A Novel Inhibitor of Ceramide Trafficking from the Endoplasmic Reticulum to the Site of Sphingomyelin Synthesis

Abstract

Ceramide produced at the endoplasmic reticulum (ER) is transported to the lumen of the Golgi apparatus for conversion to sphingomyelin (SM). N-(3-HydroxySphingolipids are ubiquitous constituents of membrane lipids in mammalian cells and play important roles in cell growth, differentiation, and apoptosis (1-3). Moreover, together with cholesterol, sphingolipids in plasma membrane constitute detergent-resistant microdomains termed lipids rafts, which are involved in membrane trafficking and cell signaling (4,5).De novo biosynthesis of sphingolipids in mammalian cells proceeds as follows (6). The first step is the condensation of L-serine and palmitoyl-CoA, a reaction catalyzed by serine palmitoyl transferase, to generate 3-ketodihydrosphingosine, which is reduced to dihydrosphingosine. Dihydrosphingosine undergoes N-acylation followed by desaturation to generate ceramide (Cer).1 These reactions to produce Cer occur at the cytosolic surface of the endoplasmic reticulum (ER) (7-9). Then, Cer is delivered to the luminal side of the Golgi apparatus and converted to sphingomyelin (SM) by SM synthase catalyzing the transfer of phosphocholine from phosphatidylcholine to Cer (10,11). Cer is also converted to glucosylceramide (GlcCer) by GlcCer synthase catalyzing the transfer of glucose from UDP-glucose to Cer. Although the catalytic site of GlcCer synthase appears to be oriented to the cytoplasm (12, 13), it is controversial whether GlcCer synthase is localized to the Golgi apparatus or more broadly distributed to microsomes. After translocation to the luminal side of the Golgi apparatus, GlcCer is further converted to more complex glycosphingolipids (14).We have previously showed that there are two pathways for transport of de novo synthesized Cer from the ER to the site of SM synthesis in various types of cultured cells, including Chinese hamster ovary (CHO) cells, HeLa cells, and human skin fibroblasts (15). The main pathway of the two is ATP-dependent, and the minor pathway is ATP-independent (or less dependent). In contrast to the synthesis of SM, the access of Cer to the site of GlcCer synthesis is largely independent of ATP. Analysis by in vitro assay of Cer transport in semi-intact CHO cells further revealed that the ATP-dependent transport of Cer, from the ER to the site of SM synthesis, requires cytosol but that cytosol is not required for transport of Cer to the site of GlcCer synthesis (16). In addition, that a CHO cell mutant defective in the ATP-dependent pathway of Cer transport exhibits no defect in transport of proteins from the ER to the Golgi apparatus has suggested that the mechanism of Cer transport differs from that of ER-to-Golgi trafficking of proteins (15).Inhibitors of de novo sphingolipid biosynthesis are useful tools to investigate the metabolism and functions of sphingolipids in cultured cells and in living animals. Various natural and chemically synthesized compounds have been found to be * The costs of publication of this article were defrayed in part by the payment of page charges. This ar...

Dynamic Modification of Sphingomyelin in Lipid Microdomains Controls Development of Obesity, Fatty Liver, and Type 2 Diabetes

“…HPA-12 reduced SM synthesis by about half compared with controls in ZS2/SMS1, agreeing with a previous report (Fig. 6D) (27). However, there was no effect from HPA-12 on SM synthesis in ZS2/SMS2 cells.…”

“…Experiments using the CERT inhibitor HPA-12 were performed as described by Yasuda et al (27). For the recovery experiment of SM, cells were labeled with [ 14 C]choline for 5 h and preincubated for 30 min with 80 M dynasore (a dynamin II inhibitor; Sigma) in DMEM containing 10% FBS.…”

Regulation and Traffic of Ceramide 1-Phosphate Produced by Ceramide Kinase

“…In COS-CerK cells, pretreatment with 5 M (1R,3R) HPA-12 decreased the levels of SM synthesized from DMB-Cer 15 min after incubation at 37°C compared with untreated controls (Fig. 5A, top panel), findings consistent with data from Hanada and co-workers (38). Unexpectedly, the levels of C1P did not decrease with drug treatment but in fact increased (Fig.…”

“…5A, lower panel). In parallel to the active (1R,3R) HPA-12 compound, we tested a 5 M racemic mixture of (1S,3R) (3S,1R) enantiomers as an inactive control (38). This inactive compound had no effect on the amount of C1P and SM synthesized from DMB-C5 Cer in either COSCerK cells or in BMDM compared with the untreated samples (Fig.…”

“…To test this hypothesis we used HPA-12, a ceramide analog synthesized by Hanada and co-workers (38) that is a direct antagonist of the CERT protein. In Chinese hamster ovary cells, the (1R,3R) HPA-12 enantiomer inhibited the transport of exogenous DMB-Cer and endogenous Cer for conversion to DMB-SM and SM, respectively (38). We observed that HPA-12 also inhibits DMB-Cer transport in COS-cells (Fig.…”