Developing long bones respond to surrounding tissues by trans-pairing of periosteal osteoclasts and endocortical osteoblasts.

Developing long bones alter their shape while maintaining uniform cortical thickness via coordinated activity of bone-forming osteoblasts and bone-resorbing osteoclasts at periosteal and endosteal surfaces, a process we designate trans-pairing. Two types of trans-pairing shift cortical bone in opposite orientations: peri-forming trans-pairing (peri-t-p) increases bone marrow space and endo-forming trans-pairing (endo-t-p) decreases it, via paired activity of bone resorption and formation across the cortex. Here, we focused on endo-t-p in growing bones. Analysis of endo-t-p activity in the cortex of mouse fibulae revealed osteoclasts under the periosteum compressed by muscles, and expression of RANKL in periosteal cells of the cambium layer. Furthermore, mature osteoblasts were localized on the endosteum, while preosteoblasts were at the periosteum and within cortical canals. X-ray tomographic microscopy revealed the presence of cortical canals more closely associated with endo- than with peri-t-p. Sciatic nerve transection followed by muscle atrophy and unloading induced circumferential endo-t-p with concomitant spread of cortical canals. Such canals likely supply the endosteum with preosteoblasts from the periosteum under endo-t-p, allowing bone shape to change in response to mechanical stress or nerve injury.

Eribulin mesylate induces bone mass loss by promoting osteoclastic bone resorption in mice.

Over the past few decades, the clinical outcomes of patients with cancer have significantly improved mostly owing to the development of effective chemotherapeutic treatments. However, chronic health conditions such as bone mass loss and risk of fragility fractures caused by chemotherapy have also emerged as crucial issues in patients treated for cancer. In this study, we aimed to understand the effect of eribulin mesylate (ERI), a microtubule-targeting agent currently used to treat metastatic breast cancer and certain subtypes of advanced sarcomas, on bone metabolism in mice. The administration of ERI reduced bone mass in mice, mainly by promoting osteoclast activity. Gene expression analysis of skeletal tissues revealed no change in the expression levels of the transcripts for RANK ligand, one of the master regulators of osteoclastogenesis; however, the transcript levels of osteoprotegerin, which neutralizes RANK ligand, were significantly reduced in ERI-treated mice compared with those in vehicle-treated controls, indicating a relative increase in RANK ligand availability after ERI treatment. In line with the increased bone resorption in ERI-treated mice, we found that zoledronate administration effectively suppressed bone loss in these mice. These results reveal a previously unrecognized effect of ERI on bone metabolism and suggest the application of bisphosphonates for patients with cancer undergoing treatment with ERI.

Study of the Acquisition of Sensitivity to Paclitaxel Plus Cetuximab by the Addition of Low-dose Proteasome Inhibitor.

BACKGROUND/AIM: Although paclitaxel plus cetuximab for recurrent/metastatic oral squamous cell carcinoma (OSCC) has a relatively high success rate, many cases are refractory. We investigated the change in nuclear factor-kappa B (NF-B) expression after this combination therapy using microcollagen 3D cell culture. We also investigated changes in antitumor efficacy using low doses of paclitaxel-cetuximab combined with the proteasome inhibitor bortezomib on a cell line with low sensitivity to paclitaxel plus cetuximab. MATERIALS AND METHODS: Eight human OSCC cell lines were cultured in 3D and exposed to paclitaxel-cetuximab. real-time polymerase chain reaction was used to evaluate NF-B mRNA expression in OSCC cell lines in vivo and in vitro after exposure to anticancer agents. Activity at the protein level was confirmed using western blotting. Bortezomib (0.002-0.4 µg/ml) was added to paclitaxel-cetuximab and its effects assessed in OSCC cell lines with low paclitaxel-cetuximab sensitivity. RESULTS: mRNA and protein expression of NF-B was significantly reduced after treatment with paclitaxel-cetuximab in cell lines sensitive to this combination. In contrast, both mRNA and protein expression significantly increased in the cell lines with low sensitivity to paclitaxel plus cetuximab. The addition of low concentrations of bortezomib to cell lines with low sensitivity to paclitaxel-cetuximab was found to enhance antitumor efficacy. CONCLUSION: Increased NF-B expression strongly contributes to resistance to paclitaxel-cetuximab, suggesting that the administration of small doses of bortezomib, which inhibits NF-B, combined with paclitaxel-cetuximab may enhance antitumor efficacy against cancer cells with low sensitivity to the combination therapy.

