Identification of an acid sphingomyelinase ceramide kinase pathway in the regulation of the chemokine CCL5 [S]
2018; Elsevier BV; Volume: 59; Issue: 7 Linguagem: Inglês
10.1194/jlr.m084202
ISSN1539-7262
AutoresBenjamin Newcomb, Cosima Rhein, Izolda Mileva, Rasheed Ahmad, Christopher J. Clarke, Justin M. Snider, Lina M. Obeid, Yusuf A. Hannun,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoAcid sphingomyelinase (ASM) hydrolyzes sphingomyelin to produce the biologically active lipid ceramide. Previous studies have implicated ASM in the induction of the chemokine CCL5 in response to TNF-α however, the lipid mediator of this effect was not established. In the present study, we identified a novel pathway connecting ASM and ceramide kinase (CERK). The results show that TNF-α induces the formation of ceramide 1-phosphate (C-1-P) in a CERK-dependent manner. Silencing of CERK blocks CCL5 production in response to TNF-α. Interestingly, cells lacking ASM have decreased C-1-P production following TNF-α treatment, suggesting that ASM may be acting upstream of CERK. Functionally, ASM and CERK induce a highly concordant program of cytokine production and both are required for migration of breast cancer cells. Taken together, these data suggest ASM can produce ceramide which is then converted to C-1-P by CERK, and that C-1-P is required for production of CCL5 and several cytokines and chemokines, with roles in cell migration. These results highlight the diversity in action of ASM through more than one bioactive sphingolipid. Acid sphingomyelinase (ASM) hydrolyzes sphingomyelin to produce the biologically active lipid ceramide. Previous studies have implicated ASM in the induction of the chemokine CCL5 in response to TNF-α however, the lipid mediator of this effect was not established. In the present study, we identified a novel pathway connecting ASM and ceramide kinase (CERK). The results show that TNF-α induces the formation of ceramide 1-phosphate (C-1-P) in a CERK-dependent manner. Silencing of CERK blocks CCL5 production in response to TNF-α. Interestingly, cells lacking ASM have decreased C-1-P production following TNF-α treatment, suggesting that ASM may be acting upstream of CERK. Functionally, ASM and CERK induce a highly concordant program of cytokine production and both are required for migration of breast cancer cells. Taken together, these data suggest ASM can produce ceramide which is then converted to C-1-P by CERK, and that C-1-P is required for production of CCL5 and several cytokines and chemokines, with roles in cell migration. These results highlight the diversity in action of ASM through more than one bioactive sphingolipid. Bioactive sphingolipids are a diverse group of signaling molecules that includes ceramide, sphingosine, ceramide 1-phosphate (C-1-P), and sphingosine 1-phosphate, with more than 40 metabolic enzymes that generate, catabolize, or interconvert them (1.Schiffmann S. Sandner J. Birod K. Wobst I. Angioni C. Ruckhaberle E. Kaufmann M. Ackermann H. Lotsch J. Schmidt H. et al.Ceramide synthases and ceramide levels are increased in breast cancer tissue.Carcinogenesis. 2009; 30: 745-752Crossref PubMed Scopus (160) Google Scholar, 2.Ruckhäberle E. Karn T. Rody A. Hanker L. Gatje R. Metzler D. Holtrich U. Kaufmann M. Gene expression of ceramide kinase, galactosyl ceramide synthase and ganglioside GD3 synthase is associated with prognosis in breast cancer.J. Cancer Res. Clin. Oncol. 2009; 135: 1005-1013Crossref PubMed Scopus (76) Google Scholar, 3.Ramírez de Molina A. de la Cueva A. Machado-Pinilla R. Rodriguez-Fanjul V. Gomez del Pulgar T. Cebrian A. Perona R. Lacal J.C. Acid ceramidase as a chemotherapeutic target to overcome resistance to the antitumoral effect of choline kinase alpha inhibition.Curr. Cancer Drug Targets. 