Factors secreted from the stem cells of human exfoliated deciduous teeth inhibit osteoclastogenesis through the activation of the endogenous antioxidant system

Systemic administration of conditioned medium (CM) from stem cells derived from human exfoliated deciduous teeth (SHED-CM) in mouse models of rheumatoid arthritis, osteoporosis, and osteoarthritis suppresses excessive osteoclast activity and restores bone integrity. However, the mechanism through which SHED-CM regulates osteoclastogenesis remains largely unknown. In the present study, we examined the anti-osteoclastogenic mechanism of SHED-CM in vitro. Bone marrow macrophages and RAW264.7 cells were treated with receptor activator of nuclear factor kappa-Β ligand (RANKL) in the presence of SHED-CM or CM from bone marrow mesenchymal stem cells (BMSC-CM). Osteoclast differentiation was assessed using tartrate-resistant acid phosphatase staining, actin ring formation, and expression of osteoclast-specific markers. RANKL-induced reactive oxygen species (ROS) production was analyzed as a critical mediator of osteoclastogenesis. The activation of endogenous antioxidant gene expression was examined using reverse transcription quantitative PCR. Liquid chromatography with tandem mass spectrometry (LC-MS) was used to identify proteins enriched in SHED-CM, and neutralizing antibodies were used to evaluate their functional roles. Compared to BMSC-CM, SHED-CM effectively inhibited RANKL-induced early osteoclast differentiation and late maturation. Notably, SHED-CM but not BMSC-CM suppressed RANKL-induced ROS production. SHED-CM increased the expression of genes encoding antioxidant enzymes. The LC-MS analysis identified seven proteins uniquely enriched in SHED-CM that activated the endogenous antioxidant system. Neutralizing antibodies against some of these proteins restore RANKL-induced ROS production and osteoclast differentiation. SHED-CM inhibited osteoclastogenesis, partially through the activation of multiple antioxidant enzymes in osteoclast precursors, highlighting its potential for treating bone-destructive diseases.


Bidirectional signals generated by Siglec-7 and its crucial ligand tri-sialylated T to escape of cancer cells from immune surveillance.

Siglec-7, an inhibitory receptor expressed on natural killer (NK) cells, recognizes sialic acid-containing glycans. However, the ligand glycan structures of Siglec-7 and its carrier proteins have not been comprehensively investigated. Here, we identified four sialyltransferases that are used for the synthesis of ligand glycans of Siglec-7 and two ligand O-glycan-carrier proteins, PODXL and MUC13, using a colon cancer line. Upon binding of these ligand glycans, Siglec-7-expressing immune cells showed reduced cytotoxic activity, whereas cancer cells expressing ligand glycans underwent signal activation, leading to enhanced invasion activity. To clarify the structure of the ligand glycan, podoplanin (PDPN) identified as a Siglec-7 ligand-carrier protein, was transfected into HEK293T cells using sialyltransferase cDNAs. Mass spectrometry of the products revealed a ligand glycan, tri-sialylated T antigen. These results indicate that Siglec-7 interaction with its ligand generates bidirectional signals in NK and cancer cells, leading to the efficient escape of cancers from host immune surveillance.


Secreted factors from stem cells of human exfoliated deciduous teeth directly activate endothelial cells to promote all processes of angiogenesis

Diabetes is a major risk factor for atherosclerosis and ischemic vascular diseases. Recently, regenerative medicine is expected to be a novel therapy for ischemic diseases. Our previous studies have reported that transplantation of stem cells promoted therapeutic angiogenesis for diabetic neuropathy and ischemic vascular disease in a paracrine manner, but the precise mechanism is unclear. Therefore, we examined whether secreted factors from stem cells had direct beneficial effects on endothelial cells to promote angiogenesis. The soluble factors were collected as conditioned medium (CM) 48 h after culturing stem cells from human exfoliated deciduous teeth (SHED) in serum-free DMEM. SHED-CM significantly increased cell viability of human umbilical vein endothelial cells (HUVECs) in MTT assays and accelerated HUVECs migration in wound healing and Boyden chamber assays. In a Matrigel plug assay of mice, the migrated number of primary endothelial cells was markedly increased in the plug containing SHED-CM or SHED suspension. SHED-CM induced complex tubular structures of HUVECs in a tube formation assay. Furthermore, SHED-CM significantly increased neovascularization from the primary rat aorta, indicating that SHED-CM stimulated primary endothelial cells to promote comprehensive angiogenesis processes. The angiogenic effects of SHED-CM were the same or greater than the effective concentration of VEGF. In conclusion, SHED-CM directly stimulates vascular endothelial cells to promote angiogenesis and is promising for future clinical application.


