C/EBPβ Regulation in Lipopolysaccharide-Stimulated Macrophages

Abstract

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/EBPβ binds the p40 promoter following lipopolysaccharide stimulation of peritoneal macrophages. However, three modes of C/EBPβ 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/EBPβ concentrations greatly increased following stimulation via MAP kinase-dependent induction of C/EBPβ gene transcription. Increased C/EBPβ concentrations were unimportant for p40 induction, however, as transcription of the p40 gene initiated before C/EBPβ concentrations increased. Furthermore, disruption of C/EBPβ upregulation by a MAP kinase inhibitor only slightly diminished p40 induction. Phosphopeptide mapping revealed that endogenous C/EBPβ 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/EBPβ activity in macrophages does not require an inducible posttranslational modification. These findings suggest that, although C/EBPβ 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.

ACKNOWLEDGMENTS

We thank Martina Buck, Philip Cohen, Roger Davis, Tony Hunter, Peter Johnson, and Valeria Poli for helpful suggestions and Valeria Poli, Xiao-Hong Sun, and Gretchen Darlington for providing C/EBPβ-deficient mice. We are also grateful to Valerie Schuman and Dana Russell for performing the thioglycolate injections and to Peter Johnson, Shomyseh Sanjabi, and Tianyi Wang for critical reading of the manuscript.

This work was supported by the USPHS National Research Service Award GM07185, a Warsaw Fellowship, and a UCLA Dissertation Year Fellowship to M.N.B. S.T.S. is an Investigator of the Howard Hughes Medical Institute.