2012; 12: 617-624Crossref PubMed Scopus (22) Google Scholar, 4.Korbelik M. Zhang W. Saw K.M. Szulc Z.M. Bielawska A. Separovic D. Cationic ceramides and analogues, LCL30 and LCL85, as adjuvants to photodynamic therapy of tumors.J. Photochem. Photobiol. B. 2013; 126: 72-77Crossref PubMed Google Scholar, 5.Hankins J.L. Ward K.E. Linton S.S. Barth B.M. Stahelin R.V. Fox T.E. Kester M. Ceramide 1-phosphate mediates endothelial cell invasion via the annexin a2-p11 heterotetrameric protein complex.J. Biol. Chem. 2013; 288: 19726-19738Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 6.Gangoiti P. Bernacchioni C. Donati C. Cencetti F. Ouro A. Gomez-Munoz A. Bruni P. Ceramide 1-phosphate stimulates proliferation of C2C12 myoblasts.Biochimie. 2012; 94: 597-607Crossref PubMed Scopus (55) Google Scholar, 7.Realini N. Solorzano C. Pagliuca C. Pizzirani D. Armirotti A. Luciani R. Costi M.P. Bandiera T. Piomelli D. Discovery of highly potent acid ceramidase inhibitors with in vitro tumor chemosensitizing activity.Sci. Rep. 2013; 3: 1035Crossref PubMed Scopus (114) Google Scholar). Due to the large number of bioactive lipids and enzymes involved, the sphingolipid metabolic network is highly regulated and compartmentalized within the cell (8.Adada M. Luberto C. Canals D. Inhibitors of the sphingomyelin cycle: sphingomyelin synthases and sphingomyelinases.Chem. Phys. Lipids. 2016; 197: 45-59Crossref PubMed Scopus (75) Google Scholar, 9.Rovina P. Schanzer A. Graf C. Mechtcheriakova D. Jaritz M. Bornancin F. Subcellular localization of ceramide kinase and ceramide kinase-like protein requires interplay of their Pleckstrin Homology domain-containing N-terminal regions together with C-terminal domains.Biochim. Biophys. Acta. 2009; 1791: 1023-1030Crossref PubMed Scopus (34) Google Scholar, 10.Lamour N.F. Stahelin R.V. Wijesinghe D.S. Maceyka M. Wang E. Allegood J.C. Merrill Jr., A.H. Cho W. Chalfant C.E. Ceramide kinase uses ceramide provided by ceramide transport protein: localization to organelles of eicosanoid synthesis.J. Lipid Res. 2007; 48: 1293-1304Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Sphingolipid metabolism can be segregated into the de novo, hydrolytic, and salvage pathways. Sphingomyelin and other complex sphingolipids form the substrates for the sphingolipid hydrolytic and salvage pathways. Catabolism of sphingomyelin is mediated by the activity of the sphingomyelin phosphodiesterases, which generate ceramide, and subsequent metabolism of ceramide can generate sphingosine (11.Hannun Y.A. Obeid L.M. Sphingolipids and their metabolism in physiology and disease.Nat. Rev. Mol. Cell Biol. 2018; 19: 175-191Crossref PubMed Scopus (830) Google Scholar). Ceramide can also serve as a substrate for the formation of C-1-P through the action of ceramide kinase (CERK) (12.Sugiura M. Kono K. Liu H. Shimizugawa T. Minekura H. Spiegel S. Kohama T. Ceramide kinase, a novel lipid kinase. Molecular cloning and functional characterization.J. Biol. Chem. 2002; 277: 23294-23300Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). Ceramide has been implicated in multiple cellular functions, including apoptosis, response to chemotherapeutics, and other anti-mitogenic activities. In contrast, C-1-P has many important signaling functions in the mediation of several inflammatory processes that promote tumor formation and progression (6.Gangoiti P. Bernacchioni C. Donati C. Cencetti F. Ouro A. Gomez-Munoz A. Bruni P. Ceramide 1-phosphate stimulates proliferation of C2C12 myoblasts.Biochimie. 