Involvement of N-terminal-extended Form of Sphingosine Kinase 2 in Serum-dependent Regulation of Cell Proliferation and Apoptosis
2005; Elsevier BV; Volume: 280; Issue: 43 Linguagem: Inglês
10.1074/jbc.m504507200
ISSN1083-351X
AutoresTaro Okada, Guo Ding, Hirofumi Sonoda, Taketoshi Kajimoto, Yuki Haga, Ali Khosrowbeygi, Sanyang Gao, Noriko Miwa, Saleem Jahangeer, Shun‐ichi Nakamura,
Tópico(s)Ion Transport and Channel Regulation
ResumoSphingosine kinase (SPHK) 1 is implicated in the regulation of cell proliferation and anti-apoptotic processes by catalyzing the formation of an important bioactive messenger, sphingosine 1-phosphate. Unlike the proliferative action of SPHK1, another isozyme, SPHK2, has been shown to possess anti-proliferative or pro-apoptotic action. Molecular mechanisms of SPHK2 action, however, are largely unknown. The present studies were undertaken to characterize the N-terminal-extended form of SPHK2 (SPHK2-L) by comparing it with the originally reported form, SPHK2-S. Real-time quantitative PCR analysis revealed that SPHK2-L mRNA is the major form in several human cell lines and tissues. From sequence analyses it was concluded that SPHK2-L is a species-specific isoform that is expressed in human but not in mouse. At the protein level it has been demonstrated by immunoprecipitation studies that SPHK2-L is the major isoform in human hepatoma HepG2 cells. SPHK2-L, when expressed in human embryonic kidney (HEK) 293 cells, did not show any inhibition of DNA synthesis in the presence of serum, whereas it showed marked inhibition in the absence of serum. Moreover, serum deprivation resulted in the translocation of SPHK2-L into the nuclei. In addition, serum deprivation induced SPHK2-L expression in HEK293 cells. Furthermore, suppression of SPHK2 by small interfering RNA treatment prevented serum deprivation- or drug-induced apoptosis in HEK293 cells. Taken together, these results indicate that a major form of SPHK2 splice variant, SPHK2-L, in human cells does not inhibit DNA synthesis under normal conditions and that SPHK2-L accumulation in the nucleus induced by serum deprivation may be involved in the cessation of cell proliferation or apoptosis depending on the cell type. Sphingosine kinase (SPHK) 1 is implicated in the regulation of cell proliferation and anti-apoptotic processes by catalyzing the formation of an important bioactive messenger, sphingosine 1-phosphate. Unlike the proliferative action of SPHK1, another isozyme, SPHK2, has been shown to possess anti-proliferative or pro-apoptotic action. Molecular mechanisms of SPHK2 action, however, are largely unknown. The present studies were undertaken to characterize the N-terminal-extended form of SPHK2 (SPHK2-L) by comparing it with the originally reported form, SPHK2-S. Real-time quantitative PCR analysis revealed that SPHK2-L mRNA is the major form in several human cell lines and tissues. From sequence analyses it was concluded that SPHK2-L is a species-specific isoform that is expressed in human but not in mouse. At the protein level it has been demonstrated by immunoprecipitation studies that SPHK2-L is the major isoform in human hepatoma HepG2 cells. SPHK2-L, when expressed in human embryonic kidney (HEK) 293 cells, did not show any inhibition of DNA synthesis in the presence of serum, whereas it showed marked inhibition in the absence of serum. Moreover, serum deprivation resulted in the translocation of SPHK2-L into the nuclei. In addition, serum deprivation induced SPHK2-L expression in HEK293 cells. Furthermore, suppression of SPHK2 by small interfering RNA treatment prevented serum deprivation- or drug-induced apoptosis in HEK293 cells. Taken together, these results indicate that a major form of SPHK2 splice variant, SPHK2-L, in human cells does not inhibit DNA synthesis under normal conditions and that SPHK2-L accumulation in the nucleus induced by serum deprivation may be involved in the cessation of cell proliferation or apoptosis depending on the cell type. The