ABSTRACT
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.
ABSTRACT
Removal of fuel debris is planned to start at Unit 2 of the Fukushima Daiichi Nuclear Power Plant. During the removal, it is desirable to distinguish fuel debris from radioactive wastes and to sort the fuel debris accordingly to the amounts of nuclear material contained. Muon scattering tomography invented at Los Alamos in the early 2000s is highly sensitivity to high-atomic-number materials such as uranium. A muon scanner to sort the debris is designed and currently in production. One of the challenges is to operate the muon scanner in the presence of high γ-ray radiations from the debris: muon-event-identification electronics and a muon-tracking algorithm in the presence of high γ-ray radiations were developed.
ABSTRACT
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.
Subject(s)
Rotator Cuff Injuries , Adipose Tissue/pathology , Aging , Animals , Female , Mice , Mice, Inbred C57BL , Muscular Atrophy , Rotator Cuff/pathology , Rotator Cuff Injuries/pathologyABSTRACT
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.
Subject(s)
ADAM10 Protein/metabolism , Amyloid Precursor Protein Secretases/metabolism , Arginase/biosynthesis , Influenza A Virus, H1N1 Subtype/metabolism , Macrophages/immunology , Membrane Proteins/metabolism , Myeloid Cells/immunology , Orthomyxoviridae Infections/pathology , ADAM10 Protein/genetics , Adoptive Transfer/methods , Amyloid Precursor Protein Secretases/genetics , Animals , Arginase/antagonists & inhibitors , Bronchoalveolar Lavage Fluid/chemistry , Bronchoalveolar Lavage Fluid/cytology , Cytokines/analysis , Immunity, Innate/immunology , Macrophages/transplantation , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Myeloid Cells/transplantation , Orthomyxoviridae Infections/mortality , Orthomyxoviridae Infections/prevention & control , Prognosis , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/metabolismABSTRACT
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.
Subject(s)
ADAM17 Protein/metabolism , L-Selectin/metabolism , Leukocytes/metabolism , Pulmonary Emphysema/chemically induced , Pulmonary Emphysema/immunology , Animals , Antioxidants/metabolism , Apoptosis , Bronchoalveolar Lavage Fluid , Cell Count , Chemokines/metabolism , Epithelial Cells/metabolism , Gene Expression Regulation , Lung/pathology , Macrophages/pathology , Matrix Metalloproteinase 12/metabolism , Mice, Inbred C57BL , Neutralization Tests , Oxidants/metabolism , Pancreatic Elastase , Pulmonary Emphysema/genetics , Pulmonary Emphysema/pathologyABSTRACT
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.
Subject(s)
Granulocyte Colony-Stimulating Factor/pharmacology , Muscles/pathology , Animals , Cell Line , Cell Size/drug effects , Disease Models, Animal , Female , Granulocyte Colony-Stimulating Factor/administration & dosage , Hypertrophy , Immobilization , MAP Kinase Signaling System/drug effects , Mice, Inbred C57BL , Muscle Fibers, Skeletal/drug effects , Muscle Fibers, Skeletal/metabolism , Muscle Fibers, Skeletal/pathology , Muscles/drug effects , Phosphorylation/drug effects , Receptors, Granulocyte Colony-Stimulating Factor/metabolism , TOR Serine-Threonine Kinases/metabolism , Tenotomy , Weight-BearingABSTRACT
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.
