C/EBPbeta regulation in lipopolysaccharide-stimulated macrophages.
C/EBP family members contribute to the induction of the interleukin-12 p40 gene and the genes encoding several other mediators of inflammation. Here, we show by chromatin immunoprecipitation that C/EBPbeta binds the p40 promoter following lipopolysaccharide stimulation of peritoneal macrophages. However, three modes of C/EBPbeta regulation reported in other cell types were not detected, including alternative translation initiation, nuclear translocation, and increased DNA binding following posttranslational modification. In contrast, C/EBPbeta concentrations greatly increased following stimulation via MAP kinase-dependent induction of C/EBPbeta gene transcription. Increased C/EBPbeta concentrations were unimportant for p40 induction, however, as transcription of the p40 gene initiated before C/EBPbeta concentrations increased. Furthermore, disruption of C/EBPbeta upregulation by a MAP kinase inhibitor only slightly diminished p40 induction. Phosphopeptide mapping revealed that endogenous C/EBPbeta in macrophages is phosphorylated on only a single tryptic peptide containing 14 potential phosphoacceptors. This peptide was constitutively phosphorylated in primary and transformed macrophages, in contrast to its inducible phosphorylation in other cell types in response to Ras and growth hormone signaling. Altered-specificity experiments supported the hypothesis that C/EBPbeta activity in macrophages does not require an inducible posttranslational modification. These findings suggest that, although C/EBPbeta contributes to the induction of numerous proinflammatory genes, it is fully active in unstimulated macrophages and poised to stimulate transcription in conjunction with other factors whose activities are induced.
Bradley
Michelle N
MN
Howard Hughes Medical Institute and Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, California 90095-1662, USA.
Zhou
Liang
L
Smale
Stephen T
ST
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000235:genetics* / Q000378:metabolism*; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D018664:Interleukin-12 / Q000235:genetics / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism*; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D021381:Protein Transport; D011725:Pyridines / Q000494:pharmacology; D015398:Signal Transduction; D014158:Transcription, Genetic; D015854:Up-Regulation; D048051:p38 Mitogen-Activated Protein Kinases
D022782:CCAAT-Enhancer-Binding Protein-beta; D004791:Enzyme Inhibitors; D007093:Imidazoles; D008070:Lipopolysaccharides; D010446:Peptide Fragments; D011725:Pyridines; D018664:Interleukin-12; D004247:DNA; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C093642:SB 203580