Microarray analyses of the effects of NF-<tex>$\kappa$</tex>B or PI3K pathway inhibitors on the LPS-induced gene expression profile in RAW264.7 cells: Synergistic effects of rapamycin on LPS-induced MMP9-overexpression
Boudjeltia, Karim Zouaoui
Faculty of Medicine and Health Sciences
Cellular signalling. - Oxford
, p. 1109-1122
University of Antwerp
Lipopolysaccharide (LPS) activates a broad range of signalling pathways including mainly NF-êB and the MAPK cascade, but recent evidence suggests that LPS stimulation also activates the PI3K pathway. To unravel the specific roles of both pathways in LPS signalling and gene expression profiling, we investigated the effects of different inhibitors of NF-êB (BAY 11-7082), PI3K (wortmannin and LY294002) but also of mTOR (rapamycin), a kinase acting downstream of PI3K/Akt, in LPS-stimulated RAW264.7 macrophages, analyzing their effects on the LPS-induced gene expression profile using a low density DNA microarray designed to monitor the expression of pro-inflammatory genes. After statistical and hierarchical cluster analyses, we determined five clusters of genes differentially affected by the four inhibitors used. In the fifth cluster corresponding to genes upregulated by LPS and mainly affected by BAY 11-7082, the gene encoding MMP9 displayed a particular expression profile, since rapamycin drastically enhanced the LPS-induced upregulation at both the mRNA and protein levels. Rapamycin also enhanced the LPS-induced NF-êB transactivation as determined by a reporter assay, phosphorylation of the p38 and Erk1/2 MAPKs, and counteracted PPAR activity. These results suggest that mTOR could negatively regulate the effects of LPS on the NF-êB and MAPK pathways. We also performed real-time RT-PCR assays on mmp9 expression using rosiglitazone (agonist of PPARã), PD98059 (inhibitor of Erk 1/2) and SB203580 (inhibitor of p38MAPK), that were able to counteract the rapamycin mediated overexpression of mmp9 in response to LPS. Our results suggest a new pathway involving mTOR for regulating specifically mmp9 in LPS-stimulated RAW264.7 cells.