Sphingosine-1-phosphate and ceramide-1-phosphate promote migration, pro-inflammatory and pro-fibrotic responses in retinal pigment epithelium cells.

Retinal pigment epithelium (RPE) cells, essential for preserving retina homeostasis, also contribute to the development of retina proliferative diseases, through their exacerbated migration, epithelial to mesenchymal transition (EMT) and inflammatory response. Uncovering the mechanisms inducing these changes is crucial for designing effective treatments for these pathologies. Sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) are bioactive sphingolipids that promote migration and inflammation in several cell types; we recently established that they stimulate the migration of retina Müller glial cells (Simón et al., 2015; Vera et al., 2021). We here analyzed whether S1P and C1P regulate migration, inflammation and EMT in RPE cells. We cultured two human RPE cell lines, ARPE-19 and D407 cells, and supplemented them with either 5 µM S1P or 10 µM C1P, or their vehicles, for 24 h. Analysis of cell migration by the scratch wound assay showed that S1P addition significantly enhanced migration in both cell lines. Pre-treatment with W146 and BML-241, antagonists for S1P receptor 1 (S1P1) and 3 (S1P3), respectively, blocked exogenous S1P-induced migration. Inhibiting sphingosine kinase 1 (SphK1), the enzyme involved in S1P synthesis, significantly reduced cell migration and exogenous S1P only partially restored it. Addition of C1P markedly stimulated cell migration. Whereas inhibiting C1P synthesis did not affect C1P-induced migration, inhibiting S1P synthesis strikingly decreased it; noteworthy, addition of C1P promoted the transcription of SphK1. These results suggest that S1P and C1P stimulate RPE cell migration and their effect requires S1P endogenous synthesis. Both S1P and C1P increase the transcription of pro-inflammatory cytokines IL-6 and IL-8, and of EMT marker α-smooth muscle actin (α-SMA) in ARPE-19 cells. Collectively, our results suggest new roles for S1P and C1P in the regulation of RPE cell migration and inflammation; since the deregulation of sphingolipid metabolism is involved in several proliferative retinopathies, targeting their metabolism might provide new tools for treating these pathologies.

Permissive Modulation of Sphingosine-1-Phosphate-Enhanced Intracellular Calcium on BKCa Channel of Chromaffin Cells.

Sphingosine-1-phosphate (S1P), is a signaling sphingolipid which acts as a bioactive lipid mediator. We assessed whether S1P had multiplex effects in regulating the large-conductance Ca2+-activated K+ channel (BKCa) in catecholamine-secreting chromaffin cells. Using multiple patch-clamp modes, Ca2+ imaging, and computational modeling, we evaluated the effects of S1P on the Ca2+-activated K+ currents (IK(Ca)) in bovine adrenal chromaffin cells and in a pheochromocytoma cell line (PC12). In outside-out patches, the open probability of BKCa channel was reduced with a mean-closed time increment, but without a conductance change in response to a low-concentration S1P (1 µM). The intracellular Ca2+ concentration (Cai) was elevated in response to a high-dose (10 µM) but not low-dose of S1P. The single-channel activity of BKCa was also enhanced by S1P (10 µM) in the cell-attached recording of chromaffin cells. In the whole-cell voltage-clamp, a low-dose S1P (1 µM) suppressed IK(Ca), whereas a high-dose S1P (10 µM) produced a biphasic response in the amplitude of IK(Ca), i.e., an initial decrease followed by a sustained increase. The S1P-induced IK(Ca) enhancement was abolished by BAPTA. Current-clamp studies showed that S1P (1 µM) increased the action potential (AP) firing. Simulation data revealed that the decreased BKCa conductance leads to increased AP firings in a modeling chromaffin cell. Over a similar dosage range, S1P (1 µM) inhibited IK(Ca) and the permissive role of S1P on the BKCa activity was also effectively observed in the PC12 cell system. The S1P-mediated IK(Ca) stimulation may result from the elevated Cai, whereas the inhibition of BKCa activity by S1P appears to be direct. By the differentiated tailoring BKCa channel function, S1P can modulate stimulus-secretion coupling in chromaffin cells.

