Sphingosine kinase-1 (SPHK1) is a key enzyme catalyzing the formation of an important bioactive lipid messenger, sphingosine 1-phosphate, and is implicated in the regulation of cell proliferation and antiapoptotic processes. Biological features of another isozyme SPHK2, however, remain unclear. The present studies were undertaken to characterize SPHK2 by comparison with SPHK1. When SPHK2 was transiently expressed in various cell lines, it was localized in the nuclei as well as in the cytosol, whereas SPHK1 was distributed in the cytosol but not in the nucleus. We have mapped a functional nuclear localization signal (NLS) to the N-terminal region of SPHK2. We have observed that the expression of SPHK2 in various cell types causes inhibition of DNA synthesis, resulting in the cell cycle arrest at G 1 /S phase. We have also demonstrated that an NLS mutant of SPHK2, SPHK2R93E/R94E, failed to enter the nucleus and to inhibit DNA synthesis. Moreover, a fusion protein, NLS-SPHK1, where SPHK1 was fused to the NLS sequence of SPHK2 acquired the ability to enter nuclei and inhibited DNA synthesis. These results indicate that SPHK2 localizes in the nuclei and causes inhibition of DNA synthesis, and this may affect subsequent cellular events.Sphingosine 1-phosphate (SPP) 1 is a bioactive lipid that regulates diverse biological processes such as calcium mobilization, cell growth, differentiation, survival, motility, and cytoskeletal reorganization, acting both inside and outside the cells (1, 2). Recently, SPP was identified as the ligand for a family of G protein-coupled receptors known as the endothelial differentiation gene-1 family, now collectively renamed SPP receptors (3-6), supporting a role for SPP as an extracellular ligand. However, the intracellular targets of SPP have not yet been identified.Sphingosine kinase (SPHK), the enzyme that catalyzes the phosphorylation of sphingosine, regulates the intracellular levels of SPP. Two isoforms of mammalian SPHK (SPHK1 and SPHK2) have been cloned and characterized (7,8). SPHK1 predominantly localizes in the cytosol, and its overexpression induces cell proliferation by promoting the G 1 to S transition of the cell cycle as well as by inhibiting the apoptotic response to serum deprivation or ceramide treatment (9). Several cellular proteins have recently been identified as SPHK1-interacting molecules, namely TRAF2 (10), RPK118 (11), and AKAP-related protein (12), which should help facilitate the understanding of the regulation and intracellular site of action of SPHK1.In contrast to SPHK1, little is known about the cellular actions of the other isozyme, SPHK2. In the present studies, we investigated the biological features of SPHK2. We have discovered that SPHK2 localizes in the nuclei of cells through its novel nuclear localization signal (NLS) sequence, depending on cell type and cell density. We have also demonstrated that nuclear localization of SPHK2 causes inhibition of DNA synthesis in various cell types.

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. The lipid second messenger sphingosine 1-phosphate (S1P) 2 has been implicated in the regulation of a variety of important mammalian cell processes, including proliferation, differentiation, and apoptosis (1-3). 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 (4). Noticeably, some of the diverse signaling roles attributed to elevated cellular S1P levels include prevention of ceramide-induced apoptosis (5, 6) and calcium mobilization (7).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 (8, 9). 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 (8). Overexpression of SPHK1 induces cell proliferation by promoting the G 1 to S transition of the cell cycle as well as by inhibiting the apoptotic response to serum deprivation or ceramide treatment (10). 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 (11), 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 overexpressed in mammalian cells (12). Similarly, Liu et al. (13) 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 (11,14), or association wit...

