ORMDL/serine palmitoyltransferase stoichiometry determines effects of ORMDL3 expression on sphingolipid biosynthesis

Abstract

The ORM1 (Saccharomyces cerevisiae)-like proteins (ORMDLs) and their yeast orthologs, the Orms, are negative homeostatic regulators of the initiating enzyme in sphingolipid biosynthesis, serine palmitoyltransferase (SPT). Genome-wide association studies have established a strong correlation between elevated expression of the endoplasmic reticulum protein ORMDL3 and risk for childhood asthma. Here we test the notion that elevated levels of ORMDL3 decrease sphingolipid biosynthesis. This was tested in cultured human bronchial epithelial cells (HBECs) (an immortalized, but untransformed, airway epithelial cell line) and in HeLa cells (a cervical adenocarcinoma cell line). Surprisingly, elevated ORMDL3 expression did not suppress de novo biosynthesis of sphingolipids. We determined that ORMDL is expressed in functional excess relative to SPT at normal levels of expression. ORMDLs and SPT form stable complexes that are not increased by elevated ORMDL3 expression. Although sphingolipid biosynthesis was not decreased by elevated ORMDL3 expression, the steady state mass levels of all major sphingolipids were marginally decreased by low level ORMDL3 over-expression in HBECs. These data indicate that the contribution of ORMDL3 to asthma risk may involve changes in sphingolipid metabolism, but that the connection is complex.

Keywords: ORM1 (Saccharomyces cerevisiae)-like protein; Orm; asthma; ceramides; endoplasmic reticulum; enzymology/enzyme regulation; lung; sphingosine phosphate.

Fig. 1.

All three ORMDL isoforms, as well as subunit 1 of SPT, are expressed at similar levels in multiple cell types. Real-time PCR was performed on RNA isolated from immortalized HBECs, HeLa cells, and A549 adenocarcinoma cells as described in the Materials and Methods. Results are normalized to levels of 18S internal control and represented as ΔC_T values.

Fig. 2.

Overexpression of ORMDL3, both transiently and in stably transfected cells, does not depress de novo ceramide biosynthesis. A: Stable cell lines overexpressing either empty vector or human ORMDL3 (not epitope tagged) were generated in HBECs using lentiviral constructs as described in the Materials and Methods. Stable lines were chosen based on levels of ORMDL3 protein expression and data is reported for low (light gray bar) and high (dark gray bar) expressing lines. Cell monolayers were incubated for 60 min in the presence (+C6) or absence (NT) of 10 μM C6-ceramide after which they were labeled for 60 min with ³H-serine and incorporation of ³H-serine into ceramide was determined as described in the Materials and Methods. HeLa cells were transiently transfected with either empty vector or mouse ORMDL3 (not epitope tagged) for 24 h prior to treatment with 10 μM C6-ceramide, after which they were labeled for 60 min with ³H-serine as described above. Results for all data shown are the means ± SD of replicates (n = 4–6). Shown are representative results from at least three independent experiments. *Significant at P < 0.05, **significant at P < 0.005 by Student’s t-test. B: Permeabilized HBEC cultures were assayed for SPT activity. ³H-serine incorporation into total sphingolipids was measured as described in the Materials and Methods using HBEC cell lines expressing either a vector control [ORMDL3 (−)] or moderately or highly elevated levels of ORMDL3 [ORMDL3 (+)]. Shown are results from four experiments in which counts for each sample have been normalized to the vector-control samples. Each experiment included four replicates of each condition. Results for all data shown are the means ± SD of the normalized data. Replicate wells were also incubated in the presence of 1 μM myriocin as a control (not shown), which eliminated labeling by over 90%. *Significant at P < 0.05 by Student’s t-test. ORMDL3-High did not differ significantly from control. C: Cell lysates were generated from both stably transfected HBECs and transiently transfected HeLa cells that were transfected with either empty vector (V) or mouse ORMDL3 (not epitope tagged) as described in the Materials and Methods. Western blotting and densitometry were performed as described in the Materials and Methods. Numerical values are relative expression levels as compared with vector-only HBECs. D: One well of each 12-well plate used for ceramide labeling studies [see (A)] was lifted and gene expression was determined by quantitative real-time PCR as described in the Materials and Methods. Results are normalized to levels of 18S internal control and represented as fold change in mRNA expression levels as calculated by the ΔΔC_T formula.

