Progression of albuminuria and podocyte injury in focal segmental glomerulosclerosis inhibited by enhanced glycosphingolipid GM3 via valproic acid.

Focal segmental glomerulosclerosis, characterized by decreased numbers of podocytes in glomeruli, is a common cause of refractory nephrotic syndrome. Recently, we showed that enhanced glycosphingolipid GM3 expression after administration of valproic acid, an upregulator of ST3GAL5/St3gal5, was effective in preventing albuminuria and podocyte injury. We also revealed the molecular mechanism for this preventive effect, which involves GM3 directly binding nephrin that then act together in glycolipid-enriched membrane (GEM) fractions under normal conditions and in non-GEM fractions under nephrin injury conditions. Kidney disease is frequently referred to as a "silent killer" because it is often difficult to detect subjective symptoms. Thus, primary treatment for these diseases is initiated after the onset of disease progression. Consequently, the efficacy of enhanced levels of GM3 induced by valproic acid needs to be evaluated after the onset of the disease with severe albuminuria such as focal segmental glomerulosclerosis. Here, we report the therapeutic effect of enhanced GM3 expression induced via administration of valproic acid on albuminuria and podocyte injury after the onset focal segmental glomerulosclerosis in anti-nephrin antibody treated mice. Our findings suggest elevated levels of GM3 following treatment with valproic acid has therapeutic utility for kidney disease associated with severe albuminuria and podocyte injury.

Enhanced Levels of Glycosphingolipid GM3 Delay the Progression of Diabetic Nephropathy.

We recently found that albuminuria levels in patients with minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) inversely correlate with glycosphingolipid GM3 expression levels in glomerular podocytes. Moreover, we showed enhanced expression of GM3 via activation of the GM3 synthase gene upon administration of valproic acid (VPA) is effective in suppressing albuminuria and podocyte injury in mice with anti-nephrin antibody-induced podocytopathy. However, the therapeutic effect of GM3 on diabetic nephropathy, which is the most common underlying disease in patients undergoing dialysis and with podocyte injury, remains unclear. Here, we investigated the therapeutic effect of enhanced GM3 expression via VPA on podocyte injury using streptozotocin-induced diabetic nephropathy model mice. Administration of VPA clearly decreased levels of albuminuria and glomerular lesions and inhibited the loss of podocytes and expansion in the mesangial area. Furthermore, we found that albuminuria levels in patients with diabetic nephropathy inversely correlate with the expression of GM3 in podocytes. These results indicate that maintaining GM3 expression in podocytes by administration of VPA may be effective in treating not only podocyte injury, such as MCD and FSGS, but also the late stage of diabetic nephropathy.

Ganglioside GM3 deficiency enhances mast cell sensitivity.

Mast cells are a significant source of cytokines and chemokines that play a role in pathological processes. Gangliosides, which are complex lipids with a sugar chain, are present in all eukaryotic cell membranes and comprise lipid rafts. Ganglioside GM3, the first ganglioside in the synthetic pathway, is a common precursor of the specifying derivatives and is well known for its various functions in biosystems. Mast cells contain high levels of gangliosides; however, the involvement of GM3 in mast cell sensitivity is unclear. Therefore, in this study, we elucidated the role of ganglioside GM3 in mast cells and skin inflammation. GM3 synthase (GM3S)-deficient mast cells showed cytosolic granule topological changes and hyperactivation upon IgE-DNP stimulation without affecting proliferation and differentiation. Additionally, inflammatory cytokine levels increased in GM3S-deficient bone marrow-derived mast cells (BMMC). Furthermore, GM3S-KO mice and GM3S-KO BMMC transplantation showed increased skin allergic reactions. Besides mast cell hypersensitivity caused by GM3S deficiency, membrane integrity decreased and GM3 supplementation rescued this loss of membrane integrity. Additionally, GM3S deficiency increased the phosphorylation of p38 mitogen-activated protein kinase. These results suggest that GM3 increases membrane integrity, leading to the suppression of the p38 signalling pathway in BMMC and contributing to skin allergic reaction.

Glycosphingolipid GM3 prevents albuminuria and podocytopathy induced by anti-nephrin antibody.