Aging Aggravates the Progression of Muscle Degeneration After Rotator Cuff Tears in Mice.

PURPOSE: The purpose of this study was to evaluate the impact of aging on muscle degeneration after rotator cuff tear (RCT) in mice. METHODS: Young (12-week-old) and aged (50-to-60-week-old) female C57BL/6 mice were used (n = 29 for each group). The rotator cuff was transected, and the proximal humerus was removed to induce degeneration of the rotator cuff muscles. The mice were euthanized 4 and 12 weeks after the procedure (referred to as RCT-4wk mice and RCT-12wk mice, respectively) and compared with the sham-treated mice. The supraspinatus muscles were collected for histology, Western blot analysis, and gene expression analyses. RESULTS: There was a significant increase in fat tissue in aged RCT-4wk mice (P = .001) and aged RCT-12wk mice (P < .001) compared with sham-treated aged mice, and aged RCT-12wk mice had a significantly increased fat area ratio compared with aged RCT-4wk mice (P < .001). The fat area was significantly larger in both the aged RCT-4wk (P = .002) and RCT-12wk mice (P < .001) than in the corresponding young mice. Muscular fibrosis was significantly increased in aged RCT-12wk mice compared with aged sham-treated mice (P = .005) and young RCT-12wk mice (P = .016). There were also significant increases in the expression of perilipin and transcripts of adipogenic and fibrogenic differentiation markers in aged RCT mice compared with young RCT mice. CONCLUSION: The present results show that aging is critically involved in the pathology of muscular fatty infiltration and fibrosis after RCT, and muscular degeneration progresses over time in aged mice. CLINICAL RELEVANCE: Aging promotes the progression of muscle degeneration in a mouse RCT model. Furthermore, this study shows that muscle degeneration occurs in aged mice even without denervation and that the model described in the present study is a useful tool for studying the pathology of muscle degeneration.

ADAM10 partially protects mice against influenza pneumonia by suppressing specific myeloid cell population.

The influenza virus infection poses a serious health threat worldwide. Myeloid cells play pivotal roles in regulating innate and adaptive immune defense. A disintegrin and metalloproteinase (ADAM) family of proteins contributes to various immune responses; however, the role of a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) in influenza virus infection remains largely unknown. Herein, we investigated its role, focusing on myeloid cells, during influenza virus infection in mice. ADAM10 gene (Adam10)flox/flox/Lyz2-Cre (Adam10ΔLyz2) and control Adam10flox/flox mice were intranasally infected with 200 plaque-forming units of influenza virus A/H1N1/PR8/34. Adam10ΔLyz2 mice exhibited a significantly higher mortality rate, stronger lung inflammation, and a higher virus titer in the lungs than control mice. Macrophages and inflammatory cytokines, such as TNF-α, IL-1ß, and CCL2, were increased in bronchoalveolar lavage fluid from Adam10ΔLyz2 mice following infection. CD11b+Ly6G-F4/80+ myeloid cells, which had an inflammatory monocyte/macrophage-like phenotype, were significantly increased in the lungs of Adam10ΔLyz2 mice. Adoptive transfer experiments suggested that these cells likely contributed to the poorer prognosis in Adam10ΔLyz2 mice. Seven days after infection, CD11b+Ly6G-F4/80+ lung cells exhibited significantly higher arginase-1 expression levels in Adam10ΔLyz2 mice than in control mice, whereas an arginase-1 inhibitor improved the prognosis of Adam10ΔLyz2 mice. Enhanced granulocyte-macrophage colony-stimulating factor (GM-CSF)/GM-CSF receptor signaling likely contributed to this process. Collectively, these results indicate that myeloid ADAM10 protects against influenza virus pneumonia and may be a promising therapeutic target.

Retinoic Acid Receptor Agonists Suppress Muscle Fatty Infiltration in Mice.

