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• W2088514755 abstract "Chronic inflammation and inflammatory cytokines have recently been implicated in the development and progression of various types of cancer. In the brain, neuroinflammatory cytokines affect the growth and differentiation of both normal and malignant glial cells, with interleukin 1 (IL-1) shown to be secreted by the majority of glioblastoma cells. Recently, elevated levels of sphingosine kinase 1 (SphK1), but not SphK2, were correlated with a shorter survival prognosis for patients with glioblastoma multiforme. SphK1 is a lipid kinase that produces the pro-growth, anti-apoptotic sphingosine 1-phosphate, which can induce invasion of glioblastoma cells. Here, we show that the expression of IL-1 correlates with the expression of SphK1 in glioblastoma cells, and neutralizing anti-IL-1 antibodies inhibit both the growth and invasion of glioblastoma cells. Furthermore, IL-1 up-regulates SphK1 mRNA levels, protein expression, and activity in both primary human astrocytes and various glioblastoma cell lines; however, it does not affect SphK2 expression. The IL-1-induced SphK1 up-regulation can be blocked by the inhibition of JNK, the overexpression of the dominant-negative c-Jun(TAM67), and the down-regulation of c-Jun expression by small interference RNA. Activation of SphK1 expression by IL-1 occurs on the level of transcription and is mediated via a novel AP-1 element located within the first intron of the sphk1 gene. In summary, our results suggest that SphK1 expression is transcriptionally regulated by IL-1 in glioblastoma cells, and this pathway may be important in regulating survival and invasiveness of glioblastoma cells. Chronic inflammation and inflammatory cytokines have recently been implicated in the development and progression of various types of cancer. In the brain, neuroinflammatory cytokines affect the growth and differentiation of both normal and malignant glial cells, with interleukin 1 (IL-1) shown to be secreted by the majority of glioblastoma cells. Recently, elevated levels of sphingosine kinase 1 (SphK1), but not SphK2, were correlated with a shorter survival prognosis for patients with glioblastoma multiforme. SphK1 is a lipid kinase that produces the pro-growth, anti-apoptotic sphingosine 1-phosphate, which can induce invasion of glioblastoma cells. Here, we show that the expression of IL-1 correlates with the expression of SphK1 in glioblastoma cells, and neutralizing anti-IL-1 antibodies inhibit both the growth and invasion of glioblastoma cells. Furthermore, IL-1 up-regulates SphK1 mRNA levels, protein expression, and activity in both primary human astrocytes and various glioblastoma cell lines; however, it does not affect SphK2 expression. The IL-1-induced SphK1 up-regulation can be blocked by the inhibition of JNK, the overexpression of the dominant-negative c-Jun(TAM67), and the down-regulation of c-Jun expression by small interference RNA. Activation of SphK1 expression by IL-1 occurs on the level of transcription and is mediated via a novel AP-1 element located within the first intron of the sphk1 gene. In summary, our results suggest that SphK1 expression is transcriptionally regulated by IL-1 in glioblastoma cells, and this pathway may be important in regulating survival and invasiveness of glioblastoma cells. Glioblastoma multiforme (GBM) 3The abbreviations used are: GBM, glioblastoma multiforme; AP-1, -2, activating proteins 1 and 2; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay; IL-1, interleukin-1; PMA, phorbol 12-myristate 13 acetate; S1P, sphingosine 1-phosphate; SphK1, sphingosine kinase 1; JNK, c-Jun N-terminal kinase; ACT, α1-antichymotrypsin; siRNA, small interference RNA; CREB, cAMP-response element-binding protein. 