SphK1 Activity Research Articles

Potentiation of Growth Factor Signaling by Insulin-like Growth Factor-binding Protein-3 in Breast Epithelial Cells Requires Sphingosine Kinase Activity

We have investigated the mechanism underlying potentiation of epidermal growth factor receptor (EGFR) and type 1 insulin-like growth factor receptor (IGFR1) signaling by IGF-binding protein-3 (IGFBP-3) in MCF-10A breast epithelial cells, focusing on a possible involvement of the sphingosine kinase (SphK) system. IGFBP-3 potentiated EGF-stimulated EGF receptor activation and DNA synthesis, and this was blocked by inhibitors of SphK activity or small interference RNA-mediated silencing of SphK1, but not SphK2, expression. Similarly, IGFR1 phosphorylation and DNA synthesis stimulated by LR3-IGF-I (an IGF-I analog not bound by IGFBP-3), were enhanced by IGFBP-3, and this was blocked by SphK1 silencing. SphK1 expression and activity were stimulated by IGFBP-3 approximately 2-fold over 24 h. Silencing of sphingosine 1-phosphate receptor 1 (S1P1) or S1P3, but not S1P2, abolished the effect of IGFBP-3 on EGF-stimulated EGFR activation. The effects of IGFBP-3 could be reproduced with exogenous S1P or medium conditioned by cells treated with IGFBP-3, and this was also blocked by inhibition of S1P1 and S1P3. These data indicate that potentiation of growth factor signaling by IGFBP-3 in MCF-10A cells requires SphK1 activity and S1P1/S1P3, suggesting that S1P, the product of SphK activity and ligand for S1P1 and S1P3, is the "missing link" mediating IGF and EGFR transactivation and cell growth stimulation by IGFBP-3.

Restoring Endocrine Response in Breast Cancer Cells by Inhibition of the Sphingosine Kinase-1 Signaling Pathway

We previously demonstrated that sphingosine kinase-1 (SphK1) is an important mediator in the cytoplasmic signaling of estrogens, including Ca(2+) mobilization, ERK1/2 activation, and the epidermal growth factor receptor transactivation. Here we report for the first time that SphK1 activity is causally associated with endocrine resistance in MCF-7 human breast cancer cells. Enforced overexpression of human SphK1 in MCF-7 cells resulted in enhanced cell proliferation and resistance to tamoxifen-induced cell growth arrest and apoptosis. Tamoxifen-resistant (TamR) MCF-7 cells selected by prolonged exposure to 4-hydroxytamoxifen, exhibited higher levels in SphK1 expression and activity, compared with the control cells. Inhibition of SphK1 activity by either specific pharmaceutical inhibitors or the dominant-negative mutant SphK1(G82D) restored the antiproliferative and proapoptotic effects of tamoxifen in the TamR cells. Furthermore, silencing of SphK1, but not SphK2, expression by the specific small interference RNA also restored the tamoxifen responsiveness in the TamR cells. Thus, blockade of the SphK1 signaling pathway may reprogram cellular responsiveness to tamoxifen and abrogate antiestrogen resistance in human breast cancer cells.

