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Suppression of PMN apoptosis by hypoxia is dependent on Mcl-1 and MAPK activity
S J Leuenroth1, P S Grutkoski, A Ayala
1Brown University School of Medicine and Rhode Island Hospital, Providence 02903, USA.
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.
Insights
Hypoxia delays neutrophil apoptosis by upregulating Mcl-1. This process involves p38 MAPK signaling, which is crucial for Mcl-1 expression and neutrophil survival in wound sites.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Hypoxia is known to delay neutrophil (polymorphonuclear leukocytes [PMNs]) apoptosis.
- Mcl-1 expression is essential for this hypoxia-mediated delay in PMN apoptosis.
- Previous studies utilized antisense oligonucleotides to establish Mcl-1's necessity.
Purpose of the Study:
- To further characterize Mcl-1 expression under hypoxic conditions.
- To investigate the specific signaling pathways involved in hypoxia-induced delay of PMN apoptosis.
- To elucidate the mechanism by which hypoxia influences Mcl-1 and PMN survival.
Main Methods:
- Neutrophils (PMNs) were incubated with kinase inhibitors (MEK, p38 MAPK, PI3K) for 12 hours.
- Apoptosis was assessed using morphologic features and Western blot analysis.
- Mcl-1 expression was quantified via Western blot and immunofluorescent staining.
Main Results:
- Hypoxia upregulated Mcl-1 expression in PMNs after 12 hours.
- Inhibition of MEK or p38 MAPK signaling during hypoxia increased PMN apoptosis to normoxic levels.
- Inhibition of p38 MAPK significantly decreased Mcl-1 expression.
Conclusions:
- Hypoxia delays PMN apoptosis through the activation of p38 MAPK signaling.
- This signaling pathway induces the expression of the anti-apoptotic protein Mcl-1.
- This mechanism is relevant for PMN function and survival at wound sites.