Neuronal activity greatly influences the formation and stabilization of synapses. Although receptors for sphingosine-1-phosphate (S1P), a lipid mediator regulating diverse cellular processes, are abundant in the central nervous system, neuron-specific functions of S1P remain largely undefined. Here, we report two novel actions of S1P using primary hippocampal neurons as a model system: (i) as a secretagogue where S1P triggers glutamate secretion and (ii) as an enhancer where S1P potentiates depolarization-evoked glutamate secretion. Sphingosine kinase 1 (SK1), a key enzyme for S1P production, was enriched in functional puncta of hippocampal neurons. Silencing SK1 expression by small interfering RNA as well as SK1 inhibition by dimethylsphingosine resulted in a strong inhibition of depolarization-evoked glutamate secretion. Fluorescence recovery after photobleaching analysis showed translocation of SK1 from cytosol to membranes at the puncta during depolarization, which resulted in subsequent accumulation of S1P within cells. Fluorescent resonance energy transfer analysis demonstrated that the S1P 1 receptor at the puncta was activated during depolarization and that depolarization-induced S1P 1 receptor activation was inhibited in SK1-knock-down cells. Importantly, exogenously added S1P at a nanomolar concentration by itself elicited glutamate secretion from hippocampal cells even when the Na ؉ -channel was blocked by tetrodotoxin, suggesting that S1P acts on presynaptic membranes. Furthermore, exogenous S1P at a picomolar level potentiated depolarization-evoked secretion in the neurons. These findings indicate that S1P, through its autocrine action, facilitates glutamate secretion in hippocampal neurons both by secretagogue and enhancer actions and may be involved in mechanisms underlying regulation of synaptic transmission.One of the remarkable features of the central nervous system (CNS) is its ability to integrate and store enormous information. Neuronal information is rapidly transferred through the chemical synapse to specialized regions of the postsynaptic plasma membrane, where neurotransmitter receptors are concentrated. Neurotransmitter secretion in the CNS shares many features with constitutive membrane trafficking (4, 19); however, it also exhibits several unique features, including storage of enormous amounts of information and plasticity, that indicate the presence of additional regulators. There is considerable interest in identifying such regulators that modulate the speed and potency of neurotransmitter release. Among various regulators sphingolipid metabolites such as sphingosine-1-phosphate (S1P) have recently attracted attention for their role in the regulation of neuronal function (9). S1P, a phosphorylated product of sphingosine catalyzed by sphingosine kinase (SK), has been implicated as an important lipid mediator acting both inside and outside the cells (26,33). Extracellular S1P binds to members of GTP-binding protein (G-protein)-coupled S1P receptor family (S1P 1-5 ), triggering diverse...

Sphingosine kinase (SPHK) is a key enzyme producing important messenger sphingosine 1-phosphate and is implicated in cell proliferation and suppression of apoptosis. Because the extent of agonist-induced activation of SPHK is modest, signaling via SPHK may be regulated through its localization at specific intracellular sites. Although the SPHK1 isoform has been extensively studied and characterized, the regulation of expression and function of the other isoform, SPHK2, remain largely unexplored. Here we describe an important post-translational modification, namely, phosphorylation of SPHK2 catalyzed by protein kinase D (PKD), which regulates its localization. Upon stimulation of HeLa cells by tumor promoter phorbol 12-myristate 13-acetate, a serine residue in a novel and putative nuclear export signal, identified for the first time, in SPHK2 was phosphorylated followed by SPHK2 export from the nucleus. Constitutively active PKD phosphorylated this serine residue in the nuclear export signal both in vivo and in vitro. Moreover, downregulation of PKDs through RNA interference resulted in the attenuation of both basal and phorbol 12-myristate 13-acetateinduced phosphorylation, which was followed by the accumulation of SPHK2 in the nucleus in a manner rescued by PKD overexpression. These results indicate that PKD is a physiologically relevant enzyme for SPHK2 phosphorylation, which leads to its nuclear export for subsequent cellular signaling. Sphingosine kinases (SPHKs)3 catalyze the formation of sphingosine 1-phosphate, a bioactive lipid that regulates a diverse range of cellular processes, including cell growth, survival, differentiation, motility, and cytoskeletal organization (1, 2). Some of these cellular processes are mediated by five sphingosine 1-phosphatespecific G protein-coupled receptors, whereas others appear to be controlled by intracellular sphingosine 1-phosphate through as yet unidentified intracellular targets (2, 3).Two distinct SPHK isoforms, SPHK1 and SPHK2, have been cloned and characterized (4, 5). Diverse external stimuli, particularly growth and survival factors, stimulate SPHK1, and intracellularly generated sphingosine 1-phosphate has been implicated in their mitogenic and anti-apoptotic effects (6 -15). Expression of SPHK1 enhanced proliferation, promoted the G 1 /S transition, protected cells from apoptosis (6,8,16), and induced tumor formation in mice (8, 9).In contrast to SPHK1 much less is known about SPHK2. Although highly similar in amino acid sequence and possessing five evolutionarily conserved domains found in all SPHKs (17), SPHK2 diverges in its N terminus and central regions. These two isoforms have different kinetic properties and differ in developmental and tissue expression (5) implying that they may have distinct physiological functions. In fact, studies from our laboratory have demonstrated that, in contrast to cytosolic distribution of SPHK1, SPHK2 enters nuclei and inhibits DNA synthesis or induces apoptosis under stressful conditions such as serum deprivation (18,19)....

