ORMDL/serine palmitoyltransferase stoichiometry determines effects of ORMDL3 expression on sphingolipid biosynthesis
2015; Elsevier BV; Volume: 56; Issue: 4 Linguagem: Inglês
10.1194/jlr.m057539
ISSN1539-7262
AutoresDeanna Siow, Manjula Sunkara, Teresa Dunn, Andrew J. Morris, Binks W. Wattenberg,
Tópico(s)Cellular transport and secretion
ResumoThe 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. 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. The ORM1 (Saccharomyces cerevisiae)-like proteins (ORMDLs) and their yeast orthologs, the ORMs, regulate the initiating and rate-limiting enzyme in sphingolipid biosynthesis, serine palmitoyltransferase (SPT) (1Breslow D.K. Collins S.R. Bodenmiller B. Aebersold R. Simons K. Shevchenko A. Ejsing C.S. Weissman J.S. Orm family proteins mediate sphingolipid homeostasis.Nature. 2010; 463: 1048-1053Crossref PubMed Scopus (433) Google Scholar, 2Han S. Lone M.A. Schneiter R. Chang A. Orm1 and Orm2 are conserved endoplasmic reticulum membrane proteins regulating lipid homeostasis and protein quality control.Proc. Natl. Acad. Sci. USA. 2010; 107: 5851-5856Crossref PubMed Scopus (202) Google Scholar, 3Siow D.L. Wattenberg B.W. Mammalian ORMDL proteins mediate the feedback response in ceramide biosynthesis.J. Biol. Chem. 2012; 287: 40198-40204Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 4Kiefer K. Carreras-Sureda A. Garcia-López R. Rubio-Moscardó F. Casas J. Fabriás G. Vicente R. Coordinated regulation of the orosomucoid-like gene family expression controls de novo ceramide synthesis in mammalian cells.J. Biol. Chem. 2015; 290: 2822-2830Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). The three ORMDL isoforms (ORMDL1–3) are small (17.5 kDa) hydrophobic membrane proteins that are situated in the endoplasmic reticulum along with SPT. The three isoforms are highly homologous and their functions appear to be redundant at the cellular level (3Siow D.L. Wattenberg B.W. Mammalian ORMDL proteins mediate the feedback response in ceramide biosynthesis.J. Biol. Chem. 2012; 287: 40198-40204Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 4Kiefer K. Carreras-Sureda A. Garcia-López R. Rubio-Moscardó F. Casas J. Fabriás G. Vicente R. Coordinated regulation of the orosomucoid-like gene family expression controls de novo ceramide synthesis in mammalian cells.J. Biol. Chem. 2015; 290: 2822-2830Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). The ORMDLs are negative regulators of SPT and mediate the homeostatic regulation of SPT in response to cellular sphingolipid levels. siRNA depletion of the ORMDLs results in elevated sphingolipid synthesis under control conditions and reverses the homeostatic inhibition of sphingolipid synthesis resulting from elevated cellular sphingolipid content (1Breslow D.K. Collins S.R. Bodenmiller B. Aebersold R. Simons K. Shevchenko A. Ejsing C.S. Weissman J.S. Orm family proteins mediate sphingolipid homeostasis.Nature. 2010; 463: 1048-1053Crossref PubMed Scopus (433) Google Scholar, 3Siow D.L. Wattenberg B.W. Mammalian ORMDL proteins mediate the feedback response in ceramide biosynthesis.J. Biol. Chem. 2012; 287: 40198-40204Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 4Kiefer K. Carreras-Sureda A. Garcia-López R. Rubio-Moscardó F. Casas J. Fabriás G. Vicente R. Coordinated regulation of the orosomucoid-like gene family expression controls de novo ceramide synthesis in mammalian cells.J. Biol. Chem. 2015; 290: 2822-2830Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). The Orms and ORMDLs form stable physical complexes with SPT (1Breslow D.K. Collins S.R. Bodenmiller B. Aebersold R. Simons K. Shevchenko A. Ejsing C.S. Weissman J.S. Orm family proteins mediate sphingolipid homeostasis.Nature. 