Nupr1 deficiency downregulates HtrA1, enhances SMAD1 signaling, and suppresses age-related bone loss in male mice.

Nuclear protein 1 (NUPR1) is a stress-induced protein activated by various stresses, such as inflammation and oxidative stress. We previously reported that Nupr1 deficiency increased bone volume by enhancing bone formation in 11-week-old mice. Analysis of differentially expressed genes between wild-type (WT) and Nupr1-knockout (Nupr1-KO) osteocytes revealed that high temperature requirement A 1 (HTRA1), a serine protease implicated in osteogenesis and transforming growth factor-ß signaling was markedly downregulated in Nupr1-KO osteocytes. Nupr1 deficiency also markedly reduced HtrA1 expression, but enhanced SMAD1 signaling in in vitro-cultured primary osteoblasts. In contrast, Nupr1 overexpression enhanced HtrA1 expression in osteoblasts, suggesting that Nupr1 regulates HtrA1 expression, thereby suppressing osteoblastogenesis. Since HtrA1 is also involved in cellular senescence and age-related diseases, we analyzed aging-related bone loss in Nupr1-KO mice. Significant spine trabecular bone loss was noted in WT male and female mice during 6-19 months of age, whereas aging-related trabecular bone loss was attenuated, especially in Nupr1-KO male mice. Moreover, cellular senescence-related markers were upregulated in the osteocytes of 6-19-month-old WT male mice but markedly downregulated in the osteocytes of 19-month-old Nupr1-KO male mice. Oxidative stress-induced cellular senescence stimulated Nupr1 and HtrA1 expression in in vitro-cultured primary osteoblasts, and Nupr1 overexpression enhanced p16ink4a expression in osteoblasts. Finally, NUPR1 expression in osteocytes isolated from the bones of patients with osteoarthritis was correlated with age. Collectively, these results indicate that Nupr1 regulates HtrA1-mediated osteoblast differentiation and senescence. Our findings unveil a novel Nupr1/HtrA1 axis, which may play pivotal roles in bone formation and age-related bone loss.

A novel role of helix-loop-helix transcriptional factor Bhlhe40 in osteoclast activation.

The basic helix-loop-helix transcriptional factor, Bhlhe40 has been shown as a crucial regulator of immune response, tumorigenesis, and circadian rhythms. We identified Bhlhe40 as a possible regulator of osteoclast differentiation and function by shRNA library screening and found that Bhlhe40 was required for osteoclast activation. Bhlhe40 expression was induced in bone marrow macrophages (BMMs) by RANKL, whereas the expression of its homolog Bhlhe41 was decreased in osteoclastogenesis. µCT analysis of tibias revealed that Bhlhe40 knockout (KO) mice exhibited increased bone volume phenotype. Bone morphometric analysis showed that osteoclast number and bone resorption were decreased in Bhlhe40 KO mice, whereas significant differences in the osteoblast parameters were not seen between wild-type (WT) and Bhlhe40 KO mice. In vitro culture of BMMs showed that Bhlhe40 deficiency did not cause difference in osteoclast formation. In contrast, bone resorption activity of Bhlhe40 KO osteoclasts was markedly reduced in comparison with that of WT osteoclasts. Analysis of potential target genes of Bhlhe40 using data-mining platform ChIP-Atlas (http://chip-atlas.org) revealed that predicted target genes of Bhlhe40 were related to proton transport and intracellular vesicle acidification. We then analyzed the expression of proton pump, the vacuolar (V)-ATPases which are responsible for bone resorption. The expression of V-ATPases V1c1 and V0a3 was suppressed in Bhlhe40 KO osteoclasts. In addition, Lysosensor yellow/blue DND 160 staining demonstrated that vesicular acidification was attenuated in vesicles of Bhlhe40 KO osteoclasts. Furthermore, analysis with pH-sensitive fluorescent probe showed that proton secretion was markedly suppressed in Bhlhe40 KO osteoclasts compared to that in WT osteoclasts. Our findings suggest that Bhlhe40 plays a novel important role in the regulation of acid production in osteoclastic bone resorption.

Leukemia/lymphoma-related factor (LRF) or osteoclast zinc finger protein (OCZF) overexpression promotes osteoclast survival by increasing Bcl-xl mRNA: A novel regulatory mechanism mediated by the RNA binding protein SAM68.

