription units. However, IL-4, IL-5, IRF-1, IL-3, GM-CSF, and EGR-1 genes were not detected in the YAC clone spanning D5S89, implying that none of these genes are in the vicinity of the D5S89 marker. Further characterization of these YACs should facilitate the isolation of novel candidate genes that may play a role in the evolution of the abnormal phenotype associated with 5q- chromosome.

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