Expression Of Sphingosine Kinase Research Articles

Isoflurane Post-Treatment Ameliorates GMH-Induced Brain Injury in Neonatal Rats

This study investigated whether isoflurane ameliorates neurological sequelae after germinal matrix hemorrhage (GMH) through activation of the cytoprotective sphingosine kinase/sphingosine-1-phosphate receptor/Akt pathway. GMH was induced in P7 rat pups by intraparenchymal infusion of bacterial collagenase (0.3 U) into the right hemispheric germinal matrix. GMH animals received 2% isoflurane either once 1 hour after surgery or every 12 hours for 3 days. Isoflurane treatment was then combined with sphingosine-1-phosphate receptor-1/2 antagonist VPC23019 or sphingosine kinase 1/2 antagonist N,N-dimethylsphingosine. Brain protein expression of sphingosine kinase-1 and phosphorylated Akt were significantly increased after isoflurane post-treatment, and cleaved caspase-3 was decreased at 24 hours after surgery, which was reversed by the antagonists. Isoflurane significantly reduced posthemorrhagic ventricular dilation and improved motor, but not cognitive, functions in GMH animals 3 weeks after surgery; no improvements were observed after VPC23019 administration. Isoflurane post-treatment improved the neurological sequelae after GMH possibly by activation of the sphingosine kinase/Akt pathway.

Increased SPHK1 expression is associated with poor prognosis in bladder cancer

Upregulation of sphingosine kinase 1 (SPHK1) protein has been reported to be associated with a poor prognosis in a variety of malignant tumors. However, the role of SPHK1 in bladder cancer (BC) has not been thoroughly elucidated. The purpose of this study was to assess SPHK1 expression and to explore its contribution to BC. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was conducted to detect SPHK1 mRNA expression in 37 pairs of fresh-frozen BC tissues and corresponding noncancerous tissues. Results showed that SPHK1 mRNA expression level in BC tissues was significantly higher than that in corresponding noncancerous tissues. To investigate the association between SPHK1 protein expression and clinicopathological characteristics of BC, immunohistochemistry (IHC) was performed in 153 archived paraffin-embedded BC samples. Interestingly, high SPHK1 expression was significantly associated with histologic grade (P = 0.045) and tumor stage (P < 0.001) of patients with BC. The Kaplan-Meier survival curve showed that patients with high SPHK1 expression had significantly reduced overall 5-year survival rates (P < 0.001). Multivariate Cox regression analysis further suggested that the increased expression of SPHK1 was an independent poor prognostic factor for this disease. In conclusion, our data offer the convincing evidence for the first time that the increased expression of SPHK1 may be involved in the pathogenesis and progression of BC. SPHK1 might be a potential marker to predict the prognosis in BC.

Dual role of sphingosine kinase-1 in promoting the differentiation of dermal fibroblasts and the dissemination of melanoma cells

Despite progress in the understanding of the biology and genetics of melanoma, no effective treatment against this cancer is available. The adjacent microenvironment has an important role in melanoma progression. Defining the molecular signals that control the bidirectional dialog between malignant cells and the surrounding stroma is crucial for efficient targeted therapy. Our study aimed at defining the role of sphingosine-1-phosphate (S1P) in melanoma-stroma interactions. Transcriptomic analysis of human melanoma cell lines showed increased expression of sphingosine kinase-1 (SPHK1), the enzyme that produces S1P, as compared with normal melanocytes. Such an increase was also observed by immunohistochemistry in melanoma specimens as compared with nevi, and occurred downstream of ERK activation because of BRAF or NRAS mutations. Importantly, migration of melanoma cells was not affected by changes in SPHK1 activity in tumor cells, but was stimulated by comparable modifications of S1P-metabolizing enzymes in cocultured dermal fibroblasts. Reciprocally, incubation of fibroblasts with the conditioned medium from SPHK1-expressing melanoma cells resulted in their differentiation to myofibroblasts, increased production of matrix metalloproteinases and enhanced SPHK1 expression and activity. In vivo tumorigenesis experiments showed that the lack of S1P in the microenvironment prevented the development of orthotopically injected melanoma cells. Finally, local tumor growth and dissemination were enhanced more efficiently by coinjection of wild-type skin fibroblasts than by fibroblasts from Sphk1(-/-) mice. This report is the first to document that SPHK1/S1P modulates the communication between melanoma cells and dermal fibroblasts. Altogether, our findings highlight SPHK1 as a potential therapeutic target in melanoma progression.