RANKL induces NFATc1, a key transcriptional factor to induce osteoclast-specific genes such as cathepsin K, whereas transcriptional control of osteoclast survival is not fully understood. Leukemia/lymphoma-related factor (LRF) in mouse and osteoclast zinc finger protein (OCZF) in rat are zinc finger and BTB domain-containing protein (zBTB) family of transcriptional regulators, and are critical regulators of hematopoiesis. We have previously shown that differentiation and survival were enhanced in osteoclasts from OCZF-Transgenic (Tg) mice. In the present study, we show a possible mechanism of osteoclast survival regulated by LRF/OCZF and the role of OCZF overexpression in pathological bone loss. In the in vitro cultures, LRF was highly colocalized with NFATc1 in cells of early stage in osteoclastogenesis, but only LRF expression persisted after differentiation into mature osteoclasts. LRF expression was further enhanced in resorbing osteoclasts formed on dentin slices. Osteoclast survival inhibitor such as alendronate, a bisphosphonate reduced LRF expression. Micro CT evaluation revealed that femurs of OCZF-Tg mice showed significantly lower bone volume compared to that of WT mice. Furthermore, OCZF overexpression markedly promoted bone loss in ovariectomy-induced osteolytic mouse model. The expression of anti-apoptotic Bcl-xl mRNA, which is formed by alternative splicing, was enhanced in the cultures in which osteoclasts are formed from OCZF-Tg mice. In contrast, the expression of pro-apoptotic Bcl-xs mRNA was lost in the culture derived from OCZF-Tg mice. We found that the expression levels of RNA binding splicing regulator, Src substrate associated in mitosis of 68 kDa (Sam68) protein were markedly decreased in OCZF-Tg mice-derived osteoclasts. In addition, shRNA-mediated knockdown of Sam68 expression increased the expression of Bcl-xl mRNA, suggesting that SAM68 regulates the expression of Bcl-xl. These results indicate that OCZF overexpression reduces protein levels of Sam68, thereby promotes osteoclast survival, and suggest that LRF/OCZF is a promising target for regulating pathological bone loss.

miR-92a-3p encapsulated in bone metastatic mammary tumor cell-derived extracellular vesicles modulates mature osteoclast longevity.

Aberrant osteoclast formation and activation are the hallmarks of osteolytic metastasis. Extracellular vesicles (EVs), released from bone metastatic tumor cells, play a pivotal role in the progression of osteolytic lesions. However, the mechanisms through which tumor cell-derived EVs regulate osteoclast differentiation and function have not been fully elucidated. In this study, we found that 4T1 bone metastatic mouse mammary tumor cell-derived EVs (4T1-EVs) are taken up by mouse bone marrow macrophages to facilitate osteoclastogenesis. Furthermore, treatment of mature osteoclasts with 4T1-EVs promoted bone resorption, which was accompanied by enhanced survival of mature osteoclasts through the negative regulation of caspase-3. By comparing the miRNA content in 4T1-EVs with that in 67NR nonmetastatic mouse mammary tumor cell-derived EVs (67NR-EVs), miR-92a-3p was identified as one of the most enriched miRNAs in 4T1-EVs, and its transfer into mature osteoclasts significantly reduced apoptosis. Bioinformatic and Western blot analyses revealed that miR-92a-3p directly targeted phosphatase and tensin homolog (PTEN) in mature osteoclasts, resulting in increased levels of phospho-Akt. Our findings provide novel insights into the EV-mediated regulation of osteoclast survival through the transfer of miR-92a-3p, which enhances mature osteoclast survival via the Akt survival signaling pathway, thus promoting bone resorption.

PMEPA1 and NEDD4 control the proton production of osteoclasts by regulating vesicular trafficking.