2010; 463: 1048-1053Crossref PubMed Scopus (433) Google Scholar), even under conditions in which the ORMDLs are minimally inhibiting SPT. In yeast, the Orm regulatory activity is controlled by Orm phosphorylation. This may not be the case for the mammalian ORMDLs, which lack the sequences that are phosphorylated in the yeast Orms. Clinical interest in ORMDL function has been sparked by the observation that SNPs adjacent to the ORMDL3 gene are highly correlated with risk for childhood asthma (5Moffatt M.F. Kabesch M. Liang L. Dixon A.L. Strachan D. Heath S. Depner M. von Berg A. Bufe A. Rietschel E. et al.Genetic variants regulating ORMDL3 expression contribute to the risk of childhood asthma.Nature. 2007; 448: 470-473Crossref PubMed Scopus (1258) Google Scholar, 6Zhang Y. Moffatt M.F. Cookson W.O. Genetic and genomic approaches to asthma: new insights for the origins.Curr. Opin. Pulm. Med. 2012; 18: 6-13Crossref PubMed Scopus (79) Google Scholar). Cookson and colleagues determined that the risk allele correlates with elevated ORMDL3 mRNA expression (5Moffatt M.F. Kabesch M. Liang L. Dixon A.L. Strachan D. Heath S. Depner M. von Berg A. Bufe A. Rietschel E. et al.Genetic variants regulating ORMDL3 expression contribute to the risk of childhood asthma.Nature. 2007; 448: 470-473Crossref PubMed Scopus (1258) Google Scholar, 7Dixon A.L. Liang L. Moffatt M.F. Chen W. Heath S. Wong K.C. Taylor J. Burnett E. Gut I. Farrall M. et al.A genome-wide association study of global gene expression.Nat. Genet. 2007; 39: 1202-1207Crossref PubMed Scopus (789) Google Scholar). It has been suggested that elevated ORMDL3 increases the risk for asthma by depressing sphingolipid biosynthesis (8Levy B.D. Sphingolipids and susceptibility to asthma.N. Engl. J. Med. 2013; 369: 976-978Crossref PubMed Scopus (22) Google Scholar). Here we directly test whether ORMDL3 overexpression inhibits de novo sphingolipid biosynthesis in cells derived from one of the tissues important for the asthmatic phenotype, the airway epithelium. We find, surprisingly, that elevated ORMDL3 does not suppress de novo sphingolipid biosynthesis and present evidence that this is because ORMDLs are normally expressed in functional excess relative to SPT. Silencer® Select siRNA oligonucleotides for human ORMDL1–3 (catalog numbers s41258, s26474, and s41260, respectively) were from Ambion® (part of Life Technologies, Grand Island, NY). The gene-specific TaqMan® probes for quantitative real-time PCR, TaqMan® Gene Expression Master Mix, high-capacity reverse transcription kit, and MicroAmp® Fast Optical 96-well PCR plates were all from Applied Biosystems® (part of Life Technologies). The Lipofectamine® RNAiMax for siRNA delivery, Lipofectamine® 2000 transfection reagent, and TRIzol® were from Invitrogen (Life Technologies). The In-Fusion® HD Cloning Plus kit and CloneAmp™ HiFi PCR Premix were both from Clontech Laboratories (Mountain View, CA). L-[3H(G)]-serine, scintillation vials, and scintillation fluid were from Perkin Elmer (Waltham, MA). C16-ceramide standard was from Avanti Polar Lipids (Alabaster, AL). Whatman TLC plates were from VWR (Radnor, PA). HeLa cells were from ATCC (Manassas, VA). A549 lung adenocarcinoma cells were from ATCC. Human bronchial epithelial cells (HBECs) (9Ramirez R.D. Sheridan S. Girard L. Sato M. Kim Y. Pollack J. Peyton M. Zou Y. Kurie J.M. Dimaio J.M. et al.Immortalization of human bronchial epithelial cells in the absence of viral oncoproteins.Cancer Res. 2004; 64: 9027-9034Crossref PubMed Scopus (496) Google Scholar) were a generous gift of Dr. Jerry Shay, University of Texas, Southwestern, Department of Cell Biology. Keratinocyte serum-free medium for culturing HBECs was from Invitrogen (Life Technologies). DMEM and all other cell culture supplies were from VWR. Anti-SPT long chain base subunit 1 (SPTLC1) antibody was from Santa Cruz Biotechnology, Inc. (Dallas, TX). Anti-ORMDL antibody was from EMD Millipore (Billerica, MA). Anti-beta actin and secondary