The paired-box homeodomain transcription factor Pax6 binds to the upstream region of the TRAP gene promoter and suppresses receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation.

Osteoclast formation is regulated by balancing between the receptor activator of nuclear factor-κB ligand (RANKL) expressed in osteoblasts and extracellular negative regulatory cytokines such as interferon-γ (IFN-γ) and interferon-ß (IFN-ß), which can suppress excessive bone destruction. However, relatively little is known about intrinsic negative regulatory factors in RANKL-mediated osteoclast differentiation. Here, we show the paired-box homeodomain transcription factor Pax6 acts as a negative regulator of RANKL-mediated osteoclast differentiation. Electrophoretic mobility shift and reporter assays found that Pax6 binds endogenously to the proximal region of the tartrate acid phosphatase (TRAP) gene promoter and suppresses nuclear factor of activated T cells c1 (NFATc1)-induced TRAP gene expression. Introduction of Pax6 retrovirally into bone marrow macrophages attenuates RANKL-induced osteoclast formation. Moreover, we found that the Groucho family member co-repressor Grg6 contributes to Pax6-mediated suppression of the TRAP gene expression induced by NFATc1. These results suggest that Pax6 interferes with RANKL-mediated osteoclast differentiation together with Grg6. Our results demonstrate that the Pax6 pathway constitutes a new aspect of the negative regulatory circuit of RANKL-RANK signaling in osteoclastogenesis and that the augmentation of Pax6 might therefore represent a novel target to block pathological bone resorption.

Tumor-induced osteomalacia: benign tumor recurrence after two surgical resections at two different medical institutions.

OBJECTIVE: To describe an exceedingly rare case of tumor-induced osteomalacia (TIO) caused by a benign phosphaturic mesenchymal tumor that recurred after two surgical resections at two different medical institutions. METHODS: A 69-year-old man complained of a 3-year history of persistent whole body pain and presented with hypophosphatemia, elevated serum levels of bone-specific alkaline phosphatase and fibroblast growth factor-23 (FGF-23), and multiple fractures. The patient was suspected of having TIO. We conducted the following diagnostic modalities considered useful to detect the tumor: serum FGF-23 level measurement in the extremities, positron emission tomography (PET)-computed tomography (CT),and magnetic resonance imaging (MRI). RESULTS: The causative tumor could be detected in the right humerus not by venous catheterization for serum FGF-23 level measurement but by the combination of PET-CT and MRI. The authors, who had successfully treated two patients with TIO, visually confirmed the absence of any tumor residue during tumorectomy. Nevertheless, the tumor recurred after surgery. The residual tumor could be localized in the right humerus not by PET-CT but by the combination of superficial venous sampling at 10 sites and MRI. The residual tumor recurred after the second tumorectomy at another hospital. This patient indicates that the possibility--a benign causative tumor may not be completely resected by surgery--cannot be ruled out thoroughly. CONCLUSION: Superficial venous sampling at multiple sites may be a surrogate for venous catheterization. Patients with TIO should be meticulously followed-up after surgery to detect any residual tumor by periodic biochemical monitoring and by imaging modalities accordingly.

Catalytic conversion of carbon dioxide into dimethyl carbonate using reduced copper-cerium oxide catalysts as low as 353 K and 1.3 MPa and the reaction mechanism.

