Sphingosylphosphorylcholine Research Articles

Increased Levels of Sphingosylphosphorylcholine (SPC) in Plasma of Metabolic Syndrome Patients.

Recent developments in lipid mass spectrometry enable extensive lipid class and species analysis in metabolic disorders such as diabesity and metabolic syndrome. The minor plasma lipid class sphingosylphosphorylcholine (SPC) was identified as a ligand for lipid sensitive G-protein coupled receptors playing a key role in cell growth, differentiation, motility, calcium signaling, tissue remodeling, vascular diseases and cancer. However, information about its role in diabesity patients is sparse. In this study, we analyzed plasma lipid species in patients at risk for diabesity and the metabolic syndrome and compared them with healthy controls. Our data show that SPC is significantly increased in plasma samples from metabolic syndrome patients but not in plasma from patients at risk for diabesity. Detailed SPC species analysis showed that the observed increase is due to a significant increase in all detected SPC subspecies. Moreover, a strong positive correlation is observed between total SPC and individual SPC species with both body mass index and the acute phase low grade inflammation marker soluble CD163 (sCD163). Collectively, our study provides new information on SPC plasma levels in metabolic syndrome and suggests new avenues for investigation.

Reprint of: Connections between single-cell biomechanics and human disease states: gastrointestinal cancer and malaria

We investigate connections between single-cell mechanical properties and subcellular structural reorganization from biochemical factors in the context of two distinctly different human diseases: gastrointestinal tumor and malaria. Although the cell lineages and the biochemical links to pathogenesis are vastly different in these two cases, we compare and contrast chemomechanical pathways whereby intracellular structural rearrangements lead to global changes in mechanical deformability of the cell. This single-cell biomechanical response, in turn, seems to mediate cell mobility and thereby facilitates disease progression in situations where the elastic modulus increases or decreases due to membrane or cytoskeleton reorganization. We first present new experiments on elastic response and energy dissipation under repeated tensile loading of epithelial pancreatic cancer cells in force- or displacement-control. Energy dissipation from repeated stretching significantly increases and the cell’s elastic modulus decreases after treatment of Panc-1 pancreatic cancer cells with sphingosylphosphorylcholine (SPC), a bioactive lipid that influences cancer metastasis. When the cell is treated instead with lysophosphatidic acid, which facilitates actin stress fiber formation, neither energy dissipation nor modulus is noticeably affected. Integrating recent studies with our new observations, we ascribe these trends to possible SPC-induced reorganization primarily of keratin network to perinuclear region of cell; the intermediate filament fraction of the cytoskeleton thus appears to dominate deformability of the epithelial cell. Possible consequences of these results to cell mobility and cancer metastasis are postulated. We then turn attention to progressive changes in mechanical properties of the human red blood cell (RBC) infected with the malaria parasite Plasmodium falciparum. We present, for the first time, continuous force–displacement curves obtained from in-vitro deformation of RBC with optical tweezers for different intracellular developmental stages of parasite. The shear modulus of RBC is found to increase up to 10-fold during parasite development, which is a noticeably greater effect than that from prior estimates. By integrating our new experimental results with published literature on deformability of Plasmodium-harbouring RBC, we examine the biochemical conditions mediating increases or decreases in modulus, and their implications for disease progression. Some general perspectives on connections among structure, single-cell mechanical properties and biological responses associated with pathogenic processes are also provided in the context of the two diseases considered in this work.

Sphingosylphosphorylcholine protects cardiomyocytes against ischemic apoptosis via lipid raft/PTEN/Akt1/mTOR mediated autophagy

Autophagy, evoked by diverse stresses including myocardial ischemia/reperfusion (I/R), profoundly affects the development of heart failure. However, the specific molecular basis of autophagy remains to be elucidated. Here we report that sphingosylphosphorylcholine (SPC), a bioactive sphingolipid, significantly suppressed apoptosis and induced autophagy in cardiomyocytes. Blocking this SPC evoked autophagy by 3-methyladenine (3MA)-sensitized cardiomyocytes to serum deprivation-induced apoptosis. Subsequent studies revealed that SPC downregulated the phosphorylation of p70S6K and 4EBP1 (two substrates of mTOR) but enhanced that of JNK when inducing autophagy. We identified SPC as a switch for the activity of Akt1, a supposed upstream modulator of both mTOR and JNK. Furthermore, β-cyclodextrin, which destroys membrane cholesterol, abolished the SPC-reduced phosphorylation of both Akt and PTEN, thus inhibiting SPC-induced autophagy. In conclusion, SPC is a novel molecule protecting cardiomyocytes against apoptosis by promoting autophagy. The lipid raft/PTEN/Akt1/mTOR signal pathway is the underlying mechanism and might provide novel targets for cardiac failure therapy.

