Mammalian ORMDL proteins mediate the feedback response in ceramide biosynthesis

Abstract

Background: The yeast Orm1/2 proteins regulate ceramide biosynthesis.

Results: Depletion of the mammalian Orm1/2 homologues, ORMDL1-3, eliminates the negative feedback of exogenous ceramide on ceramide biosynthesis in HeLa cells.

Conclusion: ORMDL proteins are the primary regulators of ceramide biosynthesis in mammalian cells.

Significance: Therapeutically manipulating levels of the pro-death lipid, ceramide, requires a molecular understanding of its regulation. The mammalian ORMDL proteins are orthologues of the yeast Orm proteins (Orm1/2), which are regulators of ceramide biosynthesis. In mammalian cells, ceramide is a proapoptotic signaling sphingolipid, but it is also an obligate precursor to essential higher order sphingolipids. Therefore levels of ceramide are expected to be tightly controlled. We tested the three ORMDL isoforms for their role in homeostatically regulating ceramide biosynthesis in mammalian cells. Treatment of cells with a short chain (C6) ceramide or sphingosine resulted in a dramatic inhibition of ceramide biosynthesis. This inhibition was almost completely eliminated by ORMDL knockdown. This establishes that the ORMDL proteins mediate the feedback regulation of ceramide biosynthesis in mammalian cells. The ORMDL proteins are functionally redundant. Knockdown of all three isoforms simultaneously was required to alleviate the sphingolipid-mediated inhibition of ceramide biosynthesis. The lipid sensed by the ORMDL-mediated feedback mechanism is medium or long chain ceramide or a higher order sphingolipid. Treatment of permeabilized cells with C6-ceramide resulted in ORMDL-mediated inhibition of the rate-limiting enzyme in sphingolipid biosynthesis, serine palmitoyltransferase. This indicates that C6-ceramide inhibition requires only membrane-bound elements and does not involve diffusible proteins or small molecules. We also tested the atypical sphingomyelin synthase isoform, SMSr, for its role in the regulation of ceramide biosynthesis. This unusual enzyme has been reported to regulate ceramide levels in the endoplasmic reticulum. We were unable to detect a role for SMSr in regulating ceramide biosynthesis. We suggest that the role of SMSr may be in the regulation of downstream metabolism of ceramide.

FIGURE 1.

Mammalian ORMDL proteins mediate the response of cells to C6-ceramide. A–C, effect of ORMDL and SMSr depletion on ceramide biosynthesis. [³H]Serine incorporation into ceramide was measured in HeLa (A), A549 (B) and HMEC (C) cells 48 h after transfection with either scrambled siRNA oligonucleotides or gene-specific siRNAs for all three ORMDL isoforms, SMSr alone, or a combination of siRNAs targeting ORMDL1/2/3 and SMSr. After transfection, cells were treated with 10 μm C6-ceramide for 2 h and then labeled with [³H]serine for 1 h. Cells were then harvested, and lipids were extracted as detailed under “Experimental Procedures.” Data are reported as mean cpm/mg of protein ± S.D. (n = 5) and are representative of at least three independent experiments for each cell type tested. D, uptake of [³H]serine. HeLa cells were transfected with siRNA oligonucleotides as described above, treated with 10 μm C6-ceramide for 2 h, and then labeled with [³H]serine for 1 h. Cells were then harvested with trypsin, and total intracellular ³H was quantitated on 10% of cells using liquid scintillation counting. Data are reported as mean cpm/well ± S.D. (n = 6) and are representative of three independent experiments. E, combinatorial knockdown of ORMDL isoforms. HeLa cells were transfected with either scrambled siRNA oligonucleotides as a control or gene-specific siRNAs for: (i) all three ORMDL isoforms at once; (ii) paired combinations targeting two ORMDL isoforms at the same time; or (iii) each ORMDL isoform singly. 48 h after transfection, cells were treated with 10 μm C6-ceramide for 2 h and then labeled with [³H]serine for 1 h. Cells were then harvested, and lipids were extracted as detailed under “Experimental Procedures.” Data are reported as mean cpm/well ± S.D. (n = 5) and are representative of at least three independent experiments. F, serum-responsive ceramide biosynthesis. Monolayers of cells were transfected with either scrambled siRNA or siRNA targeting all three ORMDL isoforms. 24 h after transfection, culture medium was replaced with serum-free medium containing 0.1% fatty-acid free BSA. Serum starvation was carried out for ∼30 h, after which time the medium was removed and the cells were labeled with [³H]serine for 1 h. Lipids were extracted, and [³H]serine incorporation into ceramide was measured as detailed under “Experimental Procedures.” Data are reported as -fold change ± S.D. (n = 5) from cells transfected with scrambled siRNA in the presence of serum for A549, HeLa, and PC-3 cells (Control, mean [³H]serine incorporation was 1420 cpm/mg of protein for A549, 6392 cpm/mg of protein for HeLa, and 176 cpm/mg of protein for PC-3). Results for all cell lines are representative of at least three independent experiments.

FIGURE 2.

ORMDL proteins regulate SPT in permeabilized and intact cells, but do not regulate ceramide synthases. C6-ceramide and sphingosine require metabolism by ceramide synthases to activate ORMDL inhibition of SPT. A, SPT activity in permeabilized cells. HeLa cell monolayers were treated with either scrambled siRNA or siRNA targeting all three ORMDL isoforms. 24 h after transfection, cells were permeabilized with digitonin and preincubated in the presence or absence of 20 μm C6-ceramide for 30 min as described under “Experimental Procedures.” Buffer containing [³H]serine and palmitoyl-CoA was added, and the incubation was continued for an additional 60 min. Selected wells were treated with 1 μm myriocin during both incubation periods. Sphingolipids were then extracted, and incorporation of [³H]serine into total sphingolipids was determined by liquid scintillation counting. Each point represents the mean ± S.D. of eight replicates, with the exception of myriocin-treated wells, which utilized four replicates. A result representative of four independent experiments is presented. B, effect of blocking ceramide synthase activity on C6-ceramide and sphingosine inhibition of total sphingolipid synthesis. Intact HeLa cell monolayers were treated overnight with FB1 where indicated, treated with either 10 μm C6-ceramide or 10 μm sphingosine for 2 h, and then labeled with [³H]serine to measure de novo biosynthesis of total sphingolipid as described under “Experimental Procedures.” Shown is myriocin-inhibitable incorporation of [³H]serine into total sphingolipids. C, C6-ceramide and sphingosine inhibition of ceramide synthesis. Lipid extracts generated as described for panel A were analyzed for [³H]serine incorporation into ceramide by thin layer chromatography. Shown is myriocin-inhibitable incorporation of [³H]serine into ceramide. D, ceramide synthase activity in intact cells. HeLa cells were transfected with siRNA oligonucleotides as described above, treated with 10 μm C6-ceramide for 2 h, and then labeled with d-[erythro-3-³H]sphingosine for 30 min. Cells were then harvested, and lipids were extracted as detailed under “Experimental Procedures.” Data in panels B–D are reported as mean cpm/well ± S.D. (n = 3–6) and are representative of at least three independent experiments.