Regulation of Glycosylation in Bone Metabolism.

Glycosylation plays a crucial role in the maintenance of homeostasis in the body and at the onset of diseases such as inflammation, neurodegeneration, infection, diabetes, and cancer. It is also involved in bone metabolism. N- and O-glycans have been shown to regulate osteoblast and osteoclast differentiation. We recently demonstrated that ganglio-series and globo-series glycosphingolipids were essential for regulating the proliferation and differentiation of osteoblasts and osteoclasts in glycosyltransferase-knockout mice. Herein, we reviewed the importance of the regulation of bone metabolism by glycoconjugates, such as glycolipids and glycoproteins, including our recent results.

Signaling domains of cancer-associated glycolipids.

Immunotherapy of malignant cancers is now becoming one of representative approaches to overcome cancers. To construct strategies for immunotherapy, presence of tumor-specific antigens should be a major promise. A number of cancer specific- or cancer-associated antigens have been reported based on various experimental sets and various animal systems. The most reasonable strategy to define tumor-specific antigens might be "autologous typing" performed by Old's group, proposing three classes of tumor-antigens recognized by host immune systems of cancer patients. Namely, class 1, individual antigens that is present only in the patient's sample analyzed; class 2, shared antigens that can be found only in some group of cancers in some patients, but not in normal cells and tissues; class 3, universal antigens that are present in some cancers but also in normal cells and tissues with different densities. Sen Hakomori reported there were novel carbohydrates in cancers that could not be detected in normal cells mainly by biochemical approaches. Consequently, many of class 2 cancer-specific antigens have been revealed to be carbohydrate antigens, and been used for cancer diagnosis and treatment. Not only as cancer markers, but roles of those cancer-associated carbohydrates have also been recognized as functional molecules in cancer cells. In particular, roles of complex carbohydrates in the regulation of cell signaling on the cell surface microdomains, glycolipid-enriched microdomain (GEM)/rafts have been reported by Hakomori and many other researchers including us. The processes and present status of these studies on cancer-associated glycolipids were summarized.

Attenuation of Bone Formation through a Decrease in Osteoblasts in Mutant Mice Lacking the GM2/GD2 Synthase Gene.

The ganglioside GD1a has been reported to promote the differentiation of mesenchymal stem cells to osteoblasts in cell culture systems. However, the involvement of gangliosides, including GD1a, in bone formation in vivo remains unknown; therefore, we herein investigated their roles in GM2/GD2 synthase-knockout (GM2/GD2S KO) mice without GD1a. The femoral cancellous bone mass was analyzed using three-dimensional micro-computed tomography. A histomorphometric analysis of bone using hematoxylin and eosin (HE) and tartrate-resistant acid phosphatase was performed to examine bone formation and resorption, respectively. Calcein double labeling was also conducted to evaluate bone formation. Although no significant differences were observed in bone mass or resorption between GM2/GD2S KO mice and wild-type (WT) mice, analyses of the parameters of bone formation using HE staining and calcein double labeling revealed less bone formation in GM2/GD2S KO mice than in WT mice. These results suggest that gangliosides play roles in bone formation.

Orthodontic tooth movement-activated sensory neurons contribute to enhancing osteoclast activity and tooth movement through sympathetic nervous signalling.

OBJECTIVES: Orthodontic tooth movement (OTM) increases sympathetic and sensory neurological markers in periodontal tissue. However, the relationship between the sympathetic and sensory nervous systems during OTM remains unclear. Therefore, the present study investigated the relationship between the sympathetic and sensory nervous systems activated by OTM using pharmacological methods. MATERIALS AND METHODS: We compared the effects of sympathectomy and sensory nerve injury during OTM in C57BL6/J mice. Capsaicin (CAP) was used to induce sensory nerve injury. Sympathectomy was performed using 6-hydroxydopamine. To investigate the effects of a ß-agonist on sensory nerve injury, isoproterenol (ISO) was administered to CAP-treated mice. Furthermore, to examine the role of the central nervous system in OTM, the ventromedial hypothalamic nucleus (VMH) was ablated using gold thioglucose. RESULTS: Sensory nerve injury and sympathectomy both suppressed OTM and decreased the percent of the alveolar socket covered with osteoclasts (Oc.S/AS) in periodontal tissue. Sensory nerve injury inhibited increases in OTM-induced calcitonin gene-related peptide (CGRP) immunoreactivity (IR), a marker of sensory neurons, and tyrosine hydroxylase (TH) IR, a marker of sympathetic neurons, in periodontal tissue. Although sympathectomy did not decrease the number of CGRP-IR neurons in periodontal tissue, OTM-induced increases in the number of TH-IR neurons were suppressed. The ISO treatment restored sensory nerve injury-inhibited tooth movement and Oc.S/AS. Furthermore, the ablation of VMH, the centre of the sympathetic nervous system, suppressed OTM-induced increases in tooth movement and Oc.S/AS. CONCLUSIONS: The present results suggest that OTM-activated sensory neurons contribute to enhancements in osteoclast activity and tooth movement through sympathetic nervous signalling.

