Coordinated regulation of the orosomucoid-like gene family expression controls de novo ceramide synthesis in mammalian cells

Abstract

The orosomucoid-like (ORMDL) protein family is involved in the regulation of de novo sphingolipid synthesis, calcium homeostasis, and unfolded protein response. Single nucleotide polymorphisms (SNPs) that increase ORMDL3 expression have been associated with various immune/inflammatory diseases, although the pathophysiological mechanisms underlying this association are poorly understood. ORMDL proteins are claimed to be inhibitors of the serine palmitoyltransferase (SPT). However, it is not clear whether individual ORMDL expression levels have an impact on ceramide synthesis. The present study addressed the interaction with and regulation of SPT activity by ORMDLs to clarify their pathophysiological relevance. We have measured ceramide production in HEK293 cells incubated with palmitate as a direct substrate for SPT reaction. Our results showed that a coordinated overexpression of the three isoforms inhibits the enzyme completely, whereas individual ORMDLs are not as effective. Immunoprecipitation and fluorescence resonance energy transfer (FRET) studies showed that mammalian ORMDLs form oligomeric complexes that change conformation depending on cellular sphingolipid levels. Finally, using macrophages as a model, we demonstrate that mammalian cells modify ORMDL genes expression levels coordinately to regulate the de novo ceramide synthesis pathway. In conclusion, we have shown a physiological modulation of SPT activity by general ORMDL expression level regulation. Moreover, because single ORMDL3 protein alteration produces an incomplete inhibition of SPT activity, this work argues against the idea that ORMDL3 pathophysiology could be explained by a simple on/off mechanism on SPT activity.

Keywords: Ceramide; Enzyme Inhibitor; Macrophage; Serine Palmitoyltransferase; Sphingolipid.

FIGURE 1.

ORMDLs modulate ceramide production. Ceramide content from HEK293 cells was quantified by mass spectrometry after incubation with 500 μm PA conjugated with 0.5% BSA or with vehicle (0.5% BSA alone). A–C, effect of myriocin on ceramide production under PA treatment. A, absolute numbers of total ceramides detected. B, difference in total ceramide (Cer) levels between cells incubated with PA and vehicle in the presence of myriocin versus the difference in the DMSO control value. C, same as in B but for the different ceramide species (n = 3; *, p < 0.05; **, p < 0.01; ***, p < 0.001). D and E, ceramide content quantification of cells transiently transfected for 36 h with scrambled siRNA (siControl) or siRNA against the three members of the ORMDL family (si123). D, representative Western blot and total quantification of the ORMDL expression repression. E, difference in total ceramide levels between cells incubated with PA and vehicle (0.5% BSA) of cells treated with si123 versus the difference in the scrambled siRNA value. F, same as in E but for the different ceramide species (n = 6; *, p < 0.05). G and H, ceramide content of cells transiently transfected with empty vector (Control), ORMDL1, ORMDL2, ORMDL3, or the same total amount of the three plasmids together (ORMDL123). G, difference in total ceramide levels between cells incubated with PA and vehicle (0.5% BSA) after transfection with the indicated plasmids versus the difference in the control value. H, same as in G but for the different ceramide species (n = 6–8; analysis of variance test compared with control; #, p < 0.05; ##, p < 0.01). Error bars represent S.E.

FIGURE 2.

ORMDLs form a complex with SPT. HEK293 cells were transfected with ORMDL1, ORMDL2, or ORMDL1 + ORMDL2 and the indicated tagged constructs to ensure every condition had all three members of the family. A and B, Western blot analysis of immunoprecipitations (IP) with an anti-myc antibody for the different conditions as indicated. C and D, FRET acceptor photobleaching studies of homomeric pairs, ORMDL1 versus ORMDL1 (ORMDL1-CFP + ORMDL1-YFP), ORMDL2 versus ORMDL2 (ORMDL2-CFP + ORMDL2-YFP), ORMDL3 versus ORMDL3 (ORMDL3-CFP + ORMDL3-YFP), and heteromeric pairs, ORMDL1 versus ORMDL3 (ORMDL1-CFP + ORMDL3-YFP), ORMDL2 versus ORMDL3 (ORMDL2-CFP + ORMDL3-YFP), and ORMDL1 versus ORMDL2 (ORMDL2-CFP + ORMDL1-YFP). Globular YFP, ORMDL3-CFP + PCDNA3-YFP. The data were obtained from three independent experiments. Error bars represent S.E. (n = 24; *, p < 0.05).

FIGURE 3.

ORMDLs rearrange under different sphingolipid environments. HEK293 cells were transfected with ORMDL1, ORMDL2, or ORMDL1 + ORMDL2 and the indicated tagged constructs to ensure every condition had all three members of the family. Myriocin (Myr), C6-ceramide (C6), or DMSO (V) was applied to cells for 4 h. A, Western blot analysis of immunoprecipitations (IP) with an anti-myc antibody for the different conditions as indicated. B, FRET acceptor photobleaching studies between ORMDL members: pairs ORMDL1 versus ORMDL1 (ORMDL1-CFP + ORMDL1-YFP), ORMDL2 versus ORMDL2 (ORMDL2-CFP + ORMDL2-YFP), and ORMDL3 versus ORMDL3 (ORMDL3-CFP + ORMDL3-YFP). Globular YFP, ORMDL3-CFP + PCDNA3-YFP. The data were obtained from three independent experiments. Error bars represent S.E. (n = 24; *, p < 0.05).

FIGURE 4.

Reduced ORMDL/SPT ratio promotes de novo ceramide synthesis in activated macrophages. RAW267.4 cells were treated with 100 ng/ml LPS for 24 h. A, time course of total ceramide content in activated RAW cells. Bars are divided into the different ceramide species detected. Statistical analysis considers the total ceramide content. B, time course of sphinganine content in activated RAW267.4 cells. C and D, gene expression analyzed by RT-PCR of ORMDLs during activation. Data are normalized to β-actin (C) and SPTLC2 (D). Error bars represent S.E. (n = 5; *, p < 0.05; **, p < 0.01; ***, p < 0.001).