1,720,964 research outputs found
Biomodulatory role of ceramide in basic fibroblast growth factor-induced proliferation of cerebellar astrocytes in primary culture
No full textTo evaluate the role of ceramide in glial growth, primary cultures of quiescent astrocytes from rat cerebellum were stimulated to proliferate by mitogenic doses of basic fibroblast growth factor (bFGF). Parallel to the bFGF mitogenic effect was a marked, and persistent, decrease in cellular ceramide levels. Both in vitro and in culture metabolic studies have led us to exclude both sphingomyelinase and ceramidase involvement in ceramide level variation. Instead, we found evidence of a functional connection between the decrease in ceramide levels and astrocyte proliferation. In fact, cell growth in bFGF-stimulated astrocytes was inhibited by exogenous ceramide and C2-ceramide, maximal inhibition being obtained at a ceramide concentration of 5-10 M. Under the same conditions, the dihydroderivatives of ceramides were without effect. Following ceramide treatment, the phosphorylation of the MAP kinase isoforms ERK1/2, key components in bFGF-induced cell proliferation, was examined. The administration of antiproliferative doses of ceramide or C2-ceramide, but not of their dihydroderivatives, resulted in a significant inhibition of ERK1/2 activation. In conclusion, our data indicate that the prompt modulation of ceramide levels by bFGF is an early step associated with the signaling pathways responsible for the mitogenic activity of bFGF in astrocyte
Alteration of glycosphingolipid levels and overexpression of GlcCer synthase are associated to acquired resistance to unrelated drugs in human glioma cells
No full textDifferent studies support that glycosphingolipids (GSLs) act as important players in tumor biology, and emerging evidence suggests their involvement in drug resistance too. In tumor specimens from patients with scarce response to chemotherapy and in drug-resistant cancer cells, the activity of glucosylceramide synthase (GCS) and the levels of glucosylceramide (GlcCer) were found elevated. Moreover, inhibitors of GCS have been shown to increase the cellular levels of ceramide, a key player in the regulation of apoptosis, and reverse drug resistance. Notwithstanding, little is known on the involvement of GSLs in malignant gliomas. These are the most frequent and deadly human primary brain tumors, their intrinsic or acquired resistance limiting therapy effectiveness. In this study we investigated the possible involvement of GSLs in the resistance of T98G human glioblastoma cells to two unrelated drugs with clinical activity against gliomas, i.e. the microtubule-disrupting agent paclitaxel and the alkylating drug temozolomide. By selection with gradually increasing drug concentrations, we generated both a paclitaxel (Ptx-R) and a temozolomide (Tmz-
R) resistant cell line. The analysis of resistance markers by immunoblotting showed that MDR1 (P-glycoprotein) was highly expressed in Ptx-R cells, but undetectable in T98G and
Tmz-R ones. O6-methylguanine-DNA methyltransferase was expressed in all cells, with marked overexpression exclusively in Tmz-R cells. After labeling at equilibrium with 3H-sphingosine
or 3H-serine, both Ptx-R and Tmz-R cells were characterized, with respect to sensitive T98G, by increased levels of GlcCer and gangliosides. In addition, the ceramide levels were found lower
in resistant vs. sensitive cells. A semi-quantitative RT-PCR analysis showed that a higher expression of the mRNA for the GCS gene was evident in both Ptx-R and Tmz-R cells. The in
vitro activity of GCS in both resistant cells was found significantly higher than in sensitive ones. Noteworthy, the levels of lactosyl-ceramide, the major neutral glycosphingolipid of T98G cells, were significantly lower in Tmz-R, but not in Ptx-
R cells. This finding supports the role for MDR1 as the major GlcCer translocase required for neutral glycosphingolipid anabolism, as well as the existence of distinct metabolic pathways for ganglioside and neutral glycolipid biosynthesis.
Altogether our data demonstrate that overexpression of GCS and alterations of glycosphingolipid level, with increased GlcCer
and ganglioside content, are associated to T98G resistance to unrelated cytotoxic drugs. These variations occur independently of MDR1 expression, and are associated to the attenuation of
ceramide levels. This suggests that the increase of specific glycosphingolipids might offer glioma cells a survival advantage,resulting in resistance to chemotherapeutic drugs
Sphingosine-1-phosphate as survival factor in etoposide-induced cytotoxicity in glioma cells: role of PI3K/Akt-dependent stimulation of endoplasmic reticulum (ER) to Golgi apparatus ceramide traffic
No full textSphingosine-1-phosphate and calcium signaling in cerebellar astrocytes and differentiated granule cells
No full textS1P is involved in the regulation of multiple biological processes (cell survival, growth, migration and differentiation) both in neurons and glial cells. The study was aimed at investigating the possible effects of S1P on calcium signaling in cerebellar astrocytes and differentiated granule cells. In cerebellar astrocytes S1P is able to mediate calcium signaling mainly through Gi protein coupled receptors, whereas in differentiated neurons it failed to evoke any calcium signaling, despite acting both extracellularly and intracellularly. The data indicate strict cell specificity in S1P-evoked calcium response, which could be relevant to communication between neurons and glial cells in the cerebellum
Sphingosine-1-phosphate promotes survival against etoposide by enhancing ceramide transport from ER to Golgi apparatus in T98G glioma cells
No full textExtracellular sphingosine-1-phosphate acts as a survival factor for human glioblastoma stem cells
No full textExtracellular release of newly synthesized sphingosine-1-phosphate by cerebellar granule cells and astrocytes
No full textSphingosine-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
Sphingosine-1-phosphate secretion and signaling provide survival advantages to human glioblastoma cells
No full textSphingosine-1-phosphate is released by cerebellar astrocytes in response to bFGF and induces astrocyte proliferation through Gi-protein-coupled receptors
No full textThe 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 G(i)/G(o)-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
Ceramide traffic in C6 glioma cells: evidence for CERT-dependent and independent transport from ER to the Golgi apparatus
No full textIntracellular movements of ceramide are strongly limited by its hydrophobic nature, and the mechanisms involved in ceramide transport can
represent a crucial aspect of sphingolipid metabolism and signaling. The recent identification of the ceramide specific carrier protein CERT has
revealed a novel pathway for the delivery of ceramide to the Golgi apparatus for sphingomyelin biosynthesis. In this study we investigated the
metabolic and functional role of CERT in C6 glioma cells. These cells were found to constitutively express CERT, the protein being mainly
associated with the cytosolic fraction. Metabolic experiments performed with different radioactive metabolic precursors of sphingolipids
demonstrated that the down regulation of CERT by RNAi technology resulted in a significant but not complete reduction of ceramide metabolism
to sphingomyelin, without affecting its utilization for glycosphingolipid biosynthesis. Since nitric oxide is an inhibitor of ceramide ER-to-Golgi
traffic and metabolism in C6 glioma cells, we evaluated the possibility that the CERT-mediated transport of ceramide might represent a target for
nitric oxide. The data obtained demonstrate that CERT down regulation does not affect the inhibitory activity of nitric oxide on Cer metabolism,
and the effects of nitric oxide and CERT silencing on ceramide utilization were additive. These results strongly suggest that a CERT-mediated and
a CERT-independent, nitric oxide-sensitive Cer transport coexist in C6 glioma cells and can separately contribute to the control of sphingolipid metabolism and Cer levels in these cells
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