Masaki Yanagishita

Handbook of glycosyltransferases and related genes

The CHST14 gene, localized at 15q14, is a single exon gene with an open reading frame of 1131 base pairs, encoding a 43 kDa protein dermatan-4-Osulfotransferase-1 (D4ST1) that catalyzes the 4-O-sulfation of N-acetyl-D-galactosamine residues in dermatan sulfate (DS). Both nearly exhaustively desulfated DS and partially desulfated DS serve as excellent substrates for the enzyme. Chst14/D4st1-deficient mice showed growth retardation as well asmultiple system abnormalities including neurology such as decreased neurogenesis and diminished T. Kosho (*) School of Medicine, Department of Medical Genetics, Shinshu University, Matsumoto, Japan e-mail: ktomoki@shinshu-u.ac.jp S. Mizumoto • K. Sugahara Laboratory of Proteoglycan Signaling and Therapeutics, Hokkaido University Graduate School of Life Science, Kita-ku, Sapporo, Japan e-mail: mizumoto@sci.hokudai.ac.jp; k-sugar@sci.hokudai.ac.jp N. Taniguchi et al. (eds.), Handbook of Glycosyltransferases and Related Genes, DOI 10.1007/978-4-431-54240-7_156, # Springer Japan 2014 1135 proliferation of neural stem cells. Recently, recessive loss-of-function mutations in the CHST14 gene were found to cause a specific form of Ehlers-Danlos syndrome (EDS) designated as D4ST1-deficient EDS (DD-EDS). The disorder is characterized by progressive multisystem fragility-related manifestations (skin hyperextensibilty and fragility, progressive spinal and foot deformities, large subcutaneous hematoma) and various malformations (facial features, congenital eye/heart/gastrointestinal defects, congenital multiple contractures). Glycosaminoglycan (GAG) chains from the affected skin fibroblasts were composed of a negligible amount of DS and excess chondroitin sulfate (CS), which was suggested to result from an impaired lock by 4-O-sulfation due to D4ST1 deficiency followed by back epimerization from L-iduronic acid to D-glucuronic acid. GAG chains of decorin from the affected skin fibroblasts were composed exclusively of CS and no DS, the opposite features observed in normal controls. Thus, skin fragility in the disorder was supposed to be caused by impaired assembly of collagen fibrils mediated by decorin bearing a CS chain that replaced a DS chain. The disorder stresses the importance of the role of CHST14/ D4ST1 and DS in human development and maintenance of extracellular matrices.

Loss of Syndecan‐1 and Increased Expression of Heparanase in Invasive Esophageal Carcinomas

Heparan sulfate proteoglycans play important biological roles in cell‐cell and cell‐matrix adhesion, and are closely associated with growth factor actions. Loss of syndecan‐1, a cell surface‐bound heparan sulfate proteoglycan, has been reported for advanced head and neck carcinomas, and expression of endoglycosidic heparanase, which cleaves heparan sulfate glycosaminoglycans (HS‐GAGs), is associated with invasion and metastatic potential of malignant tumors. Paraffin sections of 103 primary esophageal squamous cell carcinomas were immunohistochemically examined for the expression of syndecan‐1 core protein, HS‐GAGs and heparanase protein, and the results were compared with various clinicopathological parameters, such as invasion depth. For 16 cases, fresh tumor samples were quantitatively analyzed for heparanase and syndecan‐1 mRNA expression by real‐time RT‐PCR in addition to the immunohistochemical studies. Syndecan‐1 core protein and HS‐GAGs expression was significantly decreased in pT2 and pT3 cases compared with their pTis and pTl counterparts. Decreased expression of core protein and HS‐GAGs was correlated with the incidence of lymphatic invasion, and venous involvement. Furthermore, decreased expression of HS‐GAGs was correlated positively with the incidence of nodal metastasis and distant organ metastasis, and negatively with the grade of tumor cell differentiation. The percentage of cytoplas‐mic heparanase protein‐positive cases increased significantly in pT2 and pT3 cases compared to that in pTis and pTl cases, and this was associated with lymphatic invasion, and venous and lymph nodal involvement. The level of heparanase mRNA was inversely correlated with the degree of HS‐GAGs expression rather than core protein. In conclusion, loss of syndecan‐1 and heparanase overexpression in esophageal squamous cell carcinomas are closely associated with malignant potential. Regarding the mechanism of loss of HS‐GAGs, heparanase upregulation appears to play an important role.