antibodies were all from Pierce (part of Thermo Scientific, Rockford, IL). Organic solvents were from Thermo Fisher Scientific (Pittsburgh, PA). Phosphorous standard and all other chemicals used were from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated. Measurement of de novo ceramide biosynthesis in intact cells was accomplished by incorporation of 3H-serine into ceramide during a 60 minute incubation, as previously described (3Siow D.L. Wattenberg B.W. Mammalian ORMDL proteins mediate the feedback response in ceramide biosynthesis.J. Biol. Chem. 2012; 287: 40198-40204Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Briefly, HeLa cells and HBECs were plated in 12-well plates at various densities depending on the protocol in complete medium overnight. The next day, the HeLa cells were transfected as described below and the stable HBECs were cultured until desired confluence was reached. Prior to labeling, cells were treated with 10 μM ceramide-N-hexanoyl-D-erythro-sphingosine (C6)-ceramide for 60 minutes, where indicated. Cells were labeled in serine-depleted medium (MEM containing 1% dialyzed FBS and 10 μCi/ml 3H-serine) using 1 ml/well for 60 minutes. Total lipids were extracted by a modification of the Bligh-Dyer extraction (3Siow D.L. Wattenberg B.W. Mammalian ORMDL proteins mediate the feedback response in ceramide biosynthesis.J. Biol. Chem. 2012; 287: 40198-40204Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) and the organic phase was dried under nitrogen. The entire organic phase was then subjected to TLC using a solvent system of chloroform:acetic acid:methanol (90:10:2, v/v) and C16-ceramide was spotted in each lane as a standard. After visualizing the ceramide band by exposure in an iodine tank, the ceramide bands were scraped and incorporated 3H-serine was measured by liquid scintillation counting. HBECs were plated into collagen-coated 24-well plates at 0.3 × 106 cells/well overnight. Cells were then washed with PBS and permeabilized by treatment with 200 μg/ml digitonin for 3 minutes. SPT activity was measured in a buffer containing 50 mM HEPES (pH 8.0), 1 mM MgCl2, 1 mM ATP, 20 μM 5′ pyridoxyl phosphate, 50 μM palmitoyl CoA, 1 mM serine, and 3H-serine at 10 μCi/ml in a volume of 400 μl. Incubations were performed for 60 minutes at 37°C. Following incubation, cells were washed with PBS and 400 μl PBS was added to each well. Alkaline methanol (400 μl) (7 g KOH/l) was added and cells were harvested into microcentrifuge tubes. CHCl3 (100 μl) was added and extracts were vortexed. Phases were broken by the addition of 500 μl CHCl3, 500 μl alkaline water (100 μl 2 N NH4OH/100 ml water), and 100 μl 2 N NH4OH. The top aqueous layer was aspirated and the remaining organic layer was washed two times with 1 ml alkaline water. Three hundred and fifty microliters of the organic layer was dried under N2 in a scintillation vial and incorporated 3H-serine was measured by liquid scintillation counting. HeLa cells were plated at 8 × 104 cells/well in collagen-coated 12-well plates overnight in complete medium. siRNA oligonucleotide (5 nmol) for each ORMDL isoform targeted was combined with Lipofectamine® RNAiMax transfection reagent per the manufacturer's recommendations, added to medium, and cells were incubated 24–48 hours before measurement of de novo ceramide biosynthesis, as described above. The effect of siRNA treatment of expression of ORMDL1–3 was determined by quantitative real-time PCR using gene-specific TaqMan® probes, as previously described (3Siow D.L. Wattenberg B.W. Mammalian ORMDL proteins mediate the feedback response in ceramide biosynthesis.J. Biol. Chem. 2012; 287: 40198-40204Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) and detailed below. Experiments illustrated in Figs. 2, 3, 5, and 6 utilized mouse ORMDL3 (not epitope tagged), subcloned into the XbaI site of pCMV6-XL5 (Origene, Rockville, MD) by In-Fusion® cloning (Clontech) according to the manufacturer's instructions using the following primers: forward, 5'-GCT TGT CGA CTC TAG ACC ACC ATG AAT GTG GGC A-3' reverse, 5'-GCG GCC GCA ATC TAG ATC AGT ACT TAT TGA TTC CAA AG-3'. The immunoprecipitation (IP) experiments illustrated in Fig. 5 utilized human ORMDL3 in pCMV6 (epitope-tagged with Myc and FLAG, both at the carboxy-terminus) purchased from Origene (catalog number RC202279). Note that the amino acid sequences of the mouse (NCBI reference sequence NP_079937.1) and human (NCBI reference sequence ID AAM43507.1) ORMDL3s are 96% identical and of the exact same length. Single-chain SPT (scSPT) consists of a fusion of SPT long chain base subunit 2 (SPTLC2), SPT small subunit a (ssSPTa), and SPTLC1, in that order as described (10Gable K. Gupta S.D. Han G. Niranjanakumari S. Harmon J.M. Dunn T.M. A disease-causing mutation in the active site of serine palmitoyltransferase causes catalytic promiscuity.J. Biol. Chem. 2010; 285: 22846-22852Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), subcloned into the Xba1 site of pCMV6-XL5 (Origene) by In-Fusion® cloning (Clontech) according to the manufacturer's instructions using the following primers: forward, 5′-GCT TGT CGA CTC TAG ACC ACC ATG GCT AGG CGG CCG GAG C-3′ reverse, 5′-GCG GCC GCA ATC TAG ATC AGA GCA GGA CGG CCT GGG-3′. HeLa cells were plated at 1 × 105 cells/well in collagen-coated 12-well plates overnight in complete medium. The next day, medium was replaced with a transfection mix containing 1.6 μg of total plasmid and 4 μl of Lipofectamine 2000® (Invitrogen) according to the manufacturer's instructions.Fig. 3ORMDL3 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5ORMDL3 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 6Steady 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) For siRNA of ORMDL1–3, HeLa cells were plated at 1 × 106 cells in collagen-coated p100 dishes overnight in complete medium. The next day, cells were transfected using 5 nM siRNA oligonucleotides for each ORMDL isoform being targeted and Lipofectamine RNAiMax transfection solution following the manufacturer's instructions. Forty-eight hours after siRNA treatment, cells were lifted with trypsin, pelleted, washed with 1× PBS then solubilized on ice for 60 minutes with 1% digitonin IP buffer [25 mM Tris (pH 7.5), 150 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 15% glycerol, and 1% digitonin (w/v)]. For overexpression of recombinant ORMDL3, HeLa cells were plated at 3 × 106 cells in collagen-coated p100 dishes overnight in complete medium. The next day, cells were transfected with 25 μg plasmid DNA and 50 μl Lipofectamine® 2000 per p100 dish using the manufacturer's instructions. Twenty-four hours after transfection, cells were lifted with trypsin, pelleted, washed with 1× PBS then solubilized for 60 minutes on ice, as described above. Solubilized cells were centrifuged at 10,000 rpm for 10 minutes at 4°C and lysates were moved to clean microfuge tubes. Lysates were then pre-cleared with Protein A beads for 1 hour at 4°C, after which beads were pelleted and discarded and cleared lysates were transferred to clean microfuge tubes. Cleared lysates were incubated on rotator at 4°C overnight with anti-ORMDL polyclonal antibody (Millipore). Following IP, samples were centrifuged at 10,000 rpm for 10 minutes at 4°C and supernatants were moved to clean microfuge tubes. Supernatants were incubated with Protein A beads (blocked with 5% BSA in IP buffer) on a rotator at 4°C for 2 hours. Beads were then pelleted, post-IP supernatants saved for Western blot analysis, and beads were washed four times with IP buffer containing 0.1% digitonin. The 2× Laemmli sample buffer was added to the washed beads and they were heated at 60°C for 30 minutes prior to SDS-PAGE (see Western blot protocol