Osteoclast bone resorption activity is critically regulated to maintain bone homeostasis. Osteoclasts resorb bone by producing protons and acid hydrolase via lysosomal secretion, however, a detailed mechanism remains elusive. PMEPA1 is a vesicular membrane protein, which binds to the NEDD4 family member of ubiquitin ligases. We have previously reported that Pmepa1 is highly expressed in bone resorbing osteoclasts, and regulates bone resorption. Here, we investigated the mechanism of bone resorption regulated by PMEPA1. Mutant mice lacking NEDD4-binding domains of PMEPA1 displayed enhanced bone volume, and reduced bone resorption activity in comparison with those of WT mice. Analysis with pH-sensitive fluorescence probe revealed that proton secretion from osteoclasts significantly decreased in Pmepa1 mutant osteoclasts. Immunofluorescence analysis revealed that PMEPA1 was colocalized with NEDD4, V0A3, and V0D2 subunits of vacuolar ATPase, which regulate the proton production of osteoclasts. In addition, Nedd4 knockdown reduced bone resorption and proton secretion of osteoclasts. Furthermore, Pmepa1 mutation and Nedd4 knockdown altered the cytoplasmic distribution of components of V-ATPase and expression of autophagy-related proteins, suggesting that lysosomal secretion is affected. Collectively, these findings indicate that PMEPA1 controls proton secretion from osteoclasts via NEDD4 by regulating vesicular trafficking, and NEDD4 is an important regulator of bone resorption.

In vitro and in vivo detection of tunneling nanotubes in normal and pathological osteoclastogenesis involving osteoclast fusion.

Osteoclasts are multinucleated cells formed through specific recognition and fusion of mononuclear osteoclast precursors derived from hematopoietic stem cells. Detailed cellular events concerning cell fusion in osteoclast differentiation remain ambiguous. Tunneling nanotubes (TNTs), actin-based membrane structures, play an important role in intercellular communication between cells. We have previously reported the presence of TNTs in the fusion process of osteoclastogenesis. Here we analyzed morphological details of TNTs using scanning electron microscopy. The osteoclast precursor cell line RAW-D was stimulated to form osteoclast-like cells, and morphological details in the appearance of TNTs were extensively analyzed. Osteoclast-like cells could be classified into three types; early osteoclast precursors, late osteoclast precursors, and multinucleated osteoclast-like cells based on the morphological characteristics. TNTs were frequently observed among these three types of cells. TNTs could be classified into thin, medium, and thick TNTs based on the diameter and length. The shapes of TNTs were dynamically changed from thin to thick. Among them, medium TNTs were often observed between two remote cells, in which side branches attached to the culture substrates and beaded bulge-like structures were often observed. Cell-cell interaction through TNTs contributed to cell migration and rapid transport of information between cells. TNTs were shown to be involved in cell-cell fusion between osteoclast precursors and multinucleated osteoclast-like cells, in which movement of membrane vesicles and nuclei was observed. Formation of TNTs was also confirmed in primary cultures of osteoclasts. Furthermore, we have successfully detected TNTs formed between osteoclasts observed in the bone destruction sites of arthritic rats. Thus, formation of TNTs may be important for the differentiation of osteoclasts both in vitro and in vivo. TNTs could be one target cellular structure for the regulation of osteoclast differentiation and function in bone diseases.

Modulation of osteoclastogenesis through adrenomedullin receptors on osteoclast precursors: initiation of differentiation by asymmetric cell division.

Adrenomedullin (ADM), a member of the calcitonin family of peptides, is a potent vasodilator and was shown to have the ability to modulate bone metabolism. We have previously found a unique cell surface antigen (Kat1 antigen) expressed in rat osteoclasts, which is involved in the functional regulation of the calcitonin receptor (CTR). Cross-linking of cell surface Kat1 antigen with anti-Kat1 antigen monoclonal antibody (mAbKat1) stimulated osteoclast formation only under conditions suppressed by calcitonin. Here, we found that ADM provoked a significant stimulation in osteoclastogenesis only in the presence of calcitonin; a similar biological effect was seen with mAbKat1 in the bone marrow culture system. This stimulatory effect on osteoclastogenesis mediated by ADM was abolished by the addition of mAbKat1. 125I-labeled rat ADM (125I-ADM)-binding experiments involving micro-autoradiographic studies demonstrated that mononuclear precursors of osteoclasts abundantly expressed ADM receptors, and the specific binding of 125I-ADM was markedly inhibited by the addition of mAbKat1, suggesting a close relationship between the Kat1 antigen and the functional ADM receptors expressed on cells in the osteoclast lineage. ADM receptors were also detected in the osteoclast progenitor cells in the late mitotic phase, in which only one daughter cell of the dividing cell express ADM receptors, suggesting the semiconservative cell division of the osteoclast progenitors in the initiation of osteoclastogenesis. Messenger RNAs for the receptor activity-modifying-protein 1 (RAMP1) and calcitonin receptor-like receptor (CRLR) were expressed in cells in the osteoclast lineage; however, the expression of RAMP2 or RAMP3 was not detected in these cells. It is suggested that the Kat1 antigen is involved in the functional ADM receptor distinct from the general ADM receptor, consisting of CRLR and RAMP2 or RAMP3. Modulation of osteoclastogenesis through functional ADM receptors abundantly expressed on mononuclear osteoclast precursors is supposed to be important in the fine regulation of osteoclast differentiation in a specific osteotrophic hormonal condition with a high level of calcitonin in blood.