BACKGROUND: The infiltration of fat tissue into skeletal muscle, a condition referred to as muscle fatty infiltration or fatty degeneration, is regarded as an irreversible event that significantly compromises the motor function of skeletal muscle. PURPOSE: To investigate the effect of retinoic acid receptor (RAR) agonists in suppressing the adipogenic differentiation of fibroadipogenic progenitors (FAPs) in vitro and fatty infiltration after rotator cuff tear in mice. STUDY DESIGN: Controlled laboratory study. METHODS: FAPs isolated from mouse skeletal muscle were cultured in adipogenic differentiation medium in the presence or absence of an RAR agonist. At the end of cell culture, adipogenic differentiation was evaluated by gene expression analysis and oil red O staining. A mouse model of fatty infiltration-which includes the resection of the rotator cuff, removal of the humeral head, and denervation the supraspinatus muscle-was used to induce fatty infiltration in the supraspinatus muscle. The mice were orally or intramuscularly administered with an RAR agonist after the surgery. Muscle fatty infiltration was evaluated by histology and gene expression analysis. RESULTS: RAR agonists effectively inhibited the adipogenic differentiation of FAPs in vitro. Oral and intramuscular administration of RAR agonists suppressed the development of muscle fatty infiltration in the mice after rotator cuff tear. In accordance, we found a significant decrease in the number of intramuscular fat cells and suppressed expression in adipogenic markers. RAR agonists also increased the expression of the transcripts for collagens; however, an accumulation of collagenous tissues was not histologically evident in the present model. CONCLUSION: Muscle fatty infiltration can be alleviated by RAR agonists through suppressing the adipogenic differentiation of FAPs. The results also suggest that RAR agonists are potential therapeutic agents for treating patients who are at risk of developing muscle fatty infiltration. The consequence of the increased expression of collagen transcripts by RAR agonists needs to be clarified. CLINICAL RELEVANCE: RAR agonists can be used to prevent the development of muscle fatty infiltration after rotator cuff tear. Nevertheless, further studies are mandatory in a large animal model to examine the safety and efficacy of intramuscular injection of RAR agonists.

ADAM17 protects against elastase-induced emphysema by suppressing CD62L+ leukocyte infiltration in mice.

Pulmonary emphysema is a major manifestation of chronic obstructive pulmonary disease and is associated with chronic pulmonary inflammation caused by cigarette smoking, with contributions from immune cells such as neutrophils, macrophages, and lymphocytes. Although matrix metalloproteinases are well known to contribute to emphysema progression, the role of a disintegrin and metalloproteinase (ADAM) family proteins, other major metalloproteinases, in disease pathogenesis is largely unknown. ADAM17 is a major sheddase that cleaves various cell surface proteins, including CD62L, an adhesion molecule that plays a critical role in promoting the migration of immune cells to the site of inflammation. In the present study, we aimed to investigate the potential role of ADAM17 and CD62L in the development of elastase-induced emphysema. Control and Adam17flox/flox/Mx1-Cre (Adam17ΔMx1) mice (8-10 wk old) were intratracheally injected with 5 units of porcine pancreas elastase and monitored for 35 days after injection. Lung alveolar destruction was evaluated by analyzing the mean linear intercepts of lung tissue specimens and by histopathological examination. Mean linear intercepts data indicated that the degree of elastase-induced emphysema was significantly more severe in Adam17ΔMx1 mice. Furthermore, flow cytometry showed that CD62L+ neutrophil, CD62L+ macrophage, and CD62L+ B lymphocyte numbers were significantly increased in Adam17ΔMx1 mice. Moreover, the pharmacological depletion of CD62L+ cells with a CD62L-neutralizing antibody ameliorated the extent of emphysema in Adam17ΔMx1 mice. Collectively, these results suggest that ADAM17 possibly suppresses the progression of emphysema by proteolytically processing CD62L in immune cells and that ADAM17 and CD62L could be novel therapeutic targets for treating pulmonary emphysema.

Trans-pairing between osteoclasts and osteoblasts shapes the cranial base during development.

