Viviana Anelli

Sphingosine Kinase 1 Is Up-regulated during Hypoxia in U87MG Glioma Cells ROLE OF HYPOXIA-INDUCIBLE FACTORS 1 AND 2

Sphingosine 1-phosphate (S1P), a sphingolipid metabolite that plays an important role in the regulation of cell survival, growth, migration, and angiogenesis, acts both inside the cells and as an extracellular mediator through binding to five G protein-coupled receptors (S1P1-5). Sphingosine kinase 1 (SK1), the enzyme responsible for S1P production, is overexpressed in many solid tumors, including gliomas. One common feature of these tumors is the presence of “hypoxic regions,” characterized by cells expressing high levels of hypoxia-inducible factors HIF-1α and HIF-2α, two transcription regulators that modulate the levels of proteins with crucial roles in tumor progression. So far, nothing is known about the role and the regulation of SK1 during tumor-induced hypoxia or about SK1 regulation and HIFs. Here we investigated the role of HIF-1α and HIF-2α in the regulation of SK1 during hypoxic stress in glioma-derived U87MG cells. We report that hypoxia increases SK1 mRNA levels, protein expression, and enzyme activity, followed by intracellular S1P production and S1P release. Interestingly, knockdown of HIF-2α by small interfering RNA abolished the induction of SK1 and the production of extracellular S1P after CoCl2 treatment, whereas HIF-1α small interfering RNA resulted in an increase of HIF-2α and of SK1 protein levels. Moreover, using chromatin immunoprecipitation analysis, we demonstrate that HIF-2α binds the SK1 promoter. Functionally, we demonstrate that conditioned medium from hypoxia-treated tumor cells results in neoangiogenesis in human umbilical vein endothelial cells in a S1P receptor-dependent manner. These studies provide evidence of a link between S1P production as a potent angiogenic agent and the hypoxic phenotype observed in many tumors.

Sphingosine-1-phosphate is released by cerebellar astrocytes in response to bFGF and induces astrocyte proliferation through Gi-protein-coupled receptors.

The mitogenic role of sphingosine‐1‐phosphate (S1P) and its involvement in basic fibroblast growth factor (bFGF)‐induced proliferation were examined in primary cultures of cerebellar astrocytes. Exposure to bFGF resulted in a rapid increase of extracellular S1P formation, bFGF inducing astrocytes to release S1P, but not sphingosine kinase, in the extracellular milieu. The SK inhibitor N,N‐dimethylsphingosine inhibited S1P release as well as bFGF‐induced growth stimulation. S1P application in quiescent astrocytes caused a dose‐dependent increase in DNA synthesis. This gliotrophic effect was induced by a brief exposure to low nanomolar S1P, mimicked by the S1P receptor agonist dihydro‐S1P, and inhibited by pertussis toxin (PTX), an inactivator of Gi/Go‐proteins. S1P also induced activation of extracellular signal‐regulated kinase that was inhibited again by PTX. Moreover, the S1P lyase inhibitor 4‐deoxypyridoxine induced the cellular accumulation of S1P but did not affect DNA synthesis. These results support the view that S1P exerted a mitogenic effect on cerebellar astrocytes extracellularly, most likely through cell surface S1P receptors. In agreement, mRNAs for S1P1, S1P2, and S1P3 receptors are expressed in cerebellar astrocytes (Anelli et al., 2005. J Neurochem 92:1204–1215). Ceramide, a negative regulator of astrocyte proliferation and down‐regulated by bFGF (Riboni et al., 2002. Cerebellum 1:129–135), efficiently inhibited S1P‐induced proliferation. The S1P action appears to be part of an autocrine/paracrine cascade stimulated by bFGF and, together with ceramide down‐regulation, essential for astrocytes to respond to bFGF. The results suggest that S1P and bFGF/S1P may play an important role in physiopathological glial proliferation, such as brain development, reactive gliosis and brain tumor formation.

Extracellular release of newly synthesized sphingosine-1-phosphate by cerebellar granule cells and astrocytes.

Sphingosine‐1‐phosphate (S1P) is a potent biomediator that can act as either an intracellular or an intercellular messenger. In the nervous system it exerts a wide range of actions, and specific membrane receptors for it have been identified in various regions. However, the physiological origin of extracellular S1P in the nervous system is largely unknown. We investigated cerebellar granule cells at different stages of differentiation and astrocytes in primary cultures as possible origins of extracellular S1P. Although these cells show marked differences in S1P metabolism, we found that they can all release S1P and express mRNAs for S1P specific receptors. Extracellular S1P derives from the export of newly synthesized intracellular S1P, and not from the action of a released sphingosine kinase. S1P release is rapid, efficient, and can be regulated by exogenous stimuli. Phorbol ester treatment resulted in an increase in sphingosine kinase 1 activity in the membranes, accompanied by a significant increase in extracellular S1P. S1P release in cells from the cerebellum emerges as a regulated mechanism, possibly related to a specific pool of newly synthesized S1P. To our knowledge, this is the first evidence of the extracellular release of S1P by primary cells from the CNS, which supports a role of S1P as autocrine/paracrine physiological messenger in the cerebellum.

Role of sphingosine kinase-1 in paracrine/transcellular angiogenesis and lymphangiogenesis in vitro

Sphingosine‐1‐phosphate (S1P) is an important bioactive sphingolipid involved in angiogenesis and lymphangiogenesis, 2 important processes that influence the growth, survival, and spread of tumors. S1P acts as an extracellular mediator through binding to 5 highly specific S1P receptors, S1P1–5. Sphingosine kinase‐1 (SK1), one of 2 known sphingosine kinase enzymes responsible for S1P production, appears to be overexpressed in many tumors. Although a role for S1P in angiogenesis and lymphangiogenesis has been established, it is unclear whether S1P secreted from cancer cells has a paracrine function in a tumor environment. Here we investigated whether modulation of cellular SK1 could initiate a paracrine angiogenic and lymphangiogenic switch. We found that SK1 overexpression in HEK cells or its down‐regulation in glioma or breast cancer cells modulated extracellular S1P levels accordingly, which in turn increased or decreased both migration and tube formation in cocultured vascular or lymphatic endothelial cells. In contrast, down‐regulation of sphingosine kinase 2 in both glioma and breast cancer cells had no appreciable effect on cellular or secreted S1P levels. In addition, vascular endothelial growth factors VEGF and VEGF‐C down‐regulation in cancer cells appeared insufficient to block the angiogenic and lymphangiogenic switch triggered by these cells. Moreover, S1P initiated endothelial cell sprouting in 3‐dimensional collagen matrices, which is representative of a multistep angiogenic process. Our data collectively demonstrate for the first time that SK1 plays an essential role in regulating in vitro paracrine angiogenesis and lymphangiogenesis.—Anelli, V., Gault, C. R., Snider, A. J., Obeid, L. M. Role of sphingosine kinase‐1 in paracrine/transcellular angiogenesis and lymphangiogenesis in vitro. FASEB J. 24, 2727–2738 (2010). www.fasebj.org

HMGB1 as an autocrine stimulus in human T98G glioblastoma cells: role in cell growth and migration

HMGB1 (high mobility group box 1 protein) is a nuclear protein that can also act as an extracellular trigger of inflammation, proliferation and migration, mainly through RAGE (the receptor for advanced glycation end products); HMGB1–RAGE interactions have been found to be important in a number of cancers. We investigated whether HMGB1 is an autocrine factor in human glioma cells. Western blots showed HMGB1 and RAGE expression in human malignant glioma cell lines. HMGB1 induced a dose-dependent increase in cell proliferation, which was found to be RAGE-mediated and involved the MAPK/ERK pathway. Moreover, in a wounding model, it induced a significant increase in cell migration, and RAGE-dependent activation of Rac1 was crucial in giving the tumour cells a motile phenotype. The fact that blocking DNA replication with anti-mitotic agents did not reduce the distance migrated suggests the independence of the proliferative and migratory effects. We also found that glioma cells contain HMGB1 predominantly in the nucleus, and cannot secrete it constitutively or upon stimulation; however, necrotic glioma cells can release HMGB1 after it has translocated from the nucleus to cytosol. These findings provide the first evidence supporting the existence of HMGB1/RAGE signalling pathways in human glioblastoma cells, and suggest that HMGB1 may play an important role in the relationship between necrosis and malignancy in glioma tumours by acting as an autocrine factor that is capable of promoting the growth and migration of tumour cells.