Possible involvement of sphingomyelin in the regulation of the plasma sphingosine 1-phosphate level in human subjects

ObjectivesSphingosine 1-phosphate (S1P) is a bioactive sphingolipid mediator. Although the plasma S1P concentration is reportedly determined by cellular components, including erythrocytes, platelets, and vascular endothelial cells, the possible involvement of other factors, such as serum sphingomyelin (SM) and autotaxin (ATX), remains to be elucidated. Design and methodsWe measured S1P using high-performance liquid chromatography (HPLC), SM and lysophosphatidic acid (LPA) using enzymatic assays, ATX antigen using a two-site enzyme immunoassay, and ATX activity using a lysophospholipase D activity assay. To fractionate the lipoproteins, plasma samples were separated using fast protein liquid chromatography (FPLC) utilizing a Superose 6 column. ResultsThe plasma S1P level was positively correlated with the levels of SM and lysophosphatidylcholine, but not with the level of phosphatidylcholine. Although SM was present in the very low-density lipoprotein (VLDL) fraction, neither the plasma S1P level nor the SM level was affected by feeding. The plasma S1P level was negatively correlated with the ATX activity. Although the incubation of 100μmol/L of sphingosylphosphorylcholine (SPC) with the serum resulted in a significant increase in the S1P level because of the presence of ATX, the physiological concentration of SPC did not mimic this effect. ConclusionThe plasma S1P level was affected by the serum SM level, while the possibility of ATX involvement in the increase in the plasma S1P level was considered to be remote at least in healthy human subjects.

Sphingosylphosphorylcholine potentiates vasoreactivity and voltage-gated Ca2+ entry via NOX1 and reactive oxygen species.

AimsSphingosylphosphorylcholine (SPC) elicits vasoconstriction at micromolar concentrations. At lower concentrations (≤1 µmol/L), however, it does not constrict intrapulmonary arteries (IPAs), but strongly potentiates vasoreactivity. Our aim was to determine whether this also occurs in a systemic artery and to delineate the signalling pathway.Methods and resultsRat mesenteric arteries and IPAs mounted on a myograph were challenged with ∼25 mmol/L [K+] to induce a small vasoconstriction. SPC (1 µmol/L) dramatically potentiated this constriction in all arteries by ∼400%. The potentiation was greatly suppressed or abolished by inhibition of phospholipase C (PLC; U73122), PKCε (inhibitory peptide), Src (PP2), and NADPH oxidase (VAS2870), and also by Tempol (superoxide scavenger), but not by inhibition of Rho kinase (Y27632). Potentiation was lost in mesenteric arteries from p47phox–/–, but not NOX2−/–, mice. The intracellular superoxide generator LY83583 mimicked the effect of SPC. SPC elevated reactive oxygen species (ROS) in vascular smooth muscle cells, and this was blocked by PP2, VAS2870, and siRNA knockdown of PKCε. SPC (1 µmol/L) significantly reduced the EC50 for U46619-induced vasoconstriction, an action ablated by Tempol. In patch-clamped mesenteric artery cells, SPC (200 nmol/L) enhanced Ba2+ current through L-type Ca2+ channels, an action abolished by Tempol but mimicked by LY83583.ConclusionOur results suggest that low concentrations of SPC activate a PLC-coupled and NOX1-mediated increase in ROS, with consequent enhancement of voltage-gated Ca2+ entry and thus vasoreactivity. We speculate that this pathway is not specific for SPC, but may also contribute to vasoconstriction elicited by other G-protein coupled receptor and PLC-coupled agonists.

A novel trigger for cholesterol-dependent smooth muscle contraction mediated by the sphingosylphosphorylcholine-Rho-kinase pathway in the rat basilar artery: a mechanistic role for lipid rafts.