Podocytopathy, which is characterized by injury to podocytes, frequently causes proteinuria or nephrotic syndrome. There is currently a paucity of effective therapeutic drugs to treat proteinuric kidney disease. Recent research suggests the possibility that glycosphingolipid GM3 maintains podocyte function by acting on various molecules including nephrin, but its mechanism of action remains unknown. Here, various analyses were performed to examine the potential relationship between GM3 and nephrin, and the function of GM3 in podocytes using podocytopathy mice, GM3 synthase gene knockout mice, and nephrin injury cells. Reduced amounts of GM3 and nephrin were observed in podocytopathy mice. Intriguingly, this reduction of GM3 and nephrin, as well as albuminuria, were inhibited by administration of valproic acid. However, when the same experiment was performed using GM3 synthase gene knockout mice, valproic acid administration did not inhibit albuminuria. Equivalent results were obtained in model cells. These findings indicate that GM3 acts with nephrin in a collaborative manner in the cell membrane. Taken together, elevated levels of GM3 stabilize nephrin, which is a key molecule of the slit diaphragm, by enhancing the environment of the cell membrane and preventing albuminuria. This study provides novel insight into new drug discovery, which may offer a new therapy for kidney disease with albuminuria.

Decreased GM3 correlates with proteinuria in minimal change nephrotic syndrome and focal segmental glomerulosclerosis.

BACKGROUND: Glycolipids on cell membrane rafts play various roles by interacting with glycoproteins. Recently, it was reported that the glycolipid GM3 is expressed in podocytes and may play a role in podocyte protection. In this report, we describe the correlation between changes in GM3 expression in glomeruli and proteinuria in minimal change nephrotic syndrome (MCNS) and focal segmental glomerulosclerosis (FSGS) patients. METHODS: We performed a case-control study of the correlation between nephrin/GM3 expression levels and proteinuria in MCNS and FSGS patients who underwent renal biopsy at our institution between 2009 and 2014. Normal renal tissue sites were used from patients who had undergone nephrectomy at our institution and gave informed consent. RESULTS: Both MCNS and FSGS had decreased GM3 and Nephrin expression compared with the normal (normal vs. MCNS, FSGS; all p < 0.01). Furthermore, in both MCNS and FSGS, GM3 expression was negatively correlated with proteinuria (MCNS: r = - 0.61, p < 0.01, FSGS: r = - 0.56, p < 0.05). However, nephrin expression had a trend to correlate with proteinuria in FSGS (MCNS: r = 0.19, p = 0.58, FSGS: r = - 0.48, p = 0.06). Furthermore, in a simple linear regression analysis, GM3 expression also correlated with proteinuric change after 12 months of treatment (MCNS: r = 0.40, p = 0.38, FSGS: r = 0. 68, p < 0.05). CONCLUSION: We showed for the first time that decreased GM3 expression correlates with proteinuria in MCNS and FSGS patients. Further studies are needed on the podocyte-protective effects of GM3.

Endothelial ganglioside GM3 regulates angiogenesis in solid tumors.

Cancer cells require oxygen and nutrients for growth, making angiogenesis one of the essential components of tumor growth. Gangliosides, constituting membrane lipid rafts, regulate intracellular signal transduction and are involved in the malignancy of cancer cells. While endothelial cells, as well as cancer cells, express vast amounts of gangliosides, the precise function of endothelial gangliosides in angiogenesis remains unclear. In this study, we focused on gangliosides of vascular endothelial cells and analyzed their functions on tumor angiogenesis. In human breast cancer, GM3 synthase was highly expressed in vascular endothelial cells as well as immune cells. Angiogenesis increased in GM3S-KO mice. In BAEC, RNA interference of GM3S showed increased cellular invasion and oxidative stress tolerance through activation of ERK. In the breast cancer model, GM3-KO mice showed an increase in tumor growth and angiogenesis. These results suggest that the endothelial ganglioside GM3 regulates tumor angiogenesis by suppressing cellular invasion and oxidative stress tolerance in endothelial cells.

Tumor hypoxia regulates ganglioside GM3 synthase, which contributes to oxidative stress resistance in malignant melanoma.

