Expression and subcellular localization of BNIP3 in hypoxic hepatocytes and liver stress.
We have previously demonstrated that the Bcl-2/adenovirus EIB 19-kDa interacting protein 3 (BNIP3), a cell death-related member of the Bcl-2 family, is upregulated in vitro and in vivo in both experimental and clinical settings of redox stress and that nitric oxide (NO) downregulates its expression. In this study we sought to examine the expression and localization of BNIP3 in murine hepatocytes and in a murine model of hemorrhagic shock (HS) and ischemia-reperfusion (I/R). Freshly isolated mouse hepatocytes were exposed to 1% hypoxia for 6 h followed by reoxygenation for 18 h, and protein was isolated for Western blot analysis. Hepatocytes grown on coverslips were fixed for localization studies. Similarly, livers from surgically cannulated C57Bl/6 mice and from mice cannulated and subjected to 1-4 h of HS were processed for protein isolation and Western blot analysis. In hepatocytes, BNIP3 was expressed constitutively but was upregulated under hypoxic conditions, and this upregulation was countered by treatment with a NO donor. Surprisingly, BNIP3 was localized in the nucleus of normoxic hepatocytes, in the cytoplasm following hypoxia, and again in the nucleus following reoxygenation. Upregulation of BNIP3 partially required p38 MAPK activation. BNIP3 contributed to hypoxic injury in hepatocytes, since this injury was diminished by knockdown of BNIP3 mRNA. Hepatic BNIP3 was also upregulated in two different models of liver stress in vivo, suggesting that a multitude of inflammatory stresses can lead to the modulation of BNIP3. In turn, the upregulation of BNIP3 appears to be one mechanism of hepatocyte cell death and liver damage in these settings.
Metukuri
Mallikarjuna R
MR
Department of Surgery, Center for Biologic Imaging, University of Pittsburgh School of Medicine, W943 Biomedical Sciences Tower, 200 Lothrop St., Pittsburgh, PA 15213, USA.
Beer-Stolz
Donna
D
Namas
Rajaie A
RA
Dhupar
Rajeev
R
Torres
Andres
A
Loughran
Patricia A
PA
Jefferson
Bahiyyah S
BS
Tsung
Allan
A
Billiar
Timothy R
TR
Vodovotz
Yoram
Y
Zamora
Ruben
R
D000818:Animals; D016923:Cell Death / Q000502:physiology; D002478:Cells, Cultured; D015870:Gene Expression / Q000502:physiology; D006505:Hepatitis / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D022781:Hepatocytes / Q000166:cytology / Q000502:physiology*; D000860:Hypoxia / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology*; D008099:Liver / Q000378:metabolism / Q000473:pathology; D020935:MAP Kinase Signaling System / Q000502:physiology; D008297:Male; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D024101:Mitochondrial Proteins / Q000235:genetics* / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D018384:Oxidative Stress / Q000502:physiology*; D010100:Oxygen / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015854:Up-Regulation / Q000502:physiology
C505650:BNIP3 protein, rat; C508686:BNip3 protein, mouse; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D011518:Proto-Oncogene Proteins; D009569:Nitric Oxide; D010100:Oxygen