Hyperlipidemia is a risk factor for abnormal cerebrovascular events. Rafts are cholesterol-enriched membrane microdomains that influence signal transduction. We previously showed that Rho-kinase-mediated Ca(2+) sensitization of vascular smooth muscle (VSM) induced by sphingosylphosphorylcholine (SPC) has a pivotal role in cerebral vasospasm. The goals of the study were to show SPC-Rho-kinase-mediated VSM contraction in vivo and to link this effect to cholesterol and rafts. The SPC-induced VSM contraction measured using a cranial window model was reversed by Y-27632, a Rho-kinase inhibitor, in rats fed a control diet. The extent of SPC-induced contraction correlated with serum total cholesterol. Total cholesterol levels in the internal carotid artery (ICA) were significantly higher in rats fed a cholesterol diet compared with a control diet or a β-cyclodextrin diet, which depletes VSM cholesterol. Western blotting and real-time PCR revealed increases in flotillin-1, a raft marker, and flotillin-1 mRNA in the ICA in rats fed a cholesterol diet, but not in rats fed the β-cyclodextrin diet. Depletion of cholesterol decreased rafts in VSM cells, and prevention of an increase in cholesterol by β-cyclodextrin inhibited SPC-induced contraction in a cranial window model. These results indicate that cholesterol potentiates SPC-Rho-kinase-mediated contractions of importance in cerebral vasospasm and are compatible with a role for rafts in this process.

Sphingolipids inhibit vimentin-dependent cell migration.

The sphingolipids, sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine (SPC), can induce or inhibit cellular migration. The intermediate filament protein vimentin is an inducer of migration and a marker for epithelial-mesenchymal transition. Given that keratin intermediate filaments are regulated by SPC, with consequences for cell motility, we wanted to determine whether vimentin is also regulated by sphingolipid signalling and whether it is a determinant for sphingolipid-mediated functions. In cancer cells where S1P and SPC inhibited migration, we observed that S1P and SPC induced phosphorylation of vimentin on S71, leading to a corresponding reorganization of vimentin filaments. These effects were sphingolipid-signalling-dependent, because inhibition of either the S1P2 receptor (also known as S1PR2) or its downstream effector Rho-associated kinase (ROCK, for which there are two isoforms ROCK1 and ROCK2) nullified the sphingolipid-induced effects on vimentin organization and S71 phosphorylation. Furthermore, the anti-migratory effect of S1P and SPC could be prevented by expressing S71-phosphorylation-deficient vimentin. In addition, we demonstrated, by using wild-type and vimentin-knockout mouse embryonic fibroblasts, that the sphingolipid-mediated inhibition of migration is dependent on vimentin. These results imply that this newly discovered sphingolipid-vimentin signalling axis exerts brake-and-throttle functions in the regulation of cell migration.

Inhibition of autophagy promoted sphingosylphosphorylcholine induced cell death in non-small cell lung cancer cells

Sphingosylphosphorylcholine (SPC) is a bioactive lipid mediated popular cell apoptosis in cancer cells. As a cell-specific sphingolipid, its function in lung cancer cells is unknown. Here we showed that SPC treatment triggered necrosis and autophagy but inhibited apoptosis in two non-small cell lung cancer cell lines: A549 cell line and H157 cell line. Then 3-methyladenine (3-MA), an autophagy inhibitor, was introduced to clarify the relationships between autophagy and necrosis or apoptosis. 3MA suppressed the survival furtherly by promoting apoptosis while had no influence on necrosis. Subsequent studies revealed that activity of AKT and mammalian target of rapamycin (mTOR) complex 1 (mTORC1) were downregulated during autophagy. Furthermore, SPC failed to promote autophagy in p53 deleted cells. Thus SPC induced autophagy in non-small cell lung cancer cells was through AKT/mTORC1 and P53 signal pathway. Besides, SPC reduced both the mitochondria membrane potential and ROS level in A549 cells. These findings provided a molecular basis of SPC-stimulated A549 cell death and support the notion that inhibition of autophagy is likely a novel anticancer mechanism.

