Chronic kidney disease (CKD) is a significant global health issue and often involves CKD-mineral and bone disorder (MBD) and sarcopenia. Plasminogen activator inhibitor-1 (PAI-1) is an inhibitor of fibrinolysis. PAI-1 has been implicated in the pathogenesis of osteoporosis and muscle wasting induced by inflammatory conditions. However, the roles of PAI-1 in CKD-MBD and sarcopenia remain unknown. Therefore, the present study investigated the roles of PAI-1 in bone loss and muscle wasting induced by adenine in PAI-1-deficient mice. CKD was induced in PAI-1+/+ and PAI-1-/- mice by administration of adenine for ten weeks. Muscle wasting was assessed by grip strength test, quantitative computed tomography (CT) analysis and muscle weight measurement. Osteoporosis was assessed by micro-CT analysis of femoral microstructural parameters. PAI-1 deficiency did not affect adenine-induced decreases in body weight and food intake or renal dysfunction in male or female mice. PAI-1 deficiency also did not affect adenine-induced decreases in grip strength, muscle mass in the lower limbs, or the tissue weights of the gastrocnemius, soleus, and tibialis anterior muscles in male or female mice. PAI-1 deficiency aggravated trabecular bone loss in CKD-induced male mice, but significantly increased trabecular bone in CKD-induced female mice. On the other hand, PAI-1 deficiency did not affect cortical bone loss in CKD-induced mice. In conclusion, PAI-1 is not critical for the pathophysiology of CKD-MBD or CKD-induced sarcopenia in mice. However, PAI-1 may be partly related to bone metabolism in trabecular bone in the CKD state with sex differences.

Matrix vesicles (MtVs) are one of the extracellular vesicles (EVs) secreted by osteoblasts. Although MtVs have a classically-defined function as an initiator of ossification and recent findings suggest a role for MtVs in the regulation of bone cell biology, the effects of MtVs on bone repair remain unclear. In the present study, we employed collagenase-released EVs (CREVs) containing abundant MtVs from mouse osteoblasts. CREVs were administered locally in gelatin hydrogels to damaged sites after a femoral bone defect in mice. CREVs exhibited the characteristics of MtVs with a diameter <200 nm. The local administration of CREVs significantly promoted the formation of new bone with increases in the number of alkaline phosphatase (ALP)-positive cells and cartilage formation at the damaged site after the femoral bone defect. However, the addition of CREVs to the medium did not promote the osteogenic differentiation of ST2 cells or the ALP activity or mineralization of mouse osteoblasts in vitro. In conclusion, we herein showed for the first time that MtVs enhanced bone repair after a femoral bone defect partly through osteogenesis and chondrogenesis in mice. Therefore, MtVs have potential as a tool for bone regeneration.

Extracellular vesicles (EVs) play crucial roles in physiological and pathophysiological processes. Although studies have described muscle-bone interactions via humoral factors, we reported that EVs from C2C12 muscle cells (Myo-EVs) suppress osteoclast formation. Current clinical evidence suggests that inflammation induces both sarcopenia and osteoporosis. Although tumor necrosis factor-α (TNF-α) is a critical proinflammatory factor, the influences of TNF-α on muscle-bone interactions and Myo-EVs are still unclear. In the present study, we investigated the effects of TNF-α stimulation of C2C12 cells on osteoclast formation and osteoblastic differentiation modulated by Myo-EVs in mouse cells. TNF-α significantly decreased the protein amount in Myo-EVs, but did not affect the Myo-EV size distribution. TNF-α treatment of C2C12 myoblasts significantly decreased the suppression of osteoclast formation induced by Myo-EVs from C2C12 myoblasts in mouse bone marrow cells. Moreover, TNF-α treatment of C2C12 myoblasts in mouse preosteoclastic Raw 264.7 cells significantly limited the Myo-EV-induced suppression of osteoclast formation and decreased the Myo-EV-induced increase in mRNA levels of osteoclast formation-related genes. On the other hand, TNF-α treatment of C2C12 muscle cells significantly decreased the degree of Myo-EV-promoted mRNA levels of Osterix and osteocalcin, as well as ALP activity in mouse mesenchymal ST-2 cells. TNF-α also significantly decreased miR196-5p level in Myo-EVs from C2C12 myoblasts in quantitative real-time PCR. In conclusion, TNF-α stimulation of C2C12 muscle cells blunts both the osteoclast formation suppression and the osteoblastic differentiation promotion that occurs due to Myo-EVs in mouse cells. Thus, TNF-α may disrupt the muscle-bone interactions by direct Myo-EV modulation.

INTRODUCTION: Irisin is a proteolytic product of fibronectin type II domain-containing 5, which is related to the improvement in glucose metabolism. Numerous studies have suggested that irisin is a crucial myokine linking muscle to bone in physiological and pathophysiological states. MATERIALS AND METHODS: We examined the effects of local irisin administration with gelatin hydrogel sheets and intraperitoneal injection of irisin on the delayed femoral bone repair caused by streptozotocin (STZ)-induced diabetes in female mice. We analyzed the femurs of mice using quantitative computed tomography and histological analyses and then measured the mRNA levels in the damaged mouse tissues. RESULTS: Local irisin administration significantly blunted the delayed bone repair induced by STZ 10 days after a femoral bone defect was generated. Local irisin administration significantly blunted the number of Osterix-positive cells that were suppressed by STZ at the damaged site 4 days after a femoral bone defect was generated, although it did not affect the mRNA levels of chondrogenic and adipogenic genes 4 days after bone injury in the presence or absence of diabetes. On the other hand, intraperitoneal injection of irisin did not affect delayed bone repair induced by STZ 10 days after bone injury. Irisin significantly blunted the decrease in Osterix mRNA levels induced by advanced glycation end products or high-glucose conditions in ST2 cells in the presence of bone morphogenetic protein-2. CONCLUSIONS: We first showed that local irisin administration with gelatin hydrogel sheets improves the delayed bone repair induced by diabetic state partially by enhancing osteoblastic differentiation.