IgG immune complexes with Staphylococcus aureus protein A enhance osteoclast differentiation and bone resorption by stimulating Fc receptors and TLR2.

Staphylococcus aureus is a main pathogen of osteomyelitis and protein A is a virulence factor with high affinity for IgG. In this study, we investigated whether S. aureus affects the differentiation and bone resorption of osteoclasts through the IgG-binding capacity of protein A. Staphylococcus aureus pre-treated with serum or IgG showed marked enhancement in osteoclastogenesis and bone resorption compared to non-treated S. aureus or a protein A-deficient mutant. Blocking of the Fc receptor and deletion of the Fcγ receptor gene in osteoclast precursor cells showed that enhanced osteoclastogenesis stimulated by S. aureus IgG immune complexes (ICs) was mediated by the Fc receptor on osteoclast precursor cells. In addition, osteoclastogenesis stimulated by S. aureus ICs but not the protein A-deficient mutant was markedly reduced in osteoclast precursor cells of Myd88-knockout mice. Moreover, NFATc1, Syk and NF-κB signals were necessary for osteoclastogenesis stimulated by S. aureus ICs. The results suggest the contribution of a of Toll-like receptor 2 (TLR2)-Myd88 signal to the activity of S. aureus ICs. We further examined the expression of pro-inflammatory cytokines that is known to be enhanced by FcγR-TLR cross-talk. Osteoclasts induced by S. aureus ICs showed higher expression of TNF-α and IL-1ß, and marked stimulation of proton secretion of osteoclasts activated by pro-inflammatory cytokines. Finally, injection of S. aureus, but not the protein A-deficient mutant, exacerbated bone loss in implantation and intra-peritoneal administration mouse models. Our results provide a novel mechanistic aspect of bone loss induced by S. aureus in which ICs and both Fc receptors and TLR pathways are involved.

Prostate transmembrane protein androgen induced 1 is induced by activation of osteoclasts and regulates bone resorption.

Osteoclasts derived from hematopoietic cells are activated on bone surface. To resorb bone, osteoclasts release acid and lysosome acid hydrolase via membrane transport. Prostate transmembrane protein androgen induced 1 (Pmepa1) is a type I transmembrane protein that regulates proliferation, migration, and metastasis of cancer cells. Because recent reports showed that Pmepa1 is involved in membrane transport in cancer cells, we investigated the role of Pmepa1 in osteoclast function. Pmepa1 expression was barely detected in osteoclasts formed on plastic surfaces in vitro, but was markedly increased in activated osteoclasts formed on calcified matrix. Inhibitors of bone resorption, such as alendronate, bafilomycin A1, and the PI3K inhibitor LY294002, suppressed the expression of Pmepa1 in osteoclasts. Knockdown of Pmepa1 expression impaired bone resorption activity and inhibited formation of a ring-like, actin-rich podosome belt that is essential for osteoclast function. Pmepa1 protein localized to lysosomes in osteoclasts. In addition, in sites of bone destruction observed in rats with adjuvant-induced arthritis, a marked high level of Pmepa1 expression was associated with the osteoclasts' resorbing bone. Our results suggest that Pmepa1 is a critical regulator of bone resorption and is a promising marker for activated osteoclasts and a potential therapeutic target in pathologic bone destruction.-Xu, X., Hirata, H., Shiraki, M., Kamohara, A., Nishioka, K., Miyamoto, H., Kukita, T., Kukita, A. Prostate transmembrane protein androgen induced 1 is induced by activation of osteoclasts and regulates bone resorption.

