Role of peripheral myelin protein 22 in chronic exercise-induced interactions of muscle and bone in 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.

Role of tissue factor in delayed bone repair induced by diabetic state 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.

Serpinb1a suppresses osteoclast formation.

Serpinb1a, a serine protease inhibitor family protein, has been implicated in immunoregulation and several metabolic disorders, such as diabetes and obesity; however, its roles in bone remain unknown. Therefore, we herein investigated the physiological functions of Serpinb1a in osteoclastic and osteoblastic differentiation using mouse cell lines. Serpinb1a overexpression markedly reduced the number of tartrate-resistant acid phosphatase (TRAP)- and calcitonin receptor-positive multinucleated cells increased by receptor activator nuclear factor κB ligand (RANKL) in mouse preosteoclastic RAW 264.7 cells. Moreover, it significantly decreased the mRNA levels of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), TRAP and cathepsin K in these cells. Regarding osteoblasts, Serpinb1a overexpression significantly reduced the mRNA levels of alkaline phosphatase (ALP) and osteocalcin as well as ALP activity induced by bone morphogenetic protein-2 (BMP-2) in mouse mesenchymal ST2 cells, although it did not alter osteoblast differentiation in mouse osteoblastic MC3T3-E1 cells. Concerning the pathophysiological relevance of Serpinb1a, Serpinb1a mRNA levels were decreased in the soleus and gastrocnemius muscles of mice 4 weeks after bilateral sciatic nerve resection. In conclusion, we herein revealed for the first time that Serpinb1a inhibited osteoclast formation induced by RANKL in RAW 264.7 cells and suppressed BMP-2-induced ALP activity in ST2 cells.

Influence of Angptl1 on osteoclast formation and osteoblastic phenotype in mouse cells.

BACKGROUND: Osteoblasts and osteoclasts play important roles during the bone remodeling in the physiological and pathophysiological states. Although angiopoietin family Angiopoietin like proteins (Angptls), including Angptl1, have been reported to be involved in inflammation, lipid metabolism and angiogenesis, the roles of Angptl1 in bone have not been reported so far. METHODS: We examined the effects of Angptl1 on the osteoblast and osteoclast phenotypes using mouse cells. RESULTS: Angptl1 significantly inhibited the osteoclast formation and mRNA levels of tartrate-resistant acid phosphatase and cathepsin K enhanced by receptor activator of nuclear factor κB ligand in RAW 264.7 and mouse bone marrow cells. Moreover, Angptl1 overexpression significantly enhanced Osterix mRNA levels, alkaline phosphatase activity and mineralization induced by bone morphogenetic protein-2 in ST2 cells, although it did not affect the expression of osteogenic genes in MC3T3-E1 and mouse osteoblasts. On the other hand, Angptl1 overexpression significantly reduced the mRNA levels of peroxisome proliferator-activated receptor γ and adipocyte protein-2 as well as the lipid droplet formation induced by adipogenic medium in 3T3-L1 cells. CONCLUSIONS: The present study first indicated that Angptl1 suppresses and enhances osteoclast formation and osteoblastic differentiation in mouse cells, respectively, although it inhibits adipogenic differentiation of 3T3-L1 cells. These data suggest the possibility that Angptl1 might be physiologically related to bone remodeling.

Myonectin inhibits the differentiation of osteoblasts and osteoclasts in mouse cells.

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-1ß 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.

Roles of Olfactomedin 1 in Muscle and Bone Alterations Induced by Gravity Change in Mice.

Microgravity causes both muscle and bone loss. Although we previously revealed that gravity change influences muscle and bone through the vestibular system in mice, its detailed mechanism has not been elucidated. In this study, we investigated the roles of olfactomedin 1 (OLFM1), whose expression was upregulated during hypergravity in the soleus muscle, in mouse bone cells. Vestibular lesion significantly blunted OLFM1 expression in the soleus muscle and serum OLFM1 levels enhanced by hypergravity in mice. Moreover, a phosphatidylinositol 3-kinase inhibitor antagonized shear stress-enhanced OLFM1 expression in C2C12 myotubes. As for the effects of OLFM1 on bone cells, OLFM1 inhibited osteoclast formation from mouse bone marrow cells and mouse preosteoclastic RAW264.7 cells. Moreover, OLFM1 suppressed RANKL expression and nuclear factor-κB signaling in mouse osteoblasts. Serum OLFM1 levels were positively related to OLFM1 mRNA levels in the soleus muscle and trabecular bone mineral density of mice. In conclusion, we first showed that OLFM1 suppresses osteoclast formation and RANKL expression in mouse cells.

Roles of Dkk2 in the Linkage from Muscle to Bone during Mechanical Unloading in Mice.

