Suppression of PMN apoptosis by hypoxia is dependent on Mcl-1 and MAPK activity.
BACKGROUND
Hypoxia has been shown to delay the onset of neutrophil (polymorphonuclear leukocytes [PMNs]) apoptosis. With the use of antisense oligonucleotides, we have previously demonstrated that Mcl-1 is necessary for this effect. We wanted to further characterize the expression of Mcl-1 and examine signaling pathways required for the delay in apoptosis that is mediated by hypoxia.


METHODS
For kinase signaling inhibition, PMNs were incubated for 12 hours with the following inhibitors: PD98059 Mitogen Activated Protein Kinase Kinase (MEK), SB202190 (p38 mitogen-activated protein kinase [MAPK]), and LY294002 (phosphatidyl inositol-3-kinase [PI3K]). PMNs that were treated with inhibitors were assessed for apoptosis by morphologic features or were lysed for Western blot analysis.


RESULTS
Western blot analyses, immunofluorescent staining, and quantification showed an upregulation of Mcl-1 expression after 12 hours of incubation in response to hypoxia. When inhibitors of either MEK or p38 MAPK were incubated with PMNs during hypoxia, apoptosis increased to similar levels as normoxia. We further wanted to determine whether signaling through p38 MAPK or MEK led to increased Mcl-1 expression. Western blot analysis confirmed that the inhibition of p38 MAPK led to a significant decrease in Mcl-1 expression.


CONCLUSIONS
We have documented a novel mechanism by which hypoxia can modify PMN apoptosis in the wound site by the activation of p38 MAPK signaling, thereby inducing the anti-apoptotic protein Mcl-1.
Leuenroth
S J
SJ
Brown University School of Medicine and Rhode Island Hospital, Providence 02903, USA.
Grutkoski
P S
PS
Ayala
A
A
Simms
H H
HH
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D015687:Cell Hypoxia / Q000502:physiology*; D002867:Chromones / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D005786:Gene Expression Regulation; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007700:Kinetics; D020929:Mitogen-Activated Protein Kinase Kinases / Q000097:blood; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000097:blood*; D009025:Morpholines / Q000494:pharmacology; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009363:Neoplasm Proteins / Q000097:blood* / Q000235:genetics; D009504:Neutrophils / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D019253:Proto-Oncogene Proteins c-bcl-2; D011725:Pyridines / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases
D002867:Chromones; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007093:Imidazoles; D009025:Morpholines; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009363:Neoplasm Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011725:Pyridines; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; C090942:4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)imidazole; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one