Laura Riboni

A Mediator Role of Ceramide in the Regulation of Neuroblastoma Neuro2a Cell Differentiation

Current studies indicate that ceramide is involved in the regulation of important cell functions, namely cell growth, differentiation, and apoptosis. In the present study, the possible role of ceramide in the differentiation of neuroblastoma Neuro2a cells was investigated. The following results were obtained. (a) Ceramide content of Neuro2a cells, induced to differentiate by retinoic acid (RA) treatment rapidly increased after addition of RA, was maintained at high levels in RA-differentiated cells and returned to the starting levels with removal of RA and reversal of differentiation; under the same conditions, the sphingosine content remained unchanged. (b) After a short pulse with [H]sphingomyelin or [H]sphingosine or L-[H]serine, the metabolic formation of ceramide was markedly higher and more rapid in RA-differentiated than undifferentiated cells. (c) Inhibitors of ceramide biosynthesis (Fumonisin B1, β-chloroalanine and L-cycloserine) diminished the extent of the differentiating effect of RA and concomitantly Cer content decreased. (d) The activity of neutral sphingomyelinase increased after addition of RA, maintained high levels in RA-differentiated cells, and returned to the initial levels with removal of RA. (e) Experimental conditions that cause an elevation of ceramide content (treatment with sphingosine or ceramide or C-ceramide or bacterial sphingomyelinase) inhibited cell proliferation and stimulated neurite outgrowth; dihydro-analogues of sphingosine, ceramide, and C-ceramide had no effect on differentiation. (f) treatment with Fumonisin B1 completely inhibited sphingosine-induced differentiation. These data suggest a specific bioregulatory function of ceramide in the control of Neuro2a cell growth and differentiation and pose the general hypothesis of a mediator role of ceramide in the differentiation of cells of neural origin.

Salvage pathways in glycosphingolipid metabolism

In this review, the focus is on the role of salvage pathways in glycosphingolipid, particularly, ganglioside metabolism. Ganglioside de novo biosynthesis, that begins with the formation of ceramide and continues with the sequential glycosylation steps producing the oligosaccharide moieties, is briefly outlined in its enzymological and cell-topological aspects. Neo-synthesized gangliosides are delivered to the plasma membrane, where their oligosaccharide chains protrude toward the cell exterior. The metabolic fate of gangliosides after internalization via endocytosis is then described, illustrating: (a) the direct recycling of gangliosides to the plasma membrane through vesicles gemmated from sorting endosomes; (b) the sorting through endosomal vesicles to the Golgi apparatus where additional glycosylations may take place; and (c) the channelling to the endosomal/lysosomal system, where complete degradation occurs with formation of the individual sugar (glucose, galactose, hexosamine, sialic acid) and lipid (ceramide, sphingosine, fatty acid) components of gangliosides. The in vivo and in vitro evidence concerning the metabolic recycling of these components is examined in detail. The notion arises that these salvage pathways, leading to the formation of gangliosides and other glycosphingolipids, sphingomyelin, glycoproteins and glycosaminoglycans, represent an important saving of energy in the cell economy and constitute a relevant event in overall ganglioside (or glycosphingolipid, in general) turnover, covering from 50% to 90% of it, depending on the cell line and stage of cell life. Sialic acid is the moiety most actively recycled for metabolic purposes, followed by sphingosine, hexosamine, galactose and fatty acid. Finally, the importance of salvage processes in controlling the active concentrations of ceramide and sphingosine, known to carry peculiar bioregulatory/signalling properties, is discussed.

Ceramide levels are inversely associated with malignant progression of human glial tumors

Ceramide represents an important sphingoid mediator involved in the signaling pathways that control cell proliferation, differentiation, and death. To determine whether ceramide levels correlate with the malignant progression of human astrocytomas, we investigated these levels in surgical specimens of glial tumors of low‐grade and high‐grade malignancy. Tumor samples obtained from 52 patients who underwent therapeutic removal of primary brain tumors were used. The tumors were classified according to standard morphologic criteria and were grouped into tumors of low‐grade and high‐grade malignancy. Sections of normal brain tissue adjacent to the tumor were also analyzed in 11 of the 52 patients. After extraction and partial purification, ceramide was measured by quantitative derivatization to ceramide‐1‐phosphate using diacylglycerol kinase and [γ‐32P]ATP. Ceramide levels were significantly lower in the combined high‐grade tumors compared with low‐grade tumors and in both tumor groups compared with peritumoral tissue. The results indicate an inverse correlation between the amount of ceramide and tumor malignancy as assessed by both the histological grading and ganglioside pattern. Moreover, overall survival analysis of 38 patients indicates that ceramide levels are significantly associated with patient survival. The present findings indicate that ceramide is inversely associated with malignant progression of human astrocytomas and poor prognosis. The downregulation of ceramide levels in human astrocytomas emerges as a novel alteration that may contribute to glial neoplastic transformation. The low ceramide levels in high‐grade tumors may provide an advantage for their rapid growth and apoptotic resistant features. This study appears to support the rationale for the potential benefits of a ceramide‐based chemotherapy. GLIA 39:105–113, 2002.