Effect of compressive forces on extracellular matrix in rat mandibular condylar cartilage.

To reveal the effect of compressive force on the mandibular condylar cartilage, an appliance was set on 8-week-old Wistar rats to load continuous compressive force. Immunohistochemical and histochemical analyses were performed using toluidine blue, antibodies, and probes for aggrecan, hyaluronan, type II collagen, type X collagen, and 5-bromo-2″-deoxyuridine (BrdU). Histomorphometry and statistical analyses were also performed for aggrecan and BrdU immunostaining. In toluidine blue staining, tissue metachromasia was observed in the transitional zone and the hypertrophic zone of the mandibular condylar cartilage. In histomorphometry and statistical analysis, thickness of the cartilage decreased significantly in all regions in the 3-day experimental group. However, the thickness of the cartilage in the anterior region showed recovery while it decreased continuously in the posterior region. Distributional changes of aggrecan, hyaluronan, type II collagen, and type X collagen in the experimental groups were similar to those for toluidine blue staining. The immunostained area of all these molecules decreased as a result of the decrement of the cartilage area. However, enhanced immunostaining for aggrecan in the proliferative zone was observed only in the 1-day experimental group. BrdU-positive cells, observed in the proliferating zone and the transitional zone, decreased significantly in the experimental group 3 days after force was applied. These results demonstrate that continuous compressive forces on the mandibular condylar cartilage decrease the proliferation of chondrocytes and the amount of extracellular matrices.

Expression of heparanase in oral cancer cell lines and oral cancer tissues

In the process of metastasis, cancer cells secrete several enzymes which degrade extracellular matrices (ECMs) and basement membranes (BMs) of blood vessels. One of them, heparanase, has been reported to be an important enzyme when metastatic cancer cells invade blood vessels. The enzyme cleaves heparan sulfate (HS), a main component of ECM and BM. In the present study, HS-degrading ability of several human oral cancer cell lines (HSC2, HSC3, HSC4, Ca9-22, NA, ACC3 and Ab-J) and tissues derived from human oral squamous cell carcinomas (both metastatic and non-metastatic) were investigated by measuring heparanase activities and levels of heparanase mRNA by a quantitative reverse transcriptase-polymerase chain reaction. The catalytic activities and the mRNA levels of heparanase showed a good agreement. Clinical demonstration of cancer metastasis generally correlated with high levels of heparanase activity and its mRNA. The results suggest that heparanase activity and its mRNA level are good diagnostic parameters for evaluating the metastatic properties of human oral cancer cells.

Oligomers of glycamino acid

Glycamino acids, a family of sugar amino acids, are derivatives of C-glycosides that possesses a carboxyl group at the C-1 position and an amino group replacing one of the hydroxyl groups at either the C-2, 3, 4, or 6 position. We have prepared a series of glucose-type glycamino acids as monomeric building blocks: these are derivatives of 2-NH(2)-Glc-beta-CO(2)H 1, 3-NH(2)-Glc-beta-CO(2)H 2, 4-NH(2)-Glc-beta-CO(2)H 3, and 6-NH(2)-Glc-beta-CO(2)H 4 and constructed four types of homo-oligomers, beta(1-->2)-linked I, beta(1-->3)-linked II, beta(1-->4)-linked III, and beta(1-->6)-linked IV, employing the well-established N-Boc and BOP strategy. CD and NMR spectral studies of these oligomers suggested that only the beta(1-->2)-linked homo-oligomer possessed a helical structure that seems to be predetermined by the linkage position. Homo-oligomers with beta(1-->2)-linkages I and beta(1-->6)-linkages IV were also subjected to O-sulfation, and these O-sulfated oligomers were found to be able, in a linkage-specific manner, to effectively inhibit L-selectin-mediated cell adhesion, HIV infection, and heparanase activity without the anticoagulant activity associated with naturally occurring sulfated polysaccharides such as heparin.