Humoral factors that are secreted from skeletal muscles can regulate bone metabolism and contribute to muscle-bone relationships. Although extracellular vesicles (EVs) play important roles in physiological and pathophysiological processes, the roles of EVs that are secreted from skeletal muscles in bone repair have remained unclear. In the present study, we investigated the effects of the local administration of muscle cell-derived EVs on bone repair in control and streptozotocin-treated diabetic female mice. Muscle cell-derived EVs (Myo-EVs) were isolated from the conditioned medium from mouse muscle C2C12 cells by ultracentrifugation, after which Myo-EVs and gelatin hydrogel sheets were transplanted on femoral bone defect sites. The local administration of Myo-EVs significantly improved delayed bone repair that was induced by the diabetic state in mice 9 days after surgery. Moreover, this administration significantly enhanced the ratio of bone volume to tissue volume at the damaged sites 9 days after surgery in the control mice. Moreover, the local administration of Myo-EVs significantly blunted the number of Osterix-positive cells that were suppressed by the diabetic state at the damage sites after bone injury in mice. Additionally, Myo-EVs significantly blunted the mRNA levels of Osterix and alkaline phosphatase (ALP), and ALP activity was suppressed by advanced glycation end product 3 in ST2 cells that were treated with bone morphogenetic protein-2. In conclusion, we have shown for the first time that the local administration of Myo-EVs improves delayed bone repair that is induced by the diabetic state through an enhancement of osteoblastic differentiation in female mice.

Exercise is important for the prevention and treatment of sarcopenia and osteoporosis. Although the interactions between skeletal muscles and bone have recently been reported, the myokines linking muscle to bone during exercise remain unknown. We previously revealed that chronic exercise using treadmill running blunts ovariectomy-induced osteopenia in mice. We herein performed an RNA sequence analysis of the gastrocnemius and soleus muscles of male mice with or without chronic exercise to identify the myokines responsible for the effects of chronic exercise on the muscle/bone relationship. We extracted peripheral myelin protein 22 (PMP22) as a humoral factor that was putatively induced by chronic exercise in the soleus and gastrocnemius muscles of mice from the RNA sequence analysis. Chronic exercise significantly enhanced the expression of PMP22 in the gastrocnemius and soleus muscles of female mice. PMP22 suppressed macrophage-colony stimulating factor and receptor activator factor κB ligand-induced increases in the expression of osteoclast-related genes and osteoclast formation from mouse bone marrow cells. Moreover, PMP22 significantly inhibited osteoblast differentiation, alkaline phosphatase activity, and mineralization in mouse osteoblast cultures; however, the overexpression of PMP22 did not affect muscle phenotypes in mouse muscle C2C12 cells. A simple regression analysis revealed that PMP22 mRNA levels in the gastrocnemius and soleus muscles were positively related to cortical bone mineral density at the femurs of mice with or without chronic exercise. In conclusion, we identified PMP22 as a novel myokine induced by chronic exercise in mice. We first showed that PMP22 suppresses osteoclast formation and the osteoblast phenotype in vitro.

Glucocorticoids delay fracture healing and induce osteoporosis. However, the mechanisms by which glucocorticoids delay bone repair have yet to be clarified. Plasminogen activator inhibitor-1 (PAI-1) is the principal inhibitor of plasminogen activators and an adipocytokine that regulates metabolism. We herein investigated the roles of macrophages in glucocorticoid-induced delays in bone repair after femoral bone injury using PAI-1-deficient female mice intraperitoneally administered with dexamethasone (Dex). Dex significantly decreased the number of F4/80-positive macrophages at the damaged site two days after femoral bone injury. It also attenuated bone injury-induced decreases in the number of hematopoietic stem cells in bone marrow in wild-type and PAI-1-deficient mice. PAI-1 deficiency significantly weakened Dex-induced decreases in macrophage number and macrophage colony-stimulating factor (M-CSF) mRNA levels at the damaged site two days after bone injury. It also significantly ameliorated the Dex-induced inhibition of macrophage phagocytosis at the damaged site. In conclusion, we herein demonstrated that Dex decreased the number of macrophages at the damaged site during early bone repair after femoral bone injury partly through PAI-1 and M-CSF in mice.

BACKGROUND: Tissue factor (TF) is the primary activator of the extrinsic coagulation protease cascade. Although TF plays roles in various pathological states, such as thrombosis, inflammatory diseases, cancer, and atherosclerosis, its involvement in bone metabolism remains unknown. MATERIALS AND METHODS: The present study examined the roles of TF in delayed bone repair induced by a diabetic state in mice using wild-type (WT) and low TF-expressing (LTF) male mice. A diabetic state was induced by intraperitoneal injections of streptozotocin (STZ). RESULTS: A prolonged diabetic state significantly reduced total and trabecular bone mineral densities (BMD) as well as cortical bone thickness in WT and LTF mice; these BMD parameters were similar between WT and LTF mice treated with or without STZ. The diabetic state induced in WT mice delayed the repair of the femur following injury. The diabetic state induced in LTF mice was associated with further delays in bone repair. In in vitro experiments, TF significantly decreased receptor activator of nuclear factor-κB ligand-induced osteoclast formation and osteoclastogenic gene expression in RAW264.7 cells. However, it did not affect the gene expression levels of runt-related transcription factor 2 and osterix as well as alkaline phosphatase activity in mouse primary osteoblasts. CONCLUSION: Low TF state was associated with enhanced bone repair delay induced by diabetic state in mice. The TF-induced suppression of bone remodeling may be a contributing factor to the protective effects of TF against delayed bone repair in a diabetic state.