details below). Proteins were separated on 12% SDS polyacrylamide gels. Following SDS-PAGE, proteins were transferred to polyvinylidene difluoride membranes and the membranes were blocked overnight with 5% milk in TBS with 0.1% Tween20 (TBST). Blots were cut horizontally using molecular weight markers as a guide depending on the size of proteins to be probed. This allowed blots to be probed with multiple antibodies without the necessity of stripping and reprobing the blots multiple times. Anti-ORMDL (1:2,000) and anti-SPTLC1 (1:1,000) primary antibodies were incubated overnight at 4°C in TBST containing 3% milk; anti-beta actin (1:10,000) primary antibody was incubated for 1 hour at room temperature. After incubation with primary antibodies, blots were washed three times with TBST for 10 minutes per wash. Secondary antibodies (1:20,000) were incubated for 1 hour at room temperature in TBST containing 1% milk. Blots were washed three times with TBST for 10 minutes per wash. Protein bands were visualized using ECL Prime solution (GE Life Sciences, Piscataway, NJ) and exposed to film (Denville Scientific, Metuchen, NJ). Densitometry values were measured using Quantity One software and ORMDL3 levels were normalized to beta-actin for each sample. pENTR1A Gateway® entry vector (#17398) and pLenti-CMV-puro lentiviral Gateway® destination vector (#17452) were purchased from Addgene (Cambridge, MA), as described (11Campeau E. Ruhl V.E. Rodier F. Smith C.L. Rahmberg B.L. Fuss J.O. Campisi J. Yaswen P. Cooper P.K. Kaufman P.D. A versatile viral system for expression and depletion of proteins in mammalian cells.PLoS ONE. 2009; 4: e6529Crossref PubMed Scopus (588) Google Scholar). Human ORMDL3 plasmid #RC202279 (Origene, representing NM_139280) was linearized by cutting with EcoRI after which the open reading frame for ORMDL3 was amplified using CloneAmp™ HiFi PCR Premix and primers designed for use with the In-Fusion® HD Cloning Plus kit (forward, ATC CGG TAC CGA ATT GCC ATG AAT GTG GGC ACA; reverse, GTG CGG CCG CGA ATT TTA AAC GTA CTT ATT GAT TCC A). Insert and vector were gel purified prior to subcloning human ORMDL3 into pENTR1A using the In-Fusion® HD Cloning Plus kit following the manufacturer's instructions. The human ORMDL3 open reading frame was transferred from pENTR1A entry vector to pLenti-CMV-puro destination vector using the Gateway® LR Clonase™ II enzyme mix following the manufacturer's instructions. Sequences of all subsequent clones were verified by the University of Louisville core DNA laboratory. Human 293T cells were used to package pLenti-CMV-ORMDL3-puro into viral particles. Additional packaging plasmids expressing the viral structural proteins Gag, Pol, and Env were generous gifts of Dr. Chi Li, University of Louisville, Louisville, KY. Viral supernatants were collected every 12 hours after transfection of 293T cells, filtered to remove cell debris, then stored at −20°C until use. HBECs immortalized with telomerase (hTERT) and cyclin-dependent kinase 4 (Cdk) (9Ramirez R.D. Sheridan S. Girard L. Sato M. Kim Y. Pollack J. Peyton M. Zou Y. Kurie J.M. Dimaio J.M. et al.Immortalization of human bronchial epithelial cells in the absence of viral oncoproteins.Cancer Res. 2004; 64: 9027-9034Crossref PubMed Scopus (496) Google Scholar) were plated in p100 dishes at 3 × 106 cells in complete medium overnight. The next day, cells were transduced with 5 ml of viral supernatant containing either empty vector or human ORMDL3 plasmid. This was delivered in 5 ml of medium containing 8 μg/ml Polybrene® and cells were incubated at 37°C with 5% CO2 for 12 hours, after which this transduction mix was removed and fresh viral particles were added to the cells for an additional 12 hours. Viral supernatants were removed and complete medium was added to cells for overnight incubation. The next day, medium was replaced with