3The abbreviations used are: GBM, glioblastoma multiforme; AP-1, -2, activating proteins 1 and 2; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay; IL-1, interleukin-1; PMA, phorbol 12-myristate 13 acetate; S1P, sphingosine 1-phosphate; SphK1, sphingosine kinase 1; JNK, c-Jun N-terminal kinase; ACT, α1-antichymotrypsin; siRNA, small interference RNA; CREB, cAMP-response element-binding protein. is the most common malignant glioma, which has an extremely invasive phenotype (1Belda-Iniesta C. de Castro Carpeno J. Casado Saenz E. Cejas Guerrero P. Perona R. Gonzalez Baron M. Clin. Transl. Oncol.. 2006; 8: 635-641Google Scholar, 2Gilbertson R.J. Rich J.N. Nat. Rev. Cancer.. 2007; 7: 733-736Google Scholar). The ability of glioblastoma cells to diffusely infiltrate deeply into healthy brain tissue is the major obstacle for successful treatments, including surgery and radiotherapy, thus leading to the death of the patients within 10–12 months (3Hill C. Hunter S.B. Brat D.J. Adv. Anat. Pathol.. 2003; 10: 212-217Google Scholar, 4Deb P. Sharma M.C. Mahapatra A.K. Agarwal D. Sarkar C. Neurol. India.. 2005; 53: 329-332Google Scholar). Over the years, ample studies have aimed at understanding the molecular mechanisms of invasion of glioblastoma cells; however, they still remain elusive. Recently, the enhanced expression of several GBM biomarkers, such as EGFR, uPAR, uPA, PAI-1, and MMP-9, attracted significant attention (5Gondi C.S. Lakka S.S. Dinh D.H. Olivero W.C. Gujrati M. Rao J.S. Neuron Glia Biol.. 2004; 1: 165-176Google Scholar, 6Lakka S.S. Gondi C.S. Dinh D.H. Olivero W.C. Gujrati M. Rao V.H. Sioka C. Rao J.S. J. Biol. Chem.. 2005; 280: 21882-21892Google Scholar, 7Muracciole X. Romain S. Dufour H. Palmari J. Chinot O. Ouafik L. Grisoli F. Branger D.F. Martin P.M. Int. J. Radiat. Oncol. Biol. Phys.. 2002; 52: 592-598Google Scholar, 8Laerum O.D. Nygaar S.J. Steine S. Mork S.J. Engebraaten O. Peraud A. Kleihues P. Ohgaki H. J. Neurooncol.. 2001; 54: 1-8Google Scholar). More recently, a shorter prognosis for survival of patients with GBM was correlated with the elevated expression of SphK1, an enzyme that generates S1P by phosphorylating sphingosine (9Van Brocklyn J.R. Jackson C.A. Pearl D.K. Kotur M.S. Snyder P.J. Prior T.W. J. Neuropathol. Exp. Neurol.. 2005; 64: 695-705Google Scholar). S1P is a potent lipid mediator of various cell processes, including cell proliferation, differentiation, survival, and migration (10Spiegel S. Milstien S. Nat. Rev. Mol. Cell. Biol.. 2003; 4: 397-407Google Scholar). In the brain, S1P is present at high concentrations (11Edsall L.C. Spiegel S. Anal. Biochem.. 1999; 272: 80-86Google Scholar), and it has been shown to stimulate the migration and invasion of glioblastoma cells (12Van Brocklyn J.R. Young N. Roof R. Cancer Lett.. 2003; 199: 53-60Google Scholar). S1P can either be secreted by the cells to activate G-coupled S1P receptors (S1P1–5), or act via unknown intracellular processes (10Spiegel S. Milstien S. Nat. Rev. Mol. Cell. Biol.. 2003; 4: 397-407Google Scholar, 13Sanchez T. Hla T. J. Cell. Biochem.. 2004; 92: 913-922Google Scholar). In addition to SphK1, S1P can also be generated by SphK2, nevertheless the expression of SphK2 does not correlate with prognosis of GBM patients.In the past decade, it has been shown that the activity of SphK1 is stimulated by various growth factors and cytokines, including epidermal growth factor, tumor necrosis factor-α, IL-1, vascular endothelial growth factor, transforming growth factor-β, and nerve growth factor (10Spiegel S. Milstien S. Nat. Rev. Mol. Cell. Biol.. 2003; 4: 397-407Google Scholar). These factors induce rapid and transient activation of SphK1 and its