2012; 94: 597-607Crossref PubMed Scopus (55) Google Scholar, 13.Pettus B.J. Chalfant C.E. Hannun Y.A. Sphingolipids in inflammation: roles and implications.Curr. Mol. Med. 2004; 4: 405-418Crossref PubMed Scopus (111) Google Scholar, 14.Pastukhov O. Schwalm S. Zangemeister-Wittke U. Fabbro D. Bornancin F. Japtok L. Kleuser B. Pfeilschifter J. Huwiler A. The ceramide kinase inhibitor NVP-231 inhibits breast and lung cancer cell proliferation by inducing M phase arrest and subsequent cell death.Br. J. Pharmacol. 2014; 171: 5829-5844Crossref PubMed Scopus (52) Google Scholar). Therefore, CERK may have a dual function in tumor promotion involving both the production of mitogenic C-1-P as well as clearance of apoptotic ceramide (15.Simanshu D.K. Zhai X. Munch D. Hofius D. Markham J.E. Bielawski J. Bielawska A. Malinina L. Molotkovsky J.G. Mundy J.W. et al.Arabidopsis accelerated cell death 11, ACD11, is a ceramide-1-phosphate transfer protein and intermediary regulator of phytoceramide levels.Cell Reports. 2014; 6: 388-399Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Previous work from our laboratory and others has revealed a pro-inflammatory role for the sphingolipid salvage pathway, with a particular focus on acid sphingomyelinase (ASM) (16.Jenkins R.W. Clarke C.J. Canals D. Snider A.J. Gault C.R. Heffernan-Stroud L. Wu B.X. Simbari F. Roddy P. Kitatani K. et al.Regulation of CC ligand 5/RANTES by acid sphingomyelinase and acid ceramidase.J. Biol. Chem. 2011; 286: 13292-13303Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 17.Perry D.M. Newcomb B. Adada M. Wu B.X. Roddy P. Kitatani K. Siskind L. Obeid L.M. Hannun Y.A. Defining a role for acid sphingomyelinase in the p38/interleukin-6 pathway.J. Biol. Chem. 2014; 289: 22401-22412Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 18.Kitatani K. Sheldon K. Anelli V. Jenkins R.W. Sun Y. Grabowski G.A. Obeid L.M. Hannun Y.A. Acid beta-glucosidase 1 counteracts p38delta-dependent induction of interleukin-6: possible role for ceramide as an anti-inflammatory lipid.J. Biol. Chem. 2009; 284: 12979-12988Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 19.Ali M. Saroha A. Pewzner-Jung Y. Futerman A.H. LPS-mediated septic shock is augmented in ceramide synthase 2 null mice due to elevated activity of TNFalpha-converting enzyme.FEBS Lett. 2015; 589: 2213-2217Crossref PubMed Scopus (24) Google Scholar). ASM is a 631 amino acid protein, encoded by the SMPD1 gene. It mediates several downstream signaling responses initiated by lipopolysaccharides, oxidative stress, ionizing radiation, IL-1β, TNF-α, and phorbol 12-myristate 13-acetate, including roles in induction of protein kinase C, IL-6, and interferon γ (INF-γ) (20.Jenkins R.W. Canals D. Idkowiak-Baldys J. Simbari F. Roddy P. Perry D.M. Kitatani K. Luberto C. Hannun Y.A. Regulated secretion of acid sphingomyelinase: implications for selectivity of ceramide formation.J. Biol. Chem. 2010; 285: 35706-35718Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 21.Zeidan Y.H. Hannun Y.A. Activation of acid sphingomyelinase by protein kinase Cdelta-mediated phosphorylation.J. Biol. Chem. 2007; 282: 11549-11561Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 22.Zhu H. Deng K. Zhao Y.Q. Wang X. Shen Y.L. Liu T.G. Cui D.D. Xu F. The effects of ASMase mediated endothelial cell apoptosis in multiple hypofractionated irradiations in CT26 tumor bearing mice.Asian Pac. J. Cancer Prev. 2015; 16: 4543-4548Crossref PubMed Scopus (6) Google Scholar, 23.Li X. Gulbins E. Zhang Y. Oxidative stress triggers Ca-dependent lysosome trafficking and activation of acid sphingomyelinase.Cell. Physiol. Biochem . 