Osteoclasts are multinucleated cells that differentiate from hematopoietic precursors 1) and possess characteristics to resorb mineralized bone. Osteoclast-like multinucleated cells (OCLs) can be differentiated in vitro from cocultures of mouse bone marrow cells and calvarial osteoblastic cells by treatment with osteotropic factors such as 1a,25-dihydroxyvitamin D 3 , prostaglandin E 2 , interleukin-1 (IL-1) or parathyroid hormone.2,3) Osteoblasts or stromal cells are the target cells for these factors in bone. Recently, an essential factor provided by osteoblasts or stromal cells has been identified and named osteoclast differentiation factor (ODF)/osteoprotegerin ligand (OPGL)/tumor necrosis factor-related activation-induced cytokine (TRANCE)/receptor activator of nuclear factor-kB (NF-kB) ligand (RANKL). 4,5) It has been shown that RANKL induces OCL formation in cultures of bone marrow cells in the presence of M-CSF without requiring osteoblasts or stromal cells.6) RAW cells are also known to differentiate into osteoclasts in the presence of RANKL. ; rats fed onions increased their bone mass. 8) Onion also inhibits bone resorption stimulated in ovariectomized rat, 9,10) and an extract from onion is known to prevent tibial cortical and cancellous bone loss induced by a combination of low protein intake and diet-mediated mild hyperparathyroidism in rats.11) Rutin (quercetin-3-O-glucose rhamnose) ( Fig. 1) has recently been reported to inhibit ovariectomy-stimulated bone resorption in rats.12) These facts taken together suggest that rutin is one of the principal components of onions that effectively facilitate bone resorption, and that its inhibitory effect on bone loss could in part be responsible for its effects on increasing bone mass.Quercetin (Fig. 1) is the major representative of the flavonoid subclass of flavonols commonly found in fruits and vegetables, 13,14) and is also abundant in onion extracts (200-600 mg quercetin/kg onion) and primarily in the form of glycoside (rutin).14) Dietary glycosides like rutin are thought to be converted into aglycone (like quercetin) in the large intestine by the glycosidase activity of intestinal bacteria.15) While these facts suggest that quercetin inhibits bone resorption in animals, its target cells for bone resorption and its mode of action has not been fully elucidated. We investigated the effects of quercetin on the differentiation and activation of osteoclasts, and on bone resorption in cultures. We show here that quercetin inhibits pOC formation induced by sRANKL Although quercetin has suppressed bone resorption in several animal studies, its target cells and the mechanism of its action related to bone resorption has not been fully elucidated. We investigated the effect of quercetin on the differentiation and activation of osteoclasts. We used cocultures of mouse spleen cells and ST2 cells, and cultures of osteoclast progenitor cells {M-CSF-dependent (MD) cells from mouse bone marrow and murine monocytic RAW 264 (RAW) cells}. Quercetin dose-dependently inhibite...

A mechanism by which ubiquitinated cargo proteins are sorted into multivesicular bodies (MVBs) from plasma and trans-Golgi network (TGN) membranes is well established in yeast and mammalian somatic cells. However, the ubiquitin-dependent sorting pathway has not been clearly defined in germ cells. In this study we identified a novel member of the transmembrane RING-finger family of proteins, termed membraneassociated RING-CH (MARCH)-XI, that is expressed predominantly in developing spermatids and weakly in brain and pituitary. MARCH-XI possesses an E3 ubiquitin ligase activity that targets CD4 for ubiquitination. Immunoelectron microscopy of rat round spermatids showed that MARCH-XI is localized to TGN-derived vesicles and MVBs. Fluorescence staining of rat round spermatids and immunoprecipitation of rat testis demonstrated that MARCH-XI forms complexes with the adaptor protein complex-1 and with fucose-containing glycoproteins including ubiquitinated forms. Furthermore, the C-terminal region of MARCH-XI mediates its interaction with 1-adaptin and Veli through a tyrosine-based motif and a PDZ binding motif, respectively. Our data suggest that MARCH-XI acts as a ubiquitin ligase with a role in ubiquitin-mediated protein sorting in the TGN-MVB transport pathway, which may be involved in mammalian spermiogenesis.