Synthesis of dimethyl carbonate (DMC) from CO2 and methanol under milder reaction conditions was performed using reduced cerium oxide catalysts and reduced copper-promoted Ce oxide catalysts. Although the conversion of methanol was low (0.005-0.11%) for 2 h of reaction, DMC was synthesized as low as 353 K and at total pressure of as low as 1.3 MPa using reduced Cu-CeO2 catalyst (0.5 wt% of Cu). The apparent activation energy was 120 kJ mol(-1) and the DMC synthesis rates were proportional to the partial pressure of CO2. An optimum amount of Cu addition to CeO2 was 0.1 wt% for DMC synthesis under the conditions at 393 K and total pressure of 1.3 MPa for 2 h (conversion of methanol: 0.15%) due to the compromise of two effects of Cu: the activation of H2 during reduction prior to the kinetic tests and the block (cover) of the surface active site. The reduction effects in H2 were monitored through the reduction of Ce(4+) sites to Ce(3+) based on the shoulder peak intensity at 5727 eV in the Ce L3-edge X-ray absorption near-edge structure (XANES). The Ce(3+) content was 10% for reduced CeO2 catalyst whereas it increased to 15% for reduced Cu-CeO2 catalyst (0.5 wt% of Cu). Moreover, the content of reduced Ce(3+) sites (10%) associated with the surface O vacancy (defect sites) decreased to 5% under CO2 at 290 K for reduced Cu-CeO2 catalyst (0.1 wt% of Cu). The adsorption step of CO2 on the defect sites might be the key step in DMC synthesis and thus the DMC synthesis rate dependence on the partial pressure of CO2 was proportional. Subsequent H atom subtraction steps from methanol at the neighboring surface Lewis base sites should combine two methoxy species to the adsorbed CO2 to form DMC, water, and restore the surface O vacancy.

IL-27 suppresses RANKL expression in CD4+ T cells in part through STAT3.

The receptor activator of NF-κB ligand (RANKL), which is expressed by not only osteoblasts but also activated T cells, plays an important role in bone-destructive diseases such as rheumatoid arthritis. IL-27, a member of the IL-6/IL-12 family cytokines, activates STAT1 and STAT3, promotes early helper T (Th)1 differentiation and generation of IL-10-producing type 1 regulatory T (Tr1) cells, and suppresses the production of inflammatory cytokines and inhibits Th2 differentiation. In addition, IL-27 was recently demonstrated to not only inhibit Th17 differentiation but also directly act on osteoclast precursor cells and suppress RANKL-mediated osteoclastogenesis through STAT1-dependent inhibition of c-Fos, leading to amelioration of the inflammatory bone destruction. In the present study, we investigated the effect of IL-27 on the expression of RANKL in CD4(+) T cells. We found that IL-27 greatly inhibits cell surface expression of RANKL on naive CD4(+) T cells activated by T cell receptor ligation and secretion of its soluble RANKL as well. The inhibitory effect was mediated in part by STAT3 but not by STAT1 or IL-10. In contrast, in differentiated Th17 cells, IL-27 much less efficiently inhibited the RANKL expression after restimulation. Taken together, these results indicate that IL-27 greatly inhibits primary RANKL expression in CD4(+) T cells, which could contribute to the suppressive effects of IL-27 on the inflammatory bone destruction.

Caspase 8 and menin expressions are not correlated in human parathyroid tumors.

Menin is lost by the sequential inactivation of both MEN1 alleles in subsets of non-hereditary endocrine tumors as well as those associated with multiple endocrine neoplasia type 1 (MEN1), an autosomal dominant hereditary cancer syndrome characterized by multiple tumors including parathyroid, pituitary and enteropancreatic endocrine tumors. Loss of menin has been reported to be associated with lowered caspase 8 expression and resistance to apoptosis in murine fibroblasts and in pancreatic islet tumors arising in heterozygous MEN1 gene knockout mice, the animal model of the human MEN1 syndrome. We confirmed by menin-knockdown experiments with specific siRNA that menin is crucial for caspase 8 expression in human culture cells while overexpression of menin did not increase caspase 8 protein over basal levels. We then examined expression of menin, caspase 8 and cyclin-dependent kinase inhibitors p27(Kip1) and p15(Ink4b) by Western blotting in human parathyroid tumors surgically resected from patients with MEN1 and those with non-hereditary primary hyperparathyroidism. The menin and p27(Kip1) expression levels were correlated with MEN1 mutation status that was confirmed by DNA analysis. The caspase 8 and p15(Ink4b) protein levels were variable among tumors, and were not correlated with menin protein levels. These findings suggest that human endocrine tumors lacking menin may not always exhibit lowered caspase 8 expression and hence may not be resistant to apoptosis-inducing therapy.