Effects of low-dose unfractionated heparin on early brain injury after subarachnoid hemorrhage in mice.

BACKGROUND: Sphingosine kinase (SphK) 1 has been reported as an important signaling node in anti-apoptotic signaling. Heparin is a pleiotropic drug that antagonizes many pathophysiological mechanisms. In this study, we evaluated if heparin prevents early brain injury (EBI) after subarachnoid hemorrhage (SAH) by anti-apoptotic mechanisms including SphK1. METHODS: SAH was induced by endovascular perforation in mice, which were randomly assigned to sham-operated (n = 23), SAH + vehicle (n = 36), SAH + 10U heparin pretreatment (n = 13), SAH + 30U heparin pretreatment (n = 15), SAH + 10U heparin posttreatment (n = 31), and SAH + 30U heparin posttreatment (n = 23). At 24 hours post-SAH, neurological scores, brain water content and Evans blue extravasation were evaluated. Also, the expression of SphK, phosphorylated Akt, and cleaved caspase-3 was determined by Western blotting, and cell death was examined by terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling staining. RESULTS: Low-dose heparin posttreatment improved neurobehavioral function, brain edema, blood-brain barrier disruption and cell death in the cortex, associated with an increase in SphK1 and phosphorylated Akt, and a decrease in cleaved caspase-3. High-dose heparin had a tendency for increased SAH severity, which obscured the neuroprotective effects by heparin. CONCLUSIONS: Low-dose heparin posttreatment may decrease the development of post-SAH EBI through anti-apoptotic mechanisms including sphingosine-related pathway activation.

The phytosphingosine-CD300b interaction promotes zymosan-induced, nitric oxide-dependent neutrophil recruitment.

Zymosan is a glucan that is a component of the yeast cell wall. Here, we determined the mechanisms underlying the zymosan-induced accumulation of neutrophils in mice. Loss of the receptor CD300b reduced the number of neutrophils recruited to dorsal air pouches in response to zymosan, but not in response to lipopolysaccharide (LPS), a bacterial membrane component recognized by Toll-like receptor 4 (TLR4). An inhibitor of nitric oxide (NO) synthesis reduced the number of neutrophils in the zymosan-treated air pouches of wild-type mice to an amount comparable to that in CD300b-/- mice. Treatment with clodronate liposomes decreased the number of NO-producing, CD300b+ inflammatory dendritic cells (DCs) in wild-type mice, thus decreasing NO production and neutrophil recruitment. Similarly, CD300b deficiency decreased the NO-dependent recruitment of neutrophils to zymosan-treated joint cavities, thus ameliorating subsequent arthritis. We identified phytosphingosine, a lipid component of zymosan, as a potential ligand of CD300b. Phytosphingosine stimulated NO production in inflammatory DCs and promoted neutrophil recruitment in a CD300b-dependent manner. Together, these results suggest that the phytosphingosine-CD300b interaction promotes zymosan-dependent neutrophil accumulation by inducing NO production by inflammatory DCs and that CD300b may contribute to antifungal immunity.

Metabolic profiling of hypoxia/reoxygenation injury in H9c2 cells reveals the accumulation of phytosphingosine and the vital role of Dan-Shen in Xin-Ke-Shu.