Sphingosine kinase 1 plays a role in the upregulation of CD44 expression through extracellular signal-regulated kinase signaling in human colon cancer cells

Our previous study has shown that the activity and expression of sphingosine kinase (SPHK) regulated the sensitivity of human colon cancer cells to the chemotherapeutic oxaliplatin (L-OHP). In addition, the cancer stem cell marker CD44 increases cell resistance to anticancer drugs. Here, we use colon cancer cell lines to examine the relationship between SPHK1 activity and CD44 expression.CD44 expression was measured by western blotting and quantitative PCR in two human colon cancer cell lines: L-OHP-resistant RKO and L-OHP-sensitive HCT116. The regulation of CD44 by SPHK1 was examined by either blocking or overexpressing SPHK1 and by using an L-OHP-resistant HCT116 clone (HCT116-R).The levels of SPHK1, CD44, phosphorylated-Akt, and phosphorylated-extracellular signal-regulated kinase (ERK) were much higher in the RKO cells than in the HCT116 cells. The treatment of RKO cells with the SPHK inhibitor or SPHK1 silencing by RNA interference suppressed CD44 protein expression. SPHK1 and CD44 levels were much higher in HCT116-R cells compared with the parental HCT116 cells. Transfection of HCT116 cells with SPHK1 cDNA enhanced the expression of both CD44 and phosphorylated-ERK. The increase in the CD44 protein level was abolished by the inhibition of ERK phosphorylation. Treatment of RKO cells with the sphingosine-1-phosphate (S1P)2 receptor antagonist suppressed ERK phosphorylation and the expression of CD44 mRNA and protein. Exogenous stimulation with S1P increased ERK phosphorylation and CD44 protein expression in HCT116 cells, but treatment with an MEK inhibitor and S1P2 receptor antagonist blocked this effect.These findings indicate that SPHK1 and its product, S1P, contribute toward the regulation of CD44 protein expression through the ERK signaling pathway through S1P2 in human colon cancer cells.

Factor-Xa-induced mitogenesis and migration require sphingosine kinase activity and S1P formation in human vascular smooth muscle cells

Sphingosine-1-phosphate (S1P) is a cellular signalling lipid generated by sphingosine kinase-1 (SPHK1). The aim of the study was to investigate whether the activated coagulation factor-X (FXa) regulates SPHK1 transcription and the formation of S1P and subsequent mitogenesis and migration of human vascular smooth muscle cells (SMC). FXa induced a time- (3-6 h) and concentration-dependent (3-30 nmol/L) increase of SPHK1 mRNA and protein expression in human aortic SMC, resulting in an increased synthesis of S1P. FXa-stimulated transcription of SPHK1 was mediated by the protease-activated receptor-1 (PAR-1) and PAR-2. In human carotid artery plaques, expression of SPHK1 was observed at SMC-rich sites and was co-localized with intraplaque FX/FXa content. FXa-induced SPHK1 transcription was attenuated by inhibitors of Rho kinase (Y27632) and by protein kinase C (PKC) isoforms (GF109203X). In addition, FXa rapidly induced the activation of the small GTPase Rho A. Inhibition of signalling pathways which regulate SPHK1 expression, inhibition of its activity or siRNA-mediated SPHK1 knockdown attenuated the mitogenic and chemotactic response of human SMC to FXa. These data suggest that FXa induces SPHK1 expression and increases S1P formation independent of thrombin and that this involves the activation of Rho A and PKC signalling. In addition to its key function in coagulation, this direct effect of FXa on human SMC may increase cell proliferation and migration at sites of vessel injury and thereby contribute to the progression of vascular lesions.

