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• W1992044297 abstract "Sphingosine 1-phosphate (S1P), a sphingolipid metabolite that plays an important role in the regulation of cell survival, growth, migration, and angiogenesis, acts both inside the cells and as an extracellular mediator through binding to five G protein-coupled receptors (S1P1-5). Sphingosine kinase 1 (SK1), the enzyme responsible for S1P production, is overexpressed in many solid tumors, including gliomas. One common feature of these tumors is the presence of “hypoxic regions,” characterized by cells expressing high levels of hypoxia-inducible factors HIF-1α and HIF-2α, two transcription regulators that modulate the levels of proteins with crucial roles in tumor progression. So far, nothing is known about the role and the regulation of SK1 during tumor-induced hypoxia or about SK1 regulation and HIFs. Here we investigated the role of HIF-1α and HIF-2α in the regulation of SK1 during hypoxic stress in glioma-derived U87MG cells. We report that hypoxia increases SK1 mRNA levels, protein expression, and enzyme activity, followed by intracellular S1P production and S1P release. Interestingly, knockdown of HIF-2α by small interfering RNA abolished the induction of SK1 and the production of extracellular S1P after CoCl2 treatment, whereas HIF-1α small interfering RNA resulted in an increase of HIF-2α and of SK1 protein levels. Moreover, using chromatin immunoprecipitation analysis, we demonstrate that HIF-2α binds the SK1 promoter. Functionally, we demonstrate that conditioned medium from hypoxia-treated tumor cells results in neoangiogenesis in human umbilical vein endothelial cells in a S1P receptor-dependent manner. These studies provide evidence of a link between S1P production as a potent angiogenic agent and the hypoxic phenotype observed in many tumors. Sphingosine 1-phosphate (S1P), a sphingolipid metabolite that plays an important role in the regulation of cell survival, growth, migration, and angiogenesis, acts both inside the cells and as an extracellular mediator through binding to five G protein-coupled receptors (S1P1-5). Sphingosine kinase 1 (SK1), the enzyme responsible for S1P production, is overexpressed in many solid tumors, including gliomas. One common feature of these tumors is the presence of “hypoxic regions,” characterized by cells expressing high levels of hypoxia-inducible factors HIF-1α and HIF-2α, two transcription regulators that modulate the levels of proteins with crucial roles in tumor progression. So far, nothing is known about the role and the regulation of SK1 during tumor-induced hypoxia or about SK1 regulation and HIFs. Here we investigated the role of HIF-1α and HIF-2α in the regulation of SK1 during hypoxic stress in glioma-derived U87MG cells. We report that hypoxia increases SK1 mRNA levels, protein expression, and enzyme activity, followed by intracellular S1P production and S1P release. Interestingly, knockdown of HIF-2α by small interfering RNA abolished the induction of SK1 and the production of extracellular S1P after CoCl2 treatment, whereas HIF-1α small interfering RNA resulted in an increase of HIF-2α and of SK1 protein levels. Moreover, using chromatin immunoprecipitation analysis, we demonstrate that HIF-2α binds the SK1 promoter. Functionally, we demonstrate that conditioned medium from hypoxia-treated tumor cells results in neoangiogenesis in human umbilical vein endothelial cells in a S1P receptor-dependent manner. These studies provide evidence of a link between S1P production as a potent angiogenic agent and the hypoxic phenotype observed in many tumors. Sphingosine 1-phosphate (S1P), 2The abbreviations used are: S1Psphingosine 1-phosphateSKsphingosine kinasehSKhuman sphingosine