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      Osteopontin Traffic in Hypoxic Renal Epithelial Cells

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          Osteopontin (OPN), a secretory RGD-containing phosphoprotein, is induced in acute renal injury where it plays a renoprotective role. To investigate in depth the mode of OPN secretion under stress conditions, we analyzed OPN traffic in human renal proximal tubular epithelial cells (RPTEC). Western blot analysis and fluorescence microscopy revealed trace amounts of OPN in intact cells, whereas cytoplasmic OPN levels were significantly increased after 24–48 h hypoxia. Immunoelectron microscopy of RPTEC showed predominantly apical localization of gold-labeled OPN under normal conditions. Hypoxia (24 h) increased 2.5-fold immunodetectable gold-labeled OPN at the apical plasma membrane; further reoxygenation (2 h) augmented apical and basolateral labeling 2- and 10-fold, respectively. Analysis of apical and basolateral medium conditioned by RPTEC grown on semipermeable membranes using a specially developed ELISA showed a global decrease in secreted OPN after hypoxia, which recovered following 2 h reoxygenation. Agents known to disrupt the function of the Golgi apparatus (brefeldin A, monensin) or actin cytoskeleton (cytochalasin B) significantly inhibited OPN-GFP secretion in normoxic cells. In cells recovering from hypoxia, however, OPN secretion required functional Golgi apparatus, but was not affected by cytochalasin B. These findings demonstrate that stress inhibits OPN secretion by the process dependent on the functional Golgi apparatus and actin cytoskeleton; recovery restores OPN secretion, although its polarity may become perturbed.

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          Most cited references 13

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          Eta-1 (osteopontin): an early component of type-1 (cell-mediated) immunity.

          Cell-mediated (type-1) immunity is necessary for immune protection against most intracellular pathogens and, when excessive, can mediate organ-specific autoimmune destruction. Mice deficient in Eta-1 (also called osteopontin) gene expression have severely impaired type-1 immunity to viral infection [herpes simplex virus-type 1 (KOS strain)] and bacterial infection (Listeria monocytogenes) and do not develop sarcoid-type granulomas. Interleukin-12 (IL-12) and interferon-gamma production is diminished, and IL-10 production is increased. A phosphorylation-dependent interaction between the amino-terminal portion of Eta-1 and its integrin receptor stimulated IL-12 expression, whereas a phosphorylation-independent interaction with CD44 inhibited IL-10 expression. These findings identify Eta-1 as a key cytokine that sets the stage for efficient type-1 immune responses through differential regulation of macrophage IL-12 and IL-10 cytokine expression.
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            NF-κB Mediates αvβ3 Integrin-induced Endothelial Cell Survival

            The αvβ3 integrin plays a fundamental role during the angiogenesis process by inhibiting endothelial cell apoptosis. However, the mechanism of inhibition is unknown. In this report, we show that integrin-mediated cell survival involves regulation of nuclear factor-kappa B (NF-κB) activity. Different extracellular matrix molecules were able to protect rat aorta- derived endothelial cells from apoptosis induced by serum withdrawal. Osteopontin and β3 integrin ligation rapidly increased NF-κB activity as measured by gel shift and reporter activity. The p65 and p50 subunits were present in the shifted complex. In contrast, collagen type I (a β1-integrin ligand) did not induce NF-κB activity. The αvβ3 integrin was most important for osteopontin-mediated NF-κB induction and survival, since adding a neutralizing anti-β3 integrin antibody blocked NF-κB activity and induced endothelial cell death when cells were plated on osteopontin. NF-κB was required for osteopontin- and vitronectin-induced survival since inhibition of NF-κB activity with nonphosphorylatable IκB completely blocked the protective effect of osteopontin and vitronectin. In contrast, NF-κB was not required for fibronectin, laminin, and collagen type I–induced survival. Activation of NF-κB by osteopontin depended on the small GTP-binding protein Ras and the tyrosine kinase Src, since NF-κB reporter activity was inhibited by Ras and Src dominant-negative mutants. In contrast, inhibition of MEK and PI3-kinase did not affect osteopontin-induced NF-κB activation. These studies identify NF-κB as an important signaling molecule in αvβ3 integrin-mediated endothelial cell survival.
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              Brefeldin A: insights into the control of membrane traffic and organelle structure

               R D Klausner (1992)

                Author and article information

                Nephron Exp Nephrol
                Cardiorenal Medicine
                S. Karger AG
                June 2003
                17 November 2004
                : 94
                : 2
                : e66-e76
                aDepartments of Medicine and Physiology, Division of Nephrology and Hypertension, State University of New York at Stony Brook, Stony Brook, N.Y., and bDepartment of Cell Biology and Neuroscience, Rutgers University, Piscataway, N.J., USA; cCharité, Campus Virchow-Klinikum, Humboldt University at Berlin, Department of Medicine, Division of Nephrology and Medical Intensive Care, Berlin, Germany; dDepartment of Medicine, Nephrology Division and Renal Research Institute, New York Medical College, Valhalla, N.Y., USA
                71285 Nephron Exp Nephrol 2003;94:e66–e76
                © 2003 S. Karger AG, Basel

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                Page count
                Figures: 6, References: 54, Pages: 1
                Self URI (application/pdf): https://www.karger.com/Article/Pdf/71285
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