Chondroitin Sulfate-E Binds to Both Osteoactivin and Integrin αVß3 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 αVß3, and inhibited the direct binding of OA to integrin αVß3. 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 αVß3 and HSPG.

Short-chain fatty acids and ketones directly regulate sympathetic nervous system via G protein-coupled receptor 41 (GPR41).

The maintenance of energy homeostasis is essential for life, and its dysregulation leads to a variety of metabolic disorders. Under a fed condition, mammals use glucose as the main metabolic fuel, and short-chain fatty acids (SCFAs) produced by the colonic bacterial fermentation of dietary fiber also contribute a significant proportion of daily energy requirement. Under ketogenic conditions such as starvation and diabetes, ketone bodies produced in the liver from fatty acids are used as the main energy sources. To balance energy intake, dietary excess and starvation trigger an increase or a decrease in energy expenditure, respectively, by regulating the activity of the sympathetic nervous system (SNS). The regulation of metabolic homeostasis by glucose is well recognized; however, the roles of SCFAs and ketone bodies in maintaining energy balance remain unclear. Here, we show that SCFAs and ketone bodies directly regulate SNS activity via GPR41, a Gi/o protein-coupled receptor for SCFAs, at the level of the sympathetic ganglion. GPR41 was most abundantly expressed in sympathetic ganglia in mouse and humans. SCFA propionate promoted sympathetic outflow via GPR41. On the other hand, a ketone body, ß-hydroxybutyrate, produced during starvation or diabetes, suppressed SNS activity by antagonizing GPR41. Pharmacological and siRNA experiments indicated that GPR41-mediated activation of sympathetic neurons involves Gßγ-PLCß-MAPK signaling. Sympathetic regulation by SCFAs and ketone bodies correlated well with their respective effects on energy consumption. These findings establish that SCFAs and ketone bodies directly regulate GPR41-mediated SNS activity and thereby control body energy expenditure in maintaining metabolic homeostasis.

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 ß-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.

New frontiers in gut nutrient sensor research: free fatty acid sensing in the gastrointestinal tract.

Utilizing the human genome database, the recently developed G-protein-coupled receptors (GPCRs) deorphanizing strategy successfully identified multiple receptors of free fatty acids (FFAs). FFAs have been demonstrated to act as ligands of several GPCRs (FFAR1, FFAR2, FFAR3, and GPR120). These fatty acid receptors are proposed to play critical roles in various types of physiological homeostases. FFAR1 and GPR120 are activated by medium- and long-chain FFAs. In contrast, FFAR2 and FFAR3 are activated by short-chain FFAs. It has been elucidated that these four receptors are expressed in the gastrointestinal tract and have many essential roles as sensors of FFA. In this review, we summarize the physiological and pharmacological function of the receptors in the gastrointestinal tract.

Differentiation of mouse-induced pluripotent stem cells into dental epithelial-like cells in the absence of added serum.

Recent studies have successfully generated tooth-like structure by mimicking the reciprocal interaction between dental epithelial and mesenchymal cells in tooth organogenesis. However, clinical applications of these methods are limited primarily due to the lack of appropriate sources for dental epithelial cells. Induced pluripotent stem cells (iPSCs) are attractive as a source for dental epithelial cells due to their unique characteristics. In this study, we examined the effect of neurotrophin-4 (NT-4) on the differentiation of mouse iPSCs (miPSCs) into dental epithelial cells. Our results showed that the addition of NT-4 during the formation of embryoid body significantly triggered the upregulation of epithelial markers such as p63 and CK14, suggesting that NT-4 provides an inductive condition for the differentiation of miPSCs into epithelial cells. Expansion of the NT-4-treated cells under serum-free culture conditions improves the formation of cells with cobblestone-like morphology and significantly downregulated the expression of pluripotent and ectodermal markers. Phenotypic analysis revealed that a dental epithelial surface marker, CD49f, was highly expressed on these cells. Formation of miPSCs-derived dental epithelial-like cells was further confirmed by the expression of ameloblast-specific markers. These results suggest that the addition of NT-4 during the formation of embryoid body together with the serum-free culture condition promoted the differentiation of miPSCs into dental epithelial-like cells.

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.

The gut microbiota suppresses insulin-mediated fat accumulation via the short-chain fatty acid receptor GPR43.