The interactions between skeletal muscle and bone have been recently noted, and muscle-derived humoral factors related to bone metabolism play crucial roles in the muscle/bone relationships. We previously reported that extracellular vesicles from mouse muscle C2C12 cells (Myo-EVs) suppress osteoclast formation in mice. Although mechanical stress is included in extrinsic factors which are important for both muscle and bone, the detailed roles of mechanical stress in the muscle/bone interactions have still remained unknown. In present study, we examined the effects of fluid flow shear stress (FFSS) to C2C12 cells on the physiological actions of muscle cell-derived EV. Applying FFSS to C2C12 cells significantly enhanced muscle cell-derived EV-suppressed osteoclast formation and several osteoclast-related gene levels in mouse bone marrow cells in the presence of receptor activator nuclear factor κB ligand (RANKL). Moreover, FFSS to C2C12 cells significantly enhanced muscle cell-derived EV-suppressed mitochondria biogenesis genes during osteoclast formation with RANKL treatment. In addition, FFSS to C2C12 cells significantly enhanced muscle cell-derived EV-suppressed osteoclast formation and several osteoclast-related gene levels in Raw264.7 cells in the presence of RANKL. Small RNA-seq-analysis showed that FFSS elevated the expression of miR196a-5p and miR155-5p with the suppressive actions of osteoclast formation and low expression in mouse bone cells. On the other hand, muscle cell-derived EVs with or without FFSS to C2C12 cells did not affect the expression of osteogenic genes, alkaline phosphatase activity and mineralization in mouse osteoblasts. In conclusion, we first showed that FFSS to C2C12 cells enhances the suppressive effects of muscle cell-derived EVs on osteoclast formation in mouse cells. Muscle cell-derived EVs might be partly involved in the effects of mechanical stress on the muscle/bone relationships.

In bone tissues, metabolic turnover through bone resorption by osteoclasts and bone formation by osteoblasts, termed bone remodeling, is strictly controlled and maintains homeostasis. Fibrinolytic factors are expressed in osteoclasts and osteoblasts, and are involved in bone remodeling through bone resorption and formation. The repair/regeneration process after bone injury is divided into the acute inflammatory, repair, and remodeling stages. Osteoblasts, osteoclasts, chondrocytes, and macrophages involved in the bone repair process originate from hematopoietic stem/progenitor cells (HSPCs) and mesenchymal stem cells (MSCs) in the bone marrow. Therefore, stem cells in the bone marrow may be strongly influenced by bone injury. The urokinase-type PA (u-PA)/plasminogen (Plg) system functions in macrophage accumulation/phagocytosis through chemokines in the acute inflammatory stage, and Plg increases blood vessel-related growth factor expression, being involved in vascularization in mice. Plasminogen activator inhivitor-1 (PAI-1) causes bone loss and delayed bone repair through the inhibition of osteoblast differentiation in a drug-induced diabetes model in mice. Plg is considered to induce transforming growth factor-β (TGF-β) production in macrophages in the bone repair process, TGF-β release from the extracellular matrix through the activation of matrix metalloproteinase-9 (MMP-9), and stromal cell-derived factor-1 (SDF-1) expression in endosteal preosteoblasts, leading to the induction of bone marrow HSPCs in mice. Based on the above, establishment of a fibrinolytic factor-targeting method efficiently promoting bone repair/regeneration and fracture healing, and development of a new osteoporosis treatment method and diagnostic marker are awaited.

Myonectin is a myokine, which is involved in the pathophysiology of diabetes and obesity, and various myokines are involved in the interactions between skeletal muscle and bone. However, roles of myonectin in bone have still remained unknown. We therefore examined the effects of myonectin on mouse osteoblast and osteoclast differentiation in vitro. Myonectin significantly suppressed the mRNA levels of osteogenic genes and alkaline phosphatase (ALP) activity in mouse osteoblasts. As for osteoclasts, myonectin significantly suppressed osteoclast formation as well as the mRNA levels of osteoclast-related genes enhanced by receptor activator nuclear factor κB ligand (RANKL) from mouse monocytic RAW264.7 cells. Moreover, myonectin significantly suppressed osteoclast formation from mouse bone marrow cells in the presence of macrophage-colony stimulating factor and RANKL. On the other hand, myonectin significantly suppressed RANKL-induced oxygen consumption rate and peroxisome proliferator-activated receptor γ coactivator-1β mRNA levels in RAW264.7 cells, although myonectin did not affect these mitochondrial biogenesis parameters in mouse osteoblasts. In conclusion, the present study demonstrated that myonectin suppresses the differentiation and ALP activity in mouse osteoblasts. Moreover, myonectin suppressed osteoclast differentiation from mouse bone marrow and RAW264.7 cells partly through an inhibition of mitochondrial biogenesis.

Mechanical unloading simultaneously induces muscle and bone loss, but its mechanisms are not fully understood. The interactions between skeletal muscle and bone have been recently noted. Although canonical wingless-related integration site (Wnt)/β-catenin signaling is crucial for bone metabolism, its roles in the muscle and bone interactions have remained unknown. Here, we performed comprehensive DNA microarray analyses to clarify humoral factors linking muscle to bone in response to mechanical unloading and hypergravity with 3 g in mice. We identified Dickkopf (Dkk) 2, a Wnt/β-catenin signaling inhibitor, as a gene whose expression was increased by hindlimb unloading (HU) and reduced by hypergravity in the soleus muscle of mice. HU significantly elevated serum Dkk2 levels and Dkk2 mRNA levels in the soleus muscle of mice whereas hypergravity significantly decreased those Dkk2 levels. In the simple regression analyses, serum Dkk2 levels were negatively and positively related to trabecular bone mineral density and mRNA levels of receptor activator of nuclear factor-kappa B ligand (RANKL) in the tibia of mice, respectively. Moreover, shear stress significantly suppressed Dkk2 mRNA levels in C2C12 cells, and cyclooxygenase inhibitors significantly antagonized the effects of shear stress on Dkk2 expression. On the other hand, Dkk2 suppressed the mRNA levels of osteogenic genes, alkaline phosphatase activity and mineralization, and it increased RANKL mRNA levels in mouse osteoblasts. In conclusion, we showed that muscle and serum Dkk2 levels are positively and negatively regulated during mechanical unloading and hypergravity in mice, respectively. An increase in Dkk2 expression in the skeletal muscle might contribute to disuse- and microgravity-induced bone and muscle loss.

The peripheral vestibular organs are sensors for linear acceleration (gravity and head tilt) and rotation. Further, they regulate various body functions, including body stability, ocular movement, autonomic nerve activity, arterial pressure, body temperature, and muscle and bone metabolism. The gravitational environment influences these functions given the highly plastic responsiveness of the vestibular system. This review demonstrates that hypergravity or microgravity induces changes in vestibular-related physiological functions, including arterial pressure, muscle and bone metabolism, feeding behavior, and body temperature. Hopefully, this review contributes to understanding how human beings can adapt to a new gravitational environment, including the moon and Mars, in future.

