Premium
TRANSLATIONAL REGULATION OF HYPOXIA‐INDUCIBLE FACTOR 2α IN RESPONSE TO IRON AND CELLULAR STRESSORS
Author(s) -
Lin Willey,
Deck Kathryn,
Eisenstein Richard
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.599.2
Subject(s) - ubiquitin ligase , translational regulation , messenger rna , hypoxia inducible factors , erythropoiesis , translation (biology) , untranslated region , transcription factor , microbiology and biotechnology , chemistry , ubiquitin , hepcidin , hypoxia (environmental) , biology , biochemistry , medicine , inflammation , anemia , gene , immunology , oxygen , organic chemistry
Hypoxia‐inducible factor 2α (HIF‐2α) is a heterodimeric helix‐loop‐helix transcription factor that plays an integral role in the regulation of erythropoiesis and iron metabolism and is a key player in the oxygen‐sensing and signaling pathways. HIF‐2α also stimulates lipid synthesis and storage in liver suggesting a role in controlling fuel metabolism in hypoxia and iron deficiency. HIF‐2α's regulatory role in iron metabolism makes it a subject of interest in relation to blood disorders, in particular anemia induced by iron deficiency or inflammatory disorders. HIF‐2α expression has been thought to be primarily controlled via oxygen‐ and iron‐dependent degradation by the action of prolyl hydroxylases and the von Hippel Lindau (VHL) E3 ubiquitin ligase. However, our lab and others have shown that dysregulation of HIF‐2α mRNA translation leads to hematological disorders. We are interested in examining the controls of HIF‐2α mRNA translation in response to iron and the presence of different stressors including the inflammation associated with diet‐induced obesity. Our research currently focuses on the 5′ untranslated region (UTR) of the mRNA of HIF‐2α. In the 5′ UTR of HIF‐2α mRNA is an inhibitory regulatory element called the iron responsive element (IRE). Iron regulatory proteins (IRP) bind to the IRE and are important regulators of HIF‐2α mRNA translation. The IRE impedes translation when bound to IRP as during iron deficiency. When iron levels are elevated, the IRP is inactivated and HIF‐2α mRNA is translated. To study translational regulation of HIF‐2α we created a reporter construct with the HIF‐2α 5′ UTR linked to GFP. Our plasmid, obtained from R. Tsien, also encodes RFP to control for transfection efficiency. Constructs containing the full length and truncated versions (all lacking the IRE) of the HIF‐2α 5′ UTR were used to investigate the role of the IRE and possibly other translational regulatory elements. Plasmids were transfected into HEK 293 cells and subjected to iron in the form of hemin (H), or an iron chelator, desferal (D). The wildtype HIF‐2α 5′UTR‐GFP construct was induced by hemin and repressed by desferal with a H/D ratio of 2.6. The deletion mutant with the shortest 5′ UTR (80% deletion) directed more than a 2‐fold higher level of GFP activity compared the wildtype and other truncation mutants indicating the presence of a negative regulatory element that is 3′ of the IRE. Surprisingly, all deletion mutants exhibited modest (~1.4 fold H/D ratio) response to iron suggesting the presence of additional regulatory elements responsive to iron that are independent of the IRE. Additional studies are underway to examine the translational regulation of HIF‐2α in animals. Support or Funding Information Support NIH DK66600