Sphingosine kinases regulate ER contacts with late endocytic organelles and cholesterol trafficking

Membrane contact sites (MCS), close membrane apposition between organelles, are platforms for interorganellar transfer of lipids including cholesterol, regulation of lipid homeostasis, and co-ordination of endocytic trafficking. Sphingosine kinases (SphKs), two isoenzymes that phosphorylate sphingosine to the bioactive sphingosine-1-phosphate (S1P), have been implicated in endocytic trafficking. However, the physiological functions of SphKs in regulation of membrane dynamics, lipid trafficking and MCS are not known. Here, we report that deletion of SphKs decreased S1P with concomitant increases in its precursors sphingosine and ceramide, and markedly reduced endoplasmic reticulum (ER) contacts with late endocytic organelles. Expression of enzymatically active SphK1, but not catalytically inactive, rescued the deficit of these MCS. Although free cholesterol accumulated in late endocytic organelles in SphK null cells, surprisingly however, cholesterol transport to the ER was not reduced. Importantly, deletion of SphKs promoted recruitment of the ER-resident cholesterol transfer protein Aster-B (also called GRAMD1B) to the plasma membrane (PM), consistent with higher accessible cholesterol and ceramide at the PM, to facilitate cholesterol transfer from the PM to the ER. In addition, ceramide enhanced in vitro binding of the Aster-B GRAM domain to phosphatidylserine and cholesterol liposomes. Our study revealed a previously unknown role for SphKs and sphingolipid metabolites in governing diverse MCS between the ER network and late endocytic organelles versus the PM to control the movement of cholesterol between distinct cell membranes

A New Mixed-Backbone Oligonucleotide against Glucosylceramide Synthase Sensitizes Multidrug-Resistant Tumors to Apoptosis

Enhanced ceramide glycosylation catalyzed by glucosylceramide synthase (GCS) limits therapeutic efficiencies of antineoplastic agents including doxorubicin in drug-resistant cancer cells. Aimed to determine the role of GCS in tumor response to chemotherapy, a new mixed-backbone oligonucleotide (MBO-asGCS) with higher stability and efficiency has been generated to silence human GCS gene. MBO-asGCS was taken up efficiently in both drug-sensitive and drug-resistant cells, but it selectively suppressed GCS overexpression, and sensitized drug-resistant cells. MBO-asGCS increased doxorubicin sensitivity by 83-fold in human NCI/ADR-RES, and 43-fold in murine EMT6/AR1 breast cancer cells, respectively. In tumor-bearing mice, MBO-asGCS treatment dramatically inhibited the growth of multidrug-resistant NCI/ADR-RE tumors, decreasing tumor volume to 37%, as compared with scrambled control. Furthermore, MBO-asGCS sensitized multidrug-resistant tumors to chemotherapy, increasing doxorubicin efficiency greater than 2-fold. The sensitization effects of MBO-asGCS relied on the decreases of gene expression and enzyme activity of GCS, and on the increases of C18-ceramide and of caspase-executed apoptosis. MBO-asGCS was accumulation in tumor xenografts was greater in other tissues, excepting liver and kidneys; but MBO-asGCS did not exert significant toxic effects on liver and kidneys. This study, for the first time in vivo, has demonstrated that GCS is a promising therapeutic target for cancer drug resistance, and MBO-asGCS has the potential to be developed as an antineoplastic agent

Alterations of ceramide/sphingosine 1-phosphate rheostat involved in the regulation of resistance to imatinib-induced apoptosis in K562 human chronic myeloid leukemia cells

