ARTICLE |
Correspondence to: Takashi Murate, Nagoya U. School of Health Science, Daiko-minami, 1-1-20, Higashi-ku, Nagoya 461-8673, Japan. E-mail: murate@met.nagoya-u.ac.jp
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Summary |
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Cell type-specific localization of sphingosine kinase 1a (SPHK1a) in tissues was analyzed with a rabbit polyclonal antibody against the 16 C-terminal amino acids derived from the recently reported mouse cDNA sequence of SPHK1a. This antibody (anti-SPHK1a antibody) can react specifically with SPHK1a of mouse, rat, and human tissues. Utilizing its crossreactivity to human SPHK1a, the cell-specific localization of SPHK1a in human tissues was histochemically examined. Strong positive staining for SPHK1a was observed in the white matter in the cerebrum and cerebellum, the red nucleus and cerebral peduncle in the midbrain, the uriniferous tubules in the kidney, the endothelial cells in vessels of various organs, and in megakaryocytes and platelets. The lining cells of sinusoids in the liver and splenic cords in the spleen showed moderate staining. Columnar epithelia in the intestine and Leydig's cells in the testis showed weak staining patterns. In addition, TPA-treated HEL cells, a human leukemia cell line, showed a megakaryocytic phenotype accompanied with increases in immunostaining of both SPHK1a and SPHK enzyme activity, suggesting that SPHK1a may be a novel marker of megakaryocytic differentiation and that this antibody is also useful for in vitro study of differentiation models.
(J Histochem Cytochem 49:845855, 2001)
Key Words: sphingosine-1-phosphate, sphingosine kinase, polyclonal antibody, C-terminal amino acids, immunohistochemistry, tissue distribution
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Introduction |
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THE SPHINGOLIPID METABOLITES have recently been a focus of intense study in various fields of cell biology, such as apoptosis, cell growth, and cell movement, and in some pathological states. Next to ceramide and sphingosine, sphingosine-1-phosphate (Sph-1-P), sphingosine phosphorylated by sphingosine kinase (SPHK), has been added to the list of bioactive sphingolipids (R I antigen receptor signaling (
A method for measuring SPHK enzyme activity was reported by
The tissue-specific distribution of SPHK has mainly been analyzed by measuring enzyme activity (
Genomic information on SPHK has recently become available. Mouse SPHK1, which was cloned as the first mammalian form of SPHK, has an apparent molecular weight of 49 kD and has two very similar types, 1a and 1b (
Based on the mouse cDNA sequence of SPHK1a (
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Materials and Methods |
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Antibody Preparation
The antibody against SPHK1a (anti-SPHK1a antibody) was generated in New Zealand White rabbits by injecting the synthetic oligopeptide corresponding to the 16 C-terminal residues (APSGRDSRRGPPPEEP) of the mouse SPHK1a sequence (
Cell Line and Culture Conditions
A human erythroleukemia cell line, HEL, was cultured in 10% FCS in RPMI 1640 medium and treated with 10-7 M of phorbol ester, TPA (phorbol 12-myristate 13 acetate), as described previously (
Immunoprecipitation and Western Blotting Analysis of Human Tissue
Outdated human platelets were supplied from Aichi Red Cross (Aichi, Japan). Human kidney tissue was obtained from a small part of total nephrectomized kidneys with renal tumors resected from patients who had given their informed consent. The sample collection was in accordance with the Helsinki Declaration of 1975. Human tissues or platelets were suspended in lysis buffer (50 mM Tris/HCl, 1% (v/v) Triton X-100, 1% sodium cholate, 0.1% sodium dodecyl sulfate (SDS), 10 mM EGTA, 150 mM NaCl, pH 8.8) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and 20 µg/ml leupeptin. The lysate was then sonicated and centrifuged at 100,000 x g for 60 min, and the resulting supernatant (500 µg of total protein) was incubated with 2 µg of anti-SPHK1a and 40 µl of a 1:1 slurry of protein ASepharose for 16 hr at 4C. The immunoprecipitates were collected by centrifugation at 5000 x g for 5 min, washed three times with lysis buffer, and suspended in SDS sample buffer.
The immunoprecipitated proteins were separated by SDS-PAGE on a 10% gel and transferred to an Immobilon (Millipore; Bedford, MA) membrane in solution containing 25 mM Tris base, 192 mM glycine, and 20% methanol. After blocking with 5% skim milk in TBS-T buffer (0.05% Tween-20, 137 mM NaCl, 20 mM Tris-HCl, pH 7.5), membranes were incubated with the anti-SPHK1a antibody (final concentration 500 ng/ml) at 4C for 15 hr and then washed with TBS-T, followed by incubation for 1 hr at room temperature with 125I labeled protein A. The blots were again washed with TBS-T and detected using a Hamamatsu DVS 3000 image analyzer system (Hamamatsu Photonics; Hamamatsu, Japan).
