Caveolin-1 influences mitochondrial plasticity and function in hepatic stellate cell activation.

Caveolin-1 (Cav-1) is an integral membrane protein present in all organelles, responsible for regulating and integrating multiple signals as a platform. Mitochondria are extremely adaptable to external cues in chronic liver diseases, and expression of Cav-1 may affect mitochondrial flexibility in hepatic stellate cells (HSCs) activation. We previously demonstrated that exogenous expression of Cav-1 was sufficient to increase some classical markers of activation in HSCs. Here, we aimed to evaluate the influence of exogenous expression and knockdown of Cav-1 on regulating the mitochondrial plasticity, metabolism, endoplasmic reticulum (ER)-mitochondria distance, and lysosomal activity in HSCs. To characterize the mitochondrial, lysosomal morphology, and ER-mitochondria distance, we perform transmission electron microscope analysis. We accessed mitochondria and lysosomal networks and functions through a confocal microscope and flow cytometry. The expression of mitochondrial machinery fusion/fission genes was examined by real-time polymerase chain reaction. Total and mitochondrial cholesterol content was measured using Amplex Red. To define energy metabolism, we used the Oroboros system in the cells. We report that GRX cells with exogenous expression or knockdown of Cav-1 changed mitochondrial morphometric parameters, OXPHOS metabolism, ER-mitochondria distance, lysosomal activity, and may change the activation state of HSC. This study highlights that Cav-1 may modulate mitochondrial function and structural reorganization in HSC activation, being a potential candidate marker for chronic liver diseases and a molecular target for therapeutic intervention.

Biological activities of ACL-I and physicochemical properties of ACL-II, lectins isolated from the marine sponge Axinella corrugata.

Lectin II from the marine sponge Axinella corrugata (ACL-II) was purified by affinity chromatography on rabbit erythrocytic stroma incorporated into a polyacrylamide gel, followed by gel filtration on Ultrogel AcA 44 column. Purified ACL-II is a lectin with an Mr of 80 kDa and 78 kDa, estimated by SDS-PAGE and by FPLC on Superose 12 HR column, respectively. ACL-II mainly agglutinates native rabbit erythrocytes and this hemagglutinating activity is independent of Ca(2+), Mg(2+) and Mn(2+), but is inhibited by d-galactose, chitin and N-acetyl derivatives, with the exception of GalNAc. ACL-II is stable for up to 65 °C for 30 min, with a better stability at a pH range of 2 to 6. In contrast, ACL-I displays a strong mitogenic and cytotoxic effect.

Anchored and soluble gangliosides contribute to myelosupportivity of stromal cells.

Stroma-mediated myelopoiesis depends upon growth factors and an appropriate intercellular microenvironment. Previous studies have demonstrated that gangliosides, produced by hepatic stromal cell types, are required for optimal myelosupportive function. Here, we compared the mielossuportive functions of a bone marrow stroma (S17) and skin fibroblasts (SF) regarding their ganglioside pattern of synthesis and shedding. The survival and proliferation of a myeloid precursor cell (FDC-P1) were used as reporter. Although the ganglioside synthesis of the two stromal cells was similar, their relative content and shedding were distinct. The ganglioside requirement for mielossuportive function was confirmed by the decreased proliferation of FDC-P1 cells in ganglioside synthesis-inhibited cultures and in presence of an antibody to GM3 ganglioside. The distinct mielossuportive activities of the S17 and SF stromata may be related to differences on plasma membrane ganglioside concentrations or to differences on the gangliosides shed and their subsequent uptake by myeloid cells, specially, GM3 ganglioside.

ACL-I, a lectin from the marine sponge Axinella corrugata: isolation, characterization and chemotactic activity.

The lectin from the marine sponge Axinella corrugata (ACL-I) was purified by affinity chromatography on rabbit erythrocytic stroma incorporated into a polyacrylamide gel followed by gel filtration on Ultrogel AcA 44 column. Purified ACL-I is a hexameric glycoprotein with a Mr of 82.3 kDa estimated by SDS-PAGE and 78.5 kDa by FPLC on Superose 12 HR column. The pI of lectin is 6.3 and ACL-I is constituted of 13.9 kDa similar subunits some of them linked by disulphide bridges. This lectin agglutinates native rabbit, goat and dog erythrocytes and in less extent human erythrocytes. The hemagglutinating activity is independent of Ca(2+), Mg(2+) and Mn(2+), but it is strongly inhibited by carbohydrates containing N-acetyl groups. ACL-I is stable up to 70 degrees C for 30 min, with optimum pH between 7 and 8, and it is also resistant to enzymatic proteolysis in vitro. In the presence of reducing or denaturant agents, the lectin activity decreases. ACL-I displays chemotactic effect on rat neutrophil in vitro which is inhibited by N-acetyl-d-glucosamine.