In this study, mechanisms of resistance to imatinib-induced apoptosis in human K562 cells were examined. Continuous exposure to stepwise increasing concentrations of imatinib resulted in the selection of K562/IMA-0.2 and -1 cells, which expressed similar to 2.3- and 19-fold resistance, respectively. Measurement of endogenous ceramides by high performance liquid chromatography/mass spectroscopy showed that treatment with imatinib increased the generation of ceramide, mainly C-18-ceramide, which is generated by the human longevity assurance gene 1 (hLASS1), in sensitive, but not in resistant cells. Inhibition of hLASS1 by small interfering RNA partially prevented imatinib-induced cell death in sensitive cells. In reciprocal experiments, overexpression of hLASS1, and not hLASS6, in drug-resistant cells caused a marked increase in imatinib-induced C-18-ceramide generation, and enhanced apoptosis. Interestingly, there were no defects in the levels of mRNA and enzyme activity levels of hLASS1 for ceramide generation in K562/IMA-1 cells. However, expression levels of sphingosine kinase-1 (SK1) and generation of sphingosine 1-phosphate (S1P) were increased significantly in K562/IMA-1 cells, channeling sphingoid bases to the sphingosine kinase pathway. The partial inhibition of SK1 expression by small interference RNA modulated S1P levels and increased sensitivity to imatinib-induced apoptosis in resistant cells. On the other hand, forced expression of SK1 in K562 cells increased the ratio between total S1P/C-18-ceramide levels similar to 6-fold and prevented apoptosis significantly in response to imatinib. Additional data indicated a role for SK1/S1P signaling in the up-regulation of the Bcr-Abl expression at the post-transcriptional level, which suggested a possible mechanism for resistance to imatinib-mediated apoptosis. In conclusion, these data suggest a role for endogenous C-18-ceramide synthesis mainly via hLASS1 in imatinib-induced apoptosis in sensitive cells, whereas in resistant cells, alterations of the balance between the levels of ceramide and S1P by overexpression of SK1 result in resistance to imatinib-induced apoptosis

Antiapoptotic roles of ceramide-synthase-6-generated C16-ceramide via selective regulation of the ATF6/CHOP arm of ER-stress-response pathways

Emerging results suggest that ceramides with different fatty acid chain lengths might play distinct functions in the regulation of tumor growth and therapy. Here we report that de novo-generated C18- and C16-ceramides by ceramide synthases 1 and 6 (CerS1 and CerS6) play opposing proapoptotic and prosurvival roles, respectively, in human head and neck squamous cell carcinomas (HNSCCs). Unexpectedly, knockdown of CerS6/C16-ceramide using small interfering RNA induced endoplasmic reticulum (ER)-stress-mediated apoptosis. Reconstitution of C16-ceramide generation by induced expression of wild-type CerS6, but not its catalytically inactive mutant, protected cells from cell death induced by knockdown of CerS6. Moreover, using molecular tools coupled with analysis of sphingolipid metabolism showed that generation of C16-ceramide, and not dihydro-C16-ceramide, by induced expression of CerS6 rescued cells from ER stress and apoptosis. Mechanistically, regulation of ER-stress-induced apoptosis by CerS6/C16-ceramide was linked to the activation of a specific arm, ATF6/CHOP, of the unfolded protein response pathway. Notably, while expression of CerS1/C18-ceramide inhibited HNSCC xenograft growth, CerS6/C16-ceramide significantly protected ER stress, leading to enhanced tumor development and growth in vivo, consistent with their pro- and antiapoptotic roles, respectively. Thus, these data reveal an unexpected and novel prosurvival role of CerS6/C16-ceramide involved in the protection against ER-stress-induced apoptosis and induction of HNSCC tumor growth.—Senkal, C. E., Ponnusamy, S., Bielawski, J., Hannun, Y. A., Ogretmen, B. Antiapoptotic roles of ceramide-synthase-6-generated C16-ceramide via selective regulation of the ATF6/CHOP arm of ER-stress-response pathways

Targeting defective sphingosine kinase 1 in Niemann–Pick type C disease with an activator mitigates cholesterol accumulation