Deficiency of stress-associated gene Nupr1 increases bone volume by attenuating differentiation of osteoclasts and enhancing differentiation of osteoblasts.

Nuclear protein 1 (NUPR1) is a multifunctional stress-induced protein involved in regulating tumorigenesis, apoptosis, and autophagy. Bone homeostasis is maintained by bone-resorbing osteoclasts and bone-forming osteoblasts and osteocytes. We aimed to determine the role of NUPR1 in bone metabolism. Using microcomputed tomography, we found that mice lacking Nupr1 exhibited increased bone volume. Histologic analysis showed that Nupr1 deficiency decreased osteoclast numbers but increased osteoblast numbers and osteoid formation. In vitro culture of bone marrow macrophages showed that receptor activator of NF-κB ligand-induced osteoclastogenesis was down-regulated in Nupr1-deficient mice. In contrast, primary osteoblasts from Nupr1-deficient mice revealed that proliferation of osteoblasts and expression of bone matrix proteins were markedly enhanced. In addition, expression of autophagy-related genes, formation of autophagosomes, and cell survival were up-regulated in Nupr1-deficient osteoblasts. In contract, deletion of Nupr1 reduced the formation of osteocyte cellular projection, which is an indicator of mature osteocytes. Importantly, we found that the expression of sclerostin (Sost), an inhibitor of bone formation, was down-regulated in the osteoblasts and osteocytes of Nupr1-deficient mice. Conversely, Nupr1 overexpression enhanced Sost expression in primary osteoblasts. Collectively, these results indicate that Nupr1 deficiency increases bone volume by attenuating production of Sost and osteoclastogenesis and enhancing differentiation of osteoblasts.-Shiraki, M., Xu, X., Iovanna, J. L., Kukita, T., Hirata, H., Kamohara, A., Kubota, Y., Miyamoto, H., Mawatari, M., Kukita, A. Deficiency of stress-associated gene Nupr1 increases bone volume by attenuating differentiation of osteoclasts and enhancing differentiation of osteoblasts.

IL-1ß Induces Pathologically Activated Osteoclasts Bearing Extremely High Levels of Resorbing Activity: A Possible Pathological Subpopulation of Osteoclasts, Accompanied by Suppressed Expression of Kindlin-3 and Talin-1.

As osteoclasts have the central roles in normal bone remodeling, it is ideal to regulate only the osteoclasts performing pathological bone destruction without affecting normal osteoclasts. Based on a hypothesis that pathological osteoclasts form under the pathological microenvironment of the bone tissues, we here set up optimum culture conditions to examine the entity of pathologically activated osteoclasts (PAOCs). Through searching various inflammatory cytokines and their combinations, we found the highest resorbing activity of osteoclasts when osteoclasts were formed in the presence of M-CSF, receptor activator of NF-κB ligand, and IL-1ß. We have postulated that these osteoclasts are PAOCs. Analysis using confocal laser microscopy revealed that PAOCs showed extremely high proton secretion detected by the acid-sensitive fluorescence probe Rh-PM and bone resorption activity compared with normal osteoclasts. PAOCs showed unique morphology bearing high thickness and high motility with motile cellular processes in comparison with normal osteoclasts. We further examined the expression of Kindlin-3 and Talin-1, essential molecules for activating integrin ß-chains. Although normal osteoclasts express high levels of Kindlin-3 and Talin-1, expression of these molecules was markedly suppressed in PAOCs, suggesting the abnormality in the adhesion property. When whole membrane surface of mature osteoclasts was biotinylated and analyzed, the IL-1ß-induced cell surface protein was detected. PAOCs could form a subpopulation of osteoclasts possibly different from normal osteoclasts. PAOC-specific molecules could be an ideal target for regulating pathological bone destruction.

Osteoblast lineage-specific cell-surface antigen (A7) regulates osteoclast recruitment and calcification during bone remodeling.

