Polyunsaturated fatty acid elongation and desaturation in activated human T-cells: ELOVL5 is the key elongase
2018; Elsevier BV; Volume: 59; Issue: 12 Linguagem: Inglês
10.1194/jlr.m090050
ISSN1539-7262
AutoresPhilippe-Pierre Robichaud, Jean éric Munganyiki, Éric Boilard, Marc E. Surette,
Tópico(s)Cancer, Lipids, and Metabolism
ResumoPUFAs are important constituents of membrane glycerophospholipids. However, changes in the capacities to incorporate and metabolize PUFAs when cells enter the cell cycle have not been thoroughly studied. In this study, differences in the incorporation and metabolism of exogenous PUFAs in resting and proliferating primary human T-cells and in the Jurkat cell line were measured. Overall, proliferating T-cells and Jurkat cells had a greater capacity to incorporate and elongate exogenous 18- and 20-carbon PUFAs compared with resting T-cells. Proliferating T-cells and Jurkat cells also showed a greater capacity to desaturate 18-carbon PUFA substrates. Consistent with these observations, a significant increase in the expression of fatty acid desaturase (FADS) 1, FADS2, and elongation of very long chain fatty acids protein (ELOVL) 5 was measured in proliferating T-cells compared with resting T-cells. No quantifiable ELOVL2 was measured. Knockdown of ELOVL5 in T-cells and Jurkat cells significantly affected cellular monounsaturated and PUFA profiles and strongly impaired the elongation of 18- and 20-carbon PUFAs. In conclusion, the induction of proliferation in human T-cells is associated with a significant increase in the capacity to take up and metabolize exogenous PUFAs, and ELOVL5 is responsible for the elongation of 18- and 20-carbon PUFAs in these cells. PUFAs are important constituents of membrane glycerophospholipids. However, changes in the capacities to incorporate and metabolize PUFAs when cells enter the cell cycle have not been thoroughly studied. In this study, differences in the incorporation and metabolism of exogenous PUFAs in resting and proliferating primary human T-cells and in the Jurkat cell line were measured. Overall, proliferating T-cells and Jurkat cells had a greater capacity to incorporate and elongate exogenous 18- and 20-carbon PUFAs compared with resting T-cells. Proliferating T-cells and Jurkat cells also showed a greater capacity to desaturate 18-carbon PUFA substrates. Consistent with these observations, a significant increase in the expression of fatty acid desaturase (FADS) 1, FADS2, and elongation of very long chain fatty acids protein (ELOVL) 5 was measured in proliferating T-cells compared with resting T-cells. No quantifiable ELOVL2 was measured. Knockdown of ELOVL5 in T-cells and Jurkat cells significantly affected cellular monounsaturated and PUFA profiles and strongly impaired the elongation of 18- and 20-carbon PUFAs. In conclusion, the induction of proliferation in human T-cells is associated with a significant increase in the capacity to take up and metabolize exogenous PUFAs, and ELOVL5 is responsible for the elongation of 18- and 20-carbon PUFAs in these cells. 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The cellular PUFAs can be elongated by the elongation of very long chain fatty acids proteins (ELOVLs), of which seven have been identified in humans (19.Gregory M.K. Gibson R.A. Cook-Johnson R.J. Cleland L.G. James M.J. Elongase reactions as control points in long-chain polyunsaturated fatty acid synthesis.PLoS One. 2011; 6: e29662Crossref PubMed Scopus (125) Google Scholar, 20.Guillou H. Zadravec D. Martin P.G. Jacobsson A. The key roles of elongases and desaturases in mammalian fatty acid metabolism: Insights from transgenic mice.Prog. Lipid Res. 2010; 49: 186-199Crossref PubMed Scopus (568) Google Scholar, 21.Jakobsson A. Westerberg R. Jacobsson A. Fatty acid elongases in mammals: their regulation and roles in metabolism.Prog. Lipid Res. 2006; 45: 237-249Crossref PubMed Scopus (635) Google Scholar, 23.Zhang J.Y. Kothapalli K.S. Brenna J.T. Desaturase and elongase-limiting endogenous long-chain polyunsaturated fatty acid biosynthesis.Curr. Opin. Clin. Nutr. Metab. 