Stromal cell-derived factor-1 accelerates bone regeneration through multiple regenerative mechanisms

Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology


Monocyte chemoattractant protein-1 and secreted ectodomain of sialic acid-binding Ig-like lectin-9 enhance bone regeneration by inducing M2 macrophages


Secreted Ectodomain of SIGLEC-9 and MCP-1 Synergistically Improve Acute Liver Failure in Rats by Altering Macrophage Polarity.

Effective treatments for acute liver failure (ALF) are still lacking. We recently reported that a single intravenous administration of serum-free conditioned medium from stem cells derived from human exfoliated deciduous teeth (SHED-CM) into the D-galactosamine (D-Gal)-induced rat ALF model improves the liver injury. However, the specific factors in SHED-CM that are responsible for resolving ALF remain unclear. Here we found that depleting SHED-CM of two anti-inflammatory M2 macrophage inducers-monocyte chemoattractant protein-1 (MCP-1) and the secreted ectodomain of sialic acid-binding Ig-like lectin-9 (sSiglec-9)-abolished its ability to resolve rat ALF. Furthermore, treatment with MCP-1/sSiglec-9 alone dramatically improved the survival of ALF rats. This treatment induced anti-inflammatory M2, suppressed hepatocyte apoptosis, and promoted hepatocyte proliferation. Treatment with an M2-depletion reagent (mannosylated clodronate liposomes) suppressed the recovery. In addition, MCP-1 and sSiglec-9 synergistically promoted the M2 differentiation of bone marrow-derived macrophages via CCR2, accompanied by the production of multiple liver-regenerating factors. The conditioned medium from MCP-1/sSiglec-9-activated M2 macrophages, but not from interleukin-4-induced ones, suppressed the D-Gal- and LPS-induced apoptosis of primary hepatocytes and promoted their proliferation in vitro. The unique combination of MCP-1/sSiglec-9 ameliorates rat ALF by inhibiting hepatocellular apoptosis and promoting liver regeneration through the induction of anti-inflammatory/tissue-repairing M2 macrophages.


Role of Protein Phosphatase 2A in Osteoblast Differentiation and Function.

The reversible phosphorylation of proteins plays hugely important roles in a variety of cellular processes, such as differentiation, proliferation, and apoptosis. These processes are strictly controlled by protein kinases (phosphorylation) and phosphatases (de-phosphorylation). Here we provide a brief history of the study of protein phosphorylation, including a summary of different types of protein kinases and phosphatases. One of the most physiologically important serine/threonine phosphatases is PP2A. This review provides a description of the phenotypes of various PP2A transgenic mice and further focuses on the known functions of PP2A in bone formation, including its role in osteoblast differentiation and function. A reduction in PP2A promotes bone formation and osteoblast differentiation through the regulation of bone-related transcription factors such as Osterix. Interestingly, downregulation of PP2A also stimulates adipocyte differentiation from undifferentiated mesenchymal cells under the appropriate adipogenic differentiation conditions. In osteoblasts, PP2A is also involved in the ability to control osteoclastogenesis as well as in the proliferation and metastasis of osteosarcoma cells. Thus, PP2A is considered to be a comprehensive factor in controlling the differentiation and function of cells derived from mesenchymal cells such as osteoblasts and adipocytes.


Multifaceted Therapeutic Benefits of Factors Derived From Dental Pulp Stem Cells for Mouse Liver Fibrosis.