Reduction of gangliosides, phospholipids and cholesterol content in cerebral cortex of rats caused by chronic hypermethioninemia.

Neurological dysfunction is observed in patients with severe hypermethioninemia, whose physiopathology is still poorly understood. In the current study we investigated the effect of chronic administration of methionine on the content and species of gangliosides and phospholipids, as well as on the concentration of cholesterol in rat cerebral cortex. Wistar rats received subcutaneous injections of methionine (1.34-2.68 micromol/g of body weight), twice a day, from the 6th to the 28th day of age and controls received saline. Animals were killed 12h after the last injection. Results showed that methionine administration significantly decreased the total content of lipids in cerebral cortex of rats. We also observed that this amino acid significantly reduced the absolute quantity of the major brain gangliosides (GM1, GD1a, GD1b and GT1b) and phospholipids (sphingomyelin, phosphatidylcholine and phosphatidylethanolamine). We also showed that Na+,K+-ATPase activity and TBARS were changed in cerebral cortex of rats subjected to hypermethioninemia. If confirmed in human beings, these data could suggest that the alteration in lipid composition, Na+,K+-ATPase activity and TBARS caused by methionine might contribute to the neurophysiopathology observed in hypermethioninemic patients.

Variations of ganglioside biosynthetic pathways in the phenotype conversion from myofibroblasts to lipocytes in murine hepatic stellate cell line.

GRX cell line represents hepatic stellate cell and can be transformed from an actively proliferation myofibroblast phenotype into a quiescent fat-storing lipocyte phenotype. Both express the same gangliosides (GM3, GM2, GM1 and GD1a), which are resolved as doublets on HPTLC. Upper/lower band ratio is increased in lipocyte-like cells and the upper band is composed by ceramides with long-chain fatty acids. This study evaluated the contribution of de novo synthesis, sphingosine and Golgi recycling pathways on ganglioside biosynthesis, in both phenotypes. Cells were preincubated with 5 mM beta-chloroalanine (SPT: serine palmitoyltransferase inhibitor) or with 25 muM fumonisin B1 (ceramide synthase inhibitor) and then radiolabeled with [U-(14)C]galactose in the continued presence of inhibitors. Gangliosides were extracted, purified and analyzed by HPTLC. In myofibroblast-like cells, simple gangliosides use the de novo pathway while complex gangliosides are mainly synthesized by recycling pathways. In lipocyte-like cells, de novo pathway has a lesser contribution and this is in agreement with the lower activity of the committed enzyme of sphingolipid synthesis (SPT) detected in this phenotype. SPT mRNA has an identical expression in both phenotypes. It was also observed that gangliosides doublets from myofibroblast-like cells have the same distribution between triton soluble and insoluble fractions (upper band > lower band) while the gangliosides doublets from lipocyte-like cells show an inversion in the insoluble fraction (lower band > upper band) in comparison to soluble fraction. These results indicate that myofibroblast- and lipocyte-like cells have important differences between the glycosphingolipid biosynthetic pathways, which could contribute with the respective glycosphingolipid-enriched membrane microdomain's composition.

Gangliosides of the stroma layer participate in the interferon-gamma receptor-dependent controls of myelopoiesis.