Guanabenz inhibits alveolar bone resorption in a rat model of periodontitis.

Increase of sympathetic activity has been known to exacerbate osteoporosis through promotion of bone resorption. However, it is largely unknown about involvement of sympathetic activity in exacerbation of periodontitis. In this study, we investigated whether α2-adrenergic receptor (α2-AR) agonist guanabenz which decreases sympathetic activity, attenuates alveolar bone resorption in rats having high sympathetic activity with periodontitis. Volumes of residual alveolar bone and attachment levels in periodontium were examined using micro-computed tomography and hematoxylin-eosin staining, respectively. Furthermore, osteoclast numbers per bone surface and osteoclast surface per bone surface were measured using tartrate-resistant acid phosphatase staining. To examine the suppressive effects of guanabenz on pro-inflammatory cytokines, expression levels of tyrosine hydroxylase (TH), TNF-α, IL1-ß, and IL-6 in periodontium were measured using immunohistostaining. Administration of guanabenz attenuated loss of alveolar bone and attachment levels in rats having high sympathetic activity. Furthermore, its administration suppressed osteoclast numbers in rats having high sympathetic activity. TH, TNF-α, IL-1ß, and IL-6 positive cells in periodontium in rats treated with guanabenz for 12 weeks, were lower than those in control rats having high sympathetic activity. This study demonstrated administration of α2-AR agonist guanabenz attenuates alveolar bone resorption through decrease of sympathetic activity in rats.

Ganglioside GD2 Enhances the Malignant Phenotypes of Melanoma Cells by Cooperating with Integrins.

Gangliosides have been considered to modulate cell signals in the microdomain of the cell membrane, lipid/rafts, or glycolipid-enriched microdomain/rafts (GEM/rafts). In particular, cancer-associated gangliosides were reported to enhance the malignant properties of cancer cells. In fact, GD2-positive (GD2+) cells showed increased proliferation, invasion, and adhesion, compared with GD2-negative (GD2-) cells. However, the precise mechanisms by which gangliosides regulate cell signaling in GEM/rafts are not well understood. In order to analyze the roles of ganglioside GD2 in the malignant properties of melanoma cells, we searched for GD2-associating molecules on the cell membrane using the enzyme-mediated activation of radical sources combined with mass spectrometry, and integrin ß1 was identified as a representative GD2-associating molecule. Then, we showed the physical association of GD2 and integrin ß1 by immunoprecipitation/immunoblotting. Close localization was also shown by immuno-cytostaining and the proximity ligation assay. During cell adhesion, GD2+ cells showed multiple phospho-tyrosine bands, i.e., the epithelial growth factor receptor and focal adhesion kinase. The knockdown of integrin ß1 revealed that the increased malignant phenotypes in GD2+ cells were clearly cancelled. Furthermore, the phosphor-tyrosine bands detected during the adhesion of GD2+ cells almost completely disappeared after the knockdown of integrin ß1. Finally, immunoblotting to examine the intracellular distribution of integrins during cell adhesion revealed that large amounts of integrin ß1 were localized in GEM/raft fractions in GD2+ cells before and just after cell adhesion, with the majority being localized in the non-raft fractions in GD2- cells. All these results suggest that GD2 and integrin ß1 cooperate in GEM/rafts, leading to enhanced malignant phenotypes of melanomas.

New era of research on cancer-associated glycosphingolipids.

