Hiroshi Kaji

Inactivation of menin, a Smad3-interacting protein, blocks transforming growth factor type β signaling

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by endocrine tumors of parathyroids, pancreatic islets, and anterior pituitary. The MEN1 gene encodes a nuclear protein called menin. In MEN1 carriers inactivating mutations give rise to a truncated product consistent with menin acting as a tumor suppressor gene. However, the role of menin in tumorigenesis and its physiological functions are not known. Here, we show that menin inactivation by antisense RNA antagonizes transforming growth factor type β-mediated cell growth inhibition. Menin interacts with Smad3, and antisense menin suppresses transforming growth factor type β-induced and Smad3-induced transcriptional activity by inhibiting Smad3/4-DNA binding at specific transcriptional regulatory sites. These results implicate a mechanism of tumorigenesis by menin inactivation.

Interaction between Muscle and Bone

The clinical significance of sarcopenia and osteoporosis has increased with the increase in the population of older people. Sarcopenia is defined by decreased muscle mass and impaired muscle function, which is related to osteoporosis independently and dependently. Numerous lines of clinical evidence suggest that lean body mass is positively related to bone mass, which leads to reduced fracture risk. Genetic, endocrine and mechanical factors affect both muscle and bone simultaneously. Vitamin D, the growth hormone/insulin-like growth factor I axis and testosterone are physiologically and pathologically important as endocrine factors. These findings suggest the presence of interactions between muscle and bone, which might be very important for understanding the physiology and pathophysiology of sarcopenia and osteoporosis. Muscle/bone relationships include two factors: local control of muscle to bone and systemic humoral interactions between muscle and bone. As a putative local inducer of muscle ossification, we found Tmem119, a parathyroid hormone-responsive osteoblast differentiation factor. Moreover, osteoglycin might be one of the muscle-derived humoral bone anabolic factors. This issue may be important for the development of novel drugs and biomarkers for osteoporosis and sarcopenia. Further research will be necessary to clarify the details of the linkage of muscle and bone.

Menin Inactivation Leads to Loss of Transforming Growth Factor β Inhibition of Parathyroid Cell Proliferation and Parathyroid Hormone Secretion

Primary hyperparathyroidism is a common endocrine disorder caused by parathyroid gland enlargement and excessive parathyroid hormone (PTH) secretion. However, the precise mechanisms of tumorigenesis of the parathyroids are unknown. Here we have investigated the roles of transforming growth factor (TGF)-β and menin, the product of the multiple endocrine neoplasia type 1 (Men1) gene, in the proliferation and PTH production of parathyroid cells from either patients with secondary hyperparathyroidism or Men1. TGF-β was expressed in the parathyroid endocrine cells. Addition of TGF-β to parathyroid cells from patients with secondary hyperparathyroidism inhibited their proliferation and PTH secretion. These responses to TGF-β were lost when menin was specifically inactivated by antisense oligonucleotides. Moreover, TGF-β did not affect the proliferation and PTH production of parathyroid cells from a Men1 patient. These results indicate that menin is required for TGF-β action in the parathyroid. We conclude that TGF-β is an important autocrine/paracrine negative regulator of parathyroid cell proliferation and PTH secretion and that loss of TGF-β signaling due to menin inactivation contributes to parathyroid tumorigenesis.

