title: Bax/Mcl-1 balance affects neutrophil survival in intermittent hypoxia and obstructive sleep apnea: effects of p38MAPK and ERK1/2 signaling.
abstract: BACKGROUND
  Prolonged neutrophil survival is evident in various cardiovascular and respiratory morbidities, in hypoxic conditions in-vitro and in patients with obstructive sleep apnea (OSA) characterized by nightly intermittent hypoxia (IH). This may lead to persistent inflammation, tissue injury and dysfunction. We therefore investigated by a translational approach the potential contribution of the intrinsic stress-induced mitochondrial pathway in extending neutrophil survival under IH conditions. Thus, neutrophils of healthy individuals treated with IH in-vitro and neutrophils of OSA patients undergoing nightly IH episodes in-vivo were investigated. Specifically, the balance between pro-apoptotic Bax and anti-apoptotic Mcl-1 protein expression, and the potential involvement of p38MAPK and ERK1/2 signaling pathways in the control of Mcl-1 expression were investigated.
  
  
  METHODS
  Purified neutrophils were exposed to IH and compared to normoxia and to sustained hypoxia (SH) using a BioSpherix-OxyCycler C42 system. Bax and Mcl-1 levels, and p38MAPK and ERK1/2 phosphorylation were determined by western blotting. Also, Bax/Mcl-1 expression and Bax translocation to the mitochondria were assessed by confocal microscopy in pre-apoptotic neutrophils, before the appearance of apoptotic morphology. Co-localization of Bax and mitochondria was quantified by LSM 510 CarlZeiss MicroImaging using Manders Overlap Coefficient. A paired two-tailed t test, with Bonferroni correction for multiple comparisons, was used for statistical analysis.
  
  
  RESULTS
  Compared to normoxia, IH and SH up-regulated the anti-apoptotic Mcl-1 by about 2-fold, down-regulated the pro-apoptotic Bax by 41% and 27%, respectively, and inhibited Bax co-localization with mitochondria before visible morphological signs of apoptosis were noted. IH induced ERK1/2 and p38MAPKs phosphorylation, whereas SH induced only p38MAPK phosphorylation. Accordingly, both ERK and p38MAPK inhibitors attenuated the IH-induced Mcl-1 increase. In SH, only p38MAPK inhibition decreased Mcl-1 expression. Similar to neutrophils of healthy subjects exposed to IH (0.97± 0.2), in OSA neutrophils, Bax/Mcl-1 ratio was significantly lower compared to normoxic controls (1.0±0.5 vs.1.99±0.3, p=0.015), and Bax did not co-localize with mitochondria.
  
  
  CONCLUSIONS
  These findings suggest that decreased Bax/Mcl-1 balance promotes neutrophil survival in IH in-vitro as well as in OSA patients. Moreover, Bax/Mcl-1 protein function in IH and SH might be regulated by different signal transduction pathways, highlighting a novel regulatory function through ERK1/2 signaling in IH.
authors.affiliation: The Lloyd Rigler Sleep Apnea Research Laboratory, The Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel.
authors.forename: Larissa
authors.initials: L
authors.lastname: Dyugovskaya
authors.forename: Andrey
authors.initials: A
authors.lastname: Polyakov
authors.forename: Victoria
authors.initials: V
authors.lastname: Cohen-Kaplan
authors.forename: Peretz
authors.initials: P
authors.lastname: Lavie
authors.forename: Lena
authors.initials: L
authors.lastname: Lavie
mesh_terms: D000328:Adult; D016022:Case-Control Studies; D002470:Cell Survival / Q000187:drug effects; D003710:Demography; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D000860:Hypoxia / Q000150:complications / Q000201:enzymology / Q000473:pathology*; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008875:Middle Aged; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009504:Neutrophils / Q000187:drug effects / Q000473:pathology*; D010100:Oxygen / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D020181:Sleep Apnea, Obstructive / Q000150:complications / Q000201:enzymology / Q000473:pathology*; D015854:Up-Regulation / Q000187:drug effects; D051028:bcl-2-Associated X Protein / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D064549:Myeloid Cell Leukemia Sequence 1 Protein; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D010100:Oxygen