Cancer-associated glycosphingolipids have been used as markers for diagnosis and targets for immunotherapy of malignant tumors. Recent progress in the analysis of their implications in the malignant properties of cancer cells revealed that cancer-associated glycosphingolipids are not only tumor markers, but also functional molecules regulating various signals introduced by membrane microdomains, lipid rafts. In particular, a novel approach, enzyme-mediated activation of radical sources combined with mass spectrometry, has enabled us to clarify the mechanisms by which cancer-associated glycosphingolipids regulate cell signals based on the interaction with membrane molecules and formation of molecular complexes on the cell surface. Novel findings obtained from these approaches are now providing us with insights into the development of new anticancer therapies targeting membrane molecular complexes consisting of cancer-associated glycolipids and their associated membrane molecules. Thus, a new era of cancer-associated glycosphingolipids has now begun.

Deficiency of GD3 Synthase in Mice Resulting in the Attenuation of Bone Loss with Aging.

Gangliosides are widely expressed in almost all tissues and cells and are also considered to be essential in the development and maintenance of various organs and tissues. However, little is known about their roles in bone metabolism. In this study, we investigated the effects of genetic deletion of ganglioside D3 (GD3) synthase, which is responsible for the generation of all b-series gangliosides, on bone metabolism. Although b-series gangliosides were not expressed in osteoblasts, these gangliosides were expressed in pre-osteoclasts. However, the expression of these gangliosides was decreased after induction of osteoclastogenesis by receptor activator of nuclear factor kappa-B ligand (RANKL). Three-dimensional micro-computed tomography (3D-µCT) analysis revealed that femoral cancellous bone mass in GD3 synthase-knockout (GD3S KO) mice was higher than that in wild type (WT) mice at the age of 40 weeks, although there were no differences in that between GD3S KO and WT mice at 15 weeks old. Whereas bone formation parameters (osteoblast numbers/bone surface and osteoblast surface/bone surface) in GD3S KO mice did not differ from WT mice, bone resorption parameters (osteoclast numbers/bone surface and osteoclast surface/bone surface) in GD3S KO mice became significantly lower than those in WT mice at 40 weeks of age. Collectively, this study demonstrates that deletion of GD3 synthase attenuates bone loss that emerges with aging.

Deletion of Gb3 Synthase in Mice Resulted in the Attenuation of Bone Formation via Decrease in Osteoblasts.

Glycosphingolipids are known to play a role in developing and maintaining the integrity of various organs and tissues. Among glycosphingolipids, there are several reports on the involvement of gangliosides in bone metabolism. However, there have been no reports on the presence or absence of expression of globo-series glycosphingolipids in osteoblasts and osteoclasts, and the involvement of their glycosphingolipids in bone metabolism. In the present study, we investigated the presence or absence of globo-series glycosphingolipids such as Gb3 (globotriaosylceramide), Gb4 (globoside), and Gb5 (galactosyl globoside) in osteoblasts and osteoclasts, and the effects of genetic deletion of Gb3 synthase, which initiates the synthesis of globo-series glycosphingolipids on bone metabolism. Among Gb3, Gb4, and Gb5, only Gb4 was expressed in osteoblasts. However, these glycosphingolipids were not expressed in pre-osteoclasts and osteoclasts. Three-dimensional micro-computed tomography (3D-µCT) analysis revealed that femoral cancellous bone mass in Gb3 synthase-knockout (Gb3S KO) mice was lower than that in wild type (WT) mice. Calcein double labeling also revealed that bone formation in Gb3S KO mice was significantly lower than that in WT mice. Consistent with these results, the deficiency of Gb3 synthase in mice decreased the number of osteoblasts on the bone surface, and suppressed mRNA levels of osteogenic differentiation markers. On the other hand, osteoclast numbers on the bone surface and mRNA levels of osteoclast differentiation markers in Gb3S KO mice did not differ from WT mice. This study demonstrated that deletion of Gb3 synthase in mice decreases bone mass via attenuation of bone formation.

A therapeutic trial of human melanomas with combined small interfering RNAs targeting adaptor molecules p130Cas and paxillin activated under expression of ganglioside GD3.

