Ceramide-induced and age-associated increase in macrophage COX-2 expression is mediated through up-regulation of NF-κB activity

We have shown that the age-associated increase in lipopolysaccharide (LPS)-stimulated macrophages (Mphi) prostaglandin E-2 (PGE(2)) 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-kappaB, but not AP-1 and CREB, were significantly higher in Mphi from old mice than those from young mice. We further showed that the higher NF-kappaB activation in old Mphi was because of greater IkappaB degradation in the cytoplasm and p65 translocation to the nucleus. An IkappaB phosphorylation inhibitor, Bay 11-7082, inhibited NF-kappaB activation, as well as PGE(2) production, COX activity, COX-2 protein, and mRNA expression in both young and old M. Similar results were obtained by blocking NF-kappaB binding activity using a NF-kappaB decoy. Furthermore, NF-kappaB inhibition resulted in significantly greater reduction in PGE(2) production and COX activity in old compared with young Mphi. Addition of ceramide to the young Mphi, in the presence or absence of LPS, increased NF-kappaB activation in parallel with PGE(2) production. Bay 11-7082 or NF-kappaB decoy prevented this ceramide-induced increase in NF-kappaB binding activity and PGE(2) production. These findings strongly suggest that the age-associated and ceramide-induced increase in COX-2 transcription is mediated through higher NF-kappaB activation, which is, in turn, because of a greater IkappaB degradation in old Mphi.