p38 MAP kinase and MKK‐1 co‐operate in the generation of GM‐CSF from LPS‐stimulated human monocytes by an NF‐κB‐independent mechanism

The extent to which the p38 mitogen‐activated protein (MAP) kinase and MAP kinase kinase (MKK)‐1‐signalling pathways regulate the expression of granulocyte/macrophage colony‐stimulating factor (GM‐CSF) from LPS‐stimulated human monocytes has been investigated and compared to the well studied cytokine tumour necrosis factor‐α (TNFα). Lipopolysaccharide (LPS) evoked a concentration‐dependent generation of GM‐CSF from human monocytes. Temporally, this effect was preceded by an increase in GM‐CSF mRNA transcripts and abolished by actinomycin D and cycloheximide. LPS‐induced GM‐CSF release and mRNA expression were associated with a rapid and time‐dependent activation of p38 MAP kinase, ERK‐1 and ERK‐2. The respective MKK‐1 and p38 MAP kinase inhibitors, PD 098059 and SB 203580, maximally suppressed LPS‐induced GM‐CSF generation by >90%, indicating that both of these signalling cascades co‐operate in the generation of this cytokine. Electrophoretic mobility shift assays demonstrated that LPS increased nuclear factor κB (NF‐κB) : DNA binding. SN50, an inhibitor of NF‐κB translocation, abolished LPS‐induced NF‐κB : DNA binding and the elaboration of TNFα, a cytokine known to be regulated by NF‐κB in monocytes. In contrast, SN50 failed to affect the release of GM‐CSF from the same monocyte cultures. Collectively, these results suggest that the generation of GM‐CSF by LPS‐stimulated human monocytes is regulated in a co‐operative fashion by p38 MAP kinase‐ and MKK‐1‐dependent signalling pathways independently of the activation of NF‐κB. British Journal of Pharmacology (2000) 131 , 1143–1153; doi: 10.1038/sj.bjp.0703684