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      Oxidant Stress Activates AP-1 and Heparin-Binding Epidermal Growth Factor-Like Growth Factor Transcription in Renal Epithelial Cells


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          Ischemia/reperfusion injury increases the expression of bioactive heparin-binding epidermal growth factor-like growth factor (HB-EGF) in the rat kidney, suggesting that oxidant stress or cell injury related to oxidant stress might affect HB-EGF expression in the injured renal parenchyma. We utilized a nontransformed rat renal epithelial cell line (NRK-52E cells) to investigate whether reactive oxygen species induced transcriptional activation of HB-EGF mRNA. Hypoxia/reoxygenation increased HB-EGF expression in NRK-52E cells, and at concentrations that induced sublethal cell injury, hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) increased HB-EGF mRNA expression 4.7-fold. The free radical scavengers, dimethylthiourea and N-acetylcysteine inhibited HB-EGF mRNA induction. In contrast, another free radical scavenger, pyrrolidine thiocarbamate (PDTC), augmented H<sub>2</sub>O<sub>2</sub>-mediated HB-EGF expression. Since PDTC has been reported to augment AP-1-mediated transcriptional activation, we utilized an electrophoretic mobility shift assay to confirm that H<sub>2</sub>O<sub>2</sub> administration to NRK-52E cells did increase nuclear extract DNA-binding activity to a consensus AP-1 sequence. Using a CAT reporter assay coupled to the proximal 2,000 bp of the human HB-EGF 5′-untranslated region, we determined that H<sub>2</sub>O<sub>2</sub> administration increased CAT activity 5.5-fold. Truncation or deletion mutations of a putative AP-1-binding site reduced the H<sub>2</sub>O<sub>2</sub>-stimulated activity by >60%, and there was increased DNA binding of nuclear extracts from H<sub>2</sub>O<sub>2</sub>-treated cells to a 24-bp oligonucleotide containing this putative AP-1 site. Anti- fos and jun antibodies inhibited this binding, and there was no binding to an oligonucleotide in which the putative AP-1 site was mutated.The site of the residual activation was found to exist in the most proximal 5′-untranslated region (–121 to +60), which contains two putative SP1 sites. Timing and localization of AP-1-binding activity from nuclear extracts from the post-ischemic tissue correlated with HB-EGF mRNA expression. Therefore, in renal epithelial cells, oxidant stress increases HB-EGF expression, which appears to be mediated in part by an increase in AP-1 binding. This activation may play an important role in the induction of HB-EGF mRNA in response to tissue injury and may regulate early stages of recovery following ischemic damage.

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          Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.

          A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
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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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              A role for oxygen radicals as second messengers.

              All cells seem to produce oxygen radicals. Recent results suggest that small nontoxic amounts of these radicals are released by various cell types in response to stimulation with tumour necrosis factor (TNF), interleukin 1 (IL-1) and phorbol esters, all of which activate a cytoplasmic form of the transcription factor NF-kappa B by releasing an inhibitory protein subunit. Activation is inhibited by agents that remove oxygen radicals, and mimicked by exposure to mild oxidant stress. This article proposes that oxygen radicals act as second messengers for a variety of agents, including the immunomodulatory cytokines TNF and IL-1, in at least one type of regulatory pathway activating NF-kappa B.

                Author and article information

                Nephron Exp Nephrol
                Cardiorenal Medicine
                S. Karger AG
                October 2000
                06 October 2000
                : 9
                : 1
                : 28-39
                Department of Medicine and Department of Veterans Affairs Medical Center, Nashville, Tenn., USA
                20705 Exp Nephrol 2001;9:28–39
                © 2000 S. Karger AG, Basel

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                Page count
                Figures: 8, References: 69, Pages: 12
                Self URI (application/pdf): https://www.karger.com/Article/Pdf/20705
                Self URI (text/html): https://www.karger.com/Article/FullText/20705
                Self URI (journal page): https://www.karger.com/SubjectArea/Nephrology
                Original Paper

                Cardiovascular Medicine,Nephrology
                Oxidant stress,Growth factor,Gene transcription,Epidermal growth factor,Heparin-binding epidermal growth factor-like growth factor,Acute renal injury


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