BACKGROUND: Xin-Ke-Shu (XKS), a patent medicine consisting of five commonly used traditional Chinese herbs, is used for the treatment of coronary heart diseases. A previous study showed that XKS has protective effects for ameliorating myocardial ischemia/reperfusion (I/R) injury. PURPOSE: This study was aimed to deeply understand the mechanisms and compatible principle of XKS against hypoxia/reoxygenation (H/R) injury and the contribution of each single herb to the efficacy of XKS. METHODS: An H/R model in H9c2 cardiomyocytes was applied to mimic I/R injury observed in vivo. The cell viability, the levels of LDH, MDA, SOD, and apoptosis were determined to evaluate the cardioprotection of XKS and its subtracted formula (knocked out one herb) in H/R injury. Cell metabolomics, combined with western blot analysis, was performed to uncover the inert molecular mechanism of XKS against H/R injury. RESULTS: Significant protective effects of XKS against oxidative stress and apoptosis induced by H/R injury were found in the pharmacodynamic evaluation. Moreover, the metabolic profile deviation of the H/R group from the control group was mainly ascribed to thirteen metabolites involved in four aberrant pathways, in which sphingolipid metabolism was revealed as the most relevant pathway involved in H/R injury (impact > 0.1). Notably, the accumulation of phytosphingosine (VIP = 5.84) was considered the most likely characteristic in H/R injury, which is well known to promote the opening of the mitochondrial permeability transition pore (mPTP) and activate cell apoptosis. Furthermore, XKS ameliorated all the abnormalities of the metabolic network in response to H/R injury. In agreement with this, a western blot analysis showed that XKS markedly regulated the over-expression of CaMK II and cleaved caspase-3. However, the subtracted formula showed no significant difference in comparison with the XKS group on protecting H/R injury except for QDS (subtracted Dan-Shen from XKS). CONCLUSION: The roots of Salvia miltiorrhiza Bge. (Dan-Shen) play an important role in the regulation of Ca2+ overloading, oxidative stress and apoptosis in H/R injury. Our study enabled information from holistic cell metabolomics to be used for mechanism and compatibility rule elucidations of TCMs.

Sphingosine-1-phosphate activates the AKT pathway to inhibit chemotherapy induced human granulosa cell apoptosis.

To investigate whether sphingosine-1-phosphate (S1P), an apoptosis-inhibitor would be able to inhibit chemotherapy induced human granulosa cell apoptosis. Cultures of primary granulosa cells were isolated from women undergoing in vitro fertilization (IVF). MTT assay was used to measure the optimum concentration of CTX and S1P acts on human granulosa cells. Granulosa cells were added with pertussis toxin (PTX), the PI3K inhibitor LY294002. Western blot analysis was used to analyze the signaling pathway of proteins and cell apoptosis. We found that S1P (10 mm) statistically significantly decreased granulosa cell apoptosis after cyclophosphamide (CTX) treatment. The decreased cell apoptosis induced by S1P was abolished after treatment with LY294002, PI3K inhibitor. CONCLUSIONS: Treatment with S1P can inhibit the CTX-induced granulosa cell apoptosis. The S1P protective effect is mediated by activating the PI3K/Akt pathway.

Transcriptional modulation of SLC26A3 (DRA) by sphingosine-1-phosphate.

SLC26A3 or Downregulated in adenoma (DRA) is the major Cl(-)/HCO3 (-) exchanger involved in electroneutral NaCl absorption in the mammalian intestine. Alterations in DRA function and expression have been implicated in diarrheal diseases associated with inflammation or infection. Therefore, agents that upregulate DRA activity may serve as potential antidiarrheals. In this regard, sphingosine-1-phosphate (S1P), a member of the bioactive sphingolipid family, has been shown to modulate various cellular processes including improvement of intestinal barrier function. However, the role of S1P in modulating intestinal chloride absorption by regulating DRA is not known. Therefore, the present studies were designed to examine the direct effects of S1P on apical Cl(-)/HCO3 (-) exchange activity and DRA expression. S1P significantly increased Cl(-)/HCO3 (-) exchange activity and also significantly increased DRA mRNA and protein expression. Increased DRA mRNA by S1P was accompanied by enhanced DRA promoter activity, indicating involvement of transcriptional mechanisms. The specific S1P receptor subtype-2 (S1PR2) antagonist JTE-013 blocked the stimulatory effects of S1P on DRA promoter activity, indicating the involvement of S1PR2 S1P-mediated increase in DRA promoter activity involved PI3K/Akt pathway. Progressive deletions of the DRA promoter indicated that the putative S1P-responsive elements are present in the -790/-398 region of the DRA promoter. Furthermore, results obtained from electrophoretic mobility shift assay showed that S1P stimulated DRA promoter activity via increased binding of Ying-Yang1 (YY1) in the S1P-responsive region. In conclusion, transcriptional modulation of DRA expression and function in response to S1P through a PI3/Akt pathway represents a novel role of S1P as a potential proabsorptive agent.