subsequent translocation to the plasma membrane. In contrast to these observations, knowledge about the transcriptional regulation of the sphk1 gene is limited. The intrinsic expression of SphK1 depends on the specificity protein 1 binding elements located within the 5′-flanking region of the sphk1 gene (14Imamura T. Miyauchi-Senda N. Tanaka S. Shiota K. J. Vet. Med. Sci.. 2004; 66: 1387-1393Google Scholar). In addition, the expression of SphK1 is enhanced by epidermal growth factor, IL-1, tumor necrosis factor-α, histamine, prolactin, and glial cell-derived neurotrophic factor in different cell types (15Doll F. Pfeilschifter J. Huwiler A. Biochim. Biophys. Acta.. 2005; 1738: 72-81Google Scholar, 16Sobue S. Hagiwara K. Banno Y. Tamiya-Koizumi K. Suzuki M. Takagi A. Kojima T. Asano H. Nozawa Y. Murate T. J. Neurochem.. 2005; 95: 940-949Google Scholar, 17Huwiler A. Doll F. Ren S. Klawitter S. Greening A. Romer I. Bubnova S. Reinsberg L. Pfeilschifter J. Biochim. Biophys. Acta.. 2006; 1761: 367-376Google Scholar, 18Francy J.M. Nag A. Conroy E.J. Hengst J.A. Yun J.K. Biochim. Biophys. Acta.. 2007; 1769: 253-265Google Scholar, 19Murakami M. Ichihara M. Sobue S. Kikuchi R. Ito H. Kimura A. Iwasaki T. Takagi A. Kojima T. Takahashi M. Suzuki M. Banno Y. Nozawa Y. Murate T. J. Neurochem.. 2007; 102: 1585-1594Google Scholar); however, molecular mechanisms controlling the transcription of sphk1 gene in response to these factors are not known.Accordingly, the factors responsible for the enhanced SphK1 expression in GBM patients have not been identified. However, it recently became apparent that inflammation can be directly linked to the development and progression of many cancers (20Coussens L.M. Werb Z. Nature.. 2002; 420: 860-867Google Scholar, 21Karin M. Greten F.R. Nat. Rev. Immunol.. 2005; 5: 749-759Google Scholar), with inflammatory cytokines involved in these processes. IL-1 is one of the major regulators of inflammation, immune responses, and is a major neuroinflammatory cytokine in the brain that is released in response to injury or a growing tumor (22Rothwell N.J. Luheshi G.N. Trends Neurosci.. 2000; 23: 618-625Google Scholar, 23Basu A. Krady J.K. Levison S.W. J. Neurosci. Res.. 2004; 78: 151-156Google Scholar). It can induce the secretion of other proinflammatory cytokines, such as IL-6 and IL-8, as well as promote proliferation of glioblastoma cells and astrocytes (24Tada M. de Tribolet N. J. Neurooncol.. 1993; 17: 261-271Google Scholar, 25Cinque S. Willems J. Depraetere S. Vermeire L. Joniau M. Immunol. Lett.. 1992; 34: 267-271Google Scholar, 26Giulian D. Lachman L.B. Science.. 1985; 228: 497-499Google Scholar, 27Giulian D. Young D.G. Woodward J. Brown D.C. Lachman L.B. J. Neurosci.. 1988; 8: 709-714Google Scholar). Moreover, IL-1 has been implicated in tumorigenesis, tumor invasiveness, and metastasis of various cancer cells (23Basu A. Krady J.K. Levison S.W. J. Neurosci. Res.. 2004; 78: 151-156Google Scholar, 28Apte R.N. Dotan S. Elkabets M. White M.R. Reich E. Carmi Y. Song X. Dvozkin T. Krelin Y. Voronov E. Cancer Metastasis Rev.. 2006; 25: 387-408Google Scholar, 29Apte R.N. Krelin Y. Song X. Dotan S. Recih E. Elkabets M. Carmi Y. Dvorkin T. White R.M. Gayvoronsky L. Segal S. Voronov E. Eur. J. Cancer.. 