Tmem119 is involved in bone anabolic effects of PTH through enhanced osteoblastic bone formation in mice.

The intermittent administration of parathyroid hormone (PTH) exerts potent bone anabolic effects, which increase bone mineral density (BMD) and reduce fracture risk in osteoporotic patients. However, the underlying mechanisms remain unclear. Tmem119 has been proposed as a factor that is closely linked to the osteoblast phenotype, and we previously reported that PTH enhanced the expression of Tmem119 in mouse osteoblastic cells. However, roles of Tmem119 in the bone anabolic effects of PTH in vivo remain unknown. We herein investigated the roles of Tmem119 in bone anabolic effects of PTH using Tmem119-deficient mice. Tmem119 deficiency significantly reduced PTH-induced increases in trabecular bone volume and cortical BMD of femurs. Effects of Tmem119 deficiency on bone mass seemed predominant in female mice. Histomorphometric analyses with calcein labeling showed that Tmem119 deficiency significantly attenuated PTH-induced increases in the rates of bone formation and mineralization as well as numbers of osteoblasts. Moreover, Tmem119 deficiency significantly blunted PTH-induced decreases in phosphorylation of ß-catenin and increases in alkaline phosphatase activity in osteoblasts. In conclusion, the present results indicate that Tmem119 is involved in bone anabolic effects of PTH through osteoblastic bone formation partly related to canonical Wnt-ß-catenin signaling in mice.

Effects of elastase-induced emphysema on muscle and bone in mice.

Chronic obstructive pulmonary disease (COPD) causes sarcopenia and osteoporosis. However, the mechanisms underlying muscle and bone loss as well as the interactions between muscle and bone in the COPD state remain unclear. Therefore, we herein investigated the effects of the COPD state on muscle and bone in mice intratracheally administered porcine pancreatic elastase (PPE). The intratracheal administration of PPE to mice significantly reduced trabecular bone mineral density (BMD), trabecular bone volume, trabecular number, cortical BMD and cortical area. It also significantly decreased grip strength, but did not affect muscle mass or the expression of myogenic differentiation-, protein degradation- or autophagy-related genes in the soleus and gastrocnemius muscles. Among the myokines examined, myostatin mRNA levels in the soleus muscles were significantly elevated in mice treated with PPE, and negatively related to grip strength, but not bone parameters, in mice treated with or without 2 U PPE in simple regression analyses. Grip strength positively related to bone parameters in mice treated with or without PPE. In conclusion, we showed that a PPE model of COPD in mice exerts dominant effects on bone rather than skeletal muscles. Increased myostatin expression in the soleus muscles of mice in the COPD state may negatively relate to a reduction in grip strength, but not bone loss.

Irisin improves delayed bone repair in diabetic female mice.

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.

Role of Macrophages and Plasminogen Activator Inhibitor-1 in Delayed Bone Repair Induced by Glucocorticoids in Mice.

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.

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.

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.

Role of irisin in androgen-deficient muscle wasting and osteopenia in mice.

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.

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.

Plasminogen activator inhibitor-1 deficiency enhances subchondral osteopenia after induction of osteoarthritis in mice.

BACKGROUND: Subchondral osteopenia is important for the pathophysiology of osteoarthritis (OA). Although previous studies suggest that plasminogen activator inhibitor-1 (PAI-1), an inhibitor of fibrinolysis, is related to bone metabolism, its role in OA remains unknown. We therefore investigated the roles of PAI-1 in the subchondral bone in OA model mice. METHODS: Wild type (WT) and PAI-1-deficient (KO) mice were ovariectomized (OVX), and then destabilization of the medial meniscus (DMM) surgery was performed. RESULTS: DMM and OVX significantly decreased the trabecular bone mineral density of the subchondral bone evaluated by quantitative computed tomography in PAI-1 KO mice. The effects of OVX and/or PAI-1 deficiency on the OARSI score for the evaluation of the progression of knee degeneration were not significant. PAI-1 deficiency significantly augmented receptor activator nuclear factor κB ligand mRNA levels enhanced by IL-1ß in mouse primary osteoblasts, although it did not affect osteoblast differentiation. Moreover, PAI-1 deficiency significantly increased osteoclast formation from mouse bone marrow cells. CONCLUSION: We showed that PAI-1 deficiency accelerates the subchondral osteopenia after induction of OA in mice. PAI-1 might suppress an enhancement of bone resorption and subsequent subchondral osteopenia after induction of OA in mice.