Androgen deficiency plays a crucial role in the pathogenesis of male osteoporosis and sarcopenia. Myokines have recently been identified as humoral factors that are involved in the interactions between muscle and bone; however, the influence of androgen deficiency on these interactions remains unclear. Therefore, we herein investigated the roles of humoral factors linking muscle to bone using orchidectomized mice with sarcopenia and osteopenia. Orchidectomy (ORX) significantly reduced muscle mass, grip strength, and trabecular bone mineral density (BMD) in mice. Among the myokines examined, ORX only significantly reduced fibronectin type III domain-containing 5 (Fndc5) mRNA levels in both the soleus and gastrocnemius muscles of mice. In simple regression analyses, Fndc5 mRNA levels in the soleus muscle positively correlated with trabecular BMD, but not cortical BMD. The administration of irisin, a product of Fndc5, significantly protected against the decrease induced in trabecular BMD, but not muscle mass, by androgen deficiency in mice. In conclusion, the present results demonstrated that androgen deficiency decreases the expression of irisin in the skeletal muscle of mice. Irisin may be involved in muscle/bone relationships negatively affected by androgen deficiency.

The vestibular system controls balance, posture, blood pressure, and gaze. However, the roles of the vestibular system in energy and glucose metabolism remain unknown. We herein examined the roles of the vestibular system in obesity and impaired glucose metabolism using mice with vestibular lesions (VL) fed a high-sucrose/high-fat diet (HSHFD). VL was induced by surgery or arsenic. VL significantly suppressed body fat enhanced by HSHFD in mice. Glucose intolerance was improved by VL in mice fed HSHFD. VL blunted the levels of adipogenic factors and pro-inflammatory adipokines elevated by HSHFD in the epididymal white adipose tissue of mice. A β-blocker antagonized body fat and glucose intolerance enhanced by HSHFD in mice. The results of an RNA sequencing analysis showed that HSHFD induced alterations in genes, such as insulin-like growth factor-2 and glial fibrillary acidic protein, in the vestibular nuclei of mice through the vestibular system. In conclusion, we herein demonstrated that the dysregulation of the vestibular system influences an obese state and impaired glucose metabolism induced by HSHFD in mice. The vestibular system may contribute to the regulation of set points under excess energy conditions.

Objectives: Interleukin (IL)-1β and matrix metalloproteinases (MMPs) play important roles in the pathogenesis of osteoarthritis. On the other hand, plasminogen activator inhibitor-1 (PAI-1), an inhibitor of fibrinolysis, exerts functions in the pathogenesis of various diseases. However, the functional roles of PAI-1 in the chondrocytes have been still remained unknown.Methods: In the present study, we investigated the roles of PAI-1 in the effects of IL-1β on the chondrocytes using wild-type and PAI-1-deficient mice.Results: IL-1β significantly elevated PAI-1 mRNA levels in the chondrocytes from wild-type mice. PAI-1 deficiency significantly blunted the mRNA levels of TGF-β and IL-6 enhanced by IL-1β in murine chondrocytes. Moreover, PAI-1 deficiency significantly decreased the mRNA levels of MMP-13, -3 and -9 as well as MMP-13 activity enhanced by IL-1β in the chondrocytes. In addition, PAI-1 deficiency significantly reversed type II collagen mRNA levels suppressed by IL-1β in the chondrocytes. On the other hand, active PAI-1 treatment significantly enhanced the mRNA levels of MMP-13, -3 and -9 as well as decreased type II collagen mRNA levels in the chondrocytes from wild-type mice.Conclusion: We first demonstrated that PAI-1 is involved in MMP expression enhanced by IL-1β in murine chondrocytes. PAI-1 might be crucial for the cartilage matrix degradation and the impaired chondrogenesis by IL-1β in mice.

Muscle and bone masses are elevated by the increased mechanical stress associated with body weight gain in obesity. However, the mechanisms by which obesity affects muscle and bone remain unclear. We herein investigated the roles of obesity and humoral factors from adipose tissue in the recovery phase after reloading from disuse-induced muscle wasting and bone loss using normal diet (ND)- or high fat diet (HFD)-fed mice with hindlimb unloading (HU) and subsequent reloading. Obesity did not affect decreases in trabecular bone mineral density (BMD), muscle mass in the lower leg, or grip strength in HU mice. Obesity significantly increased trabecular BMD, muscle mass in the lower leg, and grip strength in reloading mice over those in reloading mice fed ND. Among the humoral factors in epididymal and subcutaneous adipose tissue, leptin mRNA levels were significantly higher in reloading mice fed HFD than in mice fed ND. Moreover, circulating leptin levels were significantly higher in reloading mice fed HFD than in mice fed ND. Leptin mRNA levels in epididymal adipose tissue or serum leptin levels positively correlated with the increases in trabecular BMD, total muscle mass, and grip strength in reloading mice fed ND and HFD. The present study is the first to demonstrate that obesity enhances the recovery of bone and muscle masses as well as strength decreased by disuse after reloading in mice. Leptin may contribute to the recovery of muscle and bone enhanced by obesity in mice.

It is well known that sex differences exist concerning the severity of osteoporosis and bone metabolism, suggesting that factors other than sex hormones might be responsible for sex differences of bone metabolism. We therefore examined sex differences of osteoblast phenotypes of mouse osteoblasts and then performed comparative gene expression analyses using a comprehensive DNA microarray between female and male osteoblasts. Alkaline phosphatase (ALP) activity, mineralization, and the expression of Osterix, ALP, and bone sialoprotein were significantly lower in mouse female osteoblasts compared with male osteoblasts. We identified Serpina3n, a novel serine protease inhibitor, as the gene whose expression has the highest ratio of females to males. A reduction in endogenous levels of Serpina3n by small interfering RNA significantly enhanced the mRNA levels of Runx2, ALP, osteocalcin, and type I collagen (Col1a1) in both male and female osteoblasts. Moreover, Serpina3n overexpression significantly suppressed the mRNA levels of Osterix, ALP, osteocalcin, and Col1a1 in MC3T3-E1 cells. Serpina3n overexpression did not affect Osterix, ALP, and osteocalcin mRNA levels enhanced by bone morphogenetic protein (BMP)-2 in ST2 cells, adipogenic differentiation in ST2 and 3T3-L1 cells, and receptor activator of nuclear factor κB ligand-induced osteoclast formation in RAW264.7 cells, although it significantly suppressed mineralization in ST2 cells differentiated into osteoblasts by BMP-2. In conclusion, we found Serpina3n as the most female osteoblast-dominant gene. Serpina3n exerts a suppression of the osteoblast phenotypes such as Col1a1 expression and ALP activity in differentiated osteoblasts, which might partly explain sex differences of the osteoblast phenotypes in mice.

