Effect of Chitosan Degradation Products, Glucosamine and Chitosan Oligosaccharide, on Osteoclastic Differentiation.

Chitosan, a natural cationic polysaccharide derived from crustaceans and shellfish shells, is known for its advantageous biological properties, including biodegradability, biocompatibility, and antibacterial activity. Chitosan and its composite materials are studied for their potential for bone tissue repair. However, the effects of chitosan degradation products, glucosamine (GlcN) and chitosan oligosaccharide (COS), on osteoclasts remain unclear. If these chitosan degradation products promote osteoclastic differentiation, careful consideration is required for the use of chitosan and related materials in bone repair applications. Here, we assessed the effects of high (500 µg/mL) and low (0.5 µg/mL) concentrations of GlcN and COS on osteoclastic differentiation in human peripheral blood mononuclear cells (PBMCs) and murine macrophage-like RAW264 cells. A tartrate-resistant acid phosphatase (TRAP) enzyme activity assay, TRAP staining, and actin staining were used to assess osteoclastic differentiation. High concentrations of GlcN and COS, but not low concentrations, suppressed macrophage colony-stimulating factor (M-CSF)- and RANKL-dependent increases in TRAP enzyme activity, TRAP-positive multinuclear osteoclast formation, and actin ring formation in PBMCs without cytotoxicity. Similar effects were observed in the RANKL-dependent osteoclastic differentiation of RAW264 cells. In conclusion, chitosan degradation products do not possess osteoclast-inducing properties, suggesting that chitosan and its composite materials can be safely used for bone tissue repair.

Reduced form of Galectin-1 Suppresses Osteoclastic Differentiation of Human Peripheral Blood Mononuclear Cells and Murine RAW264 Cells In Vitro.

Galectin-1 (Gal-1) is an evolutionarily conserved sugar-binding protein found in intra- and extracellular spaces. Extracellularly, it binds to glycoconjugates with ß-galactoside(s) and functions in various biological phenomena, including immunity, cancer, and differentiation. Under extracellular oxidative conditions, Gal-1 undergoes oxidative inactivation, losing its sugar-binding ability, although it exhibits sugar-independent functions. An age-related decrease in serum Gal-1 levels correlates with decreasing bone mass, and Gal-1 knockout promotes osteoclastic bone resorption and suppresses bone formation. However, the effect of extracellular Gal-1 on osteoclast differentiation remains unclear. Herein, we investigated the effects of extracellular Gal-1 on osteoclastogenesis in human peripheral blood mononuclear cells (PBMCs) and mouse macrophage RAW264 cells. Recombinant Gal-1 suppressed the macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand-dependent osteoclast formation, actin ring formation, and bone-resorption activity of human PBMCs. Similar results were obtained for RAW264 cells. Gal-1 knockdown increased osteoclast-like cell formation, suggesting that it affected differentiation in an autocrine-like manner. Oxidized Gal-1 slightly affected differentiation, and in the presence of lactose, the differentiation inhibitory effect of galectin-1 was not observed. These findings suggest that extracellular Gal-1 inhibits osteoclast differentiation in a ß-galactoside-dependent manner, and an age-related decrease in serum Gal-1 levels may contribute to reduced osteoclast activity and decreasing bone mass.

In vitro evaluation of the effect of galectins on Schistosoma mansoni motility.

OBJECTIVE: Galectins are sugar-binding proteins that participate in many biological processes, such as immunity, by regulating host immune cells and their direct interaction with pathogens. They are involved in mediating infection by Schistosoma mansoni, a parasitic trematode that causes schistosomiasis. However, their direct effects on schistosomes have not been investigated. RESULTS: We found that galectin-2 recognizes S. mansoni glycoconjugates and investigated whether galectin-1, 2, and 3 can directly affect S. mansoni in vitro. Adult S. mansoni were treated with recombinant galectin-1, 2, and 3 proteins or praziquantel, a positive control. Treatment with galectin-1, 2, and 3 had no significant effect on S. mansoni motility, and no other differences were observed under a stereoscopic microscope. Hence, galectin-1, 2, and 3 may have a little direct effect on S. mansoni. However, they might play a role in the infection in vivo via the modulation of the host immune response or secretory molecules from S. mansoni. To the best of our knowledge, this is the first study to investigate the direct effect of galectins on S. mansoni and helps in understanding the roles of galectins in S. mansoni infection in vivo.

Potential UV-Protective Effect of Freestanding Biodegradable Nanosheet-Based Sunscreen Preparations in XPA-Deficient Mice.

