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1 Cytoplasmic Region 1
* Intractable Disease Research Center, Tokyo Medical University, Tokyo, Japan;
Department of Immunology, Tokyo Medical University, Tokyo, Japan;
Department of Hematology and Oncology, Tokyo Medical and Dental University, Tokyo, Japan; and
Department of Oncology, Osaka University Graduate School of Medicine, Osaka, Japan
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1 cytoplasmic region. Analyses of various mutants of each molecule revealed that the region including the proline-rich domain in SPHK2 is probably responsible for the binding to IL-12R1, while the regions including the carboxyl terminus and Box II in the IL-12R1 cytoplasmic region appear to be involved in the binding to SPHK2. Transient expression of wild-type SPHK2 in T cell hybridoma augmented IL-12-induced STAT4-mediated transcriptional activation. Ectopic expression of dominant-negative SPHK2 in Th1 cell clone significantly reduced IL-12-induced IFN-
production, while that of wild-type SPHK2 enhanced it. In contrast, the expression minimally affected IL-12-induced proliferation. A similar decrease in IL-12-induced IFN- production was observed when dominant-negative SPHK2 was expressed in activated primary T cells using a retroviral expression system. These results suggest that SPHK2 associates with the IL-12R1 cytoplasmic region and probably plays a role in modulating IL-12 signaling. |
Introduction |
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production, and cytotoxic activity of NK and T cells and promoting the differentiation of naive CD4+ T cells into the Th1 subset of Th cells (1). IL-12 signals through IL-12R, which is composed of two noncovalently linked subunits, 1 and 2 (2, 3). Binding of IL-12 to the IL-12R leads to activation of members of the Janus kinase (JAK)3 family of tyrosine kinases, TYK2 and JAK2, which are associated with the cytoplasmic regions (cyt) of IL-12R1 and -2, respectively (4, 5). Activation of JAKs results in tyrosine phosphorylation of IL-12R2 and recruitment of STAT4 (6, 7). STAT4 is then phosphorylated, dimerized, and translocated to the nucleus to bind to IL-12-responsive genes. STAT4 is an important mediator of IL-12 signaling, because IL-12-induced IFN- production and T cell proliferation are impaired in STAT4-deficient mice (8, 9) as reported in IL-12- and IL-12R-deficient mice (10, 11, 12).
In addition to tyrosine phosphorylation, it has been recently demonstrated that STAT4 is phosphorylated on serine residue in response to IL-12 (13). The IL-12-dependent STAT4 serine phosphorylation is mediated by stimulation of p38 mitogen-activated protein kinase (MAPK) through its upstream activators, MAPK kinase (MKK) 3/6 and growth arrest and DNA damage inducible (GADD)45- and - (13, 14), but not of extracellular signal-regulated kinases 1/2 or c-Jun N-terminal kinase. It is required for full transcriptional activity of STAT4 and IFN- production, but not for proliferation. Moreover, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway has been demonstrated to be activated by IL-12 and play an important role in proliferation, but not in IFN- production (15). However, the molecular mechanisms underlying these pathways, especially how these pathways are molecularly linked to IL-12R1 and -2, remain to be elucidated.
Sphingosine kinase (SPHK) is a key enzyme catalyzing phosphorylation of sphingosine to form sphingosine 1-phosphate (S1P), an important lipid messenger that is implicated in the regulation of a wide variety of important cellular events, including cell growth, survival, motility, cytoskeletal changes, and the release of calcium from intracellular stores (16). S1P acts not only as an extracellular agonist, but also presumably as an intracellular messenger (16). SPHK is activated by a variety of stimuli, including platelet-derived growth factor, epidermal growth factor, nerve growth factor, muscarinic acetylcholine agonists, vitamin D3, cytokines such as TNF-
and IL-1, and cross-linking of Fc
RI and FcRI (reviewed in Refs. 17 and 18). Recently, S1P was identified as the ligand for a family of G protein-coupled receptors known as the endothelial differentiation gene-1 (EDG-1/S1PRs) family (17, 18, 19, 20). To date, two kinds of isotypes of SPHK, SPHK1 and SPHK2, have been cloned with a high degree of homology in amino acid composition and sequences (21, 22, 23, 24). SPHK1 is expressed mainly in cytosol and at much lower levels than SPHK2, which additionally has a proline-rich domain and several putative transmembrane domains, implying a different subcellular location (16, 21, 22, 23). In addition, these two isoenzymes have different tissue distributions, temporal developmental expressions, and kinetics properties, suggesting that they perform distinct cellular functions and regulate levels of S1P in a different manner.
