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 nitrogen-activated protein kinase [MAPK]), and LY294002 (phosphatidyl inositol-3-kinase: [PI3K]). PMNs that were treated with inhibitors were assessed for apoptosis by morphological 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 no I moxia. We further wanted to determine whether signaling through p38 il 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 I the wound site by the activation of p38 MAPK signaling thereby inducing the anti-apoptotic protein Mcl-1.