[Biochemical markers of bone turnover. New aspect. Bone metabolic markers available in daily practice].

Changes of bone remodeling markers reflect bone growth and bone turnover. Information on bone metabolism can be attained by blood and urine laboratory tests. Recently developed bone specific markers are categorized by bone remodeling process, i.e. bone formation and resorption. The formation markers include bone-specific alkaline phosphatase (BAP), osteocalcin (OC), undercarboxylated osteocalcin (ucOC), procollagene type I C- and N-terminal peptides (P1CP and P1NP). Bone resorption markers include deoxypyridinoline, collagen I C- and N-terminal telopeptides (CTX and NTX) , and tartrate resistent acid phosphatase (TRACP) isoform 5b. These laboratory tests offer lots of advantages for the diagnosis of bone metabolic disorders and for the evaluation of clinical states of primary osteoporosis and other metabolic skeletal diseases.

[Newly developed drugs for osteoporosis in overseas and their future roles for the therapy].

Bisphosphonates are widely used, though gastrointestinal tolerance is a problem on daily administration. Intermittent regimen, from once weekly to once yearly, is now available in overseas and can overcome GI adverse events. New generation of anti-resorptive agents (anti-RANKL antibody and a new SERM, bazedoxifene) are promising and will be soon available for the treatment of osteoporosis. Anabolic agents such as teriparatide and strontium ranelate have marked effects on BMD and reduction on fracture risk. While none of these options is suitable for everyone, the range of future available therapies does mean that most patients can find an intervention that is effective and acceptable.

[Bone quality changes in diabetes].

Diabetes-related bone fragility has recently drawn many researchers' attention. Diabetes would affect bone remodeling by various mechanisms, including deficiency of insulin actions, increased accumulation of advanced glycation end products and microangiopathy. The combination of poor bone quality of microstructure and nanoarchitecture (type I collagen and non-collageous proteins) would reduce bone strength. Bone mineral density is the best predictor for fractures of primary osteoporosis, but presumably not for type 2 diabetes. Quality changes of diabetic bone, therefore, should be more thoroughly studied.

[Parathyroid and bone. Evidence and perspective of parathyroid therapy for patients with osteoporosis].

Parathyroid hormone (PTH) is a new management option for patients with osteoporosis. As an anabolic agent that affects bone remodeling and modeling, a novel approach to reducing fracture risk could be considered for patients with severe conditions. A number of trials have shown that increases in spine and hip bone mineral density (BMD), and reduction of fracture risk in postmenopausal women. Although the combination of PTH and alendronate does not seem to be additive, PTH followed by alendronate would yield maximum increase in BMD. Treatment with PTH can change the course of osteoporosis by directly stimulating formation of new bone, and its application should be explored in daily clinical practice.

[Osteocalcin and bone].

Osteocalcin (OC) is a product of osteoblasts and accumulated in the extracellular matrix of bone. It has been recognized that serum OC is a marker of osteoblast activity, and the levels reflect the rate of bone formation. The present assay system was developed to assess the major circulating forms of intact and the large N-terminal fragments. OC binds to the crystal of hydroxyapatite, at least partly, through gamma-carboxylation of three residues. Increased rate of immature undercarboxylated osteocalcin, therefore, might display risks for osteoporotic fractures in clinical studies. However, at present, measurement of OC does not substitute for bone mass measurement and only provide limited values to evaluate the conditions of patients with primary osteoporosis.

Effects of IL-23 and IL-27 on osteoblasts and osteoclasts: inhibitory effects on osteoclast differentiation.