FTY720 mitigates torsion/detorsion-induced testicular injury in rats.

BACKGROUND: FTY720, a sphingosine-1-phosphate (S1P) receptor agonist, possesses potent anti-inflammation capacity. We evaluated the therapeutic potentials of FTY720 against testicular injury induced by testicular torsion and/or detorsion (T/D). MATERIALS AND METHODS: Young adult male Sprague-Dawley rats were allocated to receive T/D (the T/D group) and T/D plus FTY720 (4 mg/kg, the T/D-FTY group, n = 6 in each group). To investigate the possible roles of the S1P receptors, another group of rats received T/D plus FTY720 plus the potent S1P receptor antagonist VPC23019 (1 mg/kg, the T/D-FTY-VPC group, n = 6). FTY720 was administered immediately before testicular detorsion, and VPC23019 was administered 30 min before FTY720. Another set of rats that received sham operation, immediately followed by injection of normal saline, FTY720, or FTY720 plus VPC23019, served as control groups. Sham control groups were run simultaneously. After euthanization, levels of testicular injury were measured. RESULTS: Histologic findings revealed severe testicular injury changes in both the T/D and T/D-FTY-VPC groups and moderate testicular injury changes in the T/D-FTY group. In addition, malondialdehyde activity (oxidative status), concentration of interleukin-1ß (inflammation index), myeloperoxidase activity (neutrophil infiltration index), and wet-to-dry weight ratio (tissue edema index) of both the T/D and T/D-FTY-VPC groups were significantly higher than those of the T/D-FTY group. These data confirmed the protective effects of FTY720 against testicular T/D. Moreover, antagonizing the S1P receptors could reverse the protective effects of FTY720. CONCLUSIONS: FTY720 significantly mitigated testicular injury induced by testicular T/D. The mechanisms may involve activating the S1P receptors.

Differential activation of receptors and signal pathways upon stimulation by different doses of sphingosine-1-phosphate in endothelial cells.

NEW FINDINGS: What is the central question of this study? Why do different doses of sphingosine-1-phosphate (S1P) induce distinct biological effects in endothelial cells? What is the main finding and its importance? S1P at physiological concentrations preserved endothelial barrier function by binding to S1P receptor 1, then triggering Ca(2+) release from endoplasmic reticulum through phosphoinositide phospholipase C and inositol triphosphate, and consequently strengthening tight junction and F-actin assembly through Rac1 activation. Excessive S1P induced endothelial malfunction by activating S1P receptor 2 and RhoA/ROCK pathway, causing F-actin and tight junction disorganisation. Extracellular Ca(2+) influx was involved in this process. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid in plasma, and its plasma concentration can be adjusted through a complex metabolic process. The alterations in S1P levels and the activation of receptors collaboratively regulate distinct biological effects. This study was performed to investigate comparatively the effect of different concentrations of S1P on endothelial barrier function and to explore the roles of S1P receptors (S1PRs), Rho GTPases and calcium in S1P-induced endothelial responses. Endothelial barrier function was studied using transendothelial electric resistance and a resistance meter in human umbilical vein endothelial cells. Specific agonists or antagonists were applied to control the activation of S1P receptors and the release of calcium from different cellular compartments. The results indicated that at physiological concentrations, S1P preserved endothelial barrier function by binding with S1PR1. The activation of S1PR1 triggered the release of intracellular Ca(2+) from the endoplasmic reticulum through the PI-phospholipase C and inositol trisphosphate pathways. Consequently, the Rho GTPase Rac1 was activated, strengthening the assembly of tight junction proteins and F-actin. However, excessive S1P induced endothelial barrier dysfunction by activating S1PR2 followed by the RhoA/RhoA kinase pathway, causing the disorganization of F-actin and the disassembly of the tight junction protein ZO-1. An influx of extracellular Ca(2+) was involved in this process. These data suggest that physiological and excessive amounts of S1P induce different responses in human umbilical vein endothelial cells; the activation of the 1PR1-PLC-IP3 R-Ca(2+) -Rac1 pathway governs the low-dose S1P-enhanced endothelial barrier integrity, and the activation of S1PR2-calcium influx-RhoA/ROCK dominates the high-dose S1P-induced endothelial monolayer hyperpermeability response.

