A glycosphingolipid/caveolin-1 signaling complex inhibits motility of human ovarian carcinoma cells.

The genetic (stable overexpression of sialyltransferase I, GM3 synthase) or pharmacological (selective pressure by N-(4-hydroxyphenyl)retinamide)) manipulation of A2780 human ovarian cancer cells allowed us to obtain clones characterized by higher GM3 synthase activity compared with wild-type cells. Clones with high GM3 synthase expression had elevated ganglioside levels, reduced in vitro cell motility, and enhanced expression of the membrane adaptor protein caveolin-1 with respect to wild-type cells. In high GM3 synthase-expressing clones, both depletion of gangliosides by treatment with the glucosylceramide synthase inhibitor D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol and silencing of caveolin-1 by siRNA were able to strongly increase in vitro cell motility. The motility of wild-type, low GM3 synthase-expressing cells was reduced in the presence of a Src inhibitor, and treatment of these cells with exogenous gangliosides, able to reduce their in vitro motility, inactivated c-Src kinase. Conversely, ganglioside depletion by D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol treatment or caveolin-1 silencing in high GM3 synthase-expressing cells led to c-Src kinase activation. In high GM3 synthase-expressing cells, caveolin-1 was associated with sphingolipids, integrin receptor subunits, p130(CAS), and c-Src forming a Triton X-100-insoluble noncaveolar signaling complex. These data suggest a role for gangliosides in regulating tumor cell motility by affecting the function of a signaling complex organized by caveolin-1, responsible for Src inactivation downstream to integrin receptors, and imply that GM3 synthase is a key target for the regulation of cell motility in human ovarian carcinoma.

Ionizing radiations increase the activity of the cell surface glycohydrolases and the plasma membrane ceramide content.

We detected significant levels of ß-glucosidase, ß-galactosidase, sialidase Neu3 and sphingomyelinase activities associated with the plasma membrane of fibroblasts from normal and Niemann-Pick subjects and of cells from breast, ovary, colon and neuroblastoma tumors in culture. All of the cells subjected to ionizing radiations showed an increase of the activity of plasma membrane ß-glucosidase, ß-galactosidase and sialidase Neu3, in addition of the well known increase of activity of plasma membrane sphingomyelinase, under similar conditions. Human breast cancer cell line T47D was studied in detail. In these cells the increase of activity of ß-glucosidase and ß-galactosidase was parallel to the increase of irradiation dose up to 60 Gy and continued with time, at least up to 72 h from irradiation. ß-glucosidase increased up to 17 times and ß-galactosidase up to 40 times with respect to control. Sialidase Neu3 and sphingomyelinase increased about 2 times at a dose of 20 Gy but no further significant differences were observed with increase of radiation dose and time. After irradiation, we observed a reduction of cell proliferation, an increase of apoptotic cell death and an increase of plasma membrane ceramide up to 3 times, with respect to control cells. Tritiated GM3 ganglioside has been administered to T47D cells under conditions that prevented the lysosomal catabolism. GM3 became component of the plasma membranes and was transformed into LacCer, GlcCer and ceramide. The quantity of ceramide produced in irradiated cells was about two times that of control cells.

Plasma membrane-associated glycohydrolases along differentiation of murine neural stem cells.

The activities of plasma membrane associated sialidase Neu3, total ß-glucosidase, CBE-sensitive ß-glucosidase, non-lysosomal ß-glucosyl ceramidase GBA2, ß-galactosidase, ß-hexosaminidase and sphingomyelinase were determined at three different stages of differentiation of murine neural stem cell cultures, corresponding to precursors, commited progenitors, and differentiated cells. Cell immunostaining for specific markers of the differentiation process, performed after 7 days in culture in presence of differentiating agents, clearly showed the presence of oligodendrocytes, astrocytes and neurons. Glial cells were the most abundant. Sialidase Neu3 after a decrease from progenitors to precursors, showed an increase parallel to the differentiation process. All the other glycosidases increased their activity along differentiation. The activity of CBE-sensitive ß-glucosidase and GBA2 were very similar at the precursor stage, but CBE-sensitive ß-glucosidase increased 7 times while GBA2 only two in the differentiated cells. In addition, we analysed also sphingomyelinase as enzyme specifically associated to sphingolipids. The activity of this enzyme increased from precursors to differentiated cells.

Plasma membrane-associated glycohydrolases activation by extracellular acidification due to proton exchangers.