In the present study we used a yeast-two hybrid screening system to identify molecules that associate with the IL-12R1 cytoplasmic region and potentially regulate IL-12 signaling. We have found that SPHK2 associates with the IL-12R1 cytoplasmic region and probably plays a role in modulating IL-12 signaling.
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Materials and Methods |
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Mouse rIL-12 was obtained from R&D Systems (Minneapolis, MN). Human rIL-2 was provided by Shionogi (Osaka, Japan). The following Abs were purchased: anti-STAT4 Ab was from Santa Cruz Biotechnology (Santa Cruz, CA), anti-phosphotyrosine mAb (4G10) and anti-GST Ab were from Upstate Biotechnology (Lake Placid, NY), anti-FLAG mAb (M2) and anti-actin Ab were from Sigma-Aldrich (St. Louis, MO), and anti-hemagglutinin (anti-HA) mAb (3F10) was from Roche (Indianapolis, IN).
Cell culture
HEK293T cells and PLAT-E, a packaging cell line provided by Dr. T. Kitamura (University of Tokyo, Tokyo, Japan) (25), were maintained in DMEM supplemented with 10% FBS. The IL-12-responsive mouse Th1 cell clone, 2D6 (26), was maintained in RPMI 1640 medium supplemented with 10% FBS, 50 µM 2-ME, and mouse rIL-12 (250 pg/ml). Mouse T cell hybridoma 68-41 (provided by Dr. M. Kubo, Tokyo University of Science, Chiba, Japan) (27) and primary T cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 50 µM 2-ME.
Yeast two-hybrid screening
Two-hybrid screening was performed using the Matchmaker III system (Clontech, Palo Alto, CA) according to the manufacturer’s instructions. The cytoplasmic region of mouse IL-12R1 cDNA (American Type Culture Collection, Manassas, VA) was cloned into pGBKT7 in-frame with the GAL4 DNA-binding domain. The bait was transformed into yeast strain AH109 together with a mouse C57 Black Kaplan T cell lymphoma cDNA library (Clontech) fused to the GAL4 activation domain in the pACT. DNA from positive clones was prepared from yeast and transformed into competent KC8 Escherichia coli. Sequence analysis of positive clones revealed that one of these clones encoded a partial sequence of mouse SPHK2. Then a cDNA encompassing the entire coding region of mouse SPHK2 was obtained by RT-PCR using mRNA prepared from Con A-activated spleen cells and subcloned into a CMV promoter-driven mammalian expression vector, p3xFLAG-CMV-7.1 (Sigma-Aldrich), with a FLAG-epitope tag to express N-terminally 3xFLAG-tagged fusion proteins (p3xFLAG-wild-type (wt) SPHK2).
Transfection
Mouse IL-12R1 cDNA was tagged with HA epitope at its C terminus and ligated into an SR promoter-driven expression vector, pME18S (pME18S-IL-12R1-HA). The cytoplasmic region of IL-12R1 cDNA was subcloned into an SR promoter-driven mammalian GST fusion protein expression vector, pMEG (provided by Drs. T. Ishida and T. Watanabe, University of Tokyo, Tokyo, Japan; pMEG-IL-12R1cyt). Plasmids encoding various mutants or deletions of SPHK2 or IL-12R1 cytoplasmic region were generated by PCR-based mutagenesis. These plasmids were transiently cotransfected into HEK293T cells using Fugene 6 (Roche), and resultant cells were harvested after 36–48 h. Dominant-negative (dn) SPHK2, which contains amino acid substitution of the glycine residue at amino acid position 212 to aspartate, was generated by PCR-based mutagenesis and subcloned into an expression vector, p3xFLAG-CMV-10 (Sigma-Aldrich), containing a neomycin-resistant gene. 2D6 cells were transfected with p3xFLAG-dnSPHK2, p3xFLAG-wtSPHK2, or the empty vector by electroporation using Gene Pulser II (Bio-Rad, Hercules, CA) and were selected with Geneticin (G418).