Subject(s)
Anterior Cruciate Ligament Reconstruction/instrumentation , Anterior Cruciate Ligament Reconstruction/methods , Knee Joint/physiopathology , Patellar Ligament/physiopathology , Patellofemoral Joint/physiopathology , Adolescent , Adult , Female , Humans , Male , Middle Aged , Young AdultABSTRACT
Satellite cells (SCs) are muscle-specific stem cells that are essential for the regeneration of damaged muscles. Although SCs have a robust capacity to regenerate myofibers, the number of SCs decreases with aging, leading to insufficient recovery after muscle injury. We herein show that ADAM10 (a disintegrin and metalloprotease 10), a membrane-bound proteolytic enzyme with a critical role in Notch processing (S2 cleavage), is essential for the maintenance of SC quiescence. We generated mutant mice in which ADAM10 in SCs can be conditionally abrogated by tamoxifen injection. Tamoxifen-treated mutant mice did not show any apparent defects and grew normally under unchallenged conditions. However, these mice showed a nearly complete loss of muscle regeneration after chemically induced muscle injury. In situ hybridization and flow cytometric analyses revealed that the mutant mice had significantly less SCs compared with wild type controls. Of note, we found that inactivation of ADAM10 in SCs severely compromised Notch signaling and led to dysregulated myogenic differentiation, ultimately resulting in deprivation of the SC pool in vivo. Taken together, the present findings underscore the role of ADAM10 as an indispensable component of Notch signaling in SCs and for maintaining the SC pool.
Subject(s)
ADAM Proteins/metabolism , Amyloid Precursor Protein Secretases/metabolism , Membrane Proteins/metabolism , Satellite Cells, Skeletal Muscle/metabolism , ADAM Proteins/genetics , ADAM10 Protein , Amyloid Precursor Protein Secretases/genetics , Animals , Cell Differentiation , Membrane Proteins/genetics , Mice , Mice, Transgenic , Receptors, Notch/metabolism , Satellite Cells, Skeletal Muscle/cytology , Signal TransductionABSTRACT
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.
Subject(s)
ADAM Proteins/metabolism , Interleukin-1/metabolism , Receptors, Interleukin-1 Type II/metabolism , Signal Transduction , ADAM Proteins/genetics , ADAM17 Protein , Amino Acid Sequence , Animals , Binding Sites/genetics , Blotting, Western , COS Cells , Cells, Cultured , Chlorocebus aethiops , Embryo, Mammalian/cytology , Fibroblasts/cytology , Fibroblasts/metabolism , Mice, Knockout , Molecular Sequence Data , Proteolysis/drug effects , Receptors, Interleukin-1 Type I/genetics , Receptors, Interleukin-1 Type I/metabolism , Receptors, Interleukin-1 Type II/genetics , Sequence Homology, Amino Acid , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
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.
Subject(s)
DNA-Binding Proteins/genetics , Endoribonucleases/genetics , Osteoblasts/metabolism , Parathyroid Hormone-Related Protein/agonists , Parathyroid Hormone/agonists , Protein Serine-Threonine Kinases/genetics , Receptor, Parathyroid Hormone, Type 1/genetics , Transcription Factors/genetics , Animals , Binding Sites , Bone Morphogenetic Protein 2/pharmacology , Cell Differentiation/drug effects , Cells, Cultured , DNA-Binding Proteins/antagonists & inhibitors , Embryo, Mammalian , Endoplasmic Reticulum Stress/drug effects , Endoplasmic Reticulum Stress/genetics , Endoribonucleases/antagonists & inhibitors , Endoribonucleases/deficiency , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/metabolism , Gene Expression Regulation, Developmental/drug effects , Mice , Osteoblasts/cytology , Osteoblasts/drug effects , Parathyroid Hormone/genetics , Parathyroid Hormone/metabolism , Parathyroid Hormone-Related Protein/genetics , Parathyroid Hormone-Related Protein/metabolism , Promoter Regions, Genetic , Protein Binding , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/deficiency , RANK Ligand/antagonists & inhibitors , RANK Ligand/genetics , Receptor, Parathyroid Hormone, Type 1/metabolism , Regulatory Factor X Transcription Factors , Signal Transduction/drug effects , Transcription Factors/antagonists & inhibitors , Transcription, Genetic/drug effects , Unfolded Protein Response/drug effects , X-Box Binding Protein 1ABSTRACT
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.