Discovery of a Novel 2,6-Difunctionalized 2H-Benzopyran Inhibitors Toward Sphingosylphosphorylcholine Synthetic Pathway as New Anti-inflammatory Target

Novel 2,6-difuctionalized 2H-benzopyrans were synthesized and evaluated for a sphingosylphosphorylcholine(SPC) inhibitor. The synthetic 2H-benzopyrans 1c and 3a showed high potency in SPC-induced cell proliferation assay (<TEX>$IC_{50}$</TEX> < 20 nM). Neither hERG <TEX>$K^+$</TEX> channel binding (> <TEX>$10{\mu}M$</TEX>) nor CYP inhibitions (> <TEX>$10{\mu}M$</TEX>) were observed. Also, the simple structure-activity relationship (SAR) results were obtained from analysis of 2H-benzopyran derivatives 1-3 and the anti-SPC effect of 2H-benzopyran 1c was confirmed by a HUVEC tube formation assay.

Effects of sphingosylphosphorylcholine against oxidative stress and acute lung ınjury ınduced by pulmonary contusion in rats

Background/purposeThe goal of this study was to evaluate effects of exogenous sphingosylphosphorylcholine (SPC) administration on acute lung injury induced by pulmonary contusion in rats. MethodsEight animals were included in each of the following five groups: control, contusion, contusion phosphate-buffered solution (PBS), contusion SPC 2, contusion SPC 10. SPC was administered 3days at a daily two different doses of 2μm/ml and 10μm/ml intraperitoneally. The severity of lung injury was determined by the neutrophil activation and histological and immunohistochemical changes in the lung. Malondialdehyde (MDA), nitric oxide (NO), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione (GSH) were determined to evaluate the oxidative status in the lung tissue. ResultsTreatment with 2μM SPC inhibited the increase in lung MDA and NO levels significantly and also attenuated the depletion of SOD, GPx, and GSH in the lung injury induced by pulmonary contusion. These data were supported by histopathological findings. The inducible nitric oxide synthase (iNOS) positive cells and apoptotic cells in the lung tissue were observed to be reduced with the 2μM SPC treatment. But, the 10μM SPC treatment did not provide similar effects. ConclusionsIn conclusion, these findings suggested that 2μM SPC can attenuate lung damage in pulmonary contusion by prevention of oxidative stress, inflammatory process and apoptosis. All these findings suggest that low dose SPC may be a promising new therapeutic agent for acute lung injury.

Relations between GPR4 Expression, Microvascular Density (MVD) and Clinical Pathological Characteristics of Patients with Epithelial Ovarian Carcinoma (EOC)

G-protein coupled receptor 4 (GPR4) is a G protein-coupled receptor (GPCR) activated by sphingosylphosphorylcholine (SPC) and lysophosphatidylcholine (LPC). Later studies indicated that GPR4 can serve as a proton sensor. GPR4 has been known to play a critical role in the tube formation of vascular endothelial cells, and GPR4 overexpression is observed in various types of malignancies, suggesting its involvement in the cancer-related angiogenesis. In this study, we examined the GPR4 expression levels in blood vessels of ovarian cancer, and analyzed the relationship between GPR4 expression and the clinical and pathological characteristics of patients with epithelial ovarian carcinomas (EOC). Results from immunohistochemistry showed that GPR4 is detectable in the endothelium of vessels of both EOC and benign ovarian tumor tissue, but the expression levels were significantly increased in EOC. Moreover the increased expression is accompanied by a higher microvascular density (MVD) in EOC compared to that in the benign ovarian tumors. We demonstrated a positive correlation between GPR4 expression density and MVD in EOC, but not benign ovarian tumor tissues. Further analyses indicated that GPR4 expression and MVD in EOC were correlated to the status of lymph node metastasis and clinical stage, but not significantly correlated to the pathological classifications, histopathological grades, the amounts of ascites, status of peritoneal cytology, tumor sizes, or patients' ages. These results suggested that GPR4 may play an important role in the development of EOC, and its overexpression might be required for the angiogenesis, tumor growth, and metastasis of EOC.