Western Blotting Analysis of Rat Tissue
Whole rat tissue lysate without immunoprecipitation treatment was directly subjected to Western blotting analysis. The blots were detected by HRP-conjugated anti-rabbit IgG (Amersham-Pharmacia; Poole, UK) and an ECL Western blotting system (Amersham-Pharmacia).
Preparation of Recombinant Murine SPHK1a cDNA
The recombinant murine SPHK1a (
Expression and Purification of Maltose Binding Protein (MBP)-tagged Recombinant SPHK1a
The cDNA amplified as described above was subcloned into the BamHI site and EcoRI sites of a bacterial expression vector pMALc2 (New England Biolabs; Beverly, MA). Maltose binding protein (MBP)SPHK1a was expressed in E. coli strain BL21 (DE3) by incubating with 0.3 mM isopropyl ß-thiogalactopyranoside (IPTG) for 5 hr. The cells were pelleted by centrifugation, washed, and homogenized in 50 mM Tris-HCl, pH 7.5, 2 mM MgCl2, 2 mM EGTA, 1% Triton X-100, 2 mM dithiothreitol, 1 mM PMSF, and 20 µg/ml aprotinin. Purification of MBP-SPHK1a was performed according to the manufacturer's recommended procedure.
Expression of FLAG-tagged Recombinant SPHK1a
To express the recombinant SPHK1a in mammalian cells, the mammalian expression plasmid pcDNA3 (Invitrogen; San Diego, CA) was ligated with the oligonucleotides 5'-AGCTTGCCACCATGGATTACAAGGATGACGACGAT- AAGG-3' and 5'-GATCCCTTATCGTCGTCATCCTTGTAATCCATGGTGGCA-3', yielding pcDNA3-FLAG1. Then, PCR-amplified SPHK1a was subcloned into the BamHI site and the EcoRI site of pcDNA3-FLAG1, yielding pcDNA3-FLAG-SPHK1a. A FLAG-tagged SPHK1a was then expressed in CHO cells or COS 7 cells with either stable or transient transfection by LipofectAMINE PLUS (Life Technologies; Gaithersburg, MD) according to the manufacturer's procedure.
Detection of Expressed Murine SPHK1a cDNA
Purified MBP protein from bacterial lysate or crude cellular extracts of FLAG transfectants were separated by SDS-PAGE, transferred to an Immobilon membrane, and then probed with anti-SPHK1a antibody or anti-FLAG M2 antibody (Sigma; St Louis, MO). The signals were detected on an ECL+Plus Western blotting detection system (Amersham-Pharmacia).
Enzyme Activity of SPHK
The enzyme activity of SPHK was measured according to the procedure of
Immunostaining
For the immunohistochemical identification of transfected FLAG-tagged SPHK1a, COS 7 cells were transfected transiently using LipofectAMINE PLUS as described above and grown on glass coverslips to 40% confluency. Coverslips were fixed in 3.7% formaldehyde in PBS at 37C for 30 min. Coverslips were rinsed in PBS before cells were permeabilized in PBS + 0.2% Triton X-100 for 5 min. After blocking for 10 min in 1% bovine serum albumin in PBS, coverslips were incubated with either 1 µg/ml of rabbit anti-mouse SPHK1a antibody or 25 µg/ml of mouse anti-FLAG M2 antibody (Sigma) in 1% bovine serum albumin in PBS. After washing coverslips three times with PBS, Alexa 488-conjugated anti-rabbit antibody (7 µg/ml) and Alexa 594-conjugated anti-mouse secondary antibody (7 µg/ml) (Molecular Probes; Eugene, OR) were used to visualize the antigens. Coverslips were washed in PBS three times and in deionized water twice. Then they were mounted with Mowiol 4-88 (Calbiochem; Darmstadt, Germany). For the fluorescent microscopy, a Carl Zeiss Axiophot 2 (Carl Zeiss; Mainz, Germany) equipped with a x40 objective lens was used. The images were recorded in grayscale by a CCD camera, MicoMAX-1300YHS (Princeton Instruments; Trenton, NJ) and MetaMorph software (Universal Imaging; West Chester, PA). Merged images were produced in pseudo-color (green and red) artificially with MetaMorph according to the manufacturer's instructions.