Hypoxic Preconditioning-Induced Cerebral Ischemic Tolerance

The importance of bioactive lipid signaling under physiological and pathophysiological conditions is progressively becoming recognized. The disparate distribution of sphingosine kinase (SphK) isoform activity in normal and ischemic brain, particularly the large excess of SphK2 in cerebral microvascular endothelial cells, suggests potentially unique cell- and region-specific signaling by its product sphingosine-1-phosphate. The present study sought to test the isoform-specific role of SphK as a trigger of hypoxic preconditioning (HPC)-induced ischemic tolerance. Temporal changes in microvascular SphK activity and expression were measured after HPC. The SphK inhibitor dimethylsphingosine or sphingosine analog FTY720 was administered to adult male Swiss-Webster ND4 mice before HPC. Two days later, mice underwent a 60-minute transient middle cerebral artery occlusion and at 24 hours of reperfusion, infarct volume, neurological deficit, and hemispheric edema were measured. HPC rapidly increased microvascular SphK2 protein expression (1.7+/-0.2-fold) and activity (2.5+/-0.6-fold), peaking at 2 hours, whereas SphK1 was unchanged. SphK inhibition during HPC abrogated reductions in infarct volume, neurological deficit, and ipsilateral edema in HPC-treated mice. FTY720 given 48 hours before stroke also promoted ischemic tolerance; when combined with HPC, even greater (and dimethylsphingosine-reversible) protection was noted. These findings indicate hypoxia-sensitive increases in SphK2 activity may serve as a proximal trigger that ultimately leads to sphingosine-1-phosphate-mediated alterations in gene expression that promote the ischemia-tolerant phenotype. Thus, components of this bioactive lipid signaling pathway may be suitable therapeutic targets for protecting the neurovascular unit in stroke.

Distinct Roles of Sphingosine Kinase 1 and 2 in Murine Collagen-Induced Arthritis

Sphingosine kinase (SphK) phosphorylates sphingosine into sphingosine-1-phosphate (S1P). S1P plays a critical role in angiogenesis, inflammation, and various pathologic conditions. To date, two mammalian isoenzymes, SphK1 and SphK2, have been identified. Although both SphK1 and SphK2 share overall homology and produce the common product, S1P, it has been proposed they display different unique and separate functions. In this study, we examined the role of SphK1 and SphK2 in a murine collagen-induced arthritis model by down-regulating each isoenzyme via specific small interfering RNA (siRNA). Prophylactic i.p. administration of SphK1 siRNA significantly reduced the incidence, disease severity, and articular inflammation compared with control siRNA recipients. Treatment of SphK1 siRNA also down-regulated serum levels of S1P, IL-6, TNF-alpha, IFN-gamma, and IgG2a anti-collagen Ab. Ex vivo analysis demonstrated significant suppression of collagen-specific proinflammatory/Th1 cytokine (IL-6, TNF-alpha, IFN-gamma) release in SphK siRNA-treated mice. Interestingly, mice received with SphK2 siRNA develop more aggressive disease; higher serum levels of IL-6, TNF-alpha, and IFN-gamma; and proinflammatory cytokine production to collagen in vitro when compared with control siRNA recipients. Together, these results demonstrate the distinct immunomodulatory roles of SphK1 and SphK2 in the development of inflammatory arthritis by regulating the release of proinflammatory cytokines and T cell responses. These findings raise the possibility that drugs which specifically target SphK1 activity may play a beneficial role in the treatment of inflammatory arthritis.

Decrease of Tau Hyperphosphorylation by 17β Estradiol Requires Sphingosine Kinase in a Glutamate Toxicity Model

Several studies have linked estrogens with sphingosine kinase (SphK) activity, enzyme responsible of sphingosine-1-phosphate synthesis (S-1P), however their possible interaction in the nervous system is not documented yet. In the present study, we developed a glutamate toxicity model in SH-SY5Y cells to evaluate the possible effect of the inhibition of SphK activity on the protective capability of 17β-estradiol (E2). Glutamate induced cytoskeletal actin changes associated to cytotoxic stress, significant increase of apoptotic-like nuclear fragmentation, Tau hyperphosphorylation and increase of p25/p35 cleavage. These effects were prevented by E2 pre-treatment during 24 h. Although the inhibition of SphK did not block this protective effect, significantly increased Tau hyperphosphorylation by glutamate, in a way that was not reverted by E2. Our results suggest that the decrease of glutamate-induced Tau hyperphosphorylation by 17β-estradiol requires SphK.

