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      Inhibition with Antisense Oligonucleotide Suggests that IκB-α Does Not Form a Negative Autoregulatory Loop for NF-κB in Mesangial Cells

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          The IκB proteins are important in the regulation of the NF-κB/Rel group of transcription factors which are pivotal in the inflammatory response. IκB-α is itself upregulated by activation of NF-κB and is postulated to be part of a negative feedback loop. This role of IκB-α has been challenged, however, by recent evidence that demonstrates (1) continued activation of NF-κB in mesangial and endothelial cells despite the resynthesis of IκB-α protein and (2) that inhibition of the transactivating activity of NF-κB by corticosteroids can be dissociated from a rise in IκB-α protein. We investigated the role of IκB-α in mesangial cells using a phosphorothioate antisense oligonucleotide directed against the translational start point of IκB-α. If IκB-α does function as a negative feedback inhibitor in these cells, then reducing IκB-α levels should lead to an increase in NF-κB activity. We first demonstrated that IκB-α protein resynthesis following stimulation could be specifically reduced. We then showed that NF-κB DNA binding was not increased with antisense treatment following stimulation. Finally, NF- κB-dependent gene signalling after stimulation (determined through an NF-κB luciferase reporter and upregulation of the mRNA of known NF-κB-responsive genes MCP-1 and IκB-α) was reduced rather than increased. These data suggest that IκB-α does not form a negative autoregulatory loop for NF-κB in mesangial cells and may actually reduce NF-κB activity. This may have relevance to therapies directed at inhibition of NF-κB activity in mesangial cell diseases.

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          Most cited references 10

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          Dithiocarbamates as potent inhibitors of nuclear factor kappa B activation in intact cells

          Dithiocarbamates and iron chelators were recently considered for the treatment of AIDS and neurodegenerative diseases. In this study, we show that dithiocarbamates and metal chelators can potently block the activation of nuclear factor kappa B (NF-kappa B), a transcription factor involved in human immunodeficiency virus type 1 (HIV-1) expression, signaling, and immediate early gene activation during inflammatory processes. Using cell cultures, the pyrrolidine derivative of dithiocarbamate (PDTC) was investigated in detail. Micromolar amounts of PDTC reversibly suppressed the release of the inhibitory subunit I kappa B from the latent cytoplasmic form of NF-kappa B in cells treated with phorbol ester, interleukin 1, and tumor necrosis factor alpha. Other DNA binding activities and the induction of AP-1 by phorbol ester were not affected. The antioxidant PDTC also blocked the activation of NF-kappa B by bacterial lipopolysaccharide (LPS), suggesting a role of oxygen radicals in the intracellular signaling of LPS. This idea was supported by demonstrating that treatment of pre-B and B cells with LPS induced the production of O2- and H2O2. PDTC prevented specifically the kappa B-dependent transactivation of reporter genes under the control of the HIV-1 long terminal repeat and simian virus 40 enhancer. The results from this study lend further support to the idea that oxygen radicals play an important role in the activation of NF-kappa B and HIV-1.
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            The Transcriptional Activity of NF-κB Is Regulated by the IκB-Associated PKAc Subunit through a Cyclic AMP–Independent Mechanism

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              Activation of nuclear transcription factor NF-kappaB by interleukin-1 is accompanied by casein kinase II-mediated phosphorylation of the p65 subunit.

               G Virca,  S Dower,  T. Bird (1997)
              In fibroblasts and hepatoma cells, interleukin-1 (IL-1) treatment results in the rapid nuclear accumulation of the transcription factor NF-kappaB, present largely as p65 (RelA)/p50 heterodimers. It is well established that this process is dependent in large part upon the phosphorylation and subsequent degradation of the cytosolic inhibitor IkappaB. We looked for other IL-1-induced modifications of NF-kappaB components and found that, in both cell types, IL-1 stimulation led, within minutes, to phosphorylation of both NF-kappaB p65 and p50. Phosphorylation of p65 was sustained for at least 30 min after addition of the cytokine and occurred principally upon serine residues. Immunoprecipitates of NF-kappaB complexes contained an associated protein kinase, the biochemical characteristics of which were indistinguishable from casein kinase II (CKII). Purified CKII efficiently phosphorylated p65 in vitro, apparently on the same major sites that became phosphorylated in intact IL-1-treated cells. Although IL-1 treatment caused little apparent stimulation of total cellular CKII activity, the fraction that was specifically associated with NF-kappaB complexes was markedly elevated by the cytokine. The association of CKII with NF-kappaB occurred in the cytoplasm, suggesting that this phosphorylation might be involved either in control of translocation of the activated complex or in modulation of its DNA binding properties.

                Author and article information

                Nephron Exp Nephrol
                Cardiorenal Medicine
                S. Karger AG
                June 2000
                10 May 2000
                : 8
                : 3
                : 144-151
                Immunology Research Centre, St. Vincent’s Hospital, Melbourne, Vic., Australia
                20662 Exp Nephrol 2000;8:144–151
                © 2000 S. Karger AG, Basel

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                Page count
                Figures: 5, References: 39, Pages: 8
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                Original Paper

                Cardiovascular Medicine, Nephrology

                NF-ĸB, IĸB-α, Antisense oligonucleotides, MCP-1, Mesangial cells


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