This study demonstrated that a single intravenous administration of stem cells from human exfoliated deciduous teeth (SHEDs) or of the serum-free conditioned medium (CM) derived from SHEDs markedly improved mouse liver fibrosis (LF). SHED-CM suppressed chronic inflammation, eliminated activated hepatic stellate cells by inducing their apoptosis, protected hepatocytes from undergoing apoptosis, and induced differentiation of tissue-repairing macrophages expressing high levels of the profibrinolytic factor matrix metalloproteinase 13. Furthermore, hepatocyte growth factor played a central role in the SHED-CM-mediated resolution of LF. This is the first report demonstrating the multifaceted therapeutic benefits of secreted factors derived from SHEDs for LF.


Dental pulp-derived stem cell conditioned medium reduces cardiac injury following ischemia-reperfusion.

Stem cells from human exfoliated deciduous teeth (SHEDs) can regenerate various tissues. We investigated the impact of SHED-conditioned medium (SHED-CM) on myocardial injury in a mouse model of ischemia-reperfusion (I/R). Wild-type (WT) mice were subjected to myocardial ischemia followed by reperfusion. SHED-CM was intravenously injected at 5 min after reperfusion. Administration of SHED-CM reduced myocardial infarct size as well as decreased apoptosis and inflammatory cytokine levels, such as TNF-, IL-6, and IL-, in the myocardium following I/R. In cultured cardiac myocytes, SHED-CM significantly suppressed apoptosis under hypoxia/serum-deprivation and reduced LPS-induced expression of pro-inflammatory genes. Furthermore, anti-apoptotic action of SHED-CM was stronger than bone marrow-derived stem cell (BMSC)-CM or adipose-derived stem cell (ADSC)-CM in cardiac myocytes. SHED-CM contains a higher concentration of hepatocyte growth factor (HGF) than BMSC-CM and ADSC-CM, and neutralization of HGF attenuated the inhibitory actions of SHED-CM on apoptosis in cardiac myocytes. Finally, WT mice were intravenously treated with an HGF-depleted SHED-CM, followed by myocardial I/R. HGF depletion significantly attenuated the inhibitory actions of SHED-CM on myocardial infarct size and apoptosis after I/R. SHED-CM protects the heart from acute ischemic injury because it suppresses inflammation and apoptosis. SHED-CM could be a useful treatment option for acute myocardial infarction.


Therapeutic Potential of Stem Cells from Human Exfoliated Deciduous Teeth in Models of Acute Kidney Injury.

SHED attenuated the levels of inflammatory cytokines and improved kidney function in AKI induced by IRI. SHED secreted factors reduced MCP-1 and increased HGF expression, which promoted wound healing. These results suggest that SHED might provide a novel stem cell resource, which can be applied for the treatment of ischemic kidney injury.


Secreted factors from dental pulp stem cells improve glucose intolerance in streptozotocin-induced diabetic mice by increasing pancreatic -cell function.

These findings suggest that SHED-CM provides direct protection and encourages the propagation of -cells, and has potential as a novel strategy for treatment of diabetes.

Administration of 1 mL of SHED-CM twice a day improved glucose intolerance in STZ-induced diabetic mice and the effect continued for 20 days after the end of treatment. SHED-CM treatment increased pancreatic insulin content and β-cell mass through proliferation and an intraperitoneal glucose tolerance test revealed enhanced insulin secretion. Incubation of MIN6 cells (a mouse pancreatic β-cell line) with SHED-CM enhanced insulin secretion in a glucose concentration-dependent manner and reduced STZ-induced cell death, indicating that the amelioration of hyperglycemia was caused by the direct effects of SHED-CM on β-cell function and survival. These effects were more pronounced than with the use of Ex-4, a conventional incretin-based drug, and BM-CM, which is a medium derived from other stem cells.


Expression and roles of a xenopus head-forming gene homologue in human cancer cell lines.

