Differential profile of Nix upregulation and translocation during hypoxia/ischaemia in vivo versus in vitro.
Nix, a hypoxia-sensitive member of the Bcl-2 family, is upregulated at the mRNA level during hypoxia through induction of a hypoxia-inducible factor-1 alpha (HIF-1 alpha) response element in its promoter sequence. However, the mechanism(s) regulating Nix protein activation remain unclear. The present studies examine Nix protein expression and subcellular distribution in response to hypoxic stimuli in vivo and in culture and to two disparate apoptotic stimuli in vitro. Upregulation and translocation of Nix (by day 5) in hypoxic/serum-deprived CHO-K1 cells, was preceded by Bax activation (by day 4) and caspase-3 processing (by day 2), suggesting that initiation of cell death in vitro is a Nix-independent event. In contrast, an early Nix response (upregulation and translocation to the mitochondria) was observed after 6 h of middle cerebral artery occlusion in the rat. Nix translocation was observed in the ipsilateral cortex and striatum before other histological (infarct development, neuronal loss, apoptotic body formation) or biochemical (Bax activation or caspase-3 cleavage) markers of damage were detected. While fundamental differences between hypoxia/ischaemia in culture and in vivo likely explain the different temporal profiles of Nix, Bax, and caspase-3 activation observed, these studies show that like Bax, mitochondrial accumulation is a common event during Nix activation. These are the first studies to show upregulation and translocation of Nix in the ischaemic brain and suggest Nix to be a novel therapeutic target in ischaemic research. Moreover, Nix upregulation in staurosporine-treated SH-SY5Y cells and dexamethasone-treated A1.1 cells supports a more generalized role for Nix in apoptotic cell death.
Birse-Archbold
Jui-Lee A
JL
Fujisawa Institute of Neuroscience in Edinburgh, University of Edinburgh, Edinburgh, UK.
Kerr
Lorraine E
LE
Jones
Paul A
PA
McCulloch
James
J
Sharkey
John
J
D000818:Animals; D017209:Apoptosis; D016466:CHO Cells; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D006224:Cricetinae; D020925:Hypoxia-Ischemia, Brain / Q000378:metabolism*; D020244:Infarction, Middle Cerebral Artery; D008297:Male; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D021381:Protein Transport; D011518:Proto-Oncogene Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D015854:Up-Regulation / Q000235:genetics
C505659:BNIP3L protein, rat; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases