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.

Genetic stability of Schmallenberg virus in vivo during an epidemic, and in vitro, when passaged in the highly susceptible porcine SK-6 cell line

Schmallenberg virus (SBV), an arthropod-borne orthobunyavirus was first detected in 2011 in cattle suffering from diarrhea and fever. The most severe impact of an SBV infection is the induction of malformations in newborns and abortions. Between 2011 and 2013 SBV spread throughout Europe in an unprecedented epidemic wave. SBV contains a tripartite genome consisting of the three negative-sense RNA segments L, M, and S. The virus is usually isolated from clinical samples by inoculation of KC (insect) or BHK-21 (mammalian) cells. Several virus passages are required to allow adaptation of SBV to cells in vitro. In the present study, the porcine SK-6 cell line was used for isolation and passaging of SBV. SK-6 cells proved to be more sensitive to SBV infection and allowed to produce higher titers more rapidly as in BHK-21 cells after just one passage. No adaptation was required. In order to determine the in vivo genetic stability of SBV during an epidemic spread of the virus the nucleotide sequence of the genome from seven SBV field isolates collected in summer 2012 in Switzerland was determined and compared to other SBV sequences available in GenBank. A total of 101 mutations, mostly transitions randomly dispersed along the L and M segment were found when the Swiss isolates were compared to the first SBV isolated late 2011 in Germany. However, when these mutations were studied in detail, a previously described hypervariable region in the M segment was identified. The S segment was completely conserved among all sequenced SBV isolates. To assess the in vitro genetic stability of SBV, three isolates were passage 10 times in SK-6 cells and sequenced before and after passaging. Between two and five nt exchanges per genome were found. This low in vitro mutation rate further demonstrates the suitability of SK-6 cells for SBV propagation.

Sphingosine kinase 2 deficiency increases proliferation and migration of renal mouse mesangial cells and fibroblasts

Both of the sphingosine kinase (SK) subtypes SK-1 and SK-2 catalyze the production of the bioactive lipid molecule sphingosine 1-phosphate (S1P). However, the subtype-specific cellular functions are largely unknown. In this study, we investigated the cellular function of SK-2 in primary mouse renal mesangial cells (mMC) and embryonic fibroblasts (MEF) from wild-type C57BL/6 or SK-2 knockout (SK2ko) mice. We found that SK2ko cells displayed a significantly higher proliferative and migratory activity when compared to wild-type cells, with concomitant increased cellular activities of the classical extracellular signal regulated kinase (ERK) and PI3K/Akt cascades, and of the small G protein RhoA. Furthermore, we detected an upregulation of SK-1 protein and S1P3 receptor mRNA expression in SK-2ko cells. The MEK inhibitor U0126 and the S1P1/3 receptor antagonist VPC23019 blocked the increased migration of SK-2ko cells. Additionally, S1P3ko mesangial cells showed a reduced proliferative behavior and reduced migration rate upon S1P stimulation, suggesting a crucial involvement of the S1P3 receptor. In summary, our data demonstrate that SK-2 exerts suppressive effects on cell growth and migration in renal mesangial cells and fibroblasts, and that therapeutic targeting of SKs for treating proliferative diseases requires subtype-selective inhibitors.

Increased cytotoxicity of cisplatin in SK-MEL 28 melanoma cells upon down-regulation of melanoma inhibitor of apoptosis protein

BACKGROUND: Malignant melanoma is a highly metastatic cutaneous cancer and typically refractory to chemotherapy. Deregulated apoptosis has been identified as a major cause of cancer drug resistance, and upregulated expression of the inhibitor of apoptosis protein melanom, an inhibitor of apoptosis (ML-IAP) is frequent in melanoma. METHODS: Based on the conclusion that ML-IAP expression contributes to a malignant phenotype, we down-regulated the ML-IAP mRNA using sequence optimized antisense oligonucleotides. RESULTS: As measured by real-time PCR, oligonucleotides M706 and M711 inhibited ML-IAP mRNA expression by 47% and 52%, respectively in the highly metastatic and drug resistant SK-MEL28 cell line. Oligonucleotide M706, which was previously evaluated in G361 cells as the most efficient inhibitor of ML-IAP expression, was chosen to compare cell viability and drug sensitivity of these two melanoma cell lines with different p53 functionality. Protein expression was reduced by oligonucleotide M706 to 49% of the normal level and resulted in a dose-dependent specific reduction of cell viability with a maximum of 39% at 600 nM. Typical morphological changes showed that loss of viability was mainly due to cell death. In combination experiments, the use of oligonucleotide M706 resulted in a two-fold increase of cisplatin cytotoxicity at different concentrations of oligonucleotide and cisplatin (P<0.05). This is in line with our previous findings in G361 melanoma cell line, in which oligonucleotide M706 caused a 3-fold increase in cisplatin cytotoxicity. CONCLUSION: Our data suggest the use of ML-IAP antisense oligonucleotides to overcome drug resistance in metastatic melanoma, in spite of its p53 status.

