The induction of cyclooxygenase-2 mRNA in macrophages is biphasic and requires both CCAAT enhancer-binding protein β (C/EBPβ) and C/EBPδ transcription factors

Prostaglandins are important mediators of activated macrophage functions, and their inducible synthesis is mediated by cyclooxygenase-2 (COX-2). Here, we make use of the murine macrophage cells RAW264 as well as of immortalized macrophages derived from mice deficient for the transcription factor CCAAT enhancer-binding protein beta (C/EBPbeta) to explore the molecular mechanisms regulating COX-2 induction in activated macrophages. We demonstrate that lipopolysaccharide-mediated COX-2 mRNA induction is biphasic. The initial phase is independent of de novo protein synthesis, correlates with cAMP-response element-binding protein (CREB) activation, is inhibited by treatments that abolish CREB phosphorylation and reduce NF-kappaB-mediated gene activation, and requires the presence of the transcription factor C/EBPbeta. On the other hand, C/EBPdelta appears to be essential in addition to C/EBPbeta to effect the second phase of COX-2 gene transcription, which is important for maintaining the induced state and requires de novo protein synthesis. Indeed, both phases of COX-2 induction were defective in C/EBPbeta-/- macrophages. Moreover, the synthesis of C/EBPdelta was increased dramatically by treatment with lipopolysaccharide and, like COX-2 induction, repressed by combined inhibition of the MAPK and of the SAPK2/p38 cascades. Taken together, these data identify CREB, NF-kappaB, and both C/EBPbeta and -delta as key factors in coordinately orchestrating transcription from the COX-2 promoter in activated macrophages.