Xeroderma pigmentosum (XP) is a rare autosomal recessive hereditary disorder. As patients with XP are deficient in nucleotide excision repair, they show severe photosensitivity symptoms. Although skin protection from ultraviolet (UV) radiation is essential to improve the life expectancy of such patients, the optimal protective effect is not achieved even with sunscreen application, owing to the low usability of the preparations. Nanosheets are two-dimensional nanostructures with a thickness in the nanometer range. The extremely large aspect ratios of the nanosheets result in high transparency, flexibility, and adhesiveness. Moreover, their high moisture permeability enables their application to any area of the skin for a long time. We fabricated preparations containing avobenzone (BMDBM) based on freestanding poly (L-lactic acid) (PLLA) nanosheets through a spin-coating process. Although monolayered PLLA nanosheets did not contain enough BMDBM to protect against UV radiation, the layered nanosheets, consisting of five discrete BMDBM nanosheets, showed high UV absorbance without lowering the adhesive strength against skin. Inflammatory reactions in XPA-deficient mice after UV radiation were completely suppressed by the application of BMDBM-layered nanosheets to the skin. Thus, the BMDBM layered nanosheet could serve as a potential sunscreen preparation to improve the quality of life of patients with XP.

Phosphorylated Osteopontin Secreted from Cancer Cells Induces Cancer Cell Motility.

Osteopontin (OPN) plays a pivotal role in cancer cell invasion and metastasis. Although OPN has a large number of phosphorylation sites, the functional significance of OPN phosphorylation in cancer cell motility remains unclear. In this study, we attempted to investigate whether phosphorylated OPN secreted from cancer cells affect cancer cell migration. Quantitative PCR and Western blot analyses revealed that MDA-MB435S, A549, and H460 cells highly expressed OPN, whereas the OPN expression levels in H358, MIAPaca-2, and Panc-1 cells were quite low or were not detected. Compared with the cancer cell lines with a low OPN expression, the high OPN-expressing cancer cell lines displayed a higher cell migration, and the cell migration was suppressed by the anti-OPN antibody. This was confirmed by the OPN overexpression in H358 cancer cells with a low endogenous OPN. Phos-tag ELISA showed that phosphorylated OPN was abundant in the cell culture media of A549 and H460 cells, but not in those of MDA-MB435S cells. Moreover, the A549 and H460 cell culture media, as well as the MDA-MB435S cell culture media with a kinase treatment increased cancer cell motility, both of which were abrogated by phosphatase treatment or anti-OPN antibodies. These results suggest that phosphorylated OPN secreted from cancer cells regulates cancer cell motility.

αvß3 Integrin induces partial EMT independent of TGF-ß signaling.

Epithelial-mesenchymal transition (EMT) plays a pivotal role for tumor progression. Recent studies have revealed the existence of distinct intermediate states in EMT (partial EMT); however, the mechanisms underlying partial EMT are not fully understood. Here, we demonstrate that αvß3 integrin induces partial EMT, which is characterized by acquiring mesenchymal phenotypes while retaining epithelial markers. We found αvß3 integrin to be associated with poor survival in patients with lung adenocarcinoma. Moreover, αvß3 integrin-induced partial EMT promoted migration, invasion, tumorigenesis, stemness, and metastasis of lung cancer cells in a TGF-ß-independent fashion. Additionally, TGF-ß1 promoted EMT progression synergistically with αvß3 integrin, while a TGF-ß signaling inhibitor showed no effect on αvß3 integrin-induced partial EMT. Meanwhile, the microRNA-200 family abolished the αvß3 integrin-induced partial EMT by suppressing αvß3 integrin cell surface expression. These findings indicate that αvß3 integrin is a key inducer of partial EMT, and highlight a new mechanism for cancer progression.

Osteoclast Differentiation Is Suppressed by Increased O-GlcNAcylation Due to Thiamet G Treatment.

Osteoclasts are the only bone-resorbing cells in organisms and understanding their differentiation mechanism is crucial for the treatment of osteoporosis. In the present study, we investigated the effect of Thiamet G, an O-GlcNAcase specific inhibitor, on osteoclastogenic differentiation. Thiamet G treatment increased global O-GlcNAcylation in murine RAW264 cells and suppressed receptor activator of nuclear factor-κB ligand (RANKL)-dependent formation in tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells, thereby suppressing the upregulation of osteoclast specific genes. Meanwhile, knockdown of O-linked N-acetylglucosamine (O-GlcNAc) transferase promoted the formation TRAP-positive multinuclear cells. Thiamet G treatment also suppressed RANKL and macrophage colony-stimulating factor (M-CSF) dependent osteoclast formation and bone-resorbing activity in mouse primary bone marrow cells and human peripheral blood mononuclear cells. These results indicate that the promotion of O-GlcNAc modification specifically suppresses osteoclast formation and its activity and suggest that chemicals affecting O-GlcNAc modification might potentially be useful in the prevention or treatment of osteoporosis in future.