The gut microbiota affects nutrient acquisition and energy regulation of the host, and can influence the development of obesity, insulin resistance, and diabetes. During feeding, gut microbes produce short-chain fatty acids, which are important energy sources for the host. Here we show that the short-chain fatty acid receptor GPR43 links the metabolic activity of the gut microbiota with host body energy homoeostasis. We demonstrate that GPR43-deficient mice are obese on a normal diet, whereas mice overexpressing GPR43 specifically in adipose tissue remain lean even when fed a high-fat diet. Raised under germ-free conditions or after treatment with antibiotics, both types of mice have a normal phenotype. We further show that short-chain fatty acid-mediated activation of GPR43 suppresses insulin signalling in adipocytes, which inhibits fat accumulation in adipose tissue and promotes the metabolism of unincorporated lipids and glucose in other tissues. These findings establish GPR43 as a sensor for excessive dietary energy, thereby controlling body energy utilization while maintaining metabolic homoeostasis.

Formation of proteoglycan and collagen-rich scaffold-free stiff cartilaginous tissue using two-step culture methods with combinations of growth factors.

Tissue-engineered cartilage may be expected to serve as an alternative to autologous chondrocyte transplantation treatment. Several methods for producing cartilaginous tissue have been reported. In this study, we describe the production of scaffold-free stiff cartilaginous tissue of pig and human, using allogeneic serum and growth factors. The tissue was formed in a mold using chondrocytes recovered from alginate bead culture and maintained in a medium with transforming growth factor-beta and several other additives. In the case of porcine tissue, the tear strength of the tissue and the contents of proteoglycan (PG) and collagen per unit of DNA increased dose-dependently with transforming growth factor-beta. The length of culture was significantly and positively correlated with thickness, tear strength, and PG and collagen contents. Tear strength showed positive high correlations with both PG and collagen contents. A positive correlation was also seen between PG content and collagen content. Similar results were obtained with human cartilaginous tissue formed from chondrocytes expanded in monolayer culture. Further, an in vivo pilot study using pig articular cartilage defect model demonstrated that the cartilaginous tissue was well integrated with surrounding tissue at 13 weeks after the implantation. In conclusion, we successfully produced implantable scaffold-free stiff cartilaginous tissue, which characterized high PG and collagen contents.

Effect of chondroitin sulfate-E on the osteoclastic differentiation of RAW264 cells.

The present study was designed to investigate whether chondroitin sulfate (CS)-E, a CS structural isomer variant, alter the differentiation of macrophage cell line RAW264 cells to osteoclast-like cells. CS-B, CS-E, low molecular weight CS-E, synthetic chondroitin polysulfate (CPS) and heparin significantly inhibited the formation of tartrate-resistant acid phosphatase-positive multinuclear cells and pit formation on calcium phosphate (CaP)-coated plates. CS-E pre-coated on the CaP plate also inhibited pit formation. Digestion of CS on the cell surface by chondroitinase showed no effect on the osteoclastic differentiation of RAW264 cells whereas inhibitory effect on the differentiation of osteoblastic cell line MC3T3-E1. On the other hand, exogenously added fluorescein-labeled CS-E directory bound to fibronectin and RAW264 cells. These results suggest that CS-E structure on the surface of osteoblasts or bone matrix binds to cell adhesion molecule such as integrin on the pre-osteoclastic cells and inhibits the differentiation into osteoclasts. CS-E may have a potential in treating bone defect if combined with CaP materials.

Distribution and regulation of protein expression of the free fatty acid receptor GPR120.

GPR120 is a G-protein-coupled receptor whose endogenous ligands have recently been identified as free fatty acids. It has been implicated as playing an important role in the control of lipid and glucose metabolism by regulating the secretion of glucagon-like peptide-1 and cholecystokinin. We have developed an antibody against the extracellular domain of GPR120. The specificity of the antibody was demonstrated by immunoprecipitation, Western blotting, flow cytometry, and immunocytochemistry using GPR120-transfected cells. Immunoreactivity for GPR120 was abundant in the mouse large intestine, lung, and adipose tissue. Furthermore, we found that the expression of GPR120 protein was up-regulated during the adipogenic differentiation of 3T3-L1 cells, which corresponded well with changes in mRNA expression. The anti-GPR120 antibody will be of value for the further study of the function of this nutrient-sensing receptor.

Effect of chondroitinase ABC on matrix metalloproteinases and inflammatory mediators produced by intervertebral disc of rabbit in vitro.