2006; 42: 751-759Google Scholar). More importantly, glioblastoma cells have recently been shown to secrete substantial, physiologically relevant amounts of IL-1 (30Lu T. Tian L. Han Y. Vogelbaum M. Stark G.R. Proc. Natl. Acad. Sci. U. S. A.. 2007; 104: 4365-4370Google Scholar).We report here that the expression level of IL-1 correlates with the levels of SphK1, but not SphK2 in glioblastoma cell lines. IL-1 also activates the expression of SphK1, but not SphK2 in both glioblastoma cells and primary human astrocytes. We show that the response to IL-1 is mediated by the activation of the JNK-c-jun pathway, and subsequent binding of c-Jun to a novel AP-1 element located within the first intron of the sphk1 gene. Moreover, depletion of IL-1 or SphK1 levels decreases the growth and invasiveness of glioblastoma cells. Therefore, both IL-1 and SphK1 may be considered as future therapeutic targets for GBM.EXPERIMENTAL PROCEDURESCell Culture—Human glioblastoma U373-MG cells were obtained from American Type Culture Collection (Rockville, MD), whereas human glioblastoma A172, U87, U251, and T98G cells were obtained from Dr. Jaharul Haque (Cleveland Clinic Foundation, Cleveland, OH). The U373-TAM67 cells expressing the dominant-negative c-Jun(TAM67) were described previously (31Gopalan S.M. Wilczynska K.M. Konik B.S. Bryan L. Kordula T. J. Biol. Chem.. 2006; 281: 1956-1963Google Scholar). Human cortical astrocyte cultures were established using dissociated human cerebral tissue established exactly as previously described (32Wilczynska K.M. Gopalan S.M. Bugno M. Kasza A. Konik B.S. Bryan L. Wright S. Griswold-Prenner I. Kordula T. J. Biol. Chem.. 2006; 281: 34955-34964Google Scholar). Cortical tissue was provided by Advanced Bioscience Resources (Alameda, CA), and the protocol for obtaining postmortem fetal neural tissue complied with the federal guidelines for fetal research, and with the Uniformed Anatomical Gift Act. Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, antibiotics, sodium pyruvate, and non-essential amino acids.Cytokines and Cell Stimulation—Cells were stimulated with 10 ng/ml IL-1 (a gift from Immunex Corp., Seattle, WA), 5 μm S1P (Biomol Research Laboratories, Plymouth Meeting, PA) or 100 nm phorbol 12-myristate 13-acetate (PMA, Fisher Scientific Inc., Waltham, MA). For inhibitor studies, cells were pretreated with 1 μm SP600125, 10 μm BAY-117082, 10 μm SB202190, 5 μm parthenolide, 5 μg/ml Actinomycin D (Sigma), 1 μm CAY10470, 10 μm JNK peptide inhibitor I, 10 μm, JNK negative control peptide inhibitor I (EMD Biosciences, Inc., San Diego, CA), 10 μm LY294002, 1 μm U0126 (Cell Signaling Technology, Inc., Beverly, MA), 500 nm TAK-1 inhibitor (AnalytiCon Discovery, GmbH, Potsdam, Germany), 10 μm sphingosine kinase-1 inhibitor SK1-I (33Paugh S.W. Paugh B.S. Rahmani M. Kapitonov D. Almenara J.A. Kordula T. Milstien S. Adams J.K. Zipkin R.E. Grant S. Spiegel S. Blood.. 2008; 112: 1382-1391Google Scholar), and then stimulated with IL-1 as described in the figure legends.RNA Preparation and Quantitative PCR—Total RNA was prepared by phenol extraction, exactly as described previously (34Gopalan S. Kasza A. Xu W. Kiss D.L. Wilczynska K.M. Rydel R.E. Kordula T. J. Neurochem.. 2005; 94: 763-773Google Scholar). SphK1, SphK2, IL-1β, α1-antichymotrypsin (ACT), c-Jun, and 18 S mRNA levels were measured using TaqMan technology (Applied Biosystems, Foster City, CA), according to the supplier's instructions. Briefly, 1 μg of total RNA was reverse-transcribed using the High-Capacity cDNA archive kit (Applied Biosystems). Subsequently, the cDNAs were diluted 10-fold (Sphk1, SphK2, IL-1β, ACT, and c-jun), or 10,000-fold (18 S rRNA). For real-time PCR, pre-mixed primer-probe sets, and TaqMan Universal PCR Master Mix were purchased from Applied Biosystems and cDNAs were amplified using ABI 7900HT cycler. SphK1a, SphK1c, and glyceraldehyde-3-phosphate dehydrogenase mRNA levels were measured using DyNAmo™ SYBR Green quantitative PCR Kit (New England Biolabs, Inc., Ipswich, MA). To measure the expression of mRNAs for SphK1-spliced isoforms, specific primers located on the boundaries of the exons were designed and cDNA was amplified using ABI 7900HT cycler.Semiquantitative PCR—The expression