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Kazuya Kabayama

Publications and source records attributed to Kazuya Kabayama.

10 recordsLinked to original sources
PubMed2006

Endogenously produced ganglioside GM3 endows etoposide and doxorubicin resistance by up-regulating Bcl-2 expression in 3LL Lewis lung carcinoma cells.

The ganglioside patterns have been shown to dramatically change during cell proliferation and differentiation and in certain cell-cycle phases, brain development, and cancer malignancy. To investigate the significance of the ganglioside GM3 in cancer malignancy, we established GM3-reconstituted cells by transfecting the cDNA of GM3 synthase into a GM3-deficient subclone of the 3LL Lewis lung carcinoma cell line (Uemura, S. (2003) Glycobiology, 13, 207-216). The GM3-reconstituted cells were resistant to apoptosis induced by etoposide and doxorubicin. There were no changes in the expression levels of topoisomerase IIalpha or P-glycoprotein, or in the uptake of doxorubicin between the GM3-reconstituted cells and the mock-transfected cells. To understand the mechanism of the etoposide-resistant phenotype acquired in the GM3-reconstituted cells, we investigated their apoptotic signaling. Although no difference was observed in the phosphorylation of p53 at serine-15-residue site by etoposide between the GM3-reconstituted cells and mock-transfected cells, the activation of both caspase-3 and caspase-9 was specifically inhibited in the former. We found that the anti-apoptotic protein B-cell leukemia/lymphoma 2 (Bcl-2) was increased in the GM3-reconstituted cells. Moreover, wild-type 3LL Lewis lung carcinoma cells, which have an abundance of GM3, exhibited no DNA fragmentation following etoposide treatment and expressed higher levels of the Bcl-2 protein compared with the J5 subclone. Thus, these results support the conclusion that endogenously produced GM3 is involved in malignant phenotypes, including anticancer drug resistance through up-regulating the Bcl-2 protein in this lung cancer cell line.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Structure and function of lipid rafts in human activated T cells.

Lipid rafts, specialized membrane microdomains enriched in sphingolipids and cholesterol, have been shown to function as signaling platforms in T cells. Surface raft expression is known to be increased in human T cells upon activation, and this increased raft expression may account for efficient signaling capability and decreased dependency for co-stimulation in effector and/or activated T cells. However, raft-mediated signaling ability in activated T cells remains to be clarified. In this study, we analyzed the structure and function of lipid rafts in human activated T cells. We demonstrated that raft protein constituents are dramatically changed after activation along with an increase in lipid contents. T cells stimulated with anti-CD3 plus anti-CD28 antibodies showed an increase not only in surface monosialoganglioside GM1 expression but also in total amounts of raft-associated lipids such as sphingomyelin, cholesterol and glycosphingolipids. Raft proteins increased after activation include Csk, Csk-binding protein and Fyn, the molecules known to be involved in negative regulation of T cell activation. Consistent with the increase in expression of these proteins, TCR-mediated Ca(2+) response, a response dependent on raft integrity, was clearly inhibited in activated T cells. Thus, the structure and function of lipid rafts in human activated T cells seem to be quite distinct from those in naive T cells. Further, human activated T cells are relatively resistant to signaling, at least transiently, by TCR re-stimulation even though their raft expression is increased.

Adaptor Proteins, Signal Transducing↗
PubMed2004

TNFalpha-induced insulin resistance in adipocytes as a membrane microdomain disorder: involvement of ganglioside GM3.

Membrane microdomains (lipid rafts) are now recognized as critical for proper compartmentalization of insulin signaling, but their role in the pathogenesis of insulin resistance has not been investigated. Detergent-resistant membrane microdomains (DRMs), isolated in the low-density fractions, are highly enriched in cholesterol, glycosphingolipids and various signaling molecules. Tumor necrosis factor alpha (TNFalpha) induces insulin resistance in type 2 diabetes, but its mechanism of action is not fully understood. In other studies we have found a selective increase in the acidic glycosphingolipid ganglioside GM3 in 3T3-L1 adipocytes treated with TNFalpha, suggesting a specific function for GM3. In the DRMs from TNFalpha-treated 3T3-L1 adipocytes, GM3 levels were doubled compared with results in normal adipocytes. Additionally, insulin receptor (IR) accumulations in the DRMs were diminished, whereas caveolin and flotillin levels were unchanged. Furthermore, insulin-dependent IR internalization and intracellular movement of the IR substrate 1(IRS-1) were both greatly suppressed in the treated cells, leading to an uncoupling of IR-IRS-1 signaling. GM3 depletion was able to counteract the TNFalpha-induced inhibitions of IR internalization and accumulation into DRMs. Together, these findings provide compelling evidence that in insulin resistance the insulin metabolic signaling defect can be attributed to a loss of IRs in the microdomains due to an accumulation of GM3.