Sphingosylphosphorylcholine induces α-smooth muscle actin expression in human lung fibroblasts and fibroblast-mediated gel contraction via S1P2 receptor and Rho/Rho-kinase pathway

Chronic airway diseases like COPD and asthma are usually accompanied with airway fibrosis. Myofibroblasts, which are characterized by expression of smooth muscle actin (α-SMA), play an important role in a variety of developmental and pathological processes, including fibrosis and wound healing. Sphingosylphosphorylcholine (SPC), a sphingolipid metabolite, has been implicated in many physiological and pathological conditions. The current study tested the hypothesis that SPC may modulate tissue remodeling by affecting the expression of α-SMA in human fetal lung fibroblast (HFL-1) and fibroblast mediated gel contraction. The results show that SPC stimulates α-SMA expression in HFL-1 and augments HFL-1 mediated collagen gel contraction in a time- and concentration-dependent manner. The α-SMA protein expression and fibroblast gel contraction induced by SPC was not blocked by TGF-β1 neutralizing antibody. However, it was significantly blocked by S1P2 receptor antagonist JTE-013, the Rho-specific inhibitor C3 exoenzyme, and a Rho-kinase inhibitor Y-27632. These findings suggest that SPC stimulates α-SMA protein expression and HFL-1 mediated collagen gel contraction via S1P2 receptor and Rho/Rho kinase pathway, and by which mechanism, SPC may be involved in lung tissue remodeling.

Ethacrynic Acid Inhibits Sphingosylphosphorylcholine-Induced Keratin 8 Phosphorylation and Reorganization via Transglutaminase-2 Inhibition

Sphingosylphosphorylcholine (SPC) is significantly increased in the malicious ascites of tumor patients and induces perinuclear reorganization of keratin 8 (K8) filaments in PANC-1 cells. The reorganization contributes to the viscoelasticity of metastatic cancer cells resulting in increased migration. Recently, we reported that transglutaminase-2 (Tgase-2) is involved in SPC-induced K8 phosphorylation and reorganization. However, effects of Tgase-2 inhibitors on SPC-induced K8 phosphorylation and reorganization were not clearly studied. We found that ethacrynic acid (ECA) concentration-dependently inhibited Tgase-2. Therefore, we examined the effects of ECA on SPC-induced K8 phosphorylation and reorganization. ECA concentration-dependently suppressed the SPC-induced phosphorylation and perinuclear reorganization of K8. ECA also suppressed the SPC-induced migration and invasion. SPC induced JNK activation through Tgase-2 expression and ECA suppressed the activation and expression of JNK in PANC-1 cells. These results suggested that ECA might be useful to control Tgase-2 dependent metastasis of cancer cells such as pancreatic cancer and lung cancers.

Sustained activation of sphingomyelin synthase by 2-hydroxyoleic acid induces sphingolipidosis in tumor cells

The mechanism of action of 2-hydroxyoleic acid (2OHOA), a potent antitumor drug, involves the rapid and specific activation of sphingomyelin synthase (SMS), leading to a 4-fold increase in SM mass in tumor cells. In the present study, we investigated the source of the ceramides required to sustain this dramatic increase in SM. Through radioactive and fluorescent labeling, we demonstrated that sphingolipid metabolism was altered by a 24 h exposure to 2OHOA, and we observed a consistent increase in the number of lysosomes and the presence of unidentified storage materials in treated cells. Mass spectroscopy revealed that different sphingolipid classes accumulated in human glioma U118 cells after exposure to 2OHOA, demonstrating a specific effect on C16-, C20-, and C22-containing sphingolipids. Based on these findings, we propose that the demand for ceramides required to sustain the SMS activation (ca. 200-fold higher than the basal level) profoundly modifies both sphingolipid and phospholipid metabolism. As the treatment is prolonged, tumor cells fail to adequately metabolize sphingolipids, leading to a situation resembling sphingolipidosis, whereby cell viability is compromised.

Abstract WP437: The Relationship Between Cholesterol And Cerebral Vasospasm In A Rat Model Of Subarachnoid Hemorrhage: A Mechanistic Role For Lipid Rafts