A novel member of the G protein-coupled receptor (GPCR) family was cloned and characterized, which is unique, among the members, in its long extracellular domain comprising Ig-like repeats and in its high expression predominantly in the lung. The clone (IgHepta) was first identified as a polymerase chain reaction product generated with primers designed to amplify secretin receptor family members including the parathyroid hormone-related peptide receptors. Analysis of the open reading frame of cDNAs isolated from a rat lung cDNA library indicated that Ig-Hepta is a protein of 1389 amino acid residues and has two Ig-like repeats in the N-terminal extracellular domain (exodomain) of 1053 amino acid residues and 7 transmembrane spans in the C-terminal region. Northern blot analysis revealed very high expression of its mRNA in the lung and low but detectable levels in the kidney and heart. The mRNA expression in the lung was found to be strongly induced postnatally. Biochemical analysis indicated that Ig-Hepta is a highly glycosylated protein and exists as a disulfide-linked dimer. Immunohistochemistry on rat lung and kidney sections revealed dense localization of Ig-Hepta in alveolar walls and intercalated cells in the collecting duct, respectively, suggesting a role in the regulation of acid-base balance. Ig-Hepta defines a new subfamily of GPCRs.Since the cloning of rhodopsin (1) and ␤-adrenergic receptor (2), more than 1000 G protein-coupled receptors (GPCRs) 1 have been cloned and characterized (3-5). They constitute one of the largest family of proteins, which have in common seven transmembrane domains, and are involved in broad spectrum of biological processes by mediating the signal of a wide variety of stimuli such as hormones, neurotransmitters, cytokines, light, and odorants. GPCRs can be grouped into various subfamilies based on their amino acid sequences. Recently a subfamily has emerged that shares the seven-transmembrane topology but has a low overall amino acid sequence similarity with other members of the GPCR superfamily. This subfamily, now referred to as the class II GPCR family (Figs. 2 and 3), comprises receptors for secretin, glucagon, VIP, calcitonin, PTH, PTHrP, glucagon-like peptide 1, gastric inhibitory polypeptide, growth hormone-releasing hormone, corticotropin-releasing factor, pituitary adenylate cyclase-activating peptide, and an insect diuretic hormone and is therefore also called the secretin receptor family or the glucagon/VIP/calcitonin receptor family (for review see Refs. 6 -8). The class II receptors are characterized not only by the lack of the structural signature sequences present in the class I rhodopsin/␤-adrenergic receptor family but also by the presence of a large N-terminal extracellular domain (exodomain).Recently Baud et al. (9), Hamann et al. (10), and McKnight et al. (11) have identified a novel subtype of the class II receptors through the structural analyses of a cDNA clone (EMR1) of neuroectodermal origin, of a leukocyte activation antigen (CD97), and of a macr...

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The interaction of osteoclast precursors with osteoblasts and/or stromal cells is essential for the formation of mature osteoclasts and the resorption of bone. We found that myoblastic C2C12 cells induced the differentiation of mouse spleen cells into tartrate-resistant acid phosphatase-positive (TRAP-positive) multinucleated cells in the presence of 10(-7) M 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] and that C2C12 cells that had been treated with bone morphogenetic protein-2 (BMP-2) dose-dependently stimulated the formation of osteoclasts. The newly developed TRAP-positive multinucleated cells were capable of resorbing mineralized tissues. Treatment of C2C12 cells with BMP-2 for 24 h enhanced the subsequent expression in C2C12 cells of mRNA for the receptor activator of nuclear factor-kappaB ligand (RANKL) in the presence of 1alpha,25(OH)2D3. Since the formation of osteoclasts was inhibited dose-dependently by exogenous OPG, the expression of RANKL in response to BMP-2 appeared to be critical for the formation of osteoclasts. Our findings suggest that BMP-2 might play an important role in the differentiation of cells that support osteoclastogenesis.

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Diffuse large B-cell lymphoma (DLBCL) is the most common type of adult non-Hodgkin lymphoma and is treated with chemotherapy in combination with rituximab. Despite this aggressive therapy, the disease is fatal in 30-40% of patients. Inhibition of the apoptosis signalling pathways is strongly related to response to chemotherapy and eventual clinical outcome. In order to survive, lymphoma cells depend on disruption of the apoptosis pathway by mutations in apoptosis inducing genes or by continuous expression of anti-apoptotic proteins. The development of molecules targeting these apoptosis inhibitors provides a very promising opportunity to specifically target tumour cells without toxicity to non-malignant cells in DLBCL patients. Sensitivity for most of these antagonists can be predicted based on biological markers, suggesting the possibility of pre-defining patients who will most likely benefit from these targeted therapies. Experimental therapies aimed at restoring the upstream apoptosis pathway or targeting apoptosis inhibitors are currently being tested in clinical trials and are expected to be effective particularly in chemotherapy-refractory DLBCL, providing hope for patients who are refractory to current therapies.