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 (M) 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-B, but not AP-1 and CREB, were significantly higher in M from old mice than those from young mice. We further showed that the higher NF-B activation in old M was because of greater IB degradation in the cytoplasm and p65 translocation to the nucleus. An IB phosphorylation inhibitor, Bay 11-7082, inhibited NF-B 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-B binding activity using a NF-B decoy. Furthermore, NF-B inhibition resulted in significantly greater reduction in PGE 2 production and COX activity in old compared with young M. Addition of ceramide to the young M, in the presence or absence of LPS, increased NF-B activation in parallel with PGE 2 production. Bay 11-7082 or NF-B decoy prevented this ceramide-induced increase in NF-B 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-B activation, which is, in turn, because of a greater IB degradation in old M.It is well documented that T cell-mediated immune function declines in old animals and elderly humans compared with their young counterparts (1, 2). The age-associated dysregulation in macrophages (M) 1 contributes to the impaired T cell function with aging. We, as well as others, have demonstrated that immune cells, including M, from old animals and humans produced more PGE 2 than those from their young counterparts (3-7). We further showed that the increased PGE 2 production by M contributes to the decline in T cell-mediated function with aging (8).Cyclooxygenase (COX) is the rate-limiting enzyme that catalyzes the conversion of arachidonic acid (AA) to PG endoperoxide (PGH 2 ), which is further converted to different PGs and thromboxane. COX is hence a key factor in PG synthesis. Two isoforms of COX have been identified: a constitutive form, COX-1 (9, 10), and the inducible counterpart, COX-2 (11, 12). We have demonstrated that the age-associated increase in M PGE 2 production is because of higher COX activity in M from old mice compared with those from young mice. This increased COX activity is, in turn, a result of increased expression of COX-2 protein and mRNA (13). In a recent study, we further demonstrated that the age-related increase in COX-2 mRNA was because of a higher level of ceramide in old M compared with those of young, which induced up-regu...

Link between plasma ceramides, inflammation and insulin resistance: association with serum IL-6 concentration in patients with coronary heart disease

“…The sphingomyelin pathway and ceramides themselves are reported to play a role in the regulation of IL6 gene expression [11], probably through the activation of transcription factors such as the nuclear factor kappa-B (NFκB) [11,12]. Ceramides can act as inducers of proinflammatory cytokines through the Log value for serum IL-6 (pg/ml) * Fig.…”

Dysregulated expression of miR-101b and miR-26b lead to age-associated increase in LPS-induced COX-2 expression in murine macrophage

“…Indeed, many studies confirmed that NF-κB signaling pathways modulate COX-2 expression (Kim et al 2012;Wu et al 2003). To validate whether the increased expression of COX-2 in aged mouse peritoneal macrophages is associated with the activation of NF-κB, the level of phosphorylated IκBα (p-IκBα) expression was detected by Western blot assay.…”

“…Therefore, there might exist disordered regulation of COX-2 expression in the aging body. Wu et al (2003) demonstrated that NF-κB is a critical positive regulator in the age-associated LPS-induced upregulation of COX-2 activation in macrophages. To confirm this and to also ascertain the role of NF-κB in the age-associated increase of COX-2 expression, we demonstrated that NF-κB activation caused by LPS stimulation in macrophages was mirrored by those in COX-2 expression.…”

“…However, the molecular mechanism underlying the upregulation of COX-2 in the macrophages of aged animals/individuals is presently unclear. A study has suggested that LPS upregulates COX-2 transcription in the macrophages of aged mice by increasing the activation of transcription factor nuclear factor kappa B (NF-κB) (Wu et al 2003). However, to date, the regulation of COX-2 at the level of post-transcription in macrophages has not been demonstrated.…”

Ceramide triggers an NF-κB-dependent survival pathway through calpain

“…In support of the hypothesis that ceramide could play a role as inducer of survival pathways, C2 ceramide was shown to upregulate the expression of antiapoptotic genes such as Bcl-2 and Bcl-w in HC11 mammary epithelial cells 15 and cyclooxygenase (COX-2) in mouse macrophages. 16 Moreover, C2 ceramide-induced expressions of inducible nitric oxide synthase (iNos) in rat primary astrocytes 17 and in rat vascular smooth muscle cells 18 were reported to occur via NF-kB activation. The activation of the transcription factor NF-kB by other apoptotisinducing stimuli such as TNF-a, ionizing radiation or certain chemotherapeutic compounds (e.g.…”