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)/ß-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/ß-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.

PAI-1 is involved in delayed bone repair induced by glucocorticoids in mice.

Glucocorticoid (GC) treatments induce osteoporosis and chronic GC treatments have been suggested to induce delayed bone repair; however, the mechanisms by which GC induces delayed bone repair remain unclear. We herein investigated the roles of plasminogen activator inhibitor-1 (PAI-1) in GC-induced effects on bone repair after femoral bone injury using female mice with a PAI-1 deficiency and their wild-type counterparts. Dexamethasone (Dex) increased plasma PAI-1 levels as well as PAI-1 mRNA levels in the adipose tissues and muscles of wild-type mice. PAI-1 deficiency significantly blunted Dex-induced delayed bone repair in mice. Moreover, PAI-1 deficiency significantly blunted Runx2 mRNA levels suppressed by Dex as well as Dex-induced osteoblast apoptosis at the damaged site 7 days after bone injury in mice. On the other hand, PAI-1 deficiency did not affect adipogenic gene expression enhanced by Dex at the damaged site 7 days after bone injury in mice. In conclusion, we herein showed for the first time that PAI-1 is involved in delayed bone repair after bone injury induced by GC in mice. PAI-1 may influence early stage osteoblast differentiation and apoptosis during the osteoblastic restoration phase of the bone repair process.

Extracellular vesicles secreted from mouse muscle cells suppress osteoclast formation: Roles of mitochondrial energy metabolism.

Recent reports have described the interactions of muscle and bone. Various muscle-derived humoral factors, known as myokines, affect bone. Although extracellular vesicles (EVs) play a vital role in physiological and pathophysiological processes by transferring their contents to distant tissues during bone metabolism, the roles of EVs in the muscle-bone interactions remain unknown. In the present study, we investigated the effects of EVs secreted from mouse muscle C2C12 cells on mouse bone cells and mitochondrial biogenesis. EVs secreted from C2C12 cells (Myo-EVs) were isolated from the conditioned medium of C2C12 cells by ultracentrifugation. Myo-EVs suppressed osteoclast formation as well as the expression of tartrate-resistant acid phosphatase, cathepsin K, nuclear factor of activated T-cells cytoplasmic 1 and dendritic cell-specific transmembrane protein induced by receptor activator of nuclear factor κB ligand (RANKL) in mouse bone marrow cells and preosteoclastic Raw264.7 cells. Moreover, Myo-EVs suppressed oxygen consumption and mRNA expression of the mitochondrial biogenesis markers enhanced by RANKL in these cells. However, Myo-EVs did not affect the phenotypes or mitochondrial biogenesis of mouse primary osteoblasts. In conclusion, the present study showed for the first time that Myo-EVs suppress osteoclast formation and mitochondrial energy metabolism in mouse bone marrow and Raw264.7 cells. EVs secreted from skeletal muscles might be a crucial mediator of muscle-bone interactions.

Adipose Tissue-Derived Stem Cell Sheet Improves Glucose Metabolism in Obese Mice.

Previous studies indicate that the administration of adipose tissue-derived stem cells (ADSCs) through the venous route improves insulin resistance partly through a reduction in the proinflammatory cytokines in diabetic animals. However, the effects of ADSC sheet transplantation for the treatment of diabetes and obesity still remained unknown. In this study, we investigated the effects of ADSC sheet transplantation into the subcutaneous sites on the diabetic state of mice fed high-fat and high-sucrose diet (HF/HSD). ADSCs were isolated and propagated from subcutaneous adipose tissues of non-diabetic intact mice. We used the thermoresponsive designated cell culture dishes to fabricate ADSC cell sheets. ADSC sheet transplantation into the subcutaneous sites significantly improved glucose intolerance induced by HF/HSD in mice. ADSC-conditioned medium (CM) augmented the phosphorylation of Akt with or without insulin in mouse C2C12 myotubes and mouse 3T3-L1 adipocytes. Plasma adiponectin and tumor necrosis factor-α (TNF-α) levels were significantly increased and decreased by ADSC sheet transplantation in mice with or without HF/HSD, respectively. Moreover, ADSC sheet enhanced adiponectin expression in the subcutaneous adipose tissues in HF/HSD-fed mice, whereas it reduced TNF-α expression in the visceral adipose tissues. ADSC-CM enhanced and reduced the protein levels of adiponectin and TNF-α in 3T3-L1 adipocytes, respectively. In conclusion, we first revealed that ADSC sheet transplantation into the subcutaneous sites improves glucose intolerance in mice fed with HF/HSD. Changes of adiponectin and TNF-α production from the host adipose tissues might be involved in the effects of ADSC sheet on glucose metabolism in mice. ADSC sheet transplantation therapy may be a novel clinical application for diabetes.

