Tumor necrosis factor-α-induced cyclooxygenase-2 expression via sequential activation of ceramide-dependent mitogen-activated protein kinases, and IκB kinase 1/2 in human alveolar epithelial cells

The role of p44/42 mitogen-activated protein kinase (MAPK), p38, and c-Jun NH2-terminal kinase (JNK) in tumor necrosis factor (TNF)-alpha -induced cyclooxygenase (COX)-2 expression was studied in NCl-H292 epithelial cells. TNF-alpha -mediated COX-2 expression and COX-2 promoter activity were inhibited by the MAPK kinase inhibitor PD98059 or the p38 inhibitor SB203580. Treatment of cells for 10 min with TNF-alpha resulted in activation of p44/42 MAPK, p38, and JNK. C2-ceramide (a cell-permeable ceramide analog), bacterial neutral sphingomyelinase (Smase; an enzyme that degrades sphingomyelin to ceramide), and N-oleoylethanolamine (a ceramidase inhibitor) all induced activation of MAPKs, COX-2 expression, nuclear factor (NF)-kappaB DNA-protein binding, and COX-2 promoter activity. The inactive analog, dihydro-C2-ceramide, had no effect. SMase- or C2-ceramide-induced COX-2 expression and COX-2 promoter activity were also inhibited by PD98059 or SB203580. Glutathione, a neutral SMase inhibitor, attenuated TNF-alpha- or SMase-induced activation of MAPKs, COX-2 expression, and COX-2 promoter activity. TNF-alpha- or C2- ceramide- induced COX-2 promoter activity was inhibited by the dominant negative mutant of extracellular signal-regulated kinase 2, p38, JNK, I kappaB kinase (IKK)1, or IKK2. IKK activity was stimulated by either TNF-alpha or C2- ceramide, and these effects were inhibited by PD98059 or SB203580. All these results suggest that, in NCl-H292 epithelial cells, activation of MAPKs by ceramide contributes to the TNF-alpha signaling that occurs downstream of neutral SMase activation and results in the stimulation of IKK1/2, and NF-kappaB in the COX-2 promoter, followed by initiation of COX-2 expression.