In this paper, we show that the pH optimum for the plasma membrane (PM)-associated activity of four glycohydrolases (conduritol B epoxide sensitive ß-glucosidase, ß-glucosidase GBA2, ß-hexosaminidase and ß-galactosidase) measured on intact cells is acidic. Moreover, we show that drugs able to modify the efflux of protons across the PM, thus locally affecting the extracellular proton concentration close to the PM, are able to modulate the activities of these enzymes. These data strongly suggest that pH-dependent modulation of PM-associated glycohydrolases activities could be an effective way to locally modulate the cell surface glycoconjugate composition.

Cell surface associated glycohydrolases in normal and Gaucher disease fibroblasts.

Gaucher disease (GD) is the most common lysosomal disorder and is caused by an inherited autosomal recessive deficiency in ß-glucocerebrosidase. This enzyme, like other glycohydrolases involved in glycosphingolipid (GSL) metabolism, is present in both plasma membrane (PM) and intracellular fractions. We analyzed the activities of CBE-sensitive ß-glucosidase (GBA1) and AMP-DNM-sensitive ß-glucosidase (GBA2) in total cell lysates and PM of human fibroblast cell lines from control (normal) subjects and from patients with GD clinical types 1, 2, and 3. GBA1 activities in both total lysate and PM of GD fibroblasts were low, and their relative percentages were similar to those of control cells. In contrast, GBA2 activities were higher in GD cells than in control cells, and the degree of increase differed among the three GD types. The increase of GBA2 enzyme activity was correlated with increased expression of GBA2 protein as evaluated by QRT-PCR. Activities of ß-galactosidase and ß-hexosaminidase in PM were significantly higher for GD cells than for control cells and also showed significant differences among the three GD types, suggesting the occurrence of cross-talk among the enzymes involved in GSL metabolism. Our findings indicate that the profiles of glycohydrolase activities in PM may provide a valuable tool to refine the classification of GD into distinct clinical types.

Sphingosine kinase mediates resistance to the synthetic retinoid N-(4-hydroxyphenyl)retinamide in human ovarian cancer cells.

A2780 human ovarian carcinoma cells respond to treatment with the synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR) with the production of dihydroceramide and with a concomitant reduction of cell proliferation and induction of apoptosis. The derived HPR-resistant clonal cell line, A2780/HPR, is less responsive to HPR in terms of dihydroceramide generation. In this report, we show that the production of sphingosine 1-phosphate (S1P) is significantly higher in A2780/HPR versus A2780 cells due to an increased sphingosine kinase (SK) activity and SK-1 mRNA and protein levels. Treatment of A2780 and A2780/HPR cells with a potent and highly selective pharmacological SK inhibitor effectively reduced S1P production and resulted in a marked reduction of cell proliferation. Moreover, A2780/HPR cells treated with a SK inhibitor were sensitized to the cytotoxic effect of HPR, due to an increased dihydroceramide production. On the other hand, the ectopic expression of SK-1 in A2780 cells was sufficient to induce HPR resistance in these cells. Challenge of A2780 and A2780/HPR cells with agonists and antagonists of S1P receptors had no effects on their sensitivity to the drug, suggesting that the role of SK in HPR resistance in these cells is not mediated by the S1P receptors. These data clearly demonstrate a role for SK in determining resistance to HPR in ovarian carcinoma cells, due to its effect in the regulation of intracellular ceramide/S1P ratio, which is critical in the control of cell death and proliferation.

Cell surface sphingolipid glycohydrolases in neuronal differentiation and aging in culture.

Qualitative and quantitative changes in glycosphingolipids, together with changes in the expression of the corresponding glycosyltransferases, have been reported along neuronal differentiation and aging. Plasma membrane (PM) glycosphingolipid pattern and content are the result of a complex network of metabolic pathways, including those potentially involving the activity of PM glycohydrolases. We analyzed the total cell activities of sialyltransferase I, II and IV, sialidase, ß-galactosidase and ß-glucosidase, and the PM-associated activities of sialidase Neu3, ß-galactosidase, Conduritol B Epoxide-sensitive ß-glucosidase and ß-glucosidase GBA2 in rat cerebellar granule cells along differentiation and aging in culture. Sialyltransferase activities increased during cell differentiation, in agreement with the known increase of the total ganglioside content during neuronal maturation. The remodeling of ganglioside pattern could be because of the augmented activities of total sialidase and, within PM, to the action of the cell surface associated sialidase Neu3. Sialidase activities remained high during aging, in agreement with the known progressive ganglioside reduction in brain senescence. As PM ß-galactosidase and ß-glucosidase activities and parallely ceramide levels markedly increased along in vitro aging, PM ceramide production in neurons might be because of local catabolism of glycosphingolipids and not only to that of sphingomyelin, as already reported in human fibroblasts.