Sphingosine-1-phosphate stimulated connective tissue growth factor expression in human buccal fibroblasts: Inhibition by epigallocatechin-3-gallate.

BACKGROUND/PURPOSE: Connective tissue growth factor (CCN2) has been associated with the pathogenesis of various fibrotic diseases, including oral submucous fibrosis (OSF). The chemical constituents of areca nut along with the mechanical trauma cause OSF. The coarse fibers of areca nut injure the mucosa and hence sphingosine-1-phosphate (S1P) is released at the wounded sites. Recent studies have shown that S1P is involved in wound healing and the development of fibrosis. The aims of this study were to investigate the effects of S1P on CCN2 expression in human buccal fibroblasts (HBFs) and identify the potential targets for drug intervention or chemoprevention of OSF. METHODS: Western blot analyses were used to study the effects of S1P on CCN2 expression and its signaling pathways in HBFs and whether epigallocatechin-3-gallate (EGCG), the main and most significant polyphenol in green tea, could inhibit this pathway. RESULTS: S1P significantly enhanced CCN2 synthesis in HBFs. This effect can be inhibited by c-Jun NH2-terminal kinase (JNK) inhibitor and extracellular signal-regulated kinase inhibitor but not by P38 mitogen-activated protein kinase inhibitor. Interestingly, EGCG completely blocked S1P-induced CCN2 expression via suppressing S1P-induced JNK phosphorylation. CONCLUSION: S1P released by repetitive mechanical trauma during AN chewing may contribute to the pathogenesis of OSF through upregulating CCN2 expression in HBFs. EGCG could be an adjuvant to the current offered therapy options or the prevention of OSF through suppression of JNK activation.

Sphingosine 1-phosphate receptor-1 enhances mitochondrial function and reduces cisplatin-induced tubule injury.

Sphingosine 1-phosphate (S1P), the natural sphingolipid ligand for a family of five G protein- coupled receptors (S1P1-S1P5Rs), regulates cell survival and lymphocyte circulation. We have shown that the pan-S1PR agonist, FTY720, attenuates kidney ischemia-reperfusion injury by directly activating S1P1 on proximal tubule (PT) cells, independent of the canonical lymphopenic effects of S1P1 activation on B and T cells. FTY720 also reduces cisplatin-induced AKI. Therefore, in this study, we used conditional PT-S1P1-null (PepckCreS1pr1(fl/fl)) and control (PepckCreS1pr1(w/wt)) mice to determine whether the protective effect of FTY720 in AKI is mediated by PT-S1P1. Cisplatin induced more renal injury in PT-S1P1-null mice than in controls. Although FTY720 produced lymphopenia in both control and PT-S1P1-null mice, it reduced injury only in control mice. Furthermore, the increase in proinflammatory cytokine (CXCL1, MCP-1, TNF-α, and IL-6) expression and infiltration of neutrophils and macrophages induced by cisplatin treatment was attenuated by FTY720 in control mice but not in PT-S1P1-null mice. Similarly, S1P1 deletion rendered cultured PT cells more susceptible to cisplatin-induced injury, whereas S1P1 overexpression protected PT cells from injury and preserved mitochondrial function. We conclude that S1P1 may have an important role in stabilizing mitochondrial function and that FTY720 administration represents a novel strategy in the prevention of cisplatin-induced AKI.

Preclinical evaluation of the immunomodulatory lymphocyte trafficking drug FTY720 for HIV prevention in the female genital mucosa of macaques.

FTY720 has been shown to reduce inflammatory cytokines and immune cells in the genital mucosa of macaques. This pilot study examined the ability of FTY720 to inhibit HIV acquisition. Systemic treatment with FTY720 failed to prevent or delay vaginal SHIV transmission.