For the immunohistochemical examination of human tissues, paraffin blocks stored in the First Department of Pathology, Nagoya University School of Medicine, were used throughout the present study. After removal of paraffin, the section was dipped in 0.01 M EDTA in Tris buffer (pH 8.0) and was microwave-treated for 10 min. After washing three times with PBS, immunostaining was performed using the avidinbiotinperoxidase complex (ABC) method as described previously (
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Results |
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As the first step in the characterization of our polyclonal antibody against C-terminal amino acids (16 residues) of mouse SPHK1a, anti-SPHK1a antibody, homogenates of bacteria or cells transfected with tagged-mouse SPHK1a cDNA expression vectors were analyzed. Fig 1A shows the Western blotting analysis of purified MBP-tagged mouse SPHK1a protein expressed in E. coli BL21 (DE3) and the crude extract of CHO cells transfected with FLAG-tagged mouse SPHK1a cDNA. Bands consistent with the respective molecular weight (49 kD for FLAG-tagged SPHK1a and 83 kD for purified MBP-tagged SPHK1a) were clearly seen with anti-SPHK1a. The extract of mock transfectant (Fig 1A, Lane 4) did not produce any band with anti-SPHK 1a antibody. Coomassie Brilliant Blue staining of the same membrane (Fig 1B) identified this band of 83 kD as the purified MBP-tagged SPHK1a protein (Fig 1B, Lane 2). Fig 1C illustrates the staining pattern with anti-FLAG antibody. It is clear that Lane 3 only showed the band around 49 kD of SPHK1a protein.
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The specificity of our anti-SPHK1a antibody was examined for tissues of the rat liver, spleen, kidney, and brain. All rat tissues examined by anti-SPHK1a showed a single band at 4549 kD (Fig 1D). In contrast, human tissue showed a weaker band at around 45 kD (data not shown). Therefore, immunoprecipitation was performed before the Western blotting (Fig 1E). A reactive band was clearly visible at around 4449 kD with human samples. The band with a molecular weight higher than 49 kD was immunoglobulin, and no other bands were observed. When the sample was incubated with anti-SPHK1a antibody adsorbed with the antigen peptide, the band of 49 kD disappeared (Fig 1E, Lane 5).
Fig 2 provides additional proof that our antibody indeed recognized SPHK1a protein in the immunohistochemical analysis. The FLAG-tagged mouse SPHK1a expression vector was transiently expressed in COS 7 cells. The localization of each antigen (SPHK 1a protein and FLAG tag) was detected with anti-SPHK1a antibody and anti-FLAG antibody, respectively (Fig 2A and Fig 2B). As shown in the merged photos (Fig 2C), both anti-FLAG antibody and anti-SPHK1a antibody could visualize the co-localization of both FLAG tag and mouse SPHK1a proteins in the same cells and the same intracellular localization.
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Fig 3A shows the immunostaining of a human leukemia cell line, HEL cells, by anti-SPHK1a antibody. TPA-treated HEL cells (open arrow in the middle of Fig 3A) were stained more strongly in the cytoplasm than were non-treated control cells. This positive staining was diminished by absorbing the antibody with the same peptide that was used for immunization (open arrow on the lower part of Fig 3A). Fig 3B and Fig 3C show the simultaneous increase in SPHK enzyme activity in HEL cells treated with TPA compared with the activity in control cells.
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We analyzed cell-specific localization of SPHK1a in human tissue using this antibody. As shown in Fig 4, anti-SPHK1a stained almost all human tissues including the brain, kidney, lung, liver, spleen, intestine, testis, and bone marrow. In bone marrow, megakaryocytes and platelets were strongly positive (Fig 4A and Fig 4C). Neither myeloid nor erythroid cells in the bone marrow showed positive staining. Some megakaryocytes exhibited similar intense staining in the margin of the cytoplasm (data not shown), suggesting the release of platelets into the extracellular space or their preferential localization in the membrane area. This positive staining of megakaryocytes disappeared with the absorption of anti-SPHK1a antibody with the original 16 amino acids used for the immunization (arrow in Fig 4B).