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Munganyiki J.E. Surette M.E. Fatty acid remodeling in cellular glycerophospholipids following the activation of human T cells.J. Lipid Res. 2013; 54: 2665-2677Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), the differential expression of the enzymes linked to PUFA elongation and desaturation following the induction of cell proliferation is not known. In this study, we report significant changes in the capacity of primary human T-cells to metabolize PUFAs when stimulated to undergo cell proliferation, and these changes are associated with enhanced expression of FADS1, FADS2, and ELOVL5. Human recombinant interleukin-2 (IL-2), boron trifluoride (14% in methanol), 2,3,4,5,6-pentafluorobenzyl bromide, N,N-diisopropylethylamine, horseradish peroxidase-conjugated anti-β-actin, and the horseradish peroxidase-conjugated anti-rabbit antibodies were from Sigma-Aldrich (Oakville, Canada). The 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine was from Biolynx (Brockville, Canada). Fatty acid methyl esters (FAMEs) and FFAs were obtained from Nu-Check Prep (Elysian, MN). Deuterated AA (5,8,11,14-eicosatetraenoic-5,6,8,9,11,12,14,15-d8 acid [AA-d8]) and deuterated EPA [5,8,11,14,17-eicosapentaenoic-19,19,20,20,20-d5 acid (EPA-d5)] were from Cayman Chemical (Ann Arbor, MI). Antibodies against acyl-CoA synthetase (ACSL) 4, Δ5-desaturase, Δ6-desaturase, and ELOVL2 were from Abcam (Toronto, Canada). Antibody against ELOVL5 was from OriGene Technologies (Rockville, MD), phycoerythrin-conjugated anti-CD69 was from Biolegend (San Diego, CA), PC7-conjugated anti-CD25 was from Beckman-Coulter (Indianapolis, IN), and the goat anti-rabbit horseradish peroxidase-conjugated secondary antibody was from Jackson ImmunoResearch Laboratories (West Grove, PA). The nonsilencing (NS) negative control siRNA and the siRNA against ELOVL5 were from OriGene Technologies. Human peripheral blood mononuclear cells were obtained from blood following centrifugation in Lymphocyte Separation Solution (Wisent Inc., St-Bruno, Canada) as previously described (24.Böyum A. Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g.Scand. J. Clin. Lab. Invest. Suppl. 1968; 97: 77-89PubMed Google Scholar). Blood donors were male and female subjects between 18 and 65 years of age who had indicated that they had no known health issues and who had fasted for 12 h prior to the blood draw. T-cells were then isolated by negative selection using the human T-cell enrichment kit from Stem Cell Technologies (Vancouver, Canada) following the manufacturer's instructions. Primary T-cells and the Jurkat cell line (ATCC, Manassas, VA) were cultured in RPMI-1640 supplemented with 10% FBS, 100 U/ml penicillin, 10 µg/ml streptomycin, 10 mM HEPES, d-glucose (up to 25 mM), and 1 mM sodium pyruvate at 37°C in a 5% CO2 atmosphere. Jurkat cells are a human leukemic T-cell lymphoblast cell line that is often used to study T-cell functions. T-cells were stimulated with anti-CD3/anti-CD28 Dynabeads (1 × 106 beads/ml) (Invitrogen) according to the manufacturer's instructions in the presence of 30 U/ml IL-2 (Sigma-Aldrich) for up to 72 h before all experiments. HepG2 cells (ATCC) were cultured in Eagle's minimal essential medium supplemented with 10% FBS, 100 U/ml penicillin, and 