Immunoprecipitation, GST pull-down assay, and Western blotting
Cells were lysed in 1% Nonidet P-40 lysis buffer (10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 mM sodium orthovanadate, and 1 mM PMSF), followed by centrifugation. Cell lysate was incubated with Ab conjugated to protein G-Sepharose (Amersham Pharmacia Biotech, Little Chalfont, U.K.) or glutathione-Sepharose (Amersham Pharmacia Biotech) for 2 h at 4°C. After washing the beads, the complexes were separated on an SDS-PAGE under reducing conditions and transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA). The membrane was then blocked, probed with first Ab and then with appropriate secondary Ab conjugated to HRP, and visualized with the ECL detection system (Amersham Pharmacia Biotech) according to the manufacturer’s instructions.
Reporter gene assay
T cell hybridoma 68-41 was transiently transfected by electroporation with a STAT4 reporter construct (p3xGAS-Luc, firefly luciferase; provided by Dr. R. Pine, The Public Health Research Institute, New York, NY, and Dr. J. J. O’Shea, National Institutes of Health, Bethesda, MD) (13, 28), p3xFLAG-wtSPHK2, pME18S-IL-12R1, pME18S-IL-12R2, and pRL-TK (sea pansy (Renilla reniformis) luciferase expression plasmid under control of thymidine kinase promoter) as an internal control. The total amount of DNA was adjusted with empty vector. After 
40 h half the cells were harvested and used for Western blotting with anti-FLAG, and the rest were stimulated with 5 ng/ml rIL-12 for 6 h and then harvested for measurement of firefly and sea pansy luciferase activities using a Dual-Luciferase Reporter Assay System (Promega, Madison, WI) following the manufacturer’s instructions. Firefly luciferase activity in each sample was normalized by the corresponding sea pansy luciferase activity to correct the transfection efficiency. Data are shown as normalized firefly luciferase activity.
IFN- production and proliferation assays
For IFN- production assay the Th1 cell clone 2D6 was starved of IL-12 overnight, and resultant cells (2 x 105 cells/200 µl/well) were stimulated with rIL-12. After 24 h, culture supernatants were harvested, and IFN- concentrations were measured by ELISA using anti-IFN- mAb (XMG1.2; BD PharMingen, San Diego, CA) and biotinylated anti-IFN- mAb (R4-6A2; BD PharMingen). For proliferation assay, starved 2D6 cells (2 x 104 cells/200 µl/well) were stimulated with rIL-12 for 48 h and pulsed with [3H]thymidine for last 4 h.
Retroviral infection
The dn SPHK2 cDNA was subcloned into a bicistronic retroviral vector pMX-internal ribosome entry site (IRES)/enhanced green fluorescence protein (EGFP) (29) provided by Dr. T. Kitamura. The PLAT-E cell line was transfected with pMX-dnSPHK2/IRES/EGFP or the empty vector using Fugene 6 and was cultured to generate the retroviral supernatant. Primary T cells were purified by passing C57BL/6 mouse spleen cells depleted of erythrocytes through nylon wool and Sephadex G-10 columns. These purified T cells (2 x 106 cells/ml) were activated with plate-coated 2 µg/ml anti-CD3 (145-2C11; American Type Culture Collection) and 2 µg/ml anti-CD28 (Pv-1; American Type Culture Collection) for 2 days and then infected with the viral supernatant in the presence of 10 µg/ml polybrene by centrifugation at 1800 rpm for 90 min at 30°C. After incubation with 50 U/ml human IL-2 for 2 days, GFP-positive cells were purified by sorting using a FACSCaliber (BD Biosciences, Mountain View, CA). For IFN- production assay, GFP-positive cells (2 x 105/200 µl/well) were stimulated with rIL-12, and culture supernatants were harvested after 24 h and assayed for IFN- concentrations by ELISA. For proliferation assay, GFP-positive cells (4 x 104/200 µl/well) were stimulated with rIL-12 for 48 h and pulsed with [3H]thymidine for last 24 h.
Statistical analysis
Statistical analysis was performed using Student’s t test. A value of p < 0.05 was statistically significant.
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1 in vivo
We performed a yeast two-hybrid screening of a mouse C57BL Kaplan T cell lymphoma cDNA library using the cytoplasmic region of mouse IL-12R1 cDNA as bait and found that one of the positive clones contained a partial sequence of mouse SPHK2 corresponding to amino acid positions 357 to the last 617. Then a cDNA encompassing the entire coding region of mouse SPHK2 was obtained by RT-PCR using mRNA prepared from Con A-activated spleen cells.