We recently reported that hypergravity with 3 g for 4 weeks affects muscle and bone through the vestibular system in mice. The purpose of this study was to investigate the effects of hypergravity with 2 g, which had no influence on circulating glucocorticoid level, on the gene levels in muscle and bone, as well as the roles of the vestibular system in those changes using vestibular lesioned (VL) mice. Hypergravity for 2 and 8 weeks or VL exerted little effects on the mRNA levels of muscle differentiation factors and myokines in the soleus muscle. Although hypergravity for 2 weeks significantly elevated alkaline phosphatase (ALP) and type I collagen mRNA levels in the tibia, VL significantly attenuated the levels of ALP mRNA enhanced by hypergravity. In conclusion, the present study suggests that a 2-g load for 2 weeks enhances osteoblast differentiation partly through the vestibular system in mice.

Mechanical unloading induces disuse muscle atrophy and bone loss, but the details in mechanism involved in those pathophysiological conditions are not fully understood. Interaction between muscle and bone has been recently noted. Here, we investigated the roles of humoral factors linking muscle to bone during mechanical unloading using mice with hindlimb unloading (HU) and sciatic neurectomy (SNX). HU and SNX reduced muscle volume surrounding the tibia, tissue weights of soleus and gastrocnemius muscle, and trabecular bone mineral density (BMD) in the tibia of mice. Among humoral factors linking muscle to bone, HU and SNX reduced fibronectin type III domain-containing 5 (FNDC5) mRNA levels in the soleus muscle of mice. Simple regression analysis revealed that FNDC5 mRNA levels in the soleus muscle were positively related to trabecular BMD in the tibia of control and HU mice as well as sham and SNX mice. Moreover, FNDC5 mRNA levels were negatively correlated with receptor activator of nuclear factor-κB ligand (RANKL) mRNA levels in the tibia of control and HU mice. Irisin, a product of FNDC5, suppressed osteoclast formation from mouse bone marrow cells and RANKL mRNA levels in primary osteoblasts. FNDC5 mRNA levels elevated by fluid shear stress were antagonized by bone morphogenetic protein (BMP) and phosphatidylinositol 3-kinase (PI3K) signaling inhibitors in myoblastic C2C12 cells. In conclusion, the present study first showed that mechanical unloading reduces irisin expression in the skeletal muscle of mice presumably through BMP and PI3K pathways. Irisin might be involved in muscle/bone relationships regulated by mechanical stress in mice.

We previously revealed that stromal cell-derived factor-1 (SDF-1) is involved in the changes in the number of bone marrow stem cells during the bone repair process in mice. Moreover, we reported that plasminogen (Plg) deficiency delays bone repair and the accumulation of macrophages at the site of bone damage in mice. We investigated the roles of Plg in the changes in bone marrow stem cells during bone repair. We analyzed the numbers of hematopoietic stem cells (HSC) and mesenchymal stem cells (MSCs) within bone marrow from Plg-deficient and wild-type mice after a femoral bone injury using flow cytometric analysis. Plg deficiency significantly blunted a decrease in the number of HSCs after bone injury in mice, although it did not affect an increase in the number of MSCs. Plg deficiency significantly blunted the number of SDF-1- and Osterix- or SDF-1- and alkaline phosphatase-double-positive cells in the endosteum around the lesion as well as matrix metalloprotainase-9 (MMP-9) activity and mRNA levels of SDF-1 and transforming growth factor-β (TGF-β) elevated by bone injury. TGF-β signaling inhibition significantly blunted a decrease in the number of HSCs after bone injury. The present study showed that Plg is critical for the changes in bone marrow HSCs through MMP-9, TGF-β, and SDF-1 at the damaged site during bone repair in mice.

We recently revealed that plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor, is involved in diabetes, osteoporosis and muscle wasting induced by glucocorticoid (GC) treatment in mice. In the present study, we investigated the detailed mechanisms by which GC induces muscle wasting through PAI-1 in vivo and in vitro. PAI-1 deficiency suppressed the mRNA levels of atrogin1 and muscle RING-Finger Protein 1 (MuRF1), ubiquitin ligases leading to muscle degradation, elevated by GC treatment in the gastrocnemius muscle of mice. In vitro study revealed that active PAI-1 treatment augmented the increase in atrogin1 mRNA levels enhanced by dexamethasone (Dex) in mouse myoblastic C2C12 cells. Moreover, a reduction in endogenous PAI-1 level by siRNA suppressed the mRNA levels of atrogin1 and MuRF1 enhanced by Dex in C2C12 cells. In contrast, a reduction in endogenous PAI-1 levels and active PAI-1 did not affect the phosphorylations of Akt and p70S6 kinase nor myogenic differentiation with or without Dex in C2C12 cells. In addition, PAI-1 deficiency blunted IGF-1 mRNA levels decreased by GC treatment in the gastrocnemius muscle of mice, although neither active PAI-1 nor a reduction in endogenous PAI-1 levels affected the levels of IGF-1 mRNA in C2C12 cells in the presence of Dex. In conclusion, our data suggest that paracrine PAI-1 is involved in GC-induced muscle wasting through the enhancement of muscle degradation in mice.

[Abstract]Glucocorticoid(GC) therapy leads to an increase in fracture risk, and it is well recognized that bisphosphonates are effective for the treatment of GC-induced osteoporosis. We performed a non-randomized prospective study to clarify the effects of alendronate or alfacalcidol on the change in the trabecular bone score(TBS) at the lumbar spine and bone metabolic indices in 94 patients receiving high-dose GC therapy. The mean initial daily GC dose of predonisolone was 27.6±6.9 mg/day. Although the bone mineral density(BMD) at the lumbar spine continued to significantly decrease for 12 months in the alfacalcidol group, BMD increased above the baseline level at 3 and 6 months in the alendronate group. Alendronate significantly reduced urinary NTx levels from the baseline for 12 months, although alfacalcidol did not affect them. TBS was significantly decreased at 6 and 9 months in the alfacalcidol group. There were no significant differences in TBS between the alfacalcidol and alendronate groups for 12 months. TBS was significantly correlated with BMD at the lumbar spine in both groups for 12 months. In conclusion, alendronate treatment did not affect TBS at the lumbar spine in patients treated with GC, although TBS decreased with alfacalcidol.