of SphK1 isoforms were analyzed by PCR using isoform specific primers, shown below. The PCR products were analyzed by agarose gel electrophoresis after staining with ethidium bromide.Synthetic Oligonucleotides—The following oligonucleotides were synthesized to amplify the DNA fragments from the 5′-flanking region of the SphK1 gene: (300), 5′-GGGGGTCGACGCAGCTCGTCCCAAGCTC-3′ and 5′-GCTTTCTAGAACCCGGGCGGGAACCAGCTC-3′; (1200), 5′-TAACGTCGACCCGCGGCTCCCGC-3′ and 5′-GCTTTCTAGAACCCGGGCGGGAACCAGCTC-3′; (2400), 5′-GGGGGTCGACGCAGCTCGTCCCAAGCTC-3′ and 5′-GGCATCTAGAGAGATCCAAGTGGCCCGCC-3′; (L3R3), 5′-TCCCTCTAGATCACACCTTGGCAAG-3′ and 5′-CTGGGGATCCAGGTCTCTTCTGGGC-3′; (L4R4), 5′-GGCTTCTAGAAGCCAGAAAAG-3′ and 5′-GTAGGATCCTAAGGGTACAGGAGG-3′; (L5R5), 5′-GAGGTCTAGAAGAGACCTGGGTCC-3′ and 5′-GATGGATCCCCTTCCCACGGTTGC-3′. The mutations within the AP-1 and CRE elements were generated using the following primers: AP-1 mutant, 5′-GCTCTCTAGAATCCGTCGGGCCGGAACC-3′ and 5′-GGATTCTAGAGAGCCCCGTGGCTCCC-3′; CRE mutant, 5′-GGAAGGATCCCGGTGCTCCTGCAGCCAC-3′ and 5′-ACCGGGATCCTTCCCTGCGGCGGCTGG-3′. The AP-1 double-stranded oligonucleotides used in EMSA had the following sequence: 5′-GATCTGGGGCTCTGACTCATCCGTCGA-3′ and 5′-GATCTCGACGGATGAGTCAGAGCCCCA-3′. The expression of SphK1 isoforms was assayed using the following isoform specific primers: (AB-R), 5′-CCTGCCTTCAGCTCCTTATC-3′; (AB-F), 5′-CCGGACCGACTGGGTCCAG-3′; (R), 5′-CCTTGCCGCCGCGCGGG-3′; (A-R), 5′-GGCCGCCCGCTGGATCC-3′; (B-R), 5′-TTCCGCCGCTCAGTGAGCA-3′; (B-F), 5′-GGTTATGGATCCAGTGGTCG-3′; (C-F), 5′-GGGATTTTTACGCAGCTGGAC-3′; and (AC-F), 5′-GGTTATGGATCCAGCGGG CG-3′.Plasmid Construction—Plasmids pSphK1(300)CAT, pSphK1(1200)CAT, and pSphK1(2400)CAT were generated by cloning the XbaI/SalI-digested PCR products into the XbaI/XhoI sites of ptkCATΔEH. pSphK1(L3R3)CAT, pSphK1(L4R4)CAT, and pSphK1(L5R5)CAT plasmids were generated by cloning the XbaI/BamHI-digested PCR products into the XbaI/BamHI sites of pSphK1(1200)CAT. pSphK1(L4R4)CAT plasmid was utilized as the template to construct plasmids with mutated AP-1 and CRE sites using the QuikChange XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions.Transient Transfections—Primary human astrocytes were transfected in 12-well clusters using FuGENE6 transfection reagent (Roche Applied Science), with 300 ng of the reporter CAT plasmid and 100 ng of the expression plasmid encoding β-galactosidase. One day after transfection, the cells were stimulated with IL-1 or PMA, cultured another 24 h, and harvested. Extracts were prepared by freeze thawing. Chloramphenicol acetyltransferase (CAT) and β-galactosidase assays were performed as described (35Seed B. Sheen J.Y. Gene (Amst.).. 1988; 67: 271-277Google Scholar, 36Delegeane A.M. Ferland L.H. Mellon P.L. Mol. Cell. Biol.. 1987; 7: 3994-4002Google Scholar). CAT activities were normalized to β-galactosidase activity and are means ± S.E. (3–5 determinations).EMSA—Nuclear extracts were prepared as described (37Baeuerle P.A. Baltimore D. Science.. 1988; 242: 540-546Google Scholar). The oligonucleotides used for EMSA were designed to contain four base-long single-stranded 5′-overhangs at each end after annealing. Double-stranded DNA fragments were labeled by filling in the 5′-protruding ends with Klenow enzyme using [α-32P]dCTP (3000 Ci/mmol). EMSA was carried out according to the published procedures (38Sawadogo M. Van Dyke M.W. Gregor P.D. Roeder R.G. J. Biol. Chem.. 1988; 263: 11985-11993Google Scholar). Briefly, 5 μg of nuclear extracts and ∼10 fmol (10,000 cpm) of probe were used. The competition experiment was performed in the presence of a 100-fold excess of the cold oligonucleotides. Polyclonal anti-c-Fos, anti-FosB, anti-Fra1, anti-Fra2, anti-c-Jun, anti-JunB, and anti-JunD antisera