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Rationale: Granulocyte-macrophage colony stimulating factor (GM-CSF) is one of the recognized contributors in the pathogenesis of lung diseases. It evokes a dramatic increase of phosphorylation events in the inflammatory cells after stimulation. Lipoxin A4 (LxA4) is a novel lipid mediator with putative proresolution properties that appear to function as inhibitory signal during the time course of inflammation. We investigated the influence of LxA4 on intracellular events during stimulation with GM-CSF.
Methods: 2D electrophoresis was used to resolve whole-cell proteome, subcellular compartments, and GM-CSF signaling-specific proteome, using biotinylated ligand immunoprecipitation and GM-CSF receptor immunoprecipitation as well. Overall protein phosphorylation was assessed on the gels with phospho-specific stain Pro-Q-Diamond and on the Western blot using anti-phosphotyrosine immunostaining. Phosphorylation of ERK, SHP-2, and STAT-5 was revealed with specific antibodies. Protein interactions were assessed by co-immunoprecipitation. Cytokine profile was measured with multiplex assay.
Results: Pretreatment of the cells with LxA4 before GM-CSF stimulation led to the decrease of cytokine production as well as to changes in overall protein phosphorylation and tyrosine phosphorylation. Significant changes were also observed in phosphorylation of key kinases of GM-CSF signaling. Among signaling molecules affected by LxA4, STAT-5 transcription factor and SHP-2 phosphatase were detected. 2D electrophoresis showed that LxA4 pretreatment changed GM-CSF signaling in the proteome.
Conclusions: The inhibitory effect of Lipoxin A4 on GM-CSF-induced cellular function may indicate its potential for downregulation of GM-CSF-driven inflammatory processes. We propose mechanisms of inhibition of GM-CSF action by Lipoxin A4 involving different cornerstones of GM-CSF signaling, including protein phosphatases and transcription factor STAT-5.