Sphingosine kinase expression is associated with poor prognosis in resectable pancreatic ductal adenocarcinoma

Sphingosine kinase expression is associated with poor prognosis in resectable pancreatic ductal adenocarcinoma

Increased expression of sphingosine kinase in the amnion during labor

IntroductionSphingosine-1-phosphate (S1P), a bioactive lipid, has been reported to regulate inflammation processes. The onset of labor is thought to be related to inflammation. We therefore hypothesized that S1P might be involved in the onset of labor. MethodsThe expression of sphingosine kinase (SPHK)-1, which produces S1P, and S1P lyase (SPL)-1, which irreversibly inactivates S1P, were examined in the fetal membranes. The expression levels were compared between amnions from cases of elective Caesarean deliveries (pre-labor) and those from vaginal deliveries (post-labor). In primary cultured human amnion cells, the expression levels of prostaglandin-endoperoxide synthase (PTGS)-2 were examined in the presence or absence of S1P treatment. ResultsSPHK-1 and SPL-1 were both expressed in the amnion. The expression of SPHK-1 in the post-labor amnions increased compared with that in the pre-labor amnions. The expression of PTGS-2, a key regulator of labor, also increased in the post-labor amnion. However, the SPL-1 expression in the pre-labor amnion was not significantly different from that in the post-labor amnion. S1P1-3 and 5, which were coupled with Gi protein, were consistently found in the amnion cells. The treatment with S1P increased the expression of PTGS-2, and this was completely suppressed by a Gi inhibitor in the amnion cells. DiscussionWe are herein provide the first evidence of increased SPHK-1 expression in post-labor amnions, and that S1P increases the PTGS-2 expression in amnion cells. ConclusionsOur results suggest that S1P might play a role in the onset of labor via the induction of PTGS-2.

Sphingosine kinase 1 expression is downregulated during differentiation of Friend cells due to decreased c-MYB

Sphingosine kinase 1 (SPHK1) overexpression in malignant cells has been reported. Mouse Friend cells showed higher SPHK1 but not SPHK2 expression compared with other mouse cell lines. A Sphk1 promoter analysis demonstrated the region between −53bp and the first exon as the minimal promoter. Further promoter truncation revealed the importance of a MYB-binding site. EMSA using this region as the probe demonstrated one band containing c-MYB protein, and its intensity decreased during erythroid differentiation with hexamethylane bisacetamide (HMBA), a potent inducer of erythroid differentiation of Friend cells. ChIP assay also revealed in vivo binding of c-MYB. c-MYB overexpression and siRNA for c-Myb affected SPHK1 expression, confirming the important regulatory role of c-MYB in SPHK1 expression. HMBA reduced c-MYB expression rapidly. Induced differentiation by HMBA caused a marked and rapid reduction of SPHK1 mRNA, protein and enzyme activity leading to the rapid decrease of cellular sphingosine 1-phosphate level. Moreover, terminally differentiated cells did not resume SPHK1 expression. Compared with original Friend cells, stable overexpression of wild-type SPHK1 showed higher cell proliferation, resistance to cell death by serum depletion. Interestingly, HMBA-induced differentiation of these cells was delayed but not completely suppressed. In contrast, SPHK inhibitor and its siRNA inhibited cell growth and enhanced HMBA-induced differentiation significantly, suggesting that SPHK1 delayed HMBA-induced differentiation by its cell proliferation-promoting activity. Effects of pertussis toxin, a G-protein-coupled receptor inhibitor, and S1P receptor antagonist on Friend cell growth and differentiation were negligible, suggesting the importance of the intracellular SPHK1/S1P signaling in Friend cells.