Objective: Rho-kinase (ROK)-mediated vascular smooth muscle (VSM) contraction plays a pivotal role in cerebral vasospasm (CV). Sphingosylphosphorylcholine (SPC), a sphingolipid, induces ROK-mediated VSM contraction, and hypercholesterolemia increases SPC-ROK-mediated VSM contraction. Lipid rafts are cholesterol-enriched membrane microdomains that influence signal transduction. In this study, we report the effect of cholesterol and lipid rafts on CV. Methods: Adult male Sprague-Dawley rats (400-500g, n=53) were fed control, 1% cholesterol or 1% cholesterol +5% β-cyclodextrin (β-CD), which depletes VSM cholesterol, diets for 8 weeks. We used a double-hemorrhage rat subarachnoid hemorrhage (SAH) model, and investigated CV of the basilar artery (BA) at Day5 using a cranial window. T-Cho levels in the internal carotid artery (ICA) were measured using Gas Chromatography. The expression of Flotillin-1, a raft marker, and Flotillin-1 mRNA in the ICA were analysed by Western blot and real-time RT-PCR, respectively. Results: The BA diameters were 358.94±32.05μm and 302.21±38.36μm in control diet (non-treated) and SAH rats, respectively (n=9/group, p&lt;0.05 vs. non-treated). SAH-induced BA contraction was markedly reversed by intracisternal infusion of 10μM Y27632 (n=4, p&lt;0.05), a ROK inhibitor. The BA diameter in SAH rats fed the cholesterol diet was 248.11±34.56μm (n=6, p&lt;0.05 vs. control SAH-rats), compared with 305±25.28μm in SAH rats fed a β-CD diet (n=6, p&lt;0.05 vs. SAH-rat fed a cholesterol diet). T-Cho levels in the ICA were significantly higher in rats fed a cholesterol diet compared with the control diet (0.82±0.24 vs. 0.49±0.09mg/g, n=5/group, p=0.028) or the β-CD diet (0.47±0.06 mg/g, n=5, p=0.016 vs. cholesterol diet). Western blot analysis and real-time RT-PCR revealed increases in Flotillin-1 and Flotillin-1 mRNA expression in the ICA from rats fed a cholesterol diet, whereas Flotillin-1 and Flotillin-1 mRNA expression were not altered in the ICA from rats fed the β-CD diet. Conclusions: These results indicate that cholesterol potentiates SAH-induced CV and are compatible with a role for lipid rafts in this process.

Plasma choline-containing phospholipids: potential biomarkers for colorectal cancer progression

Colorectal cancer (CRC) is believed to progress through the adenoma–carcinoma sequence. The adenoma–carcinoma transition is an important window for early detection and intervention of CRC. In the present study, plasma samples from patients with CRC (n = 120), patients with adenomatous polyps (AP) (n = 120), and healthy controls (n = 120) were collected. Plasma phospholipid levels were analyzed with liquid chromatography–tandem mass spectrometry. It was found that the plasma levels of major lysophosphatidylcholine (LPC) species were gradationally decreased from healthy controls, AP to CRC subjects. A formula including total saturated LPCs, 18:2 LPC and sphingosylphosphorylcholine (SPC) yielded a sensitivity and specificity of 88.3 and 80 % for separating CRC from healthy controls. An optimized model with total saturated LPCs, 20:4 LPC and sphingomyelins (SM) as markers yielded a sensitivity and specificity of 89 and 80 % for separating AP from the healthy controls. Moreover, with SM, SPC and saturated LPCs as markers, a model was made to separate CRC from AP with the sensitivity and specificity of 90 and 92.5 %, respectively. These data indicate that the plasma choline-containing phospholipid levels represent potential biomarkers to distinguish between healthy controls, AP and CRC cases, implying their clinical usage in CRC and/or AP-CRC progression detection.

The G Protein Coupled Receptor 3 Is Involved in cAMP and cGMP Signaling and Maintenance of Meiotic Arrest in Porcine Oocytes

The arrest of meiotic prophase in mammalian oocytes within fully grown follicles is dependent on cyclic adenosine monophosphate (cAMP) regulation. A large part of cAMP is produced by the Gs-linked G-protein-coupled receptor (GPR) pathway. In the present study, we examined whether GPR3 is involved in the maintenance of meiotic arrest in porcine oocytes. Expression and distribution of GPR3 were examined by western blot and immunofluorescence microscopy, respectively. The results showed that GPR3 was expressed at various stages during porcine oocyte maturation. At the germinal vesicle (GV) stage, GPR3 displayed a maximal expression level, and its expression remained stable from pro-metaphase I (MI) to metaphase II (MII). Immunofluorescence staining showed that GPR3 was mainly distributed at the nuclear envelope during the GV stage and localized to the plasma membrane at pro-MI, MI and MII stages. RNA interference (RNAi) was used to knock down the GPR3 expression within oocytes. Injection of small interfering double-stranded RNA (siRNA) targeting GPR3 stimulated meiotic resumption of oocytes. On the other hand, overexpression of GPR3 inhibited meiotic maturation of porcine oocytes, which was caused by increase of cGMP and cAMP levels and inhibition of cyclin B accumulation. Furthermore, incubation of porcine oocytes with the GPR3 ligand sphingosylphosphorylcholine (SPC) inhibited oocyte maturation. We propose that GPR3 is required for maintenance of meiotic arrest in porcine oocytes through pathways involved in the regulation of cAMP and cGMP.