Bone remodeling is a continuous process characterized by highly coordinated cell-cell interactions in distinct multi-cellular units. Osteoclasts, which are specialized bone resorbing cells, play a central role in bone remodeling. Although the RANKL/RANK axis determines the gross number of osteoclasts present in bone tissue, detailed molecular events regulating bone remodeling related to osteoclast recruitment, initiation of bone remodeling, and coupling of bone resorption and bone formation are still ambiguous. We hypothesized that osteoblast-specific cell-surface molecules contribute to the molecular modulation of bone remodeling. Therefore, we searched for regulatory cell-surface molecules expressed on osteoblasts by use of B-cell hybridoma technology. We obtained a monoclonal antibody A7 (A7 MAb) highly specific to cells of osteoblast-lineage. Here we describe the expression pattern and possible role of A7 antigen specifically recognized by A7 MAb. In vitro, A7 antigen was expressed on cell-surface of osteoblasts and osteoblast-like bone marrow stromal cells. In vivo, A7 antigen was detected in a subset of bone surface osteoblasts and in osteocytes, with a typical cell membrane expression pattern. Tissue array analysis showed only a limited expression of A7 antigen in osteocytes close to the bone surface. Immunoblotting and immunoprecipitation analysis showed that A7 antigen is a lineage-specific cell-surface protein with an approximate molecular weight of 45 KDa. Cross-linking of cell-surface A7 antigen in cultures of osteoclastogenesis showed stimulation of osteoclast formation. Marked suppression of calcification in primary osteoblast cultures was observed when A7 antigen was cross-linked with anti-A7 antigen MAb, A7 MAb. These data suggest that A7 antigen regulates recruitment of osteoclasts and triggering of calcification. A7 antigen may be an important molecule involved in the precise regulation of bone remodeling.

Pmepa1 induced by RANKL-p38 MAPK pathway has a novel role in osteoclastogenesis.

Osteoclasts are multinucleated cells formed by fusion of preosteoclasts (POCs) derived from cells of the monocyte/macrophage lineage. We have reported a culture system that supports the formation of POCs from stroma-depleted rat bone marrow cells. Global gene expression analysis of this culture system identified genes highly expressed in POCs. Here, we have analyzed the expression and function of one of these highly expressed genes, prostate transmembrane protein androgen induced 1 (Pmepa1), a target of TGF-ß and binds Nedd4 ubiquitin ligase, which plays a role in intracellular trafficking. We show here that the expression of Pmepa1 was strongly induced by RANKL in mouse bone marrow macrophage and in the osteoclast precursor cell line RAW-D. The expression of Pmepa1 was increased at 24 hr of culture, but was decreased at 72 hr. Pmepa1 protein was localized to intracellular vesicle membrnane of mononuclear cells, some of which were cathepsin-K positive. RANKL-induced expression of Pmepa1 was significantly reduced by inhibitors of p38 MAPK signaling. Pmepa1 siRNA suppressed the formation of osteoclasts in RAW-D cells, and inhibited the expression of cathepsin K and c-fos but not RANK. In addition, inhibition of Pmepa1 expression reduced the surface expression of RANK in RAW-D cells induced by RANKL. These results demonstrate that Pmepa1 is induced by RANK-p38 MAPK pathway signaling, and upregulates cell surface expression of RANK, suggesting that Pmepa1 plays a role in osteoclastogenesis and osteoclast signaling.

Osteoblast-derived Laminin-332 is a novel negative regulator of osteoclastogenesis in bone microenvironments.

Laminin-332 (Lm-332), a major basement membrane protein, has been shown to provide a niche for some stem cells. Here, we found that Lm-332 was expressed in osteoblasts, and is implicated in the regulation of osteoclast differentiation. Immunofluorescence analysis of laminin-ß3, a unique component of Lm-332, indicated specific expression of laminin-ß3 in osteoblast-like cells localized on bone surface. RT-PCR analysis confirmed that α3, ß3, and γ2 chains of Lm-332 were all expressed in primary osteoblasts prepared from mouse calvaria. Lm-332 markedly inhibited osteoclastogenesis induced by receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL) when bone marrow-derived macrophages (BMMs) were cultured on Lm-332-coated plates. Lm-332 also blocked RANKL-induced activation of mitogen-activated protein kinases (MAPKs) (ERK, JNK, and p38) and expression of NFATc1, c-Fos, and c-Jun. Lm-332 suppressed osteoclast differentiation while retaining macrophage phenotypes, including nonspecific esterase activity and gene expression of lysozyme and EGF-like module-containing mucin-like hormone receptor-like 1 (Emr1). Furthermore, the treatment of primary osteoblasts with osteoclastogenic factors dramatically suppressed expression of Lm-332. These findings suggest that Lm-332 produced by osteoblasts in bone tissues has a pivotal role in controlling normal bone remodeling through suppressing osteoclastogenesis.