lipid second messenger sphingosine 1-phosphate (S1P) 2The abbreviations used are:S1Psphingosine 1-phosphateSPHKsphingosine kinasehSPHK2human SPHK2SPHK2-LN-terminal-extended form of SPHK2SPHK2-Soriginally reported form of SPHK2HAinfluenza hemagglutininFCSfetal calf serumBrdUrdbromodeoxyuridinesiRNAsmall interfering RNAHEKhuman embryonic kidneyGAPDHglyceraldehyde 3-phosphate dehydrogenase 2The abbreviations used are:S1Psphingosine 1-phosphateSPHKsphingosine kinasehSPHK2human SPHK2SPHK2-LN-terminal-extended form of SPHK2SPHK2-Soriginally reported form of SPHK2HAinfluenza hemagglutininFCSfetal calf serumBrdUrdbromodeoxyuridinesiRNAsmall interfering RNAHEKhuman embryonic kidneyGAPDHglyceraldehyde 3-phosphate dehydrogenase has been implicated in the regulation of a variety of important mammalian cell processes, including proliferation, differentiation, and apoptosis (1Le Stunff H. Peterson C. Liu H. Milstien S. Spiegel S. Biochim. Biophys. Acta. 2002; 1582: 8-17Crossref PubMed Scopus (93) Google Scholar, 2Pyne S. Pyne N.J. Biochem. J. 2000; 349: 385-402Crossref PubMed Scopus (659) Google Scholar, 3Igarashi Y. J. Biochem. (Tokyo). 1997; 122: 1080-1087Crossref PubMed Scopus (131) Google Scholar). Interest in S1P has focused recently on two distinct cellular actions of this lipid, namely the function of S1P as an extracellular ligand activating specific G protein-coupled receptors and the role of S1P as an intracellular second messenger (4Meyer zu Heringdorf D. van Koppen C.J. Jakobs K.H. FEBS Lett. 1997; 410: 34-38Crossref PubMed Scopus (120) Google Scholar). Noticeably, some of the diverse signaling roles attributed to elevated cellular S1P levels include prevention of ceramide-induced apoptosis (5Xia P. Wang L. Gamble J.R. Vadas M.A. J. Biol. Chem. 1999; 274: 34499-34505Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 6Cuvillier O. Pirianov G. Kleuser B. Vanek P.G. Coso O.A. Gutkind S. Spiegel S. Nature. 1996; 381: 800-803Crossref PubMed Scopus (1337) Google Scholar) and calcium mobilization (7Mattie M. Brooker G. Spiegel S. J. Biol. Chem. 1994; 269: 3181-3188Abstract Full Text PDF PubMed Google Scholar).Sphingosine kinase (SPHK), the enzyme that catalyzes the phosphorylation of sphingosine, plays a central role in the regulation of intracellular levels of S1P. Two isoforms of mammalian SPHK (SPHK1 and SPHK2) have been cloned and characterized (8Kohama T. Olivera A. Edsall L. Nagiec M.M. Dickson R. Spiegel S. J. Biol. Chem. 1998; 273: 23722-23728Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar, 9Liu 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 (558) Google Scholar). SPHK1 is a cytosolic enzyme with an apparent molecular mass of 49 kDa and contains five conserved domains, the second of which has a conserved ATP binding motif found in diacylglycerol kinases (8Kohama T. Olivera A. Edsall L. Nagiec M.M. Dickson R. Spiegel S. J. Biol. Chem. 1998; 273: 23722-23728Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar). Overexpression of SPHK1 induces cell proliferation by promoting the G1 to S transition of the cell cycle as well as by inhibiting the apoptotic response to serum deprivation or ceramide treatment (10Olivera A. Kohama T. Edsall L. Nava V. Cuvillier O. Poulton S. Spiegel S. J. Cell Biol. 1999; 147: 545-557Crossref PubMed Scopus (460) Google Scholar). SPHK2 contains the same five conserved domains found in SPHK1 while also having divergent sequences at the N-terminal and in the middle regions, resulting in a protein 200 amino acids larger than SPHK1. In addition, heterogeneity in the N terminus was found in SPHK2 (11Billich A. Bornancin F. Devay P. Mechtcheriakova D. Urtz N. Baumruker T. J. Biol. Chem. 2003; 278: 47408-47415Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar), whose mechanism of generation remains unknown. The role of SPHK2, however, has not been elucidated until recently. Studies from our laboratory have demonstrated that SPHK2 is a nuclear protein and inhibits DNA synthesis when over-expressed in