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In the cerebral cortex, white matter was diffusely immunostained (Fig 4D). In the midbrain, only the red nucleus and cerebral peduncle were positive (Fig 4F). In the cerebellum, the white matter was positive, and the gray matter was negative (Fig 4H). Higher magnifications (Fig 4E, Fig 4G, and Fig 4I) show that the axon areas were strongly positive. In the kidney, uriniferous tubules were strongly positive, but Bowman's capsule was negative (Fig 4J). In the lung, endothelial cells of the vessels and submucosal bronchial glands were moderately positive, whereas alveoli were negative (Fig 4K). In the spleen, endothelial cells and splenic cords were positive but most lymphocytes in the follicle were negative (Fig 4L). In the liver, the lining cells of the sinusoids were positive, but hepatocytes were negative (Fig 4M). In the testis, endothelial cells and smooth muscle cells of the vessels were positive, and interstitial tissue, such as Leydig's cells, was weakly positive (Fig 4N). In the intestine, columnar epithelia were weakly positive (Fig 4O).
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Discussion |
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Our polyclonal antibody against C-terminal amino acids (16 residues) of mouse SPHK1a, anti-SPHK1a antibody, was assessed to be reactive for the recombinant mouse SPHK1a. Using a tagged mouse SPHK1a cDNA expression vector, we tried to detect the MBP-tagged mouse SPHK1a protein of transfected E. coli BL21 and FLAG-tagged mouse SPHK 1a protein of transfected CHO cells in Western blotting. Anti-SPHK1a antibody could detect respective bands with the consistent molecular weight. In Fig 1A, a band with an approximate molecular weight of 200 kD observed in Lane 1 of MBP-tagged SPHK1a may be an aggregation of proteins, but this remains to be determined. Anti-FLAG antibody could detect the band around 49 kD (Fig 1C) as well as a band with the molecular weight of 32.5 kD. The latter band appears to be nonspecific because it was observed in both FLAG-tagged SPHK1a transfectant and FLAG-tagged mock transfectant.
We analyzed rat tissue homogenates to assess the reactivity of anti-SPHK1a antibody because the full cDNA sequences of rat and mouse are very similar (Kohama T, Sankyo Pharmaceutical Co. Tokyo, Japan, personal communication). Under our experimental conditions, a single band with the molecular weight of 4549 kD was observed. However, it is reported that mouse SPHK1 has two isoforms, SPHK1a and 1b. The difference between them is only 7 amino acids located at its N-terminal (
Human tissue homogenates were used to prove the crossreactivity of our anti-SPHK1a antibody. Immunoprecipitation followed by Western blotting was effective in detecting the band at around 4449 kD with human samples. Prior incubation of anti-SPHK1a antibody with the antigen peptide erased the band at around 4449 kD, supporting the identity of this band as SPHK1a. From these results, it was concluded that anti-SPHK1a specifically reacted with SPHK1a in mouse, rat, and human tissues.
During preparation of this manuscript, the cDNA sequence of human SPHK1a was reported (
It is reported that TPA-treated HEL cells are induced to a megakaryocytic lineage (
SPHK activity is reportedly high in the testis, intestine, kidney (
The most remarkable finding in the present study is the localized distribution of SPHK1a in the central nervous system. The diffuse and dense distribution of SPHK1a in the axons (white matter of the cerebrum and cerebellum as well as the red nucleus and cerebral peduncle in the midbrain) is of particular interest. It suggests that SPHK1a is concentrated in axons but not in neurons overall and that the possible increase of Sph-1-P (the product of SPHK) in these axons might play roles either in signal transduction of the nervous system or in preventing apoptosis of the axon. It is also interesting that hepatocytes in the liver and lymphocytes in the spleen were not immunostained by anti-SPHK1a, although SPHK activity has been reported in their whole homogenates (
Very recently, several isoforms of SPHK have been reported (
The physiological significance of SPHK1a will be explained finally by future knockout mouse experiments. However, it is noteworthy that endothelial cells (and/or smooth muscle cells of the vessels) were immunostained with anti-SPHK1a in almost all human tissues examined. FCS or PDGF is reported to increase Sph-1-P by increasing SPHK activity (
Taken together, the findings of present study provide basic necessary information for understanding the roles of SPHK1a in cell responses. This approach is also promising as a potentially useful method to elucidate the pathogenesis of various diseases.
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Acknowledgments |
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We gratefully thank Drs C. Inoue and T. Kamiya (Aichi Red Cross Center) for providing the outdated platelets. We also thank Dr T. Kinoshita (First Department of Internal Medicine, Nagoya University School of Medicine), Dr M. Suzuki (Disease Mechanism and Control, Nagoya University School of Medicine), Mr T. Yamada, and Mr Y. Nakade (Nagoya University School of Health Science) for helpful discussion and technical advice.
Received for publication October 2, 2000; accepted February 14, 2001.
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