Extremely High Expression of Antisense RNA for Wilms' Tumor 1 in Active Osteoclasts: Suppression of Wilms' Tumor 1 Protein Expression during Osteoclastogenesis.

Wilms' tumor 1 (WT1), a zinc-finger transcription regulator of the early growth response family, identified as the product of a tumor suppressor gene of Wilms' tumors, bears potential ability to induce macrophage differentiation in blood cell differentiation. Herein, we examined the involvement of WT1 in the regulation of osteoclastogenesis. We detected a high level of WT1 protein expression in osteoclast precursors; however, WT1 expression was markedly suppressed during osteoclastogenesis. We examined expression of WT1 transcripts in bone tissue by RNA in situ hybridization. We found a high level of antisense transcripts in osteoclasts actively resorbing bone in mandible of newborn rats. Expression of antisense WT1 RNA in mandible was also confirmed by Northern blot analysis and strand-specific RT-PCR. Overexpression of antisense WT1 RNA in RAW-D cells, an osteoclast precursor cell line, resulted in a marked enhancement of osteoclastogenesis, suggesting that antisense WT1 RNA functions to suppress expression of WT1 protein in osteoclastogenesis. High level expression of antisense WT1 RNA may contribute to commitment to osteoclastogenesis, and may allow osteoclasts to maintain or stabilize their differentiation state.

Stage-specific functions of leukemia/lymphoma-related factor (LRF) in the transcriptional control of osteoclast development.

Cell fate determination is tightly regulated by transcriptional activators and repressors. Leukemia/lymphoma-related factor (LRF; encoded by Zbtb7a), known as a POK (POZ/BTB and Krüppel) family transcriptional repressor, is induced during the development of bone-resorbing osteoclasts, but the physiological significance of LRF in bone metabolism and the molecular mechanisms underlying the transcriptional regulation of osteoclastogenesis by LRF have not been elucidated. Here we show that LRF negatively regulates osteoclast differentiation by repressing nuclear factor of activated T cells c1 (NFATc1) induction in the early phase of osteoclast development, while positively regulating osteoclast-specific genes by functioning as a coactivator of NFATc1 in the bone resorption phase. The stage-specific distinct functions of LRF were demonstrated in two lines of conditional knockout mice in which LRF was deleted in the early or late phase of osteoclast development. Thus, this study shows that LRF plays stage-specific distinct roles in osteoclast differentiation, exemplifying the delicate transcriptional regulation at work in lineage commitment.

Regulation of osteoclastogenesis through Tim-3: possible involvement of the Tim-3/galectin-9 system in the modulation of inflammatory bone destruction.

Galectins are a unique family of lectins bearing one or two carbohydrate recognition domains (CRDs) that have the ability to bind molecules with ß-galactoside-containing carbohydrates. It has been shown that galectins regulate not only cell growth and differentiation but also immune responses, as well as inflammation. Galectin-9, a tandem repeat type of galectin, was originally identified as a chemotactic factor for eosinophils, and is also involved in the regulatory process of inflammation. Here, we examined the involvement of galectin-9 and its receptor, T-cell immunoglobulin- and mucin-domain-containing molecule 3 (Tim-3), in the control of osteoclastogenesis and inflammatory bone destruction. Expression of Tim-3 was detected in osteoclasts and its mononuclear precursors in vivo and in vitro. Galectin-9 markedly inhibited osteoclastogenesis as evaluated in osteoclast precursor cell line RAW-D cells and primary bone marrow cells of mice and rats. The inhibitory effects of galectin-9 on osteoclastogenesis was negated by the addition of ß-lactose, an antagonist for galectin binding, suggesting that the inhibitory effect of galectin-9 was mediated through CRD. When galectin-9 was injected into rats with adjuvant-induced arthritis, marked suppression of bone destruction was observed. Inflammatory bone destruction could be efficiently ameliorated by controlling the Tim-3/galectin-9 system in rheumatoid arthritis.