kinaseS1P1-5sphingosine 1-phosphate receptors 1-5HIFhypoxia-inducible factorVEGFvascular endothelial growth factorEGFepidermal growth factorDMEMDulbecco's modified Eagle's mediumBSAbovine serum albuminHUVEChuman umbilical vein endothelial cellssiRNAsmall interfering RNACMFDA5-chloromethylfluorescein diacetate. a phosphorylated derivative of sphingosine, the structural backbone of all sphingolipids, is a bioactive lipid that regulates different biological processes, such as cell growth, differentiation, survival, and motility (1Leclercq T.M. Pitson S.M. IUBMB Life. 2006; 58: 467-472Crossref PubMed Scopus (51) Google Scholar). Moreover, a growing body of recent evidence has implicated S1P as one of the most potent proangiogenic agents (2Wang F. Van Brocklyn J.R. Hobson J.P. Movafagh S. Zukowska-Grojec Z. Milstien S. Spiegel S. J. Biol. Chem. 1999; 274: 35343-35350Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar, 3Liu C.H. Thangada S. Lee M.J. Van Brocklyn J.R. Spiegel S. Hla T. Mol. Biol. Cell. 1999; 10: 1179-1190Crossref PubMed Scopus (168) Google Scholar, 4Spiegel S. English D. Milstien S. Trends Cell Biol. 2002; 12: 236-242Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), enhancing tube formation and migration of endothelial cells (4Spiegel S. English D. Milstien S. Trends Cell Biol. 2002; 12: 236-242Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). S1P is a peculiar molecule acting both inside the cell on as yet undefined targets and as an extracellular mediator through binding to one of five cell surface G-protein-coupled receptors of the EDG family, recently renamed S1P1-5 receptors (5Payne S.G. Milstien S. Spiegel S. FEBS Lett. 2002; 531: 54-57Crossref PubMed Scopus (184) Google Scholar). Each S1P receptor subtype activates a unique set of G proteins with varying preferences (6Young N. Van Brocklyn J.R. Sci. World J. 2006; 6: 946-966Crossref Scopus (57) Google Scholar, 7Taha T.A. Argraves K.M. Obeid L.M. Biochim. Biophys. Acta. 2004; 1682: 48-55Crossref PubMed Scopus (165) Google Scholar). sphingosine 1-phosphate sphingosine kinase human sphingosine kinase sphingosine 1-phosphate receptors 1-5 hypoxia-inducible factor vascular endothelial growth factor epidermal growth factor Dulbecco's modified Eagle's medium bovine serum albumin human umbilical vein endothelial cells small interfering RNA 5-chloromethylfluorescein diacetate. S1P is produced by the action of sphingosine kinases. In mammals, two isoforms of this enzyme have been identified and cloned, namely sphingosine kinase 1 (SK1) (8Nava V.E. Lacana E. Poulton S. Liu H. Sugiura M. Kono K. Milstien S. Kohama T. Spiegel S. FEBS Lett. 2000; 473: 81-84Crossref PubMed Scopus (89) Google Scholar, 9Pitson S.M. D'Andrea R.J. Vandeleur L. Moretti P.A. Xia P. Gamble J.R. Vadas M.A. Wattenberg B.W. Biochem. J. 2000; 350: 429-441Crossref PubMed Scopus (166) Google Scholar) and sphingosine kinase 2 (SK2) (10Liu H. Sugiura M. Nava V.E. Edsall L.C. Kono K. Poulton S. Milstien S. Kohama T. Spiegel S. J. Biol. Chem. 2000; 275: 19513-19520Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar). These two proteins differ in their catalytic properties, cellular localization, and functions (11Maceyka M. Sankala H. Hait N.C. Le Stunff H. Liu H. Toman R. Collier C. Zhang M. Satin L.S. Merrill Jr., A.H. Milstien S. Spiegel S. J. Biol. Chem. 2005; 280: 37118-37129Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar); SK1 is mainly cytosolic and mediates prosurvival functions (12Olivera A. Kohama T. Edsall L. Nava V. Cuvillier O. Poulton S. Spiegel S. J. Cell Biol. 1999; 147: 545-558Crossref PubMed Scopus (462) Google Scholar, 13Olivera A. Rosenfeldt H.M. Bektas M. Wang F. Ishii I. Chun J. Milstien S. Spiegel S. J. Biol. Chem. 2003; 278: 