Adipocytes↗
PubMed2004

Convenient synthesis and evaluation of glycosidase inhibitory activity of alpha- and beta-galactose-type valienamines, and some N-alkyl derivatives.

Valienamine analogues having alpha- and beta-galactose-type structures were synthesized by racemic modification from (1SR,2RS,3SR)-6-methylenecyclohex-4-ene-1,2,3-triol. Four N-alkyl derivatives of the beta-anomer were readily prepared selectively by treatment of key intermediate 2,6-di-O-acetyl-3,4-O-isopropylidene-5a-carba-alpha- and beta-l-arabino-hex-5(5a)-enopyranosyl bromides with alkyl amines. All compounds were assayed for inhibitory activity against six glycosidases, and the N-dodecyl derivative was shown to be a very strong inhibitor of beta-galactosidase (IC(50) 0.01 microM, bovine liver).

Animals↗
PubMed2004

Glycosphingolipids govern gene expression.

To elucidate the biological significance of the lactosylceramide (LacCer) branching in glycosphingolipid (GSL) biosynthesis, we established ganglioside GM3- and lactosylsulfatide SM3-reconstituted cells by introducing the GM3 synthase gene and the sulfotransferase gene, respectively. In SM3-expressing cells, the reduction of beta1 integrin mRNA expression, the reduced adhesivity to fibronectin and laminin, and the suppression of anchorage-independent growth (tumorigenic potential) were observed. On the other hand, in GM3-expressing cells, anchorage-independent growth was promoted and the expression of PDGF alpha receptor mRNA was specifically reduced. Interestingly enough, no change in anchorage-dependent growth was observed in these cells, and tumorigenic signals were controlled selectively in both positive and negative directions. Thus, the spatio-temporal, gene expression control mechanism by individual GSL molecules accumulating in the cell membrane microdomain (raft) has been proven.

Animals↗
PubMed2004

Triglyceride accumulation and altered composition of triglyceride-associated fatty acids in the skin of tenascin-X-deficient mice.

Tenascin-X (TNX) is a member of the tenascin family of glycoproteins of the extracellular matrix. Here, we observed abnormalities in the skin of TNX-deficient mice in comparison with that of wild-type mice. Histological analysis with Oil Red O staining demonstrated that there was considerable accumulation of lipid in the skin of TNX-deficient (TNX-/-) mice. By thin-layer chromatography of total lipids, it was found that the level of triglyceride was significantly increased in TNX-/- mice. The mRNA levels of most of the lipogenic enzyme genes examined were remarkably increased in TNX-/- mice. By gas chromatography-mass spectrometry analysis of triglyceride-associated fatty acids in the skin, saturated fatty acid palmitoic acid was decreased, whereas unsaturated fatty acids palmitoleic acid and oleic acid were increased in TNX-/- mice compared with those in wild-type mice. Conversely, fibroblast cell lines transfected with TNX showed a significant decrease in the amount of triglyceride. An increase in the saturated fatty acid stearic acid and decreases in the unsaturated fatty acids palmitoleic acid, oleic acid and linoleic acid, compared to those in mock-transfected cells were also caused by over-expression of TNX. These results indicate that TNX is involved in the regulation of triglyceride synthesis and the regulation of composition of triglyceride-associated fatty acids.

Adipose Tissue↗
PubMed2003

Reduction of glycosphingolipid levels in lipid rafts affects the expression state and function of glycosylphosphatidylinositol-anchored proteins but does not impair signal transduction via the T cell receptor.