10 µg/ml streptomycin at 37°C in a 5% CO2 atmosphere. Total mRNA was extracted from resting and stimulated T-cells using Ribozol reagent (AMRESCO), and the extracted mRNA was purified with the Direct-zol kit (Zymo Research). mRNA was quantified by absorbance at 260 nm, and its purity was evaluated by the ratios of absorbance at 260/280 nm (∼2.0) and 260/230 nm (2.0–2.2). RNA integrity was also evaluated by electrophoretic migration on a 1% agarose gel. mRNA reverse transcription was performed on 1 µg RNA using the QuantiTect Reverse Transcription Kit (QIAGEN). Gene expression was evaluated by quantitative PCR (ABI 7500; Applied Biosystems) using PrimeTime assays (Integrated DNA Technologies) with PerfeCTa qPCR SuperMix Low ROX (Quanta Biosciences). The efficiency of primer pairs (Table 1) was evaluated using a standard curve, and the stability of the RN18S1 reference gene expression between treatments (Ct < 1) was verified. This confirms previous reports of RN18S1 as an appropriate reference gene in stimulated human T-cells (25.Bas A. Forsberg G. Hammarstrom S. Hammarstrom M.L. Utility of the housekeeping genes 18S rRNA, beta-actin and glyceraldehyde-3-phosphate-dehydrogenase for normalization in real-time quantitative reverse transcriptase-polymerase chain reaction analysis of gene expression in human T lymphocytes.Scand. J. Immunol. 2004; 59: 566-573Crossref PubMed Scopus (342) Google Scholar). Specific primers for ELOVL2, ELOVL5, FADS1, FADS2, and RN18S1 were created by using PrimerQuest (Integrated DNA Technologies).TABLE 1List of primer sequences used in quantitative PCR experiments and product size for each of the indicated transcriptsTranscript (Accession)PrimersSequencesProducts (bp)ELOVL2ForwardTTGGAATCACACTTCTCTCCGCGT141NM_017770Probe56-FAM/TCCACTTGG/ZEN/GAAGGAGGCTACAACTT/3IABkFQReverseAGTACCACCAAAGCACCTTGGCTAELOVL2ForwardTGTGTCCAGGAACTCTACTGA111NM_017770Probe56-FAM/TTGGCTACC/ZEN/CGGATGTCAGCTTC/3IABkFQReverseGGCTACAACTTACAGTGTCAAGAELOVL5ForwardTTCATCCTGCGCAAGAACAACCAC188NM_021814Probe56-FAM/TACCACCAT/ZEN/GCCTCGATGCTGAACAT/3IABkFQReverseATGGAAGGGACTGACGACAAACCAFADS1ForwardAAGCAACTGGTTTGTGTGGGTGAC198NM_013402Probe56-FAM/AGGCCACAT/ZEN/GCAATGTCCACAAGTCT/3IABkFQReverseTAATTGTGTCGAGGCATCGTGGGAFADS2ForwardTACGCTGGAGAAGATGCAACGGAT139NM_004265Probe56-FAM/TGACCTGGA/ZEN/ATTCGTGGGCAAGTTCT/3IABkFQReverseTCTTTGAGTTCTTGCCGTGGTCCTRN18S1ForwardGAGACTCTGGCATGCTAACTAG129NR_003286Probe56-FAM/TGCTCAATC/ZEN/TCGGGTGGCTGAA/3IABkFQReverseGGACATCTAAGGGCATCACAG Open table in a new tab Cells were washed twice with PBS, and lysis buffer (150 mM NaCl; 1% Nonidet P-40; 2 mM EDTA; and 50 mM Tris-HCL, pH 7.6) containing protease inhibitor cocktail (Roche) was added to the pellets. After complete homogenization, proteins were quantified by the Microplate BCA Protein Assay Kit (Pierce). 5× Laemmli sample buffer was added, and samples were heated 10 min at 40°C (9.Robichaud P.P. Boulay K. Munganyiki J.E. Surette M.E. Fatty acid remodeling in cellular glycerophospholipids following the activation of human T cells.J. Lipid Res. 2013; 54: 2665-2677Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Cellular proteins (10 μg) were separated on Criterion 4–15% polyacrylamide gels (Bio-Rad) and transferred onto a PVDF membrane. Membranes were incubated with indicated primary antibodies and horseradish peroxidase-conjugated secondary antibodies. Western blots were developed using Amersham ECL Prime (GE Healthcare), and images were captured using an Alpha Innotech FluorChem imager. After 3 days of incubation for primary T-cells or 2 days of incubation for Jurkat and HepG2 cells since the last split, the incorporation and metabolism of PUFAs were evaluated by incubating resting