Heparanase and tumor invasion patterns in human oral squamous cell carcinoma xenografts

The role of heparanase, an endo‐β‐glucuronidase specifically degrading heparan sulfate (HS) glycosaminoglycans, in the mechanism of cancer cell invasion was investigated. Three human oral squamous cell carcinoma (SCC) cell lines (i.e., HSC‐2, HSC‐3 and LMF4), exhibiting various degrees of invasiveness to their surrounding tissues, were xenografted to the tongue of SCID mice in order to establish experimental cancer foci. Cancer cells and their surrounding tissues were examined for the expression of heparanase mRNA by an in situ hybridization technique, and for various basement membrane (BM)‐associated molecules (i.e., perlecan, laminins and type IV collagen) by immunohistochemical procedures. BM structures surrounding cancer tissues were also examined by electron microscopy. Increasing levels of heparanase mRNA expression were observed with the progression of cancer invasiveness, as manifested by the destruction of BM structures. Enhanced heparanase enzyme activities in cancer tissues with more invasive properties were demonstrated by the disappearance of HS glycosaminoglycans in the face of retained HS pro‐teoglycan core proteins. These results demonstrated a positive correlation between the heparanase enzyme activities and the invasiveness of human oral SCC. The roles of heparanase in cancer cell invasion were not precisely clarified by the present morphological study, but the enhanced heparanase activity in an early phase of BM destruction by cancer cells suggested the participation of this enzyme from the early phase of cancer invasion. (Cancer Sci 2003; 94: 277–285)

Characterization of Heparanase from a Rat Parathyroid Cell Line

Cell surface heparan sulfate proteoglycans undergo unique intracellular degradation pathways after they are endocytosed from the cell surface. Heparanase, an endo-β-glucuronidase capable of cleaving heparan sulfate, has been demonstrated to contribute to the physiological degradation of heparan sulfate proteoglycans and therefore regulation of their biological functions. A rat parathyroid cell line was found to produce heparanase with an optimal activity at neutral and slightly acidic conditions suggesting that the enzyme participates in heparan sulfate proteoglycan metabolism in extralysosomal compartments. To elucidate the detailed properties of the purified enzyme, the substrate specificity against naturally occurring heparan sulfates and chemically modified heparins was studied. Cleavage sites of rat heparanase were present in heparan sulfate chains obtained from a variety of animal organs, but their occurrence was infrequent (average, 1–2 sites per chain) requiring recognition of both undersulfated and sulfated regions of heparan sulfate. On the other hand intact and chemically modified heparins were not cleaved by heparanase. The carbohydrate structure of the newly generated reducing end region of heparan sulfate cleaved by the enzyme was determined, and it represented relatively undersulfated structures. O-Sulfation of heparan sulfate chains also played important roles in substrate recognition, implying that rat parathyroid heparanase acts near the boundary of highly sulfated and undersulfated domains of heparan sulfate proteoglycans. Further elucidation of the roles of heparanase in normal physiological processes would provide an important tool for analyzing the regulation of heparan sulfate-dependent cell functions.

