INTERACTION OF CERAMIDES, SPHINGOSINE, AND SPHINGOSINE 1-PHOSPHATE IN REGULATING DNA-SYNTHESIS AND PHOSPHOLIPASE-D ACTIVITY

C-2- and C-6-ceramides (N-acetylsphingosine and N-hexanoylsphingosine, respectively) abolished the stimulation of DNA synthesis by sphingosine 1-phosphate in rat fibroblasts. This inhibition by ceramide was partially prevented by insulin C-2-ceramide did not alter the stimulation of DNA synthesis by insulin and decreased the sphingosine-induced stimulation by only 16%. The ceramides did not significantly modify the actions of sphingosine or sphingosine l-phosphate in decreasing cAMP concentrations. C-2- and C-6-ceramides blocked the activation of phospholipase D by sphingosine l-phosphate, and this inhibition was not affected by insulin. Okadaic acid decreased the activation of phospholipase D by sphingosine l-phosphate and did not reverse the inhibitory effect of C-2-ceramide on this activation. Therefore, this effect of C-2-ceramide is unlikely to involve the stimulation of phosphoprotein phosphatase activity. Sphingosine did not activate phospholipase D activity significantly after 10 min. C-2-ceramide stimulated the conversion of exogenous [H-3]sphingosine 1-phosphate to sphingosine and ceramide in fibroblasts. Ceramides can inhibit some effects of sphingosine l-phosphate by stimulating its degradation via a phosphohydrolase that also hydrolyzes phosphatidate. Furthermore, C-2- and C-6-ceramides stimulated ceramide production from endogenous lipids, and this could propagate the intracellular signal. This work demonstrates that controlling the production of ceramide versus sphingosine and sphingosine l-phosphate after sphingomyelinase activation could have profound effects on signal transduction.