2012; 30: 815-826Crossref PubMed Scopus (51) Google Scholar). ASM has also been implicated in viral and bacterial uptake and infection (24.Grassmé H. Gulbins E. Brenner B. Ferlinz K. Sandhoff K. Harzer K. Lang F. Meyer T.F. Acidic sphingomyelinase mediates entry of N. gonorrhoeae into nonphagocytic cells.Cell. 1997; 91: 605-615Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 25.Grassmé H. Jendrossek V. Riehle A. von Kurthy G. Berger J. Schwarz H. Weller M. Kolesnick R. Gulbins E. Host defense against Pseudomonas aeruginosa requires ceramide-rich membrane rafts.Nat. Med. 2003; 9: 322-330Crossref PubMed Scopus (462) Google Scholar). In particular, our previous studies disclosed an important role for ASM in mediating the induction of CCL5/RANTES in response to the action of IL-1 and TNF-α. CCL5 has been implicated as a key chemokine in the regulation of the tumor microenvironment, and along with other cytokines, including TNF-α, has been implicated in tumor progression and development of intratumoral heterogeneity (26.Serrels A. Lund T. Serrels B. Byron A. McPherson R.C. von Kriegsheim A. Gomez-Cuadrado L. Canel M. Muir M. Ring J.E. et al.Nuclear FAK controls chemokine transcription, Tregs, and evasion of anti-tumor immunity.Cell. 2015; 163: 160-173Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 27.Bonapace L. Coissieux M.M. Wyckoff J. Mertz K.D. Varga Z. Junt T. Bentires-Alj M. Cessation of CCL2 inhibition accelerates breast cancer metastasis by promoting angiogenesis.Nature. 2014; 515: 130-133Crossref PubMed Scopus (461) Google Scholar, 28.Joyce J.A. Fearon D.T. T cell exclusion, immune privilege, and the tumor microenvironment.Science. 2015; 348: 74-80Crossref PubMed Scopus (1296) Google Scholar, 29.Marusyk A. Tabassum D.P. Altrock P.M. Almendro V. Michor F. Polyak K. Non-cell-autonomous driving of tumour growth supports sub-clonal heterogeneity.Nature. 2014; 514: 54-58Crossref PubMed Scopus (418) Google Scholar). In breast cancer cells, TNF-α induces not only CCL5, but also NFkB, MAPK/AKT, AP1, JNK, Ras, as well as many other mitogenic pathways (30.Soria G. Ofri-Shahak M. Haas I. Yaal-Hahoshen N. Leider-Trejo L. Leibovich-Rivkin T. Weitzenfeld P. Meshel T. Shabtai E. Gutman M. et al.Inflammatory mediators in breast cancer: coordinated expression of TNFalpha & IL-1beta with CCL2 & CCL5 and effects on epithelial-to-mesenchymal transition.BMC Cancer. 2011; 11: 130Crossref PubMed Scopus (207) Google Scholar, 31.Leibovich-Rivkin T. Liubomirski Y. Meshel T. Abashidze A. Brisker D. Solomon H. Rotter V. Weil M. Ben-Baruch A. The inflammatory cytokine TNFalpha cooperates with Ras in elevating metastasis and turns WT-Ras to a tumor-promoting entity in MCF-7 cells.BMC Cancer. 2014; 14: 158Crossref PubMed Scopus (16) Google Scholar, 32.Rivas M.A. Carnevale R.P. Proietti C.J. Rosemblit C. Beguelin W. Salatino M. Charreau E.H. Frahm I. Sapia S. Brouckaert P. et al.TNF alpha acting on TNFR1 promotes breast cancer growth via p42/P44 MAPK, JNK, Akt and NF-kappa B-dependent pathways.Exp. Cell Res. 2008; 314: 509-529Crossref PubMed Scopus (119) Google Scholar, 33.Qiao Y. He H. Jonsson P. Sinha I. Zhao C. Dahlman-Wright K. AP-1 is a key regulator of proinflammatory cytokine TNFalpha-mediated triple-negative breast cancer progression.J. Biol. Chem. 2016; 291: 5068-5079Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Interestingly, C-1-P and CERK are known to play roles in many of the same pathways induced by TNF-α and regulated by ASM. In nonsmall cell lung cancer cell lines, C-1-P has been found to be a potent activator of invasion and of MAKP and AKT signaling (34.Schneider G. Sellers Z.P. Bujko K. Kakar S.S. Kucia M. Ratajczak M.Z. Novel pleiotropic effects of bioactive phospholipids in human lung cancer metastasis.Oncotarget. 2017; 8: 58247-58263Crossref PubMed Scopus (23) Google Scholar). Furthermore, CERK has been found to modulate NFkB activity in neutrophils from the lungs of mice challenged with lipopolysaccharides (35.Baudiß K. de Paula Vieira R. Cicko S. Ayata K. Hossfeld M. Ehrat N. Gomez-Munoz A. Eltzschig H.K. Idzko M. C1P attenuates lipopolysaccharide-induced acute lung injury by preventing NF-kappaB activation in neutrophils.Journal of immunology (Baltimore. 