Acid β-Glucosidase 1 Counteracts p38δ-dependent Induction of Interleukin-6: POSSIBLE ROLE FOR CERAMIDE AS AN ANTI-INFLAMMATORY LIPID*

Activation of protein kinase C (PKC) by the phorbol ester (phorbol 12-myristate 13-acetate) induces ceramide formation through the salvage pathway involving, in part, acid β-glucosidase 1 (GBA1), which cleaves glucosylceramide to ceramide. Here, we examine the role of the GBA1-ceramide pathway, in regulating a pro-inflammatory pathway initiated by PKC and leading to activation of p38 and induction of interleukin 6 (IL-6). Inhibition of ceramide formation by fumonisin B1 or down-regulation of PKCδ potentiated PMA-induced activation of p38 in human breast cancer MCF-7 cells. Similarly, knockdown of GBA1 by small interfering RNAs or pharmacological inhibition of GBA1 promoted further activation of p38 after PMA treatment, implicating the GBA1-ceramide pathway in the termination of p38 activation. Knockdown of GBA1 also evoked the hyperproduction of IL-6 in response to 4β phorbol 12-myristate 13-acetate. On the other hand, increasing cellular ceramide with cell-permeable ceramide treatment resulted in attenuation of the IL-6 response. Importantly, silencing the δ isoform of the p38 family significantly attenuated the hyperproduction of IL-6. Reciprocally, p38δ overexpression induced IL-6 biosynthesis. Thus, the GBA1-ceramide pathway is suggested to play an important role in terminating p38δ activation responsible for IL-6 biosynthesis. Furthermore, the p38δ isoform was identified as a novel and predominant target of ceramide signaling as well as a regulator of IL-6 biosynthesis.Activation of protein kinase C (PKC) by the phorbol ester (phorbol 12-myristate 13-acetate) induces ceramide formation through the salvage pathway involving, in part, acid beta-glucosidase 1 (GBA1), which cleaves glucosylceramide to ceramide. Here, we examine the role of the GBA1-ceramide pathway, in regulating a pro-inflammatory pathway initiated by PKC and leading to activation of p38 and induction of interleukin 6 (IL-6). Inhibition of ceramide formation by fumonisin B1 or down-regulation of PKCdelta potentiated PMA-induced activation of p38 in human breast cancer MCF-7 cells. Similarly, knockdown of GBA1 by small interfering RNAs or pharmacological inhibition of GBA1 promoted further activation of p38 after PMA treatment, implicating the GBA1-ceramide pathway in the termination of p38 activation. Knockdown of GBA1 also evoked the hyperproduction of IL-6 in response to 4beta phorbol 12-myristate 13-acetate. On the other hand, increasing cellular ceramide with cell-permeable ceramide treatment resulted in attenuation of the IL-6 response. Importantly, silencing the delta isoform of the p38 family significantly attenuated the hyperproduction of IL-6. Reciprocally, p38delta overexpression induced IL-6 biosynthesis. Thus, the GBA1-ceramide pathway is suggested to play an important role in terminating p38delta activation responsible for IL-6 biosynthesis. Furthermore, the p38delta isoform was identified as a novel and predominant target of ceramide signaling as well as a regulator of IL-6 biosynthesis.