Molecular mechanisms for both morphogenesis and carcinogenesis have frequently overlapped, and similar signaling pathways are often involved in these processes. Yamamoto et al. identified a novel protein that induces head formation in Xenopus (Yamamoto et al. Cell, 120, 223-225, 2005). This new protein, named Shisa, plays unique roles in head formation by suppressing the maturation processes of receptors for Wnt and FGF at the endoplasmic reticulum. Here, we have identified a human homologue of the shisa gene (hu-shisa-2), and analyzed its expression in various human cancer cell lines by real-time reverse transcription polymerase chain reaction. High levels of mRNA expression were observed in some neuroectoderm-derived human cancer cell lines and small cell lung cancer cell lines. Intracellular localization of hu-Shisa-2 protein was also analyzed, indicating that it is present in the endoplasmic reticulum. Over-expression of hu-Shisa-2 resulted in increased cell growth and invasion, suggesting that hu-Shisa-2 is involved in the evolution and/or progression of human cancers.


Mouse homologues of Shisa antagonistic to Wnt and Fgf signalings.

In an effort to identify Otx2 targets in mouse anterior neuroectoderm we identified a gene, mShisa, which is homologous to xShisa1 that we previously reported as a head inducer in Xenopus. mShisa encodes an antagonist against both Wnt and Fgf signalings; it inhibits these signalings cell-autonomously as xShisa1 does. The mShisa expression is lost or greatly reduced in Otx2 mutant visceral endoderm, anterior mesendoderm and anterior neuroectoderm. However, mShisa mutants exhibited no defects in head development. Shisa is composed of five subfamilies, but normal head development in mShisa mutants is unlikely to be explained in terms of the compensation of mShisa deficiency by its paralogues or by known Wnt antagonists in anterior visceral endoderm and/or anterior mesendoderm.


Shisa2 promotes the maturation of somitic precursors and transition to the segmental fate in Xenopus embryos.

In vertebrate somitogenesis, FGF and Wnt signals constitute a morphogenetic gradient that controls the maturation of the presomitic mesoderm (PSM) as well as the transition to segmental units. It remains unclear, however, whether there is a regulatory mechanism that promotes the transition by a direct regulation of FGF and Wnt signaling in the PSM. Here we show that Shisa2, a member of a novel Shisa gene family, plays an essential role in segmental patterning during Xenopus somitogenesis. Shisa2 encodes an endoplasmic reticulum (ER) protein that cell-autonomously inhibits FGF and Wnt signaling by preventing the maturation and the cell-surface expression of their receptors. Shisa2 is expressed in the PSM and its knockdown caused a reduction in somite number by the delayed maturation of PSM and anterior shift of the transition; however, the phase of the segmental clock remained intact. These phenotypes were abolished by the inhibition of both FGF and Wnt signals, but by neither alone. We therefore propose that the individual inhibition of both types of signaling by the regulation of receptor maturation in the ER plays an essential role in the establishment of proper segmental patterning.


Shisa promotes head formation through the inhibition of receptor protein maturation for the caudalizing factors, Wnt and FGF.

Head formation requires simultaneous inhibition of multiple caudalizing signals during early vertebrate embryogenesis. We identified a novel antagonist against Wnt and FGF signaling for head formation, Shisa, which functions cell autonomously in the endoplasmic reticulum (ER). Shisa is specifically expressed in the prospective head ectoderm and the Spemann organizer of Xenopus gastrulae. Overexpression of Shisa inhibited both Wnt and FGF signaling in Xenopus embryos and in a cell line. Loss of Shisa function sensitized the neuroectoderm to Wnt signaling and suppressed head formation during gastrulation. Shisa physically interacted with immature forms of the Wnt receptor Frizzled and the FGF receptor within the ER and inhibited their posttranslational maturation and trafficking to the cell surface. Taken together, these findings indicate that Shisa is a novel molecule that controls head formation by regulating the establishment of the receptors for caudalizing factors.


Mouse paraxial protocadherin is expressed in trunk mesoderm and is not essential for mouse development.