mammalian cells (12Igarashi 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 (346) Google Scholar). Similarly, Liu et al. (13Liu 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 (305) Google Scholar) have reported that SPHK2 induces apoptosis through its putative BH3 domain. More recently, SPHK2 has been postulated to function as a potential immunomodulator either through phosphorylation of an immunosuppressant drug, FTY720 (11Billich A. Bornancin F. Devay P. Mechtcheriakova D. Urtz N. Baumruker T. J. Biol. Chem. 2003; 278: 47408-47415Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar, 14Paugh S.W. Payne S.G. Barbour S.E. Milstien S. Spiegel S. FEBS Lett. 2003; 554: 189-193Crossref PubMed Scopus (267) Google Scholar), or association with inter-leukin-12 receptor in T cells (15Yoshimoto T. Furuhata M. Kamiya S. Hisada M. Miyaji H. Magami Y. Yamamoto K. Fujiwara H. Mizuguchi J. J. Immunol. 2003; 171: 1352-1359Crossref PubMed Scopus (60) Google Scholar). However, the physiological role of SPHK2 remains largely unknown.The present studies were undertaken to determine the physiological role of N-terminal-extended SPHK2 (SPHK2-L) in comparison with the originally reported form (SPHK2-S). Here we have shown that SPHK2-L is the predominant form, at least in human cell lines and tissues tested. We have also presented data that SPHK2-L is involved in the regulation of cell proliferation and apoptosis in concert with serum.EXPERIMENTAL PROCEDUREScDNA Cloning and Mammalian Expression Vectors—The human SPHK2-L cDNA (DDBJ/EMBL/GenBank™ accession number NM_020126) was amplified from a human liver cDNA library by PCR using KOD-PLUS polymerase (Toyobo, Tokyo) with 5′-AC AGA TCT AAC AGA GCA GAG GAC CAG CAG-3′ and 5′-AC AGA TCT AAG CTT GTT TAG TTT CAG GGC-3′ sense and antisense primers, respectively. The PCR product was subcloned into pCMV5 to make an N-terminal influenza hemagglutinin (HA) epitope-tagged construct. Construction of human SPHK2-S expression vectors has been described previously (12Igarashi 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 (346) Google Scholar).Real-time Quantitative Reverse Transcription-PCR—Total RNA was isolated from HEK293 cells using the Nucleospin RNA II kit (Macherey-Nagel, Duven, Germany). Total RNAs from various human tissues were purchased from Ambion (Austin, TX). cDNA synthesis was performed with 1 μg of total RNA using MuLV reverse transcriptase (Applied Biosystems, Foster City, CA) priming with random hexamers. Quantitative PCR was performed by applying the real-time SYBR Green PCR technology with the use of an ABI PRISM 7000 sequence detection system (Applied Biosystems). The human SPHK2-specific primers were designed by using Primer Express software (Applied Biosystems), and their sequences were as follows: SPHK2-L mono-specific, 5′-ATG AAT GGA CAC CTT GAA GCA G-3′ and 5′-CAT GGC CTT AGC CCT GAC CAG-3′, located in exon 1 and exon 2, respectively (Fig. 2A, Pair 1); SPHK2-L and SPHK2-S duo-specific, 5′-CTG TCT GCT CCG AGG ACT GC-3′ and 5′-CAA AGG GAT TGA CCA ATA GAA GC-3′, located in exon 2 and exon 3, respectively (Fig. 2A, Pair 2); and for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 5′-GCC ATC AAT GAC CCC TTC ATT-3′ and 5′-TCT CGC TCC TGG AAG ATG G-3′. Amplification reaction was performed with SYBR Premix Ex Taq (Takara, Otsu, Japan). Thermal cycling conditions were as follows: 10 s at 95 °C, 40 cycles of 5 s at 95 °C, and 31 s at 60 °C. The expression of human SPHK2 (hSPHK2) mRNA was normalized to GAPDH mRNA expression.Immunoprecipitation and SPHK Assay—Preparation of an antibody against hSPHK2 has been described previously (12Igarashi 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 (346) Google Scholar). An antibody against the N terminus of SPHK2-L (anti-SPHK2-L) was generated by immunizing rabbits with an oligopeptide (MNGHLEAEEQQDQRPDQELTGSWGHGPRSTLVRAKA) containing the N-terminal 36 amino acids of SPHK2-L fused with glutathione S-transferase. The antibody was affinity purified by