Bone growth is linked to expansion of nearby organs, as is the case for the cranial base and the brain. Here, we focused on development of the mouse clivus, a sloping surface of the basioccipital bone, to define mechanisms underlying morphological changes in bone in response to brain enlargement. Histological analysis indicated that both endocranial and ectocranial cortical bone layers in the basioccipital carry the osteoclast surface dorsally and the osteoblast surface ventrally. Finite element analysis of mechanical stress on the clivus revealed that compressive and tensile stresses appeared mainly on respective dorsal and ventral surfaces of the basioccipital bone. Osteoclastic bone resorption occurred primarily in the compression area, whereas areas of bone formation largely coincided with the tension area. These data collectively suggest that compressive and tensile stresses govern respective localization of osteoclasts and osteoblasts. Developmental analysis of the basioccipital bone revealed the clivus to be angled in early postnatal wild-type mice, whereas its slope was less prominent in Tnfsf11-/- mice, which lack osteoclasts. We propose that osteoclast-osteoblast "trans-pairing" across cortical bone is primarily induced by mechanical stress from growing organs and regulates shape and size of bones that encase the brain.

Innervation of the tibial epiphysis through the intercondylar foramen.

The periosteum and mineralized bone are innervated by nerves that sense pain. These include both myelinated and unmyelinated neurons with either free nerve endings or bearing nociceptors. Parasympathetic and sympathetic autonomic nerves also innervate bone. However, little is known about the route sensory nerves take leaving the epiphyses of long bones at the adult knee joint. Here, we used transgenic mice that express fluorescent Venus protein in Schwann cells (Sox10-Venus mice) to visualize myelinated and unmyelinated nerves in the tibial epiphysis. Immunofluorescence to detect a pan-neuronal marker and the sensory neuron markers calcitonin gene-related peptide (CGRP) and tropomyosin receptor kinase A (TrkA) also revealed Schwann cell-associated sensory neurons. Foramina in the intercondylar area of the tibia were conserved between rodents and primates. Venus-labeled fibers were detected within bone marrow of the proximal epiphysis, exited through foramina along with blood vessels in the intercondylar area of the tibia, and joined Venus-labeled fibers of the synovial membrane and meniscus. These data suggest that innervation of the subchondral plate and trabecular bone within the tibial epiphysis carries pain signals from the knee joint to the brain through intercondylar foramina.

Granulocyte-colony stimulating factor enhances load-induced muscle hypertrophy in mice.

Granulocyte-colony stimulating factor (G-CSF) is a cytokine crucially involved in the regulation of granulopoiesis and the mobilization of hematopoietic stem cells from bone marrow. However, emerging data suggest that G-CSF exhibits more diverse functions than initially expected, such as conferring protection against apoptosis to neural cells and stimulating mitogenesis in cardiomyocytes and skeletal muscle stem cells after injury. In the present study, we sought to investigate the potential contribution of G-CSF to the regulation of muscle volume. We found that the administration of G-CSF significantly enhances muscle hypertrophy in two different muscle overload models. Interestingly, there was a significant increase in the transcripts of both G-CSF and G-CSF receptors in the muscles that were under overload stress. Using mutant mice lacking the G-CSF receptor, we confirmed that the anabolic effect is dependent on the G-CSF receptor signaling. Furthermore, we found that G-CSF increases the diameter of myotubes in vitro and induces the phosphorylation of AKT, mTOR, and ERK1/2 in the myoblast-like cell line C2C12 after differentiation induction. These findings indicate that G-CSF is involved in load-induced muscle hypertrophy and suggest that G-CSF is a potential agent for treating patients with muscle loss and sarcopenia.

Relationship between patellar mobility and patellofemoral joint cartilage degeneration after anterior cruciate ligament reconstruction.

Patellofemoral cartilage degeneration is a potential complication of anterior cruciate ligament reconstruction (ACLR) surgery. Hypomobility of the patella in the coronal plane is often observed after ACLR. Few studies, however, have examined the relationship between cartilage degeneration in the patellofemoral joint and mobility after ACLR. The present study investigated 1) the coronal mobility of the patella after ACLR, 2) the relationship between patellar mobility and cartilage degeneration of the patellofemoral joint, and 3) the relationship between patellar mobility and knee joint function after ACLR. Forty patients who underwent medial hamstring-based ACLR participated in the study. Lateral and medial patellar displacements were assessed with a modified patellofemoral arthrometer, and the absolute values of the displacements were normalized to patient height. The International Cartilage Repair Society (ICRS) cartilage injury classification of the patellar and femoral (trochlear) surfaces, and the Lysholm Knee Scoring Scale were used to evaluate knee function. Lateral and medial patellar displacements were reduced compared with the non-operated knee at the second-look arthroscopy and bone staple extraction operation (second operation; 24.4 ± 7.9 months after ACLR, P<0.01). The ICRS grades of the patellofemoral joint (patella and trochlea) were significantly worse than those pre-ACLR. Neither lateral nor medial patellar mobility, however, were significantly correlated with the ICRS grade or the Lysholm score. Although patellar mobility at approximately 2 years after ACLR was decreased compared to the non-operated knee, small displacement of the patella was not related to cartilage degeneration or knee joint function at the time of the second operation.