46452-46460Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 14Sarkar S. Maceyka M. Hait N.C. Paugh S.W. Sankala H. Milstien S. Spiegel S. FEBS Lett. 2005; 579: 5313-5317Crossref PubMed Scopus (174) Google Scholar, 15Taha T.A. Kitatani K. El-Alwani M. Bielawski J. Hannun Y.A. Obeid L.M. FASEB J. 2006; 20: 482-484Crossref PubMed Scopus (128) Google Scholar), whereas SK2 is predominantly in the nucleus, where it inhibits growth and enhances apoptosis (16Igarashi N. Okada T. Hayashi S. Fujita T. Jahangeer S. Nakamura S. J. Biol. Chem. 2003; 278: 46832-46839Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar, 17Liu H. Toman R.E. Goparaju S.K. Maceyka M. Nava V.E. Sankala H. Payne S.G. Bektas M. Ishii I. Chun J. Milstien S. Spiegel S. J. Biol. Chem. 2003; 278: 40330-40336Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, 18Okada T. Ding G. Sonoda H. Kajimoto T. Haga Y. Khosrowbeygi A. Gao S. Miwa N. Jahangeer S. Nakamura S. J. Biol. Chem. 2005; 280: 36318-36325Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). SK1 has been shown to be activated by numerous external stimuli, such as tumor necrosis factor-α (19Pettus B.J. Bielawski J. Porcelli A.M. Reames D.L. Johnson K.R. Morrow J. Chalfant C.E. Obeid L.M. Hannun Y.A. FASEB J. 2003; 17: 1411-1421Crossref PubMed Scopus (295) Google Scholar, 20Billich A. Bornancin F. Mechtcheriakova D. Natt F. Huesken D. Baumruker T. Cell. Signal. 2005; 17: 1203-1217Crossref PubMed Scopus (119) Google Scholar), platelet-derived growth factor (21Olivera A. Edsall L. Poulton S. Kazlauskas A. Spiegel S. FASEB J. 1999; 13: 1593-1600Crossref PubMed Scopus (74) Google Scholar), VEGF (22Shu X. Wu W. Mosteller R.D. Broek D. Mol. Cell Biol. 2002; 22: 7758-7768Crossref PubMed Scopus (247) Google Scholar), neural growth factor, basic fibroblast growth factor (23Rius R.A. Edsall L.C. Spiegel S. FEBS Lett. 1997; 417: 173-176Crossref PubMed Scopus (98) Google Scholar), EGF (24Meyer zu Heringdorf D. Lass H. Kuchar I. Alemany R. Guo Y. Schmidt M. Jakobs K.H. FEBS Lett. 1999; 461: 217-222Crossref PubMed Scopus (64) Google Scholar), and phorbol 12-myristate 13-acetate (25Johnson K.R. Becker K.P. Facchinetti M.M. Hannun Y.A. Obeid L.M. J. Biol. Chem. 2002; 277: 35257-35262Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar), resulting in increased intracellular S1P and increased release of S1P from certain cell types (25Johnson K.R. Becker K.P. Facchinetti M.M. Hannun Y.A. Obeid L.M. J. Biol. Chem. 2002; 277: 35257-35262Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 26Anelli V. Bassi R. Tettamanti G. Viani P. Riboni L. J. Neurochem. 2005; 92: 1204-1215Crossref PubMed Scopus (97) Google Scholar, 27Hanel P. Andreani P. Graler M.H. FASEB J. 2007; 21: 1202-1209Crossref PubMed Scopus (311) Google Scholar, 28Bassi R. Anelli V. Giussani P. Tettamanti G. Viani P. Riboni L. Glia. 2006; 53: 621-630Crossref PubMed Scopus (106) Google Scholar). SK1 has been shown to act as an oncogene, whereby its overexpression regulates cell growth in soft agar and leads to tumor formation in xenograft model (29Xia P. Gamble J.R. Wang L. Pitson S.M. Moretti P.A. Wattenberg B.W. D'Andrea R.J. Vadas M.A. Curr. Biol. 2000; 10: 1527-1530Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). Moreover, knockdown of SK1 has been shown to activate apoptotic pathways of cell death (15Taha T.A. Kitatani K. El-Alwani M. Bielawski J. Hannun Y.A. Obeid L.M. FASEB J. 2006; 20: 482-484Crossref PubMed Scopus (128) Google Scholar). Importantly, SK1 mRNA was found to be significantly higher in various tumor tissues, such as brain, breast, colon, lung, ovary, stomach, uterus, kidney, rectum, and small intestine. The elevated SK1 mRNA expression was confirmed by immunopositive staining for SK1 in lung and colon cancer (30Johnson K.R. Johnson K.Y. Crellin H.G. Ogretmen B. Boylan A.M. Harley R.A. Obeid L.M. J. Histochem. Cytochem. 