Targeting sphingosine kinase 1 in carcinoma cells decreases proliferation and survival by compromising PKC activity and cytokinesis

Sphingosine kinases (SK) catalyze the phosphorylation of proapoptotic sphingosine to the prosurvival factor sphingosine 1-phosphate (S1P), thereby promoting oncogenic processes. Breast (MDA-MB-231), lung (NCI-H358), and colon (HCT 116) carcinoma cells were transduced with shRNA to downregulate SK-1 expression or treated with a pharmacologic SK-1 inhibitor. The effects of SK-1 targeting were investigated by measuring the level of intracellular sphingosine, the activity of protein kinase C (PKC) and cell cycle regulators, and the mitotic index. Functional assays included measurement of cell proliferation, colony formation, apoptosis, and cell cycle analysis. Downregulation of SK-1 or its pharmacologic inhibition increased intracellular sphingosine and decreased PKC activity as shown by reduced phosphorylation of PKC substrates. In MDA-MB-231 cells this effect was most pronounced and reduced cell proliferation and colony formation, which could be mimicked using exogenous sphingosine or the PKC inhibitor RO 31-8220. SK-1 downregulation in MDA-MB-231 cells increased the number of cells with 4N and 8N DNA content, and similar effects were observed upon treatment with sphingosine or inhibitors of SK-1 or PKC. Examination of cell cycle regulators unveiled decreased cdc2 activity and expression of Chk1, which may compromise spindle checkpoint function and cytokinesis. Indeed, SK-1 kd cells entered mitosis but failed to divide, and in the presence of taxol also failed to sustain mitotic arrest, resulting in further increased endoreduplication and apoptosis. Our findings delineate an intriguing link between SK-1, PKC and components of the cell cycle machinery, which underlines the significance of SK-1 as a target for cancer therapy.

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.

Central Europeans towards Integration with the European Union: A Study of Public Opinion, Social Attitudes and National Identities

Since the turbulence of 1989, the countries of Central and Eastern Europe have striven to "return to Europe". Agreements have been signed with ten post-communist countries, beginning in 1991 with Czechoslovakia (before its division), Hungary and Poland. Since that time several countries have expressed a desire to become members of the EU. In 1997 the European Commission announced its opinion on the applications for EU membership of the Czech Republic, Hungary, Poland, Slovakia, and seven other applicant countries. The Commission recommended the commencement of negotiations on accession with the Czech Republic, Estonia, Hungary, Poland, and Slovenia. Mr. Kucia's report, presented in the form of a series of manuscripts totalling 91 pages, written in English and Polish and including many pages of tables and graphs, presents the results of a study of public opinion on European integration in four countries of Central Europe (CE): the Czech Republic (CZ), Hungary (H), Poland (PL), and Slovakia (SK). The research results are primarily based on a public-opinion survey known as the Central and Eastern Eurobarometer (CEEB). CEEB has been conducted on behalf of the European Commission in the Central and Eastern European countries each year in autumn since 1990. Below is a very small selection of Mr. Kucia's research findings. Throughout the 90s people in the four countries increasingly saw their countries' future tied up with the EU, since economic and political connections to the EU were growing and prospects for EU membership were increasing. Regional co-operation within CE did not gain much popular recognition. However, initially high levels of enthusiasm for the EU were gradually superseded by a more realistic approach or even scepticism. Poland was the exception in this respect; its population was more positive about the EU in 1996 than ever before. Mr. Kucia concludes that, since the political "elites" in CE are more positive about the EU than the people they serve, they should do their best to bring people round to their beliefs, lest the project of European integration become purely the business of the elites, as Mr. Kucia claims it has been in the EU up till now. He accuses the governments of the region, the EU authorities and the media of failing to provide appropriate information, especially about the two subjects which most affect them, association with the EU and the PHARE assistance programme. Respondents were asked to rank in order the countries or regions they saw their country's future most closely tied up with. In the period 92-96 the EU received the highest ratings in all of CE. The ratings were highest in CZ in 92 and 93 (46%) and in Poland in 96 (46%). They were the lowest in Hungary (22% in 94). After the EU came "Other Western European countries (non EU)", that is Austria, Sweden and Finland (before they joined the EU in 1995), Switzerland and Norway. Mr. Kucia puts the high ratings of these countries down to historical connections and geographical proximity, particularly in the case of Austria. The USA always came second in Poland, and in Hungary too its standing has always been higher than in CZ or SK. Indeed Mr. Kucia suggests that the USA's standing is disproportionately low in especially the CZ. Germany was nominated frequently by Hungarians, though in the CZ and SK, figures have been consistently low (1-2%). "Other CE/EE countries" increased their ratings in all of CE except Poland between 92 and 96. With regard to these last figures, Mr. Kucia makes an interesting note. Assuming that for the respondents in the four countries this category covered the Visegrad 4, least support was found in Poland, whose government was the most in favour of close political co-operation within the V4, while most support was in evidence in CZ and SK, for whose governments V4 was simply not a priority. Again, there is evidence of a divide between the political elites and the people. Russia has occupied a consistently modest rank. It was the highest in PL, fairly low in H and SK and the lowest in CZ. The Slovak government's policy of closer ties with Russia is reflected in a growth in the figures from 2% in 93 to 6% in 95. Every year the spontaneous answer "we should depend on ourselves" appeared, which Mr. Kucia interprets as either a sign of isolationism and disillusionment or as a call for self-reliance. Unfortunately he regards both these tendencies as unfeasible in the uniting Europe. Moving to more general conclusions, Mr. Kucia finds that the concept "Central Europe" does not have much meaning for Central Europeans. He believes that this is probably due to the failure to establish a viable regional co-operation network. Group discussions also revealed that people thought themselves European as a consequence of being Czech or Polish etc. Thus European identity is based on national identities. Generally within the surveyed period, the numbers of those who said they often think themselves European decreased, while the numbers of those who said they never think themselves European increased from 41% in PL, 36% in CZ, and 30% in H in 1990, to 67% in CZ, 58% in PL, and 51% in H in 1995.