BACKGROUND: Tumor hypoxia drastically changes cancer phenotypes, including angiogenesis, invasion, and cell death. Gangliosides are sialic acid-containing glycosphingolipids that are ubiquitously distributed on plasma membranes and are involved in many biological processes, such as the endoplasmic reticulum stress response and apoptosis. In this study, we investigated the regulation and function of glycosphingolipids, which associate with lipid raft on mammalian plasma membranes under hypoxic condition. METHODS: B16F10 melanoma cells were subjected to chemical hypoxia and low pO2 condition, and the effect of hypoxia on expression of GM3 synthase were analyzed. Cellular resistance to oxidative stress was analyzed in GM3S-KO B16F10 cells. RESULTS: Hypoxia treatment decreased the expression of ganglioside GM3 synthase (GM3S; ST3GAL5), which synthesizes the common substrate of ganglioside biosynthesis. RNA interference of hypoxia inducible factor 1 subunit alpha (HIF-1α) inhibited hypoxia-induced GM3S suppression. Additionally, GM3S deficiency increased cellular resistance to oxidative stress and radiation therapy via upregulation of ERK. CONCLUSIONS: Altered synthesis of glycosphingolipids downstream of HIF-1α signaling increased the resistance of melanoma cells to oxidative stress. Furthermore, GM3 has important role on cellular adaptive response to hypoxia. GENERAL SIGNIFICANCE: This study indicates that tumor hypoxia regulates therapy-resistance via modulation of ganglioside synthesis.

New Anti-Nephrin Antibody Mediated Podocyte Injury Model Using a C57BL/6 Mouse Strain.

BACKGROUND: Focal segmental glomerulosclerosis (FSGS) is considered a subset of the podocytopathies. The molecular pathogenesis of podocytopathy is still unknown. There has not been an experimental animal model of isolated podocytopathy induced by antibody in C57BL/6 strain, which is widely used as the genetic background. Nephrin is closely associated with the slit diaphragm of the glomerular podocyte, and has recently received attention as a potential therapeutic target. The function of nephrin, especially its role in FSGS development via podocytopathy, is being elucidated. We report our experience with a C57BL/6 FSGS model induced by polyclonal rabbit anti-mouse nephrin antibody (α-mNep Ab). METHODS: α-mNep Ab, which was generated by genetic immunization, was administered into C57BL/6 mice at once, intravenously. Urinary protein excretion, the development of glomerulosclerosis and the number of podocyte in mouse kidney were evaluated. RESULTS: The α-mNep Ab-induced FSGS was associated with massive proteinuria and nephrotic syndrome. In periodic acid-Schiff staining, FSGS was observed from day 7 after antibody injection. Podocyte numbers and podocyte marker (anti-Wilms tumor 1 and anti-synaptopodin)-positive areas were clearly decreased. These results suggest that this FSGS mouse model reliably reproduces the human nephrotic syndrome and FSGS. CONCLUSION: We succeeded in making the nephrotic syndrome model mice induced by α-mNep Ab using C57BL/6. This model may be useful for studying the mechanisms of podocytopathy.

Induction of Glycosphingolipid GM3 Expression by Valproic Acid Suppresses Cancer Cell Growth.

Glycosphingolipid GM3, a known suppressor of epidermal growth factor receptor (EGFR) phosphorylation, inhibits cell proliferation. Valproic acid, conversely, is known as an up-regulator of GM3 synthase gene (ST3GAL5). To test the possibility that valproic acid could inhibit EGFR phosphorylation by increasing the level of GM3 in cells, we treated A431 epidermoid carcinoma cells with valproic acid and found that valproic acid treatment caused an about 6-fold increase in the GM3 level but only a marginal increase in the GM2 level in these cells and that the observed increase in GM3 level was valproic acid dose-dependent. Consistent with this observation, valproic acid treatment induced GM3 synthase gene expression by about 8-fold. Furthermore, phosphorylation of EGFR was reduced, and cell proliferation was inhibited following valproic acid treatment. Consistent with these results, transient expression of GM3 synthase gene in A431 cells also increased cellular level of GM3, reduced phosphorylation of EGFR, and inhibited cell proliferation. Treatment with l-phenyl-2-decanoylamino-3-morpholino-l-propanol, an inhibitor of glucosylceramide synthesis, decreased the cellular level of GM3 and reduced the inhibitory effects of valproic acid on EGFR phosphorylation and cell proliferation. These results suggested that induction of GM3 synthesis was enough to inhibit proliferation of cancer cells by suppressing EGFR activity. Valproic acid treatment similarly increased the GM3 level and reduced phosphorylation of EGFR in U87MG glioma cells and inhibited their proliferation. These results suggested that up-regulators of GM3 synthase gene, such as valproic acid, are potential suppressors of cancer cell proliferation.