Stroma-mediated myelopoiesis depends upon growth-factors and an appropriate intercellular microenvironment, whose polarity is relevant for granulocyte-macrophage colony stimulating factor (GM-CSF) mediated myeloid cell proliferation. Here we have studied qualitative and quantitative aspects of ganglioside participation in controls of the microenvironment required to sustain myelopoiesis. We analysed ganglioside synthesis, expression and shedding by two primary liver stromal cell cultures isolated from wild type and interferon-gamma (IFNgamma) receptor knockout mice. The latter one has a higher capacity to sustain myelopoiesis. FDC-P1 myeloid growth factor-dependent cell line was used as the reporter system, monitoring the cell survival and proliferation that reflect the bio-availability and the activity of GM-CSF. Although the two stromal cells synthesised the same gangliosides their relative content was quite different. FDC-P1 proliferation decreased in cultures in which ganglioside synthesis was inhibited in the stroma, as well as in presence of stroma cell supernatants in which GM3 was neutralised by the anti-GM3 monoclonal antibody. Addition of exogenous GM3 reverted the inhibition and sustained proliferation of FDC-P1 cells. FDC-P1 cells do not accumulate GM3, but they are able to take up the stroma-produced sphingolipids. Thus, stroma has a double role in sustaining myelopoiesis, providing both growth factor(s) and ganglioside(s) required for the optimal stimulation of the myeloid cell proliferation, and the IFNgamma mediated stroma-dependent controls of myelopoiesis are determinant for this cell interaction.

Gangliosides of myelosupportive stroma cells are transferred to myeloid progenitors and are required for their survival and proliferation.

In previous studies, we have shown that the myelopoiesis dependent upon myelosupportive stroma required production of growth factors and heparan-sulphate proteoglycans, as well as generation of a negatively charged sialidase-sensitive intercellular environment between the stroma and the myeloid progenitors. In the present study, we have investigated the production, distribution and role of gangliosides in an experimental model of in vitro myelopoiesis dependent upon AFT-024 murine liver-derived stroma. We used the FDC-P1 cell line, which is dependent upon GM-CSF (granulocyte/macrophage colony-stimulating factor) for both survival and proliferation, as a reporter system to monitor bioavailability and local activity of GM-CSF. G(M3) was the major ganglioside produced by stroma, but not by myeloid cells, and it was required for optimal stroma myelosupportive function. It was released into the supernatant and selectively incorporated into the myeloid progenitor cells, where it segregated into rafts in which it co-localized with the GM-CSF-receptor alpha chain. This ganglioside was also metabolized further by myeloid cells into gangliosides of the a and b series, similar to endogenous G(M3). In these cells, G(M1) was the major ganglioside and it was segregated at the interface by stroma and myeloid cells, partially co-localizing with the GM-CSF-receptor alpha chain. We conclude that myelosupportive stroma cells produce and secrete the required growth factors, the cofactors such as heparan sulphate proteoglycans, and also supply gangliosides that are transferred from stroma to target cells, generating on the latter ones specific membrane domains with molecular complexes that include growth factor receptors.

Ovariectomy enhances acetylcholinesterase activity but does not alter ganglioside content in cerebral cortex of female adult rats.

In the present work we investigated the effect of ovariectomy on acetylcholinesterase (AChE) activity and ganglioside content in cerebral cortex of female rats. We also studied the activity of butyrylcholinesterase (BuChE) in serum of these animals. Adult Wistar rats were divided into three groups: (1) naive females (control), (2) sham-operated females and (3) castrated females (ovariectomy). Thirty days after ovariectomy, rats were sacrificed by decapitation without anaesthesia. Blood was collected and the serum used for BuChE determination. Cerebral cortex was homogenized to determine AChE activity and extracted with chlorophorm:methanol for ganglioside evaluation. Results showed that rats subjected to ovariectomy presented a significant increase of AChE activity, but did not change the content and the profile of gangliosides in cerebral cortex when compared to sham or naive rats. BuChE activity was decreased in serum of rats ovariectomized. Our findings suggest that the alteration in the activity of brain AChE, as well as serum BuChE activity caused by ovariectomy may contribute to the impaired cognition and/or other neurological dysfunction found in post-menopausal women.

G(M1)-ganglioside degradation and biosynthesis in human and murine G(M1)-gangliosidosis.