Roles of the vestibular system in obesity and impaired glucose metabolism in high-fat diet-fed mice.

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 ß-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.

Roles of leptin in the recovery of muscle and bone by reloading after mechanical unloading in high fat diet-fed obese 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.

A New Type of Li-Rich Rock-Salt Oxide Li2 Ni1/3 Ru2/3 O3 with Reversible Anionic Redox Chemistry.

Li-rich oxide cathodes are of prime importance for the development of high-energy lithium-ion batteries (LIBs). Li-rich layered oxides, however, always undergo irreversible structural evolution, leading to inevitable capacity and voltage decay during cycling. Meanwhile, Li-rich cation-disordered rock-salt oxides usually exhibit sluggish kinetics and inferior cycling stability, despite their firm structure and stable voltage output. Herein, a new Li-rich rock-salt oxide Li2 Ni1/3 Ru2/3 O3 with Fd-3m space group, where partial cation-ordering arrangement exists in cationic sites, is reported. Results demonstrate that a cathode fabricated from Li2 Ni1/3 Ru2/3 O3 delivers a large capacity, outstanding rate capability as well as good cycling performance with negligible voltage decay, in contrast to the common cations disordered oxides with space group Fm-3m. First principle calculations also indicate that rock-salt oxide with space group Fd-3m possesses oxygen activity potential at the state of delithiation, and good kinetics with more 0-TM (TM = transition metals) percolation networks. In situ Raman results confirm the reversible anionic redox chemistry, confirming O2- /O- evolution during cycles in Li-rich rock-salt cathode for the first time. These findings open up the opportunity to design high-performance oxide cathodes and promote the development of high-energy LIBs.

Plasminogen activator inhibitor-1 is involved in interleukin-1ß-induced matrix metalloproteinase expression in murine chondrocytes.

Objectives: Interleukin (IL)-1ß 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-1ß on the chondrocytes using wild-type and PAI-1-deficient mice.Results: IL-1ß significantly elevated PAI-1 mRNA levels in the chondrocytes from wild-type mice. PAI-1 deficiency significantly blunted the mRNA levels of TGF-ß and IL-6 enhanced by IL-1ß 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-1ß in the chondrocytes. In addition, PAI-1 deficiency significantly reversed type II collagen mRNA levels suppressed by IL-1ß 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-1ß in murine chondrocytes. PAI-1 might be crucial for the cartilage matrix degradation and the impaired chondrogenesis by IL-1ß in mice.

Plasminogen activator inhibitor-1 deficiency suppresses osteoblastic differentiation of mesenchymal stem cells in mice.

Plasminogen activator inhibitor-1 (PAI-1) is known as an inhibitor of fibrinolytic system. Previous studies suggest that PAI-1 is involved in the pathogenesis of osteoporosis induced by ovariectomy, diabetes, and glucocorticoid excess in mice. However, the roles of PAI-1 in early-stage osteogenic differentiation have remained unknown. In the current study, we investigated the roles of PAI-1 in osteoblastic differentiation of mesenchymal stem cells (MSCs) using wild-type (WT) and PAI-1-deficient (PAI-1 KO) mice. PAI-1 mRNA levels were increased with time during osteoblastic differentiation of MSCs or mesenchymal ST-2 cells. However, the increased PAI-1 levels declined at the mineralization phase in the experiment using MC3T3-E1 cells. PAI-1 deficiency significantly blunted the expression of osteogenic gene, such as osterix and alkaline phosphatase enhanced by bone morphogenetic protein (BMP)-2 in bone marrow-derived MSCs (BM-MSCs), adipose-tissue-derived MSCs (AD-MSCs), and bone marrow stromal cells of mice. Moreover, a reduction in endogenous PAI-1 levels by small interfering RNA significantly suppressed the expression of osteogenic gene in ST-2 cells. Plasmin did not affect osteoblastic differentiation of AD-MSCs induced by BMP-2 with or without PAI-1 deficiency. PAI-1 deficiency and a reduction in endogenous PAI-1 levels did not affect the phosphorylations of receptor-specific Smads by BMP-2 and transforming growth factor-ß in AD-MSCs and ST-2 cells, respectively. In conclusion, we first showed that PAI-1 is crucial for the differentiation of MSCs into osteoblasts in mice.

Serpina3n, Dominantly Expressed in Female Osteoblasts, Suppresses the Phenotypes of Differentiated Osteoblasts 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.

c-Myb regulates transcriptional activation of miR-143/145 in vascular smooth muscle cells.