Epitopes in α8ß1 and other RGD-binding integrins delineate classes of integrin-blocking antibodies and major binding loops in α subunits.

Identification of epitopes for integrin-blocking monoclonal antibodies (mAbs) has aided our understanding of structure-function relationship of integrins. We mapped epitopes of chicken anti-integrin-α8-subunit-blocking mAbs by mutational analyses, examining regions that harboured all mapped epitopes recognized by mAbs against other α-subunits in the RGD-binding-integrin subfamily. Six mAbs exhibited blocking function, and these mAbs recognized residues on the same W2:41-loop on the top-face of the ß-propeller. Loop-tips sufficiently close to W2:41 (<25 Å) contained within a footprint of the mAbs were mutated, and the loop W3:34 on the bottom face was identified as an additional component of the epitope of one antibody, clone YZ5. Binding sequences on the two loops were conserved in virtually all mammals, and that on W3:34 was also conserved in chickens. These indicate 1) YZ5 binds both top and bottom loops, and the binding to W3:34 is by interactions to conserved residues between immunogen and host species, 2) five other blocking mAbs solely bind to W2:41 and 3) the α8 mAbs would cross-react with most mammals. Comparing with the mAbs against the other α-subunits of RGD-integrins, two classes were delineated; those binding to "W3:34 and an top-loop", and "solely W2:41", accounting for 82% of published RGD-integrin-mAbs.

Efficient synthesis of chloro-derivatives of sialosyllactosylceramide, and their enhanced inhibitory effect on epidermal growth factor receptor activation.

Glycosphingolipids are components of essentially all mammalian cell membranes and are involved in a variety of significant cellular functions, including proliferation, adhesion, motility and differentiation. Sialosyllactosylceramide (GM3) is known to inhibit the activation of epidermal growth factor receptor (EGFR). In the present study, an efficient method for the total chemical synthesis of monochloro- and dichloro-derivatives of the sialosyl residue of GM3 was developed. The structures of the synthesized compounds were fully characterized by high-resolution mass spectrometry and nuclear magnetic resonance. In analyses of EGFR autophosphorylation and cell proliferation ([3H]-thymidine incorporation) in human epidermoid carcinoma A431 cells, two chloro-derivatives exhibited stronger inhibitory effects than GM3 on EGFR activity. Monochloro-GM3, but not GM3 or dichloro-GM3, showed a significant inhibitory effect on ΔEGFR, a splicing variant of EGFR that lacks exons 2-7 and is often found in human glioblastomas. The chemical synthesis of other GM3 derivatives using approaches similar to those described in the present study, has the potential to create more potent EGFR inhibitors to block cell growth or motility of a variety of types of cancer that express either wild-type EGFR or ΔEGFR.

Selective detection of HbA1c using surface enhanced resonance Raman spectroscopy.

In the current work, we report on selective detection of HbA1c, a marker for glycemic control in diabetic patients, using surface enhanced resonance raman spectroscopy (SERRS). We found a characteristic band around 770-830 cm(-1) in the SERRS spectrum of HbA1c which was not present in the SERRS spectrum of HbA. To examine the contribution of glucosyl moiety to the characteristic SERRS band of HbA1c, we investigated SERRS spectra for nonenzymatically glycosylated HbA. We found that the SERRS spectral features are essentially identical for both HbA1c and nonenzymatically glycosylated HbA. Furthermore, addition of HbA into colloidal solution of silver nanoparticles (Ag NPs) resulted in the formation of large aggregates of Ag NPs and subsequent sedimentation. On the other hand, aggregation of Ag NPs was considerably low in the case of HbA1c. The differential effect of HbA and HbA1c on colloidal solution of Ag NPs, probably due to their difference in hydrophilicity, enabled us to separate them in a mixture. The separation was characterized by electrophoresis and SERRS analysis. Thus, colloidal solution of Ag NPs and SERRS would be a promising tool for the selective detection of HbA1c.