Quantitative analysis of ß1,6GlcNAc-branched N-glycans on ß4 integrin in cutaneous squamous cell carcinoma.

α6ß4 integrin plays pivotal roles in cancer progression in several types of cancers. Our previous study using N-glycan-manipulated cell lines demonstrated that defects in N-glycans or decreased ß1,6GlcNAc-branched N-glycans on ß4 integrin suppress ß4 integrin-mediated cancer cell adhesion, migration, invasion, and tumorigenesis. Furthermore, immunohistochemical analysis has shown that colocalization of ß1,6GlcNAc-branched N-glycans with ß4 integrin was observed in cutaneous squamous cell carcinoma (SCC) tissue. However, until now there has been no direct evidence that ß1,6GlcNAc-branched N-glycans are upregulated on ß4 integrin in cutaneous SCC. In the present study, we performed an ELISA analysis of ß1,6GlcNAc-branched N-glycans on ß4 integrins as well as ß4 integrins in cell lysates from human normal skin and cutaneous SCC tissues. The SCC samples showed a 4.9- to 7.4-fold increase in the ratio of ß1,6GlcNAc-branched N-glycans to ß4 integrin compared with normal skin samples. These findings suggest that the addition of ß1,6GlcNAc-branched N-glycans onto ß4 integrin was markedly elevated in cutaneous SCC tissue compared to normal skin tissue. The value of ß1,6GlcNAc-branched N-glycans on ß4 integrin may be useful as a diagnostic marker associated with cutaneous SCC tumor progression.

Galectin-2 Has Bactericidal Effects against Helicobacter pylori in a ß-galactoside-Dependent Manner.

Helicobacter pylori is associated with the onset of gastritis, peptic ulcers, and gastric cancer. Galectins are a family of ß-galactoside-binding proteins involved in diverse biological phenomena. Galectin-2 (Gal-2), a member of the galectin family, is predominantly expressed in the gastrointestinal tract. Although some galectin family proteins are involved in immunoreaction, the role of Gal-2 against H. pylori infection remains unclear. In this study, the effects of Gal-2 on H. pylori morphology and survival were examined. Gal-2 induced H. pylori aggregation depending on ß-galactoside and demonstrated a bactericidal effect. Immunohistochemical staining of the gastric tissue indicated that Gal-2 existed in the gastric mucus, as well as mucosa. These results suggested that Gal-2 plays a role in innate immunity against H. pylori infection in gastric mucus.

TAFRO Syndrome Presenting with Retroperitoneal Panniculitis-like Computed Tomography Findings at Disease Onset.

TAFRO syndrome is rare, and its pathophysiology remains unclear. We herein report the case of a 66-year-old man who presented at our emergency department with epigastric pain. Contrast-enhanced computed tomography (CT) showed high-density retroperitoneal panniculus with contrast enhancement. He was treated initially with a protease inhibitor and hydration, to little effect. Anasarca, thrombocytopenia, and renal dysfunction developed gradually, and TAFRO syndrome was diagnosed. He was treated successfully with prednisolone and cyclophosphamide, and subsequent CT findings showed improvement. Abnormal CT findings of the retroperitoneum may indicate the early stages of TAFRO syndrome before the presentation of typical ascites.

The Association between Glomerular Filtration Rate Estimated Using Different Equations and Mortality in the Japanese Community-Based Population: The Yamagata (Takahata) Study.

BACKGROUND: To evaluate renal function, the indices of estimated glomerular filtration rate (eGFR) obtained using several equations, including the Japanese versions of the serum creatinine-based MDRD equation (eGFRcreat), Chronic Kidney Disease Epidemiology Collaboration equation (eGFR-EPI), and serum cystatin C-based equation (eGFRcys), are utilized. This study prospectively examined the association between these eGFR values and all-cause mortality during a 12-year observational period in a community-based population. METHODS AND RESULTS: The subjects of this study were 1312 participants undergoing a health checkup, aged ≥40 years. In the total population, the mean eGFR values (mL·min-1·1.73 m-2) were 81.5 for eGFRcreat, 78.1 for eGFR-EPI, and 76.6 for eGFRcys. There were 141 deaths during the observation period, and the area under the receiver operating characteristic curve for predicting mortality was 0.59 for eGFRcreat, 0.67 for eGFR-EPI, and 0.70 for eGFRcys (all P < 0.01). In the Cox proportional analysis adjusted for age and sex, eGFRcys, but not eGFRcreat and eGFR-EPI, showed a significant association with all-cause mortality (per 15 mL·min-1·1.73 m-2 decrease: hazard ratio 1.40, 95% confidence interval 1.18-1.67). CONCLUSIONS: This study revealed that eGFRcys showed lower values than eGFRcreat and eGFR-EPI and was significantly associated with all-cause mortality in the Japanese community-based population.