2005; 53: 1159-1166Crossref PubMed Scopus (161) Google Scholar, 31Kawamori T. Osta W. Johnson K.R. Pettus B.J. Bielawski J. Tanaka T. Wargovich M.J. Reddy B.S. Hannun Y.A. Obeid L.M. Zhou D. FASEB J. 2006; 20: 386-388Crossref PubMed Scopus (192) Google Scholar). Glioblastoma multiforme is the most commonly occurring primary brain tumor in adults and displays aggressive growth and invasion into surrounding brain tissue, leading to a short life expectancy after the initial diagnosis. One of the key histopathological features of gliomas is an intense increased angiogenesis attributed to increased VEGF expression (32Plate K.H. Breier G. Weich H.A. Mennel H.D. Risau W. Int. J. Cancer. 1994; 59: 520-529Crossref PubMed Scopus (428) Google Scholar). S1P has been shown to enhance growth, migration, and invasiveness of glioblastoma multiforme cell lines (33Van Brocklyn J. Letterle C. Snyder P. Prior T. Cancer Lett. 2002; 181: 195-204Crossref PubMed Scopus (87) Google Scholar). Moreover, expression of SK1 in glioblastoma multiforme tissue has been shown to correlate with short patient survival (34Van Brocklyn J.R. Jackson C.A. Pearl D.K. Kotur M.S. Snyder P.J. Prior T.W. J. Neuropathol. Exp. Neurol. 2005; 64: 695-705Crossref PubMed Scopus (285) Google Scholar). Gliomas, similar to other malignant tumors, are characterized by extensive regions of low oxygen tension caused by rapid cell proliferation, leading to tissue hypoxia (35Kaur B. Khwaja F.W. Severson E.A. Matheny S.L. Brat D.J. Van Meir E.G. Neuro-oncol. 2005; 7: 134-153Crossref PubMed Scopus (527) Google Scholar). Central to the adaptive response that occurs in tumor hypoxia is a dramatic increase of protein levels of hypoxia-inducible factors (HIFs), which are key transcriptional regulators involved in the induction of a set of hypoxia-regulated genes. HIF is composed of an oxygen-regulated HIF-α subunit (HIF-1α and HIF-2α) and the ubiquitous aryl hydrocarbon receptor nuclear translocator (or HIF-1β) partner protein (36Srinivas V. Zhang L.P. Zhu X.H. Caro J. Biochem. Biophys. Res. Commun. 1999; 260: 557-561Crossref PubMed Scopus (126) Google Scholar). HIF-α protein turnover in normoxia is very rapid due to the action of HIF-α prolyl hydroxylases. These oxygen-dependent enzymes hydroxylate two conserved proline residues of HIF-α proteins, promoting binding of the Von Hippel-Lindau protein, ubiquitination, and subsequent proteosomal degradation (37Salceda S. Caro J. J. Biol. Chem. 1997; 272: 22642-22647Abstract Full Text Full Text PDF PubMed Scopus (1417) Google Scholar). Under hypoxic conditions, the hydroxylases are inhibited, and HIF-α proteins are stabilized and transcriptionally activated, leading to potent induction of target genes. An important question in HIF biology is what roles the two HIF-α subunits exert on target gene activation, particularly the extent to which they cooperate, overlap, or have distinct roles. Although they exhibit high sequence homology and seem to be regulated in a similar fashion, it has been demonstrated that there is little redundancy between the two α subunits, and they have distinct expression and activation of target genes. For example, it has been shown that HIF-1α exclusively induces the hypoxic transcription of glycolytic genes, such as phosphoglycerate kinase 1 (pgk1) and aldolase A (aldA) (38Hu C.J. Wang L.Y. Chodosh L.A. Keith B. Simon M.C. Mol. Cell Biol. 2003; 23: 9361-9374Crossref PubMed Scopus (1082) Google Scholar, 39Wang V. Davis D.A. Haque M. Huang L.E. Yarchoan R. Cancer Res. 2005; 65: 3299-3306Crossref PubMed Scopus (68) Google Scholar), whereas HIF-2α induces VEGF and transforming growth factor α expression (38Hu C.J. Wang L.Y. Chodosh L.A. Keith B. Simon M.C. Mol. Cell Biol. 2003; 23: 9361-9374Crossref PubMed Scopus (1082) Google Scholar, 40Gunaratnam L. Morley M. Franovic A. de Paulsen N. Mekhail K. Parolin D.A. Nakamura E. Lorimer I.A. Lee S. J. Biol. Chem. 2003; 278: 44966-44974Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 41Wykoff C.C. Sotiriou C. Cockman M.E. Ratcliffe P.J. Maxwell P. Liu E. Harris A.L. Br. J. Cancer. 