Inhibition of Sphingosine Kinase by Bovine Viral Diarrhea Virus NS3 Is Crucial for Efficient Viral Replication and Cytopathogenesis

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid implicated in diverse cellular functions including survival, proliferation, tumorigenesis, inflammation, and immunity. Sphingosine kinase (SphK) contributes to these functions by converting sphingosine to S1P. We report here that the nonstructural protein NS3 from bovine viral diarrhea virus (BVDV), a close relative of hepatitis C virus (HCV), binds to and inhibits the catalytic activity of SphK1 independently of its serine protease activity, whereas HCV NS3 does not affect SphK1 activity. Uncleaved NS2-3 from BVDV was also found to interact with and inhibit SphK1. We suspect that inhibition of SphK1 activity by BVDV NS3 and NS2-3 may benefit viral replication, because SphK1 inhibition by small interfering RNA, chemical inhibitor, or overexpression of catalytically inactive SphK1 results in enhanced viral replication, although the mechanisms by which SphK1 inhibition leads to enhanced viral replication remain unknown. A role of SphK1 inhibition in viral cytopathogenesis is also suggested as overexpression of SphK1 significantly attenuates the induction of apoptosis in cells infected with cytopathogenic BVDV. These findings suggest that SphK is targeted by this virus to regulate its catalytic activity.

Resveratrol attenuates C5a‐induced inflammatory responsesin vitroandin vivoby inhibiting phospholipase D and sphingosine kinase activities

The anti-inflammatory activity of the phytoalexin resveratrol (RSV) was evaluated in C5 anaphylatoxin (C5a)-stimulated primary neutrophils and in a mouse model of acute peritonitis. Pretreatment of human and mouse neutrophils with RSV significantly blocked oxidative burst, leukocyte migration, degranulation, and inflammatory cytokine production. The anti-inflammatory activity of RSV was a function of inhibition of sphingosine kinase (SphK) activity (IC(50) approximately 20 microM) within 5 min of exposure, its membrane localization, and SphK1-mediated Ca(2+) release. As an experimental control, the SphK1 pharmacological inhibitor N,N-dimethyl sphingosine (DMS) was used to compare the inhibitory effect of RSV. We also provide evidence that the SphK inhibitory effect of RSV was mediated via its ability to block phospholipase D (PLD) activity and membrane recruitment. Furthermore, RSV blocked ERK1/2 phosphorylation, which functioned independently of SphK1 in this study. To provide in vivo relevance to these data, C5a-induced model of acute peritonitis was established, and the effects of prior injection of RSV were investigated. Indeed, prior injection of RSV virtually completely attenuated the effects of C5a on vascular permeability, neutrophil migration, release of interleukin 1beta, tumor necrosis factor alpha, interleukin 6, and the chemokine MIP-1alpha. Taken together, these data demonstrate strong anti-inflammatory activity of RSV in vitro and in vivo and highlight SphK1 as a potential target of this remarkable phytoalexin. These data could have tremendous implications for the clinical use of RSV in inflammatory pathologies.

Targeting the sphingolipid metabolism to defeat pancreatic cancer cell resistance to the chemotherapeutic gemcitabine drug

Defeating pancreatic cancer resistance to the chemotherapeutic drug gemcitabine remains a challenge to treat this deadly cancer. Targeting the sphingolipid metabolism for improving tumor chemosensitivity has recently emerged as a promising strategy. The fine balance between intracellular levels of the prosurvival sphingosine-1-phosphate (S1P) and the proapoptotic ceramide sphingolipids determines cell fate. Among enzymes that control this metabolism, sphingosine kinase-1 (SphK1), a tumor-associated protein overexpressed in many cancers, favors survival through S1P production, and inhibitors of SphK1 are used in ongoing clinical trials to sensitize epithelial ovarian and prostate cancer cells to various chemotherapeutic drugs. We here report that the cellular ceramide/S1P ratio is a critical biosensor for predicting pancreatic cancer cell sensitivity to gemcitabine. A low level of the ceramide/S1P ratio, associated with a high SphK1 activity, correlates with a robust intrinsic pancreatic cancer cell chemoresistance toward gemcitabine. Strikingly, increasing the ceramide/S1P ratio, by using pharmacologic (SphK1 inhibitor or ceramide analogue) or small interfering RNA-based approaches to up-regulate intracellular ceramide levels or reduce SphK1 activity, sensitized pancreatic cancer cells to gemcitabine. Conversely, decreasing the ceramide/S1P ratio, by up-regulating SphK1 activity, promoted gemcitabine resistance in these cells. Development of novel pharmacologic strategies targeting the sphingolipid metabolism might therefore represent an interesting promising approach, when combined with gemcitabine, to defeat pancreatic cancer chemoresistance to this drug.