Fig. 3.

ORMDL3 overexpression inhibits SPT expressed at elevated levels in HeLa cells. A: Forty-eight hours prior to labeling total lipids with ³H-serine, HeLa cells were transfected with either scrambled or ORMDL-specific siRNA oligonucleotides targeting all three isoforms of ORMDL. Twenty-four hours after ORMDL knockdown, cells were transiently transfected with either empty vector or a construct expressing three subunits of SPT as a single polypeptide (scSPT) as described in the Materials and Methods. Labeling and lipid extraction/quantitation were performed exactly as outlined for Fig. 2. NT, not treated with C6-ceramide; +C6, treated with 10micromolar C-6 ceramide for 60 minutes as described in Methods. Open bars, vector/control siRNA transfected. Light grey bars, transfected with siRNA directed against all three ORMDLs as described in Methods. Dark grey bars, transfected with scSPT. Black bars, transfected with both scSPT and with ORMDL siRNA. B: Twenty-four hours prior to labeling total lipids with ³H-serine, HeLa cells were transiently transfected with either empty vector, mouse ORMDL3 (not epitope tagged), scSPT, or a combination of ORMDL3 and scSPT as described in the Materials and Methods. Labeling and lipid extraction/quantitation were performed exactly as outlined for (A). C: Cell lysates were generated from one well of each 12-well plate used in the ceramide labeling studies outlined above. SDS-PAGE electrophoresis followed by Western blot analysis, as described in the Materials and Methods, was performed to determine levels of protein expression for all cell lines used for ceramide labeling studies. D: Real-time PCR was performed as described for Fig. 2D. A, B: Results are representative of at least three experiments, at least four replicates/sample. Mean ± SD is shown. **Significant to P < 0.005, ***significant to P < 0.0005 by Student’s t-test.

Fig. 4.

Steady state levels of sphingolipids in HeLa cells resulting from SPT and ORMDL3 expression reflect changes in de novo synthesis. HeLa cells were transfected with control plasmids or plasmids encoding a scSPT construct and/or mouse ORMDL3 (not epitope tagged). Sphingolipids were extracted and subjected to analysis by mass spectroscopy as described in the Materials and Methods. *Significant to P < 0.05, **significant to P < 0.005, ***significant to P < 0.0005 by Student’s t-test. Unmarked bars did not differ from the vector control at P > 0.05.

Fig. 5.

ORMDL3 and SPT are constitutively associated and levels of the complex are not increased by ORMDL3 overexpression or affected by elevation of cellular ceramide. A: Twenty-four hours prior to harvesting and IP, HeLa cells were transiently transfected with either scrambled siRNA oligonucleotides or siRNA olignucleotides directed against all three ORMDL isoforms (siORMDLs) as a control for the specificity of IP. A separate set of cells was transfected either with empty vector or human ORMDL3 (epitope-tagged) as described in the Materials and Methods. A third set of untransfected cells was incubated either with vehicle or 10 μM C-6 ceramide for 60 min prior to harvest. ORMDLs were immunoprecipitated and Western blotting performed as described in the Materials and Methods. Note that the overexpressed ORMDL3 is epitope tagged and so migrates at an increased molecular mass as compared with endogenous ORMDLs (P, post-IP beads; S, post-IP supernatant; L, pre-IP lysate). B: Overexpression of non-epitope-tagged mouse ORMDL3 does not increase co-IP of SPTLC1 with ORMDL. Hela cells, in triplicate, were transfected either with a vector control (−) or with mouse ORMDL3 lacking any epitope tags (+). Lysates were prepared and immunoprecipitated with anti-ORMDL antibody as described in the Materials and Methods. Immunoprecipitates and lysates were immunoblotted for ORMDL (below line) and SPTLC1 (above line) as described in the Materials and Methods.

Fig. 6.

Steady state levels of sphingolipids in HBECs stably expressing ORMDL3. Stable HBEC cell lines overexpressing either empty vector or human ORMDL3 (not epitope tagged) at moderately (Low) or highly (High) elevated levels were extracted and subjected to analysis by mass spectroscopy as described in the Materials and Methods. Data represent the means ± SD of replicates (n = 6) and are representative of two independent experiments. *Significant to P < 0.05, **significant to P < 0.005 by Student’s t-test.