Gravity changes concurrently affect muscle and bone as well as induce alterations in vestibular signals. However, the role of vestibular signals in the changes in muscle and bone induced by gravity changes remains unknown. We therefore investigated the effects of vestibular lesions (VL) on the changes in muscle and bone induced by 3 g hypergravity for 4 weeks in C57BL/6J mice. Quantitative computed tomography analysis revealed that hypergravity increased muscle mass surrounding the tibia and trabecular bone mineral content, adjusting for body weight in mice. Hypergravity did not affect cortical bone and fat masses surrounding the tibia. Vestibular lesions blunted the increases in muscle and bone masses induced by hypergravity. Histological analysis showed that hypergravity elevated the cross-sectional area of myofiber in the soleus muscle. The mRNA levels of myogenic genes such as MyoD, Myf6, and myogenin in the soleus muscle were elevated in mice exposed to hypergravity. Vestibular lesions attenuated myofiber size and the mRNA levels of myogenic differentiation markers enhanced by hypergravity in the soleus muscle. Propranolol, a β-blocker, antagonized the changes in muscle induced by hypergravity. In conclusion, this study is the first to demonstrate that gravity changes affect muscle and bone through vestibular signals and subsequent sympathetic outflow in mice.

Measurement of ionized calcium is more important than measurement of total calcium in serum samples. In the present study, equations were derived from complexation and acid dissociation equilibrium equations, and were used to determine the concentration of ionized calcium from the observed serum concentrations of total calcium, albumin, total protein, and inorganic phosphate. The ionized calcium concentration was calculated in 67 serum samples from healthy subjects and 34 outpatients previously identified as having abnormal serum calcium levels. The correlation coefficient between our method (y) and the calcium-ion-selective electrode method (x) was 0.953 and the linear regression equation was y = 0.97x at pH 7.4 with a factor of α = 0.21, which was based on the differences between the concentrations of calcium phosphorus compounds obtained by the electrode method and by calculation. The developed calculation is as useful and accurate as the electrode method, and therefore extremely useful for clinical diagnoses.

国際宇宙ステーション内の微小重力または人工1-g環境で30日間飼育したマウスのヒラメ筋および腓腹筋のプロテオミクス解析を実施し、微小重力環境飼育により萎縮した両筋においてPlasminogen（Plg）が有意に増加することを明らかにした。Plgは線溶系の重要因子であり、線溶系関連因子は血管内のみならず血管外でも機能し、骨格筋の肥大や損傷からの再生過程においても重要な役割を果たすことが報告されている。しかし、線溶系と非荷重に伴う骨格筋の萎縮の関係は不明であった。そこで、Plg遺伝子欠損マウスと野生型マウスを21日間後肢懸垂飼育したところ、Plg遺伝子欠損マウスでは後肢筋の萎縮がより顕著に生じ、ヒラメ筋に比べて非荷重の影響を受けにくいとされる腓腹筋がより萎縮していることを見いだした。その後、非荷重環境下においてPlgが腓腹筋の萎縮を軽減させるメカニズムを追究し、後肢懸垂飼育したPlg遺伝子欠損マウスの腓腹筋ではオートファジー関連因子の発現が増加しており、オートファジー・リソソーム系によるタンパク分解が野生型マウスよりも亢進されていることを示唆する結果を得た。一方、非荷重に伴うユビキチンリガーゼ（Atrogin-1、MuRF1）の遺伝子発現の亢進についてはPlg遺伝子欠損マウスと野生型マウスで差がなかったことから、ユビキチン・プロテアソーム系によるタンパク質分解にPlgが関与する可能性は低いと考えられる。また、タンパク質合成に関わるAkt-mTOR経路については両マウスにおいて後肢懸垂飼育の影響はなかった。以上の結果から、非荷重環境下でPlgは腓腹筋におけるオートファジー・リソソーム系によるタンパク質分解亢進を部分的に抑制することにより、筋量減少に対して抑制的な作用を示す可能性が考えられる。

本研究は、筋と骨の連関機構(筋・骨連関)におけるエクソソーム(exo)の関与に着目し、筋由来exo(Myo-exo)の骨への作用を明らかにすることを目的としている。これまでに研究代表者は、Myo-exoが骨芽細胞の分化を促進し、破骨細胞の形成を抑制することをin vitroの実験系で明らかにしており、本研究ではMyo-exoの骨組織再生作用について、大腿骨欠損マウスモデルを用いて検討した。 研究に用いるMyo-exoはマウス筋細胞株であるC2C12細胞から回収した。コンフレントに培養したC2C12細胞を48時間培養した培養上清を回収し、遠心 (3,000×g)、フィルター濾過 (0.22 μm)によって細胞デブリなどの異物を除去した後、超遠心 (100,000×g)にて沈降したペレットをMyo-exoとして実験に用いた。得られたMyo-exoはBCAassay法によって蛋白量を定量した。また、Western blottingによるエクソソーム特異的な抗原(CD9, CD81)の発現を確認するとともに、Nanosightによる粒径解析によって、得られた粒子の大部分が200 nm以下の粒径であることを確認した。 健常マウスおよび骨修復が遅延する、ストレプトゾトシン誘発性糖尿病モデルマウスの大腿骨に0.8mm径の欠損を作製し、Myo-exoを局所移植した。移植担体として、1.5mm径に成型した生分解性のゼラチンハイドロゲルを用いた。移植9日後に欠損部近辺を動物用CT装置で撮影した。その結果、糖尿病マウスは健常マウスと比べて骨再生の遅延を認め、欠損部面積が大きかった。しかし糖尿病マウスにMyo-exoを移植することで、骨欠損部面積が有意に減少し、糖尿病によって引き起こされる骨修復の遅延がMyo-exoの局所移植によって改善することが明らかとなった。