Sphingosine Kinase Isoforms Regulate Oxaliplatin Sensitivity of Human Colon Cancer Cells through Ceramide Accumulation and Akt Activation

The relationship between sphingosine kinase (SPHK), cellular ceramide concentration and chemosensitivity was investigated in human colon cancer cell lines. Among nine colon cancer cell lines, SPHK1 and SPHK2 activity and protein expression was highest in RKO cells and lowest in HCT116 cells. A viability assay revealed that HCT116 cells were sensitive to the effects of oxaliplatin (l-OHP), whereas RKO cells were resistant to those of l-OHP. Treatment with 5microg/ml l-OHP induced a marked time-dependent increase in various ceramides (C16, C24, C24:1) in HCT116 cells but not in RKO cells, as indicated by liquid chromatography/mass spectrometry. The increase in ceramide and caspase activation induced by l-OHP in the sensitive HCT116 cells was abolished by pretreatment with a neutral sphingomyelinase inhibitor, suggesting that the ceramide formation was due to the activation of neutral, rather than acid, sphingomyelinase. In contrast, in l-OHP-resistant RKO cells, treatment with an SPHK inhibitor or SPHK1 and SPHK2 silencing by RNA interference suppressed cell viability and increased caspase activity and cellular ceramide formation after l-OHP treatment. The elevated ceramide formation induced by SPHK inhibition and l-OHP was inhibited by fumonisin B1 but not myriocin, suggesting that ceramide formation was through the salvage pathway. Endogenous phosphorylated Akt levels were much higher in the resistant RKO cells than in the sensitive HCT116 cells. Either SPHK1 or SPHK2 silencing in RKO cells decreased phosphorylated Akt levels and increased p53 and p21 protein levels as well as poly(ADP-ribose) polymerase cleavage in response to l-OHP treatment. These findings indicate that SPHK isoforms and neutral sphingomyelinase contribute to the regulation of chemosensitivity by controlling ceramide formation and the downstream Akt pathway in human colon cancer cells.

Sphingosine kinase is induced in mouse 3T3-L1 cells and promotes adipogenesis

Sphingosine 1-phosphate (S1P) is a lysophospholipid mediator that exerts numerous biological activities both as a receptor ligand and as an intracellular second messenger. In the present study, we explored roles of sphingosine kinase (SphK), an S1P-producing enzyme, in adipose tissue. We utilized mouse 3T3-L1 cells as an in vitro model of adipogenesis, using a mixture of insulin/dexamethasone/3-isobutyl-1-methylxanthine (IBMX) to induce differentiation. Real-time quantitative PCR (qRT-PCR) assays revealed that the expression levels of transcripts encoding both isoforms of SphK-1 and SphK-2 are up-regulated during adipogenesis (37.6- and 6.6-fold vs. basal, P < 0.05, respectively). Concomitantly, SphK-1/SphK-2 protein abundance and S1P contents of these cells increased at 3 days after hormonal stimulation. Loss-of-function approaches by pharmacological inhibition of SphK activity as well as by transfection with small interfering RNA (siRNA) against SphK-1 led to significant attenuation of lipid droplet accumulation and adipocyte marker gene expression. We detected marked elevation of SphK-1 mRNA in adipose tissue derived from 13-week-old ob/ob mice with obese phenotype than their lean littermates. These results suggest that increased expression of SphK, an S1P-producing enzyme, plays a significant role during adipogenesis, potentially providing a novel point of control in adipose tissue.

