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      NFκB Signaling in Posthypoxic Endothelial Cells: Relevance to E-Selectin Expression and Neutrophil Adhesion

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          Abstract

          Our previous studies have implicated the nuclear transcription factor ĸB (NFĸB) in the regulation of adhesion molecule expression in endothelial cells exposed to anoxia-reoxygenation (A/R) or a redox imbalance. The objectives of this study were (1) to define the kinetics of NFĸB activation by examining IĸBα degradation and the nuclear translocation of p65 in response to A/R or redox imbalance (induced by treatment of cells with diamide and buthionine sulfoximine) and (2) to determine whether the signal for IĸBα degradation, nuclear translocation of p65, and E-selectin-mediated neutrophil adhesion is related to the activity of protein tyrosine kinase (PTK), protein tyrosine phosphatase (PTPase) and/or protein kinase C (PKC). The results demonstrate that both A/R and redox imbalance led to IĸBα degradation within 30 min and the concomitant appearance of p65 in the nucleus, consistent with rapid cytosolic activation of NFĸB and subsequent nuclear translocation of the activated p65 subunit. Inhibition of PKC blocked IĸBα degradation and p65 translocation in A/R-challenged, but not redox-altered, endothelial cells. However, both A/R- and redox-induced NFĸB activation was blocked by inhibition of PTK. Similarly, A/R-induced E-selectin expression and neutrophil-endothelial cell adhesion were blocked by inhibition of PKC or PTK, while only PTK inhibited the redox-induced adhesion response. Pretreatment of cells with N-acetyl cysteine effectively blocked A/R- or redox-induced IĸB degradation and significantly attenuated the respective neutrophil adhesion responses. Collectively, these findings indicate that A/R-induced E-selectin expression and neutrophil-endothelial cell adhesion are mediated by both PKC and PTK, which signal rapid activation of NFĸB. This A/R-induced NFĸB signaling response appears to be mediated, at least in part, by intracellular redox imbalance.

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          Most cited references 4

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          Functional analysis of the human vascular cell adhesion molecule 1 promoter

          The vascular cell adhesion molecule 1 (VCAM-1) is a 110-kD member of the immunoglobulin gene superfamily expressed on the surface of interleukin 1 beta- or tumor necrosis factor alpha (TNF)-stimulated endothelial cells. The cell surface protein functions as an inducible adhesion receptor for circulating mononuclear leukocytes and some tumor cells. We have previously characterized the genomic organization of the VCAM1 gene and described its chromosomal localization. In this report, the promoter of the VCAM1 gene is characterized. New transcription of the VCAM1 gene occurred when endothelial cells were treated with TNF. Fusion plasmids containing the 5' flanking sequence of the VCAM1 gene and the chloramphenicol acetyltransferase reporter gene were used to identify cis-acting sequences that direct the cytokine-induced transcription. When transfected into bovine aortic endothelial cells, constructs containing 755 bp of the 5' flanking sequence were induced by TNF. Within the cytokine-responsive region of the core promoter were functional NF-kappa B and GATA elements. Upstream of the core promoter, the VCAM1 5' flanking sequence contained a negative regulatory activity. NF-kappa B-mediated activation of VCAM1 gene expression may lead to endothelial expression of a mononuclear leukocyte adhesion molecule associated with initial events in the development of an atherosclerotic lesion.
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            Glutathione regulation of tumor necrosis factor-alpha-induced NF-kappa B activation in skeletal muscle-derived L6 cells.

            TNF alpha is implicated in several skeletal muscle pathologies including muscle wasting of cachexia. Muscle wasting and other conditions such as physical exercise and immobilization are also associated with disturbances in muscle glutathione status. Hence, it was of interest to investigate the role of endogenous glutathione status in TNF alpha induced NF-kappa B activation in skeletal muscle-derived cells. TNF alpha proved to be a potent inducer of transient NF-kappa B activation in L6 myoblasts. In buthioninesulfoximine (BSO) treated cells, TNF alpha induced NF-kappa B activation was markedly potentiated suggesting that such activation is sensitive to cellular GSH, but may have been independent of high levels of intracellular GSSG. Because this activation was inhibited by the antioxidant pyrrolidinedithiocarbamate (PDTC) the involvement of reactive oxygen species in this activation system seems likely. NF-kappa B activation in L6 cells was also observed in response to direct H2O2 treatment. Results from GSSG reductase inhibited cells suggest that GSSG may participate in, but is not required for, TNF alpha induced NF-kappa B activation. The inhibitory effect of PDTC on NF-kappa B activation correlated with its effect on ICAM-1 expression suggesting that this GSH status modifying agent not only influenced nuclear translocation of NF-kappa B proteins but also regulated kappa B dependent transcription.
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              • Article: not found

              Redox regulation of lipopolysaccharide (LPS)-induced interleukin-8 (IL-8) gene expression mediated by NF kappa B and AP-1 in human astrocytoma U373 cells.

              LPS-induced expression of the IL-8 gene was markedly enhanced by H2O2 or by deprivation of the cellular antioxidant glutathione by L-buthionine-(S,R)-sulfoximine (BSO) in human astrocytoma U373 cells. In contrast, it was markedly suppressed by the reductant N-acetyl-L-cysteine (NAC) and other antioxidants. Transient expression analysis using the chloramphenicol acetyltransferase assay revealed that activation of the IL-8 promoter by LPS was stimulated by BSO and was suppressed by NAC; likewise LPS-induced activation of both NF kappa B and AP-1 was enhanced by BSO and inhibited by NAC. These results suggest that LPS-induced IL-8 gene expression is regulated by cellular redox via modulation of these transcription factors.
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                Author and article information

                Journal
                JVR
                J Vasc Res
                10.1159/issn.1018-1172
                Journal of Vascular Research
                S. Karger AG
                1018-1172
                1423-0135
                2001
                February 2001
                08 February 2001
                : 38
                : 1
                : 47-58
                Affiliations
                aDepartment of Molecular and Cellular Physiology and bCenter of Excellence in Arthritis and Rheumatism, Louisiana State University Health Sciences Center, Shreveport, La., USA; cFirst Department of Internal Medicine, Kyoto Prefectural University of Medicine, Kyoto, Japan
                Article
                51029 J Vasc Res 2001;38:47–58
                10.1159/000051029
                11173994
                © 2001 S. Karger AG, Basel

                Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                Page count
                Figures: 10, References: 37, Pages: 12
                Categories
                Research Paper

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