Histamine increases sphingosine kinase-1 expression and activity in the human arterial endothelial cell line EA.hy 926 by a PKC-alpha-dependent mechanism

Sphingosine 1-phosphate (S1P) is a potent mitogenic signal generated from sphingosine by the action of sphingosine kinases (SKs). In this study, we show that in the human arterial endothelial cell line EA.hy 926 histamine induces a time-dependent upregulation of the SK-1 mRNA and protein expression which is followed by increased SK-1 activity. A similar upregulation of SK-1 is also observed with the direct protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA). In contrast, SK-2 activity is not affected by neither histamine nor TPA. The increased SK-1 protein expression is due to stimulated de novo synthesis since cycloheximide inhibited the delayed SK-1 protein upregulation. Moreover, the increased SK-1 mRNA expression results from an increased promoter activation by histamine and TPA. In mechanistic terms, the transcriptional upregulation of SK-1 is dependent on PKC and the extracellular signal-regulated protein kinase (ERK) cascade since staurosporine and the MEK inhibitor U0126 abolish the TPA-induced SK-1 induction. Furthermore, the histamine effect is abolished by the H1-receptor antagonist diphenhydramine, but not by the H2-receptor antagonist cimetidine. Parallel to the induction of SK-1, histamine and TPA stimulate an increased migration of endothelial cells, which is prevented by depletion of the SK-1 by small interfering RNA (siRNA). To appoint this specific cell response to a specific PKC isoenzyme, siRNA of PKC-alpha, -delta, and -epsilon were used to selectively downregulate the respective isoforms. Interestingly, only depletion of PKC-alpha leads to a complete loss of TPA- and histamine-triggered SK-1 induction and cell migration. In summary, these data show that PKC-alpha activation in endothelial cells by histamine-activated H1-receptors, or by direct PKC activators leads to a sustained upregulation of the SK-1 protein expression and activity which, in turn, is critically involved in the mechanism of endothelial cell migration.

Extracellular nucleotides induce migration of renal mesangial cells by upregulating sphingosine kinase-1 expression and activity