Sphingolipid signaling and treatment during remodeling of the uninfarcted ventricular wall after myocardial infarction

The sphingosine kinase (SphK)/sphingosine 1-phosphate (S1P) pathway, known to determine the fate and growth of various cell types, can enhance cardiac myocyte survival in vitro and provide cardioprotection in acute ex vivo heart preparations. However, the relevance of these findings to chronic cardiac pathology has never been demonstrated. We hypothesized that S1P signaling is impaired during chronic remodeling of the uninfarcted ventricle during the evolution of post-myocardial infarction (MI) cardiomyopathy and that a therapeutic enhancement of S1P signaling would ameliorate ventricular dysfunction. SphK expression and activity were measured in the remote, uninfarcted myocardium (RM) of C57Bl/6 mice subjected to coronary artery ligation. The mRNA expression of S1P receptor isoforms was also measured, as was the activation of the downstream S1P receptor mediators. A cardioprotective role for S1P(1) receptor agonism was tested via the administration of the S1P(1)-selective agonist SEW2871 during and after MI. As a result, the expression data suggested that a dramatic reduction in SphK activity in the RM early after MI may reflect a combination of posttranscriptional and posttranslational modulation. SphK activity continued to decline gradually during chronic post-MI remodeling, when S1P(1) receptor mRNA also fell below baseline. The S1P(1)-specific agonism with oral SEW2871 during the first 2-wk after MI reduced apoptosis in the RM and resulted in improved myocardial function, as reflected in the echocardiographic measurement of fractional shortening. In conclusion, these results provide the first documentation of alterations in S1P-mediated signaling during the in situ development of cardiomyopathy and suggest a possible therapeutic role for the pharmacological S1P receptor agonism in the post-MI heart.

Gα q-mediated plasma membrane translocation of sphingosine kinase-1 and cross-activation of S1P receptors

Sphingosine-1-phosphate (S1P), formed by sphingosine kinases (SphKs), regulates cellular proliferation and migration by acting as an agonist at specific receptors or intracellularly. Since S1P's effects are probably dependent on subcellular localization of its formation and degradation, we have studied the influence of G protein-coupled receptors on the localization of SphK1. Activation of G q-coupled receptors induced a profound, rapid (half-life 3–5 s) and long-lasting (> 2 h) translocation of SphK1 to the plasma membrane. This was mimicked by expression of constitutively active G protein α-subunits specifically of the G q family. Classical G q signalling pathways, or phosphorylation at Ser 225, phospholipase D and Ca 2+/calmodulin were not involved in M 3 receptor-induced SphK1 translocation in HEK-293 cells. Translocation was associated with S1P receptor internalization, which was dependent on catalytic activity of SphK1 and S1P receptor binding and thus resulted from S1P receptor cross-activation. It is concluded that SphK1 is an important effector of G q-coupled receptors, linking them via cross-activation of S1P receptors to G i and G 12/13 signalling pathways.

Interleukin-1 Regulates the Expression of Sphingosine Kinase 1 in Glioblastoma Cells

Chronic inflammation and inflammatory cytokines have recently been implicated in the development and progression of various types of cancer. In the brain, neuroinflammatory cytokines affect the growth and differentiation of both normal and malignant glial cells, with interleukin 1 (IL-1) shown to be secreted by the majority of glioblastoma cells. Recently, elevated levels of sphingosine kinase 1 (SphK1), but not SphK2, were correlated with a shorter survival prognosis for patients with glioblastoma multiforme. SphK1 is a lipid kinase that produces the pro-growth, anti-apoptotic sphingosine 1-phosphate, which can induce invasion of glioblastoma cells. Here, we show that the expression of IL-1 correlates with the expression of SphK1 in glioblastoma cells, and neutralizing anti-IL-1 antibodies inhibit both the growth and invasion of glioblastoma cells. Furthermore, IL-1 up-regulates SphK1 mRNA levels, protein expression, and activity in both primary human astrocytes and various glioblastoma cell lines; however, it does not affect SphK2 expression. The IL-1-induced SphK1 up-regulation can be blocked by the inhibition of JNK, the overexpression of the dominant-negative c-Jun(TAM67), and the down-regulation of c-Jun expression by small interference RNA. Activation of SphK1 expression by IL-1 occurs on the level of transcription and is mediated via a novel AP-1 element located within the first intron of the sphk1 gene. In summary, our results suggest that SphK1 expression is transcriptionally regulated by IL-1 in glioblastoma cells, and this pathway may be important in regulating survival and invasiveness of glioblastoma cells.

