Involvement of Acid β-Glucosidase 1 in the Salvage Pathway of Ceramide Formation

Abstract

In the present study, we examined whether acid ␤-glucosidase 1 (GBA1), which hydrolyzes glucosylceramide to form lysosomal ceramide, was involved in PKC-regulated formation of ceramide from recycled sphingosine. Glucosylceramide levels declined after treatment of MCF-7 cells with a potent PKC activator, phorbol 12-myristate 13-acetate (PMA). Silencing GBA1 by small interfering RNAs significantly attenuated acid glucocerebrosidase activity and decreased PMA-induced formation of ceramide by 50%. Silencing GBA1 blocked PMA-induced degradation of glucosylceramide and generation of sphingosine, the source for ceramide biosynthesis. Reciprocally, forced expression of GBA1 increased ceramide levels. These observations indicate that GBA1 activation can generate the source (sphingosine) for PMA-induced formation of ceramide through the salvage pathway. Next, the role of PKC␦, a direct effector of PMA, in the formation of ceramide was determined. By attenuating expression of PKC␦, cells failed to trigger PMA-induced alterations in levels of ceramide, sphingomyelin, and glucosylceramide. Thus, PKC␦ activation is suggested to stimulate the degradation of both sphingomyelin and glucosylceramide leading to the salvage pathway of ceramide formation. Collectively, GBA1 is identified as a novel source of regulated formation of ceramide, and PKC␦ is an upstream regulator of this pathway.

Defining a Role for Acid Sphingomyelinase in the p38/Interleukin-6 Pathway

“…This relates to previous work from our laboratory, which showed that the novel isoform PKC␦ is required for ASM activation leading to ceramide accumulation (9). Moreover, ASM and GBA1 both contribute to PKC␦-dependent ceramide accumulation, which is predominantly C 16 -ceramide (8). However, unique biological roles for ASM-and GBA1-derived ceramide had not been defined in the same context.…”

“…1B) (8). These findings raise the possibility of a novel paradigm that not only the specific identity of a bioactive sphingolipid is important, but the enzyme from which it is produced within a subcellular location may dictate its biologic function.…”

“…in our laboratory (8,17) and were further validated either by Western blot or real-time PCR (Fig. 1, E and F).…”

“…Activation by Sphingolipid Salvage Enzymes-Stimulation of the breakdown of complex sphingolipids by PMA through the salvage pathway has been shown to be dependent on PKC␦, a novel PKC, in MCF-7 breast cancer cells (8). Moreover, activation of ASM, leading to ceramide accumulation, has been linked to PKC␦ activity (9).…”

Regulated Secretion of Acid Sphingomyelinase

“…Although several mammalian SMases have been identified and characterized (5), neutral sphingomyelinase 2 (nSMase 2, SMPD3) and acid sphingomyelinase (aSMase, SMPD1) are the most extensively studied enzymes in regulated Cer generation (6). Recently, glucosylceramidase was also implicated in the regulated formation of ceramide in the salvage pathway (7,8).…”