Microarray analyses of the effects of NF-kappaB or PI3K pathway inhibitors on the LPS-induced gene expression profile in RAW264.7 cells: synergistic effects of rapamycin on LPS-induced MMP9-overexpression.
Lipopolysaccharide (LPS) activates a broad range of signalling pathways including mainly NF-kappaB 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-kappaB (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-kappaB 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-kappaB and MAPK pathways. We also performed real-time RT-PCR assays on mmp9 expression using rosiglitazone (agonist of PPARgamma), PD98059 (inhibitor of Erk 1/2) and SB203580 (inhibitor of p38(MAPK)), 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.
Mendes
Sofia Dos Santos
Sdos S
University of Namur-FUNDP, Research Unit in Cellular Biology, Rue de Bruxelles 61, Namur, Belgium.
Candi
Aurélie
A
Vansteenbrugge
Martine
M
Pignon
Marie-Rose
MR
Bult
Hidde
H
Boudjeltia
Karim Zouaoui
KZ
Munaut
Carine
C
Raes
Martine
M
D000818:Animals; D002460:Cell Line; D004357:Drug Synergism; D020869:Gene Expression Profiling; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D008070:Lipopolysaccharides / Q000494:pharmacology*; D020780:Matrix Metalloproteinase 9 / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D005810:Multigene Family; D016328:NF-kappa B / Q000037:antagonists & inhibitors*; D020411:Oligonucleotide Array Sequence Analysis; D047495:PPAR gamma / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020123:Sirolimus / Q000494:pharmacology*; D058570:TOR Serine-Threonine Kinases; D015533:Transcriptional Activation / Q000187:drug effects; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D008070:Lipopolysaccharides; D016328:NF-kappa B; D047495:PPAR gamma; D000081082:Phosphoinositide-3 Kinase Inhibitors; D012333:RNA, Messenger; D011494:Protein Kinases; C546843:mTOR protein, mouse; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases; D020780:Matrix Metalloproteinase 9; D020123:Sirolimus