IRE1α/XBP1-mediated branch of the unfolded protein response regulates osteoclastogenesis.

The unfolded protein response (UPR) is a cellular adaptive mechanism that is activated in response to the accumulation of unfolded proteins in the endoplasmic reticulum. The inositol-requiring protein-1α/X-box-binding protein-mediated (IRE1α/XBP1-mediated) branch of the UPR is highly conserved and has also been shown to regulate various cell-fate decisions. Herein, we have demonstrated a crucial role for the IREα/XBP1-mediated arm of the UPR in osteoclast differentiation. Using murine models, we found that the conditional abrogation of IRE1α in bone marrow cells increases bone mass as the result of defective osteoclastic bone resorption. In osteoclast precursors, IRE1α was transiently activated during osteoclastogenesis, and suppression of the IRE1α/XBP1 pathway in these cells substantially inhibited the formation of multinucleated osteoclasts in vitro. We determined that XBP1 directly binds the promoter and induces transcription of the gene encoding the master regulator of osteoclastogenesis nuclear factor of activated T cells cytoplasmic 1 (NFATc1). Moreover, activation of IRE1α was partially dependent on Ca2+ oscillation mediated by inositol 1,4,5-trisphosphate receptors 2 and 3 (ITPR2 and ITPR3) in the endoplasmic reticulum, as pharmacological inhibition or deletion of these receptors markedly decreased Xbp1 mRNA processing. The present study thus reveals an intracellular pathway that integrates the UPR and osteoclast differentiation through activation of the IRE1α/XBP1 pathway.

ADAM17 regulates IL-1 signaling by selectively releasing IL-1 receptor type 2 from the cell surface.

Interleukin (IL)-1 is one of the most evolutionarily conserved cytokines and plays an essential role in the regulation of innate immunity. IL-1 binds to two different receptors, IL-1R1 and IL-1R2, which share approximately 28% amino acid homology. IL-1R1 contains a cytoplasmic domain and is capable of transducing cellular signals; by contrast, IL-1R2 lacks a functional cytoplasmic domain and serves as a decoy receptor for IL-1. Interestingly, IL-1R2 is proteolytically cleaved and also functions as a soluble receptor that blocks IL-1 activity. In the present study, we examined the shedding properties of IL-1R2 and demonstrate that ADAM17 is de facto the major sheddase for IL-1R2 and that introducing a mutation into the juxta-membrane domain of IL-1R2 significantly desensitizes IL-1R2 to proteolytic cleavage. IL-1R1 was almost insensitive to ADAM17-dependent cleavage; however, the replacement of the juxta-membrane domain of IL-R1 with that of IL-1R2 significantly increased the sensitivity of IL-1R1 to shedding. Furthermore, we demonstrate that ADAM17 indirectly enhances IL-1 signaling in a cell-autonomous manner by selectively cleaving IL-1R2. Taken together, the data collected in the present study indicate that ADAM17 affects sensitivity to IL-1 by changing the balance between IL-1R1 and the decoy receptor IL-1R2.

Conditional inactivation of TNFα-converting enzyme in chondrocytes results in an elongated growth plate and shorter long bones.