Niemann–Pick type C (NPC) disease is a lysosomal storage disorder arising from mutations in the cholesterol-trafficking protein NPC1 (95%) or NPC2 (5%). These mutations result in accumulation of low-density lipoprotein-derived cholesterol in late endosomes/lysosomes, disruption of endocytic trafficking, and stalled autophagic flux. Additionally, NPC disease results in sphingolipid accumulation, yet it is unique among the sphingolipidoses because of the absence of mutations in the enzymes responsible for sphingolipid degradation. In this work, we examined the cause for sphingosine and sphingolipid accumulation in multiple cellular models of NPC disease and observed that the activity of sphingosine kinase 1 (SphK1), one of the two isoenzymes that phosphorylate sphingoid bases, was markedly reduced in both NPC1 mutant and NPC1 knockout cells. Conversely, SphK1 inhibition with the isotype-specific inhibitor SK1-I in WT cells induced accumulation of cholesterol and reduced cholesterol esterification. Of note, a novel SphK1 activator (SK1-A) that we have characterized decreased sphingoid base and complex sphingolipid accumulation and ameliorated autophagic defects in both NPC1 mutant and NPC1 knockout cells. Remarkably, in these cells, SK1-A also reduced cholesterol accumulation and increased cholesterol ester formation. Our results indicate that a SphK1 activator rescues aberrant cholesterol and sphingolipid storage and trafficking in NPC1 mutant cells. These observations highlight a previously unknown link between SphK1 activity, NPC1, and cholesterol trafficking and metabolism.Fil: Newton, Jason. Virginia Commonwealth University School of Medicine; Estados UnidosFil: Palladino, Elisa N.D.. Virginia Commonwealth University School of Medicine; Estados UnidosFil: Weigel, Cynthia. Virginia Commonwealth University School of Medicine; Estados UnidosFil: Maceyka, Michael. Virginia Commonwealth University School of Medicine; Estados UnidosFil: Gräler, Markus H.. Universitätsklinikum Jena; AlemaniaFil: Senkal, Can E.. Virginia Commonwealth University School of Medicine; Estados UnidosFil: Enriz, Ricardo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; ArgentinaFil: Marvanova, Pavlina. Veterinární univerzita Brno; República ChecaFil: Jampilek, Josef. Univerzita Komenského v Bratislave; EslovaquiaFil: Lima, Santiago. Virginia Commonwealth University; Estados UnidosFil: Milstien, Sheldon. Virginia Commonwealth University School of Medicine; Estados UnidosFil: Spiegel, Sarah. Virginia Commonwealth University School of Medicine; Estados Unido

Direct interaction between the inhibitor 2 and ceramide via sphingolipid-protein binding is involved in the regulation of protein phosphatase 2A activity and signaling

In this study, the inhibitor 2 of protein phosphatase 2A (I2PP2A) was identified in vitro and in situ as a ceramide-binding protein, which exhibits stereoisomer specificity and fatty acid chain length preference. Site- directed mutagenesis coupled with structural details of I2PP2A suggested that VIK 207-209 residues localized on helix 7 are important for ceramide binding and single mutation of K209D altered this interaction. Notably, I2PP2A-ceramide binding decreased the association between PP2A and the inhibitor, preventing the inhibition of PP2A activity in vitro. In addition, studies in A549 human lung cancer cells revealed that ceramide mediates c-Myc degradation via its PP2A-dependent dephosphorylation at S62, and treatment with okadaic acid and expression of c-Myc mutants with S62A or S62D conversions resulted in resistance to ceramide-mediated degradation. Importantly, whereas down-regulation of I2PP2A enhanced PP2A-mediated c-Myc degradation in response to ceramide, ectopic expression of wild-type I2PP2A but not of its K209D mutant protected this degradation in A549 cells. Moreover, expression of wild-type I2PP2A prevented the growth-inhibitory effects of ceramide both against A549 cells and xenograft-driven tumors in situ and in vivo compared with that in controls. Thus, these results suggest that direct interaction of I2PP2A with ceramide plays important biological roles via the regulation of PP2A activity and signaling, which in turn control ceramide-mediated degradation of c-Myc and antiproliferation.—Mukhopadhyay, A., Saddoughi, S. A., Song, P., Sultan, I., Ponnusamy, S., Senkal, C. E., Snook, C. F., Arnold, H. K., Sears, R. C., Hannun, Y. A., Ogretmen, B. Direct interaction between the inhibitor 2 and ceramide via sphingolipid-protein binding is involved in the regulation of protein phosphatase 2A activity and signaling