using the immunogen-immobilized Sepharose 4B.HEK293 cells were harvested and washed with phosphate-buffered saline. Crude supernatant fractions were prepared as described previously (16Fujita T. Okada T. Hayashi S. Jahangeer S. Miwa N. Nakamura S. Biochem. J. 2004; 382: 717-723Crossref PubMed Scopus (46) Google Scholar) except that detergent was excluded from the lysis buffer and cells were disrupted by sonication. SPHK2 was immunoprecipitated using anti-SPHK2 or anti-SPHK2-L antibody and assayed for SPHK activity as reported earlier (12Igarashi 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 (346) Google Scholar).Immunocytochemistry—Cells were grown on 4-chambered slides (Nalge/Nunc) and transfected with SPHK2-L using FuGENE 6 reagent according to the manufacturer's instructions (Roche Applied Science). Subcellular localization studies using confocal microscopy were performed as described previously (17Hayashi S. Okada T. Igarashi N. Fujita T. Jahangeer S. Nakamura S. J. Biol. Chem. 2002; 277: 33319-33324Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar).Small Interfering RNA (siRNA)—hSPHK2-targeted siRNA-1 (5′-UCAUCCAGACAGAACGACAGAACCA-AG-3′ and 5′-UGGUUCUGUCGUUCUGUCUGGAUGA-AU-3′), siRNA-2 (5′-GCUGGGCUGUCCUUCAACCU-3′ and 5′-AGGUUGAAGGACAGCCCAGC-3′), and control siRNA were synthesized (iGENE Therapeutics, Inc., Tsukuba, Japan). HEK293 cells were transfected with siRNA at a final concentration of 50 nm/slide chamber using Lipofectamine 2000 reagent (Invitrogen). 24 h after transfection, cells were cultured for another 72 h either with 10 or 0.1% FCS (serum deprived) and then fixed with 4% paraformaldehyde in phosphate-buffered saline. The cells were stained with Hoechst 33258 (20 μg/ml) and analyzed for morphological studies.Other Procedures—Bromodeoxyuridine (BrdUrd) incorporation was measured as described previously (12Igarashi 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 (346) Google Scholar).RESULTSmRNA for SPHK2-L Is the Predominant Form in Various Human Tissues and Cells—BLAST searches of the human genome sequence data base revealed the existence of SPHK2-L. The SPHK2-L gene is ∼10 kb long and consists of 6 exons separated by 5 introns (Fig. 1A). The deduced amino acid sequence of this cDNA contained 654 residues and a calculated molecular weight of 69,213. SPHK2-S, originally reported by Liu et al. (9Liu 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 (558) Google Scholar), lacks the exon 1, and the first initiation codon for translation was within the exon 2 (Fig. 1B). It is reasonable to assume that SPHK2-S is generated by means of alternative splicing from the same gene that encodes SPHK2-L. It should be noted that prior to the initiating codon in mouse exon 2 there is a stop codon (Fig. 1B, double underlined TGA), strongly suggesting that no SPHK2-L exists in mouse.FIGURE 1Structure of SPHK2. A, schematic representation of genomic organization of hSPHK2. Protein coding exons of hSPHK2 gene are given as boxes and introns as a thin horizontal line. Note that a large intron follows exon 1 in SPHK2-L and that exon 2 includes the first initiation codon for SPHK2-S, the previously reported SPHK2 (9Liu 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 (558) Google Scholar). B, cDNA and the deduced amino acid sequences for the N-terminal regions of hSPHK2-L and mouse SPHK2-S. The deduced amino acid sequence is indicated under the nucleotide sequence. The N-terminal-extended 36-amino acids result from genomic organization of exon 1 and exon 2 are shown in italic and bold letters. The N-terminal-initiating methionines for SPHK2-L and SPHK2-S are circled or boxed, respectively. The nucleotide (upper) and amino acid (lower) numbering is based on the hSPHK2-L sequence.