Ceramide regulates the transcription of cyclooxygenase-2 - Evidence for involvement of extracellular signal-regulated kinase c-Jun N-terminal kinase and p38 mitogen-activated protein kinase pathways

The ceramide signaling pathway is activated by the sphingomyelinase (SMase)-mediated hydrolysis of cell membrane sphingomyelin to ceramide, We determined whether ceramide, a lipid second messenger, induced cyclooxygenase-2 (COX-2) in human mammary epithelial cells. Treatment of cells with neutral SMase or C-2- or C-6-ceramide enhanced prostaglandin E-2 synthesis and increased levels of COX-2 protein and mRNA. Nuclear runoff assays revealed increased rates of COX-2 transcription after treatment with SMase and C-2- and C-6-ceramide, Transient transfections utilizing COX-2 promoter deletion constructs and COX-2 promoter constructs in which specific enhancer elements were mutagenized indicated that the effects of ceramide were mediated via a cAMP response element. The induction of COX-2 by ceramide was inhibited by calphostin C, an inhibitor of protein kinase C, Induction of COX-2 promoter activity by SMase was blocked by overexpressing kinase-deficient Raf-1, Triggering of the ceramide pathway also led to increases in extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) activities; pharmacological inhibitors of MAPK kinase (MEK) and p38 MAPK blocked the induction of COX-2 by SMase. Overexpressing ERK1, JNK, or p38 led to severalfold increases in COX-2 promoter activity. By comparison, overexpression of dominant negatives for ERK1/2, JNK, or p38 blocked the activation of COX-2 promoter activity by SMase, A dominant negative for c-Jun inhibited the activation of COX-2 promoter activity by ceramide, Thus, in response to ceramide, increased MAPK signaling activates c-Jun, which, in turn, induces COX-2 gene expression via the cAMP response element in the COX-2 promoter.