Remodeling of sphingolipids by plasma membrane associated enzymes.

The sphingolipid plasma membrane content and pattern is the result of several processes, among which the main, in term of quantity, are: neo-biosynthesis in endoplasmic reticulum and Golgi apparatus, membrane turnover with final catabolism in lysosomes and membrane shedding. In addition to this, past and recent data suggest that the head group of sphingolipids can be opportunely modified at the plasma membrane level, probably inside specific membrane lipid domains, by the action of enzymes involved in the sphingolipids metabolism, working directly at the cell surface. The number of membrane enzymes, hydrolases and transferases, acting on membrane sphingolipids is growing very rapidly. In this report we describe some properties of these enzymes.

Secondary alterations of sphingolipid metabolism in lysosomal storage diseases.

In several neurodegenerative diseases, sphingolipid metabolism is deeply deregulated, leading to the expression of abnormal membrane sphingolipid patterns and altered plasma membrane organization. In this paper, we review the potential importance of these alterations to the pathogenesis of these diseases and focus the reader's attention on some secondary alterations of sphingolipid metabolism that have been sporadically reported in the literature. Moreover, we present a detailed analysis of the lipid composition of different central nervous system and extraneural tissues from the acid sphingomyelinase-deficient mouse, the animal model for Niemann-Pick disease type A, characterized by the accumulation of sphingomyelin. Our data show an unexpected, tissue specific selection of the accumulated molecular species of sphingomyelin, and an accumulation of GM3 and GM2 gangliosides in both neural and extraneural tissues, that cannot be solely explained by the lack of acid sphingomyelinase.

Sphingolipidomics of A2780 human ovarian carcinoma cells treated with synthetic retinoids.

The dihydroceramide, ceramide, sphingomyelin, lactosylceramide, and ganglioside species of A2780 human ovarian carcinoma cells treated with the synthetic retinoids N-(4-hydroxyphenyl)retinamide (fenretinide, 4-HPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR) in culture were characterized by ESI-MS. We characterized 32 species of ceramide and dihydroceramide, 15 of sphingomyelin, 12 of lactosylceramide, 9 of ganglioside GM2, and 6 of ganglioside GM3 differing for the long-chain base and fatty acid structures. Our results indicated that treatment with both 4-HPR and 4-oxo-4-HPR led to a marked increase in dihydroceramide species, while only 4-oxo-4-HPR led to a minor increase of ceramide species. Dihydroceramides generated in A2780 cells in response to 4-HPR or 4-oxo-4-HPR differed for their fatty acid content, suggesting that the two drugs differentially affect the early steps of sphingolipid synthesis. Dihydroceramides produced upon treatments with the drugs were further used for the synthesis of complex dihydrosphingolipids, whose levels dramatically increased in drug-treated cells.

Photoactivable sphingosine as a tool to study membrane microenvironments in cultured cells.

Human fibroblasts from normal subjects and Niemann-Pick A (NPA) disease patients were fed with two labeled metabolic precursors of sphingomyelin (SM), [(3)H]choline and photoactivable sphingosine, that entered into the biosynthetic pathway allowing the synthesis of radioactive phosphatidylcholine and SM, and of radioactive and photoactivable SM ([(3)H]SM-N(3)). Detergent resistant membrane (DRM) fractions prepared from normal and NPA fibroblasts resulted as highly enriched in [(3)H]SM-N(3). However, lipid and protein analysis showed strong differences between the two cell types. After cross-linking, different patterns of SM-protein complexes were found, mainly associated with the detergent soluble fraction of the gradient containing most cell proteins. After cell surface biotinylation, DRMs were immunoprecipitated using streptavidin. In conditions that maintain the integrity of domain, SM-protein complexes were detectable only in normal fibroblasts, whereas disrupting the membrane organization, these complexes were not recovered in the immunoprecipitate, suggesting that they involve proteins belonging to the inner membrane layer. These data suggest that differences in lipid and protein compositions of these cell lines determine specific lipid-protein interactions and different clustering within plasma membrane. In addition, our experiments show that photoactivable sphingolipids metabolically synthesized in cells can be used to study sphingolipid protein environments and sphingolipid-protein interactions.

Glycosphingolipid behaviour in complex membranes.

Glycosphingolipids, due to their tendency to form laterally separated liquid-ordered phases, possess a high potential for the creation of order in biological membranes. The formation of glycosphingolipid-rich domains within the membrane has profound consequences on the membrane organization at different levels, and on the conformational and biological properties of membrane-associated proteins and multimolecular protein complexes. In this review, we will discuss 1) how glycosphingolipids influence the lateral organization of biological membranes; 2) how glycosphingolipids influence the function of membrane-associated proteins.