T-cells, stimulated T-cells, Jurkat cells, or HepG2 cells for 24 h with different PUFAs or their diluent (0.05% ethanol) in the cells' respective culture media containing 10% FBS as described above. Stimulated T-cells, Jurkat cells, and HepG2 cells were incubated with 5 µM of each PUFA, whereas resting T-cells were incubated with 15 µM of each PUFA. The PUFAs utilized were LA, γ-linolenic acid (GLA; 18:3n-6), AA, ALA, stearidonic acid (SDA; 18:4n-3), EPA, AA-d8, and EPA-d5. After 24 h of incubation with different PUFAs or their diluent, cells were washed by centrifugation using PBS containing 1 mg/ml BSA, and cellular lipids were extracted into chloroform containing 3.2 μg of the internal standard 1,2-diheptadecanoyl-sn-glycerol-3-phosphorylcholine (Biolynx) using the Bligh and Dyer method (26.Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42689) Google Scholar). The extracted lipids were saponified with 0.5 M KOH in methanol (100°C for 15 min), and FAMEs were prepared by adding 14% BF3 in methanol (100°C for 15 min) and quantified by GC with flame ionization detection (FID) as previously described (9.Robichaud P.P. Boulay K. Munganyiki J.E. Surette M.E. Fatty acid remodeling in cellular glycerophospholipids following the activation of human T cells.J. Lipid Res. 2013; 54: 2665-2677Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 27.Robichaud P.P. Poirier S.J. Boudreau L.H. Doiron J.A. Barnett D.A. Boilard E. Surette M.E. On the cellular metabolism of the click chemistry probe 19-alkyne arachidonic acid.J. Lipid Res. 2016; 57: 1821-1830Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). FAME standards (Nu-Check Prep) were used to identify peak retention times, and standard curves were used for FAME quantification. For the deuterated FA experiments, pentafluorobenzyl esters of FAs were prepared and measured by negative ion chemical ionization GC/MS using a Polaris Q mass spectrometer (Thermo Electron Corporation) as previously described (9.Robichaud P.P. Boulay K. Munganyiki J.E. Surette M.E. Fatty acid remodeling in cellular glycerophospholipids following the activation of human T cells.J. Lipid Res. 2013; 54: 2665-2677Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 27.Robichaud P.P. Poirier S.J. Boudreau L.H. Doiron J.A. Barnett D.A. Boilard E. Surette M.E. On the cellular metabolism of the click chemistry probe 19-alkyne arachidonic acid.J. Lipid Res. 2016; 57: 1821-1830Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Jurkat cells, proliferating T-cells, and HepG2 cells cultured without penicillin and streptomycin for at least 24 h before electroporation were washed twice with RPMI without l-glutamine. Electroporation (270 V and 600 μF) was done in 0.4 cm cuvettes on 1 × 107 cells in 300 μl RPMI without l-glutamine in the presence of 300 nM NS siRNA or siRNA against ELOVL5. Cells were transferred to culture flasks prewarmed with complete culture medium without penicillin and streptomycin. Cells were verified for viability after 48 h by trypan blue exclusion under a light microscope. Cell proliferation was measured by flow cytometry using the CellTrace CFSE Cell Proliferation Kit (Thermo Fisher Scientific). Briefly, wild-type Jurkat cells were stained with 1 μM CFSE in PBS for 20 min, and unincorporated CFSE was quenched and washed with complete culture medium following the manufacturer's protocol. Stained cells were incubated for 48 h before their transfection with the NS control siRNA and the siRNA against the ELOVL5. Cells were then analyzed by flow cytometry (FC500; Beckman-Coulter) at 48 and 96 h posttransfection. Cell proliferation was also measured using the Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit (Thermo