Differential regulation of sphingosine kinases 1 and 2 in lung injury

Two mammalian sphingosine kinase (SphK) isoforms, SphK1 and SphK2, possess identical kinase domains but have distinct kinetic properties and subcellular localizations, suggesting each has one or more specific roles in sphingosine-1-phosphate (S1P) generation. Although both kinases use sphingosine as a substrate to generate S1P, the mechanisms controlling SphK activation and subsequent S1P generation during lung injury are not fully understood. In this study, we established a murine lung injury model to investigate LPS-induced lung injury in SphK1 knockout (SphK1(-/-)) and wild-type (WT) mice. We found that SphK1(-/-) mice were much more susceptible to LPS-induced lung injury compared with their WT counterparts, quantified by multiple parameters including cytokine induction. Intriguingly, overexpression of WT SphK1 delivered by adenoviral vector to the lungs protected SphK1(-/-) mice from lung injury and attenuated the severity of the response to LPS. However, adenoviral overexpression of a SphK1 kinase-dead mutant (SphKKD) in SphK1(-/-) mouse lungs further exacerbated the response to LPS as well as the extent of lung injury. WT SphK2 adenoviral overexpression also failed to provide protection and, in fact, augmented the degree of LPS-induced lung injury. This suggested that, in vascular injury, S1P generated by SphK2 activation plays a distinctly separate role compared with SphK1-dependent S1P generation and survival signaling. Microarray and real-time RT-PCR analysis of SphK1 and SphK2 expression levels during lung injury revealed that, in WT mice, LPS treatment caused significantly enhanced SphK1 expression ( approximately 5x) levels within 6 h, which declined back to baseline levels by 24 h posttreatment. In contrast, expression of SphK2 was gradually induced following LPS treatment and was elevated within 24 h. Collectively, our results for the first time demonstrate distinct functional roles of the two SphK isoforms in the regulation of LPS-induced lung injury.

Divergent roles of sphingosine kinases in kidney ischemia–reperfusion injury

Sphingosine-1-phosphate (S1P), produced by sphingosine kinase 1 (SphK1) or kinase 2 (SphK2), mediates biological effects through intracellular and/or extracellular mechanisms. Here we determined a role for these kinases in kidney injury of wild-type mice following ischemia-reperfusion. SphK1 but not SphK2 mRNA expression and activity increased in the kidney following injury relative to sham-operated animals. Although SphK1(-/-) mice had no alteration in renal function following injury, mice with a disrupted SphK2 gene (SphK2(tr/tr)) had histological damage and impaired function. The immune-modulating pro-drug, FTY720, an S1P agonist failed to provide protection in SphK2(tr/tr) mice. Injured kidneys of these mice showed increased neutrophil infiltration and neutrophil chemokine expression along with a 3- to 5-fold increase in expression of the G-protein-coupled receptor S1P(3) compared to heterozygous SphK2(+/tr) mice. Kidney function and reduced vascular permeability were preserved in S1P(3)(-/-) compared to S1P(3)(+/-) mice after ischemia-reperfusion injury, suggesting increased S1P(3) mRNA may play a role in the injury of SphK2(tr/tr) mice. Our study suggests that constitutive expression of SphK2 may contribute to reduced ischemia-reperfusion injury of the kidney, and its absence may enhance injury due to increased neutrophil infiltration and S1P(3) activation. We also confirm that SphK2 is necessary to mediate the protective effects of FTY720.