were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and used for the supershift studies.Western Blotting and Antibodies—Cells were lysed in 10 mm Tris, pH 7.4, 150 mm sodium chloride, 1 mm EDTA, 0.5% Nonidet P-40, 1% Triton X-100, 1 mm sodium orthovanadate, 0.2 mm phenylmethylsulfonyl fluoride, and protease inhibitor mixture (Roche Applied Science). Equal amounts of proteins were resolved using SDS-PAGE and electroblotted onto nitrocellulose membranes (Schleicher & Schuell, Keene, NH). Polyclonal anti-IκBϵ, anti-c-fos, anti-phospho-c-jun, and anti-tubulin antisera were purchased from Santa Cruz Biotechnology, whereas anti-phospho-p38, anti-phospho-ERK, anti-phospho-JNK, anti-phospho-MKK3/6, anti-IκBα, anti-phospho-IκBα, anti-phospho-IKK, anti-phospho-p65, anti-phospho-ATF-2, and anti-phospho-Akt antisera were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Polyclonal anti-phospho-CREB/anti-phospho-ATF-1 antibodies were purchased from Upstate (Charlottesville, VA). Anti-sphingosine kinase 1 antibodies were described previously (39Hait N.C. Sarkar S. Le Stunff H. Mikami A. Maceyka M. Milstien S. Spiegel S. J. Biol. Chem.. 2005; 280: 29462-29469Google Scholar). Antigen-antibody complexes were visualized by enhanced chemiluminescence according to the manufacturer's instructions (Pierce). The neutralizing anti-IL-1β antibodies were purchased from R&D Systems (Minneapolis, MN).Down-regulation with siRNA—The expression of p65 and c-jun was down-regulated using SMARTpool siRNAs purchased from Dharmacon, Inc. (Lafayette, CO). SphK1 mRNA was down-regulated with siRNA targeted to a unique hSphK1 sequence as described previously (40Bektas M. Jolly P.S. Muller C. Eberle J. Spiegel S. Geilen C.C. Oncogene.. 2005; 24: 178-187Google Scholar). siRNAs were transfected into cells using Dharmafect 1 according to the manufacturer's instructions (Dharmacon, Inc., Lafayette, CO).Sphingosine Kinase Activity Assay—SphK activity was measured using 10-μg cell lysates with 50 μm sphingosine, in the presence of 0.25% Triton X-100 and [γ-32P]ATP (10 μCi and 1 mm) containing MgCl2 (10 mm) in buffer containing 20 mm Tris (pH 7.4), 10% glycerol, 1 mm 2-mercaptoethanol, 1 mm EDTA, 5 mm sodium orthovanadate, 40 mm β-glycerophosphate, 15 mm NaF, 10 μg/ml leupeptin, aprotinin, and soybean trypsin inhibitor, 1 mm phenylmethylsulfonyl fluoride, and 0.5 mm 4-deoxypyridoxine in a final volume of 200 μl, as described previously (41Kohama T. Olivera A. Edsall L. Nagiec M.M. Dickson R. Spiegel S. J. Biol. Chem.. 1998; 273: 23722-23728Google Scholar, 42Paugh S.W. Payne S.G. Barbour S.E. Milstien S. Spiegel S. FEBS Lett.. 2003; 554: 189-193Google Scholar). S1P was separated by TLC on silica gel G-60 with chloroform/acetone/methanol/acetic acid/water (10:4:3: 2:1, v/v) as a solvent, and the radioactive spots corresponding to S1P were quantified with a FX Molecular Imager (Bio-Rad). Background was determined in the absence of substrate. Activity is expressed as picomoles of S1P formed per min per mg of protein.Invasion Assay—The invasion of the cells was measured in a modified Boyden chamber, using polycarbonate filters (25 by 80 mm, 12-μm pore size) coated with Matrigel (BD Biosciences). IL-1 was added to both the upper and lower chamber, and the cells were also added to the upper chamber at 5 × 104 cells/well. After 7 h, non-migratory cells on the upper membrane surface were mechanically removed, and the cells that traversed and spread on the lower surface of the filter were fixed and stained with Diff-Quik (Fisher Scientific). The migrated cells were counted with a microscope and a 10× objective. Each data point is the average number of cells in five random fields, and is the average ± S.D. of three individual wells.Proliferation Assay—The number of viable cells was measured using the WST-1 assay (Roche Applied Science), according to the supplier's instructions.RESULTSCoexpression of SphK1 and IL-1β in Glioblastoma Cells; Effect of IL-1 on Proliferation and Invasion—Enhanced expression of SphK1, but not SphK2, has been correlated with the shorter survival of glioblastoma patients (9Van Brocklyn J.R. Jackson C.A. Pearl D.K. Kotur M.S. Snyder P.J. Prior T.W. J. Neuropathol. Exp. Neurol.. 