WS14-6 - Plasma Prosaposin: A New Potential Biomarker for Metastatic Prostate Cancer Regulated by Sphingosine Kinase 1

ABSTRACT Background Prosaposin is a neurotrophic secreted protein that stimulates specific G-coupled receptors and is involved in cancer progression, metastasis and chemoresistance through activation of the Raf-MAPK and uPA/uPAR pathways. Here we have investigated the crossregulation of prosaposin and sphingosine kinase 1 (SK1) pathways in hormone refractory prostate cancer (PCa) models. Methods and results Prosaposin knockout mice exhibited an involution of the prostate gland which correlated with a significant reduction of prostate SK1 activity. In PCa cell lines SK1 activity correlated with the amount of secreted, but not intracellular prosaposin, indicating a role for the prosaposin/GPCR signalling. The specific knockdown of prosaposin in PCa cells induced a significant decrease in SK1 activity and expression on mRNA and protein level. Additionally, a short prosaposin peptide TX14A enhanced SK1 activity and up-regulated SK1 mRNA expression via ERK1/2-mediated mechanism. Cytokine array showed that TX14A induced a significant upregulation of MIF and PAI-1 secretion, which was abrogated by pretreatment with psap- or SphK1-specific siRNA. Increased prosaposin secretion was detected in metastatic PCa cells lines and associated with increased cancer cell motility. Subsequently, SK1 activation was essential for prosaposin secretion and expression during serum deprivation. The effect was further amplified upon overexpression of SK1 and was drastically abrogated by specific knockdown or SK1 inhibitors, which could also abrogate prosaposin-induced cancer cell migration. Analysis of PCa patients plasma samples revealed increased levels of circulating prosaposin in comparison to cancer-free controls. Conclusions These data provide evidence that SK1-dependent prosaposin secretion promotes cancer cell migration and has potential diagnostic/prognostic and therapeutic value in PCa patients.

Sphingosine Kinase 1 Is Required for Mesothelioma Cell Proliferation: Role of Histone Acetylation

BackgroundMalignant pleural mesothelioma (MPM) is a devastating disease with an overall poor prognosis. Despite the recent advances in targeted molecular therapies, there is a clear and urgent need for the identification of novel mesothelioma targets for the development of highly efficacious therapeutics.Methodology/Principal FindingsIn this study, we report that the expression of Sphingosine Kinase 1 (SphK1) protein was preferentially elevated in MPM tumor tissues (49 epithelioid and 13 sarcomatoid) compared to normal tissue (n = 13). In addition, we also observed significantly elevated levels of SphK1 and SphK2 mRNA and SphK1 protein expression in MPM cell lines such as H2691, H513 and H2461 compared to the non-malignant mesothelial Met5 cells. The underlying mechanism appears to be mediated by SphK1 induced upregulation of select gene transcription programs such as that of CBP/p300 and PCAF, two histone acetyl transferases (HAT), and the down regulation of cell cycle dependent kinase inhibitor genes such as p27Kip1 and p21Cip1. In addition, using immunoprecipitates of anti-acetylated histone antibody from SphK inhibitor, SphK-I2 treated Met5A and H2691 cell lysates, we also showed activation of other cell proliferation related genes, such as Top2A (DNA replication), AKB (chromosome remodeling and mitotic spindle formation), and suppression of p21 CIP1 and p27KIP1. The CDK2, HAT1 and MYST2 were, however, unaffected in the above study. Using SphK inhibitor and specific siRNA targeting either SphK1 or SphK2, we also unequivocally established that SphK1, but not SphK2, promotes H2691 mesothelioma cell proliferation. Using a multi-walled carbon nanotubes induced peritoneal mesothelioma mouse model, we showed that the SphK1−/− null mice exhibited significantly less inflammation and granulamatous nodules compared to their wild type counterparts.Conclusions/SignificanceThe lipid kinase SphK1 plays a positive and essential role in the growth and development of malignant mesothelioma and is therefore a likely therapeutic target.