To examine the in vivo association between SPHK2 and IL-12R1, HEK293T cells were cotransfected with expression vectors of FLAG-tagged (FLAG-) SPHK2 and HA-tagged IL-12R1 (IL-12R1-HA), and cell lysates were immunoprecipitated with anti-FLAG, anti-HA, or control Abs. Coprecipitated FLAG-SPHK2 or IL-12R1-HA were detected by immunoblotting with anti-FLAG or anti-HA, respectively (Fig. 1). Two bands corresponding to 100 and 120 kDa of IL-12R1-HA, presumably due to glycosylation variants (30), were observed by anti-HA in the total lysate of cells transfected with the expression vector of IL-12R1-HA. Similar bands, but with much higher intensity in the lower band, were detected in immunoprecipitates with anti-FLAG, but not with control Ab, of lysates of cells cotransfected with expression vectors of FLAG-SPHK2 and IL-12R1-HA. These bands were not observed in immunoprecipitates of lysates of cells cotransfected with the expression vector of IL-12R1-HA and the empty vector. On the other hand, an 80 kDa band of FLAG-SPHK2 was observed by anti-FLAG in the total lysates of cells transfected with expression vector of FLAG-SPHK2. A similar band was detected in immunoprecipitates with anti-HA, but not with control Ab, of lysates of cells cotransfected with the expression vectors of FLAG-SPHK2 and IL-12R1-HA, but not with expression vector of FLAG-SPHK2 and the empty vector. These results suggest the association between SPHK2 and IL-12R1 in vivo.
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1 cytoplasmic region
To determine the binding domain in SPHK2 to IL-12R1 cytoplasmic region, we conducted in vivo binding experiments using GST pull-down assays. HEK293T cells were cotransfected with expression vectors of FLAG-SPHK2 or its various mutants (Fig. 2A) and GST-tagged (GST-) IL-12R1cyt, and cell lysates were precipitated with glutathione-Sepharose, followed by immunoblotting with anti-FLAG (Fig. 2B). Consistent with the finding that the positive clone obtained in the yeast two-hybrid screening contained the region at amino acid positions 357–617, the last half region of SPHK2 (Mut 291–617), which is including the proline-rich domain, was able to bind specifically to GST-IL-12R1cyt, but not to GST alone. On the other hand, the first half region of SPHK2 (Mut 1–292), which is including the catalytic domain, bound significantly to GST-IL-12R1cyt, but it bound almost equally to GST alone as well, indicating that it is a nonspecific binding to IL-12R1cyt due to the increased binding to GST alone. The SPHK2 mutant (Mut 1–492) that lacked its carboxyl terminus at amino acid positions 493–617 was still able to bind specifically to GST-IL-12R1cyt. In contrast, SPHK2 mutants (Mut 
345–492 and Mut 293–492), that lacked the region including the proline-rich domain, bound much less specifically to GST-IL-12R1cyt. Thus, these results suggest that the domain including the proline-rich domain in SPHK2 is presumably responsible for binding to the IL-12R1 cytoplasmic region.
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1 cytoplasmic region to SPHK2
To determine the binding domain in the IL-12R1 cytoplasmic region to SPHK2, HEK293T cells were cotransfected with expression vectors of FLAG-SPHK2 and GST-IL-12R1cyt or its various deletion mutants (Fig. 3A), and cell lysates were precipitated with glutathione-Sepharose, followed by immunoblotting with anti-FLAG (Fig. 3B). Deletion of the carboxyl terminus in the IL-12R1 cytoplasmic region at amino acid positions 702–738 reduced greatly the binding to SPHK2. Deletion of the domain at amino acid positions 665–701 slightly enhanced the binding, while that at amino acid positions 629–664, including Box II, almost completely abolished it. These results suggest that the domains including the carboxyl terminus and Box II in the IL-12R1 cytoplasmic region are putatively responsible for the binding to SPHK2.
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The most important signal emanating from IL-12R is activation of the transcriptional factor STAT4. To examine the possible role of SPHK2 in IL-12-induced STAT4-mediated transcriptional activation, transient transfection and reporter gene assay were performed using the T cell hybridoma 68-41. A STAT4-responsive reporter construct was cotransfected with expression vectors of FLAG-wtSPHK2 and IL-12R1 and -2 into the T cell hybridoma. Resultant cells were stimulated with rIL-12 for 6 h, and their luciferase activities were measured. The expression of FLAG-SPHK2, which was detected by immunoprecipitation and immunoblotting with anti-FLAG, was enhanced dose-dependently with an increase in the amount of p3xFLAG-wtSPHK2 plasmid added (Fig. 4A). Concomitant with the enhanced FLAG-wtSPHK2 expression, IL-12-induced STAT4-mediated transcriptional activation was augmented more than twice (Fig. 4B). These results suggest that SPHK2 is involved in the IL-12-induced STAT4-mediated transcriptional activation.