Biological role of site-specific O-glycosylation in cell adhesion activity and phosphorylation of osteopontin.

Osteopontin (OPN) is an extracellular glycosylated phosphoprotein that promotes cell adhesion by interacting with several integrin receptors. We previously reported that an OPN mutant lacking five O-glycosylation sites (Thr134/Thr138/Thr143/Thr147/Thr152) in the threonine/proline-rich region increased cell adhesion activity and phosphorylation compared with the wild type. However, the role of O-glycosylation in cell adhesion activity and phosphorylation of OPN remains to be clarified. Here, we show that site-specific O-glycosylation in the threonine/proline-rich region of OPN affects its cell adhesion activity and phosphorylation independently and/or synergistically. Using site-directed mutagenesis, we found that OPN mutants with substitution sets of Thr134/Thr138 or Thr143/Thr147/Thr152 had decreased and increased cell adhesion activity, respectively. In contrast, the introduction of a single mutation into the O-glycosylation sites had no effect on OPN cell adhesion activity. An adhesion assay using function-blocking antibodies against αvß3 and ß1 integrins, as well as αvß3 integrin-overexpressing A549 cells, revealed that site-specific O-glycosylation affected the association of OPN with the two integrins. Phosphorylation analyses using phos-tag and LC-MS/MS indicated that phosphorylation levels and sites were influenced by the O-glycosylation status, although the number of O-glycosylation sites was not correlated with the phosphorylation level in OPN. Furthermore, a correlation analysis between phosphorylation level and cell adhesion activity in OPN mutants with the site-specific O-glycosylation showed that they were not always correlated. These results provide conclusive evidence of a novel regulatory mechanism of cell adhesion activity and phosphorylation of OPN by site-specific O-glycosylation.

ß4-Integrin/PI3K Signaling Promotes Tumor Progression through the Galectin-3-N-Glycan Complex.

Malignant transformation is associated with aberrant N-glycosylation, but the role of protein N-glycosylation in cancer progression remains poorly defined. ß4-integrin is a major carrier of N-glycans and is associated with poor prognosis, tumorigenesis, and metastasis. Here, N-glycosylation of ß4-integrin contributes to the activation of signaling pathways that promote ß4-dependent tumor development and progression. Increased expression of ß1,6GlcNAc-branched N-glycans was found to be colocalized with ß4-integrin in human cutaneous squamous cell carcinoma tissues, and that the ß1,6GlcNAc residue was abundant on ß4-integrin in transformed keratinocytes. Interruption of ß1,6GlcNAc-branching formation on ß4-integrin with the introduction of bisecting GlcNAc by N-acetylglucosaminyltransferase III overexpression was correlated with suppression of cancer cell migration and tumorigenesis. N-Glycan deletion on ß4-integrin impaired ß4-dependent cancer cell migration, invasion, and growth in vitro and diminished tumorigenesis and proliferation in vivo The reduced abilities of ß4-integrin were accompanied with decreased phosphoinositol-3 kinase (PI3K)/Akt signals and were restored by the overexpression of the constitutively active p110 PI3K subunit. Binding of galectin-3 to ß4-integrin via ß1,6GlcNAc-branched N-glycans promoted ß4-integrin-mediated cancer cell adhesion and migration. In contrast, a neutralizing antibody against galectin-3 attenuated ß4-integrin N-glycan-mediated PI3K activation and inhibited the ability of ß4-integrin to promote cell motility. Furthermore, galectin-3 knockdown by shRNA suppressed ß4-integrin N-glycan-mediated tumorigenesis. These findings provide a novel role for N-glycosylation of ß4-integrin in tumor development and progression, and the regulatory mechanism for ß4-integrin/PI3K signaling via the galectin-3-N-glycan complex.Implications:N-Glycosylation of ß4-integrin plays a functional role in promoting tumor development and progression through PI3K activation via the galectin-3-N-glycan complex. Mol Cancer Res; 16(6); 1024-34. ©2018 AACR.