Lipid rafts are highly enriched in cholesterol and sphingolipids. In contrast to many reports that verify the importance of cholesterol among raft lipid components, studies that address the role of sphingolipids in raft organization and function are scarce. Here, we investigate the role of glycosphingolipids (GSLs) in raft structure and raft-mediated signal transduction in T lymphocytes by the usage of a specific GSL synthesis inhibitor, d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP). Surface GM1 expression and the expression of GSLs in rafts were profoundly reduced by D-PDMP treatment, whereas the expression of other lipid and protein constituents, such as cholesterol, sphingomyelin, Lck, and linker for activation of T cells, was not affected. T cell receptor-mediated signal transduction induced by antigen stimulation or by antibody cross-linking was normal in D-PDMP-treated T cells. In contrast, the signal through glycosylphosphatidylinositol (GPI)-anchored proteins was clearly augmented by D-PDMP treatment. Moreover, GPI-anchored proteins became more susceptible to phosphatidylinositol-specific phospholipase C cleavage in D-PDMP-treated cells, demonstrating that GSL depletion from rafts primarily influences the expression state and function of GPI-anchored proteins. Finally, by comparing the effect of D-PDMP with that of methyl-beta-cyclodextrin, we identified that compared with cholesterol depletion, GSL depletion has the opposite effect on the phosphatidylinositol-specific phospholipase C sensitivity and signaling ability of GPI-anchored proteins. These results indicate a specific role of GSLs in T cell membrane rafts that is dispensable for T cell receptor signaling but is important for the signal via GPI-anchored proteins.

Animals↗
PubMed2002

Sialylation and sulfation of lactosylceramide distinctly regulate anchorage-independent growth, apoptosis, and gene expression in 3LL Lewis lung carcinoma cells.

To investigate the significance of sialylation and sulfation of lactosylceramide in transformed cells, we established ganglioside GM3- and lactosylsulfatide (SM3)-reconstituted cells by transfecting cDNAs of GM3 synthase and cerebroside sulfotransferase into the J5 subclone of 3LL Lewis lung carcinoma cells. The J5 clone was selected for the transfection of these genes because it lacks GM3 and SM3 but accumulates lactosylceramide. The anchorage-dependent growth of both GM3- and SM3-reconstituted cells was similar. However, anchorage-independent growth (as measured by colony-forming ability in soft agar) of the SM3- reconstituted cells was almost completely lost, which supports our previous observation showing the suppression of tumorigenic potential in vivo and beta1 integrin gene expression induced by the introduction of cerebroside sulfotransferase gene (Kabayama et al. [2001] J. Biol. Chem., 276, 26777-26783). The GM3-reconstituted cells formed a significantly higher number of colonies in soft agar compared to mock-transfected cells and began to proliferate and become resistant to apoptosis when serum was depleted, indicating that endogenous GM3 is essential for maintaining these fundamental properties of malignant cells. We also found that serum-induced ERK1/2 activation was suppressed in the GM3-reconstituted cells, suggesting that anchorage-independent cell cycle initiation by endogenous GM3 is elicited through pathway(s) independent of ERK1/2 activation. The selective down-regulation of platelet-derived growth factor (PDGF)-dependent ERK1/2 activation in the GM3-reconstituted cells was due to the substantial decreases of PDGF alpha receptor mRNA and protein, but in the SM3-reconstituted cells PDGF alpha receptor expression was similar to mock cells. Thus, endogenously produced GM3 and SM3 differentially and distinctly regulate tumor-progression ability, that is, GM3 leads the transformed phenotype of J5 cells to promotion and SM3 to abrogation.

Animals↗
PubMed2001

Ganglioside GM3 participates in the pathological conditions of insulin resistance.

Gangliosides are known as modulators of transmembrane signaling by regulating various receptor functions. We have found that insulin resistance induced by tumor necrosis factor-alpha (TNF-alpha) in 3T3-L1 adipocytes was accompanied by increased GM3 ganglioside expression caused by elevating GM3 synthase activity and its mRNA. We also demonstrated that TNF-alpha simultaneously produced insulin resistance by uncoupling insulin receptor activity toward insulin receptor substrate-1 (IRS-1) and suppressing insulin-sensitive glucose transport. Pharmacological depletion of GM3 in adipocytes by an inhibitor of glucosylceramide synthase prevented the TNF-alpha-induced defect in insulin-dependent tyrosine phosphorylation of IRS-1 and also counteracted the TNF-alpha-induced serine phosphorylation of IRS-1. Moreover, when the adipocytes were incubated with exogenous GM3, suppression of tyrosine phosphorylation of insulin receptor and IRS-1 and glucose uptake in response to insulin stimulation was observed, demonstrating that GM3 itself is able to mimic the effects of TNF on insulin signaling. We used the obese Zucker fa/fa rat and ob/ob mouse, which are known to overproduce TNF-alpha mRNA in adipose tissues, as typical models of insulin resistance. We found that the levels of GM3 synthase mRNA in adipose tissues of these animals were significantly higher than in their lean counterparts. Taken together, the increased synthesis of cellular GM3 by TNF may participate in the pathological conditions of insulin resistance in type 2 diabetes.

3T3 Cells↗