One of the most potent inducers of granulocyte-macrophage colony-stimulating factor (GM-CSF) production in B cells, is bacterial lipopolysaccharides (LPS). The similar effects of LPS and the tubulin binding drug taxol implicates the microtubule network as a cellular target for LPS interaction(s). We found that pretreatment of the murine B cell line M12.4.1 with a variety of microtubule disrupting agents dramatically synergizes LPS-mediated induction of GM-CSF in these cells, both at the transcriptional and translational levels. While synergy occurred in a time-dependent manner, maximal (x10) induction of GM-CSF mRNA expression was observed when cells were pretreated with colchicine for 4 hours prior to addition of LPS. Colchicine both independently, and in combination with LPS enhanced transcriptional responsiveness of a reporter gene derived by either full-length human or murine GM-CSF promoters. Interestingly, a minimal sequence comprising of 0.06kb of DNA from upstream of the mouse GM-CSF transcription initiation site (-60bp) conferred responsiveness to colchicine. In contrast, the response to LPS appears to minimally require sequences further upstream (-200bp) in the GM-CSF promoter. Actinomycin D chase experiments revealed that colchicine pretreatment also increased the half-life of LPS induced GM-CSF mRNA. Colchicine potently activated c-Jun N-terminal Kinase (JNK1), while LPS specifically activated erk2 Kinase (MAPK) in these cells. Colchicine treatment stimulated phosphorylation of c-jun and CREB/ATF1 proteins in M12.4.1 cells, as well as the binding of nucleoprotein complexes to a consensus AP1 probe and the CLE0 motif present in the 0.06kb GM-CSF minimal promoter. Robust activation of JNK1 by colchicine preceded the phosphorylation of c-jun and sustained expression of the c jun gene, and potently stimulated expression of an AP1 reporter gene. Expression of a c-jun dominant negative mutant however, did not impair the ability of colchicine to upregulate LPS-stimulated GM-CSF promoter activity. Our finding suggest that LPS and microtubule disruption activate independent and multiple signaling pathways that appear to converge at distinct region(s) in the GM-CSF promoter, and potentiate the synthesis of GM-CSF in LPS-stimulated B cells, via both transcriptional and posttranscriptional mechanisms.