高齢化社会の進展とともに健康寿命の延伸が喫緊の課題となり、骨粗鬆症とサルコペニアの予防・治療が重要となってきた。その中で、骨格筋と骨の相互関連（筋・骨連関）が注目されている。しかし、長期的な運動が筋・骨連関におよぼす影響とマイオカインの役割は不明である。そこで私共は、マウスにおいて、慢性的な運動負荷により、筋で産生され骨に影響をおよぼす新規マイオカインを探索し、その骨代謝におよぼす影響を検討した。8週間のトレッドミル運動を行ったマウス腓腹筋とヒラメ筋の網羅的RNAシークエンス解析により、慢性運動によって筋で発現が増加する因子としてperipheral myelin protein 22 (PMP22) を抽出した。さらに、慢性運動は卵巣摘出による骨量減少を回復し、腓腹筋PMP22発現を増加させた。In vitro解析において、PMP22は骨芽細胞の分化および石灰化を減少させた。一方、マウス骨髄細胞において、PMP22はRANKLによって誘導される破骨細胞形成を有意に抑制した。実験に用いたマウスでの単相関分析によって、腓腹筋とヒラメ筋のPMP22発現量は大腿骨の皮質骨骨密度と正の相関を示したが、海綿骨骨密度とは有意な相関はみられなかった。これらの実験結果より、マウスにおいて、慢性運動は骨格筋のPMP22発現を増加させることが明らかとなった。さらに、PMP22は、破骨細胞形成を抑制する作用により、慢性運動による骨量増加作用に寄与する新規のマイオカインであることが示唆された。

微小重力に伴う医学的諸問題の原因に，重力の感知器官である耳石前庭系の可塑的変化が関与しているとの仮説のもと，前庭系を介する種々の身体活動および過重力，微小重力環境により引き起こされる耳石感覚細胞と1次ニューロンである前庭神経核の変化を調べ，以下の成果を得た。 前庭系は，これまで知られていた機能以外にも摂食，糖代謝，体温，血圧，筋量・骨量調節など多彩な身体機能調節に関与している。これらの機能は，可塑性が強く異なる重力環境に曝されるとその機能が変化する。この可塑的変化には，球形嚢および前庭神経核の遺伝子発現変化が関与する。可塑的変化の対策として前庭系を電気刺激する方法が有用である可能性が示された。

骨粗鬆症の病態には明らかな性差があり、性ホルモン以外の要因も骨代謝の性差に関与する可能性が示唆されてきた。マウス初代培養骨芽細胞表現型における性差を検討したところ、分化や石灰化は、雄と比較して雌において著明に低かった。このような骨芽細胞の性差を決定する因子の探索を行ったところ、最も雌に発現量が優位に高い遺伝子として、Serpina3nを同定した。雌の骨芽細胞に優位に発現するSerpina3nは、分化した骨芽細胞における表現型を抑制する作用を有し、マウスの骨芽細胞表現型や骨粗鬆症病態の性差を部分的に説明する可能性が考えられた。

本研究では、メカニカルストレスによって誘導される筋と骨のネットワークに寄与する因子を探索し、脂肪が筋と骨のネットワークにおよぼす影響を検討した。今回、過重力負荷によって筋で産生され、骨代謝に影響を及ぼす因子としてフォリスタチンを見出し、フォリスタチンは、骨に正の作用を有することを明らかにした。さらに、非荷重による骨量減少に筋でのアイリシン産生減少が関与することが示唆された。また、肥満は非荷重によって減少した骨量、筋量、筋力の再荷重による回復を促進させることを見出し、アディポカインとしてのレプチンが肥満による筋と骨の回復促進に寄与することが示唆された。

オキサリプラチンによる肝類洞閉塞症候群（SOS）の病態形成における凝固線溶因子の関与を明らかにすることを目的とした。 薬剤剤投与により肝臓でのPAI-1 発現の増加が見られ、TF 発現は低下した。PAI-1 発現増加と体重減少率には有意な負の相関が見られ、PAI-1増加の病態悪化への関与が示唆された。PAI-1欠損マウスを用いた実験では、肝臓での炎症性サイトカインや線維化マーカーの発現増加が軽微であった。TF欠損マウスを用いた実験では、体重減少や死亡率が高かった。以上より、肝臓の炎症・線維化亢進に加え、PAI-1発現増加等により惹起される血栓傾向が病態増悪に関与すると考えられた。

最近、骨格筋と骨の相互関連(筋・骨連関)が注目されている。私共はマウスを用いて、力学的因子の筋・骨連関に及ぼす影響を、網羅的遺伝子解析を手がかりとして検討した。その結果、重力変化は前庭系を介して筋量や骨量に影響をおよぼすこと、その作用に筋から産生される体液性因子としてフォリスタチンが関与すること、メカニカルストレスは、筋からのアイリシン産生を介して、骨密度を増加させる報告に作用することを明らかにした。

骨損傷後の修復過程で線溶系因子であるプラスミノーゲン（Plg）の関与が考えられる。本研究は、骨損傷早期の骨髄造血幹細胞の誘導における組織線溶系の関与について検討した。Plg欠損は、骨損傷後の修復を遅延させ、SDF-1とTGF-β発現増加を阻害した。さらに、野生型マウスに対するTGF-βシグナル阻害剤投与は、骨損傷で誘導される骨髄HSC数の低下を阻害した。さらに、骨損傷により誘導されるSDF-1発現増加を抑制した。以上より、骨損傷後の骨修復早期における骨局所への骨髄からのHSC誘導に、組織線溶系により誘導されるTGF-βとそれに続く骨損傷部のSDF-1が関与することが示唆された。

マウス皮下脂肪組織をコラゲナーゼ処理し遠心分離により脂肪由来幹細胞の分離方法をまず確立した。続いて脂肪由来幹細胞の持つ抗炎症性作用により脂肪組織における慢性炎症を抑制させることができるかどうかについて、試験管内で検討を行った。脂肪細胞に脂肪由来幹細胞の培養上清を添加して培養を行うと、悪玉アディポカインの発現量は有意に減少させたが、善玉アディポカインの発現量は減少させなかった。 次に、２型糖尿病モデルマウスを作製し、これらを２群に分け、偽手術群と脂肪由来幹細胞シートの皮下移植群を行い、10日目にインスリン負荷試験を行って糖代謝能の検討を行った。細胞シート移植群でインスリン抵抗性改善を認めた。