We previously demonstrated that focal adhesion kinase (FAK), p130Cas and paxillin are crucially involved in the enhanced malignant properties under expression of ganglioside GD3 in melanoma cells. Therefore, molecules existing in the GD3-mediated signaling pathway could be considered as suitable targets for therapeutic intervention in malignant melanoma. The aim of this study was to determine whether blockade of p130Cas and/or paxillin by RNAi suppresses melanoma growth. We found a suitable dose (40 µM siRNA, 25 µl/tumor) of the siRNA to suppress p130Cas in the xenografts generated in nu/nu mice. Based on these results, we performed intratumoral (i.t.) treatment with anti-p130Cas and/or anti-paxillin siRNAs mixed with atelocollagen as a drug delivery system in a xenograft tumor of a human melanoma cell line, SK-MEL-28. Mixture of atelocollagen (1.75%) and an siRNA (500 or 1000 pmol/tumor) was injected into the tumors every 3 days after the first injection. An siRNA against human p130Cas markedly suppressed tumor growth of the xenograft in a dose-dependent manner, whereas siRNA against human paxillin slightly inhibited the tumor growth. A control siRNA against firefly luciferase showed no effect. To our surprise, siRNA against human p130Cas (500 or 1000 pmol/tumor) combined with siRNA against human paxillin dramatically suppressed tumor growth. In agreement with the tumor suppression effects of the anti-p130Cas siRNA, reduction in Ki-67 positive cell number as well as in p130Cas expression was demonstrated by immunohistostaining. These results suggested that blockade of GD3-mediated growth signaling pathways by siRNAs might be a novel and promising therapeutic strategy against malignant melanomas, provided signaling molecules such as p130Cas and paxillin are significantly expressed in individual cases. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.

Salubrinal improves mechanical properties of the femur in osteogenesis imperfecta mice.

Salubrinal is an agent that reduces the stress to the endoplasmic reticulum by inhibiting de-phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α). We and others have previously shown that the elevated phosphorylation of eIF2α stimulates bone formation and attenuates bone resorption. In this study, we applied salubrinal to a mouse model of osteogenesis imperfecta (Oim), and examined whether it would improve Oim's mechanical property. We conducted in vitro experiments using RAW264.7 pre-osteoclasts and bone marrow derived cells (BMDCs), and performed in vivo administration of salubrinal to Oim (+/-) mice. The animal study included two control groups (wildtype and Oim placebo). The result revealed that salubrinal decreased expression of nuclear factor of activated T cells cytoplasmic 1 (NFATc1) and suppressed osteoclast maturation, and it stimulated mineralization of mesenchymal stem cells from BMDCs. Furthermore, daily injection of salubrinal at 2 mg/kg for 2 months made stiffness (N/mm) and elastic module (GPa) of the femur undistinguishable to those of the wildtype control. Collectively, this study supported salubrinal's beneficial role to Oim's femora. Unlike bisphosphonates, salubrinal stimulates bone formation. For juvenile OI patients who may favor strengthening bone without inactivating bone remodeling, salubrinal may present a novel therapeutic option.

Guanabenz Downregulates Inflammatory Responses via eIF2α Dependent and Independent Signaling.

Integrated stress responses (ISR) may lead to cell death and tissue degeneration via eukaryotic translation initiation factor 2 α (eIF2α)-mediated signaling. Alleviating ISR by modulating eIF2α phosphorylation can reduce the symptoms associated with various diseases. Guanabenz is known to elevate the phosphorylation level of eIF2α and reduce pro-inflammatory responses. However, the mechanism of its action is not well understood. In this study, we investigated the signaling pathway through which guanabenz induces anti-inflammatory effects in immune cells, in particular macrophages. Genome-wide mRNA profiling followed by principal component analysis predicted that colony stimulating factor 2 (Csf2, or GM-CSF as granulocyte macrophage colony stimulating factor) is involved in the responses to guanabenz. A partial silencing of Csf2 or eIF2α by RNA interference revealed that Interleukin-6 (IL6), Csf2, and Cyclooxygenase-2 (Cox2) are downregulated by guanabenz-driven phosphorylation of eIF2α. Although expression of IL1ß and Tumor Necrosis Factor-α (TNFα) was suppressed by guanabenz, their downregulation was not directly mediated by eIF2α signaling. Collectively, the result herein indicates that anti-inflammatory effects by guanabenz are mediated by not only eIF2α-dependent but also eIF2α-independent signaling.

