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      Angiotensin II Induces Hypoxia-Inducible Factor-1α in PC 12 Cells through a Posttranscriptional Mechanism: Role of AT 2 Receptors

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          Abstract

          Background: Angiotensin II (ANG II) inhibits proliferation and induces differentiation in PC 12 cells via AT<sub>2</sub> receptor activation. Using differential display analysis, we previously isolated SM-20/PHD3 as a key factor, which is downregulated by ANG II treatment. Subsequently, it turned out that SM-20/PHD3 is a rat homologue of PHD3, a key prolyl hydroxylase involved in the initial steps fostering the degradation of hypoxia-inducible factor (HIF). The present study was undertaken to investigate whether the ANG-II-mediated suppression of SM-20/PHD3/PHD3 may be associated with an increase in HIF-1α. Methods: HIF-1α protein expression was assessed by Western blots. mRNA levels for HIF-1α were measured by real-time PCR and for SM-20/PHD3 by Northern blots. Binding of HIF-1α to consensus oligonucleotides in vitro was determined with gel shift analysis. SM-20/PHD3 was transiently overexpressed in PC 12 cells using an inducible expression system. Results: ANG II stimulated HIF-1α protein expression. This effect was already detected after 30 min and peaked at 6 h, but was not detectable anymore after 24– 48 h of stimulation. PD 123177, but not losartan, antagonized this effect, indicating transduction through AT<sub>2</sub> receptors. Real-time PCR failed to show a significant increase in HIF-1α transcripts after ANG II challenge at any time point. Gel shift analysis revealed that ANG-II-induced nuclear HIF-1α protein binds to consensus sites. A reduction in SM-20/PHD3 mRNA expression paralleled the increase in HIF-1α. Overexpression of SM-20/PHD3 transiently resulted in a decrease in HIF-1α protein concentrations under basal conditions as well as after stimulation with ANG II. Conclusion: ANG II stimulates HIF-1α expression by a posttranscriptional mechanism via AT<sub>2</sub> receptors. This increase is likely caused by a downregulation of SM-20/PHD3. The ANG-II-mediated increase in HIF-1α expression could be potentially involved in physiological as well as pathophysiological processes such as differentiation, growth inhibition, and remodeling.

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          Most cited references 17

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          HIF prolyl-hydroxylase 2 is the key oxygen sensor setting low steady-state levels of HIF-1alpha in normoxia.

          Hypoxia-inducible factor (HIF), a transcriptional complex conserved from Caenorhabditis elegans to vertebrates, plays a pivotal role in cellular adaptation to low oxygen availability. In normoxia, the HIF-alpha subunits are targeted for destruction by prolyl hydroxylation, a specific modification that provides recognition for the E3 ubiquitin ligase complex containing the von Hippel-Lindau tumour suppressor protein (pVHL). Three HIF prolyl-hydroxylases (PHD1, 2 and 3) were identified recently in mammals and shown to hydroxylate HIF-alpha subunits. Here we show that specific 'silencing' of PHD2 with short interfering RNAs is sufficient to stabilize and activate HIF-1alpha in normoxia in all the human cells investigated. 'Silencing' of PHD1 and PHD3 has no effect on the stability of HIF-1alpha either in normoxia or upon re-oxygenation of cells briefly exposed to hypoxia. We therefore conclude that, in vivo, PHDs have distinct assigned functions, PHD2 being the critical oxygen sensor setting the low steady-state levels of HIF-1alpha in normoxia. Interestingly, PHD2 is upregulated by hypoxia, providing an HIF-1-dependent auto-regulatory mechanism driven by the oxygen tension.
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            Induction of hypoxia-inducible factor-1alpha by transcriptional and translational mechanisms.

            Hypoxia-inducible factor-1 (HIF-1) regulates the transcription of many genes induced by low oxygen conditions. Recent studies have demonstrated that non-hypoxic stimuli can also activate HIF-1 in a cell-specific manner. Here, we define two key mechanisms that are implicated in increasing the active subunit of the HIF-1 complex, HIF-1alpha, following the stimulation of vascular smooth muscle cells (VSMC) with angiotensin II (Ang II). We show that, in contrast to hypoxia, the induction of HIF-1alpha by Ang II in VSMC is dependent on active transcription and ongoing translation. We demonstrate that stimulation of VSMC by Ang II strongly increases HIF-1alpha gene expression. The activation of diacylglycerol-sensitive protein kinase C (PKC) plays a major role in the increase of HIF-1alpha gene transcription. We also demonstrate that Ang II relies on ongoing translation to maintain elevated HIF-1alpha protein levels. Ang II increases HIF-1alpha translation by a reactive oxygen species (ROS)-dependent activation of the phosphatidylinositol 3-kinase pathway, which acts on the 5'-untranslated region of HIF-1alpha mRNA. These results establish that the non-hypoxic induction of the HIF-1 transcription factor via vasoactive hormones (Ang II and thrombin) is triggered by a dual mechanism, i.e. a PKC-mediated transcriptional action and a ROS-dependent increase in HIF-1alpha protein expression. Elucidation of these signaling pathways that up-regulate the vascular endothelial growth factor (VEGF) could have a strong impact on different aspects of vascular biology.
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              Intracellular localisation of human HIF-1 alpha hydroxylases: implications for oxygen sensing.

              Hypoxia-inducible factor1 (HIF-1) is an essential transcription factor for cellular adaptation to decreased oxygen availability. In normoxia the oxygen-sensitive alpha-subunit of HIF-1 is hydroxylated on Pro564 and Pro402 and thus targeted for proteasomal degradation. Three human oxygen-dependent HIF-1 alpha prolyl hydroxylases (PHD1, PHD2, and PHD3) function as oxygen sensors in vivo. Furthermore, the asparagine hydroxylase FIH-1 (factor inhibiting HIF) has been found to hydroxylate Asp803 of the HIF-1 C-terminal transactivation domain, which results in the decreased ability of HIF-1 to bind to the transcriptional coactivator p300/CBP. We have fused these enzymes to the N-terminus of fluorescent proteins and transiently transfected the fusion proteins into human osteosarcoma cells (U2OS). Three-dimensional 2-photon confocal fluorescence microscopy showed that PHD1 was exclusively present in the nucleus, PHD2 and FIH-1 were mainly located in the cytoplasm and PHD3 was homogeneously distributed in cytoplasm and nucleus. Hypoxia did not influence the localisation of any enzyme under investigation. In contrast to FIH-1, each PHD inhibited nuclear HIF-1 alpha accumulation in hypoxia. All hydroxylases suppressed activation of a cotransfected hypoxia-responsive luciferase reporter gene. Endogenous PHD2mRNA and PHD3mRNA were hypoxia-inducible, whereas expression of PHD1mRNA and FIH-1mRNA was oxygen independent. We propose that PHDs and FIH-1 form an oxygen sensor cascade of distinct subcellular localisation.
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                Author and article information

                Journal
                AJN
                Am J Nephrol
                10.1159/issn.0250-8095
                American Journal of Nephrology
                S. Karger AG
                0250-8095
                1421-9670
                2004
                August 2004
                17 September 2004
                : 24
                : 4
                : 415-421
                Affiliations
                Department of Medicine, Division of Nephrology and Osteology, University of Hamburg, Hamburg, Germany
                Article
                80086 Am J Nephrol 2004;24:415–421
                10.1159/000080086
                15308873
                © 2004 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: 5, References: 26, Pages: 7
                Product
                Self URI (application/pdf): https://www.karger.com/Article/Pdf/80086
                Categories
                Original Report: Laboratory Investigation

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