Isolation of proteoglycan (versican) aggregate from rat dental pulp

Versican is a large interstitial proteoglycan that is believed to be able to bind hyaluronan to form large aggregate structures, but no study has isolated native versican aggregates from any tissue. In this study, ternary aggregate structures consisting of versican, hyaluronan, and link protein were isolated from rat dental pulp by associative extractions followed by caesium sulphate rate zonal sedimentation centrifugation. Fractions from the centrifugation were analysed by dot blot and Western blot using monoclonal antibodies and hyaluronan-binding protein. About 60% of the hexuronic acid was extracted by associative extractions. Positive reactions for versican, hyaluronan and link protein were clearly detected in the bottom fractions from the centrifugation, but were barely detectable in the top fractions. These results suggest that the majority of the versican, hyaluronan, and link protein forms ternary aggregate structures in the rat dental pulp.

Basement Membrane Matrix Modifies Cytokine Interactions between Lung Cancer Cells and Fibroblasts

Objective: Proliferation of fibroblasts (desmoplastic reaction) in the lung adenocarcinomas is an important phenomenon that correlates with metastases and poor prognosis. Because basement membranes are often involved in the desmoplastic areas and many cytokines have binding capacity to basement membrane molecules, we hypothesized that basement membrane modify the paracrine effects between cancer cells and fibroblasts via the fibrogenic cytokines and this hypothesis was experimentally investigated. Methods: The effects of conditioned media derived from ten lung carcinoma cell lines and normal airway epithelial cells on DNA synthesis of fetal lung fibroblasts were determined. We focused on fibroblast growth factor 2 (FGF-2) as the candidate paracrine cytokines and examined their diffusion through an experimental basement membrane matrix model, Matrigel™. Results: All the conditioned media promoted DNA synthesis of fetal lung fibroblasts. Detection by ELISA methods and the neutralizing antibodies suggested that FGF-2 was one of the responsible factors for the growth promotion. Diffusion of FGF-2 across the polycarbonate membrane was suppressed by coating with Matrigel. When FGF-2-secreting A549 cells were covered with Matrigel, FGF-2 was stored in Matrigel and its diffusion into the culture media was significantly reduced. Binding of FGF-2 to Matrigel was completely blocked by a basic protein, protamine sulfate. In the presence of protamine sulfate in Matrigel overlaid on A549 cells, diffusion of FGF-2 increased 7-fold as much as that without overlaid Matrigel. Conclusion: These results suggest that the basement membrane acts as a barrier to the diffusion and a reservoir of cytokines secreted by cancer cells, and that the subsequent degradation of the basement membrane by cancer cells could release the stored cytokines and promote growth of fibroblasts.

Influence of Occlusal Stimuli on the Remodelling of Alveolar Bone in a Rat Hypofunction-Recovery Model

Remodelling of alveolar bone is a crucial factor determining tooth movement against orthodontic forces. Occlusion is the most influential stimulus causing physiological remodelling of alveolar bone. A removable metal appliance was devised, which can sequentially induce hypofunctional occlusion at molar teeth and reestablish normal occlusion. Using this appliance, remodelling of the alveolar bone was examined by histological morphometric analyses, with particular attention to localized regions of bone resorption by identifying the receptor activator of NF-κB ligand (RANKL), a growth factor inducing osteoclastogenesis, and osteoclasts. In the occlusal hypofunction period, the number of TRAP-positive cells increased at the margin of marrow spaces, and the marrow space enlarged. RANKL-positive osteoblasts also increased, especially around the blood vessels. In bone morphometric parameters, BV/TV, Tb. Th and Tb. N decreased, and Tb. Sp and Oc. N increased. These results are consistent with active bone remodelling consisting of active resorption at the inner alveolar bone and bone formation on the outer alveolar bone surface. In the 3 days recovery group, many TRAP-positive cells were observed at the inner alveolar bone and around the bone surface. RANKL-positive osteoblasts were observed at the margin of the marrow space in alveolar bone. However, BV/TV, Tb. Th, and Tb. Sp returned to the control level in the 7 days recovery group, with decreased TRAP-positive cells and RANKL-positive osteoblasts. These findings suggest that occlusal stimuli play important roles in the maintenance of functional alveolar structure and the regulation of alveolar bone in rats.