STUDY DESIGN: Lumbar intervertebral discs in rabbit were cultured in the presence of chondroitinase ABC. The matrix metalloproteinases (MMPs) and inflammatory mediators produced in culture media were then analyzed. OBJECTIVES: To investigate the effect of chondroitinase ABC on MMPs and inflammatory mediators produced by intervertebral disc of rabbit in vitro. SUMMARY OF BACKGROUND DATA: The chemonucleolytic effect of chondroitinase ABC is caused by the decrease in the chondroitin sulfate, hyaluronan, and protein content of the nucleus pulposus in rabbit. The reason for the decreases in protein content remains unclear. METHODS: Anulus fibrosus and nucleus pulposus were cultured for 72 hours with or without chondroitinase ABC stimulated or not stimulated by interleukin-1 after preculture for 4 days. Subsequently, the MMPs (gelatinases MMP-2, MMP-9, and collagenase) and inflammatory mediators (prostaglandin E2 and nitric oxide) produced in the culture media were analyzed. RESULTS: In the anulus fibrosus chondroitinase ABC and interleukin-1 synergistically increased the collagenase activity, which was at a significantly higher level than the increment solely due to interleukin-1. In contrast, chondroitinase ABC counteracted the increase in nitric oxide production by interleukin-1. In the nucleus pulposus the collagenase and nitric oxide productions were not particularly affected by chondroitinase ABC and/or interleukin-1. In zymographic analysis MMP-2 was detected, but MMP-9 was only slightly detected in both tissues. There were no significant differences in both tissues for MMP-2 and prostaglandin E2 following incubation with or without chondroitinase ABC, whether stimulated by interleukin-1 or not. CONCLUSIONS: The collagenase activity in the anulus fibrosus was increased by chondroitinase ABC with interleukin-1. This finding may support the hypothesis that some proteolytic activities are involved in the chemonucleolytic process by chondroitinase ABC treatment.

Immunogenicity of hylan g-f 20 in Guinea pigs and mice.

OBJECTIVE: To determine the product-specific immunogenicity of a chemically-modified sodium hyaluronate derivative, hylan G-F 20, that is used in the treatment of osteoarthritis of the knee. METHODS: Guinea pigs were subcutaneously immunized with hylan G-F 20 (Synvisc) once a week for 3 weeks. After resting, these animals received an intradermal challenge with hylan to elicit allergic skin reactions. Animal sera were tested for the presence of hylan-specific antibodies by homologous passive cutaneous anaphylaxis (PCA) assay and of anti-hylan IgG by ELISA. Further, mice were similarly immunized with hylan, and their sera were analyzed by heterologous PCA assay in rats and by ELISA for anti-hylan Ig(G+M) and anti-hylan IgE. RESULTS: In the guinea pig studies, acute and delayed erythematous skin reactions were elicited in immunized animals after the intradermal challenge with hylan. The sera of hylan-immunized guinea pigs showed positive reaction in the homologous PCA assay and significantly high amount of anti-hylan IgG, whereas the sera did not show any cross-reactivity against sodium hyaluronate. Hylan also exhibited immunogenicity in mice of 3 inbred strains, and C3H/HeN mice showed higher production of anti-hylan antibodies than Balb/c and C57BL/6 mice. CONCLUSION: Hylan G-F 20 exhibited immunogenicity in guinea pigs and mice. Recent reported severe acute inflammatory reactions in human patients after repeated intraarticular injections of hylan G-F 20 might involve product-specific, immune-mediated mechanisms.

The effects of intraarticularly injected sodium hyaluronate on levels of intact aggrecan and nitric oxide in the joint fluid of patients with knee osteoarthritis.

OBJECTIVE: Intraarticular injections of sodium hyaluronate (Na-HA) appear effective in reducing subjective symptoms of osteoarthritis (OA) and may also have protective effects on the cartilage matrix. The present study analyzed the suppressive effects of Na-HA on the release and degradation of aggrecan and on levels of nitric oxide (NO) in the joint fluid of patients with knee OA. DESIGN: Sixteen OA patients with knee joint effusion were treated by 5 weekly intraarticular injections of Na-HA. Prior to each Na-HA injection, joint fluid was collected to determine the levels of chondroitin 4-sulfate (C4S) and chondroitin 6-sulfate (C6S), intact aggrecan and NO. RESULTS: One week after the final injection, the joint fluid levels of C4S, C6S, and NO were significantly decreased. In contrast, the joint fluid level of intact aggrecan was stable during the series of Na-HA injections. A trend was seen for a positive correlation (P < 0.1) between the clinical score and C4S or C6S joint fluid levels, and for a negative correlation between the joint fluid levels of intact aggrecan and C4S or C6S. No significant correlations were observed between joint fluid levels of NO, the clinical score, and levels of C4S, C6S, and intact aggrecan. CONCLUSION: The results of this study suggest that intraarticularly injected Na-HA is able to improve the clinical symptoms of OA partially based on its ability to reduce the release and degradation of aggrecan and/or to enhance the synthesis of aggrecan in the joint tissues of the patients with knee OA. While Na-HA also reduces the NO level in the joint fluid of patients with knee OA, this effect may be independent from the other effects of Na-HA.