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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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          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.

                Author and article information

                Am J Nephrol
                American Journal of Nephrology
                S. Karger AG
                August 2004
                17 September 2004
                : 24
                : 4
                : 415-421
                Department of Medicine, Division of Nephrology and Osteology, University of Hamburg, Hamburg, Germany
                80086 Am J Nephrol 2004;24:415–421
                © 2004 S. Karger AG, Basel

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                Page count
                Figures: 5, References: 26, Pages: 7
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                Original Report: Laboratory Investigation


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