Sphingosine-1-phosphate receptor agonist, FTY720, restores coronary flow reserve in diabetic rats.

BACKGROUND: Impairment of coronary flow reserve (CFR) has been generally demonstrated in diabetic patients and animals with microvascular complications but without obvious obstructive coronary atherosclerosis. There have been few studies investigating CFR in cases of relatively well-controlled therapy. The purpose of this study is to evaluate the effect of treatment with a Sphingosine-1-phosphate (S1P) receptor potent agonist, FTY720, on early diabetic rats in terms of CFR. METHODS AND RESULTS: Male Sprague-Dawley (SD) rats were divided into 3 groups: (1) streptozotocin-uninjected rats (control rats); (2) streptozotocin-injected hyperglycemic rats (diabetic group); and (3) FTY720-fed and streptozotocin-injected hyperglycemic rats. FTY720 (1.25 mg/kg per day orally) was administrated for 9 weeks in SD rats (from 6 weeks old to 15 weeks old). CFR was evaluated by (13)NH3-positron emission tomography. No obvious pathological changes of macrovascular atherosclerosis were observed in each group. Diabetic rats had impaired CFR compared with the control group (1.39±0.26 vs. 1.94±0.24, P<0.05). Treatment with FTY720 for 9 weeks attenuated the heart histological changes and improved CFR in 32% of diabetic rats (1.84±0.36 vs. 1.39±0.26, P<0.05). CONCLUSIONS: In summary, long-term therapy with the Sphingosine-1-phosphate receptor agonist, FTY720, improved CFR by attenuating the heart histological changes, and it might have a beneficial effect on coronary microvascular function in diabetic rats.

Cationic ceramides and analogues, LCL30 and LCL85, as adjuvants to photodynamic therapy of tumors.

Photodynamic therapy (PDT) is known to alter the expression of various genes in treated cells. This prompted us to examine the activity of genes encoding two important enzymes in sphingolipid (SL) metabolism, dihydroceramide desaturase (DES) and sphingosine kinase (SPHK), in mouse SCCVII tumor cells treated by PDT using either the porphyrin-based photosensitizer Photofrin or silicon phthalocyanine Pc4. The results revealed that PDT induced an upregulation in the expression of two major isoforms of both genes (DES1 and DES2 as well as SPHK1 and SPHK2). While the changes were generally moderate (2-3-fold gains), the increase in DES2 expression was more pronounced and it was much greater with Photofrin-PDT than with Pc4-PDT (over 23-fold vs. less than 5-fold). Combining either Photofrin-PDT or Pc4-PDT with the cationic C16-ceramide LCL30 (20mg/kg i.p.) for treatment of subcutaneously growing SCCVII tumors rendered important differences in the therapy outcome. Photofrin-PDT, used at a dose that attained good initial response but no tumor cures, produced 50% cures when combined with a single LCL30 treatment. In contrast, the same LCL30 treatment combined with Pc4-PDT had no significant effect on tumor response. The optimal timing of LCL30 injection was immediately after Photofrin-PDT. The therapeutic benefit was lost when LCL30 was given in two 20mg/kg injections encompassing intervals before and after PDT. LCL85, the cationic B13 ceramide analogue and SL-modulating agent, also increased cure rates of Photofrin-PDT treated tumors, but the therapeutic benefit was less pronounced than with LCL30. These results with LCL30 and LCL85, and our previous findings for LCL29 (another SL analogue), assert the potential of SLs for use as adjuvants to augment the efficacy of PDT-mediated tumor destruction.

Treatment with the sphingosine-1-phosphate analogue FTY 720 reduces loss of plasma volume during experimental sepsis in the rat.

