Transcriptional regulation of the cyclooxygenase-2 gene in macrophages by PU.1

Macrophages are an abundant source of cyclooxygenase-2 (COX-2) enzymatic products, but a specific mechanism for macrophage COX-2 gene expression has not been described. We examined whether PU.1, a myeloid-specific Ets family transcription factor, is involved. Sequence analysis revealed two potential c-Ets binding sites in the COX-2 promoter (COX-2p) which bind to immunoreactive PU.1. Chromatin immunoprecipitation analysis shows inducible PU.1 binding to these sites in response to lipopolysaccharide, and COX-2 protein production is augmented by ectopic expression of PU.1 but not by PU.1S148A, indicating that PU.1 phosphorylation is likely involved. Interestingly, expression of PU.1 results in acetylation of CCAAT/enhancer-binding protein-beta (C/EBP-beta) and increased production of COX-2 protein. Coimmunoprecipitation experiments suggest a role for p300 in C/EBP-beta acetylation and COX-2 expression. In contrast, E1A inhibits acetylation of C/EBP-beta and is correlated with decreased COX-2 expression. Together, these data suggest that PU.1 is activated by phosphorylation of Ser148 in response to lipopolysaccharide treatment and subsequently binds to sequences in the endogenous COX-2p in a time-dependent manner. Concomitantly, C/EBP-beta becomes acetylated, and expression of the COX-2 gene increases. We speculate that a combinatorial role of PU.1 and C/EBP-beta mediates the robust production of COX-2 products by macrophages which occurs in Gram-negative bacterial sepsis.