2004; 90: 1235-1243Crossref PubMed Scopus (86) Google Scholar). It was recently shown that hypoxic stress induced S1P formation in vascular smooth muscle cells (42Yun J.K. Kester M. Arch. Biochem. Biophys. 2002; 408: 78-86Crossref PubMed Scopus (34) Google Scholar). Moreover, acute hypoxia increased SK1 and SK2 mRNA transcript levels in human pulmonary smooth muscle cells (43Ahmad M. Long J.S. Pyne N.J. Pyne S. Prostaglandins Other Lipid Mediat. 2006; 79: 278-286Crossref PubMed Scopus (49) Google Scholar). In addition, cardiac myocytes null for the SK1 gene were shown to be more vulnerable to hypoxic stress (44Tao R. Zhang J. Vessey D.A. Honbo N. Karliner J.S. Cardiovasc. Res. 2007; 74: 56-63Crossref PubMed Scopus (82) Google Scholar). However, little is known about the role and regulation of SK during tumor-induced hypoxia or about SK regulation by HIFs. In this study, we show for the first time that SK1 protein, message, and SK activity are increased in tumor cell hypoxia and in response to CoCl2, a hypoxia-mimicking agent (45Yuan Y. Hilliard G. Ferguson T. Millhorn D.E. J. Biol. Chem. 2003; 278: 15911-15916Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar). This increase in SK1 is accompanied by an increase in cellular and released S1P. We also show that HIF-2α but not HIF-1α is required for the hypoxia-induced increases in SK1 and S1P. Moreover, we demonstrate that HIF-2α binds the SK1 promoter and that this binding is increased by hypoxia. In addition, we demonstrate that conditioned media from hypoxia-treated tumor cells results in neoangiogenesis in human umbilical vein endothelial cells in a S1P receptor-dependent manner. MaterialsTissue culture medium, heat-inactivated fetal bovine serum, penicillin/streptomycin, phosphate-buffered saline, oligofectamine, and Lipofectamine 2000 were from Invitrogen. Human glioma cells U87 and 786-O were from the American Type Culture Collection. HUVECs were from Cambrex. CoCl2, KCl, Tris-HCl, EDTA, deoxypyridoxine, sodium orthovanadate, β-glycerophosphate, phenylmethylsulfonyl fluoride, glycerol, Triton X-100, and ATP were from Sigma. d-erythro Sphingosine and d-erythro-sphingosine 1-phosphate were from Biomol. d-erythro-[3-3H]Sphingosine and [γ-32P]ATP were from PerkinElmer Life Sciences. Rabbit polyclonal anti-HIF-1α and HIF-2α were from Novus Biological. Rabbit anti-hSK1 was prepared by the Medical University of South Carolina antibody facility as previously described (39Wang V. Davis D.A. Haque M. Huang L.E. Yarchoan R. Cancer Res. 2005; 65: 3299-3306Crossref PubMed Scopus (68) Google Scholar). Cell CulturesU87 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and 100 units/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin B in a 5% CO2 incubator at 37 °C. 786-O cells were cultured under similar conditions in RPMI medium. HUVECs were cultured in endothelial cell medium-2 (EGM™-2) supplemented with 2% FCS and singleQuots®. For in vitro angiogenesis experiments, HUVECs were used between passages 2 and 8. Treatment of Cells with CoCl2 and HypoxiaCells at 70-80% confluence were serum-starved for 16 h in DMEM or RPMI containing 0.1% fatty acid-free BSA and incubated in the absence or presence of 150 μm CoCl2. For the hypoxia experiments, cells were incubated for different times in a 5% CO2, 