BACKGROUND AND PURPOSE: Extracellular nucleotides act as potent mitogens for renal mesangial cells (MC). In this study we determined whether extracellular nucleotides trigger additional responses in MCs and the mechanisms involved. EXPERIMENTAL APPROACH: MC migration was measured after nucleotide stimulation in an adapted Boyden-chamber. Sphingosine kinase-1 (SK-1) protein expression was detected by Western blot analysis and mRNA expression quantified by real-time PCR. SK activity was measured by an in vitro kinase assay using sphingosine as substrate. KEY RESULTS: Nucleotide stimulation caused biphasic activation of SK-1, but not SK-2. The first peak occurred after minutes of stimulation and was followed by a second delayed peak after 4-24 h of stimulation. The delayed activation of SK-1 is due to increased SK-1 mRNA steady-state levels and de novo synthesis of SK-1 protein, and depends on PKC and the classical MAPK cascade. To see whether nucleotide-stimulated cell responses require SK-1, we selectively depleted SK-1 from cells by using small-interference RNA (siRNA). MC migration is highly stimulated by ATP and UTP; this is mimicked by exogenously added S1P. Depletion of SK-1 by siRNA drastically reduced the effect of ATP and UTP on cell migration but not on cell proliferation. Furthermore, MCs isolated from SK-1-deficient mice were completely devoid of nucleotide-induced migration. CONCLUSIONS AND IMPLICATIONS: These data show that extracellular nucleotides besides being mitogenic also trigger MC migration and this cell response critically requires SK-1 activity. Thus, pharmacological intervention of SK-1 may have impacts on situations where MC migration is important such as during inflammatory kidney diseases.

Malignant melanoma and bone resorption

The cellular and humoral mechanisms accounting for osteolysis in skeletal metastases of malignant melanoma are uncertain. Osteoclasts, the specialised multinucleated cells that carry out bone resorption, are derived from monocyte/macrophage precursors. We isolated tumour-associated macrophages (TAMs) from metastatic (lymph node/skin) melanomas and cultured them in the presence and absence of osteoclastogenic cytokines and growth factors. The effect of tumour-derived fibroblasts and melanoma cells on osteoclast formation and resorption was also analysed. Melanoma TAMs (CD14+/CD51-) differentiated into osteoclasts (CD14-/CD51+) in the presence of receptor activator for nuclear factor kappaB ligand (RANKL) and macrophage-colony stimulating factor. Tumour-associated macrophage-osteoclast differentiation also occurred via a RANKL-independent pathway when TAMs were cultured with tumour necrosis factor-alpha and interleukin (IL)-1alpha. RT-PCR showed that fibroblasts isolated from metastatic melanomas expressed RANKL messenger RNA and the conditioned medium of cultured melanoma fibroblasts was found to be capable of inducing osteoclast formation in the absence of RANKL; this effect was inhibited by the addition of osteoprotegerin (OPG). We also found that cultured human SK-Mel-29 melanoma cells produce a soluble factor that induces osteoclast differentiation; this effect was not inhibited by OPG. Our findings indicate that TAMs in metastatic melanomas can differentiate into osteoclasts and that melanoma fibroblasts and melanoma tumour cells can induce osteoclast formation by RANKL-dependent and RANKL-independent mechanisms, respectively.

Prolactin upregulates sphingosine kinase-1 expression and activity in the human breast cancer cell line MCF7 and triggers enhanced proliferation and migration

Sphingosine kinases (SK) catalyze the formation of sphingosine-1-phosphate (S1P) which plays a crucial role in cell growth and survival. Here, we show that prolactin (PRL) biphasically activates the SK-1, but not the SK-2 subtype, in the breast adenocarcinoma cell-line MCF7. A first peak occurs after minutes of stimulation and is followed by a second delayed activation after hours of stimulation. A similar biphasic effect on SK-1 activity is seen for 17beta-estradiol (E(2)). The delayed activation of SK-1 derives from an upregulated mRNA and protein expression and is due to increased SK-1 promoter activity and mechanistically involves STAT5 activation as well as protein kinase C and the classical mitogen-activated protein kinases. Furthermore, glucocorticoids also block both hormone-induced SK-1 expression and activity. Functionally, long-term stimulation of MCF7 cells with PRL or E(2) is well known to trigger increased cell proliferation and migration. Both hormone-induced cell responses critically involve SK-1 activation since the depletion of SK-1, but not SK-2, by siRNA transfection abolishes the hormone-induced cell proliferation and migration. In summary, our data show that PRL and E(2) cause a pronounced delayed SK-1 activation which is due to increased gene transcription, and critically determines the capability of cells to grow and move. Thus, the SK-1 may represent a novel attractive target for anti-tumor therapy.