Risk Factors for Hip Fracture in Hemodialysis Patients

Background: The incidence and mortality of hip fractures were several times greater in the patients with hemodialysis (HD) than in the general population. Although patients with end-stage renal disease develop renal osteodystrophy, few published data examined the risk factor of hip fractures in the dialysis population. Methods: The present study was performed to compare various indices and bone mineral density (BMD) of HD patients with or without the history of hip fractures. Moreover, we analyzed the factors which predicted hip fractures in 183 patients with chronic HD enrolled in the cross-sectional study. Results: Serum level of alkaline phosphatase was significantly higher in HD patients with hip fractures, compared to those without hip fractures. Oral calcium carbonate dose was significantly lower in HD patients with hip fractures. 1/3 radius (R)-BMD and ultradistal (UD)R-BMD were significantly lower in HD patients with hip fractures. However, lumbar spine (LS)-BMD was comparable in HD patients with or without hip fractures. Although there was no significant differences of BMD between with and without hip fractures in diabetes mellitus, UDR-BMD of patients with hip fractures was significantly lower than that of patients without hip fractures in chronic glomerulonephritis. Radial BMD was lower in female patients with hip fractures, compared to without hip fractures, although there were no significant differences in male patients. In multiple logistic regression analysis, oral calcitriol dose and 1/3R-BMD were selected as a risk factor of hip fractures in HD patients. Conclusion: Radial BMD was lower in HD patients with hip fractures. However, its contribution is different, depending on gender and the original disease leading to HD. Radial BMD and oral calcitriol dose seemed to be important to predict the risk of hip fractures.

Interactions between muscle tissues and bone metabolism.

Sarcopenia and osteoporosis have recently been noted for their relationship with locomotive syndrome and increased number of older people. Sarcopenia is defined by decreased muscle mass and impaired muscle function, which may be associated with frailty. Several clinical data have indicated that increased muscle mass is related to increased bone mass and reduced fracture risk. Genetic, endocrine and mechanical factors as well as inflammatory and nutritional states concurrently affect muscle tissues and bone metabolism. Several genes, including myostatin and α‐actinin 3, have been shown in a genome‐wide association study (GWAS) to be associated with both sarcopenia and osteoporosis. Vitamin D, growth hormone and testosterone as well as pathological disorders, such as an excess in glucocorticoid and diabetes, affect both muscle and bone. Basic and clinical research of bone metabolism and muscle biology suggests that bone interacts with skeletal muscle via signaling from local and humoral factors in addition to their musculoskeletal function. However, the physiological and pathological mechanisms related to muscle and bone interactions remain unclear. We found that Tmem119 may play a critical role in the commitment of myoprogenitor cells to the osteoblast lineage. We also reported that osteoglycin and FAM5C might be muscle‐derived humoral osteogenic factors. Other factors, including myostatin, osteonectin, insulin‐like growth factor I, irisin and osteocalcin, may be associated with the interactions between muscle tissues and bone metabolism. J. Cell. Biochem. 116: 687–695, 2015.

Statin Suppresses Apoptosis in Osteoblastic Cells : Role of Transforming Growth Factor-β-Smad3 Pathway

Statins possess pleiotropic effects in several tissues. Among them, their bone anabolic actions have been recently noted. We have proposed that Smad3, a TGF-beta-signaling molecule, is a promoter of bone formation. However, whether statins would affect TGF-beta-Smad3 pathway in osteoblasts is still unknown. The present study was performed to examine the effects of statin on Smad3 expression and cell apoptosis by employing mouse osteoblastic MC3T3-E1 and rat osteoblastic UMR-106 cells. Statins (pitavastatin, mevastatin, and simvastatin) as well as alendronate increased the levels of Smad3 in MC3T3-E1 cells. The effects of pitavastatin on Smad3 levels were observed from 3 hours and later. Pitavastatin induced the expression of TGF-beta, and cycloheximide, a protein synthesis inhibitor, antagonized the increased levels of pitavastatin on Smad3. On the other hand, pitavastatin antagonized dexamethasone- or etoposide-induced apoptosis in a dose-dependent manner, and Smad3 inactivation by dominant negative Smad3 or an inhibition of endogenous TGF-beta action by SB431542 antagonized anti-apoptotic effects of pitavastatin, indicating that pitavastatin suppressed osteoblast apoptosis partly through TGF-beta-Smad3 pathway. In conclusion, the present study has demonstrated for the first time that statin suppressed cell apoptosis partly through TGF-beta-Smad3 pathway in osteoblastic cells.

