Accumulation of sphingomyelin in Niemann-Pick disease type C cells disrupts Rab9-dependent vesicular trafficking of cholesterol.

Niemann-Pick disease type C (NPC) is a genetic disorder in which patient cells have endosomal/lysosomal accumulation of cholesterol and sphingolipids. However, the relationship between sphingolipids and cholesterol accumulation in NPC cells has not been established. Here, we investigated the role of sphingomyelin (SM) on the accumulation of cholesterol in NPC cells. Reduction of SM by inhibition of the ceramide transfer protein CERT decreased the cholesterol accumulation in NPC cells. The accumulation of SM in NPC cells inhibited the transport of cholesterol to the endoplasmic reticulum. Overexpression of Rab9 in NPC cells reduced the cholesterol accumulation, which was recovered by treatment with SM. In NPC cells that overexpressed a Rab9 constitutively active mutant, SM treatment did not lead to the cholesterol accumulation. These results indicate that SM negatively regulates the Rab9-dependent vesicular trafficking of cholesterol, and a reduction in SM levels in NPC cells recovers the Rab9-dependent vesicular trafficking defect.

Lactosylceramide interacts with and activates cytosolic phospholipase A2α.

Lactosylceramide (LacCer) is a member of the glycosphingolipid family and is known to be a bioactive lipid in various cell physiological processes. However, the direct targets of LacCer and cellular events mediated by LacCer are largely unknown. In this study, we examined the effect of LacCer on the release of arachidonic acid (AA) and the activity of cytosolic phospholipase A2α (cPLA2α). In CHO-W11A cells, treatment with 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP), an inhibitor of glucosylceramide synthase, reduced the glycosphingolipid level, and the release of AA induced by A23187 or platelet-activating factor was inhibited. The addition of LacCer reversed the PPMP effect on the stimulus-induced AA release. Exogenous LacCer stimulated the release of AA, which was decreased by treatment with an inhibitor of cPLA2α or silencing of the enzyme. Treatment of CHO-W11A cells with LacCer induced the translocation of full-length cPLA2α and its C2 domain from the cytosol to the Golgi apparatus. LacCer also induced the translocation of the D43N mutant of cPLA2α. Treatment of L929 cells with TNF-α induced LacCer generation and mediated the translocation of cPLA2α and AA release, which was attenuated by treatment with PPMP. In vitro studies were then conducted to test whether LacCer interacts directly with cPLA2α. Phosphatidylcholine vesicles containing LacCer increased cPLA2α activity. LacCer bound to cPLA2α and its C2 domain in a Ca(2+)-independent manner. Thus, we propose that LacCer is a direct activator of cPLA2α.