Endotoxin (LPS) Stimulates 4E-BP1/PHAS-I Phosphorylation in Macrophages

Abstract

Introduction. Translational control of cytokine production in endotoxin (LPS)-stimulated macrophages is poorly characterized but likely important. An early step in protein translation is engagement of mRNA by eukaryotic initiation factor 4E (eIF-4E). Translation initiation can be prevented by small 4E-binding proteins (4E-BP1 or PHAS-I) which must be phosphorylated in order to disengage eIF-4E. We examined whether LPS alters 4E-BP1 phosphorylation in macrophages.Materials and methods. Elicited rat peritoneal macrophages and Raw 264.7 macrophages were treated with signal transduction inhibitors and then LPS. Cells were harvested and equal protein amounts were electrophoresed (SDS-PAGE). Western blots (WB) were developed with 4E-BP1 antibody. Alternatively cell lysates were exposed to 7-methyl GTP Sepharose beads in order to isolate the cap-binding protein eIF-4E. The relative amounts of 4E-BP1 associated with eIF-4E were then determined by WB.Results. Macrophage 4E-BP1 is phosphorylated upon stimulation by LPS as evidenced by the appearance of a more slowly migrating γ (hyperphosphorylated) band on gel electrophoresis. Inhibition of both the p42/p44 MAPK pathway (PD 98059) and the p38 MAPK pathway (SB 203580) failed to alter LPS-induced 4E-BP1 phosphorylation. Rapamycin (FRAP/mTOR inhibitor) blocked 4E-BP1 phosphorylation causing a predominance of the α (hypophosphorylated) band. This was confirmed further by 7-methyl-GTP Sepharose isolation of eIF-4E with which 4E-BP1 coprecipitates.Conclusion. LPS stimulates 4E-BP1 phosphorylation in macrophages through FRAP/mTOR signaling. This pathway may contribute to the translational control of cytokine gene expression in macrophages.