2016; 196: 2319-2326Crossref PubMed Scopus (43) Google Scholar). In addition to its role as a modulator of stress responses in lung tissues, CERK has been found to activate stress-activated protein kinase/c-Jun N-terminal kinase and regulate lipid droplet formation in an ERK- and p38-independent manner (36.Gubern A. Barcelo-Torns M. Barneda D. Lopez J.M. Masgrau R. Picatoste F. Chalfant C.E. Balsinde J. Balboa M.A. Claro E. JNK and ceramide kinase govern the biogenesis of lipid droplets through activation of group IVA phospholipase A2.J. Biol. Chem. 2009; 284: 32359-32369Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). CERK is also known to have a direct role in TNF-α signaling. CERK has been shown to be a downstream modulator of TNF-α-induced cytosolic phospholipases A2 and in induction of NADPH oxidase (37.Barth B.M. Gustafson S.J. Hankins J.L. Kaiser J.M. Haakenson J.K. Kester M. Kuhn T.B. Ceramide kinase regulates TNFalpha-stimulated NADPH oxidase activity and eicosanoid biosynthesis in neuroblastoma cells.Cell. Signal. 2012; 24: 1126-1133Crossref PubMed Scopus (20) Google Scholar, 38.Lamour N.F. Subramanian P. Wijesinghe D.S. Stahelin R.V. Bonventre J.V. Chalfant C.E. Ceramide 1-phosphate is required for the translocation of group IVA cytosolic phospholipase A2 and prostaglandin synthesis.J. Biol. Chem. 2009; 284: 26897-26907Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Due to the findings that ASM mediates inflammatory signaling in breast cancers, CERK promotes tumor progression, and CCL5 has a prominent role in the tumor microenvironment, we sought to investigate a possible connection between ASM, CERK, and CCL5. This was further prompted by an inability to pinpoint the role of ASM in mediating CCL5 on ceramide, sphingosine, or sphingosine 1-phosphate. Here, we present results that suggest that C-1-P mediates the production of CCL5 in response to TNF-α stimulation, and that CERK generates C-1-P from ceramide produced by ASM. MCF7 cells were obtained from ATCC (Manassas, VA). Niemann-Pick disease types A and B (NPD) (Cat GM16195, passage 11) and Lesch-Nyhan (LN) (Cat GM02226, passage 16) cells were obtained from Coriell Cell Repository (Camden, NJ). Trypsin-EDTA (0.05%) was from Gibco (Holtsville, NY, Cat 25300062). TNF-α was purchased from Peprotech (Rocky Hill, NJ). Porcine brain sphingomyelin was from Avanti Polar Lipids (Alabaster, AL, Cat 860062P). MCF7 cells were maintained in RPMI from Gibco (Holtsville, NY, Cat 11875-093) supplemented with 10% (v/v) heat-inactivated FBS from HyClone (Port Washington, NY, Cat SH30396.03). MCF7 cells were kept in culture for no longer than 30 days. All cell lines were tested monthly for mycoplasma contamination using the MycoAlert kit from Lonza (Allendale, NJ, Cat LT07-218). NPD and LN cells were maintained at less than 75% confluency. All cell treatments with TNF-α were carried out in serum free media, unless otherwise noted. The CERK-DsRed plasmid was a kind gift of Charles Chalfant. Generation of the pEF6-ASM-V5 plasmid was described previously (39.Bini F. Frati A. Garcia-Gil M. Battistini C. Granado M. Martinesi M. Mainardi M. Vannini E. Luzzati F. Caleo M. et al.New signalling pathway involved in the anti-proliferative action of vitamin D(3) and its analogues in human neuroblastoma cells. A role for ceramide kinase.Neuropharmacology. 