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Granulocyte-macrophage colony-stimulating factor enhances 5-lipoxygenase levels in human polymorphonuclear leukocytes
M Pouliot1, P P McDonald, L Khamzina
1Centre de Recherche en Inflammation, Immunologie et Rhumatologie, Université Laval, Ste-Foy, Québec, Canada.
Abstract:
Stimulation of human polymorphonuclear leukocytes (PMNL) with granulocyte-macrophage CSF (GM-CSF) results in the enhanced expression of several genes, including some coding for cytokines and enzymes. In this study, we investigated the ability of GM-CSF to up-regulate the human neutrophil 5-lipoxygenase (5-LO), a key enzyme in the leukotriene synthetic pathway. GM-CSF induced a dose- and time-dependent de novo synthesis of the 5-LO in PMNL, as determined by immunoprecipitation of 35S-methionine-labeled 5-LO. This up-regulation occurred within 30 min of treatment with GM-CSF and was observed using concentrations of GM-CSF as low as 30 pM. Prior treatment of the cells with the protein synthesis inhibitor cycloheximide abolished this effect of GM-CSF. Western blot analyses demonstrated that levels of 5-LO did not vary over a 6-h period in unstimulated PMNL treated with CX, and that GM-CSF induced a rapid increase in the total cellular level of 5-LO protein; taken together these results indicated a translational effect of GM-CSF on the expression of the 5-LO. However, GM-CSF did not significantly affect the level of 5-LO mRNA in neutrophils, as determined by Northern blot analysis. Furthermore GM-CSF did not alter the stability of 5-LO mRNA, in agreement with a posttranscriptional effect of GM-CSF on 5-LO expression in PMNL. These results show that human PMNL are capable of up-regulating the expression of the 5-LO in response to physiologic activation.
Insights
Granulocyte-macrophage colony-stimulating factor (GM-CSF) enhances the expression of 5-lipoxygenase (5-LO) in human neutrophils. This occurs via a post-transcriptional mechanism, increasing the synthesis of this key enzyme in leukotriene pathways.
Area of Science:
- Immunology and Molecular Biology
- Cellular signaling pathways
- Inflammatory response mechanisms
Background:
- Human polymorphonuclear leukocytes (PMNL) express various genes upon stimulation.
- Granulocyte-macrophage colony-stimulating factor (GM-CSF) is known to modulate gene expression in PMNL.
- The 5-lipoxygenase (5-LO) enzyme is critical for leukotriene synthesis, playing a role in inflammation.
Purpose of the Study:
- To investigate GM-CSF's effect on the expression of human neutrophil 5-lipoxygenase (5-LO).
- To determine the mechanism by which GM-CSF regulates 5-LO expression at the molecular level.
Main Methods:
- Stimulation of human PMNL with varying concentrations and durations of GM-CSF.
- Immunoprecipitation using 35S-methionine to quantify de novo 5-LO synthesis.
- Western blot analysis to assess total 5-LO protein levels.
- Northern blot analysis to determine 5-LO mRNA levels and stability.
Main Results:
- GM-CSF induced a dose- and time-dependent increase in 5-LO protein synthesis in PMNL.
- This up-regulation was observed at low GM-CSF concentrations (30 pM) and rapid onset (within 30 min).
- GM-CSF treatment did not significantly alter 5-LO mRNA levels or stability, indicating a post-transcriptional regulation.
Conclusions:
- GM-CSF up-regulates the expression of the 5-LO enzyme in human neutrophils.
- The regulation occurs at the translational level, not transcriptional, suggesting post-transcriptional control.
- Human PMNL can enhance 5-LO expression in response to physiological activation by GM-CSF.