Effects of down-regulation of sphingosine kinase-1 expression on cell proliferation and cell apoptosis of cervical carcinoma Hela cells and its molecular mechanism

Objective To study the down-regulation of sphingosine kinase 1 ( SphK1 ) expression on cell proliferation and cell apoptosis of cervical carcinoma cell line Hela cells,and to explore its possible molecular mechanisms.Methods SphK1 siRNA and control siRNA were utilized to transfect to Hela cells,which was divided into three groups including untransfected group,siRNA control group and SphK1 siRNA group.Expressions of SphK1 mRNA and protein were detected by read-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting methods after transfection with SphK1 siRNA.Cell counting Kit-8 (CCK-8) and Flow cytometry methods was used to analyze the changes of cell proliferation and cell apoptosis.Further,expressions of cell proliferation and cell apoptosis related proteins were detected by Western blotting method.Results Relative levels of SphK1 mRNA and protein in SphK1 siRNA group were 0.153 ±0.037 and 0.123 ±0.037,respectively,which was significantly lower than those in untransfected group ( 1.000 ±0.000 and 0.688 ±0.049 ) and siRNA control group (0.932 ±0.039 and 0.673 ±0.057 ) (P ＜0.05 ).The results of cell proliferation demonstrated that compared with untransfected group and siRNA control group,the proliferation of Hela cells in SphK1 siRNA group was obviously inhibited ( P ＜0.05 ).The results of Flow Cytometry revealed that early apoptotic rate of Hela cells in SphK1 siRNA group ( 23.85 ± 0.72)％ were markedly higher than those in untransfected group ( 11.67 ± 1.02 ) ％ and s iRN A control group ( 11.85 ± 0.71 ) ％,and there was significant difference ( F =211.451,P＜0.05 ).Further,the results of Western blotting demonstrated that expressions of p21,caspase-3 and caspase-9 proteins in SphK1 siRNA group (0.381±0.025,0.406 ±0.041 and 0.374 ±0.035,respectively) were obviously higher than those in nntransfected group (0.127 ±0.039,0.064 ±0.023 and 0.115 ±0.039,respectively) and siRNA control group (0.125 ±0.034,0.060±0.018 and 0.126 ±0.038,respectively)( P ＜0.05 ).Conclusion SphK1 may be closely associated with the development and occurrence of cervical carcinoma,and its expression down-regulation mediated proliferation inhibition and cell apoptosis may be tightly related to elevation of p21,caspase-3 and caspase-9 expressions. Key words: Sphingosine kinase-1; Hela cells; Small interfering RNA; Cell proliferation; Cell apoptosis

Isoflurane Attenuates Blood–Brain Barrier Disruption in Ipsilateral Hemisphere After Subarachnoid Hemorrhage in Mice

We examined effects of isoflurane, volatile anesthetics, on blood-brain barrier disruption in the endovascular perforation model of subarachnoid hemorrhage (SAH) in mice. Animals were assigned to sham-operated, SAH+vehicle-air, SAH+1%, or 2% isoflurane groups. Neurobehavioral function, brain water content, Evans blue dye extravasation, and Western blotting for sphingosine kinases, occludin, claudin-5, junctional adhesion molecule, and vascular endothelial cadherin were evaluated at 24 hours post-SAH. Effects of sphingosine kinase (N,N-dimethylsphingosine) or sphingosine-1-phosphate receptor-1/3 (S1P1/3) inhibitors (VPC23019) on isoflurane's action were also examined. SAH aggravated neurological scores, brain edema, and blood-brain barrier permeability, which were prevented by 2% but not 1% isoflurane posttreatment. Two percent isoflurane increased sphingosine kinase-1 expression and prevented a post-SAH decrease in expressions of the blood-brain barrier-related proteins. Both N,N-dimethylsphingosine and VPC23019 abolished the beneficial effects of isoflurane. Two percent isoflurane can suppress post-SAH blood-brain barrier disruption, which may be mediated by sphingosine kinase 1 expression and sphingosine-1-phosphate receptor-1/3 activation.

Isoflurane delays the development of early brain injury after subarachnoid hemorrhage through sphingosine-related pathway activation in mice*

