Abstract
Acid sphingomyelinase (ASM) is one of the key enzymes involved in regulating the metabolism of the bioactive sphingolipid ceramide in the sphingolipid salvage pathway, yet defining signaling pathways by which ASM exerts its effects has proven difficult. Previous literature has implicated sphingolipids in the regulation of cytokines such as interleukin-6 (IL-6), but the specific sphingolipid pathways and mechanisms involved in inflammatory signaling need to be further elucidated. In this work, we sought to define the role of ASM in IL-6 production because our previous work showed that a parallel pathway of ceramide metabolism, acid β-glucosidase 1, negatively regulates IL-6. First, silencing ASM with siRNA abrogated IL-6 production in response to the tumor promoter, 4β-phorbol 12-myristate 13-acetate (PMA), in MCF-7 cells, in distinction to acid β-glucosidase 1 and acid ceramidase, suggesting specialization of the pathways. Moreover, treating cells with siRNA to ASM or with the indirect pharmacologic inhibitor desipramine resulted in significant inhibition of TNFα- and PMA-induced IL-6 production in MDA-MB-231 and HeLa cells. Knockdown of ASM was found to significantly inhibit PMA-dependent IL-6 induction at the mRNA level, probably ruling out mechanisms of translation or secretion of IL-6. Further, ASM knockdown or desipramine blunted p38 MAPK activation in response to TNFα, revealing a key role for ASM in activating p38, a signaling pathway known to regulate IL-6 induction. Last, knockdown of ASM dramatically blunted invasion of HeLa and MDA-MB-231 cells through Matrigel. Taken together, these results demonstrate that ASM plays a critical role in p38 signaling and IL-6 synthesis with implications for tumor pathobiology.
Highlights
Sphingolipids are important in multiple biological processes, but there is a lack of understanding of the pathways that mediate these effects
Differential Regulation of IL-6 Production and p38 Activation by Sphingolipid Salvage Enzymes—Stimulation of the breakdown of complex sphingolipids by phorbol 12-myristate 13-acetate (PMA) through the salvage pathway has been shown to be dependent on PKC␦, a novel PKC, in MCF-7 breast cancer cells [8]
These results demonstrate that Acid sphingomyelinase (ASM) is required for the induction of IL-6, but further metabolism of the ASM product ceramide to sphingosine via acid ceramidase (ACD) may not be required in MCF-7 cells
Summary
Sphingolipids are important in multiple biological processes, but there is a lack of understanding of the pathways that mediate these effects. Results: Acid sphingomyelinase is involved in p38 activation, interleukin-6 production, and cancer cell invasion. Acid sphingomyelinase (ASM) is one of the key enzymes involved in regulating the metabolism of the bioactive sphingolipid ceramide in the sphingolipid salvage pathway, yet defining signaling pathways by which ASM exerts its effects has proven difficult. Knockdown of ASM dramatically blunted invasion of HeLa and MDA-MB-231 cells through Matrigel Taken together, these results demonstrate that ASM plays a critical role in p38 signaling and IL-6 synthesis with implications for tumor pathobiology. This work provides evidence for the involvement of ASM in the production of IL-6 and the phosphorylation of p38, in distinction to GBA1, revealing functional specificity within the sphingolipid salvage pathway. This study provides novel evidence that ASM is required for invasion of aggressive carcinoma cells The implications of these findings for sphingolipid signaling and cancer biology are further discussed
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