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production by ectopic expression of dnSPHK2 and enhancement of it by ectopic expression of wtSPHK2 in Th1 cell clone
To further investigate the possible role of SPHK2 in IL-12 signaling, we next examined the effect of one of competitive inhibitors of SPHK, N,N-dimethylsphingosine (DMS) (24), on IL-12-induced IFN- production using the IL-12-responsive Th1 cell clone 2D6. 2D6 cells were stimulated with rIL-12 (1 ng/ml) in the presence of various concentrations of DMS, and after 24 h, supernatants were harvested for measurement of IFN- by ELISA. IL-12-induced IFN- production was inhibited in a dose-dependent manner (data not shown). Approximately 50% inhibition of the IFN- production was observed when 2D6 cells were stimulated by IL-12 in the presence of 10 µM DMS.
Although DMS has been used extensively in studies examining the role of S1P and SPHK, various secondary effects on the cell have been reported as well (31, 32, 33, 34, 35). To overcome the problems associated with the use of SPHK inhibitors and to increase the specificity to SPHK, we next prepared a dominant-negative form of SPHK2 by replacing the glycine residue at amino acid position 212 to aspartate in the putative ATP binding site of the diacylglycerol kinase catalytic domain as reported for SPHK1 (36, 37). Then, the Th1 cell clone 2D6 was stably transfected with expression vectors of FLAG-dnSPHK2, FLAG-wtSPHK2, or empty vector, and resultant transfectants were compared for their abilities to induce IFN- production and proliferation in response to rIL-12. Comparable expression of FLAG-dnSPHK2 or FLAG-wtSPHK2 was observed among the three transfectants (Fig. 5A). Consistent with the results obtained using the inhibitor DMS (data not shown), IL-12-induced IFN- production was suppressed by the ectopic expression of FLAG-dnSPHK2 to 50% that of vector alone and was enhanced by the ectopic expression of FLAG-wtSPHK2 twice as much (Fig. 5B). In contrast, the ectopic expression of FLAG-dnSPHK2 or FLAG-wtSPHK2 minimally affected IL-12-induced proliferation (Fig. 5C). These results suggest that SPHK2 is likely to be more critically involved in IL-12-induced IFN- production than proliferation.
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production by retroviral expression of dnSPHK2 in activated primary T cells
To obtain more physiological evidence of the role of SPHK2 in IL-12 signaling, we prepared a retroviral expression construct of dnSPHK2 and infected activated spleen T cells with the retrovirus. Viral infection was achieved in 20–40% of primary T cells, and infected T cells were purified by sorting for expression of GFP to analyze the responsiveness to rIL-12 (Fig. 6A). Activated primary T cells expressing dnSPHK2/EGFP produced significantly less IFN- than those expressing EGFP alone in response to rIL-12 (Fig. 6B). On the other hand, IL-12-induced proliferation was not significantly reduced by retroviral expression of dnSPHK2/EGFP compared with that of EGFP alone (Fig. 6B). These results suggest that SPHK2 plays a role in modulating IL-12 signaling in primary T cells.
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1 cytoplasmic region (Fig. 2). These results may suggest a lower possibility that SPHK1 also binds to the IL-12R1 cytoplasmic region. On the other hand, two distinct domains in the IL-12R1 cytoplasmic region, one of which was the carboxyl terminus and the other was the domain including Box II, appear to be involved in the binding to SPHK2 (Fig. 3, A and B). The cytoplasmic region of mouse IL-12R1 is much longer than that of human IL-12R1, and there appears to be no region in human IL-12R1 homologous to the carboxyl terminus of the mouse IL-12R1 cytoplasmic region (30), which was assigned to one of the binding domains to SPHK2 (Fig. 3C). Although the other binding domain including Box II is conserved between mouse and human (Fig. 3C) (30), whether human SPHK2 can bind to the human IL-12R1 cytoplasmic region remains to be examined. Alternatively, it was previously reported that DCs possess IL-12R with the 1 subunit, whose amino acid sequence downstream from Box I differs from that on T cells, and that IL-12 signaling through this receptor involves members of the NF-
B, but not the STAT, family (38). Several classes of IL-12R1 cDNA clones were also found and were confirmed to result from alternative splicings by comparison with the genomic sequence (39). These results might indicate the presence of another human IL-12R1 isoform highly homologous to mouse IL-12R1, especially in the carboxyl-terminal region.