TNFα-converting enzyme (TACE) is a membrane-bound proteolytic enzyme with essential roles in the functional regulation of TNFα and epidermal growth factor receptor (EGFR) ligands. Previous studies have demonstrated critical roles for TACE in vivo, including epidermal development, immune response, and pathological neoangiogenesis, among others. However, the potential contribution of TACE to skeletal development is still unclear. In the present study, we generated a Tace mutant mouse in which Tace is conditionally disrupted in chondrocytes under the control of the Col2a1 promoter. These mutant mice were fertile and viable but all exhibited long bones that were approximately 10% shorter compared to those of wild-type animals. Histological analyses revealed that Tace mutant mice exhibited a longer hypertrophic zone in the growth plate, and there were fewer osteoclasts at the chondro-osseous junction in the Tace mutant mice than in their wild-type littermates. Of note, we found an increase in osteoprotegerin transcripts and a reduction in Rankl and Mmp-13 transcripts in the TACE-deficient cartilage, indicating that dysregulation of these genes is causally related to the skeletal defects in the Tace mutant mice. Furthermore, we also found that phosphorylation of EGFR was significantly reduced in the cartilage tissue lacking TACE, and that suppression of EGFR signaling increases osteoprotegerin transcripts and reduces Rankl and Mmp-13 transcripts in primary chondrocytes. In accordance, chondrocyte-specific abrogation of Egfr in vivo resulted in skeletal defects nearly identical to those observed in the Tace mutant mice. Taken together, these data suggest that TACE-EGFR signaling in chondrocytes is involved in the turnover of the growth plate during postnatal development via the transcriptional regulation of osteoprotegerin, Rankl, and Mmp-13.

Systemic overexpression of TNFα-converting enzyme does not lead to enhanced shedding activity in vivo.

TNFα-converting enzyme (TACE/ADAM17) is a membrane-bound proteolytic enzyme with a diverse set of target molecules. Most importantly, TACE is indispensable for the release and activation of pro-TNFα and the ligands for epidermal growth factor receptor in vivo. Previous studies suggested that the overproduction of TACE is causally related to the pathogenesis of inflammatory diseases and cancers. To test this hypothesis, we generated a transgenic line in which the transcription of exogenous Tace is driven by a CAG promoter. The Tace-transgenic mice were viable and exhibited no overt defects, and the quantitative RT-PCR and Western blot analyses confirmed that the transgenically introduced Tace gene was highly expressed in all of the tissues examined. The Tace-transgenic mice were further crossed with Tace⁻/⁺ mice to abrogate the endogenous TACE expression, and the Tace-transgenic mice lacking endogenous Tace gene were also viable without any apparent defects. Furthermore, there was no difference in the serum TNFα levels after lipopolysaccharide injection between the transgenic mice and control littermates. These observations indicate that TACE activity is not necessarily dependent on transcriptional regulation and that excess TACE does not necessarily result in aberrant proteolytic activity in vivo.

The IRE1α-XBP1 pathway positively regulates parathyroid hormone (PTH)/PTH-related peptide receptor expression and is involved in pth-induced osteoclastogenesis.

To address the "endoplasmic reticulum stress" triggered by the burden of protein synthesis, the unfolded protein response is induced during osteoblast differentiation. In this study, we show that the transcription of parathyroid hormone (PTH)/PTH-related peptide receptor (PTH1R) is regulated by one of the endoplasmic reticulum-stress mediators, the IRE1α-XBP1 pathway, in osteoblasts. We found that the increase in Pth1r transcription upon BMP2 treatment is significantly suppressed in mouse embryonic fibroblasts lacking IRE1α. As expected, gene silencing of Ire1α and Xbp1 resulted in a decrease in Pth1r transcripts in BMP2-treated embryonic fibroblasts. We identified two potential binding sites for XBP1 in the promoter region of Pth1r and found that XBP1 promotes the transcription of Pth1r by directly binding to those sites. Moreover, we confirmed that the gene silencing of Xbp1 suppresses PTH-induced Rankl expression in primary osteoblasts and thereby abolishes osteoclast formation in an in vitro model of osteoclastogenesis. Thus, the present study reveals potential involvement of the IRE1α-XBP1 pathway in PTH-induced osteoclastogenesis through the regulation of PTH1R expression.

A novel multi-kinase inhibitor pazopanib suppresses growth of synovial sarcoma cells through inhibition of the PI3K-AKT pathway.

Synovial sarcoma is an aggressive soft tissue sarcoma with only a modest response to conventional cytotoxic agents. In the present study, we evaluated the potential antitumor effects of a novel anti-angiogenesis agent, pazopanib, against synovial sarcoma cells. We found that pazopanib directly inhibited the growth of synovial sarcoma cells by inducing G1 arrest. Multiplex analyses revealed that the PI3K-AKT pathway was highly suppressed in pazopanib-sensitive synovial sarcoma cells. Furthermore, administration of pazopanib highly suppressed the tumor growth in a xenograft model. Taken together, these results suggest pazopanib as a possible agent against synovial sarcoma and may warrant further clinical studies.