Interleukin (IL)-23 and IL-27 are IL-6/IL-12 family members that play a role in the regulation of T helper 1 cell differentiation. Cytokines are known to be involved in the bone remodeling process, although the effects of IL-23 and IL-27 have not been clarified. In this study, we examined the possible roles of these cytokines on osteoblast phenotypes and osteoclastogenesis. We found that IL-27 induced signal transducers and activators of transcription 3 activation in osteoblasts. However, neither IL-23 nor IL-27 showed any significant effects on alkaline phosphatase activity, receptor activator of nuclear factor kappaB ligand (RANKL) expression, mRNA expression such as alkaline phosphatase type I procollagen, or the proliferation of osteoblasts. Osteoclastogenesis from bone marrow cells induced by soluble RANKL was partially inhibited by IL-23 and IL-27 with reduced multinucleated cell numbers, but these interleukins did not affect the proliferation of osteoclast progenitor cells. These results indicate that IL-23 and IL-27 could partly modify cell fusion or the survival of multinucleated osteoclasts. On the other hand, partially purified T cells, which are activated by 2 microg/ml anti-CD3 antibody, completely inhibited osteoclastogenesis by M-CSF/RANKL. On using T cells activated with 0.2 microg/ml anti-CD3 antibody, in which osteoclastogenesis was partially inhibited, the interleukins had additive effects for inhibiting osteoclastogenesis. Although the consequences of phosphorylated signals in osteoblasts have not been identified, IL-23 and IL-27, partly and indirectly through activated T cells, inhibited osteoclastogenesis, indicating that these interleukins may protect against bone destructive autoimmune disorders.

Antiadhesive sites present in the fibronectin type III-like repeats of human plasma fibronectin.

We have found that fibronectin (FN) has a functional cryptic site opposing cell adhesion to extracellular matrix (ECM): a synthetic FN peptide derived from the 14th FN type III-like (FN-III) repeat, termed peptide FNIII14, inhibits cell adhesion to the FN without binding to beta1 integrins. This antiadhesive activity of peptide FNIII14 depends on its C-terminal amino acid sequence YTIYVIAL. A 50-kDa membrane protein (p50) has been detected as a specific binding protein of peptide FNIII14. Here we showed that antiadhesive activity of peptide FNIII14 was depedent upon the presence of p50 on cell surfaces. Furthermore, we found that there exists a sequence, analogous to the YTIYVIAL, in the 10th FN-III repeat of the FN molecule and that a FN peptide containing this analogous sequence, termed peptide FNIII10, inhibited cell adhesion to the FN. Peptide FNIII10 appeared to share p50 with peptide FNIII14 in expressing the antiadhesive activity. As a physiological consequence of decreased adhesion, peptides FNIII10 and FNIII14 accelerated the anoikis-like apoptosis of normal fibroblasts by down-regulating Bcl-2 expression through blocking the FAK/PI3K/Akt signaling pathway. Thus, the YTIYVIAL-related sequences of the FN molecule may be involved in cell regulation by modulating negatively cell adhesion to the ECM, in which p50 probably serves as a membrane receptor.

[Management of osteoporosis in patients with inhaled glucocorticoids].

Inhaled glucocorticoids are the standard of therapy in asthma and are commonly prescribed for chronic obstructive pulmonary disease. Accumulating evidence suggests that the effect of inhaled glucocorticoids on bone is not small, especially in patients taking moderate or high doses for long periods of time. The risk of adverse events is likely to differ between inhaled glucocorticoids. Inhaled glucocorticoids should be used widely, since they reduce the need of oral corticosteroids and improve respiratory function, but that they need to be managed carefully to minimize the risk of fracture with long-term use. This article described the effects of inhaled glucocorticoids on bone and fracture risk.

[Therapeutic approaches for diabetic osteopahty].

Although the clinical manifestations of diabetic osteopahty are not fully elucidated, diabetes may affect bone remodeling by various mechanisms, including deficiency of insulin actions, increased accumulation of advanced glycation end products and microangiopahty. The combination of subsequent poor bone quality of micro- or nano-architecture and frequent injurious falls would be related to an increase of fracture incidence. Several recent reports have revealed that older women with diabetes had a particularly increased risk of fractures. Bone mineral density (BMD) is the best predictor for fractures of primary osteoporosis, and increased risk for fractures of the type 1 diabetes is associated with the decrease of BMD, but not on the type 2 diabetes. The patients frequently show an increase of BMD, but suffer from fractures. At present, there is mostly no data what kind of intervention or pharmaceutical therapy is the best to avoid the incidence. Some in vivo studies support that vitamin K(2) (menatetrenone) may ameliorate bone quality damage in diabetics.