Fisher Scientific) in combination with the FxCycle Violet Stain (Thermo Fisher Scientific) following the manufacturer's protocol. Briefly, at 72 and 96 h posttransfection, Jurkat cells were incubated with 10 µM 5-ethynyl-2′-deoxyuridine (EdU) for 2 h at 37°C, washed, fixed, and permeabilized, and the Click-iT reaction was done to conjugate the incorporated EdU molecules to the Alexa Fluor 488. All components used were provided with the kit. Cells were then resuspended in 1 ml PBS and stained with the FxCycle Violet Stain following the manufacturer's protocol before being analyzed by flow cytometry (FC500; Beckman-Coulter). Jurkat cells transfected with the NS control siRNA and the siRNA against the ELOVL5 and incubated for 72 h were stained with annexin V (BioLegend) and with propidium iodide (Invitrogen) following the manufacturer's protocol and analyzed by flow cytometry (FC500; Beckman-Coulter). T-cells were stimulated with anti-CD3/anti-CD28 as described above with or without the addition of IL-2. After 18 h, cells were transfected with NS siRNA or the siRNA against the ELOVL5, and incubation was continued for an additional 48 h as described above. Expression of the T-cell activation markers CD25 and CD69 was then measured by flow cytometry (FC500; Beckman-Coulter). GraphPad Prism version 6.0 was used to perform the statistical analyses. This study was approved by the Université de Moncton Institutional Review Committee for Research involving human subjects (approval number 1314-029). All subjects provided written informed consent prior to their participation in the study. After 3 days of incubation, the stimulated T-cells grew in clusters, and the cell size and cell counts were increased compared with resting cells (mean ± SEM: 2.4 ± 0.2-fold; P < 0.0001 as determined by Student's t-test; n = 8), in accordance with previous reports (9.Robichaud P.P. Boulay K. Munganyiki J.E. Surette M.E. Fatty acid remodeling in cellular glycerophospholipids following the activation of human T cells.J. Lipid Res. 2013; 54: 2665-2677Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 28.Boilard E. Surette M.E. Anti-CD3 and concanavalin A-induced human T cell proliferation is associated with an increased rate of arachidonate-phospholipid remodeling. Lack of involvement of group IV and group VI phospholipase A2 in remodeling and increased susceptibility of proliferating T cells to CoA-independent transacyclase inhibitor-induced apoptosis.J. Biol. Chem. 2001; 276: 17568-17575Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 29.Anel A. Naval J. Gonzalez B. Torres J.M. Mishal Z. Uriel J. Pineiro A. Fatty acid metabolism in human lymphocytes. I. Time-course changes in fatty acid composition and membrane fluidity during blastic transformation of peripheral blood lymphocytes.Biochim. Biophys. Acta. 1990; 1044: 323-331Crossref PubMed Scopus (77) Google Scholar, 30.Calder P.C. Yaqoob P. Harvey D.J. Watts A. Newsholme E.A. Incorporation of fatty acids by concanavalin A-stimulated lymphocytes and the effect on fatty acid composition and membrane fluidity.Biochem. J. 1994; 300: 509-518Crossref PubMed Scopus (188) Google Scholar). In preliminary experiments, cells were incubated with 5 µM exogenous PUFAs for 24 h. However, resting T-cells incorporated very little FAs, and thus PUFA metabolism was difficult to assess. Therefore, all further experiments with resting T-cells utilized PUFA concentrations of 15 µM. This difference in the capacity of resting and proliferating T-cells to take up exogenous AA is consistent with previous reports of a significantly enhanced capacity to incorporate [3H]AA in stimulated T-cells in pulse-labeling experiments (9.Robichaud P.P. Boulay