complete medium containing 10 μg/ml puromycin to begin the selection process. Cells were maintained under puromycin selection until used for experiments as outlined. Cells were routinely collected and ORMDL3 levels were analyzed by Western blot and real-time PCR. For each well of cells to be analyzed, 1 ml of TRIzol® was placed directly on the cells. The TRIzol®/cell mixture was transferred to a microfuge tube and stored at −80°C until analysis. To isolate RNA, the TRIzol®/cell mixture was added to prepared phase lock gels followed by 200 μl chloroform:isoamyl alcohol then vigorously shaken for 15 seconds. Tubes were incubated at room temperature for 5 minutes then centrifuged at 16,000 g for 15 minutes (4°C) to separate phases. The upper (aqueous) phase was transferred to sterile RNase-free microfuge tubes and 500 μl isopropanol was added to each tube and incubated at room temperature for 10 minutes to precipitate RNA. Tubes were spun at 16,000 g for 20 minutes (4°C) to pellet RNA. RNA was washed with 70% ethanol then air dried and resuspended in RNase-free water (30–100 μl) and quantitated using a NanoDrop 8000. cDNA was prepared from 1 μg of total RNA using a high-capacity reverse transcriptase kit from Applied Biosystems (Life Technologies, Thermo-Fisher, Grand Island, NY) following all manufacturer's instructions. Quantitative real-time PCR was performed using gene-specific TaqMan® probes for all three ORMDL isoforms and the large subunit #1 of SPT. TaqMan® Gene Expression Master Mix and MicroAmp® Fast Optical 96-well plates were used along with an Applied Biosystems® FAST 7500 instrument to perform real-time PCR amplification. Analysis was done using the ΔCT (Fig. 1) or ΔΔCT (Fig. 2, Fig. 3) formula for calculating the fold change in expression levels. Sphingolipids were extracted from lysates as previously described (12Siow D.L. Anderson C.D. Berdyshev E.V. Skobeleva A. Pitson S.M. Wattenberg B.W. Intracellular localization of sphingosine kinase 1 alters access to substrate pools but does not affect the degradative fate of sphingosine-1-phosphate.J. Lipid Res. 2010; 51: 2546-2559Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Prior to extraction, a mixture of C17 sphingolipids was added to each sample (125 pmol/sample, Avanti sphingolipid mix #1) as internal standards. Sphingolipids were quantitated by HPLC electrospray ionization tandem mass spectrometry using selected ion monitoring on an ABSciex 4000 Q-Trap instrument as described previously (13Deevska G.M. Sunkara M. Morris A.J. Nikolova-Karakashian M.N. Characterization of secretory sphingomyelinase activity, lipoprotein sphingolipid content and LDL aggregation in ldlr-/- mice fed on a high-fat diet.Biosci. Rep. 2012; 32: 479-490Crossref PubMed Scopus (33) Google Scholar, 14Salous A.K. Panchatcharam M. Sunkara M. Mueller P. Dong A. Wang Y. Graf G.A. Smyth S.S. Morris A.J. Mechanism of rapid elimination of lysophosphatidic acid and related lipids from the circulation of mice.J. Lipid Res. 2013; 54: 2775-2784Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 15Schneider G. Bryndza E. Abdel-Latif A. Ratajczak J. Maj M. Tarnowski M. Klyachkin Y.M. Houghton P. Morris A.J. Vater A. et al.Bioactive lipids S1P and C1P are prometastatic factors in human rhabdomyosarcoma, and their tissue levels increase in response to radio/chemotherapy.Mol. Cancer Res. 2013; 11: 793-807Crossref PubMed Scopus (58) Google Scholar). Total phospholipids for each sample were measured using a modified Ames and Dubin assay (16Ames B.N. Dubin D.T. The role of polyamines in the neutralization of bacteriophage deoxyribonucleic acid.J. Biol. Chem. 1960; 235: 769-775Abstract Full Text PDF PubMed Google Scholar), as previously described (12Siow D.L. Anderson C.D. Berdyshev E.V. Skobeleva A. Pitson S.M. Wattenberg B.W. Intracellular localization of sphingosine kin
Referência(s)