Role of miR-222-3p in c-Src-Mediated Regulation of Osteoclastogenesis.

MicroRNAs (miRNAs) are small non-coding RNAs that play a mostly post-transcriptional regulatory role in gene expression. Using RAW264.7 pre-osteoclast cells and genome-wide expression analysis, we identified a set of miRNAs that are involved in osteoclastogenesis. Based on in silico analysis, we specifically focused on miR-222-3p and evaluated its role in osteoclastogenesis. The results show that the inhibitor of miR-222-3p upregulated the mRNA levels of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) and tartrate-resistant acid phosphatase (TRAP), while its mimicking agent downregulated their mRNA levels. Western blot analysis showed that its inhibitor increased the protein levels of TRAP and cathepsin K, while its mimicking agent decreased their levels. Genome-wide mRNA expression analysis in the presence and absence of receptor activator of nuclear factor κ-B ligand (RANKL) predicted c-Src as a potential regulatory target of miR-222-3p. Live cell imaging using a fluorescence resonance energy transfer (FRET) technique revealed that miR-222-3p acted as an inhibitor of c-Src activity, and a partial silencing of c-Src suppressed RANKL-induced expression of TRAP and cathepsin K, as well as the number of multi-nucleated osteoclasts and their pit formation. Collectively, the study herein demonstrates that miR-222-3p serves as an inhibitor of osteoclastogenesis and c-Src mediates its inhibition of cathepsin K and TRAP.

Trimeric Tn antigen on syndecan 1 produced by ppGalNAc-T13 enhances cancer metastasis via a complex formation with integrin α5ß1 and matrix metalloproteinase 9.

We demonstrated previously that ppGalNAc-T13 (T13), identified as an up-regulated gene with increased metastasis in a DNA microarray, generated trimeric Tn (tTn) antigen (GalNAcα1-Ser/Thr)3 on Syndecan 1 in highly metastatic sublines of Lewis lung cancer. However, it is not known how tTn antigen regulates cancer metastasis. Here, we analyzed the roles of tTn antigen in cancer properties. tTn antigen on Syndecan 1 increased cell adhesion to fibronectin in an integrin-dependent manner. Furthermore, cell adhesion to fibronectin induced phosphorylation of focal adhesion kinase and paxillin in T13-transfectant cells. In the search of Syndecan 1-interacting molecules, it was demonstrated that tTn antigen-carrying Syndecan 1 interacted with integrin α5ß1 and matrix metalloproteinase 9 and that these molecules shifted to a glycolipid-enriched microdomain/rafts along with increased metastatic potential in T13-transfectant cells. We also identified a tTn substitution site on Syndecan 1, demonstrating that tTn on Syndecan 1 is essential for the interaction with integrin α5ß1 as well as for the reaction with mAb MLS128. These data suggest that high expression of the ppGalNAc-T13 gene generates tTn antigen on Syndecan 1 under reduced expression of GM1, leading to enhanced invasion and metastasis via the formation of a molecular complex consisting of integrin α5ß1, Syndecan 1, and MMP-9 in the glycolipid-enriched microdomain/rafts.

Globo-series Gb4 activates ERK and promotes the proliferation of osteoblasts.

OBJECTIVES: Globo-series Gb4 (globoside) is involved in the immune system and disease pathogenesis. We recently reported that systemic Gb4 deficiency in mice led to decreased bone formation due to a reduction in osteoblast number. However, it remains unclear whether Gb4 expressed in osteoblasts promotes their proliferation. Therefore, we investigated the role of Gb4 in osteoblast proliferation in vitro. METHODS: We examined osteoblast proliferation in Gb3 synthase knockout mice lacking Gb4. We investigated the effects of Gb4 synthase knockdown in the mouse osteoblast cell line MC3T3-E1 on its proliferation. Furthermore, we administered Gb4 to MC3T3-E1 cells in which Gb4 was suppressed by a glucosylceramide synthase (GCS) inhibitor and evaluated its effects on their proliferation. To elucidate the mechanisms by which Gb4 promotes osteoblast proliferation, the phosphorylated extracellular signal-regulated kinases 1 and 2 (ERK1/2) levels were measured in MC3T3-E1 cells. RESULTS: Osteoblast proliferation was lower in Gb3 synthase knockout mice lacking Gb4 than in wild-type mice. Proliferation was inhibited by Gb4 synthase knockdown in MC3T3-E1 cells. Furthermore, the administration of Gb4 to MC3T3-E1 cells, in which a GCS inhibitor suppressed Gb4, promoted their proliferation. Moreover, it increased the phosphorylated ERK1/2 levels in MC3T3-E1 cells. CONCLUSIONS: Our results suggest that Gb4 expressed in osteoblasts promotes their proliferation through ERK1/2 activation.