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.

Correlation between ganglioside distribution and histological grading of human astrocytomas.

Changes in membrane lipid content and composition have been repeatedly detected in experimentally induced as well as spontaneous tumors. Previous data on ganglioside analysis in human astrocytomas suggested a possible correlation between their content and pattern and the histological grading of these tumors. In our study we could definitely prove this correlation: in fact the level of malignancy, passing from grade 1 to grade IV, is associated with a statistically significant increase of a ganglioside identified as ganglioside GD3. Another important modification in the ganglioside pattern of human astrocytomas is the decrease in polysialylated species with increasing level of malignancy.

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.

Patterns of endogenous gangliosides and metabolic processing of exogenous gangliosides in cerebellar granule cells during differentiation in culture

The qualitative and quantitative pattern of endogenous gangliosides and the routes of metabolic processing of exogenous GM1,3H labeled in the sphingosine moiety (Sph-3H GM1) were studied in cerebellar granule cells during differentiation in vitro. During the first 7–8 days in culture the ganglioside content markedly increased, and the qualitative pattern showed, in percentage terms, a drastic decrease of GD3 and a marked increase of GD2, O-Ac-GT1b, O-Ac-GQ1b and GQ1b. After pulse with (Sph-3H) GM1, at all the investigated days in culture, different radiolabelled lipids were formed indicating that taken up exogenous GM1 was degraded and that its catabolic fragments, and partly GM1 itself, were used for biosynthetic purposes; moreover radioactive water was measured in the culture medium during chase indicating that labelled sphingosine underwent also degradation. The uptake of exogenous GM1 and the extent of its metabolic processing per cell unit increased during differentiation: a) GM2 was the major metabolic product and was relatively more abundant at 2 than 7 days in culture; b) the percentage of metabolites of biosynthetic origin over total metabolites increased during differentiation, especially at the short pulse times; c) among the metabolites of anabolic origin sphingomyelin equalled gangliosides at 2 days, whereas it was largely overcome by gangliosides at 7 days in culture; d) at 4 and 7 days in culture a radioactive substance, not yet identified, was present, whereas no trace of it was found at 2 days. In conclusion, cerebellar granule cells in culture feature a different pattern of endogenous gangliosides and display different ability to metabolically process exogenous GM1 ganglioside in the undifferentiated and fully differentiated stage.

Gangliosides turnover and neural cells function: a new perspective.

Publisher Summary There is a possibility that ganglioside turnover plays a major role in the formation of intracellular metabolic regulators. The chapter focuses on the evidence supporting this possibility, with particular reference to neural cells function. Gangliosides are involved in many nerve cell functions and the concept of a multifunctional role for these molecules seems the most flexible and suitable way to explain different functional behaviors. This wider view of interpreting ganglioside function includes the challenging hypothesis that gangliosides contribute to generate a new family of metabolic second messengers, comprising sphingosine, ceramide, and some of their derivatives. On the basis of the present knowledge, the only conceivable link between ganglioside metabolism and formation of compounds of a sphingoid nature is intra-lysosomal breakdown of membrane-bound gangliosides subsequent to endocytosis. Therefore, the suggestion is that external ligand binding to ganglioside affects the rate of ganglioside endocytosis and/or their sorting from endosomes to lysosomes. Consequently, their metabolic processing and the subsequent formation of second messengers of sphingoid nature turn out to be modified. The final effect is a modulation of cellular responses. The various aspects of this new perspective as well as assessment of its applicability to definite aspects of nerve cell function must be precisely defined and require the development of new experimental designs and ideas.

Formation of free sphingosine and ceramide from exogenous ganglioside GM1 by cerebellar granule cells in culture

Cerebellar granule cells differentiated in culture were incubated with ganglioside [3H‐Sph]GM1 in order to have it inserted into the plasma membrane and metabolized. Among the formed metabolites radioactive sphingosine and ceramide were identified. [3H]Ceramide started to be measurable after 10 min of incubation (pulse), and [3H]sphingosine after 15 min. Their concentrations increased with pulse time, and after a 1‐hour pulse, with chase time. After a 1‐hour pulse with 2 × 10−6 M [3H‐Sph]GM1 followed by a 4‐hour chase, the amount of [3H]sphingosine and [3H]ceramide formed were 0.04 and 0.4 pmol/106 cells, respectively. Particularly the ability to produce sphingosine was higher in differentiated than in undifferentiated cells. It is concluded that ganglioside turnover contributes to the maintenance of the intracellular levels of free sphingosine and ceramide.