BACKGROUND: Increased vascular leakage leading to hypovolaemia and tissue oedema is common in severe sepsis. Hypovolaemia together with oedema formation may contribute to hypoxia and result in multiorgan failure and death. To improve treatment during sepsis, a potential therapeutic target may be to reduce the vascular leakage. Substances affecting the endothelial barrier are interesting in this respect, as it is suggested that increase in vascular leakage depends on reorganisation of the endothelial cells and breakdown of the endothelial barrier. The agonist of the bioactive lipid sphingosine-1-phosphate, FTY720, has been shown to modulate the integrity of the endothelium and reduce permeability both in vitro and in vivo. The aim of the present study was to determine if FTY720 could reduce the loss of plasma volume during experimental sepsis in rats. METHODS: Sepsis was induced by ligation and incision of the caecum in the rat. Plasma volume was determined before and 4.5 h after induction of sepsis by a dilution technique using (125) I-labelled albumin. RESULTS: FTY720 in a dose of 0.2 mg/kg reduced the loss of plasma during sepsis by approximately 30% compared with vehicle, without any adverse effects on haemodynamic and physiological parameters. The increase in hematocrit and haemoglobin concentration was also found to be higher in the vehicle group. CONCLUSION: FTY720 in a dose without haemodynamic side effects reduces loss of plasma volume during experimental sepsis most likely because of reduction in permeability and may therefore be beneficial in sepsis.

Protein kinase D2 regulates migration and invasion of U87MG glioblastoma cells in vitro.

Glioblastoma multiforme (GBM) is the most common malignant brain tumor, which, despite combined modality treatment, reoccurs and is invariably fatal for affected patients. Recently, a member of the serine/threonine protein kinase D (PRKD) family, PRKD2, was shown to be a potent mediator of glioblastoma growth. Here we studied the role of PRKD2 in U87MG glioblastoma cell migration and invasion in response to sphingosine-1-phosphate (S1P), an activator of PRKD2 and a GBM mitogen. Time-lapse microscopy demonstrated that random cell migration was significantly diminished in response to PRKD2 silencing. The pharmacological PRKD family inhibitor CRT0066101 decreased chemotactic migration and invasion across uncoated or matrigel-coated Transwell inserts. Silencing of PRKD2 attenuated migration and invasion of U87MG cells even more effectively. In terms of downstream signaling, CRT0066101 prevented PRKD2 autophosphorylation and inhibited p44/42 MAPK and to a smaller extent p54/46 JNK and p38 MAPK activation. PRKD2 silencing impaired activation of p44/42 MAPK and p54/46 JNK, downregulated nuclear c-Jun protein levels and decreased c-Jun(S73) phosphorylation without affecting the NFκB pathway. Finally, qPCR array analyses revealed that silencing of PRKD2 downregulates mRNA levels of integrin alpha-2 and -4 (ITGA2 and -4), plasminogen activator urokinase (PLAU), plasminogen activator urokinase receptor (PLAUR), and matrix metallopeptidase 1 (MMP1). Findings of the present study identify PRKD2 as a potential target to interfere with glioblastoma cell migration and invasion, two major determinants contributing to recurrence of glioblastoma after multimodality treatment.

Fingolimod reduces cerebral lymphocyte infiltration in experimental models of rodent intracerebral hemorrhage.

T-lymphocytes promote cerebral inflammation, thus aggravating neuronal injury after stroke. Fingolimod, a sphingosine 1-phosphate receptor analog, prevents the egress of lymphocytes from primary and secondary lymphoid organs. Based on these findings, we hypothesized fingolimod treatment would reduce the number of T-lymphocytes migrating into the brain, thereby ameliorating cerebral inflammation following experimental intracerebral hemorrhage (ICH). We investigated the effects of fingolimod in two well-established murine models of ICH, implementing intrastriatal infusions of either bacterial collagenase (cICH) or autologous blood (bICH). Furthermore, we tested the long term neurological improvements by Fingolimod in a collagenase-induced rat model of ICH. Fingolimod, in contrast to vehicle administration alone, improved neurological functions and reduced brain edema at 24 and 72 h following experimental ICH in CD-1 mice (n=103; p<0.05). Significantly fewer lymphocytes were found in blood and brain samples of treated animals when compared to the vehicle group (p<0.05). Moreover, fingolimod treatment significantly reduced the expression of intercellular adhesion molecule-1 (ICAM-1), interferon-γ (INF-γ), and interleukin-17 (IL-17) in the mouse brain at 72 h post-cICH (p<0.05 compared to vehicle). Long-term neurocognitive performance and histopathological analysis were evaluated in Sprague-Dawley rats between 8 and 10 weeks post-cICH (n=28). Treated rats showed reduced spatial and motor learning deficits, along with significantly reduced brain atrophy and neuronal cell loss within the basal ganglia (p<0.05 compared to vehicle). We conclude that fingolimod treatment ameliorated cerebral inflammation, at least to some extent, by reducing the availability and subsequent brain infiltration of T-lymphocytes, which improved the short and long-term sequelae after experimental ICH in rodents.