The vestibular system is critical for the changes in muscle and bone induced by hypergravity in mice.

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 ß-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.

Role of osteoclasts in heterotopic ossification enhanced by fibrodysplasia ossificans progressiva-related activin-like kinase 2 mutation in mice.

Fibrodysplasia ossificans progressiva (FOP) is a disorder of skeletal malformations and progressive heterotopic ossification. The constitutively activating mutation (R206H) of the bone morphogenetic protein type 1 receptor, activin-like kinase 2 (ALK2), is responsible for the pathogenesis of FOP. Although transfection of the causal mutation of FOP into myoblasts enhances osteoclast formation by transforming growth factor-ß (TGF-ß), the role of osteoclasts in heterotopic ossification is unknown. We therefore examined the effects of alendronate, SB431542 and SB203580 on heterotopic ossification induced by the causal mutation of FOP. Total bone mineral content as well as numbers of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated and alkaline phosphatase (ALP)-positive cells in heterotopic bone were significantly higher in muscle tissues implanted with ALK2 (R206H)-transfected mouse myoblastic C2C12 cells than in the tissues implanted with empty vector-transfected cells in nude mice. Alendronate, an aminobisphosphonate, did not affect total mineral content or numbers of TRAP-positive multinucleated and ALP-positive cells in heterotopic bone, which were enhanced by the implantation of ALK2 (R206H)-transfected C2C12 cells, although it significantly decreased serum levels of cross-linked C-telopeptide of type I collagen, a bone resorption index. Moreover, neither SB431542, an inhibitor of TGF-ß receptor type I kinase, nor SB203580, an inhibitor of p38 mitogen-activated protein kinase, affected the increase in heterotopic ossification due to the implantation of ALK2 (R206H)-transfected C2C12 cells. In conclusion, the present study indicates that osteoclast inhibition does not affect heterotopic ossification enhanced by FOP-related mutation.

Stromal cell-derived factor-1 mediates changes of bone marrow stem cells during the bone repair process.

Osteoblasts, osteoclasts, chondrocytes, and macrophages that participate in the bone repair process are derived from hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). However, the roles of these stem cells during the repair of injured bone tissue are still unclear. In the present study, we examined the effects of bone defect on HSCs and MSCs in bone marrow and spleen in 75 mice and its mechanism. We analyzed the HSC and MSC populations in these tissues of a mouse with femoral bone damage by using flow cytometry. The number of HSCs in the bone marrow of mice with damaged femurs was significantly lower than the number of these cells in the bone marrow of the contralateral intact femurs on day 2 after injury. Meanwhile, the number of MSCs in the bone marrow of mice with damaged femurs was significantly higher than that of the contralateral femurs. Both intraperitoneal administration of AMD3100, a C-X-C chemokine receptor 4 (CXCR4) antagonist, and local treatment with an anti-stromal cell-derived factor-1 (SDF-1) antibody blunted the observed decrease in HSC and increase in MSC populations within the bone marrow of injured femurs. In conclusion, the present study revealed that there is a concurrent decrease and increase in the numbers of HSCs and MSCs, respectively, in the bone marrow during repair of mouse femoral bone damage. Furthermore, the SDF-1/CXCR4 system was implicated as contributing to the changes in these stem cell populations upon bone injury.

α2-Antiplasmin is involved in bone loss induced by ovariectomy in mice.