Lewis y antigen is expressed in oral squamous cell carcinoma cell lines and tissues, but disappears in the invasive regions leading to the enhanced malignant properties irrespective of sialyl-Lewis x.

Expression and implication of carbohydrate antigens in squamous cell carcinomas (SCCs) in oral cavity was examined. In the cell lines, type 2H and Lewis y antigens were markedly expressed. In the tissues from SCC patients and benign disorders, type 2H was highly expressed in hyperplasia (96.4 %), displasia (92.9 %) and SCC (100 %). Lewis y was, in turn, expressed mainly in cancer tissues (91.3 %), suggesting that Lewis y is a cancer-associated antigen. Normal oral mucosa showed no expression of these blood group antigens. Surprisingly, Lewis y antigen disappeared in the invasion sites where Ki-67 was definitely stained. Over-expression of Lewis y with manipulation of a fucosyltransferase cDNA resulted in suppression of cell growth and invasion, and knockdown of Lewis y also brought about increased cell growth and invasion. In either situations, no changes in the expression of sialyl-Lewis x could be found. Lowered tumor growth and invasion into surrounding tissues were also shown in Lewis y-positive SCC grafts in nu/nu mice. All these results together with alternative staining between Lewis y and Ki-67 in cancer tissues and FUT1 transfectants suggested that loss of Lewis y is a crucial event for the late stage of SCCs.

Suppression of osteoclastogenesis through phosphorylation of eukaryotic translation initiation factor 2 alpha.

In response to various stresses including viral infection, nutrient deprivation, and stress to the endoplasmic reticulum, eukaryotic translation initiation factor 2 alpha (eIF2α) is phosphorylated to cope with stress induced apoptosis. Although bone cells are sensitive to environmental stresses that alter the phosphorylation level of eIF2α, little is known about the role of eIF2α mediated signaling during the development of bone-resorbing osteoclasts. Using two chemical agents (salubrinal and guanabenz) that selectively inhibit de-phosphorylation of eIF2α, we evaluated the effects of phosphorylation of eIF2α on osteoclastogenesis of RAW264.7 pre-osteoclasts as well as development of MC3T3 E1 osteoblast-like cells. The result showed that salubrinal and guanabenz stimulated matrix deposition of osteoblasts through upregulation of activating transcription factor 4 (ATF4). The result also revealed that these agents reduced expression of the nuclear factor of activated T cells c1 (NFATc1) and inhibited differentiation of RAW264.7 cells to multi-nucleated osteoclasts. Partial silencing of eIF2α with RNA interference reduced suppression of salubrinal/guanabenz-driven downregulation of NFATc1. Collectively, we demonstrated that the elevated phosphorylation level of eIF2α not only stimulates osteoblastogenesis but also inhibit osteoclastogenesis through regulation of ATF4 and NFATc1. The results suggest that eIF2α-mediated signaling might provide a novel therapeutic target for preventing bone loss in osteoporosis.

Effects of salubrinal on development of osteoclasts and osteoblasts from bone marrow-derived cells.