Reversible dimerization of EGFR revealed by single-molecule fluorescence imaging using quantum dots.

The current work explores intermolecular interactions involved in the lateral propagation of cell-signaling by epidermal growth factor receptors (EGFRs). Activation of EGFRs by binding an EGF ligand in the extracellular domain of the EGFR and subsequent dimerization of the EGFR initiates cell-signaling. We investigated interactions between EGFRs in living cells by using single-molecule microscopy, Förster resonance energy transfer (FRET), and atomic force microscopy. By analyzing time-correlated intensity and propagation trajectories of quantum dot (QD)-labeled EGFR single molecules, we found that signaling dimers of EGFR [(EGF-EGFR)(2)] are continuously formed in cell membrane through reversible association of heterodimers [EGF(EGFR)(2)]. Also, we found that the lateral propagation of EGFR activation takes place through transient association of a heterodimer with predimers [(EGFR)(2)]. We varified the transient association between activated EGFR and predimers using FRET from QD-labeled heterodimers to Cy5-labeled predimers and correlated topography and fluorescence imaging. Without extended single-molecule fluorescence imaging and by using bio-conjugated QDs, reversible receptor dimerization in the lateral activation of EGFR remained obscured.

Mechanism of abnormal growth in astrocytes derived from a mouse model of GM2 gangliosidosis.

Sandhoff disease is a progressive neurodegenerative disorder caused by mutations in the HEXB gene which encodes the beta-subunit of N-acetyl-beta-hexosaminidase A and B, resulting in the accumulation of the ganglioside GM2. We isolated astrocytes from the neonatal brain of Sandhoff disease model mice in which the N-acetyl-beta-hexosaminidase beta-subunit gene is genetically disrupted (ASD). Glycolipid profiles revealed that GM2/GA2 accumulated in the lysosomes and not on the cell surface of ASD astrocytes. In addition, GM3 was increased on the cell surface. We found remarkable differences in the cell proliferation of ASD astrocytes when compared with cells isolated from wild-type mice, with a faster growth rate of ASD cells. In addition, we observed increased extracellular, signal-regulated kinase (ERK) phosphorylation in ASD cells, but Akt phosphorylation was decreased. Furthermore, the phosphorylation of ERK in ASD cells was not dependent upon extracellular growth factors. Treatment of ASD astrocytes with recombinant N-acetyl-beta-hexosaminidase A resulted in a decrease of their growth rate and ERK phosphorylation. These results indicated that the up-regulation of ERK phosphorylation and the increase in proliferation of ASD astrocytes were dependent upon GM2/GA2 accumulation. These findings may represent a mechanism in linking the nerve cell death and reactive gliosis observed in Sandhoff disease.

Clathrin-mediated endocytosis of quantum dot-peptide conjugates in living cells.

Efficient intracellular delivery of quantum dots (QDs) and unravelling the mechanism underlying the intracellular delivery are essential for advancing the applications of QDs toward in vivo imaging and therapeutic interventions. Here, we show that clathrin-mediated endocytosis is the most important pathway for the intracellular delivery of peptide-conjugated QDs. We selected an insect neuropeptide, namely, allatostatin (AST1, APSGAQRLYG FGL-NH(2)), conjugated it with CdSe-ZnS QDs, and investigated the intracellular delivery of the conjugate in living cells such as human epidermoid ovarian carcinoma cells (A431) and mouse embryonic fibroblast cells (3T3). We selected AST1 to investigate the intracellular delivery of QDs because we recently found it to be efficient for delivering QDs in living mammalian cells. Also, the receptors of AST1 in insects show functional and sequence similarity to G-protein-coupled galanin receptors in mammals. We employed flow cytometry and fluorescence microscopy and investigated the contributions of clathrin-mediated endocytosis, receptor-mediated endocytosis, and charge-based cell penetration or transduction to the intracellular delivery of QD-AST1 conjugates. Interestingly, the intracellular delivery was suppressed by approximately 57% when we inhibited the regulatory enzyme phosphoinositide 3-kinase (PI3K) with wortmannin and blocked the formation of clathrin-coated vesicles. In parallel, we investigated clathrin-mediated endocytosis by colocalizing QD560-labeled clathrin heavy-chain antibody and QD605-AST1. We also estimated galanin receptor-mediated endocytosis of QD-AST1 at <10% by blocking the cells with a galanin antagonist and transduction at <30% by both removing the charge of the peptide due to arginine and suppressing the cell-surface charge due to glycosaminoglycan. In short, the current work shows that multiple pathways are involved in the intracellular delivery of peptide-conjugated QDs, among which clathrin-mediated endocytosis is the most important.