BACKGROUND: Gangliosides are building blocks of cell membranes and their biosynthesis and degradation have been extensively studied in the past. Regulation of the metabolism of these glycolipids controls fundamental cell functions. G(M1)-gangliosidosis, a neurodegenerative glycosphingolipid storage disease, is caused by deficiency of lysosomal beta-galactosidase with consequent disruption of the normal degradative pathway of G(M1)-ganglioside. We studied the impact of G(M1)-ganglioside accumulation on its biosynthetic enzyme in cells and tissues from human patients and from the G(M1)-gangliosidosis mouse model. METHODS: We tested the qualitative and quantitative pattern of gangliosides by thin layer chromatography and N-acetylneuraminic acid dosage, respectively. Regulation of G(M1)-ganglioside biosynthesis was evaluated by G(M1) synthase assay in human and murine samples. RESULTS: G(M1)-ganglioside accumulation has an inhibitory effect on the human but not on the mouse G(M1) synthase. We present evidence that G(M1) synthase activity in human and murine cells are regulated by different mechanisms. CONCLUSIONS: Alternative pathways in the mouse may account for these results and possibly explain some of the phenotypical differences between the human and mouse forms of this disorder.

Neonatal hypoxia-ischemia reduces ganglioside, phospholipid and cholesterol contents in the rat hippocampus.

Hypoxia-ischemia is a common cause of neonatal brain damage producing serious impact on cerebral maturation. This report demonstrates that rats submitted to hypoxia-ischemia present a marked decrease in hippocampal gangliosides, phospholipids and cholesterol contents as from 7 days after the injury. Although chromatographic profiles of the different ganglioside species (GM1, GD1a, GD1b, and GT1b) from the hippocampus of hypoxic-ischemic hippocampi groups (HI) were apparently unaffected, as compared with controls, there were quantitative absolute reductions in HI. The phospholipid patterns were altered in HI as from the 14th to the 30th day after the injury, where phosphatidylcholine (PC) quantities were higher than phosphatidylethanolamine (PE); additionally, the cardiolipin band was detected only in hippocampi of control adult rats. In general, the absolute quantities of phospholipids were lower in HI than in correspondent controls since 7th day after the injury. Considering that reported effects were maintained, we suggest they express a late biochemical response triggered by the neonatal hypoxic-ischemic episode; the consequences would be cell death and a delay on brain development, expressed by a reduction on synaptogenesis and myelinogenesis processes.

Changes of sphingolipid species in the phenotype conversion from myofibroblasts to lipocytes in hepatic stellate cells.

Sphingolipids play a relevant role in cell-cell interaction, communication, and migration. We studied the sphingolipid content in the murine hepatic stellate cell line GRX, which expresses the myofibroblast phenotype, and can be induced in vitro to display the fat-storing phenotype. Lipid modifications along this induction were investigated by labeling sphingolipids with [(14)C]galactose, [(14)C]serine, or [(14)C]choline, and determination of fatty acid composition of sphingomyelin. The total ganglioside content and the GM2 synthase activity were lower in myofibroblasts. Both phenotypes presented similar gangliosides of the a-pathway: GM2, GM1, and GD1a as well as their precursor GM3. Sphingomyelin and all the gangliosides were expressed as doublets; the upper/lower band ratio increased in lipocytes, containing more long-chain fatty acids in retinol-induced lipocytes as compared to the insulin/indomethacin induced ones. Time-course experiments indicated a transfer of metabolic precursors from phosphatidylcholine to sphingomyelin in the two phenotypes. Taken together, these results indicate that myofibroblast and lipocytes can use distinct ceramide pools for sphingolipid synthesis. Differential ganglioside expression and presence of the long-chain saturated fatty acids suggested that they may participate in formation of distinct membrane microdomains or rafts with specific functions on the two phenotypes of GRX-cells.

GD3 expression in CHO-K1 cells increases growth rate, induces morphological changes, and affects cell-substrate interactions.

We have generated a panel of CHO-K1 cell clones with different glycolipid compositions by stable transfection of appropriate glycosyltransferases and studied the morphological and growth phenotype of a clone stably expressing Sial-T2. Compared with the GM3 expressing parental cells, Sial-T2 transfectants show low expression of GM3 and neo expression of GD3 and GT3. These cells show about 60% reduction of the mean cell area, and about 2-fold increase of the mean colony area and growth rate. Cells over expressing Sial-T2 showed a flattened appearance, and with time in culture they detached from the substrate leaving adhered material that was GD3 immunoreactive. No apoptotic or proteome differences could be detected in the Sial-T2 transfectants. Thus, increased expression of GD3 and GT3 influence parameters of growth and social behavior of CHO-K1 cells. However, the molecular and cellular basis underlying these influences requires further investigation.