2005; 64: 695-705Google Scholar); nevertheless, factors that enhance SphK1 expression in the tumors of these patients have not been identified. Because glioblastoma cells secrete IL-1 and chronic inflammation has been linked to cancer, the expression of SphK1 could be regulated by proinflammatory cytokines, including IL-1. To examine whether IL-1 is responsible for the enhanced expression of SphK1, we analyzed the expression of SphK1, SphK2, and IL-1β in several glioblastoma cell lines, and found drastic differences in their expression (Fig. 1A). More importantly, the enhanced expression of IL-1β correlated with high expression levels of SphK1, but not SphK2. Of note, U87 cells expressed the highest levels of SphK1 and IL-1β, and these cells are fairly invasive in comparison to the other cell lines (43Schichor C. Kerkau S. Visted T. Martini R. Bjerkvig R. Tonn J.C. Goldbrunner R. J. Neurooncol.. 2005; 73: 9-18Google Scholar).To determine if IL-1 can also induce the expression of SphK1 in primary cells, we analyzed the expression of both SphK1 and SphK2 in primary human astrocytes. As a control, we stimulated cells with PMA, which has previously been shown to activate SphK1 expression in other cell types (44Nakade Y. Banno Y.K.T.K. Hagiwara K. Sobue S. Koda M. Suzuki M. Kojima T. Takagi A. Asano H. Nozawa Y. Murate T. Biochim. Biophys. Acta.. 2003; 1635: 104-116Google Scholar). Similarly to the coexpression of SphK1 and IL-1 in glioblastoma cells, the expression of SphK1 was significantly up-regulated by IL-1 (and PMA) in astrocytes, while the expression of SphK2 was not changed (Fig. 1B); thus suggesting that the activation of SphK1 expression by IL-1 may occur in the brain during inflammation. We conclude that IL-1 is an important regulator of SphK1 expression in human astrocytes and glioblastoma cells.Because IL-1 is secreted by glioblastoma cells and induces the expression of SphK1, whose product S1P stimulates their growth and invasion, we analyzed whether inhibition of IL-1 and SphK1 affects growth and invasiveness of these cells. We used U373 cells as a model, because their response to S1P is well characterized (12Van Brocklyn J.R. Young N. Roof R. Cancer Lett.. 2003; 199: 53-60Google Scholar). In fact, IL-1 increased the number of viable U373 cells, while the knockdown of SphK1 had an opposite effect (Fig. 1C). Furthermore, neutralizing antibodies to IL-1β significantly decreased both the number of U373 cells and their invasion (Fig. 1, D and E). Thus, IL-1 is an important factor that can influence glioblastoma cell number and invasion, likely via the activation of Sphk1 expression.IL-1 Up-regulates SphK1 Expression in Glioblastoma Cells—Subsequently, we used U373 and A172 cells, which express low levels of SphK1 and IL-1β (Fig. 1A), and analyzed the expression of SphK1 and SphK2 in response to IL-1. In addition, we stimulated these cells with S1P to determine if this product of SphK1 can modulate SphK1 expression. We found that both IL-1 and S1P efficiently activated the expression of SphK1 on the mRNA level in U373, and they had an additive effect when used together (Fig. 2A). In contrast to SphK1, SphK2 expression was not affected by IL-1 or S1P. The up-regulation of SphK1 expression by IL-1 was time- and dose-dependent (Fig. 