The mechanism of postmenopausal osteoporosis is not fully understood. α2-Antiplasmin (α2-AP) is the primary inhibitor of plasmin in the fibrinolytic system, but is known to have activities beyond fibrinolysis. However, its role in bone metabolism and the pathogenesis of osteoporosis remains unknown. In the current study, we therefore examined the effects of α2-AP deficiency on ovariectomy (OVX)-induced bone loss by using wild-type and α2-AP-deficient mice. Quantitative computed tomography analysis revealed that α2-AP deficiency blunted OVX-induced trabecular bone loss in mice. Moreover, α2-AP deficiency significantly blunted serum levels of bone-specific alkaline phosphatase, cross-linked C-telopeptide of type I collagen, and interleukin (IL)-1ß elevated by OVX. α2-AP treatment elevated the levels of IL-1ß and tumor necrosis factor (TNF)-α mRNA in RAW 264.7 cells, although it suppressed osteoclast formation induced by receptor activator of nuclear factor-κB ligand. α2-AP treatment activated ERK1/2 and p38 MAP kinase pathways in RAW 264.7 cells, and these MAP kinase inhibitors antagonized the levels of IL-1ß mRNA elevated by α2-AP. The data demonstrate that α2-AP is linked to bone loss due to OVX, through a mechanism that depends in part on the production of IL-1ß and TNF-α in monocytes.

The Tissue Fibrinolytic System Contributes to the Induction of Macrophage Function and CCL3 during Bone Repair in Mice.

Macrophages play crucial roles in repair process of various tissues. However, the details in the role of macrophages during bone repair still remains unknown. Herein, we examined the contribution of the tissue fibrinolytic system to the macrophage functions in bone repair after femoral bone defect by using male mice deficient in plasminogen (Plg-/-), urokinase-type plasminogen activator (uPA-/-) or tissue-type plasminogen activator (tPA-/-) genes and their wild-type littermates. Bone repair of the femur was delayed in uPA-/- mice until day 6, compared with wild-type (uPA+/+) mice. Number of Osterix-positive cells and vessel formation were decreased in uPA-/- mice at the bone injury site on day 4, compared with those in uPA+/+ mice. Number of macrophages and their phagocytosis at the bone injury site were reduced in uPA-/- and Plg-/-, but not in tPA-/- mice on day 4. Although uPA or plasminogen deficiency did not affect the levels of cytokines, including TNF-α, IL-1ß, IL-6, IL-4 and IFN-γ mRNA in the damaged femur, the elevation in CCL3 mRNA levels was suppressed in uPA-/- and Plg-/-, but not in tPA-/- mice. Neutralization of CCL3 antagonized macrophage recruitment to the site of bone injury and delayed bone repair in uPA+/+, but not in uPA-/- mice. Our results provide novel evidence that the tissue fibrinolytic system contributes to the induction of macrophage recruitment and CCL3 at the bone injury site, thereby, leading to the enhancement of the repair process.

Erythropoietin Receptor Antagonist Suppressed Ectopic Hemoglobin Synthesis in Xenografts of HeLa Cells to Promote Their Destruction.

The aim of this study is to explore a cause-oriented therapy for patients with uterine cervical cancer that expresses erythropoietin (Epo) and its receptor (EpoR). Epo, by binding to EpoR, stimulates the proliferation and differentiation of erythroid progenitor cells into hemoglobin-containing red blood cells. In this study, we report that the HeLa cells in the xenografts expressed ε, γ, and α globins as well as myoglobin (Mb) to produce tetrameric α2ε2 and α2γ2 and monomeric Mb, most of which were significantly suppressed with an EpoR antagonist EMP9. Western blotting revealed that the EMP9 treatment inhibited the AKT-pAKT, MAPKs-pMAPKs, and STAT5-pSTAT5 signaling pathways. Moreover, the treatment induced apoptosis and suppression of the growth and inhibited the survival through disruption of the harmonized hemoprotein syntheses in the tumor cells concomitant with destruction of vascular nets in the xenografts. Furthermore, macrophages and natural killer (NK) cells with intense HIF-1α expression recruited significantly more in the degenerating foci of the xenografts. These findings were associated with the enhanced expressions of nNOS in the tumor cells and iNOS in macrophages and NK cells in the tumor sites. The treated tumor cells exhibited a substantial number of perforations on the cell surface, which indicates that the tumors were damaged by both the nNOS-induced nitric oxide (NO) production in the tumor cells as well as the iNOS-induced NO production in the innate immune cells. Taken together, these data suggest that HeLa cells constitutively acquire ε, γ and Mb synthetic capacity for their survival. Therefore, EMP9 treatment might be a cause-oriented and effective therapy for patients with squamous cell carcinoma of the uterine cervix.