2012; 63: 524-537Crossref PubMed Scopus (34) Google Scholar). Transient transfections were carried out in 6-well trays with 1×105 cells per well. 24 h after seeding cells, the media were changed and cells were transfected with 1 µg of plasmid DNA per well, using X-tremeGENE DNA transfection reagent from Roche (Basel, Switzerland, Cat 06365787001) according to manufacturer's instructions. Small interfering RNA (siRNA) duplexes were obtained from ThermoScientific (Rockford, IL), and were designed against the following target sequences: ASM, 5′-AACTCCTTTGGATGGGCCTGG-3′ and CERK (prevalidated, s34929). All-Star Negative Control siRNA was obtained from Qiagen. A total of 5×105 cells was plated in each well of a 6-well tray and 20 nM siRNA was transfected using Lipofectamine RNAiMAX from ThermoFisher (Cat 13778150), according to manufacturer's specifications. After 24 h, RNAi complexes were washed out, and cells were incubated with complete media for another 24 h. Media were exchanged 4 h prior to experimental manipulation. Sandwich ELISA kits for Human CCL5 were obtained from R&D Systems (Minneapolis, MN, Cat DY278-05 and DY008) and used according to the manufacturer's specifications. Briefly, cell supernatants were harvested, and centrifuged for 5 min at 21,000 g in a table top microcentrifuge, and 100 µL of supernatant was used per well of the ELISA assay. Cell lysates were prepared in buffer containing 50 mM TrisHCl, 0.2% TritonX-100, and protease inhibitor from Sigma (St. Louis, MO, Cat S8830-20TAB) (cell lysis buffer). Cell lysates were adjusted to a protein concentration of 0.5 mg/ml, and 100 µL of lysate was applied to each well of the ELISA. RNA purification was performed with the Purelink RNA Kit from ThermoFisher, according to manufacturer's protocol. Concentration of RNA was determined by nanodrop, and 500 ng of RNA was transformed into cDNA using the Quanta cDNA Kit (Gaithersburg, MD, Cat 95047) according to the manufacturer's protocol. For RT-PCR, reactions were performed in triplicate in 96-well plates with each reaction containing 10 μl 2 × iTAQ mastermix, 5 μl of diluted (1:10, v/v) cDNA, 1 μl of 20× FAM tagged Taqman gene specific primer probe, 0.3 µL of 60× VIC tagged Actin probe, and 4 μl of water. The following probes were purchased from Life Technologies: ACTB (Cat Hs01060665_g1); ASM (Cat Hs03679347_g1); CERK (Cat Hs00368483_m1); CCL5 (Cat Hs00174575_m1). MCF7 cells were plated in 10 cm dishes at 105 cells per plate. The following day, cells were transfected with the plasmids containing overexpression vectors with the indicated genes. Cells were incubated for an additional 24 h. Cells were lysed, and mRNA was harvested and converted to cDNA as above. Cytokine arrays were obtained from SA Biosciences (Cat PAHS-150Z) and used according to manufacturer's protocol. Data were analyzed using the SA Biosciences PCR Array Data Analysis Software. MCF7 or NPD or LN cells were seeded at 106 cells per plate in 10 cm plates. Following 24 h of growth in complete media, cells were treated as indicated. Following treatment, cells were scraped and pelleted in cold PBS, and lipids were extracted in 2 ml isopropanol:water:ethyl acetate (30:10:60 by vol). Cell extracts were analyzed by reverse phase high pressure liquid chromatography coupled to electrospray ionization and subsequent separation by mass spectrometry. Analysis of sphingoid bases, ceramides, and sphingomyelins was performed on a Thermo Quantum Ultra mass spectrometer, operating in a multiple reaction-monitoring positive ionization mode, as described (40.Bielawski J. Pierce J.S. Snider J. Rembiesa B. Szulc Z.M. Bielawska A. Sphingolipid analysis by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).Adv. Exp. Med. Biol. 2010; 688: 46-59Crossref PubMed Scopus (101) Google Scholar). Lipid phosphate concentrations were measured and sphingolipid levels were normalized to total lipid phosphate in each sample. ASM assays were performed as previously described (20.Jenkins R.W. Canals