95% nitrogen incubator with an oxygen sensor set at 0.5% O2. Chromatin Immunoprecipitation (ChIP) AnalysisThe ChIP assay was performed according to the protocol provided with the ChIP assay kit (Upstate Biotechnology, Inc.). Briefly, U87 and 786-O cells at 70-80% confluence were serum-starved for 16 h in DMEM or RPMI containing 0.1% fatty acid-free BSA and incubated in the absence or presence of 150 μm CoCl2 for 2 h. At the end of incubation, formaldehyde was added to the plate at 1% final concentration, and cells were incubated for 10 min at room temperature. Cells were washed twice with cold phosphate-buffered saline, lysed in 1 ml of SDS lysis buffer, and sonicated three times for 10 min to shear DNA to an average fragment size of 200-1000 bp. Ten μl of each sonicated sample of cell lysate were removed and used as input control in the final PCRs. The supernatant was recovered, diluted, and precleared using Protein A-agarose/salmon sperm DNA. The recovered supernatant was incubated with either rabbit anti-HIF-1α, anti-HIF-2α, or an isotype control IgG overnight in the presence of Protein A-agarose/salmon sperm DNA. The beads were washed with low salt, high salt, and LiCl buffers. The immunoprecipitated DNA was eluted from the beads with 0.2% SDS and 0.1 m NaHCO3 solution. Eluates were then incubated with 0.2 m NaCl for 4 h at 65 °C. DNA was then purified using a PCR purification kit (Qiagen), and PCR was done on the extracted DNA using Advantage-GC Genomic PCR (Clontech) with the following specific primers for different regions of SK1 promoter: A, 5′-gtttgagacgggtttctcca-3′ (forward) and 5′-cctttctccagacccctttc-3′ (reverse); B, 5′-tgaaccaggtgggctttatc-3′ (forward) and 5′-ctccgagaaacaggaacgag-3′ (reverse); C, 5′-tcgttcctgtttctcggagt-3′ (forward) and 5′-ggagaggaggcttgacagtg-3′ (reverse); D, 5′-ggtcctccggaagagaagac-3′ (forward) and 5′-gattggaaagccaagcatgt-3′ (reverse). siRNA Down-regulation of HIF-1α, HIF-2α, and S1P1 ExpressionHIF-1α was down-regulated using 50 nm sequence-specific siRNA from Invitrogen (Validated Stealth 46-3242; duplexes 1 and 2 showed similar knockdown results (see supplemental Fig. 2) and duplex 1 was used in all experiments). HIF-2α was down-regulated using 50 nm sequence-specific siRNA from Ambion (predesigned siRNA ID 106447 and 106448; both showed similar knockdown effectiveness (see supplemental Fig. 2), and the former was used in all experiments). S1P1 was down-regulated using 10 nm sequence-specific siRNA from Santa Cruz Biotechnology (catalog number sc37086). Scrambled siRNA was synthesized from Xeragon (5′-AATTCTCCGAACGTGTCACGT-3′) and was used as negative control. Cells were seeded in 60-mm dishes at a density of 2 × 105 24 h before down-regulation and transfected using Oligofectamine reagent according to the manufacturer's protocol. Briefly, 5 μl of 20 μm siRNA (for HIF-1α and HIF-2α) or 2 μl of 10 μm (for S1P1) were resuspended in 400 μl of Opti-MEM medium and mixed with 30 μl of oligofectamine-opti-MEM medium complex (4:11, v/v). The mixture was then incubated for 20 min at room temperature and added to the cells (incubated for 20 min in 1.6 ml of Opti-MEM medium). After 4 h, 1 ml of 30% fetal bovine serum medium (DMEM or RPMI or EGM™-2) was added to the dishes, and the cells were further incubated for 48 h. The efficiency of the knockdown was determined by quantitative real time PCR for HIF-1α, HIF-2α, and S1P1 mRNA and immunoblotting using specific antibodies 48 h after transfection. Measurement of Promoter ActivityThe hSK1 promoter was cloned from human genomic DNA through PCR amplification of a 3-kb DNA segment upstream of the SK1 5′-untranslated region. This fragment was gel-purified and cloned into TOPO-XL vector (Invitrogen) and verified by sequencing. The putative