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We quantified the amount of each splice variant in various human cell lines by real-time quantitative PCR using two pairs of primer sets (Fig. 2A). In primer set 1 the forward primer was located within exon 1 and the reverse primer on exon 2 so that the specific mRNA encoding SPHK2-L could be detected, whereas in primer set 2 the forward primer was located within exon 2 and the reverse primer on exon 3, which enabled us to recognize mRNA encoding both SPHK2-L and SPHK2-S. Assuming that the molar ratio of SPHK2-L to SPHK2-S mRNA is 1:1, the ratio of the amount of PCR products obtained with these primer pairs is anticipated to be 1:2. Surprisingly, in various human tissues and cell lines the relative amount of PCR product generated with primer Pair 1 is almost similar to that obtained with the primer Pair 2, indicating that SPHK2-L is the major form expressed in most human tissues and cell lines except brain and kidney (Fig. 2, B and C), where SPHK2-S might be expressed significantly. 3Using a human brain total RNA purchased from a different commercial source (BD Biosciences), the observed proportion between SPHK2-L and total SPHK2 mRNAs was similar to that obtained previously and shown in Fig. 2C, suggesting the possibility of expression of SPHK2-S in these tissues. Using a different pair of primers in which the forward primer was located within exon 5 and the reverse primer on exon 6 so that the mRNA encoding both the SPHK2-L and the SPHK2-S could be detected, almost the same results as with the Pair 2 were obtained (data not shown).Endogenous SPHK2-L Is the Predominant Form in Human Cell Lines—To detect endogenous SPHK2-L, an antibody against SPHK2-L was prepared. A rabbit polyclonal antibody raised against a glutathione S-transferase-fused oligopeptide containing the N-terminal-extended 36 amino acids of SPHK2-L specifically recognized SPHK2-L, but not SPHK2-S, on immunoblots (Fig. 3A). This antibody immunoprecipitated SPHK2 activity from HeLa cell lysates in a dose-dependent manner (Fig. 3B), confirming the existence of SPHK2-L in HeLa cells. Using this antibody, subcellular distribution of endogenous SPHK2-L was studied. SPHK2-L was mainly distributed in the nuclei and to a small extent diffusely in the cytosol of HeLa cells (Fig. 3C). This distribution pattern is similar to that of SPHK2-S and is quite reasonable because SPHK2-L contains the same nuclear localization signal as SPHK2-S (12Igarashi 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 (346) Google Scholar). Similarly, in human hepatoma HepG2 cells endogenous SPHK2-L was detected in the nuclei and to a similar extent in the cytosol in a punctate pattern, suggesting that nucleocytoplasmic shuttling is regulated depending on cell types. To demonstrate further that SPHK2-L is a major form at the protein level endogenous SPHK2 was immunoprecipitated and analyzed. Anti-SPHK2-L antibody immunoprecipitated the enzyme only from human hepatoma HepG2 cells but not from mouse NIH3T3 cells, although anti-SPHK2 antibody, which recognizes both SPHK2-L and SPHK2-S, immunoprecipitated it from both cell lysates (Fig. 4A). Together with results from the sequence analyses (Fig. 1B), these results indicate that SPHK2-L is a species-specific isoform that is present in human but not in mouse, as expected from the results in Fig. 1B.FIGURE 3Detection of endogenous SPHK2-L in various human cell lines. A, generation of anti-body that specifically recognizes SPHK2-L. Purified recombinant SPHK2-L and SPHK2-S were subjected to 12.5% SDS-PAGE followed by immunoblot analysis with either anti-SPHK2-L or anti-SPHK2 antibody. L, SPHK2-L; S, SPHK2-S. B, HeLa cell lysates (200 μg) were incubated with various amounts of anti-SPHK2-L antibody. After immunoprecipitation SPHK activity was measured. C, subcellular localization of SPHK2-L using immunocytochemistry. HeLa and HepG2 cells were fixed, permeabilized, and immunostained for confocal microscopic analyses using anti-SPHK2-L antibody (green). Nuclei were stained with 2 μg/ml 4,6-diamidino-2-phenylindole (DAPI) (blue). A differential interference contrast (DIC) image of each area is also presented. Bars, 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4SPHK2-L is a species-specific and major isoform in human hepatoma HepG2 cells. A, HepG2 and NIH3T3 cell lysates (200 μg each) were incubated with either anti-SPHK2 or anti-SPHK2-L antibody. After immunoprecipitation SPHK activity was measured. B, SPHK2 immunoprecipitated from HepG2 cell lysates with anti-SPHK2 anti-body was eluted by the immunogen peptide. The eluted SPHK2 was further incubated with various amounts of anti-SPHK2-L antibody. The immunoprecipitated SPHK2 and the resultant supernatants were assayed for SPHK activity. Data are means ± S.E. of three independent determinations.