TGFβ protects mesoangioblasts from apoptosis via sphingosine kinase-1 regulation

Mesoangioblasts are vessel-derived progenitor cells that can be induced to differentiate into different cell types of the mesoderm such as muscle and bone. Here we examined the role of transforming growth factor-beta (TGFβ), a pleiotropic cytokine that plays a major role in development and specifically induces smooth muscle differentiation of mesoangioblasts, in the regulation of death and survival of these cells. TGFβ exerts a marked anti-apoptotic action in mesoangioblasts with a mechanism involving regulation of sphingosine kinase 1 (SphK1), one of the isoforms responsible for S1P formation. Treatment with the cytokine efficaciously protected mesoangioblasts from apoptosis induced by serum starvation or staurosporine treatment assessed by various means such as activation of caspase-3, determination of cytoplasmic histone-associated-DNA-fragments and PE-AnnexinV staining. The protective action of TGFβ from staurosporine-induced apoptosis was strongly reduced when the SphK activity was inhibited by drugs, when SphK1 but not SphK2 was downregulated by specific siRNA and when a SphK1 dominant negative mutant was overexpressed. Staurosporine treatment induced down-regulation of both SphK isoforms and TGFβ rescued SphK1 but not SphK2 expression. Interestingly, TGFβ strongly enhanced SphK activity during staurosporine-induced cell death. Both TGFβ-induced SphK1 up-regulation and TGFβ anti-apoptotic action were found to be dependent on p42/44 MAPK activation.

Sphingosine Kinase 1: A New Modulator of Hypoxia Inducible Factor 1α during Hypoxia in Human Cancer Cells

Here, we provide the first evidence that sphingosine kinase 1 (SphK1), an oncogenic lipid kinase balancing the intracellular level of key signaling sphingolipids, modulates the transcription factor hypoxia inducible factor 1alpha (HIF-1alpha), master regulator of hypoxia. SphK1 activity is stimulated under low oxygen conditions and regulated by reactive oxygen species. The SphK1-dependent stabilization of HIF-1alpha levels is mediated by the Akt/glycogen synthase kinase-3beta signaling pathway that prevents its von Hippel-Lindau protein-mediated degradation by the proteasome. The pharmacologic and RNA silencing inhibition of SphK1 activity prevents the accumulation of HIF-1alpha and its transcriptional activity in several human cancer cell lineages (prostate, brain, breast, kidney, and lung), suggesting a canonical pathway. Therefore, we propose that SphK1 can act as a master regulator for hypoxia, giving support to its inhibition as a valid strategy to control tumor hypoxia and its molecular consequences.

Activation of Sphingosine Kinase-1 Reverses the Increase in Lung Vascular Permeability Through Sphingosine-1-Phosphate Receptor Signaling in Endothelial Cells

The lipid mediator sphingosine-1-phosphate (S1P), the product of sphingosine kinase (SPHK)-induced phosphorylation of sphingosine, is known to stabilize interendothelial junctions and prevent microvessel leakiness. Here, we investigated the role of SPHK1 activation in regulating the increase in pulmonary microvessel permeability induced by challenge of mice with lipopolysaccharide or thrombin ligation of protease-activating receptor (PAR)-1. Both lipopolysaccharide and thrombin increased mouse lung microvascular permeability and resulted in a delayed activation of SPHK1 that was coupled to the onset of restoration of permeability. In contrast to wild-type mice, Sphk1(-/-) mice showed markedly enhanced pulmonary edema formation in response to lipopolysaccharide and PAR-1 activation. Using endothelial cells challenged with thrombin concentration (50 nmol/L) that elicited a transient but reversible increase in endothelial permeability, we observed that increased SPHK1 activity and decreased intracellular S1P concentration preceded the onset of barrier recovery. Thus, we tested the hypothesis that released S1P in a paracrine manner activates its receptor S1P1 to restore the endothelial barrier. Knockdown of SPHK1 decreased basal S1P production and Rac1 activity but increased basal endothelial permeability. In SPHK1-depleted cells, PAR-1 activation failed to induce Rac1 activation but augmented RhoA activation and endothelial hyperpermeability response. Knockdown of S1P1 receptor in endothelial cells also enhanced the increase in endothelial permeability following PAR-1 activation. S1P treatment of Sphk1(-/-) lungs or SPHK1-deficient endothelial cells restored endothelial barrier function. Our results suggest the crucial role of activation of the SPHK1-->S1P-->S1P1 signaling pathway in response to inflammatory mediators in endothelial cells in regulating endothelial barrier homeostasis.