Abnormal production of macrophage inflammatory protein-1alpha by microglial cell lines derived from neonatal brains of Sandhoff disease model mice.

Sandhoff disease (SD) is a lysosomal beta-hexosaminidase deficiency involving excessive accumulation of undegraded substrates, including terminal N-acetylglucosamine-oligosaccharides and GM2 ganglioside, and progressive neurodegeneration. Our previous study demonstrated remarkable induction of macrophage inflammatory factor-1alpha (MIP-1alpha) in microglia in the brains of SD model mice as a putative pathogenic factor for SD via microglia-mediated neuroinflammation. In this study, we established microglial cell lines (WT- and SD-Mg) from wild-type and SD mice, and first demonstrated the enhanced production of MIP-1alpha in SD-Mg. Inhibitors of protein kinase C (PKC) and Akt reduced the production of MIP-1alpha by SD-Mg. Elevated activation of Akt and partial translocation of PKC isozymes (alpha, betaI, betaII, and delta) from the cytoplasm to the membrane in SD-Mg were also revealed by means of immunoblotting. Furthermore, it was demonstrated that intracellular extracellular signal-regulated kinase, c-Jun N-terminal kinase, and phospholipase C (PLC), but not phosphoinositide 3-kinase, should contribute to the induction of MIP-1alpha in SD-Mg, and that PLC could independently regulate the activation of both PKC and Akt. We proposed here that the deregulated activation of PLC should cause the enhanced MIP-1alpha production via plural signaling pathways mediated by PKC and Akt, followed by extracellular signal-regulated kinase and c-Jun N-terminal kinase, in SD-Mg.

Tyrosine kinase activity of epidermal growth factor receptor is regulated by GM3 binding through carbohydrate to carbohydrate interactions.

Epidermal growth factor receptor (EGFR), an N-glycosylated transmembrane protein with an intracellular kinase domain, undergoes dimerization by ligand binding resulting in activation of the kinase domain and phosphorylation. Ganglioside GM3 containing sialyllactose inhibits the tyrosine kinase activity of EGFR through carbohydrate to carbohydrate interactions (CCI) between N-glycans with GlcNAc termini on EGFR and oligosaccharides on GM3. In this study, we provide further evidence for CCI between EGFR and GM3. (i) In vitro and in situ, the inhibitory effect of GM3 on EGFR tyrosine kinase was much higher in A431 cells upon exposure of the GlcNAc termini of the N-glycans to glycosidase treatment (neuraminidase and beta-galactosidase) than in untreated A431 cells. Furthermore, the GM3-mediated inhibition was abrogated by co-incubation with N-glycan containing terminal GlcNAc. (ii) In situ, inhibition of EGFR phosphorylation by GM3 was not observed in alpha-mannosidase IB (ManIB)-knocked down A431 cells that accumulate high mannose-type N-glycans. (iii) EGFR binding to GM3 was enhanced in glycosidase-treated cells that accumulated GlcNAc termini, whereas GM3 did not bind to EGFR from ManIB-knocked down cells that accumulated high mannose-type N-glycans. These results indicate that GM3-mediated inhibition of EGFR phosphorylation is caused by interaction of GM3 with GlcNAc-terminated N-glycan on EGFR.