GM3 synthase overexpression results in reduced cell motility and in caveolin-1 upregulation in human ovarian carcinoma cells.

In this paper, we describe the effects of the expression of GM3 synthase at high levels in human ovarian carcinoma cells. Overexpression of GM3 synthase in A2780 cells consistently resulted in elevated ganglioside (GM3, GM2 and GD1a) levels. GM3 synthase overexpressing cells had a growth rate similar to wild-type cells, but showed a strongly reduced in vitro cell motility accompanied by reduced levels of the epithelial-mesenchymal transition marker alpha smooth muscle actin. A similar reduction in cell motility was observed upon treatment with exogenous GM3, GM2, and GM1, but not with GD1a. A photolabeling experiment using radioactive and photoactivable GM3 highlighted several proteins directly interacting with GM3. Among those, caveolin-1 was identified as a GM3-interacting protein in GM3 synthase overexpressing cells. Remarkably, caveolin-1 was markedly upregulated in GM3 synthase overexpressing cells. In addition, the motility of low GM3 synthase expressing cells was also reduced in the presence of a Src kinase inhibitor; on the other hand, higher levels of the inactive form of c-Src were detected in GM3 synthase overexpressing cells, associated with a ganglioside- and caveolin-rich detergent insoluble fraction.

Regulation of tumor phenotypes by caveolin-1 and sphingolipid-controlled membrane signaling complexes.

Aberrant (glyco)sphingolipid expression deeply affects several properties of tumor cells that are involved in tumor progression and metastasis formation: cell adhesion (to the extracellular matrix or to the endothelium of blood vessels), motility, recognition and invasion of host tissues. In particular, (glyco)sphingolipids might contribute to the modulation of integrin-dependent interactions of tumor cells (determining their adhesion, motility and invasiveness) with the extracellular matrix as well as with host cells present in the stromal compartment of the tumor. A model based on solid experimental evidence has been proposed: (glyco)sphingolipids at the cell surface interact with plasma membrane receptors (e.g., integrin receptors and growth factor receptors) and adapter molecules (including tetraspanins) forming signaling complexes that are able to influence the activity of signal transduction molecules oriented at the cytosolic surface of the plasma membrane (mainly the Src kinases pathway members). The function of these signaling complexes appears to be strictly dependent on their (glyco)sphingolipid composition, and likely on specific sphingolipid-protein interactions. From this point of view, particularly intriguing is the connection between (glyco)sphingolipids and caveolin-1, a membrane protein that plays multiple roles as a suppressor of tumor growth and metastasis in ovarian, breast and colon human carcinomas.

Thin layer chromatography of gangliosides.

Thin layer chromatography is the easiest way to analyze the total glycosphingolipid mixtures extracted, and, in some cases, partially purified from tissues and cultured cells. Several solvent systems have been introduced to separate the complex mixtures as a function of their composition, presence of contaminants and, in some cases, of their quantity. In addition, colorimetric, enzymatic, immunological and radiochemical detection procedures are available for their recognition. The method does not allow to determine the chemical structure of separated molecules, but gives a very economical and very accessible first information on their possible structure on the basis of their chromatographic mobility in comparison with standards, and of their reactivity to the staining procedures. In this paper we show how to perform mono and two-dimensional thin layer chromatography of the total lipid mixture extracted from mouse brains and, in a few cases, from cells in culture. Table 1 shows the structures of reported lipids.

Brain lipid composition in grey-lethal mutant mouse characterized by severe malignant osteopetrosis.

The grey-lethal mouse (gl/gl) mutant most closely resembles the severe human malignant autosomal recessive OSTM1-dependent form of osteopetrosis that it has been described to be associated with neurological abnormalities. For this reason, we have analyzed the brain lipid composition (gangliosides, neutral glycosphingolipids, phospholipids and cholesterol), from gl/gl mice at different ages of development and compared with wild type mice. Both cholesterol and glycerophospholipid content and pattern in the gl/gl and control mice were very similar. In contrast, significant differences were observed in the content of several sphingolipids. Higher amount of the monosialogangliosides GM2 and GM3, and lower content of sphingomyelin, sulfatide and galactosylceramide were observed in the gl/gl brain with respect to controls. The low content of sphingomyelin, sulfatide and galactosylceramide is consistent with the immunohistochemical results showing that in the grey-lethal brain significant depletion and disorganization of the myelinated fibres is present, thus supporting the hypothesis that loss of function of the OSTM1 causes neuronal impairment and myelin deficit.

