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      Expression und Regulation des Hypoxie-induzierbaren Faktors HIF-2alpha in verschienen Organen der Ratte

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          Abstract

          Sauerstoffmangel spielt in der Pathophysiologie zahlreicher Erkrankungen mit gro§er klinischer Relevanz wie z.B. Herzinfarkte und SchlaganfŠlle eine zentrale Rolle. Eine Anpassung an Hypoxie wird durch die Familie der Hypoxie-induzierbaren Faktoren (HIF) vermittelt. Als Transkriptionsfaktoren sind HIF-1alpha und HIF-2alpha entscheidende Regulatoren von Genen, die durch niedrige Sauerstoffspannung aktiviert werden und Anpassungen wie z.B. Angiogenese (VEGF) und Umstellung auf anaeroben Energiemetabolismus (glykolytische Enzyme) bewirken. HIF ist ein Heterodimer, bestehend aus einer Alpha- und Beta-Untereinheit. WŠhrend die Beta-Untereinheit konstitutiv prŠsent ist, reprŠsentiert die Alpha-Untereinheit die sauerstoffregulierte Komponente von HIF, welche unter Normoxie proteasomal degradiert wird. Unter hypoxischen Bedingungen werden beide Faktoren in Zellinien stabilisiert. Das gleiche gilt fŸr HIF-1alpha in vivo. Bisher waren die Organe und Zellen der Expression von HIF-2alpha im lebenden Organismus unbekannt. Die vorliegende Arbeit konzentrierte sich daher auf die Charakterisierung der HIF-2alpha -Expression in vivo. Unter normoxischen Kontrollbedingungen war HIF-2alpha nicht nachweisbar. Es zeigte sich jedoch nach Stimulation mit 0,1 % CO (funktionelle AnŠmie) eine Induktion von HIF-2alpha in allen untersuchten Organen einschlie§lich Hirn, Leber, Lunge, Darm, Herz und Niere. Sowohl der zeitliche Verlauf des Anstiegs (1-6 Stunden) als auch die IntensitŠt der Induktion variierten in den einzelnen Organen, was durch Immunoblotting nachgewiesen werden konnte. Durch immunhistochemische Untersuchungen konnte gezeigt werden, in welchen Zellen der verschiedenen Gewebe eine nukleŠre Akkumulation von HIF-2alpha erfolgte. In Niere und Hirn erfolgte eine Induktion in nicht-parenchymatšsen Zellen, in Leber und Darm hingegen fand sich eine vorwiegende Expression in parenchymatšsen Zellen. Eine gleiche Verteilung zwischen den beiden verschiedenen Zelltypen fand sich im Herz. Durch Untersuchungen der mRNA mittels RNA Protection Assay konnte bestŠtigt werden, dass die Regulation von HIF-2alpha posttranslational erfolgt, da keine Hochregulation der mRNA festgestellt werden konnte. Die Daten zeigen, dass HIF-2alpha eine wichtige Rolle bei der transkriptionellen Antwort auf verminderte SauerstoffverfŸgbarkeit in vivo spielt und dass seine Rolle zu der von HIF-1alpha verschieden ist.

          Abstract

          Deficiency of oxygen plays a central role in the pathophysiology of diseases with great clinical relevance like heart failure and stroke. An adaptation to hypoxia is mediated by the family of hypoxia-inducible factors (HIF). As transcription-factors HIF-1alpha and HIF-2alpha are regulators of genes that are activated by low oxygen tension and that cause adaptations to hypoxia like angiogenesis (VEGF) and change to anaerobic energy-metabolism (glycolytic enzymes). HIF is a heterodimer, consisting of an Alpha- and Beta-subunit. Whereas the Beta-subunit is constitutively present, the Alpha-subunit represents the oxygen-regulated component of HIF, which is degraded in the proteasom under normoxia. Under hypoxic conditions both subunits are stabilized in cell lines and HIF-1alpha is widely expressed in vivo. In contrast, regulation and sites of HIF-2alpha expression in vivo were unknown. Therefore this work concentrates on the characterisation of the expression of HIF-2alpha in vivo. Although HIF-2alpha was not detectable under baseline conditions, marked hypoxic induction occurred in all organs investigated, in brain, liver, kidney, lung, intestine and heart after stimulation with 0,1% CO (functional anaemia). Time course and amplitude varied between organs. Immunohistochemistry revealed nuclear accumulation in distinct cell populations of each tissue, which were exclusively non-parenchymal in kidney and brain, predominantly parenchymal in liver and intestine or equally distributed in the heart. These data indicate that HIF-2alpha plays an important role in the transcriptional response to hypoxia in vivo and is complementary to rather than redundant with HIF-1alpha.

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          HIFalpha targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing.

          HIF (hypoxia-inducible factor) is a transcription factor that plays a pivotal role in cellular adaptation to changes in oxygen availability. In the presence of oxygen, HIF is targeted for destruction by an E3 ubiquitin ligase containing the von Hippel-Lindau tumor suppressor protein (pVHL). We found that human pVHL binds to a short HIF-derived peptide when a conserved proline residue at the core of this peptide is hydroxylated. Because proline hydroxylation requires molecular oxygen and Fe(2+), this protein modification may play a key role in mammalian oxygen sensing.
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            FIH-1: a novel protein that interacts with HIF-1alpha and VHL to mediate repression of HIF-1 transcriptional activity.

            Hypoxia-inducible factor 1 (HIF-1) is a master regulator of oxygen homeostasis that controls angiogenesis, erythropoiesis, and glycolysis via transcriptional activation of target genes under hypoxic conditions. O(2)-dependent binding of the von Hippel-Lindau (VHL) tumor suppressor protein targets the HIF-1alpha subunit for ubiquitination and proteasomal degradation. The activity of the HIF-1alpha transactivation domains is also O(2) regulated by a previously undefined mechanism. Here, we report the identification of factor inhibiting HIF-1 (FIH-1), a protein that binds to HIF-1alpha and inhibits its transactivation function. In addition, we demonstrate that FIH-1 binds to VHL and that VHL also functions as a transcriptional corepressor that inhibits HIF-1alpha transactivation function by recruiting histone deacetylases. Involvement of VHL in association with FIH-1 provides a unifying mechanism for the modulation of HIF-1alpha protein stabilization and transcriptional activation in response to changes in cellular O(2) concentration.
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              Cellular adaptation to hypoxia: O2-sensing protein hydroxylases, hypoxia-inducible transcription factors, and O2-regulated gene expression.

              Although it was known for a long time that oxygen deprivation leads to the transcriptional induction of the gene encoding erythropoietin, the molecular mechanisms behind this process remained enigmatic. The cloning of the hypoxia-inducible factors (HIFs), the finding that HIF-1 regulates the expression of many more genes apart from erythropoietin, and the elucidation of the oxygen-dependent mechanisms degrading the HIF alpha subunits recently led to the spectacular discovery of the molecular principles of oxygen sensing. This review aims to summarize our current knowledge of oxygen-regulated gene expression..
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                Author and article information

                Journal
                Medizinische Fakultät - Universitätsklinikum Charité, Humboldt-Universität (kvv )
                24 February 2006
                Article
                oai:HUBerlin.de:26703
                ba034970-6738-4a27-85f1-212960af8771
                History

                Sauerstoff,oxygen,Medizin,Hypoxie,hypoxia,Hypoxie-induzierbarer Transkriptionsfaktor,Genexpression,HIF,hypoxia-inducible transcription factor,gene expression,YC 2604,YC 2621,Angiogenese,angiogenesis,VEGF

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