title: Mammalian tumor suppressor Int6 specifically targets hypoxia inducible factor 2 alpha for degradation by hypoxia- and pVHL-independent regulation.
abstract: The hypoxia-inducible factors HIF-1 alpha and HIF-2 alpha are structurally similar as regards their DNA-binding and dimerization domains, but differ in their transactivation domains and, as is shown by experiments using hif-1 alpha(-/-) and hif-2 alpha(-/-) mice, in their functions. This implies that HIF-1 alpha and HIF-2 alpha may have unique target genes. To address this discrepancy and identify HIF-2 alpha-specific target genes, we performed yeast two-hybrid analysis and identified the tumor suppressor Int6/eIF3e/p48 as a novel target gene product involved in HIF-2 alpha regulation. The int6 gene was first identified from a screen in which the mouse mammary tumor virus was employed as an insertional mutagen to identify genes whose functions are critical for breast tumor formation. Here, by using two-hybrid analysis, immunoprecipitation in mammalian cells, and HRE-reporter assays, we report the specific interaction of HIF-2 alpha (but not HIF-1 alpha or HIF-3 alpha) with Int6. The results indicate that the direct interaction of Int6 induces proteasome inhibitor-sensitive HIF-2 alpha degradation. This degradation was clearly observed in renal cell carcinoma 786-O cells, and was found to be both hypoxia- and pVHL-independent. Furthermore, Int6 protein knockdown by int6-siRNA vectors or the dominant-negative mutant Int6-Delta C increased endogenous HIF-2 alpha expression, even under normoxia, and induced sets of critical angiogenic factors comprising vascular endoplasmic growth factor, angiopoietin, and basic fibroblast growth factor mRNA. These results indicate that Int6 is a novel and critical determinant of HIF-2 alpha-dependent angiogenesis as well as cancer formation, and that int6-siRNA transfer may be an effective therapeutic strategy in pathological conditions such as heart and brain ischemia, hepatic cirrhosis, and obstructive vessel diseases.
authors.affiliation: Translation Research Project, Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan.
authors.forename: Li
authors.initials: L
authors.lastname: Chen
authors.forename: Kazuyo
authors.initials: K
authors.lastname: Uchida
authors.forename: Alexander
authors.initials: A
authors.lastname: Endler
authors.forename: Futoshi
authors.initials: F
authors.lastname: Shibasaki
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism*; D015687:Cell Hypoxia; D039621:Eukaryotic Initiation Factor-3 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D009369:Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D009389:Neovascularization, Pathologic / Q000188:drug therapy / Q000378:metabolism*; D051794:Von Hippel-Lindau Tumor Suppressor Protein / Q000378:metabolism*; D000099162:Endothelial PAS Domain-Containing Protein 1
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D039621:Eukaryotic Initiation Factor-3; D036341:Intercellular Signaling Peptides and Proteins; D051794:Von Hippel-Lindau Tumor Suppressor Protein; D000099162:Endothelial PAS Domain-Containing Protein 1