PP2A and DUSP6 are involved in sphingosylphosphorylcholine-induced hypopigmentation

Activation of extracellular signal-related kinase (ERK) is involved in decreased melanogenesis by sphingosylphosphorylcholine (SPC). In the present study, we confirmed that SPC activated ERK and that a specific inhibitor of the ERK pathway (PD98059) recovered SPC-induced hypopigmentation. Moreover, we found that SPC significantly reduces protein phosphatase 2A (PP2A) activity in Mel-Ab cells, and that PP2A activator treatment abrogated SPC-induced hypopigmentation. We determined that α-melanocyte-stimulating hormone (α-MSH) increased the expression of dual-specificity phosphatase 6 (DUSP6), an ERK phosphatase, in a time-dependent manner. In contrast, SPC decreased the level of DUSP6 in Mel-Ab cells. Furthermore, inhibiting DUSP6 increased ERK activation and subsequently augmented the SPC-induced hypopigmenting effects. Taken together, our data suggest that SPC-induced phosphatase inhibition is also responsible for the hypopigmentary effects.

Sub‐contractile sphingosylphosphorylcholine enhances vasoreactivity via reactive oxygen species

Sphingosylphosphorylcholine (SPC), at concentrations that do not cause contraction (&lt;1uM), causes potentiation of vasoconstriction to other agonists or depolarisation (Snetkov et al, Hypertension 2008;51:239–245). Our objective was to determine whether this was related to activation of NADPH oxidase and production of reactive oxygen species (ROS).METHODSSmall pulmonary or mesenteric arteries were mounted on a myograph, challenged with depolarising solutions containing ~23 mM KCl to elicit a small contraction, and incubated with SPC (1uM) before being re‐challenged.RESULTS0.2 to 1uM SPC caused substantial potentiation of depolarisation‐induced force to between 400 and 800% of control in both rat and mouse arteries. Tempol (3mM) suppressed potentiation (e.g. PA: 526 +/− 220%; +tempol: 200+/−60%, n=5). SPC enhanced ROS production estimated by lucigenin and DCF. Potentiation was abolished in p47phox −/− mice (n=6). 0.2 uM SPC enhanced voltage activated calcium current in whole cell patch by ~30% (p&lt;0.01), and this was mimicked by exogenous ROS. We conclude that low concentrations of SPC may cause hyperreactivity via NADPH oxidase and elevation of ROS, which enhances calcium entry via L‐type channels. Funded by the Wellcome Trust

Keratin 8 phosphorylation regulates keratin reorganization and migration of epithelial tumor cells

Cell migration and invasion are largely dependent on the complex organization of the various cytoskeletal components. Whereas the role of actin filaments and microtubules in cell motility is well established, the role of intermediate filaments in this process is incompletely understood. Organization and structure of the keratin cytoskeleton, which consists of heteropolymers of at least one type 1 and one type 2 intermediate filament, are in part regulated by post-translational modifications. In particular, phosphorylation events influence the properties of the keratin network. Sphingosylphosphorylcholine (SPC) is a bioactive lipid with the exceptional ability to change the organization of the keratin cytoskeleton, leading to reorganization of keratin filaments, increased elasticity, and subsequently increased migration of epithelial tumor cells. Here we investigate the signaling pathways that mediate SPC-induced keratin reorganization and the role of keratin phosphorylation in this process. We establish that the MEK-ERK signaling cascade regulates both SPC-induced keratin phosphorylation and reorganization in human pancreatic and gastric cancer cells and identify Ser431 in keratin 8 as the crucial residue whose phosphorylation is required and sufficient to induce keratin reorganization and consequently enhanced migration of human epithelial tumor cells.