Plasminogen Plays a Crucial Role in Bone Repair

The further development in research of bone regeneration is necessary to meet the clinical demand for bone reconstruction. Plasminogen is a critical factor of the tissue fibrinolytic system, which mediates tissue repair in the skin and liver. However, the role of the fibrinolytic system in bone regeneration remains unknown. Herein, we investigated bone repair and ectopic bone formation using plasminogen‐deficient (Plg–/–) mice. Bone repair of the femur is delayed in Plg–/– mice, unlike that in the wild‐type (Plg+/+) mice. The deposition of cartilage matrix and osteoblast formation were both decreased in Plg–/– mice. Vessel formation, macrophage accumulation, and the levels of vascular endothelial growth factor (VEGF) and transforming growth factor‐β (TGF‐β) were decreased at the site of bone damage in Plg–/– mice. Conversely, heterotopic ossification was not significantly different between Plg+/+ and Plg–/– mice. Moreover, angiogenesis, macrophage accumulation, and the levels of VEGF and TGF‐β were comparable between Plg+/+ and Plg–/– mice in heterotopic ossification. Our data provide novel evidence that plasminogen is essential for bone repair. The present study indicates that plasminogen contributes to angiogenesis related to macrophage accumulation, TGF‐β, and VEGF, thereby leading to the enhancement of bone repair.

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.

Plasminogen activator inhibitor-1 is involved in impaired bone repair associated with diabetes in female mice.

Previous studies suggest that fracture healing is impaired in diabetes; however, the underlying mechanism remains unclear. Here, we investigated the roles of plasminogen activator inhibitor-1 (PAI-1) in the impaired bone repair process by using streptozotocin (STZ)-induced diabetic female wild-type (PAI-1 +/+) and PAI-1-deficient (PAI-1 −/−) mice. Bone repair and the number of alkaline phosphatase (ALP)-positive cells at the site of a femoral bone damage were comparable in PAI-1 +/+ and PAI-1 −/− mice without STZ treatment. Although the bone repair process was delayed by STZ treatment in PAI-1 +/+ mice, this delayed bone repair was blunted in PAI-1 −/− mice. The reduction in the number of ALP-positive cells at the site of bone damage induced by STZ treatment was attenuated in PAI-1 −/− mice compared to PAI-1 +/+ mice. On the other hand, PAI-1 deficiency increased the levels of ALP and type I collagen mRNA in female mice with or without STZ treatment, and the levels of Osterix and osteocalcin mRNA, suppressed by diabetic state in PAI-1 +/+ mice, were partially protected in PAI-1 −/− mice. PAI-1 deficiency did not affect formation of the cartilage matrix and the levels of types II and X collagen and aggrecan mRNA suppressed by STZ treatment, although PAI-1 deficiency increased the expression of chondrogenic markers in mice without STZ treatment. The present study indicates that PAI-1 is involved in the impaired bone repair process induced by the diabetic state in part through a decrease in the number of ALP-positive cells.

Plasminogen activator inhibitor-1 is involved in streptozotocin-induced bone loss in female mice

In diabetic patients, the risk of fracture is high because of impaired bone formation. However, the details of the mechanisms in the development of diabetic osteoporosis remain unclear. In the current study, we investigated the role of plasminogen activator inhibitor (PAI)-1 in the pathogenesis of type 1 diabetic osteoporosis by using PAI-1–deficient mice. Quantitative computed tomography analysis showed that PAI-1 deficiency protected against streptozotocin-induced bone loss in female mice but not in male mice. PAI-1 deficiency blunted the changes in the levels of Runx2, osterix, and alkaline phosphatase in tibia as well as serum osteocalcin levels suppressed by the diabetic state in female mice only. Furthermore, the osteoclast levels in tibia, suppressed in diabetes, were also blunted by PAI-1 deficiency in female mice. Streptozotocin markedly elevated the levels of PAI-1 mRNA in liver in female mice only. In vitro study demonstrated that treatment with active PAI-1 suppressed the levels of osteogenic genes and mineralization in primary osteoblasts from female mouse calvaria. In conclusion, the current study indicates that PAI-1 is involved in the pathogenesis of type 1 diabetic osteoporosis in females. The expression of PAI-1 in the liver and the sensitivity of bone cells to PAI-1 may be an underlying mechanism.