Mesenchymal stem cells markedly suppress inflammatory bone destruction in rats with adjuvant-induced arthritis.

Mesenchymal stem cells (MSCs) have potential to differentiate into multiple cell lineages. Recently, it was shown that MSCs also have anti-inflammatory and immunomodulatory functions. In this report, we investigated the regulatory function of MSCs in the development of inflammatory bone destruction in rats with adjuvant-induced arthritis (AA rats). MSCs were isolated from rat bone marrow tissues, expanded in the presence of basic FGF, and intraperitoneally injected into AA rats. MSC administration significantly suppressed inflammatory parameters: swelling score, swelling width, and thickness of hind paw. Radiographic evaluation indicated that MSC significantly suppressed bone destruction. Histological analysis showed that administration of MSCs markedly suppressed osteoclastogenesis in AA rats. To further delineate their effects on osteoclastogenesis, MSCs were added to in vitro bone marrow cultures undergoing osteoclastogenesis. MSCs significantly suppressed osteoclastogenesis in this system. Chemokine receptor expression in MSCs was assessed by RT-PCR, and a chemotactic assay was performed using a transwell culture system. MSCs showed significant chemotaxis to MIP-1α (CCL3) and SDF-1α (CXCL12), chemokines preferentially expressed in the area of inflammatory bone destruction. Furthermore, MSCs expressed IL-10 and osteoprotegerin, cytokines that suppress osteoclastogenesis. These data suggest that recruitment of MSC to the area of bone destruction in AA rats could suppress inflammatory bone destruction and raise the possibility that MSCs may have potential for the treatment of inflammatory bone destruction in arthritis.

Tunneling nanotube formation is essential for the regulation of osteoclastogenesis.

Osteoclasts are the multinucleated giant cells formed by cell fusion of mononuclear osteoclast precursors. Despite the finding of several membrane proteins involving DC-STAMP as regulatory proteins required for fusion among osteoclast precursors, cellular and molecular events concerning this process are still ambiguous. Here we identified Tunneling Nanotubes (TNTs), long intercellular bridges with small diameters, as the essential cellular structure for intercellular communication among osteoclast precursors in prior to cell fusion. Formation of TNTs was highly associated with osteoclastogenesis and it was accompanied with the significant induction of the M-Sec gene, an essential gene for TNT formation. M-Sec gene expression was significantly upregulated by RANKL-treatment in osteoclast precursor cell line. Blockage of TNT formation by Latrunclin B or by M-Sec siRNA significantly suppressed osteoclastogenesis. We have detected the rapid intercellular transport of not only the membrane phospholipids labeled with DiI but also the DC-STAMP-GFP fusion protein through TNTs formed among osteoclast precursors during osteoclastogenesis. Transportation of such regulatory molecules through TNTs would be essential for the process of the specific cell fusion among osteoclast precursors.

Adenosine blocks aminopterin-induced suppression of osteoclast differentiation.

To search cell surface molecules involved in the regulation of osteoclastogenesis, especially in fusion process, it is one powerful approach to obtain monoclonal antibodies bearing ability to block formation of multinucleated osteoclasts. Ideally, direct bio-assay of hybridoma supernatants is quite convenient to screen monoclonal antibodies of interest from numerous culture wells. However, addition of hybridoma supernatant containing hypoxanthine-aminopterin-thymidine (HAT), components of the selection medium, to whole bone marrow cultures strikingly suppressed osteoclastogenesis. Here we clarified aminopterin is the responsible component in HAT medium to inhibit osteoclastogenesis. Methotrexate (MTX), mono-methylated aminopterin, showed similar suppressive effect on osteoclastogenesis. When bone marrow cells were cultured in the presence of all nucleosides, aminopterin and MTX-induced suppression of osteoclastogenesis was abrogated. Among four nucleosides only adenosine canceled aminopterin-induced suppression of osteoclastogenesis. Direct bio-assay of hybridoma supernatant containing HAT selection medium is now available to screen monoclonal antibodies if adenosine-containing culture medium was utilized for evaluating osteoclastogenesis.