Sphingosine kinase 1 and 2 regulate the capacity of mesangial cells to resist apoptotic stimuli in an opposing manner

Abstract Sphingosine kinases (SKs) are key enzymes regulating the production of sphingosine-1-phosphate (S1P), which determines important cell responses including cell growth and death. Here we show that renal mesangial cells isolated from wild-type, SK-1(-/-), and SK-2(-/-) mice show a differential response to apoptotic stimuli. Wild-type mesangial cells responded to staurosporine with increased DNA fragmentation and caspase-3 processing, which was enhanced in SK-1(-/-) cells. In contrast, SK-2(-/-) cells were highly resistant to staurosporine-induced apoptosis. Furthermore, the basal phosphorylation and activity of the anti-apoptotic protein kinase B (PKB) and of its substrate Bad were decreased in SK-1(-/-) but not in SK-2(-/-) cells. Upon staurosporine treatment, phosphorylation of PKB and Bad decreased in wild-type and SK-1(-/-) cells, but remained high in SK-2(-/-) cells. In addition, the anti-apoptotic Bcl-X(L) was significantly upregulated in SK-2(-/-) cells, which may further contribute to the protective state of these cells. In summary, our data show that SK-1 and SK-2 have opposite effects on the capacity of mesangial cells to resist apoptotic stimuli. This is due to differential modulation of the PKB/Bad pathway and of Bcl-X(L) expression. Thus, subtype-selective targeting of SKs will be critical when considering these enzymes as therapeutic targets for the treatment of inflammation or cancer.

Sphingosine kinase-1 is a hypoxia-regulated gene that stimulates migration of human endothelial cells

Sphingosine kinases (SK) catalyze the production of sphingosine-1-phosphate which in turn regulates cell responses such as proliferation and migration. Here, we show that exposure of the human endothelial cell line EA.hy 926 to hypoxia stimulates a increased SK-1, but not SK-2, mRNA, protein expression, and activity. This effect was due to stimulated SK-1 promoter activity which contains two putative hypoxia-inducible factor-responsive-elements (HRE). By deletion of one of the two HREs, hypoxia-induced promoter activation was abrogated. Furthermore, hypoxia upregulated the expression of HIF-1alpha and HIF-2alpha, and both contributed to SK-1 gene transcription as shown by selective depletion of HIF-1alpha or HIF-2alpha by siRNA. The hypoxia-stimulated SK-1 upregulation was functionally coupled to increased migration since the selective depletion of SK-1, but not of SK-2, by siRNAs abolished the migratory response. In summary, these data show that hypoxia upregulates SK-1 activity and results in an accelerated migratory capacity of endothelial cells. SK-1 may thus serve as an attractive therapeutic target to treat diseases associated with increased endothelial migration and angiogenesis such as cancer growth and progression.

Differences in endplate deformation of the adjacent and augmented vertebra following cement augmentation

Vertebral cement augmentation can restore the stiffness and strength of a fractured vertebra and relieve chronic pain. Previous finite element analysis, biomechanical tests and clinical studies have indirectly associated new adjacent vertebral fractures following augmentation to altered loading. The aim of this repeated measures in situ biomechanical study was to determine the changes in the adjacent and augmented endplate deformation following cement augmentation of human cadaveric functional spine units (FSU) using micro-computed tomography (micro-CT). The surrounding soft tissue and posterior elements of 22 cadaveric human FSU were removed. FSU were assigned to two groups, control (n = 8) (loaded on day 1 and day 2) and augmented (n = 14) (loaded on day 1, augmented 20% cement fill, and loaded on day 2). The augmented group was further subdivided into a prophylactic augmentation group (n = 9), and vertebrae which spontaneously fractured during loading on day 1 (n = 5). The FSU were axially loaded (200, 1,000, 1,500-2,000 N) within a custom made radiolucent, saline filled loading device. At each loading step, FSUs were scanned using the micro-CT. Endplate heights were determined using custom software. No significant increase in endplate deformation following cement augmentation was noted for the adjacent endplate (P > 0.05). The deformation of the augmented endplate was significantly reduced following cement augmentation for both the prophylactic and fracture group (P < 0.05, P < 0.01, respectively). Endplate deformation of the controls showed no statistically significant differences between loading on day 1 and day 2. A linear relationship was noted between the applied compressive load and endplate deflection (R (2) = 0.58). Evidence of significant endplate deformation differences between unaugmented and augmented FSU, while evident for the augmented endplate, was not present for the adjacent endplate. This non-invasive micro-CT method may also be useful to investigate endplate failure, and parameters that predict vertebral failure.