Isoflurane, a volatile anesthetic agent, has been recognized for its potential neuroprotective properties and has antiapoptotic effects. We examined whether isoflurane posttreatment is protective against early brain injury after subarachnoid hemorrhage and determined whether this effect needs sphingosine-related pathway activation. Controlled in vivo laboratory study. Animal research laboratory. One hundred seventy-nine 8-wk-old male CD-1 mice weighing 30-38 g. Subarachnoid hemorrhage was induced in mice by endovascular perforation. Animals were randomly assigned to sham-operated, subarachnoid hemorrhage-vehicle, and subarachnoid hemorrhage+2% isoflurane. Neurobehavioral function and brain edema were evaluated at 24 and 72 hrs. The expression of sphingosine kinase, phosphorylated Akt, and cleaved caspase-3 was determined by Western blotting and immunofluorescence. Neuronal cell death was examined by terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling staining. Effects of a sphingosine kinase inhibitor N, N-dimethylsphingosine or a sphingosine 1 phosphate receptor inhibitor VPC23019 on isoflurane's protective action against postsubarachnoid hemorrhage early brain injury were also examined. Isoflurane significantly improved neurobehavioral function and brain edema at 24 hrs but not 72 hrs after subarachnoid hemorrhage. At 24 hrs, isoflurane attenuated neuronal cell death in the cortex, associated with an increase in sphingosine kinase 1 and phosphorylated Akt, and a decrease in cleaved caspase-3. The beneficial effects of isoflurane were abolished by N, N-dimethylsphingosine and VPC23019. Isoflurane posttreatment delays the development of postsubarachnoid hemorrhage early brain injury through antiapoptotic mechanisms including sphingosine-related pathway activation, implying its use for anesthesia during acute aneurysm surgery or intervention.

The Reduction of Allograft Arteriosclerosis in Intestinal Transplant Is Associated With Sphingosine Kinase 1/Sphingosine-1-Phosphate Signaling After Fish Oil Treatment

Transplant arteriosclerosis is a major cause of late intestinal allograft dysfunction. However, little is known about the immunologic and molecular mechanisms underlying it, and no effective treatment is available. This study aimed to investigate the role of sphingosine kinase 1 (SPHK1)/sphingosine-1-phosphate (S1P) in transplant arteriosclerosis and find out whether fish oil (FO) attenuates allograft arteriosclerosis through S1P signaling. A rat model with orthotopic intestinal transplantation was conducted in this study. Animals received daily FO supplementation after intestinal transplant. The allogeneic recipients by phosphate-buffered saline or corn oil treatment served as controls. The allograft arteriosclerosis was characterized, and the expression of SPHK1 and S1P receptors (S1P₁, S1P₂, and S1P₃) was determined on day 190 posttransplant. The allogeneic controls presented transplant vasculopathy in mesenteric vessels, including intimal thickening, fibrosis, and leukocyte infiltration. The transplant arteriosclerosis was markedly reduced in FO-fed animals. The pression of SPHK1 and its activity were significantly augmented, and the expression of S1P₁ and S1P₃ messenger RNA was up-regulated in the allogeneic controls. FO supplementation suppressed the activation of SPHK1 and led to a decrease in the expression of S1P₁ and S1P₃ in these tissues in transplant arteriosclerosis. These results demonstrate that the activation of SPHK1/S1P signaling plays a possible role in the pathogenesis of transplant arteriosclerosis. The reduction of allograft arteriosclerosis by FO may be associated with down-regulation of SPHK1/S1P signaling. Understanding the role of FO for SPHK1/S1P may help us to identify considerable therapeutic targets for transplant arteriosclerosis.

Inhibition of serine palmitoyltransferase delays the onset of radiation-induced pulmonary fibrosis through the negative regulation of sphingosine kinase-1 expression

The enforcement of sphingosine-1-phosphate (S1P) signaling network protects from radiation-induced pneumonitis. We now demonstrate that, in contrast to early postirradiation period, late postirradiation sphingosine kinase-1 (SphK1) and sphingoid base-1-phosphates are associated with radiation-induced pulmonary fibrosis (RIF). Using the mouse model, we demonstrate that RIF is characterized by a marked upregulation of S1P and dihydrosphingosine-1-phosphate (DHS1P) levels in the lung tissue and in circulation accompanied by increased lung SphK1 expression and activity. Inhibition of sphingolipid de novo biosynthesis by targeting serine palmitoyltransferase (SPT) with myriocin reduced radiation-induced pulmonary inflammation and delayed the onset of RIF as evidenced by increased animal lifespan and decreased expression of markers of fibrogenesis, such as collagen and α-smooth muscle actin (α-SMA), in the lung. Long-term inhibition of SPT also decreased radiation-induced SphK activity in the lung and the levels of S1P-DHS1P in the lung tissue and in circulation. In vitro, inhibition or silencing of serine palmitoyltransferase attenuated transforming growth factor-β1 (TGF-β)-induced upregulation of α-SMA through the negative regulation of SphK1 expression in normal human lung fibroblasts. These data demonstrate a novel role for SPT in regulating TGF-β signaling and fibrogenesis that is linked to the regulation of SphK1 expression and S1P-DHS1P formation.