The transient appearance of zipper-like actin superstructures during the fusion of osteoclasts.

Multinucleated osteoclasts are responsible for bone resorption. Hypermultinucleated osteoclasts are often observed in some bone-related diseases such as Paget's disease and cherubism. The cellular mechanics controlling the size of osteoclasts is poorly understood. We introduced EGFP-actin into RAW 264.7 cells to monitor actin dynamics during osteoclast differentiation. Before their terminal differentiation into osteoclasts, syncytia displayed two main types of actin assembly, podosome clusters and clusters of zipper-like structures. The zipper-like structures morphologically resembled the adhesion zippers found at the initial stage of cell-cell adhesion in keratinocytes. In the zipper-like structure, Arp3 and cortactin overlapped with the distribution of dense F-actin, whereas integrin ß3, paxillin and vinculin were localized to the periphery of the structure. The structure was negative for WGA-lectin staining and biotin labeling. The zipper-like structure broke down and transformed into a large actin ring, called a podosome belt. Syncytia containing clusters of zipper-like structures had more nuclei than those with podosome clusters. Differentiated osteoclasts with a podosome belt also formed the zipper-like structure at the cell contact site during cell fusion. The breakdown of the cell contact site resulted in the fusion of the podosome belts following plasma membrane fusion. Additionally, osteoclasts in mouse calvariae formed the zipper-like structure in the sealing zone. Therefore, we propose that the zipper-like actin superstructures might be involved in cell-cell interaction to achieve efficient multinucleation of osteoclasts. Understanding of the zipper-like structure might lead to selective therapeutics for bone diseases caused by hypermultinucleated osteoclasts.

Dual functions of cell-autonomous and non-cell-autonomous ADAM10 activity in granulopoiesis.

Previous studies have revealed various extrinsic stimuli and factors involved in the regulation of hematopoiesis. Among these, Notch-mediated signaling has been suggested to be critically involved in this process. Herein, we show that conditional inactivation of ADAM10, a membrane-bound protease with a crucial role in Notch signaling (S2 cleavage), results in myeloproliferative disorder (MPD) highlighted by severe splenomegaly and increased populations of myeloid cells and hematopoietic stem cells. Reciprocal transfer of bone marrow cells between wild-type and ADAM10 mutant mice revealed that ADAM10 activity in both hematopoietic and nonhematopoietic cells is involved in the development of MPD. Notably, we found that MPD caused by lack of ADAM10 in nonhematopoietic cells was mediated by G-CSF, whereas MPD caused by ADAM10-deficient hematopoietic cells was not. Taken together, the present findings reveal previously undescribed nonredundant roles of cell-autonomous and non-cell-autonomous ADAM10 activity in the maintenance of hematopoiesis.

Receptor activator of NF-kappaB (RANK) ligand induces ectodomain shedding of RANK in murine RAW264.7 macrophages.

Osteoclastogenesis is a highly sophisticated process that involves a variety of membrane-bound proteins expressed in osteoblasts and osteoclast precursors. Over the past several years, proteolytic cleavage and release of the ectodomain of membrane-bound proteins, also referred to as ectodomain shedding, has emerged as an important posttranslational regulatory mechanism for modifying the function of cell surface proteins. In line with this notion, several membrane-bound molecules involved in osteoclastogenesis, including CSF-1R and receptor activator of NF-kappaB ligand (RANKL), are proteolytically cleaved and released from the cell surface. In this study, we investigated whether receptor activator of NF-kappaB (RANK), one of the most essential molecules in osteoclastogenesis, undergoes ectodomain shedding. The results showed that RANK is released in the form of a soluble monomeric protein and that TNF-alpha-converting enzyme is involved in this activity. We also identified potential cleavage sites in the juxtamembrane domain of RANK and found that rRANKL induces RANK shedding in a macrophage-like cell line RAW264.7 via TNFR-associated factor 6 and MAPK pathways. Furthermore, we found that RANKL-induced osteoclastogenesis is accelerated in TNF-alpha-converting enzyme-deficient osteoclast precursors. These observations suggest the potential involvement of ectodomain shedding in the regulation of RANK functions and may provide novel insights into the mechanisms of osteoclastogenesis.