Lipid content of brain, brain membrane lipid domains, and neurons from acid sphingomyelinase deficient mice.

The cholesterol, sphingolipid, and glycerophospholipid content of total brain, of detergent-resistant membranes prepared from the total brain, and of cerebellar granule cells differentiated in culture from wild type (WT) and acid sphingomyelinase knockout (ASMKO) were studied. Brains derived from 7-month-old ASMKO animals showed a fivefold higher level of sphingomyelin and a significant increase in ganglioside content, mainly because of monosialogangliosides GM3 and GM2 accumulation, while the cholesterol and glycerophospholipid content was unchanged with respect to WT animals. An increase in sphingomyelin, but not in gangliosides, was also detected in cultured cerebellar granule neurons from ASMKO mice, indicating that ganglioside accumulation is not a direct consequence of the enzyme defect. When a detergent-resistant membrane fraction was prepared from ASMKO brains, we observed that a higher detergent-to-protein ratio was needed than in WT animals. This likely reflects a reduced fluidity in restricted membrane areas because of a higher enrichment in sphingolipids in the case of ASMKO brain.

Modulation of cell functions by glycosphingolipid metabolic remodeling in the plasma membrane.

The endoplasmic reticulum and the Golgi apparatus are the subcellular sites for glycosphingolipid neobiosynthesis. In addition to this very well-established knowledge, it is now emerging that post-Golgi changes in glycosphingolipid structures occurring at the plasma membrane are an important opportunity to modulate cell glycosphingolipid composition and to affect consequently a number of signaling processes. In fact, it is possible to modify very rapidly the membrane organization by the modulation of plasma membrane-associated enzymes through external stimuli, thus affecting the membrane environment and the functional properties of plasma membrane proteins involved in cell signaling. The number of enzymes for glycosphingolipid metabolism that have been shown to be associated with the plasma membrane and the information on their features are growing very rapidly, and today some of these enzymes have been deeply characterized. In this review, we focus on the possible role and on the involvement of these plasma membrane-associated enzymes in modulating cell functions.

Reorganization of prion protein membrane environment during low potassium-induced apoptosis in primary rat cerebellar neurons.

We studied the changes occurring in the membrane environment of prion protein (PrP) during apoptosis induced by low potassium in primary rat cerebellar neurons. Ceramide levels increased during apoptosis-inducing treatment, being doubled with respect to time-matched controls after 24 h. Sphingomyelin levels were parallely decreased, while cholesterol and ganglioside contents were not affected. Changes in ceramide and sphingomyelin composition were exclusively restricted to a detergent-resistant membrane fraction. The pro-apoptotic treatment was accompanied by the down-regulation of PrP and of the non-receptor kinase Fyn. The levels of PrP and Fyn were correspondingly reduced in the detergent-resistant membrane fraction. In control cells, the membrane microenvironment separated by immunoprecipitation with anti-PrP antibody contained 80% of the detergent-resistant PrP and 35% and 38% of the sphingolipids and cholesterol respectively. Upon low potassium treatment, 20% of the PrP originally present in the detergent-resistant fraction was immunoprecipitated, together with 19% of sphingolipids and 22% of cholesterol. Thus, PrP in the immunoprecipitate from apoptotic cells was ninefold less than in control ones, while sphingolipids and cholesterol were about 50% with respect to controls cells. The molar ratio between cholesterol, sphingomyelin and ceramide was 15 : 6 : 1 in the PrP-rich environment from control neurons, and 6 : 2 : 1 in that from apoptotic cells.

Plasma membrane production of ceramide from ganglioside GM3 in human fibroblasts.

Ceramide is a key lipid molecule necessary to regulate some cellular processes, including apoptosis and cell differentiation. In this context, its production has been shown to occur via sphingomyelin hydrolysis or sphingosine acylation. Here, we show that in human fibroblasts, plasma membrane ceramide is also produced from ganglioside GM3 by detachment of sugar units. Membrane-bound glycosylhydrolases have a role in this process. In fact, the production of ceramide from GM3 has been observed even under experimental conditions able to block endocytosis or lysosomal activity, and the overexpression of the plasma membrane ganglioside sialidase Neu3 corresponded to a higher production of ceramide in the plasma membrane. The increased activity of Neu3 was paralleled by an increase of GM3 synthase mRNA and GM3 synthase activity. Neu3-overexpressing fibroblasts were characterized by a reduced proliferation rate and higher basal number of apoptotic cells in comparison with wild-type cells. A similar behavior was observed when normal fibroblasts were treated with exogenous C2-ceramide.