Filamin A Links Sphingosine Kinase 1 and Sphingosine-1-Phosphate Receptor 1 at Lamellipodia To Orchestrate Cell Migration

Sphingosine kinase 1 (SphK1) catalyzes the phosphorylation of sphingosine to produce the potent lipid mediator sphingosine-1-phosphate (S1P), which plays a critical role in cell motility via its cell surface receptors. Here, we have identified filamin A (FLNa), an actin-cross-linking protein involved in cell movement, as a bona fide SphK1-interacting protein. Heregulin stimulated SphK1 activity only in FLNa-expressing A7 melanoma cells but not in FLNa-deficient cells and induced its translocation and colocalization with FLNa at lamellipodia. SphK1 was required for heregulin-induced migration, lamellipodia formation, activation of PAK1, and subsequent FLNa phosphorylation. S1P directly stimulated PAK1 kinase, suggesting that it may be a target of intracellularly generated S1P. Heregulin also induced colocalization of S1P(1) (promotility S1P receptor) but not S1P(2), with SphK1 and FLNa at membrane ruffles. Moreover, an S1P(1) antagonist inhibited the lamellipodia formation induced by heregulin. Hence, FLNa links SphK1 and S1P(1) to locally influence the dynamics of actin cytoskeletal structures by orchestrating the concerted actions of the triumvirate of SphK1, FLNa, and PAK1, each of which requires and/or regulates the actions of the others, at lamellipodia to promote cell movement.

Signaling Pathways Involved in Sphingosine Kinase Activation and Sphingosine-1-Phosphate Release in Rat Myometrium in Late Pregnancy: Role in the Induction of Cyclooxygenase 2

We investigated the regulation of the sphingosine kinase (SphK)/sphingosine-1-phosphate (S1P) axis and its role during pregnancy in the rat myometrium. SphK1 and SphK2 were coexpressed in myometrium during gestation. The levels and activity of SphK1/2 were modest at midgestation (d 12), increased at d 19 and progressively declined to low at postpartum. Similar patterns were observed for the phosphorylation of ERK and protein kinase C (PKC). Inhibition of PKC and ERK reduced SphK1/2 activity. In late pregnancy, levels of cyclooxygenase 2 (COX2) increased in parallel to SphK levels. Using a pharmacological approach, we demonstrated that in primary cultures of myometrial cells from d-19 pregnant rats, induction of COX2 was mediated by 4beta-phorbol 12,13-dibutyrate and IL-1beta through sequential activation of PKC, ERK1/2, and SphK1. S1P produced by SphK1 was released in the medium. Addition of S1P, IL-1beta or 4beta-phorbol 12,13-dibutyrate enhanced COX2 levels via Gi protein. Interestingly, S1P was also released by myometrial tissues at late gestation. This event was dependent on PKC/ERK/SphK1. By contrast, in d-12 myometrial tissues, the release of S1P was markedly reduced in association with low levels of SphK1 and COX2. However, prolonged incubation of myometrium from midgestation led to the induction of COX2. This effect was blocked by SphK inhibitors, providing evidence of the close relationship between SphK activity and COX2 induction in rat myometrium. Overall, our findings provided insight into the physiological relevance of the SphK activation and S1P release in uterine smooth muscle during gestation.