Ceramide-mediated stimulation of inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF) accumulation in murine macrophages requires tyrosine kinase activity

In macrophages, bacterial lipopolysaccharide (LPS) has been noted to mimic certain effects of the sphingolipid ceramide, suggesting that ceramide may be involved in macrophage activation by LPS and/or that LPS utilizes ceramide‐related signaling pathways. Putative downstream targets of ceramide include a ceramide‐activated (serine/threonine) protein kinase (CAPK) and phosphatase (CAPP). However, the potential role of tyrosine phosphorylation pathways in macrophage response to ceramide has not been examined. Herein we report that cell‐permeable analogs of ceramide up‐regulate both inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF) production in RAW 264.7 murine macrophages. Herbimycin A and genistein, potent natural inhibitors of protein tyrosine (but not serine/threonine) phosphorylation, block ceramide‐induced iNOS and TNF production. Furthermore, the highly src‐family selective pyrazolopyrimidine inhibitor PP1 also blocks ceramide‐induced iNOS and TNF production in RAW 264.7 cells. We found that PP1 also inhibits ceramide‐mediated tyrosine phosphorylation of the src‐family kinase hck. These data indicate that src‐related tyrosine kinases play a critical role in macrophage activation by ceramide.