BACKGROUND: Osteoporosis is a skeletal disease leading to an increased risk of bone fracture. Using a mouse osteoporosis model induced by administration of a receptor activator of nuclear factor kappa-B ligand (RANKL), salubrinal was recently reported as a potential therapeutic agent. To evaluate the role of salubrinal in cellular fates as well as migratory and adhesive functions of osteoclast/osteoblast precursors, we examined the development of primary bone marrow-derived cells in the presence and absence of salubrinal. We addressed a question: are salubrinal's actions more potent to the cells isolated from the osteoporotic mice than those isolated from the control mice? METHODS: Using the RANKL-injected and control mice, bone marrow-derived cells were harvested. Osteoclastogenesis was induced by macrophage-colony stimulating factor and RANKL, while osteoblastogenesis was driven by dexamethasone, ascorbic acid, and ß-glycerophosphate. RESULTS: The results revealed that salubrinal suppressed the numbers of colony forming-unit (CFU)-granulocyte/macrophages and CFU-macrophages, as well as formation of mature osteoclasts in a dosage-dependent manner. Salubrinal also suppressed migration and adhesion of pre-osteoclasts and increased the number of CFU-osteoblasts. Salubrinal was more effective in exerting its effects in the cells isolated from the RANKL-injected mice than the control. Consistent with cellular fates and functions, salubrinal reduced the expression of nuclear factor of activated T cells c1 (NFATc1) as well as tartrate-resistant acid phosphatase. CONCLUSIONS: The results support the notion that salubrinal exhibits significant inhibition of osteoclastogenesis as well as stimulation of osteoblastogenesis in bone marrow-derived cells, and its efficacy is enhanced in the cells harvested from the osteoporotic bone samples.

Knee loading reduces MMP13 activity in the mouse cartilage.

BACKGROUND: Moderate loads with knee loading enhance bone formation, but its effects on the maintenance of the knee are not well understood. In this study, we examined the effects of knee loading on the activity of matrix metalloproteinase13 (MMP13) and evaluated the role of p38 MAPK and Rac1 GTPase in the regulation of MMP13. METHODS: Knee loading (0.5-3 N for 5 min) was applied to the right knee of surgically-induced osteoarthritis (OA) mice as well as normal (non-OA) mice, and MMP13 activity in the femoral cartilage was examined. The sham-loaded knee was used as a non-loading control. We also employed primary non-OA and OA human chondrocytes as well as C28/I2 chondrocyte cells, and examined MMP13 activity and molecular signaling in response to shear at 2-20 dyn/cm². RESULTS: Daily knee loading at 1 N for 2 weeks suppressed cartilage destruction in the knee of OA mice. Induction of OA elevated MMP13 activity and knee loading at 1 N suppressed this elevation. MMP13 activity was also increased in primary OA chondrocytes, and this increase was attenuated by applying shear at 10 dyn/cm². Load-driven reduction in MMP13 was associated with a decrease in the phosphorylation level of p38 MAPK (p-p38) and NFκB (p-NFκB). Molecular imaging using a fluorescence resonance energy transfer (FRET) technique showed that Rac1 activity was reduced by shear at 10 dyn/cm² and elevated by it at 20 dyn/cm². Silencing Rac1 GTPase significantly reduced MMP13 expression and p-p38 but not p-NFκB. Transfection of a constitutively active Rac1 GTPase mutant increased MMP13 activity, while a dominant negative mutant decreased it. CONCLUSIONS: Knee loading reduces MMP13 activity at least in part through Rac1-mediated p38 MAPK signaling. This study suggests the possibility of knee loading as a therapy not only for strengthening bone but also preventing tissue degradation of the femoral cartilage.

Proteomic analysis of ganglioside-associated membrane molecules: substantial basis for molecular clustering.

Ganglioside GD3 is specifically expressed in human melanomas, and plays a role in the enhancement of malignant phenotypes of melanoma cells. To analyze the mechanisms by which GD3 enhances malignant properties and signals in melanomas, it is essential to clarify how GD3 interacts with membrane molecules on the cell membrane. In this study, we performed proteomics analysis of glycolipid-enriched microdomains (GEM) with current sucrose density gradient ultracentrifugation of Triton X-100 extracts and MS. We also examined GD3-associated molecules using enzyme-mediated activation of radical sources (EMARS) reaction combined with MS. Comparison of molecules identified as residents in GEM/rafts and those detected by EMARS reaction using an anti-GD3 antibody revealed that a relatively low number of molecules is recruited around GD3, while a number of membrane and secreted molecules was defined in GEM/rafts. These results suggested that EMARS reaction is useful to identify actually interacting molecules with gangliosides such as GD3 on the cell membrane, and many other microdomains than GD3-associating rafts exist. Representative examples of GD3-associated molecules such as neogenin and MCAM were shown.