D. Idkowiak-Baldys J. Simbari F. Roddy P. Perry D.M. Kitatani K. Luberto C. Hannun Y.A. Regulated secretion of acid sphingomyelinase: implications for selectivity of ceramide formation.J. Biol. Chem. 2010; 285: 35706-35718Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In brief, 200 µM porcine brain sphingomyelin was mixed with 14C labeled sphingomyelin and Triton X-100. Micelles were formed by sonication in a buffer containing 250 mM sodium acetate (pH 5.00) and 1.0 mM EDTA. Cells were lysed in buffer containing 50 mM TrisHCl pH7.4, 0.2% Triton X-100, 1.0 mM EDTA, and protease inhibitor. An amount of 25 µg of cell lysate in 100µL lysis buffer was added to 100 µL of micelle mix and the reaction was incubated at 37°C for 30 min. The reaction was terminated with the addition of 1.5 ml of CHCl3: MeOH (2:1, v/v) followed by 0.4 ml of water. Samples were vortexed, centrifuged (5 min at 1,865 g in a table top centrifuge), and 0.8 ml of the aqueous/methanolic phase was removed for scintillation counting. Invasion of cells was measured using the Corning BioCoat Tumor Invasion System (Cat 354165), according to manufacturer's protocol. Briefly, MDA-MB-231 cells were trypsinized, and resuspended in serum free media at a concentration of 5×104 cells/ml. A total of 500 µL of cell suspension was added to the upper well of each chamber of the transwell plate. The lower chamber contained serum free media (control), complete media, or complete media plus TNF-α. Cells were allowed to invade for 48 h, and invasion of live cells was quantified by staining cells with Calcein AM and measuring fluorescence using a Spectra Max M5 mulitplate reader. MDA-231 (100K) were reverse transfected with All Star or CERK siRNA (20nM) in 6-well trays for 24 h. To initiate the time course, media were changed and viable cell number was assessed by MTT assay at 0, 24, 48 and 72 h. Briefly, 1 ml of 5 mg/ml MTT was added to 1 ml media and incubated for 30 min at 37°C. Media were aspirated and formazan dye was solubilized in 2 ml DMSO for 10 min at room temperature with gentle rocking. Absorbance of formazan product was assessed at 570 nm. Data are represented as the mean of at least three independent replicates ± standard error, unless otherwise indicated. Unpaired Student's t-test, one-way ANOVA with Dunnett's post test, and two-way ANOVA with Bonferroni post test statistical analyses were performed using Prism 6.2 software. Previously, we demonstrated a role for ASM in IL-1β− and TNF-α− induced CCL5 production in MCF7 breast carcinoma cells (16.Jenkins R.W. Clarke C.J. Canals D. Snider A.J. Gault C.R. Heffernan-Stroud L. Wu B.X. Simbari F. Roddy P. Kitatani K. et al.Regulation of CC ligand 5/RANTES by acid sphingomyelinase and acid ceramidase.J. Biol. Chem. 2011; 286: 13292-13303Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). However, in these studies the functional sphingolipid mediating those actions was not defined. Because inflammatory cytokines are known to activate CERK to induce C-1-P (37.Barth B.M. Gustafson S.J. Hankins J.L. Kaiser J.M. Haakenson J.K. Kester M. Kuhn T.B. Ceramide kinase regulates TNFalpha-stimulated NADPH oxidase activity and eicosanoid biosynthesis in neuroblastoma cells.Cell. Signal. 