[Diabetic osteopahty and vitamin K].

Diabetes mellitus is considered as a risk factor for fractures, and there are some reports showing that metabolic effects of poor glycemic control resulted in lower bone turnover. Previous studies have revealed that this reduction of bone turnover increases bone fragility, independently of bone mineral density, so that the mechanisms of diabetic osteopahty seem to be more closely related to bone quality than bone quantity. The mechanisms of the preventive effect on fractures by vitamin K treatment should be related to amelioration of bone quality via increasing amounts of carboxylated osteocalcin. Therefore, administration of vitamin K(2) (menatetrenone) to the patients with diabetic osteopathy will be beneficial in order to improve the impaired bone quality and to reduce fracture risk.

[Bone and bone related biochemical examinations. Bone and collagen related metabolites. Osteocalcin (OC)].

Osteocalcin is produced by mature osteoblasts and primarily deposited in the extracellular matrix of skeletal tissue. It has been shown that serum osteocalcin is a marker of osteoblastic activity, and the levels reflect the rate of bone formation. The first osteocalcin assays were competitive radioimmunoassays using bovine osteocalcin as a standard, and a new generation of assays was developed to measure the major circulating forms of the protein, which is the intact and the large N-terminal fragment. Since osteocalcin binds to hydroxyapatite crystal after gamma-carboxylation at three residues, increased amounts of immature undercarboxylated osteocalcin might reflect risks for fractures. However, at present, measurement of osteocalcin does not substitute for bone mass measurement and only provide limited clinical values on evaluation of patients with osteoporosis or metabolic bone diseases.

Involvement of hepatocyte growth factor in the development of bone metastasis of a mouse mammary cancer cell line, BALB/c-MC.

Some cancers frequently affect the skeleton, and the bone microenvironment supports growth of certain cancer cells. After tumors metastasize to bone, they stimulate osteoclastogenesis and expand in the bone tissue. Hepatocyte growth factor (HGF), which was originally identified as a potent mitogen for hepatocytes, promotes tumor growth, invasion and metastasis. HGF is mainly produced by cells of mesenchymal origin, and osteoblasts/osteocytes and bone marrow stromal cells originate from mesenchymal cells. However, it is not clear what effect HGF has on tumor progression in bone metastasis. In the present study, we investigated the roles of HGF in bone metastasis using the mouse mammary cancer cell line BALB/c-MC. Cancer cells injected into hearts of mice metastasized to bone in their hind limbs. HGF immunoreactivity was detected in the stroma surrounding the tumor nests, and blood vessels expressing CD31 (a marker of endothelial cells) were observed in the HGF-positive area. To identify the cells producing HGF, we measured concentration of HGF in culture media. HGF concentration was elevated in osteoblast cultures (3.13+/-0.25 ng/ml), whereas HGF was undetectable (<0.4 ng/ml) in BALB/c-MC and bone marrow cell cultures. HGF concentration in osteoblast cultures increased 2.5-fold in response to 10(-6) M PGE(2). Addition of HGF to BALB/c-MC cultures caused doubling of the cell number. Moreover, Western blot analysis revealed expression of c-Met/HGF receptor by BALB/c-MC. In the Matrigel invasion chamber assay, addition of HGF to the bottom well increased the rate at which BALB/c-MC invaded the bottom well through the membrane. Furthermore, when osteoblasts were cultured in the bottom well, the number of BALB/c-MC cells that invaded the bottom well through the membrane increased 3.7-fold, compared to assays without osteoblasts. Addition of NK4, an inhibitor of HGF, completely abolished the enhancement of the invasive potential of the BALB/c-MC cells in the presence of osteoblasts. These findings suggest that HGF produced by osteoblasts induces migration of cancer cells from sinusoidal capillaries to bone marrow space and stimulates growth of cancer cells in the bone microenvironment. Thus, osteoblasts appear to promote bone metastasis of some cancers via HGF-c-Met signaling.