Ceramide-induced and Age-associated Increase in Macrophage COX-2 Expression Is Mediated through Up-regulation of NF-κB Activity*

Abstract

We have shown that the age-associated increase in lipopolysaccharide (LPS)-stimulated macrophages (Mφ) prostaglandin E₂ (PGE₂) production is because of ceramide-induced up-regulation of cyclooxygenase (COX)-2 transcription that leads to increased COX-2 expression and enzyme activity. To determine the mechanism of the age-related and ceramide-dependent increase in COX-2 transcription, we investigated the role of various transcription factors involved in COX-2 gene expression. The results showed that LPS-initiated activations of both consensus and COX-2-specific NF-κB, but not AP-1 and CREB, were significantly higher in Mφ from old mice than those from young mice. We further showed that the higher NF-κB activation in old Mφ was because of greater IκB degradation in the cytoplasm and p65 translocation to the nucleus. An IκB phosphorylation inhibitor, Bay 11-7082, inhibited NF-κB activation, as well as PGE₂ production, COX activity, COX-2 protein, and mRNA expression in both young and old Mφ. Similar results were obtained by blocking NF-κB binding activity using a NF-κB decoy. Furthermore, NF-κB inhibition resulted in significantly greater reduction in PGE₂ production and COX activity in old compared with young Mφ. Addition of ceramide to the young Mφ, in the presence or absence of LPS, increased NF-κB activation in parallel with PGE₂ production. Bay 11-7082 or NF-κB decoy prevented this ceramide-induced increase in NF-κB binding activity and PGE₂ production. These findings strongly suggest that the age-associated and ceramide-induced increase in COX-2 transcription is mediated through higher NF-κB activation, which is, in turn, because of a greater IκB degradation in old Mφ.