SK1 promoter was excised using KpnI and NheI and ligated into the PGL3 Basic luciferase vector. U87MG cells were seeded at 2 × 105 cells in 60-mm dishes for 24 h and treated with scrambled, HIF-1α, or HIF-2α siRNA for 24 h. Cells were then co-transfected using Lipofectamine 2000 with 0.8 μg of SK1 promoter construct and 0.2 μg of β-galactosidase plasmid for 24 h. Cells were serum-starved for 16 h and treated with CoCl2 for 2 h. Cells were then lysed, and luciferase and β-galactosidase measurements were made using a luciferase and β-galactosidase enzyme assay system kit (from Stratagene) according to the manufacturer's instructions. Data are normalized with β-galactosidase activity in each sample. Quantitative Real Time PCRTotal RNA was isolated using the RNeasy minikit (Qiagen), treated with the Turbo RNase-free kit (Ambion), and measured using the Quant-iT RiboGreen RNA kit (Invitrogen). RNA (1 μg) was reverse transcribed to cDNA using the SuperScript First-Strand Synthesis System (Invitrogen). Quantitative real time PCR was performed with an iCycler Q real time detection system using the SYBR Green Supermix kit (Bio-Rad). Reactions were performed using hSK1-specific primers (forward primer, 5′-CTGGCAGCTTCCTTGAACCAT-3′; reverse primer, 5′-TGTGCAGAGACAGCAGGTTCA-3′), hSK2-specific primers (forward primer, 5′-CCAGTGTTGGAGAGCTGAAGGT-3′; reverse primer, 5′-GTCCATTCATCTGCTGGTCCTC-3′), hHIF-1α-specific primers (forward primer, 5′-GAAAGCGCAAGTCCTCAAAG-3′; reverse primer, 5′-TGGGTAGGAGATGGAGATGC-3′), hHIF-2α-specific primers (forward primer, 5′-TCCCACCAGCTTCACTCTCT-3′; reverse primer, 5′-TCAGAAAAAGGCCACTGCTT-3′), and β-actin-specific primer (forward primer, 5′-ATTGGCAATGAGCGGTTCC-3′; reverse primer, 5′-GGTAGTTTCGTGGATGCCACA-3′). Real time PCR conditions were as follows: 3 min at 95 °C followed by 40 cycles with 45 s at 60 °C, 1 min at 95 °C, 1 min at 60 °C, and 10 min at 4 °C. Real time PCR results were analyzed using Q-Gene® software, which expresses data as mean normalized expression. All of the genes were normalized to expression of β-actin as an endogenous control. Sphingosine Kinase ActivitySphingosine kinase activity was determined as described previously with minor modifications (46Olivera A. Rosenthal J. Spiegel S. J. Cell. Biochem. 1996; 60: 529-537Crossref PubMed Scopus (92) Google Scholar). After CoCl2 treatment cells were washed with cold phosphate-buffered saline and harvested in SK1 buffer (containing 20 mm Tris-HCl, pH 7.4, 1 mm EDTA, 0.5 mm deoxypyridoxine, 15 mm NaF, 1 mm β-mercaptoethanol, 1 mm sodium orthovanadate, 40 mm β-glycerophosphate, 0.4 mm phenylmethylsulfonyl fluoride, 10% glycerol, 0.5% Triton X-100, and complete protease inhibitors). After brief sonication and protein concentration (determined by the BCA method), 30 μg of proteins were incubated in 100 μl of reaction mixture containing sphingosine (50 μm, delivered in 4 mg/ml fatty acid-free bovine serum albumin), [γ-32P]ATP (5 μCi, 1 mm dissolved in 10 mm MgCl2), and SK1 buffer for 30 min at of 37 °C. The reaction was terminated by the addition of 10 μl of 1 n HCl and 400 μl of chloroform/methanol/HCl (100:200:1, v/v/v). Subsequently, 120 μl of chloroform and 120 μl of 2 m KCl were added, and samples were centrifuged at 3000 × g for 5 min. 200 μl of the organic phase was transferred to new glass tubes and dried. Samples were resuspended in chloroform/methanol/HCl (100: 100:1, v/v/v). Lipids were then resolved on silica thin layer chromatography plates using 1-butanol/methanol/acetic acid/water (8:2:1:2, v/v/v/v) as solvent system and visualized by autoradiography. The radioactive spots corresponding to S1P were scraped from the plates and counted for radioactivity. Background values were determined in negative controls in which