Paraxial protocadherin (PAPC) is a cell adhesion molecule that marks cells undergoing convergence-extension cell movements in Xenopus and zebrafish gastrulating embryos. Here a mouse homologue (mpapc) was identified and characterized. During early- to mid-gastrulation, mpapc is expressed in the primitive streak as the trunk mesoderm undergoes morphogenetic cell movements. At head-fold stage mpapc expression becomes localized to paraxial regions in which somites are formed in the segmental plate. At later stages, mpapc displays a complex expression pattern in cerebral cortex, olfactory bulb, inferior colliculus, and in longitudinal stripes in hindbrain. To analyze the effect of the loss of PAPC function during mouse development, a null allele of the mouse papc gene was generated. Homozygous animals show no defects in their skeleton and are viable and fertile.

Genesis : the journal of genetics and development


Zebrafish Mesp family genes, mesp-a and mesp-b are segmentally expressed in the presomitic mesoderm, and Mesp-b confers the anterior identity to the developing somites.

Segmentation of a vertebrate embryo begins with the subdivision of the paraxial mesoderm into somites through a not-well-understood process. Recent studies provided evidence that the Notch-Delta and the FGFR (fibroblast growth factor receptor) signalling pathways are required for segmentation. In addition, the Mesp family of bHLH transcription factors have been implicated in establishing a segmental prepattern in the presomitic mesoderm. In this study, we have characterized zebrafish mesp-a and mesp-b genes that are closely related to Mesp family genes in other vertebrates. During gastrulation, only mesp-a is expressed in the paraxial mesoderm at the blastoderm margin. During the segmentation period, both genes are segmentally expressed in one to three stripes in the anterior parts of somite primordia. In fused somites (fss) embryos, in which all early somite boundary formation is blocked, initial mesp-a expression at the gastrula stage remains intact, but the expression of mesp-a and mesp-b is not detected during the segmentation period. This suggests that these genes are downstream targets of fss at the segmentation stage. Comparison with her1 expression (Müller, M., von Weizsäcker, E. and Campos-Ortega, J. A. (1996) Development 122, 2071-2078) suggests that, like her1, mesp genes are not expressed in primordia of the first several somites. Furthermore, we found that zebrafish her1 expression oscillates in the presomitic mesoderm. The her1 stripe, which first appears in the tailbud region, moves in a caudal to rostral direction, and it finally overlaps the most rostral mesp stripe. Thus, in the trunk region, both her1 and mesp transcripts are detected in every somite primordium posterior to the forming somites. Ectopic expression of Mesp-b in embryos causes a loss of the posterior identity within the somite primordium, leading to a segmentation defect. These embryos show a reduction in expression of the posterior genes, myoD and notch5, with uniform expression of the anterior genes, FGFR1, papc and notch6. These observations suggest that zebrafish mesp genes are involved in anteroposterior specification within the presumptive somites, by regulating the essential signalling pathways mediated by Notch-Delta and FGFR.


The role of paraxial protocadherin in selective adhesion and cell movements of the mesoderm during Xenopus gastrulation.

Paraxial Protocadherin (PAPC) encodes a transmembrane protein expressed initially in Spemann's organizer and then in paraxial mesoderm. Together with another member of the protocadherin family, Axial Protocadherin (AXPC), it subdivides gastrulating mesoderm into paraxial and axial domains. PAPC has potent homotypic cell adhesion activity in cell dissociation and reaggregation assays. Gain- and loss-of-function microinjection studies indicate that PAPC plays an important role in the convergence and extension movements that drive Xenopus gastrulation. Thus, PAPC is not only an adhesion molecule but also a component of the machinery that drives gastrulation movements in Xenopus. PAPC may provide a link between regulatory genes in Spemann's organizer and the execution of cell behaviors during morphogenesis.


Complex gangliosides are essential in spermatogenesis of mice: possible roles in the transport of testosterone.

Mice, homozygous for disrupted ganglioside GM2/GD2 synthase (EC 2.4. 1.94) gene and lacking all complex gangliosides, do not display any major neurologic abnormalities. Further examination of these mutant mice, however, revealed that the males were sterile and aspermatogenic. In the seminiferous tubules of the mutant mice, a number of multinuclear giant cells and vacuolated Sertoli cells were observed. The levels of testosterone in the serum of these mice were very low, although testosterone production equaled that produced in wild-type mice. Testosterone was found to be accumulated in interstitial Leydig cells, and intratesticularly injected testosterone was poorly drained in seminiferous fluid in the mutant mice. These results suggested that complex gangliosides are essential in the transport of testosterone to the seminiferous tubules and bloodstream from Leydig cells. Our results provide insights into roles of gangliosides in vivo.