2, E and F, respectively), with a maximum stimulation at 8–18 h. The up-regulation of SphK1 expression on the mRNA level was paralleled by similar changes on the protein level (Fig. 2C) and in the enzymatic activity (Fig. 2D). Because of the presence of nonspecific bands detected by SphK1 antibodies, the identity of SphK1 bands was established by the down-regulation of SphK1 expression using specific siRNA (Fig. 2B). We found that both U373 and A172 cells express two SphK1 isoforms, migrating at 46 and 52 kDa, which likely represent SphK1a and Sphk1c splice variants described previously (45Imamura T. Ohgane J. Ito S. Ogawa T. Hattori N. Tanaka S. Shiota K. Genomics.. 2001; 76: 117-125Google Scholar, 46Venkataraman K. Thangada S. Michaud J. Oo M.L. Ai Y. Lee Y.M. Wu M. Parikh N.S. Khan F. Proia R.L. Hla T. Biochem. J.. 2006; 397: 461-471Google Scholar). These data indicate that (i) SphK1 expression is tightly regulated by IL-1, which likely provides a sustained pool of S1P, and (ii) S1P provides additional positive feedback loop, which further enhances expression of SphK1.FIGURE 2IL-1 up-regulates SphK1 expression and activity in glioblastoma cells. U373 cells were stimulated with 10 ng/ml IL-1 or 5 μm S1P for 18 h. A, SphK1 or SphK2 mRNA expression was analyzed using real-time PCR. B, A172 and U373 cells were transfected with either control or SphK1 siRNA. 48 h later, protein lysates were prepared and down-regulation of SphK1 protein expression was measured by Western blotting. C, lysates were prepared, and SphK1 protein level was determined by Western blotting. D, sphingosine kinase assays were performed in whole cell lysates, and bands corresponding to [32P]S1P were separated and quantitated. E, U373 cells were treated with 10 ng/ml IL-1 for the indicated times or for 18 h with indicated concentration of IL-1. F, subsequently, RNA was isolated, reverse-transcribed, and SphK1 mRNA expression was analyzed using real-time PCR (TaqMan). Data shown in the A, E, and F are expressed as -fold induction after normalization to 18 S rRNA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Identification of the SphK1 Isoforms Regulated by IL-1—Multiple isoforms of SphK1 have previously been shown to exist in rats, mice, and humans (45Imamura T. Ohgane J. Ito S. Ogawa T. Hattori N. Tanaka S. Shiota K. Genomics.. 2001; 76: 117-125Google Scholar, 46Venkataraman K. Thangada S. Michaud J. Oo M.L. Ai Y. Lee Y.M. Wu M. Parikh N.S. Khan F. Proia R.L. Hla T. Biochem. J.. 2006; 397: 461-471Google Scholar). The gene encoding SphK1 contains five exons and can be alternatively spliced producing SphK1 isoforms: SphK1a, SphK1b, and SphK1c. SphK1a lacks a fragment of the third exon encoding 14 amino acids, which is present in SphK1b and SphK1c, whereas SphK1c possesses an additional 86 amino acids at the N terminus (Fig. 3A). To determine which of the SphK1 isoforms are expressed in U373 cells, isoform-specific primers were designed on the boundaries of the exons to specifically detect different mRNA splice variants (Fig. 3A). We found that both SphK1a and SphK1c isoforms are expressed in U373 cells, whereas the SphK1b was not detected (Fig. 3B). Subsequently, using isoform-specific quantitative PCR, we tested whether IL-1 regulates the expression of both isoforms. In fact, the expression of both SphK1a and SphK1c was similarly up-regulated by IL-1 (Fig. 3C).FIGURE 3IL-1 regulates expression" @default.
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• W2088514755 date "2009-02-01" @default.
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• W2088514755 title "Interleukin-1 Regulates the Expression of Sphingosine Kinase 1 in Glioblastoma Cells" @default.
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