sphingosine was not added to the reaction mixture. In Vivo Assay for Sphingosine 1-Phosphate Formation and ReleaseU87MG cells were seeded at 1 × 105 cells in 35-mm dishes and treated or not with siRNA for HIF-1α or HIF-2α. After 48 h, the medium was removed, and cells were serum-starved for 16 h and treated with CoCl2 for 2 h. At the end of incubation, cells were pulsed with 20 nm d-erythro-[3-3H] sphingosine (0.5 μCi/dish). Total lipids were extracted from cells at 4 °C with chloroform/methanol as previously reported (26Anelli V. Bassi R. Tettamanti G. Viani P. Riboni L. J. Neurochem. 2005; 92: 1204-1215Crossref PubMed Scopus (97) Google Scholar, 47Riboni L. Viani P. Bassi R. Giussani P. Tettamanti G. J. Neurochem. 2000; 75: 503-510Crossref PubMed Scopus (26) Google Scholar). Briefly, the total lipid extract was partitioned by the addition of 0.15 volumes of 0.1 m NH4OH, and the phases were separated by centrifugation. S1P in the upper phase was separated by high performance thin layer chromatography using n-butanol/acetic acid/water (3:1:1, v/v/v). Bands corresponding to the S1P standard were scraped, and the radioactivity was measured using a scintillation counter. Extracellular S1P was extracted from pulse medium and partially purified as reported (26Anelli V. Bassi R. Tettamanti G. Viani P. Riboni L. J. Neurochem. 2005; 92: 1204-1215Crossref PubMed Scopus (97) Google Scholar). The final organic phase containing S1P was submitted to high performance thin layer chromatography as above, and the S1P bands were scraped and counted as described above. Western Blot AnalysisWestern blot analysis was used to evaluate hSK1, HIF-1α, and HIF-2α. Cells were lysed in buffer containing 20 mm Hepes, pH 7.4, 50 mm NaCl, 1 mm EGTA, 0.5 mm β-glycerophosphate, 30 mm sodium pyrophosphate, 0.1 mm sodium orthovanadate, 1% Triton X-100, and protease inhibitor mixture tablet (Roche Applied Science) and kept on ice for 30 min. After brief sonication, protein concentration was determined using the BCA method, and 30 μg of protein was loaded on an SDS-10% polyacrylamide gel, transferred to a nitrocellulose membrane, and blotted with anti-hSK1, HIF-1α, HIF-2α, and actin. Immunoreactive bands were detected with horseradish peroxidase-conjugated anti-rabbit IgG (Jackson) and an ECL detection kit (Pierce). In Vitro Tube Formation AssayAn in vitro tube formation assay was performed to evaluate the angiogenic properties of U87MG-conditioned medium on endothelial cells. HUVECs were seeded in 100-mm dishes at a concentration of 5 × 105 and used when they reached 80% confluence. At the day of the experiment the fluorescent cellTracker™ Green CMFDA (Molecular Probes) was added to the cells at 5 μm final concentration and incubated for 45 min. Medium was then removed, and cells were further incubated for 4 h in EGM-2 medium (without fetal bovine serum and singleQuots®) containing 0.1% fatty acid-free BSA. At the end of the incubation, cells were trypsinized and seeded at 4 × 104 cells/cm2 in a 24-well plate precoated with growth factor-reduced Matrigel™ matrix (BD Biosciences) (450 μg/well of Matrigel diluted 1:1 with serum-free EGM-2) in DMEM, DMEM containing 100 nm S1P, or DMEM conditioned from U87 (incubated in 1 or 20% oxygen for 2 h) in the absence or presence of 1 μm VPC 23019. Tube formation was observed using a laser-scanning confocal microscope (LSM 510 Meta; Carl Zeiss, Thornwood, NY) after 16 h. Cell Migrati" @default.
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• W1992044297 date "2008-02-01" @default.
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• W1992044297 title "Sphingosine Kinase 1 Is Up-regulated during Hypoxia in U87MG Glioma Cells" @default.
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