Role of plasminogen activator inhibitor-1 in glucocorticoid-induced diabetes and osteopenia in mice.

Long-term use of glucocorticoids (GCs) causes numerous adverse effects, including glucose/lipid abnormalities, osteoporosis, and muscle wasting. The pathogenic mechanism, however, is not completely understood. In this study, we used plasminogen activator inhibitor-1 (PAI-1)-deficient mice to explore the role of PAI-1 in GC-induced glucose/lipid abnormalities, osteoporosis, and muscle wasting. Corticosterone markedly increased the levels of circulating PAI-1 and the PAI-1 mRNA level in the white adipose tissue of wild-type mice. PAI-1 deficiency significantly reduced insulin resistance and glucose intolerance but not hyperlipidemia induced by GC. An in vitro experiment revealed that active PAI-1 treatment inhibits insulin-induced phosphorylation of Akt and glucose uptake in HepG2 hepatocytes. However, this was not observed in 3T3-L1 adipocytes and C2C12 myotubes, indicating that PAI-1 suppressed insulin signaling in hepatocytes. PAI-1 deficiency attenuated the GC-induced bone loss presumably via inhibition of apoptosis of osteoblasts. Moreover, the PAI-1 deficiency also protected from GC-induced muscle loss. In conclusion, the current study indicated that PAI-1 is involved in GC-induced glucose metabolism abnormality, osteopenia, and muscle wasting in mice. PAI-1 may be a novel therapeutic target to mitigate the adverse effects of GC.

Tissue-type plasminogen activator deficiency delays bone repair: roles of osteoblastic proliferation and vascular endothelial growth factor.

Further development in research of bone regeneration is necessary to meet the clinical demand for bone reconstruction. Recently, we reported that plasminogen is crucial for bone repair through enhancement of vessel formation. However, the details of the role of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) in the bone repair process still remain unknown. Herein, we examined the effects of plasminogen activators on bone repair after a femoral bone defect using tPA-deficient (tPA(-/-)) and uPA-deficient (uPA(-/-)) mice. Bone repair of the femur was delayed in tPA(-/-) mice, unlike that in wild-type (tPA(+/+)) mice. Conversely, the bone repair was comparable between wild-type (uPA(+/+)) and uPA(-/-) mice. The number of proliferative osteoblasts was decreased at the site of bone damage in tPA(-/-) mice. Moreover, the proliferation of primary calvarial osteoblasts was reduced in tPA(-/-) mice. Recombinant tPA facilitated the proliferation of mouse osteoblastic MC3T3-E1 cells. The proliferation enhanced by tPA was antagonized by the inhibition of endogenous annexin 2 by siRNA and by the inhibition of extracellular signal-regulated kinase (ERK)1/2 phosphorylation in MC3T3-E1 cells. Vessel formation as well as the levels of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) were decreased at the damaged site in tPA(-/-) mice. Our results provide novel evidence that tPA is crucial for bone repair through the facilitation of osteoblast proliferation related to annexin 2 and ERK1/2 as well as enhancement of vessel formation related to VEGF and HIF-1α at the site of bone damage.