Proceedings of the National Academy of Sciences of the United States of America


Genetic remodeling of gangliosides resulted in the enhanced reactions to the foreign substances in skin.

Several lines of transgenic mice with gangliosides GM2/GD2 synthase gene were established, and the expression levels of the transgene in brain, liver, spleen and thymus were analyzed by comparing with those in their litter mates. Among four tissues, brain and skin showed markedly high expression levels of the transgene in Northern blotting. Particularly, transgenic mice skin showed about 10-fold higher expression of GM2/GD2 synthase gene than the wild type mice skin. Therefore, alterations in the morphology, glycolipid components, and responses to the exogenous stimulations in the transgenic mice skin were examined. Gangliosides in the transgenic skin were dramatically converted from GM3 to GM1, whereas no morphological changes were observed. However, when skin flap test was performed with insertion of nylon membranes under the skin flaps, much stronger inflammatory reactions consisting of edema, marked thickness, and cell infiltration were observed in the transgenic mice compared with the wild type. Similar enhanced inflammatory reaction was also observed in the skin injected by silicon gel, and in the peritoneal reaction to the injected casein. Main cell population in these inflammatory reactions consisted of neutrophils, suggesting an increased sensitivity of neutrophils to chemotactic factors in the transgenic mice.


Patterning by genes expressed in Spemann's organizer.

Cold Spring Harbor Symposia on Quantitative Biology


Mice with disrupted GM2/GD2 synthase gene lack complex gangliosides but exhibit only subtle defects in their nervous system.

Gangliosides, sialic acid-containing glycosphingolipids, are abundant in the vertebrate (mammalian) nervous system. Their composition is spatially and developmentally regulated, and gangliosides have been widely believed to lay essential roles in establishment of the nervous system, especially in neuritogenesis and synaptogenesis. However, this has never been tested directly. Here we report the generation of mice with a disrupted beta 1,4-N-acetylgalactosaminyltransferase (GM2/GD2 synthase; EC 2.4.1.92) gene. The mice lacked all complex gangliosides. Nevertheless, they did not show any major histological defects in their nervous systems or in gross behavior. Just a slight reduction in the neural conduction velocity from the tibial nerve to the somatosensory cortex, but not to the lumbar spine, was detected. These findings suggest that complex gangliosides are required in neuronal functions but not in the morphogenesis and organogenesis of the brain. The higher levels of GM3 and GD3 expressed in the brains of these mutant mice may be able to compensate for the lack of complex gangliosides.


Site restricted and neuron dominant expression of alpha 2,8sialyltransferase gene in the adult mouse brain and retina.

Gene expression of the alpha 2,8sialyltransferase (alpha 2,8S-T) responsible for GD3 synthesis in the adult mouse brain and retina was analysed by reverse transcription-polymerase chain reaction/Southern blotting (RT-PCR/Southern) and in situ hybridization. Among various portions of the brain, high levels of 9.5 kb mRNA were observed in the retina and midbrain. Results of RT-PCR/Southern did not necessarily correlate with the enzyme activities in the individual sites. In situ hybridization analysis revealed that this gene was characteristically expressed in the inner segment of photoreceptor cells, some nuclei in the midbrain, cranial nerve nuclei in the pons-medulla, Purkinje cells in the cerebellum, pyramidal cells of the hippocampus and granular cells of the dentate gyrus. In the retina, the alpha 2,8S-T gene was broadly expressed over the layers during development, and retained high expression levels in the photoreceptor cells of adult mice consistent with high expression of GD3. Destruction of neurons in the hippocampus and dentate gyrus by injection of kainic acid and colchicine respectively resulted in the disappearance of the hybridization signal, suggesting that the alpha 2,8S-T gene was mainly expressed by neurons.


Heterogeneity in the expression pattern of two ganglioside synthase genes during mouse brain development.