Subject(s)
Actins/chemistry , Actins/metabolism , Osteoclasts/cytology , Osteoclasts/metabolism , Animals , Bone Resorption/metabolism , Bone Resorption/pathology , Cell Adhesion , Cell Compartmentation , Cell Differentiation , Cell Fusion , Cell Line , Giant Cells/cytology , Giant Cells/metabolism , Mice , Microscopy, Electron, Transmission , Models, BiologicalABSTRACT
BACKGROUND: The regenerative and adaptive capacity of skeletal muscles reduces with age, leading to severe disability and frailty in the elderly. Therefore, development of effective therapeutic interventions for muscle wasting is important both medically and socioeconomically. In the present study, we aimed to elucidate the potential contribution of fibro-adipogenic progenitors (FAPs), which are mesenchymal stem cells in skeletal muscles, to immobilization-induced muscle atrophy. METHODS: Young (2-3 months), adult (12-14 months), and aged (20-22 months) mice were used for analysis. Muscle atrophy was induced by immobilizing the hind limbs with a steel wire. FAPs were isolated from the hind limbs on days 0, 3, and 14 after immobilization for transcriptome analysis. The expression of ST2 and IL-33 in FAPs was evaluated by flow cytometry and immunostaining, respectively. To examine the role of IL-33-ST2 signaling in vivo, we intraperitoneally administered recombinant IL-33 or soluble ST2 (sST2) twice a week throughout the 2-week immobilization period. After 2-week immobilization, the tibialis anterior muscles were harvested and the cross-sectional area of muscle fibers was evaluated. RESULTS: The number of FAPs increased with the progression of muscle atrophy after immobilization in all age-groups. Transcriptome analysis of FAPs collected before and after immobilization revealed that Il33 and Il1rl1 transcripts, which encode the IL-33 receptor ST2, were transiently induced in young mice and, to a lesser extent, in aged mice. The number of FAPs positive for ST2 increased after immobilization in young mice. The number of ST2-positive FAPs also increased after immobilization in aged mice, but the difference from the baseline was not statistically significant. Immunostaining for IL-33 in the muscle sections revealed a significant increase in the number of FAPs expressing IL-33 after immobilization. Administration of recombinant IL-33 suppressed immobilization-induced muscle atrophy in aged mice but not in young mice. CONCLUSIONS: Our data reveal a previously unknown protective role of IL-33-ST2 signaling against immobilization-induced muscle atrophy in FAPs and suggest that IL-33-ST2 signaling is a potential new therapeutic target for alleviating disuse muscle atrophy, particularly in older adults.
Subject(s)
Interleukin-1 Receptor-Like 1 Protein , Interleukin-33 , Humans , Aged , Mice , Animals , Interleukin-33/metabolism , Interleukin-1 Receptor-Like 1 Protein/genetics , Interleukin-1 Receptor-Like 1 Protein/metabolism , Adipogenesis , Muscle, Skeletal/metabolism , Muscular Atrophy/etiology , Muscular Atrophy/prevention & control , Muscular Atrophy/metabolism , Cell Differentiation/physiologyABSTRACT
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.
Subject(s)
ADAM Proteins/genetics , Amyloid Precursor Protein Secretases/genetics , Hematopoiesis/genetics , Membrane Proteins/genetics , Myeloid Cells/metabolism , Myeloproliferative Disorders/genetics , ADAM Proteins/metabolism , ADAM10 Protein , Amyloid Precursor Protein Secretases/metabolism , Animals , Bone Marrow Cells/metabolism , Cytokines/blood , Cytokines/metabolism , Female , Flow Cytometry , Granulocyte Colony-Stimulating Factor/blood , Granulocyte Colony-Stimulating Factor/metabolism , Hematopoietic Stem Cells/metabolism , Male , Membrane Proteins/metabolism , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Myeloproliferative Disorders/metabolism , Receptor, Notch1/genetics , Receptor, Notch1/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Splenomegaly/genetics , Splenomegaly/metabolism , T-Lymphocytes/metabolismABSTRACT
During skeletal development, osteoblasts produce large amounts of extracellular matrix proteins and must therefore increase their secretory machinery to handle the deposition. The accumulation of unfolded protein in the endoplasmic reticulum induces an adoptive mechanism called the unfolded protein response (UPR). We show that one of the most crucial UPR mediators, inositol-requiring protein 1α (IRE1α), and its target transcription factor X-box binding protein 1 (XBP1), are essential for bone morphogenic protein 2-induced osteoblast differentiation. Furthermore, we identify Osterix (Osx, a transcription factor that is indispensible for bone formation) as a target gene of XBP1. The promoter region of the Osx gene encodes two potential binding motifs for XBP1, and we show that XBP1 binds to these regions. Thus, the IRE1α-XBP1 pathway is involved in osteoblast differentiation through promoting Osx transcription.
