The differential regulation of cyclic AMP by sphingomyelin-derived lipids and the modulation of sphingolipid-stimulated extracellular signal regulated kinase-2 in airway smooth muscle

We report that sphingosine and short-chain ceramides activate adenylate cyclase and stimulate intracellular cyclic AMP formation in airway-smooth-muscle (ASM) cells. In each case, there is a conditional requirement for GTP-G(8) alpha. Sphingosine utilizes a protein kinase C-dependent pathway to elicit activation of adenylate cyclase, whereas for short-chain ceramides the mechanism remains unidentified. In contrast, sphingosine phosphate inhibits G(s)-stimulated cyclic AMP formation via a G(i)-dependent mechanism. Therefore, the potential interconversion of sphingosine and sphingosine phosphate is a switch that can elicit reciprocal changes in cyclic AMP levels. This may have a significant impact upon the regulation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal specific kinase (JNK) by sphingolipids and may help to explain how growth factors that utilize these second messengers evoke pleiotropic responses such as proliferation and cell survival. In this context, short-chain ceramides are poor stimulators of ERKs in ASM cells, and sphingosine is inactive, whereas both sphingolipids are powerful activators of the JNK module. Activated JNK catalyses N-terminal phosphorylation of c-Jun, a kinase cascade that programmes growth arrest. Therefore, in blocking ceramide-stimulated ERK-2 activity, cyclic AMP may allow the ceramide-dependent activation of JNK to programme cells to opt out of the cell cycle. In contrast, sphingosine phosphate activates ERK-2, potentiates growth-factor-stimulated DNA synthesis and fails to activate JNK, indicating that its sequential formation from ceramide and sphingosine may commit cells to DNA synthesis. ERK-2 can be activated by both cyclic AMP-sensitive c-Raf-1 kinase-dependent and cyclic AMP-insensitive c-Raf-1 kinase-independent pathways in ASM cells. In this context, sphingosine phosphate activates ERK-2 exclusively via c-Raf-1 kinase. Sphingosine phosphate-stimulated ERK-2 activity is also abolished by pertussis toxin, indicating that c-Raf-1 kinase is activated via a G(i)-dependent mechanism.