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Tumor interstitial pressure is a fundamental feature of cancer biology. Elevation in tumor pressure affects the efficacy of cancer treatment. It causes heterogenous intratumoral distribution of drugs and macromolecules. It also causes the development of hypoxia within tumor bulk, leading to reduced efficacy of therapeutic drugs and radiotherapy. Tumor pressure has been associated with increased metastatic potential and poor prognosis in some tumors. The formation of increased pressure in solid tumors is multifactorial. Factors known to affect tumor pressure include hyperpermeable tortuous tumor vasculatures, the lack of functional intratumoral lymphatic vessels, abnormal tumor microenvironment, and the solid stress exerted by proliferating tumor cells. Reducing this pressure is known to enhance the uptake and homogenous distribution of many therapies. Pharmacologic and biologic agents have been shown to reduce tumor pressure. These include antiangiogenic therapy, vasodilatory agents, antilymphogenic therapy, and proteolytic enzymes. Physical manipulation has been shown to cause reduction in tumor pressure. These include irradiation, hyperbaric oxygen therapy, hyper-or hypothermic therapy, and photodynamic therapy. This review explores the methods to reduce tumor pressure that may open up new avenues in cancer treatment. Cancer Res; 74(10); 2655-62. Ó2014 AACR.

Sphingosine 1-phosphate (S1P) produced by sphingosine kinase (SPHK) is implicated in acute immunoresponses, however, mechanisms of SPHK/S1P signaling in the pathogenesis of bronchial asthma are poorly understood. In this study, we hypothesized that SPHK inhibition could ameliorate lung inflammation in ovalbumin (OVA)-challenged mouse lungs. Six- to eight-week-old C57BL/6J mice were sensitized and exposed to OVA for 3 consecutive days. Twenty-four hours later, mice lungs and bronchoalveolar lavage (BAL) fluid were analyzed. For an inhibitory effect, either of the two different SPHK inhibitors, N,N-dimethylsphingosine (DMS) or SPHK inhibitor [SK-I; 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole], was nebulized for 30 min before OVA inhalation. OVA inhalation caused S1P release into BAL fluid and high expression of SPHK1 around bronchial epithelial walls and inflammatory areas. DMS or SK-I inhalation resulted in a decrease in S1P amounts in BAL fluid to basal levels, accompanied by decreased eosinophil infiltration and peroxidase activity. The extent of inhibition caused by DMS inhalation was higher than that caused by SK-I. Like T helper 2 (Th2) cytokine release, OVA inhalation-induced increase in eotaxin expression was significantly suppressed by DMS pretreatment both at protein level in BAL fluid and at mRNA level in lung homogenates. Moreover, bronchial hyperresponsiveness to inhaled methacholine and goblet cell hyperplasia were improved by SPHK inhibitors. These data suggest that the inhibition of SPHK affected acute eosinophilic inflammation induced in antigen-challenged mouse model and that targeting SPHK may provide a novel therapeutic tool to treat bronchial asthma.

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