K. Munganyiki J.E. Surette M.E. Fatty acid remodeling in cellular glycerophospholipids following the activation of human T cells.J. Lipid Res. 2013; 54: 2665-2677Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). When cells were incubated with 18:2n-6 (LA), there was a significant increase in the cellular content of LA and of its elongation product 20:2n-6 in resting T-cells compared with nonsupplemented controls (Fig. 2A). The accumulation of LA compared with nonsupplemented controls that was measured in proliferating T-cells and in Jurkat cells was also accompanied by an augmentation of cellular 20:2n-6 content; however, in Jurkat cells there was also an increase in 18:3n-6 and 20:3n-6 (Fig. 2B, C).When cells were incubated with 18:3n-6 (GLA), only the accumulation of a small quantity of GLA was measured in resting T-cells that was different from controls (Fig. 2A). In proliferating T-cells a small increase in cellular GLA was also measured; however, a significant accumulation of its elongation product 20:3n-6 was measured, indicating that T-cell stimulation enhanced the cells' capacity to incorporate and elongate GLA (Fig. 2B). In Jurkat cells there was also a large increase of 20:3n-6 content compared with controls (Fig. 2C). When cells were incubated with 20:4n-6 (AA), there was no change in the n-6 PUFA content of resting T-cells compared with controls, while in proliferating T-cells and Jurkat cells a significant increase in both AA and 22:4n-6 content was measured (Fig. 2A–C). Overall, these results indicate that T-cell stimulation increases the capacity of the cells to take up and elongate these PUFAs. Indeed, these molar data demonstrate the much greater capacity of stimulated T-cells and Jurkat cells to take up exogenous FAs after a 24 h incubation based on the increase from baseline in cellular PUFA content (>100 nmol/108 cells) compared with resting T-cells (<20 nmol/108 cells) despite the resting cells having been exposed to greater concentrations of exogenous PUFAs. Importantly, the incubation of the cells with these FAs did not have an impact on the total FA pool, as the total mass of FAs per cell was not significantly changed (supplemental Table S1, supplemental Table S2). This suggests that the PUFA concentrations of 15 µM for resting cells and of 5 µM for proliferating cells did not cause an imbalance in overall cellular FA content. These patterns of changes were similar when comparisons of the percentage of total FAs were made (supplemental Fig. S1A–C). When cells were incubated with 18:3n-3 (ALA), a significant increase in cellular ALA was measured in resting and proliferating T-cells compared with controls (Fig. 2D–E). Unlike resting cells, a significant increase in cellular 20:3n-3 content was also measured in proliferating T-cells, indicating a greater capacity to elongate n-3 PUFA than in resting T-cells. In Jurkat cells incubated with ALA, significant increases in ALA, 18:4n-3, 20:3n-3, EPA, and 22:5n-3 were measured compared with controls. The extent of the metabolism of ALA to 22:5n-3 indicates that Jurkat cells have a greater capacity to elongate and desaturate PUFAs than primary T-cells (Fig. 2F). When cells were incubated with 18:4n-3 (SDA), a small accumulation of SDA and an increase in the cellular content of its elongation product 20:4n-3 was measured in resting T-cells compared with controls. The elongation of incorporated SDA to 20:4n-3 was significantly more pronounced in proliferating T-cells (an increase in 20:4n-3 of 2.4 ± 0.5 nmol/108 cells in resting cells and 100.2 ± 16.6 nmol/108 cells in proliferating cells; P < 0.05 as determined by paired Studen
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