Sphingosine-1-phosphate inhibits interleukin-1ß-induced inflammation in human articular chondrocytes.

Sphingosine-1-phosphate (S1P) is a pluripotent lipid mediator that transmits signals through a family of G-protein-coupled receptors (GPCRs) to control diverse biological processes including inflammation and wound-healing. In this study, a novel biological activity of S1P in articular chondrocytes was identified. Human primary chondrocytes were cultured in a monolayer. Reverse transcription-polymerase chain reaction (RT-PCR) and western blotting were performed to detect genes and proteins involved in inflammation and cartilage degradation when human primary chondrocytes were stimulated by interleukin (IL)-1ß. Matrix metalloproteinase (MMP)-2 and MMP-9 activity was evaluated by gelatin zymography. Glycosaminoglycan (GAG) degradation was evaluated using the dimethylene blue method. Prostaglandin E2 (PGE2) was measured by enzyme-linked immunosorbent assay (ELISA). By using the S1P1 receptor agonist and antagonist, we discovered the key role played by S1P1 in the S1P-dependent inhibition of IL-1ß-induced inflammation in human chondrocytes. S1P dose-dependently inhibited IL-1ß-induced NF-κB p65, cyclooxygenase (COX)-2, MMP-1, MMP-3, MMP-13 and MMP-14 mRNA expression in human chondrocytes and IL-1ß-induced PGE2 synthesis and GAG degradation in human cartilage explants. W146, a known S1P1 receptor antagonist, inhibited the active form of NF-κB p65 and COX-2 expression induced by IL-1ß. The anti-inflammatory action of the S1P1 receptor agonist SEW2871 was similar to that of S1P. This study demonstrates that S1P has anti-inflammatory effects on chondrocytes via the S1P1 receptor. Our data suggest that targeting S1P and S1P1 may be a potential therapy for arthritis.

Therapeutic approach to steroid-resistant dermatitis using novel immunomodulator FTY720 (Fingolimod) in combination with betamethasone ointment in NC/Nga mice.

The therapeutic efficacy of the novel immunomodulator FTY720 (Fingolimod), alone and in combination with betamethasone ointment, was examined in the NC/Nga mouse model of spontaneous steroid-resistant dermatitis. Male NC/Nga mice which had developed severe dermatitis were divided into six groups: 1) a biweekly betamethasone group (betamethasone ointment, twice a week), 2) a daily betamethasone group (betamethasone ointment, six times a week), 3) an FTY720 group (FTY720, orally, three times a week), 4) a biweekly combination group (oral FTY720 plus betamethasone ointment, twice a week), 5) a daily combination group (oral FTY720 plus betamethasone ointment, six times a week) and 6) a placebo group (vehicle alone). The therapeutic efficacy was evaluated in terms of severity of dermatitis, epidermal hypertrophy, accumulation and degranulation of mast cells and infiltrated CD3+ T cells into the dermis after 4 weeks of treatment. Biweekly and daily betamethasone treatments had little effect, confirming that the dermatitis was steroid-resistant. In the FTY720 and biweekly combination groups, the dermatitis showed no marked improvement. In the daily combination group, the dermatitis was significantly (p<0.05, Mann-Whitney U-test) improved as compared with the FTY720 group, biweekly and daily betamethasone groups and placebo group. Further, epidermal hypertrophy and accumulation of mast cells were suppressed. Therefore, combination therapy with FTY720 and daily betamethasone ointment is a promising candidate for treatment of steroid-resistant atopic dermatitis.