Involvement of Acid β-Glucosidase 1 in the Salvage Pathway of Ceramide Formation

Activation of protein kinase C (PKC) promotes the salvage pathway of ceramide formation, and acid sphingomyelinase has been implicated, in part, in providing substrate for this pathway (Zeidan, Y. H., and Hannun, Y. A. (2007) J. Biol. Chem. 282, 11549–11561). In the present study, we examined whether acid β-glucosidase 1 (GBA1), which hydrolyzes glucosylceramide to form lysosomal ceramide, was involved in PKC-regulated formation of ceramide from recycled sphingosine. Glucosylceramide levels declined after treatment of MCF-7 cells with a potent PKC activator, phorbol 12-myristate 13-acetate (PMA). Silencing GBA1 by small interfering RNAs significantly attenuated acid glucocerebrosidase activity and decreased PMA-induced formation of ceramide by 50%. Silencing GBA1 blocked PMA-induced degradation of glucosylceramide and generation of sphingosine, the source for ceramide biosynthesis. Reciprocally, forced expression of GBA1 increased ceramide levels. These observations indicate that GBA1 activation can generate the source (sphingosine) for PMA-induced formation of ceramide through the salvage pathway. Next, the role of PKCδ, a direct effector of PMA, in the formation of ceramide was determined. By attenuating expression of PKCδ, cells failed to trigger PMA-induced alterations in levels of ceramide, sphingomyelin, and glucosylceramide. Thus, PKCδ activation is suggested to stimulate the degradation of both sphingomyelin and glucosylceramide leading to the salvage pathway of ceramide formation. Collectively, GBA1 is identified as a novel source of regulated formation of ceramide, and PKCδ is an upstream regulator of this pathway.

Metabolic Formation of Ceramide-1-Phosphate in Cerebellar Granule Cells: Evidence for the Phosphorylation of Ceramide by Different Metabolic Pathways

Aiming to investigate the possible production of ceramide-1-phosphate from complex sphingolipid metabolism in neurons, we administered radiolabeled sphingolipids to cerebellar granule cells and inspected the formation of labeled ceramide-1-phosphate in different experimental conditions. We report that differentiated granule cells are capable to form Cer-1-P via ceramide derived from SM degradation at the plasma membrane level. Moreover we observed that ceramide-1-phosphate can be also produced from a metabolic pathway not involving SM degradation. In particular, we obtained evidence that ceramide, synthesized via the recycling of sphingosine produced from ganglioside catabolism, can also be the precursor of ceramide-1-phosphate. We also found that undifferentiated and differentiated granule cells display different capacities to phosphorylate Cer produced by the two different metabolic pathways. The results here obtained demonstrate that cerebellar neurons are able to metabolically produce ceramide-1-phosphate and support that this molecule may serve a potential role in sphingoid-mediated signaling in the nervous system.

A bidirectional crosstalk between glioblastoma and brain endothelial cells potentiates the angiogenic and proliferative signaling of sphingosine-1-phosphate in the glioblastoma microenvironment

Glioblastoma is one of the most malignant, angiogenic, and incurable tumors in humans. The aberrant communication between glioblastoma cells and tumor microenvironment represents one of the major factors regulating glioblastoma malignancy and angiogenic properties. Emerging evidence implicates sphingosine-1-phosphate signaling in the pathobiology of glioblastoma and angiogenesis, but its role in glioblastoma-endothelial crosstalk remains largely unknown. In this study, we sought to determine whether the crosstalk between glioblastoma cells and brain endothelial cells regulates sphingosine-1-phosphate signaling in the tumor microenvironment. Using human glioblastoma and brain endothelial cell lines, as well as primary brain endothelial cells derived from human glioblastoma, we report that glioblastoma-co-culture promotes the expression, activity, and plasma membrane enrichment of sphingosine kinase 2 in brain endothelial cells, leading to increased cellular level of sphingosine-1-phosphate, and significant potentiation of its secretion. In turn, extracellular sphingosine-1-phosphate stimulates glioblastoma cell proliferation, and brain endothelial cells migration and angiogenesis. We also show that, after co-culture, glioblastoma cells exhibit enhanced expression of S1P1 and S1P3, the sphingosine-1-phosphate receptors that are of paramount importance for cell growth and invasivity. Collectively, our results envision glioblastoma-endothelial crosstalk as a multi-compartmental strategy to enforce pro-tumoral sphingosine-1-phosphate signaling in the glioblastoma microenvironment.