2012; 24: 1126-1133Crossref PubMed Scopus (20) Google Scholar, 41.Pettus B.J. Bielawska A. Spiegel S. Roddy P. Hannun Y.A. Chalfant C.E. Ceramide kinase mediates cytokine- and calcium ionophore-induced arachidonic acid release.J. Biol. Chem. 2003; 278: 38206-38213Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar), we sought to investigate the role of CERK and C-1-P in CCL5 production and clarify the interactions between ASM and C-1-P. As expected, we observed a rapid and robust time-dependent increase in the level of CCL5 following TNF-α treatment. CCL5 message increased up to 18-fold over control, (Fig. 1A) and secreted CCL5 protein increased up to 120-fold over control (Fig. 1B) in response to TNF-α treatment (it should be noted, however, that the fold changes varied between experiments, mostly related to differences in the basal levels of CCL5). To investigate the possibility that C-1-P plays a role in the TNF-α response, we measured C-1-P levels following TNF-α stimulation, and observed a robust time dependent increase in C-1-P (Fig. 1C). Interestingly, sphingosine did not increase during TNF-α treatment (Fig. 1D, E). These data suggest TNF-α induces a C-1-P dependent signaling pathway in MCF7 cells in a relevant time frame. To date, CERK is the only identified source of C-1-P in mammalian cells (42.Mitsutake S. Yokose U. Kato M. Matsuoka I. Yoo J.M. Kim T.J. Yoo H.S. Fujimoto K. Ando Y. Sugiura M. et al.The generation and behavioral analysis of ceramide kinase-null mice, indicating a function in cerebellar Purkinje cells.Biochem. Biophys. Res. Commun. 2007; 363: 519-524Crossref PubMed Scopus (35) Google Scholar). As such, we sought to determine the role of CERK in production of C-1-P and CCL5. Using siRNA directed toward CERK, we were able to knock down expression of CERK (supplemental Fig. S1), and consequently, we measured a significant decrease in both the basal and TNF-α stimulated levels of C-1-P (Fig. 2A). To rule out a possible effect on ceramide and sphingosine levels, we measured both ceramide and sphingosine in vehicle- and TNF-α- treated cells. TNF-α induced a significant accumulation of ceramide that was not affected by ablation of CERK, albeit, there was a trend to an increase in ceramide levels that did not reach statistical significance (Fig. 2B). Additionally, CERK knockdown did not affect sphingosine levels in these cells (Fig. 2C). These data demonstrate that the increase in C-1-P in response to TNF-α is due to CERK activity, and that CERK knockdown does not affect levels of the related bioactive lipids, including ceramide and sphingosine. Cytokines, including CCL5, are regulated at many levels, including transcription, translation, and secretion (43.Frank S.P. Thon K.P. Bischoff S.C. Lorentz A. SNAP-23 and syntaxin-3 are required for chemokine release by mature human mast cells.Mol. Immunol. 2011; 49: 353-358Crossref PubMed Scopus (40) Google Scholar, 44.Lim J.K. Burns J.M. Lu W. DeVico A.L. Multiple pathways of amino terminal processing produce two truncated variants of RANTES/CCL5.J. Leukoc. Biol. 2005; 78: 442-452Crossref PubMed Scopus (28) Google Scholar, 45.Hoffmann A. Levchenko A. Scott M.L. Baltimore D. The IkappaB-NF-kappaB signaling module: temporal control and selective gene activation.Science. 2002; 298: 1241-1245Crossref PubMed Scopus (1486) Google Scholar). As such, we investigated the role of CERK in CCL5 production by measuring secreted CCL5 protein by ELISA. We observed that knockdown of CERK significantly reduced levels of CCL5 in the media of TNF-α-treated MCF7 cells (Fig. 3A). To assess the role of CERK in CCL5 expression, CCL5 mRNA was measured in cells treated with CERK siRNA. The results showed that ablation of CERK caused a significant decrease in the expression of the CCL5 message after treatment with TNF-α, commensurate with the effects on protein levels (Fig. 3B). Previous reports have shown that CCL5 is highly regulated at the secretory level, and that CCL5 is stored in secretory vesicles and vesicular fusion and subsequent CCL5 secretions are regulated by SNARE and synaptobrevin-2 (43.Frank S.P. Thon K.P. Bischoff S.C. Lorentz A. SNAP-23 and syntaxin-3 are required for chemokine release by mature human mast cells.Mol
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