Dual ligand stimulation of RAW 264.7 cells uncovers feedback mechanisms that regulate TLR-mediated gene expression.
To characterize how signaling by TLR ligands can be modulated by non-TLR ligands, murine RAW 264.7 cells were treated with LPS, IFN-gamma, 2-methyl-thio-ATP (2MA), PGE(2), and isoproterenol (ISO). Ligands were applied individually and in combination with LPS, for 1, 2, and 4 h, and transcriptional changes were measured using customized oligo arrays. We used nonadditive transcriptional responses to dual ligands (responses that were reproducibly greater or less than the expected additive responses) as a measure of pathway interaction. Our analysis suggests that cross-talk is limited; <24% of the features with significant responses to the single ligands responded nonadditively to a dual ligand pair. PGE(2) and ISO mainly attenuated, while 2MA enhanced, LPS-induced transcriptional changes. IFN-gamma and LPS cross-regulated the transcriptional response induced by each other: while LPS preferentially enhanced IFN-gamma-induced changes in gene expression at 1 h, IFN-gamma signaling primarily attenuated LPS-induced changes at 4 h. Our data suggest specific cross-talk mechanisms: 1) LPS enhances the expression of IFN-gamma-response genes by augmenting STAT1 activity and by activating NF-kappaB, which synergizes with IFN-gamma-induced transcriptional factors; 2) IFN-gamma attenuates the late LPS transcriptional response by increasing the expression of suppressor of cytokine signaling 1 and cytokine-inducible SH2-containing protein expression; 3) 2MA modulates LPS secondary transcriptional response by increasing IFN-beta and inhibiting IL-10 gene expression; 4) PGE(2) and ISO similarly regulate the LPS transcriptional response. They increase IL-10 transcription, resulting in attenuated expression of known IL-10-suppressed genes.
Zhu
Xiaocui
X
Molecular Biology Laboratory, Alliance for Cellular Signaling, Division of Biology, California Institute of Technology, Pasadena, CA 91125.
Chang
Mi Sook
MS
Hsueh
Robert C
RC
Taussig
Ron
R
Smith
Kelly D
KD
Simon
Melvin I
MI
Choi
Sangdun
S
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000276:immunology / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D015232:Dinoprostone / Q000276:immunology / Q000378:metabolism; D015870:Gene Expression; D005786:Gene Expression Regulation / Q000276:immunology*; D007371:Interferon-gamma / Q000276:immunology / Q000378:metabolism; D007545:Isoproterenol / Q000276:immunology / Q000378:metabolism; D008024:Ligands; D008070:Lipopolysaccharides / Q000276:immunology / Q000378:metabolism; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D020239:Receptor Cross-Talk / Q000276:immunology*; D015398:Signal Transduction / Q000276:immunology*; D013873:Thionucleotides / Q000276:immunology / Q000378:metabolism; D051193:Toll-Like Receptors / Q000096:biosynthesis* / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects
D008024:Ligands; D008070:Lipopolysaccharides; D013873:Thionucleotides; D051193:Toll-Like Receptors; D007371:Interferon-gamma; D000255:Adenosine Triphosphate; D015232:Dinoprostone; D007545:Isoproterenol; C008501:2-methylthio-ATP