Thiazolidinedione‐dependent activation of sphingosine kinase 1 causes an anti‐fibrotic effect in renal mesangial cells

PPARγ agonists [thiazolidinediones (TZDs)] are known to exert anti-fibrotic effects in the kidney. In addition, we previously demonstrated that sphingosine kinase 1 (SK-1) and intracellular sphingosine-1-phosphate (S1P), by reducing the expression of connective tissue growth factor (CTGF), have a protective role in the fibrotic process. Here, we investigated the effect of TZDs on intracellular sphingolipid levels and the transcriptional regulation of SK-1 in mesangial cells to evaluate potential novel aspects of the anti-fibrotic capacity of TZDs. Stimulation with the TZDs, troglitazone and rosiglitazone, led to increased S1P levels in rat mesangial cells. This was paralleled by increased SK-1 activity as a consequence of direct effects of the TZDs on SK-1 expression. GW-9662, a PPARγ antagonist, inhibited the stimulating effect of TZDs on SK-1 mRNA and activity levels and intracellular S1P concentrations. Furthermore, SK-1 up-regulation by TZDs was functionally coupled with lower amounts of pro-fibrotic CTGF. SK-1 inhibition with SKI II almost completely abolished this effect in a dose-dependent manner. Moreover, the CTGF lowering effect of TZDs was fully blocked in MC isolated from SK-1 deficient mice (SK-1(-/-) ) as well as in glomeruli of SK-1(-/-) mice compared with wild-type mice treated with TRO and RSG. These data show that TZD-induced SK-1 up-regulation results in lower amounts of CTGF, demonstrating novel facets for the anti-fibrotic effects of this class of drugs.

Isoflurane Protects Against Human Endothelial Cell Apoptosis by Inducing Sphingosine Kinase-1 via ERK MAPK

Endothelial dysfunction is a major clinical problem affecting virtually every patient requiring critical care. Volatile anesthetics are frequently used during the perioperative period and protect the heart and kidney against ischemia and reperfusion injury. We aimed to determine whether isoflurane, the most commonly used volatile anesthetic in the USA, protects against endothelial apoptosis and necrosis and the mechanisms involved in this protection. Human endothelial EA.hy926 cells were pretreated with isoflurane or carrier gas (95% room air + 5% CO2) then subjected to apoptosis with tumor necrosis factor-α or to necrosis with hydrogen peroxide. DNA laddering and in situ Terminal Deoxynucleotidyl Transferase Biotin-dUTP Nick-End Labeling (TUNEL) staining determined EA.hy926 cell apoptosis and percent LDH released determined necrosis. We also determined whether isoflurane modulates the expression and activity of sphingosine kinase-1 (SK1) and induces the phosphorylation of extracellular signal regulated kinase (ERK MAPK) as both enzymes are known to protect against cell death. Isoflurane pretreatment significantly decreased apoptosis in EA.hy926 cells as evidenced by reduced TUNEL staining and DNA laddering without affecting necrosis. Mechanistically, isoflurane induces the phosphorylation of ERK MAPK and increased SK1 expression and activity in EA.hy926 cells. Finally, selective blockade of SK1 (with SKI-II) or S1P1 receptor (with W146) abolished the anti-apoptotic effects of isoflurane. Taken together, we demonstrate that isoflurane, in addition to its potent analgesic and anesthetic properties, protects against endothelial apoptosis most likely via SK1 and ERK MAPK activation. Our findings have significant clinical implication for protection of endothelial cells during the perioperative period and patients requiring critical care.