Gangliosides are synthesized by sequential catalytic reaction of multiple glycosyltransferases. GM2/GD2 synthase and GD3 synthase are key enzymes for ganglioside synthesis, because their relative activities regulate the main profiles of ganglioside expression. Mouse GD3 synthase (EC 2.4.99.8) cDNA was cloned by eukaryotic expression cloning, and its mRNA expression as well as that of GM2/GD2 synthase gene during the development of the mouse CNS was analyzed by using northern blotting, reverse transcription-polymerase chain reaction, and in situ hybridization. When brain tissue was analyzed as a whole mass, a typical pattern corresponding to the reported findings obtained by biochemical analyses was observed, i.e., high expression of GD3 synthase gene in the early stage and gradual increase of GM2/GD2 synthase gene expression in the late stage of the development. However, the results of in situ hybridization of these two genes revealed that the expression kinetics of these two genes were heterogeneous among various sites in the brain under development. These findings suggest that various expression patterns of the two genes reflect differences in the course of the development of individual sites, and also different ganglioside components are required in individual portions of the brain for development and maintenance of the function.


Diverse expression of beta 1,4-N-acetylgalactosaminyltransferase gene in the adult mouse brain.

Among various tissues of mouse, beta 1,4-N-acetylgalactosaminyltransferase (GM2/GD2 synthase) gene is expressed predominantly in the brain. Further analysis of the gene expression in the mouse CNS was performed by northern blotting and by enzyme assays using extracts from various parts of the CNS. In situ hybridization was also done to investigate the distribution of cells generating GM2/GD2 synthase. In northern blots, diverse levels of the gene expression were observed, depending on the regions examined. By in situ hybridization, pyramidal cells in the hippocampus, granular cells in dentate gyrus and cerebral cortex, Purkinje cells in cerebellum, and mitral cells in the olfactory bulb expressed high levels of the mRNA; these results corresponded to the results obtained by northern blot. Enzyme levels in these sites were accordingly high. However, enzyme levels in certain areas with low mRNA intensities, such as thalamus and pons medulla, were higher than expected from the results of northern blotting. The significance of the high gene expression in certain areas for brain function and the reason for the discrepancy between mRNA level and enzyme activity in some regions are discussed.


Substrate specificity of beta 1,4-N-acetylgalactosaminyltransferase in vitro and in cDNA-transfected cells. GM2/GD2 synthase efficiently generates asialo-GM2 in certain cells.

The substrate specificity of beta 1,4-N-acetylgalactosaminyltransferase has been analyzed using a fusion enzyme which consisted of the catalytic domain of the enzyme and the IgG binding domain of protein A, and also by extracts from cDNA transfectants. Both enzyme sources were capable of producing not only GM2 and GD2, but also asialo-GM2, GalNAc-sialylparagloboside, and Gal-NAc-GD1a from appropriate acceptors, although the efficiencies were at most 1-3% of those of GM2/GD2. The biological significance of these low specificities was studied with transient and stable transfectant cells. From the results of transient expression of the cDNA, asialo-GM2 expression appeared to inversely correlate with GM2 synthase levels in those lines. Consequently, GM2 seemed to be preferentially synthesized when both GM3 and lactosylceramide are available, and asialo-GM2 is synthesized in the absence of GM3 synthesis. However, the results of double immunostaining of CHO transfectants with anti-GM2 and anti-asialo-GM2 antibodies indicated that another factor may be involved in asialo-GM2 synthesis. From the in vitro assay using mixed acceptors, it was concluded that the presence of certain levels of GM2 might enhance the asialo-GM2 synthesis. These results suggest that even acceptors showing low efficiencies in vitro might be used in certain cells depending on the availability of precursors, expression levels of other gangliosides, as well as the kinetic properties of the enzyme, and the compartmentation of the glycosylation machineries in the cells.