Subject(s)
Cell Differentiation/physiology , DNA-Binding Proteins/metabolism , Endoribonucleases/metabolism , Gene Expression Regulation/physiology , Osteoblasts/physiology , Protein Serine-Threonine Kinases/metabolism , Transcription Factors/metabolism , Animals , Chromatin Immunoprecipitation , Endoplasmic Reticulum/metabolism , Endoribonucleases/genetics , Humans , Luciferases , Mice , Mice, Knockout , Protein Serine-Threonine Kinases/genetics , Regulatory Factor X Transcription Factors , Reverse Transcriptase Polymerase Chain Reaction , Sp7 Transcription Factor , X-Box Binding Protein 1ABSTRACT
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.
ABSTRACT
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.
Subject(s)
ADAM Proteins/metabolism , Macrophages/metabolism , RANK Ligand/metabolism , Receptor Activator of Nuclear Factor-kappa B/metabolism , ADAM Proteins/deficiency , ADAM Proteins/genetics , ADAM17 Protein , Animals , Binding Sites , Blotting, Western , COS Cells , Cell Line , Chlorocebus aethiops , Flow Cytometry , Macrophages/cytology , Membrane Proteins/chemistry , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Knockout , Mitogen-Activated Protein Kinases/metabolism , NFATC Transcription Factors/genetics , NFATC Transcription Factors/metabolism , Osteoclasts/cytology , Osteoclasts/metabolism , RANK Ligand/genetics , Receptor Activator of Nuclear Factor-kappa B/chemistry , Receptor Activator of Nuclear Factor-kappa B/genetics , Reverse Transcriptase Polymerase Chain Reaction , Solubility , TNF Receptor-Associated Factor 6/genetics , TNF Receptor-Associated Factor 6/metabolism , Transfection , Up-RegulationABSTRACT
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.
Subject(s)
Carcinoma, Squamous Cell , Mouth Neoplasms , Antibodies, Monoclonal, Humanized/therapeutic use , Bortezomib/pharmacology , Bortezomib/therapeutic use , Carcinoma, Squamous Cell/drug therapy , Carcinoma, Squamous Cell/pathology , Cell Line, Tumor , Cetuximab/pharmacology , Cetuximab/therapeutic use , Humans , Mouth Neoplasms/drug therapy , NF-kappa B , Neoplasm Recurrence, Local , Paclitaxel , Proteasome Inhibitors/pharmacology , RNA, Messenger/geneticsABSTRACT
IL-27 was first discovered as a factor supporting initial Th1 immune responses. Subsequent studies revealed that this cytokine has pleiotropic effects, including inhibition of certain immune cells, a regulatory role in hemopoietic stem cell differentiation, and antitumor activities. However, the role of human IL (hIL)-27 in human osteoclast precursors and inflammatory bone disease is unclear. Here, we examined the direct effect of hIL-27 on human osteoclastogenesis. Human bone marrow cells cultured in MethoCult medium containing human (h) GM-CSF, human stem cell factor, and hIL-3 expressed Mac-1, c-kit, and c-Fms. These cells, called hCFU-GMs, also expressed the IL-27 receptor, an IL-27Ralpha (WSX-1)/gp130 heterodimer. Cultivation in hM-CSF and human receptor activator of NF-kappaB ligand induced the differentiation of tartrate-resistant acid phosphatase-positive multinucleated cells (osteoclasts) from hCFU-GMs, and hIL-27 inhibited this osteoclastogenesis in a dose-dependent manner. hIL-27 also repressed bone resorption by osteoclasts on a dentine slice. hIL-27 caused a remarkable increase in STAT1 phosphorylation and enhanced the STAT1 protein level. It also inhibited the expression of receptor activator of NF-kappaB ligand-induced c-Fos and cytoplasmic, calcineurin-dependent 1 NFAT (NFATc1), which are indispensable transcription factors for osteoclastogenesis. Fludarabine, a STAT1 inhibitor, and STAT1 small interfering RNA partially rescued the inhibition of osteoclastogenesis by IL-27. A WSX-1 deficiency caused severe inflammatory bone destruction primed by Escherichia coli cell wall lysate in vivo. Therefore, hIL-27 may act as an anti-inflammatory cytokine in human bone destruction, by inhibiting osteoclastogenesis from hCFU-GMs via STAT1-dependent down-regulation of the transcription factor c-Fos. Our results suggest that hIL-27 may prove useful as a therapeutic target for inflammatory bone destruction.
