title: Hypoxia-induced overexpression of BNIP3 is not dependent on hypoxia-inducible factor 1α in mouse hepatocytes.
abstract: We sought to investigate the expression of the cell death protein BNIP3 in hypoxic hepatocytes, as well as the role that hypoxia-inducible factor 1 (HIF-1α) plays in the upregulation of BNIP3 in hypoxic primary mouse hepatocytes and in the livers of mice subjected to ischemia-reperfusion. Freshly isolated mouse hepatocytes were exposed to 1% hypoxia for 1, 3, 6, 24, and 48 h, and the RNA and protein were isolated for reverse transcriptase-polymerase chain reaction and Western blot analysis. Similarly, livers from mice subjected to segmental (70%) hepatic warm ischemia for 30 min or 1 h, or to 1-h ischemia followed by 0.5- to 4-h reperfusion, were collected and subjected to Western blot analysis for HIF-1α protein. We showed that hypoxic stress increases the formation of the BNIP3 homodimer while decreasing the amount of the monomeric form of BNIP3 in primary mouse hepatocytes. In contrast to RAW264.7 macrophages, there is a basal expression of HIF-α protein in normoxic primary mouse hepatocytes that does not change significantly upon exposure to hypoxia. Using siRNA technology, we demonstrated that reduced HIF-1α protein levels did not block the hypoxia-induced overexpression of BNIP3. In contrast to the effect on BNIP3 expression reported previously, livers from ischemic animals demonstrated only a modest increase in HIF-1α protein as compared with resting livers from control animals; and this expression was not statistically different from sham controls. These results suggest that HIF-1α does not mediate the hypoxia-induced upregulation of BNIP3 in mouse hepatocytes in vitro and possibly in the liver in vivo.
authors.affiliation: Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
authors.forename: Rajaie A
authors.initials: RA
authors.lastname: Namas
authors.forename: Mallikarjuna R
authors.initials: MR
authors.lastname: Metukuri
authors.forename: Rajeev
authors.initials: R
authors.lastname: Dhupar
authors.forename: Claudia
authors.initials: C
authors.lastname: Velosa
authors.forename: Bahiyyah S
authors.initials: BS
authors.lastname: Jefferson
authors.forename: Evan
authors.initials: E
authors.lastname: Myer
authors.forename: Greg M
authors.initials: GM
authors.lastname: Constantine
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Billiar
authors.forename: Yoram
authors.initials: Y
authors.lastname: Vodovotz
authors.forename: Ruben
authors.initials: R
authors.lastname: Zamora
mesh_terms: D000818:Animals; D015153:Blotting, Western; D015687:Cell Hypoxia / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D002470:Cell Survival / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D050687:E2F1 Transcription Factor / Q000235:genetics / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering
chemical_list: C508686:BNip3 protein, mouse; D050687:E2F1 Transcription Factor; C493754:E2f1 protein, mouse; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D034741:RNA, Small Interfering