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Next, the immunoreactivity of the enzyme with anti-SPHK2 and anti-SPHK2-L antibodies was tested. First, the enzyme was immunoprecipitated from HepG2 cell lysates with anti-SPHK2 antibody and dissociated from the immune complex by incubating with the immunogen peptide. The eluted enzyme was then immunoprecipitated by anti-SPHK2-L antibody. The majority of SPHK activity was immunoprecipitated by the long form-specific antibody with a concomitant loss of the activity from the supernatant in an antibody concentration-dependent manner (Fig. 4B), suggesting that SPHK2-L is a major form in HepG2 cells. Similar results were obtained when HEK293 cell lysates were used as endogenous enzyme sources (data not shown).Inhibition of DNA Synthesis by SPHK2-L Is Overcome by Serum—We have previously shown that SPHK2 enters nucleus and inhibits DNA synthesis (12Igarashi 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 (346) Google Scholar). To determine whether this was true for the SPHK2 variant as well, DNA synthesis was measured in HEK293 cells expressing HA-SPHK2-L or HA-SPHK2-S. As shown in Fig. 5, BrdUrd incorporation was markedly suppressed in cells transiently expressing HA-SPHK2-S compared with green fluorescent protein-expressing control cells, which is consistent with our previous report (12Igarashi 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 (346) Google Scholar). In contrast, when HA-SPHK2-L was expressed the inhibition of BrdUrd uptake was minimal in 10% serum-containing medium. Even comparing the cells that expressed the recombinant proteins to a similar extent, SPHK2-L has little or no effect on BrdUrd uptake (not shown), indicating that SPHK2-L does not inhibit DNA synthesis significantly under normal culture conditions (in the presence of 10% serum). We have previously reported that inhibition of DNA synthesis in HeLa cells by SPHK2 is more easily observed when cells are cultured under low (0.1%) serum conditions (12Igarashi 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 (346) Google Scholar). To demonstrate this tendency with SPHK2-L, DNA synthesis was measured in HEK293 cells transiently expressing SPHK2-L under low or high serum conditions. Under low serum conditions, BrdUrd incorporation was strongly suppressed in cells expressing SPHK2-L when compared with control (green fluorescent protein expressing) cells (Fig. 5). The serum concentration-dependent inhibition of DNA synthesis by SPHK2-L was further demonstrated by cell cycle analysis showing that the population of G2/M cells expressing SPHK2-L was remarkably low in 0.1% but not in 10% serum-containing medium as compared with respective control cells expressing green fluorescent protein (see supplemental materials).FIGURE 5Inhibition of DNA synthesis by SPHK2-L is overcome by serum. HEK293 cells were transiently transfected with HA-SPHK2-L, HA-SPHK2-S, or green fluorescent protein plasmid (control) and cultured for 1 day in medium supplemented either with 0.1% (serum -) or 10% FCS (serum +). After adding BrdUrd, double immunofluorescence was used to visualize SPHK-transfected cells and BrdUrd incorporation, and the proportion of SPHK-expressing cells incorporating BrdUrd was determined. Data are means ± S.E. of three independent determinations.View Large Image Figure ViewerDownload Hi-res image Download (PPT)SPHK2-L Tends to Enter