SUMMARY Mesenchymal stem cell transplantation (MSCT) has been used to treat human diseases, but the detailed mechanisms underlying its success are not fully understood. Here we show that MSCT rescues bone marrow MSC (BMMSC) function and ameliorates osteopenia in Fas-deficient-MRL/lpr mice. Mechanistically, we show that Fas deficiency causes failure of miR-29b release, thereby elevating intracellular miR-29b levels, and downregulates DNA methyltransferase 1 (Dnmt1) expression in MRL/lpr BMMSCs. This results in hypomethylation of the Notch1 promoter and activation of Notch signaling, in turn leading to impaired osteogenic differentiation. Furthermore, we show that exosomes, secreted due to MSCT, transfer Fas to recipient MRL/lpr BMMSCs to reduce intracellular levels of miR-29b, which results in recovery of Dnmt1-mediated Notch1 promoter hypomethylation and thereby improves MRL/lpr BMMSC function. Collectively our findings unravel the means by which MSCT rescues MRL/lpr BMMSC function through reuse of donor exosome-provided Fas to regulate the miR-29b/Dnmt1/Notch epigenetic cascade.

Although ganglioside GD3 levels are highly elevated in malignant melanomas, the role of GD3 in melanomas' malignant properties has not been clearly shown. To investigate this problem, we genetically generated GD3-positive (GD3 ؉ ) transfectant cells from a GD3-negative (GD3 ؊ ) mutant line SK-MEL-28-N1 and analyzed the phenotypic changes in the transfected cells. GD3 ؉ cells showed markedly increased cell growth and invasive characteristics. Two bands that underwent stronger tyrosine phosphorylation in GD3 ؉ cell lines than in controls after treatment with FCS were found with molecular masses of 130 and 68 kDa. They were identified as p130Cas and paxillin by sequential immunoprecipitation. Their roles in cell growth and invasion were analyzed with a small interfering RNA (siRNA) approach. Cell growth, as analyzed by BrdUrd uptake, was strongly suppressed in GD3 ؉ cells to near the levels of GD3 ؊ cells when treated with siRNA for p130Cas but not when treated with siRNA for paxillin. However, treatment with siRNAs of either p130Cas or paxillin resulted in the marked suppression of the invasive activity of GD3 ؉ cells almost to the levels of control cells. These results suggested that these two molecules function as effectors of GD3-mediated signaling, leading to such malignant properties as rapid cell growth and invasion.small interfering RNA ͉ sialyltransferase ͉ phosphorylation

Ganglioside GD3 is widely expressed in human malignant melanoma cell lines and tumors. Previously, we reported that GD3؉ cells show stronger tyrosine phosphorylation of focal adhesion kinase (FAK), p130Cas , and paxillin when treated with fetal calf serum than GD3؊ cells. In this study, we analyzed the changes in the signals mediated by the interaction between integrins and extracellular matrices (ECM) to clarify how GD3 enhances cell signals in the vicinity of the cell membrane. An adhesion assay with a real time cell electronic sensing system revealed that GD3؉ cells had stronger adhesion to all extracellular matrices examined. In particular, GD3؉ cells attached more strongly to collagen type I and type IV than controls. Correspondingly, they showed stronger tyrosine phosphorylation of FAK and paxillin during adhesion to collagen type I. In the floating pattern of detergent extracts, a high level of integrin ␤1 was found in glycolipid-enriched microdomain (GEM)/rafts in GD3؉ cells before adhesion, whereas a smaller amount of integrin ␤1 was detected in the GEM/rafts of controls. Some phosphorylated forms of FAK as well as total FAK were found in GEM/rafts during cell adhesion only in GD3؉ cells. Another signal consisting of integrin-linked kinase/Akt was also activated during adhesion more strongly in GD3؉ cells than in controls. In double stained GD3؉ cells, GD3 and integrin ␤1 colocalized at the focal adhesion with a punctate pattern. All these results suggested that integrins assembled and formed a cluster in GEM/rafts, leading to the enhanced signaling and malignant properties under GD3 expression.