Subject(s)
Down-Regulation/immunology , Granulocyte-Macrophage Colony-Stimulating Factor/biosynthesis , Interleukins/physiology , Osteoclasts/immunology , Osteoclasts/metabolism , Proto-Oncogene Proteins c-fos/antagonists & inhibitors , RANK Ligand/antagonists & inhibitors , RANK Ligand/physiology , STAT1 Transcription Factor/physiology , Adult , Animals , Cells, Cultured , Humans , Inflammation Mediators/metabolism , Inflammation Mediators/physiology , Interleukins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Middle Aged , Osteoclasts/pathology , Proto-Oncogene Proteins c-fos/biosynthesis , Receptors, Cytokine/deficiency , Receptors, Cytokine/genetics , Receptors, Cytokine/metabolism , Receptors, Interleukin , Stem Cells/immunology , Stem Cells/metabolism , Stem Cells/pathologyABSTRACT
BACKGROUND: Osteoarthritis (OA) is a common disease, afflicting many sufferers with both pain and functional disorders. Various therapies have been attempted for OA, but no fully effective treatment has been established yet. Apoptosis of chondrocytes caused by reactive oxygen species (ROS) has been considered important in the pathogenesis of OA. The progression of OA may be prevented by suppressing apoptosis of chondrocytes. Geranylgeranylacetone (GGA) has been used as an anti-ulcer drug in Japan for more than 20 years. Several recent studies have shown that GGA can induce heat shock protein (HSP) and exert cytoprotective actions on a large variety of cells and tissues. In this study, we investigated the effects of GGA on the apoptosis of OA chondrocytes induced by hydrogen peroxide (H(2)O(2)). METHODS: Human isolated OA chondrocytes were cultured in the absence or presence of GGA. Cell viability, caspase 3/7 and 9 activities, HSP70 mRNA and protein expressions were examined, and morphological analyses were conducted after exposure of cells to H(2)O(2) to induce apoptosis. RESULTS: Geranylgeranylacetone dose-dependently reversed the H(2)O(2)-induced decrease in cell viability. It was recognized that GGA rendered OA chondrocytes resistant to H(2)O(2)-induced apoptosis from Hoechst 33342 staining and TUNEL staining. Caspases 3 and 9 were activated by addition of H(2)O(2), and GGA suppressed this H(2)O(2)-induced activation of both caspases. H(2)O(2)-induced induction of HSP70 was enhanced in OA chondrocytes by pretreatment with GGA. The results showed that GGA can suppress apoptosis of chondrocytes and enhance production of HSP70. CONCLUSIONS: This study is the first, to our knowledge, to demonstrate that GGA protects OA chondrocytes from H(2)O(2)-induced apoptosis, at least in part by enhancing HSP70 production. These results indicate that GGA is a potentially useful drug for the treatment of OA.
Subject(s)
Apoptosis/drug effects , Chondrocytes/drug effects , Chondrocytes/physiology , Diterpenes/pharmacology , Osteoarthritis/pathology , Aged , Aged, 80 and over , Cells, Cultured , Female , Humans , Hydrogen Peroxide/pharmacology , MaleABSTRACT
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.