Deletion of CD39 on natural killer cells attenuates hepatic ischemia/reperfusion injury in mice

Natural killer (NK) cells play crucial roles in innate immunity and express CD39 (Ecto-nucleoside triphosphate diphosphohydrolase 1 [E-NTPD1]), a rate-limiting ectonucleotidase in the phosphohydrolysis of extracellular nucleotides to adenosine. We have studied the effects of CD39 gene deletion on NK cells in dictating outcomes after partial hepatic ischemia/reperfusion injury (IRI). We show in mice that gene deletion of CD39 is associated with marked decreases in phosphohydrolysis of adenosine triphosphate (ATP) and adenosine diphosphate to adenosine monophosphate on NK cells, thereby modulating the type-2 purinergic (P2) receptors demonstrated on these cells. We note that CD39-null mice are protected from acute vascular injury after single-lobe warm IRI, and, relative to control wild-type mice, display significantly less elevation of aminotransferases with less pronounced histopathological changes associated with IRI. Selective adoptive transfers of immune cells into Rag2/common gamma null mice (deficient in T cells, B cells, and NK/NKT cells) suggest that it is CD39 deletion on NK cells that provides end-organ protection, which is comparable to that seen in the absence of interferon gamma. Indeed, NK effector mechanisms such as interferon gamma secretion are inhibited by P2 receptor activation in vitro. Specifically, ATPgammaS (a nonhydrolyzable ATP analog) inhibits secretion of interferon gamma by NK cells in response to interleukin-12 and interleukin-18, providing a mechanistic link between CD39 deletion and altered cytokine secretion. CONCLUSION: We propose that CD39 deficiency and changes in P2 receptor activation abrogate secretion of interferon gamma by NK cells in response to inflammatory mediators, thereby limiting tissue damage mediated by these innate immune cells during IRI.

Loss of sphingosine kinase-1 in carcinoma cells increases formation of reactive oxygen species and sensitivity to doxorubicin-induced DNA damage

Sphingosine kinases (SK) catalyse the formation of sphingosine 1-phosphate, which is a key lipid mediator regulating cell responses such as proliferation, survival and migration. Here we have investigated the effect of targeted inhibition of SK-1 on cell damage and elucidated the mechanisms involved.

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.

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.

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.

Promotion of liver regeneration by natural killer cells in a murine model is dependent on extracellular adenosine triphosphate phosphohydrolysis

Nucleotides, such as adenosine triphosphate (ATP), are released by cellular injury, bind to purinergic receptors expressed on hepatic parenchymal and nonparenchymal cells, and modulate cellular crosstalk. Liver resection and resulting cellular stress initiate such purinergic signaling responses between hepatocytes and innate immune cells, which regulate and ultimately drive liver regeneration. We studied a murine model of partial hepatectomy using immunodeficient mice to determine the effects of natural killer (NK) cell-mediated purinergic signaling on liver regeneration. We noted first that liver NK cells undergo phenotypic changes post-partial hepatectomy (PH) in vivo, including increased cytotoxicity and more immature phenotype manifested by alterations in the expression of CD107a, CD27, CD11b, and CD16. Hepatocellular proliferation is significantly decreased in Rag2/common gamma-null mice (lacking T, B, and NK cells) when compared to wildtype and Rag1-null mice (lacking T and B cells but retaining NK cells). Extracellular ATP levels are elevated post-PH and NK cell cytotoxicity is substantively increased in vivo in response to hydrolysis of extracellular ATP levels by apyrase (soluble NTPDase). Moreover, liver regeneration is significantly increased by the scavenging of extracellular ATP in wildtype mice and in Rag2/common gamma-null mice after adoptive transfer of NK cells. Blockade of NKG2D-dependent interactions significantly decreased hepatocellular proliferation. In vitro, NK cell cytotoxicity is inhibited by extracellular ATP in a manner dependent on P2Y1, P2Y2, and P2X3 receptor activation. Conclusion: We propose that hepatic NK cells are activated and cytotoxic post-PH and support hepatocellular proliferation. NK cell cytotoxicity is, however, attenuated by hepatic release of extracellular ATP by way of the activation of specific P2 receptors. Clearance of extracellular ATP elevates NK cell cytotoxicity and boosts liver regeneration.

P2X1 regulated IL-22 secretion by innate lymphoid cells is required for efficient liver regeneration.

Paracrine signalling mediated via cytokine secretion is essential for liver regeneration after hepatic resection, yet the mechanisms of cellular crosstalk between immune and parenchymal cells are still elusive. Interleukin-22 (IL-22) is released by immune cells and mediates strong hepatoprotective functions. However, it remains unclear if IL-22 is critical for the crosstalk between liver lymphocytes and parenchymal cells during liver regeneration after partial hepatectomy. Here we found that plasma levels of IL-22 and its upstream cytokine IL-23 are highly elevated in patients after major liver resection. In a mouse model of partial hepatectomy, deletion of IL-22 was associated with significantly delayed hepatocellular proliferation and an increase of hepatocellular injury and endoplasmic reticulum stress. Using Rag1-/- and Rag2-/- γc-/- mice we show that the main producers of IL-22 post partial hepatectomy are conventional natural killer cells and innate lymphoid cells type 1. Extracellular ATP, a potent danger molecule, is elevated in patients immediately after major liver resection. Antagonism of the P2 type nucleotide receptors P2X1 and P2Y6 significantly decreased IL-22 secretion ex vivo. In vivo, specific inhibition of P2X1 was associated with decreased IL-22 secretion, elevated liver injury and impaired liver regeneration.