Anti-GD2 ganglioside antibodies could be a promising, novel therapeutic approach to the eradication of human small cell lung cancers, as anti-GD2 monoclonal antibodies (mAbs) induced apoptosis of small cell lung cancer cells in culture. In this study, we analyzed the mechanisms for the apoptosis of these cells by anti-GD2 mAbs and elucidated the mechanisms by which apoptosis signals were transduced via reduction in the phosphorylation levels of focal adhesion kinase (FAK) and the activation of a MAPK family member, p38, upon the antibody binding. Knock down of FAK resulted in apoptosis and p38 activation. The inhibition of p38 activity blocked antibody-induced apoptosis, indicating that p38 is involved in this process. Immunoprecipitation-immunoblotting analysis of immune precipitates with anti-FAK or anti-integrin antibodies using an anti-GD2 mAb revealed that GD2 could be precipitated with integrin and/or FAK. These results suggested that GD2, integrin, and FAK form a huge molecular complex across the plasma membrane. Taken together with the fact that GD2؉ cells showed marked detachment from the plate during apoptosis, GD2؉ small cell lung cancer cells seemed to undergo anoikis through the conformational changes of integrin molecules and subsequent FAK dephosphorylation.Gangliosides have been considered to be tumor markers of neuroectoderm-derived human cancers, such as malignant melanomas, neuroblastomas, and gliomas (1, 2). They have been used as target molecules in antibody therapy, i.e. GD3 in malignant melanomas (3) or GD2 in neuroblastomas (4). Some patients showed significant responses to antibody therapy against gangliosides. In the extended analysis of ganglioside expression in other human cancer cells, we have demonstrated the characteristic expression of GD2 in human T lymphotropic virus type I-infected T lymphocytes (5), GD3 in acute T lymphoblastic leukemia cells (6), and GD2 in small cell lung cancer (SCLC) cells (7).In many of these studies, no clear implication of individual gangliosides have been elucidated, except that GD2 in small cell lung cancer (SCLC) 1 was shown to induce increased cell growth and invasion using the transfectant cells with GD3 synthase cDNA (7). Furthermore, it was shown that anti-GD2 monoclonal antibodies (mAbs) could suppress the cell proliferation of GD2ϩ SCLC cells and also induce apoptosis with caspase activation. These results indicated that antibody therapy might be a promising approach to overcoming the disastrous disease SCLC.As for the tumor cell apoptosis with anti-ganglioside antibodies, a few studies have been reported. Hanai and co-workers (8) report apoptosis induction of melanoma cells with anti-GM2 mAb in multicellular heterospheroids. On the other hand, in neuronal tissues, anti-ganglioside antibodies often cause tissue damage mainly in the motor neuron system and trigger serious motor neuron paralysis, such as Guillain-Barre syndrome (9). These facts suggest that gangliosides on malignant tumor cells can be good targets of antibody therapy with mAbs or...

The expression and implications of gangliosides in human osteosarcomas have not been systematically analyzed. In this study, we showed that gangliosides GD3 and GD2 are highly expressed in the majority of human osteosarcoma cell lines derived from oral cavity regions. Introduction of GD3 synthase cDNA into a GD3/GD2‐negative (GD3/GD2−) human osteosarcoma subline resulted in the establishment of GD3/GD2+ transfectant cells. They showed increased cell migration and invasion activities in wound healing and Boyden chamber invasion assays, respectively, compared to the control cells. When treated with serum, GD3/GD2+ cells showed stronger tyrosine phosphorylation of p130Cas, focal adhesion kinase, and paxillin than GD3/GD2− cells. In particular, paxillin underwent much stronger phosphorylation, suggesting its role in cell motility. Furthermore, we tried to dissect the roles of GD3 and GD2 in the malignant properties of the transfectant cells by establishing single ganglioside‐expressing cells, that is, either GD3 or GD2. Although GD3/GD2+ cells showed the most malignant properties, GD2+ cells showed almost equivalent levels to GD3/GD2+ cells in invasion and migration activities, and in the intensities of tyrosine phosphorylation of paxillin. Among Src family kinases, Lyn was expressed predominantly, and was involved in the invasion and motility of GD3‐ and/or GD2‐expressing transfectants. Furthermore, it was elucidated by gene silencing that Lyn was located in a different pathway from that of FAK to eventually lead paxillin activation. These results suggested that GD2/GD3 are responsible for the enhancement of the malignant features of osteosarcomas, and might be candidate targets in molecular‐targeted therapy.