Treatment of a Th1 cell clone with an inhibitor of SPHK, DMS (24), suppressed IL-12-induced IFN- production (data not shown). Although inhibitors of SPHK such as DMS have been used extensively in studies examining the roles of S1P and SPHK, they appear to have secondary effects on cells, including inhibition of protein kinase C (31, 32) and activation of sphingosine-dependent protein kinases (33), 3-phosphoinositide-depedent kinase 1 (34), and casein kinase II (35). To overcome these secondary effects and improve the specificity, a catalytically inactive, dominant-negative SPHK2 was prepared by replacing the glycine residue at amino acid position 212 with aspartate in the putative ATP binding site of the diacylglycerol kinase catalytic domain as reported for SPHK1 (36, 37). Consistent with the results obtained using the SPHK inhibitor DMS (data not shown), ectopic expression of dnSPHK2 significantly, but partially, reduced IL-12-induced IFN- production (Figs. 5B and 6B), and ectopic expression of wtSPHK2 enhanced IL-12-induced STAT4-mediated transcriptional activation and IFN- production (Figs. 4 and 5B). However, preliminary data suggest that no apparent difference in the tyrosine phosphorylation level of STAT4 after stimulation with rIL-12 was observed among 2D6 cells expressing dnSPHK2 or wtSPHK2, and the empty vector-transfected 2D6 cells as a control (data not shown). In contrast, the ectopic expression of dnSPHK2 minimally suppressed IL-12-induced proliferation in Th1 cell clone 2D6 and did not constantly inhibit the proliferation in activated primary T cells (Figs. 5C and 6C). Although we were unable to obtain evidence that supports a critical role for SPHK2 in IL-12-induced proliferation, overexpression of SPHK1 or stimulation with S1P in fibroblasts has been demonstrated to enhance the proliferative response (40, 41). Further studies are required to clarify whether SPHK2 is also involved in the IL-12 signaling leading to proliferation in addition to IFN- production.
The defective response to IL-12 observed in lymphocytes from STAT4 knockout mice indicates that STAT4 is essential for T cell proliferation and IFN- production mediated by IL-12 (8, 9). A previous report on a patient whose T cells were unable to produce IFN- or proliferate with impaired activation of STAT1, -3, and -5, but intact activation of STAT4 in response to IL-12, suggested that the activation of STAT4 alone is not sufficient for IL-12-induced IFN- production and proliferation, and that other signaling molecules, such as STAT1, -3, and -5, could play a role in these responses to IL-12 (42). Treatment of activated T cells with one of the protein tyrosine kinase inhibitors, tyrphostin B42 (AG490), which inhibited IL-12-induced tyrosine phosphorylation and activation of JAK2, but not those of TYK2, led to a decrease in IL-12-induced tyrosine phosphorylation of STAT3, but not that of STAT4, and in IFN- production and to programmed cell death as well (43). While treatment of activated T cells with another protein tyrosine kinase inhibitor, tyrphostin A1, which inhibited IL-12-induced tyrosine phosphorylation and activation of TYK2, but not those of JAK2, led to a decrease in IL-12-induced tyrosine phosphorylation of STAT3, but not that of STAT4, and in IFN- production without affecting IL-12-induced proliferation (43). Moreover, TYK2 knockout mice showed decreased tyrosine phosphorylation of STAT4 and STAT3 and IFN- production in response to IL-12 without an affect on proliferation (44, 45). Recently, it has been demonstrated that JAK2 activation is required for IL-12-induced T cell proliferation potentially through STAT5 and c-Myc-associated mitogenic signaling and that the TYK2/STAT4/STAT3 pathway is critical for IFN- production (46). Furthermore, it has been demonstrated that STAT4 is also phosphorylated by IL-12 on serine residue and is mediated by stimulation of p38 MAPK through its upstream activators MKK3/6 and GADD45- and - (13, 14). It is required for full transcriptional activity of STAT4 and IFN- production, but not for proliferation (13, 14). PI3K/Akt pathway has been also demonstrated to be activated by IL-12 and important for proliferation, but not for IFN- production (15). Collectively, these results suggest that the JAK2 pathway potentially involving STAT5 and c-Myc and the PI3K/Akt pathway are required for IL-12-induced T cell proliferation, while TYK2/STAT4/STAT3 pathway and serine phosphorylation of STAT4 by MKK6/p38 MAPK are critical for the IFN- production, although some minor discrepancies among each report may exist, probably due to the different experimental conditions. On the other hand, S1P was previously demonstrated to increase p38 MAPK activity in endothelial cells, which is important for S1P-induced chemotaxis (47). S1P was also shown to activate Akt via PI3K in hepatocytes, which protects cells from apoptosis (48), and in vascular endothelial cells (49). Considering that both SPHK2 and TYK2 associate with IL-12R1 cytoplasmic region and that SPHK2 appears to be involved in IL-12-induced IFN- production without apparently affecting the tyrosine phosphorylation of STAT4, it is tempting to speculate that SPHK2 might modulate the TYK2/STAT4/STAT3 pathway and/or serine phosphorylation of STAT4 by MKK6/p38 MAPK. Further studies are necessary to elucidate the molecular mechanism by which SPHK2 modulates the IL-12 signaling leading to IFN- production.