the Nucleus under Low Serum Conditions—The observation that serum (10%) overcomes the inhibition of DNA synthesis by SPHK2-L prompted us to study the regulation of nucleocytoplasmic shuttling of SPHK2-L by serum. HEK293 cells were transiently transfected with HA-SPHK2-L, and its subcellular distribution was analyzed under high or low serum conditions. Under high serum conditions (10% FCS), SPHK2-L tended to be mainly localized in the cytosol with a small amount in the nucleus (Fig. 6, A and C). Under the low serum conditions (0.1% FCS), the population of cells with predominant nuclear localization was increased (Fig. 6, B and C), indicating the regulation of nucleocytoplasmic shuttling of SPHK2-L by serum. This tendency of serum deprivation-induced accumulation of SPHK2-L in the nucleus was confirmed using endogenous enzyme in non-neoplastic human skin fibroblasts (see supplemental materials).FIGURE 6Serum-dependent regulation of nucleocytoplasmic shuttling of SPHK2-L. A and B, HEK293 cells were transiently transfected with HA-SPHK2-L and cultured for 1 day with either 10% (A, serum +) or 0.1% (B, serum -) FCS. Cells were fixed, permeabilized, and immunostained for confocal microscopic analyses using anti-SPHK2-L anti-body. C, from immunocytochemical data obtained in panels A and B, cells expressing SPHK2-L were subdivided into three populations depending on SPHK2-L staining pattern: cells with predominantly nuclear localization (N > C), cells with equal distribution between nucleus and cytosol (n = C), and cells with predominantly cytosolic localization (N < C). Five different fields were analyzed with a minimum of 100 cells. The data presented are representative of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Serum Deprivation Results in Increased SPHK2-L Expression in HEK293 Cells—During the course of studies on the regulation of nucleocytoplasmic shuttling of HA-SPHK2-L by serum in HEK293 cells, we noticed that the amount of endogenous SPHK2 tended to increase under conditions when cells were cultured in low serum medium. This observation prompted us to measure endogenous SPHK2 levels under different serum conditions. HEK293 cells were cultured for 2 days either in a low (0.1%) or high (10%) serum medium and assayed for SPHK2 activity or mRNA levels by real-time quantitative PCR using primer Pair 2 (Fig. 2A), which allows the detection of both SPHK2-S and SPHK2-L mRNAs. Low serum conditions resulted in 1.6- and 5.4-fold increases in SPHK2-specific activity and total SPHK2 mRNA levels, respectively (TABLE ONE). Similar results were obtained when real-time quantitative PCR was conducted using a primer Pair 1 that is specific to SPHK2-L, suggesting that serum deprivation induces mainly SPHK2-L expression but not SPHK2-S. During the 2-day culture of HEK293 cells in low serum medium, however, the cellular content of ceramide and S1P did not change significantly as compared with the cells cultured in high serum medium (see supplemental materials).TABLE ONESerum deprivation-induced SPHK2 expressionSPHK2 activityTotal SPHK2 mRNASPHK2-L mRNApmol/min/106 cells-FoldRelative expression-FoldRelative expression-Fold+ Serum1.33 ± 0.0611.0 ± 0.2211.0 ± 0.061− Serum2.15 ± 0.081.65.4 ± 0.375.45.5 ± 0.335.5 Open table in a new tab Suppression of SPHK2 Expression by siRNA Protects HEK293 Cells from Serum Deprivation- or Drug-induced Apoptosis—To assess the physiological role of SPHK2 we analyzed cellular responses induced by serum starvation in HEK293 cells where the level of endogenous SPHK2 was specifically suppressed by SPHK2-targeted siRNA. Compared with control siRNA, treatment with two different SPHK2-siRNAs suppressed the SPHK2 mRNA level in HEK293 cells (Fig. 7A). HEK293 cells underwent apoptosis after serum deprivation as seen in control siRNA-treated cells (Fig. 7, B and C). Importantly, serum deprivation-induced apoptosis was strongly inhibited by SPHK2-targeted siRNA treatment (Fig. 7, B an
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