BACKGROUND: MicroRNAs (miR) are small non-coding RNAs that regulate diverse biological functions. The bicistronic gene miR-143/145 determines cell fate and phenotype of vascular smooth muscle cells (VSMC), in part, by destabilizing Elk-1 mRNA. The transcription factor c-Myb also regulates differentiation and proliferation of VSMC, and here we test whether these effects may be mediated by miR-143/145. METHODS & RESULTS: Flow cytometry of cardiovascular-directed d3.75 embryoid bodies (EBs) isolated smooth muscle progenitors with specific cell surface markers. In c-myb knockout (c-myb -/-) EB, these progenitors manifest low levels of miR-143 (19%; p<0.05) and miR-145 (6%; p<0.01) expression as compared to wild-type (wt) EB. Primary VSMC isolated from transgenic mice with diminished expression (c-myblx/lx) or reduced activity (c-mybh/h) of c-Myb also manifest low levels of miR-143 (c-myblx/lx: 50%; c-mybh/h: 41%), and miR-145 (c-myblx/lx: 49%; c-mybh/h: 56%), as compared to wt (P<0.05). Sequence alignment identified four putative c-Myb binding sites (MBS1-4) in the proximal promoter (PP) of the miR-143/145 gene. PP-reporter constructs revealed that point mutations in MBS1 and MBS4 abrogated c-Myb-dependent transcription from the miR-143/145 PP (P<0.01). Chromatin immunoprecipitation (ChIP) revealed preferential c-Myb binding at MBS4 (p<0.001). By conjugating Elk-1 3'-untranslated region (UTR) to a reporter and co-transducing wt VSMC with this plus a miR-143-antagomir, and co-transducing c-myblx/lx VSMC with this plus a miR-143-mimic, we demonstrate that c-Myb's ability to repress Elk-1 is mediated by miR-143. CONCLUSION: c-Myb regulates VSMC gene expression by transcriptional activation of miR-143/145.

Roles of plasminogen in the alterations in bone marrow hematopoietic stem cells during bone repair.

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-ß (TGF-ß) elevated by bone injury. TGF-ß 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-ß, and SDF-1 at the damaged site during bone repair in mice.

Role of Macrophages and Plasminogen Activator Inhibitor-1 in Delayed Bone Repair in Diabetic Female Mice.

Delayed fracture healing is a clinical problem in diabetic patients. However, the mechanisms of diabetic delayed bone repair remain unknown. Here, we investigate the role of macrophages in diabetic delayed bone repair after femoral bone injury in streptozotocin (STZ)-treated and plasminogen activator inhibitor-1 (PAI-1)-deficient female mice. STZ treatment significantly decreased the numbers of F4/80-positive cells (macrophages) but not granulocyte-differentiation antigen-1-positive cells (neutrophils) at the damaged site on day 2 after femoral bone injury in mice. It significantly decreased the messenger RNA (mRNA) levels of macrophage colony-stimulating factor, inducible nitric oxide synthase (iNOS), interleukin (IL)-6, and CD206 at the damaged site on day 2 after bone injury. Moreover, STZ treatment attenuated a decrease in the number of hematopoietic stem cells in bone marrow induced by bone injury. On the other hand, PAI-1 deficiency significantly attenuated a decrease in the number of F4/80-positive cells induced by STZ treatment at the damaged site on day 2 after bone injury in mice. PAI-1 deficiency did not affect the mRNA levels of iNOS and IL-6 in F4/80- and CD11b-double-positive cells from the bone marrow of the damaged femurs decreased by diabetes in mice. PAI-1 deficiency significantly attenuated the phagocytosis of macrophages at the damaged site suppressed by diabetes. In conclusion, we demonstrated that type 1 diabetes decreases accumulation and phagocytosis of macrophages at the damaged site during early bone repair after femoral bone injury through PAI-1 in female mice.

Direct Visualization of the Reversible O2- /O- Redox Process in Li-Rich Cathode Materials.

Conventional cathodes of Li-ion batteries mainly operate through an insertion-extraction process involving transition metal redox. These cathodes will not be able to meet the increasing requirements until lithium-rich layered oxides emerge with beyond-capacity performance. Nevertheless, in-depth understanding of the evolution of crystal and excess capacity delivered by Li-rich layered oxides is insufficient. Herein, various in situ technologies such as X-ray diffraction and Raman spectroscopy are employed for a typical material Li1.2 Ni0.2 Mn0.6 O2 , directly visualizing O- O- (peroxo oxygen dimers) bonding mostly along the c-axis and demonstrating the reversible O2- /O- redox process. Additionally, the formation of the peroxo OO bond is calculated via density functional theory, and the corresponding OO bond length of ≈1.3 Å matches well with the in situ Raman results. These findings enrich the oxygen chemistry in layered oxides and open opportunities to design high-performance positive electrodes for lithium-ion batteries.