Inhibition of SPHK1 Suppresses Phorbol 12-Myristate 13-Acetate-Induced Metastatic Phenotype in Colorectal Cancer HT-29 Cells

Expression of sphingosine kinase 1 (SPHK1) plays a role in colorectal cancer progression. This study aimed to demonstrate the mechanism of human colorectal cancer cell metastatic phenotype through SPHK1 knockdown. Human colorectal cancer HT-29 cells were stimulated by phorbol 12-myristate 13-acetate (PMA) with or without SPHK1 siRNA transfection. Tumor cell phenotypic changes were analyzed by using invasion, motility, cell viability, and apoptosis assays. Gene expressions were assessed using Western blot. PMA induced a metastatic phenotype in colorectal cancer cells, as indicated by cell viability, migration and invasion capacity, and ERK1/2 phosphorylation, whereas SPHK1 siRNA transfection suppressed the metastatic phenotype of tumor cells and antagonized PMA's effects. SPHK1 knockdown also inhibited secretion of MMP-2 and MMP-9 into the tumor cell conditioned medium. Suppression of SPHK1 expression suppresses the PMA-induced metastatic phenotype via ERK1/2 phosphorylation in human colorectal cancer cells.

Sphingosine-1-phosphate is increased in patients with idiopathic pulmonary fibrosis and mediates epithelial to mesenchymal transition

BackgroundIdiopathic pulmonary fibrosis (IPF) is characterised by the aberrant epithelial to mesenchymal transition (EMT) and myofibroblast accumulation. Sphingosine-1-phosphate (S1P) and sphingosine kinase 1 (SPHK1) have been implicated in lung myofibroblast...

An novel role of sphingosine kinase-1 (SPHK1) in the invasion and metastasis of esophageal carcinoma

BackgroundTreatment failure for esophageal carcinoma is frequently due to lymph node metastasis and invasion to neighboring organs. The aim of the present study was to investigate invasion- and metastasis-related genes in esophageal carcinoma cells in vitro and in vivo.MethodsA metastasis model using a Matrigel invasion clonal selection approach was employed to establish a highly invasive subline EC9706-P4 from the esophageal carcinoma cell (ESCC) line EC9706. The differentially expressed genes of the subline and the parental cells determined by gene microarrays were further analyzed by RT-PCR and Western blotting.ResultsWe identified sphingosine kinase 1 (SPHK1) as an invasion and metastasis-related gene of esophageal cancer. SPHK1 was overexpressed in the EC9706-P4 subline with high invasive capacity. Among six ESCC lines tested, KYSE2 and KYSE30 cells showed the highest SPHK1 mRNA and protein expressions as well as the most invasive phenotype. By Western blotting, in 7/12 cases (58%), SPHK1 expression was higher in esophageal carcinomas than in the companion normal tissue. In 23/30 cases (76%), SPHK1 protein expression was upregulated in the tumors compared to matched normal tissue by immunohistochemistry (IHC). Esophageal carcinoma tissue microarray analysis indicated that SPHK1 expression correlated with the depth of tumor invasion (P < 0.0001) and lymph node metastasis (P = 0.016). By Kaplan-Meier analysis, strong SPHK1 expression was significantly associated with clinical failure (P < 0.01), suggesting the involvement of SPHK1 in aggressiveness of human esophageal carcinoma. SPHK1 overexpression significantly increased the invasiveness of EC9706 cells in vitro and also increased EC9706 cell growth and spontaneous metastasis in vivo, promoting significant increases in tumor growth, tumor burden and spontaneous lung metastasis in nude mice. SPHK1 expression significantly correlated with the expression of many EGFR pathway genes associated with invasion of cancer cells. SPHK1 protein expression also significantly correlated with the phosphorylation of EGFR.ConclusionIn summary, our data implicate SPHK1 in the metastasis of esophageal cancer. Our study also identified downstream mediators of SPHK1 in esophageal cancer cells that may mediate enhanced malignant behavior, and several of these mediators may be useful as therapeutic targets.