Satoshi Miyauchi

Analysis of unsaturated disaccharides from glycosaminoglycuronan by high-performance liquid chromatography☆

A rapid and simple analytical method for unsaturated disaccharide isomers formed by enzymatic digestion from hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, and heparin by high-performance liquid chromatography using an amine-bound silica column with a linear gradient of sodium dihydrogen phosphate was developed. The analyses were performed on isomers of two groups belonging to the chondroitin sulfate family and the heparin sulfate family. In both families, disaccharide isomers eluted in the order non-, mono-, di-, and trisulfated disaccharides by elevating salt concentrations. The method was applied to the analysis of constituent disaccharides of representative sulfated glycosaminoglycans, which proved that most constituents could be quantified separately. This method is advantageous in that enzymatic digests can be applied directly on a column without any pretreatment and good resolution of several disaccharides can be obtained by one chromatography.

Oversulfated chondroitin sulfate‐E binds to BMP‐4 and enhances osteoblast differentiation

Small leucine‐rich proteoglycans, such as biglycan, and their side chain sulfated glycosaminoglycans (GAGs), have been suggested to be involved in bone formation and mineralization processes. The present study was designed to investigate whether chondroitin sulfate (CS), one of the GAG, and its oversulfated structures coupled with bone morphogenetic protein‐4 (BMP‐4) alter the differentiation and subsequent mineralization of MC3T3‐E1 osteoblastic cells. CS‐E, one of the oversulfated CS structure, enhanced cell growth, alkaline phosphatase (ALP) activity, collagen deposition, and mineralization whereas heparin enhanced only ALP activity and mineralization. As well as CS‐E, CS‐H, and CPS also enhanced the mineralization of the cells. CS‐E enhanced the mineralization of the cells by interacting with protein in the conditioned medium. CS‐E induced mineralization was significantly inhibited by an antibody against BMP‐4. The addition of exogenous BMP‐4 further increased the capacity of CS‐E to enhance mineralization. Fluorescence correlation spectroscopy method using fluoresceinamine‐labeled GAG revealed that the oversulfated GAGs have a high affinity for BMP‐4. The disaccharide analysis of the cells indicated that MC3T3‐E1 cells are capable of producing oversulfated structures of CS by themselves. The lack of CS from the cells after chondroitinase treatment resulted in the inhibition of mineralization. These results in the present study indicate that oversulfated CS, which possesses 4,6‐disulfates in N‐acetyl‐galactosamine, binds to BMP‐4 and promotes osteoblast differentiation and subsequent mineralization. J. Cell. Physiol. 217: 769–777, 2008.

Evaluation of osteoclastic resorption activity using calcium phosphate coating combined with labeled polyanion.

Osteoclasts are involved in bone resorption, and its activation is considered one of the causes of osteoporosis. The pit assay is the principal method for evaluating osteoclast function by measuring hydroxyapatite resorption in vitro. However, the pit assay requires time and trained techniques, including the pit image analysis, and there is no other easy method for evaluating bone resorption. In this study, we developed a novel approach to quantify the bone resorption activity using a calcium phosphate (CaP) coating labeled with fluorescent polyanion. Fluoresceinamine-labeled chondroitin polysulfate or Hoechst 33258-labeled deoxyribonucleic acid was used for CaP labeling. When macrophage cell line RAW264 was cultured on the labeled CaP under the stimulation with the receptor activator of the NF-κB ligand (RANKL), RAW264 cells differentiated into osteoclastic cells and the fluorescence intensity of the culture supernatant and pit area increased in a time- and dose-dependent manner. Furthermore, drugs for osteoporosis treatment, such as pamidronate and β-estradiol, inhibited fluorescein release by the cells stimulated with RANKL. A positive correlation between the fluorescence intensity and pit area was observed (r=0.917). These results indicated that this new method using fluorescent polyanion-labeled CaP is a standardized useful assay system for the evaluation of bone resorption activity.

Chondroitin Sulfate-E Binds to Both Osteoactivin and Integrin αVβ3 and Inhibits Osteoclast Differentiation.

Integrins and their ligands have been suggested to be associated with osteoclast‐mediated bone resorption. The present study was designed to investigate whether chondroitin sulfate E (CS‐E), which is one of the sulfated glycosaminoglycans (GAGs), is involved in osteoactivin (OA) activity, and osteoclast differentiation. The binding affinity of sulfated GAGs to integrin and its ligand was measured using biotin‐labeled CS‐E, and the osteoclast differentiation was evaluated by tartrate‐resistant acid phosphatase staining and a pit formation assay. CS‐E as well as CS‐B, synthetic chondroitin polysulfate, and heparin inhibited osteoclast differentiation of bone marrow‐derived macrophages. Pre‐coating of OA to synthetic calcium phosphate‐coated plates enhanced the osteoclastic differentiation of RAW264 cells, and addition of a neutralizing antibody to OA inhibited its differentiation. CS‐E bound not only to OA, fibronectin, and vitronectin, but also to its receptor integrin αVβ3, and inhibited the direct binding of OA to integrin αVβ3. Furthermore, CS‐E blocked the binding of OA to cells and inhibited OA‐induced osteoclastic differentiation. On the other hand, heparinase treatment of RAW264 cells inhibited osteoclastic differentiation. Since binding of OA to the cells was inhibited by the presence of heparan sulfate or heparinase treatment of cells, heparan sulfate proteoglycan (HSPG) was also considered to be an OA receptor. Taken together, the present results suggest that CS‐E is capable of inhibiting OA‐induced osteoclast differentiation by blocking the interaction of OA to integrin αVβ3 and HSPG. J. Cell. Biochem. 116: 2247–2257, 2015.