Biochemical Mechanisms of the Generation of Endogenous Long Chain Ceramide in Response to Exogenous Short Chain Ceramide in the A549 Human Lung Adenocarcinoma Cell Line

Treatment of A549 cells with C6-ceramide resulted in a significant increase in the endogenous long chain ceramide levels, which was inhibited by fumonisin B1 (FB1), and not by myriocin (MYR). The biochemical mechanisms of generation of endogenous ceramide were investigated using A549 cells treated with selectively labeled C6-ceramides, [sphingosine-3-3H]d-erythro-, andN-[N-hexanoyl-1-14C]d-erythro-C6-ceramide. The results demonstrated that 3H label was incorporated into newly synthesized long chain ceramides, which was inhibited by FB1 and not by MYR. Interestingly, the 14C label was not incorporated into long chain ceramides. Taken together, these results show that generation of endogenous ceramide in response to C6-ceramide is due to recycling of the sphingosine backbone of C6-ceramide via deacylation/reacylation and not due to the elongation of its fatty acid moiety. Moreover, the generation of endogenous long chain ceramide in response to C6-ceramide was completely blocked by brefeldin A, which causes Golgi disassembly, suggesting a role for the Golgi in the metabolism of ceramide. In addition, the generation of endogenous ceramide in response to short chain exogenous ceramide was induced byd-erythro- but notl-erythro-C6-ceramide, demonstrating the stereospecificity of this process. Interestingly, several key downstream biological activities of ceramide, such as growth inhibition, cell cycle arrest, and modulation of telomerase activity were induced byd-erythro-C6-ceramide, and notl-erythro-C6-ceramide (and inhibited by FB1) in A549 cells, suggesting a role for endogenous long chain ceramide in the regulation of these responses.