Consequently, the present study suggests that SPHK2 associates with the IL-12R1 cytoplasmic region and appears to be involved in the positive modulation of IL-12 signaling, especially IL-12-induced IFN- production.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests Dr. Takayuki Yoshimoto, Intractable Disease Research Center, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan. E-mail address: yoshimot{at}tokyo-med.ac.jp
3 Abbreviations used in this: JAK, Janus kinase; cyt, cytoplasmic region; GADD, growth arrest and DNA damage inducible; DMS, N,N-dimethylsphingosine; dn, dominant negative; IRES, internal ribosome entry site; EGFP, enhanced green fluorescence protein; FLAG-, FLAG-tagged; HA, hemagglutinin; MAPK, mitogen-activated protein kinase; MKK, MAPK kinase; PI3K, phosphatidylinositol 3-kinase; SPHK, sphingosine kinase; S1P, sphingosine 1-phosphate; TYK, tyrosine kinase; wt, wild type.
Received for publication March 15, 2003. Accepted for publication May 29, 2003.
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M. V. Girart, M. B. Fuertes, C. I. Domaica, L. E. Rossi, and N. W. Zwirner Engagement of TLR3, TLR7, and NKG2D Regulate IFN-{gamma} Secretion but Not NKG2D-Mediated Cytotoxicity by Human NK Cells Stimulated with Suboptimal Doses of IL-12 J. Immunol., September 15, 2007; 179(6): 3472 - 3479. [Abstract] [Full Text] [PDF]
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T. Owaki, M. Asakawa, F. Fukai, J. Mizuguchi, and T. Yoshimoto IL-27 Induces Th1 Differentiation via p38 MAPK/T-bet- and Intercellular Adhesion Molecule-1/LFA-1/ERK1/2-Dependent Pathways J. Immunol., December 1, 2006; 177(11): 7579 - 7587. [Abstract] [Full Text] [PDF]
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M. Shimizu, M. Shimamura, T. Owaki, M. Asakawa, K. Fujita, M. Kudo, Y. Iwakura, Y. Takeda, A. D. Luster, J. Mizuguchi, et al. Antiangiogenic and Antitumor Activities of IL-27. J. Immunol., June 15, 2006; 176(12): 7317 - 7324. [Abstract] [Full Text] [PDF]
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T. Owaki, M. Asakawa, S. Kamiya, K. Takeda, F. Fukai, J. Mizuguchi, and T. Yoshimoto IL-27 Suppresses CD28-Medicated IL-2 Production through Suppressor of Cytokine Signaling 3. J. Immunol., March 1, 2006; 176(5): 2773 - 2780. [Abstract] [Full Text] [PDF]
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A. Olivera, N. Urtz, K. Mizugishi, Y. Yamashita, A. M. Gilfillan, Y. Furumoto, H. Gu, R. L. Proia, T. Baumruker, and J. Rivera IgE-dependent Activation of Sphingosine Kinases 1 and 2 and Secretion of Sphingosine 1-Phosphate Requires Fyn Kinase and Contributes to Mast Cell Responses J. Biol. Chem., February 3, 2006; 281(5): 2515 - 2525. [Abstract] [Full Text] [PDF]
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