The lipogenic phenotype is a metabolic hallmark of cancer cells. Sterol regulatory element-binding proteins (SREBP) are key transcriptional factors to regulate biosynthesis of cholesterol and fatty acids. It has been poorly understood how the lipogenic phenotype in cancer cells is regulated and how it augments their malignant properties. Here we describe roles of the melanoma antigen ganglioside GD3 and phosphatidylinositol 3-kinase (PI3K)-Akt-mTOR complex 1 (mTORC1) signaling in the regulation of SREBP activity, cholesterol biosynthesis, and the integrity of lipid rafts in human melanoma cells. GD3 expression induced the activation of both SREBP-1 and SREBP-2. Consequently, HMG-CoA reductase expression and cholesterol biosynthesis increased. The activation of the SREBP pathway was independent of the oncogenic BRAF mutation. On the other hand, it was regulated by PI3K-Akt-mTORC1 signaling in human melanoma cells. Disruption of the signaling pathway resulted in the reduction of cholesterol in lipid rafts. Inhibition of the SREBP pathway attenuated Akt activation in lipid rafts and suppressed the growth of human melanoma cells in vitro and in vivo. These results suggest that PI3K-Akt-mTORC1 signaling is important for the integrity of lipid rafts by regulating SREBP activation and subsequent cholesterogenesis. We thus propose a positive feedback circuit in which PI3K-Akt-mTORC1-SREBP signaling boosts Akt signaling in human melanoma cells expressing GD3. Cancer Res; 71(14); 4989-97. Ó2011 AACR.

The possible roles of Src family kinases in the enhanced malignant properties of melanomas related to GD3 expression were analyzed. Among Src family kinases only Yes, not Fyn or Src, was functionally involved in the increased cell proliferation and invasion of GD3-expressing transfectant cells (GD3؉). Yes was located upstream of p130Cas and paxillin and at an equivalent level to focal adhesion kinase. Yes underwent autophosphorylation even before serum treatment and showed stronger kinase activity in GD3؉ cells than in GD3؊ cells following serum treatment. Coimmunoprecipitation experiments revealed that Yes bound to focal adhesion kinase or p130Cas more strongly in GD3؉ cells than in GD3؊ cells. As a possible mechanism for the enhancing effects of GD3 on cellular phenotypes, it was shown that majority of Yes was localized in glycolipidenriched microdomain/rafts in GD3؉ cells even before serum treatment, whereas it was scarcely detected in glycolipid-enriched microdomain/rafts in GD3؊ cells. An in vitro kinase assay of Yes revealed that coexistence of GD3 with Yes in membranous environments enhances the kinase activity of GD3؊ cell-derived Yes toward enolase, p125, and Yes itself. Knockdown of GD3 synthase resulted in the alleviation of tumor phenotypes and reduced activation levels of Yes. Taken together, these results suggest a role of GD3 in the regulation of Src family kinases.

MMP13 is enriched in mature chondrocytes and considered a prime cause of ECM degradation in the osteoarthritic articular cartilage in temporomandibular joints. We asked whether surviving stress to the endoplasmic reticulum (ER) would upregulate transcription of MMP13, and if so, whether a cross-talk would exist between surviving ER stress and p38 MAPK pathways. Using C28/I2 chondrocyte cell line, ER stress was induced by thapsigargin and tunicamycin and upregulation of phosphorylated eIF2α and ATF4 protein was observed. Both thapsigargin and tunicamycin elevated the mRNA level of MMP13 and phosphorylation of p38 MAPK. Thapsigargin-induced MMP13 mRNA upregulation was significantly suppressed by SB203580, while its upregulation by tunicamycin was completely attenuated by SB203580. Those results support that homeostasis of chondrocytes is affected by the surviving ER stress through p38 MAPK pathways, suggesting a potential role of ER stress in joint diseases such as osteoarthritis.