本研究では骨修復・再生過程でのマクロファージの動態および活性化における組織線溶系の役割を検討した。今回、骨修復・再生過程において組織線溶系が中心的な役割を果たすことが明らかになった。さらに、組織線溶系は骨修復・再生部位にマクロファージを集積させ、それらの活性化に寄与することで骨修復に寄与することが明らかになった。今後、組織線溶系を標的としたマクロファージの機能調節により、効率よく骨修復・再生を亢進する方法の確立を目指してさらに研究を進めたい。

組織線溶系とそれにより誘導されるタンパク分解酵素系や増殖因子が組織修復過程において重要な役割をはたすことがこれまで示唆されてきた。そこで、私共は骨修復・再生過程における組織線溶系の役割を検討した。マウス大腿骨に骨欠損を作製することにより、骨修復・再生過程を検討できるモデルを確立した。プラスミノゲン欠損マウスを用いて、プラスミノゲンが骨修復過程に必須であることを示した。さらに、組織型プラスミノゲンアクチベーター(tPA)の欠損マウスを用いて、tPAも骨修復に重要で、骨芽細胞にAnexin2やMAPキナーゼを介する機序により働いて、骨芽細胞増殖を促進することを明らかにした。

筋と骨ミネラル代謝の相互関連を解明するために。筋における骨化調節因子および筋から産生され、骨形成促進的に作用する体液性因子の同定を試みた。筋由来の細胞と骨化シグナルを増強させた細胞で網羅的遺伝子解析をおこない、骨形成活性を有する因子を抽出した。そのなかで、Tmem119は筋骨化を局所性に誘導する因子として期待され、オステオグリシンおよびFAM5Cは筋から産生される新規の体液性骨形成因子の候補として、今後の骨粗鬆症治療薬開発の標的としてさらなる研究を進めたい。

種々の遺伝子の転写調節領域に存在するCpG islandのシトシンメチル化は遺伝子発現を抑制的に制御することが知られている。一方で、非CpG island領域のシトシンメチル化についても、転写制御に重要な影響を示すものがあり、私どもは、膀胱癌におけるTGF-βシグナル経路の偽受容体BAMBIと、糖尿病ラットの腎腫瘍発生とP16の発現制御を対象として、メチル化の進展が腫瘍の形態変化に関与することを示した。

1.最も骨形成促進剤として期待されている副甲状腺ホルモン(PTH)の骨芽細胞株におけるSmad3及びbeta-catenin系への影響について検討した。PTH及びSmad3はいずれもbeta-cateninの発現を促進した。PTHのその作用にcAMP系及びカルシウム/プロテインキナーゼC系の両者が関与し、さらにPTHはbeta-cateninが誘導する転写活性も増強した。PTHが誘導したbeta-catenin発現や転写活性増強はいずれもSmad3の不活化により阻害された。これらのシグナルはPTHのアポトーシス阻害作用に関与することが示唆された。2.Smad3の骨形成シグナルとしての役割を明らかにしてきた。マウス未分化間葉系幹細胞株のST-2細胞においてSmad3はBone morphogenetic protein-2が誘導する骨芽細胞への分化を阻害した。一方マウス骨芽細胞株MC3T3-E1細胞でSmad3のアルカリフォスファターゼや1型コラーゲンの発現促進作用は分化が進むと低下傾向であったが、Runx2やオステオカルシンへの作用は石灰化期では抑制から促進に転じた。種々の石灰化調節因子にも影響を及ぼした。3.Erk1/2阻害剤にて処理したSmad3過剰発現骨芽細胞とコントロール細胞で45000遺伝子のDNAマイクロアレイを行ない、骨形成の関連する可能性がある既知あるいは未知の因子を数個選び、現在その意義を解析中である。未知の因子については発現ベクターを作製し、細胞株での機能を検討している。4.Smadに対して抑制シグナルとなるSmad7の過剰発現株をMC3T3-E1細胞を用いて作った。Smad7は骨芽細胞の増殖、分化、骨基質蛋白産生、石灰化すべて抑制した。また副甲状腺ホルモンはSmad7の発現を誘導し、Smad7はホルモンの骨芽細胞を抑制的に作用するシグナルの標的になっている可能性が示唆された。

私共はこれまで多発性内分泌腫瘍症I型の原因MEN-I遺伝子産物Meninが骨芽細胞の初期分化に必要で、後期分化に関してはむしろ抑制することを明らかにしてきた。さらにTGF-beta特異的なシグナル分子のSmad3が骨芽細胞において骨形成に重要な分子であることを提唱してきた。また副甲状腺ホルモン(PTH)は最も骨形成促進の強い薬剤であるが、その作用機序は未だ不明である。今回私共は骨芽細胞におけるPTHのSmad3に及ぼす影響及びその生理的意義について検討し、PTHの骨芽細胞アポトーシス抑制作用にSmad3が関与することを示した。さらに骨芽細胞株で糖質コレチコイドのデキサメサゾンがSmad3による転写活性やアルカリフォスファターゼ活性促進を阻害し、Smad3には影響を及ぼさないことを証明し、ステロイド骨粗鬆症の骨形成抑制の機序の一つとして提唱した。一方、これまでMeninとBone morphogenetic proteinのシグナルが骨芽細胞への初期分化に関連することを検討してきたが、今回細胞株を用いて間葉系幹細胞から骨芽細胞への分化段階においてMeninはSmad1やSmad5、さらにはその下流で骨芽細胞分化に必須の転写因子であるRunx2と機能的にも物理的にも相互作用をしていることを示した。さらにこの相互作用は骨芽細胞に分化してしまうと減弱し、その機序として一部MeninとSmad3の相互作用が抑制的な作用を及ぼしていることが示唆された。また分化した骨芽細胞においては骨芽細胞の機能に重要なAP-1因子の一つJunDが分化、石灰化を促進する作用を有し、MeninはJunDと結合することによりJunDの発現、転写活性及び骨形成作用を阻害することにより骨芽細胞の分化を抑制することを明らかにした。