T cell receptor-mediated stimulation of mouse thymocytes induces up-regulation of the GM2/GD2 synthase gene.

cDNA clones of the mouse GM2/GD2 synthase (EC 2.4.1.92) gene were isolated, and their analyses revealed that the protein has a type II transmembrane structure with 533 amino acids, which was very similar to the human homolog except for the mRNA size. The mRNA level in thymocytes dramatically increased after treatment with anti-CD3 monoclonal antibody, whereas it was not elevated when treated with prostaglandin E2. In situ hybridization showed an elevation of mRNA levels in medullar thymocytes, suggesting that T cell receptor-mediated signaling induces up-regulation of the GM2/GD2 synthase gene in mature thymocytes.


Isolation of GD3 synthase gene by expression cloning of GM3 alpha-2,8-sialyltransferase cDNA using anti-GD2 monoclonal antibody.

For the isolation of ganglioside GD3 synthase (EC 2.4.99.8) cDNA, we developed an expression cloning approach that used an anti-GD2 monoclonal antibody for selection. A host recipient cell line that we have named KF3027-Hyg5 was also utilized. This cell line expresses high levels of GM2 as well as GM3 but no GD3 or GD2 and was constructed from mouse B16 melanoma cells transfected with the polyoma large tumor antigen gene (KF3027) and the previously cloned beta-1,4-N-acetylgalactosaminyltransferase (EC 2.4.1.92) cDNA. Four rounds of transfection, monoclonal antibody 3F8 panning, and Hirt extraction resulted in the isolation of two cDNA clones, transfection of which directed the expression of GD3 in KF3027 and B16 melanoma cells and GD3 and GD2 in KF3027-Hyg5 cells. The cDNA contained a 1650-bp insert and a single open reading frame. The deduced amino acid predicted a type II membrane topology consisting of cytoplasmic (14 aa), transmembrane (18 aa), and catalytic (309 aa) domains. The sequence also predicted the presence of a sialyl motif similar to that found in the other sialyltransferases cloned so far. As expected, mRNA of this gene (2.6 kb) was strongly expressed in human melanoma lines.

Proceedings of the National Academy of Sciences of the United States of America


Epoc-1: a POU-domain gene expressed in murine epidermal basal cells and thymic stromal cells.

POU-domain transcription factors are known as developmental regulators which control organ development and cell phenotypes. In order to clarify the roles of POU-domain transcription factors in cell differentiation, we cloned a novel POU family gene, Epoc-1, from a murine thymus cDNA library. The amino acid (aa) sequence of the POU-specific domain of Epoc-1 is almost identical to those of Oct-1 and Oct-2. However, within the POU-homeodomain, 13 out of 60 aa differ between Epoc-1 and Oct-2. Recombinant Epoc-1 products were found to bind specifically to the octamer sequence. Epoc-1 was found to be expressed in skin, thymus, stomach and testis. In situ hybridization experiments and RNase protection assays indicated that Epoc-1 is expressed in the epidermal basal cells of the skin, which contain stem cells unipotent for keratinocyte differentiation and in thymic stromal elements. These results suggest that Epoc-1 might be one of the developmental regulators which controls epidermal development and thymic organogenesis.


Effect of accessory cells on stimulation of murine T-cell leukemia with antibodies to the CD3/T cell antigen receptor complex.

Stimulation of EL4 and RL male 1 leukemia cells in vitro with immobilized anti-CD3 epsilon monoclonal antibody (mAb) (145-2C11) or anti-TCR beta mAb (H57-597) in the absence of accessory cells induced interleukin-2 (IL-2) production, and caused growth inhibition. The growth inhibition was, however, transient and the tumors started to grow again within 5 days in immobilizing plates treated with antibodies at concentrations of 2.5-100 micrograms/ml. Addition of mitomycin C-treated accessory cells to the culture inhibited IL-2 production and resulted in augmented and persistent growth inhibition. No recovery of tumor growth was observed. Furthermore, DNA from EL4 and RL male 1 leukemia cells stimulated with anti-CD3/TCR mAbs was fragmented even in the absence of accessory cells, but fragmentation was much greater in the presence of accessory cells. Marginal and high expression of the bcl-2 gene were observed in EL4 and RL male 1, respectively, indicating that apoptosis of these leukemias mediated by signalling through the CD3/TCR complex has no direct relationship with expression of the bcl-2 gene.

Gann : Japanese Journal of Cancer Research