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.

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.

[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.

[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.

[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.

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.

[Present development of new drugs for osteoporosis].

Bisphosphonates are widely used, though gastrointestinal tolerance is a problem on daily administration. Alendronate 35 mg administered once weekly is as effective at increasing bone mineral density (BMD) as 5 mg/day in the treatment of osteoporosis. Once weekly regimen will be soon available in Japan and can reduce adverse events. Injection therapies may also circumvent this, although this introduces the smaller problem of acute phase reactions. New generation bisphosphonates (milodronate, ibandronate and zoledronate) along with anti-receptor activator of nuclear factor-kappaB ligand (RANKL) antibody are promising and eagerly developed for the treatment of osteoporosis. Raloxifene appears to have a superior safety profile to hormone replacement therapy (HRT), though its efficacy on bone may be limited, so that various new generation of selective estrogen receptor modulator (SERMs) is now underdeveloped. Anabolic agents such as teriparatide and strontium ranelate have marked effects on BMD and subsequent 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.

[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.

[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.

A fibronectin fragment induces tumor necrosis factor production of rat basophilic leukemia cells.

Proteolytic digest of fibronectin (FN), but not intact FN, induced TNF-alpha secretion of rat basophilic leukemia (RBL-2H3) cells. As a result of the identification of FN fragment responsible for TNF-alpha secretion, a 30-kDa fragment derived from the carboxyl-terminal heparin-binding (Hep 2) domain of FN was isolated from the FN digest. The TNF-alpha secretion was abrogated by treatment of RBL-2H3 cells with cycloheximide, indicating the de novo synthesis of TNF-alpha, but not with polymyxin B, excluding the possible TNF-alpha induction by some contaminated lipopolysaccharides. A 22-mer synthetic peptide originated from the Hep 2 domain, termed FNIII14, which has been found to negatively modulate the beta1 integrin activation, had the ability to induce TNF-alpha production, whereas this activity of FNIII14 disappeared by shuffling a YTIYVIAL sequence essential for the integrin-inactivating activity. FNIII14 suppressed the spreading of RBL-2H3 cells on FN substrate, wherein RBL-2H3 cell proliferation was inhibited with FNIII14 in a dose-dependent manner. Thus, it appears that FN fragments containing the YTIYVIAL anti-adhesive site affect the activation status of RBL-2H3 mast cells, characterized by the stimulation of TNF-alpha production and growth suppression, probably due to negative regulation of beta1 integrin activity.

Stability of A+U-rich element binding factor 1 (AUF1)-binding messenger ribonucleic acid correlates with the subcellular relocalization of AUF1 in the rat uterus upon estrogen treatment.

The nucleocytoplasmic shuttling protein, A+U-rich element binding factor 1 (AUF1), is one of the RNA-binding proteins that specifically bind adenylate-uridylate rich elements (AREs) in mRNA 3'-untranslated regions (UTRs), and acts as a regulator of ARE-mediated mRNA degradation in the cytoplasm. We previously reported that in the female rat uterus, the levels of specific AUF1 isoform mRNAs (p40/p45) were increased by 17 beta-estradiol (E2) treatment. Therefore, we examined the role of AUF1 in the regulation of E2-mediated mRNA turnover in the rat uterus. We identified ABIN2 and Ier2/pip92 mRNAs as candidate targets of AUF1 in the rat uterus. We found that AUF1-binding elements were present in the 3'-UTR of both mRNAs and that the 3'-UTRs functioned as mRNA turnover regulatory elements. In the ovariectomized rat uterus, the nucleocytoplasmic localization of AUF1p40/p37 isoform proteins was regulated by E2. We also found that cytoplasmic AUF1-bound mRNA levels changed coincidentally with the cytoplasmic levels of AUF1p40/p37. Finally, we confirmed that the subcellular localization of AUF1p40 controlled the stability of target mRNAs in vitro, such that cytoplasmically localized AUF1p40 led to marked mRNA stabilization, whereas nuclear-localized AUF1p40 stabilized target mRNA only slightly. These results suggested that E2-inducible ARE-containing gene transcripts are regulated, at least in part, via mRNA stabilization through the nucleocytoplasmic relocalization of AUF1.

[Osteoporosis and alcohol intake].

Current understanding suggests that moderate quantities of alcohol use may have beneficial effects on bone mineral density, irrespective of age and sex, and would not be a risk factor of osteoporotic fracture. Heavy alcohol intake or alcoholism, however, frequently disrupts calcium and bone homeostasis, which leads to reduce bone mineral density and increase the incidence of fragility fracture. Since the habits of alcohol intake is closely associated with other diet and lifestyle factors, the exact mechanism underlying alcohol-related bone loss still remained to be clarified. The effects of alcohol use on osteoporosis should be studied more in detail.

[Treatment with bisphosphonates: daily or once-weekly oral administration and intravenous injection].

Oral bisphosphonates are among the most effective therapeutics available to manage osteoporosis, which increase bone mineral density and reduce the incidence of fractures. Although daily administration of bisphosphonates has been well tolerated in most clinical use, some patients develop upper gastrointestinal symptoms. Once-weekly bisphosphonates offers convenience to patients while showing equal efficacy, which may also provide improved compliance and a lower risk of gastrointestinal symptoms. Now, both alendronate and risedronate are available in once-weekly formulations in US and European countries, which have efficacy and tolerability possibly better than the once-daily doses. Under certain circumstances, intermittent treatment with injectable bisphosphonates could be an alternative when compliance is at risk. This type of treatment must be applicable to some Japanese osteoporotic patients in the nearest future.

[Bisphosphonates: the molecular targets and mechanisms of action].

Bisphosphonates directly act on osteoclasts to inhibit bone resorption and used most widely to treat osteoporosis. The compounds can be classified into two groups with different modes of action. Nitrogen containing bisphosphonates exert their effects by inhibiting a key enzyme in the mevalonate pathway. The specific target is the isoprenoid biosynthetic enzyme, farnesyl pyrophosphate synthase, which is indispensable for protein prenylation and activation of intracellular signalling proteins, including the small GTPases Rho, Rac, Cdc42 and Ras. The disruption of the function of these key enzymes may explain the loss of osteoclast activity and induction of apoptosis. Whereas the first generation of bisphosphonates such as etidronate and clodronate (nitrogen deficient bisphosphonates) can be incorporated into nonhydrolysable analogues of ATP that may inhibit ATP-dependent intracellular events. Bisphosphonates are highly effective to inhibit bone resorption and increase bone mineral density, although their precise mechanisms of molecular action are not completely understood.

Interaction between 3' untranslated region of calcitonin receptor messenger ribonucleic acid (RNA) and adenylate/uridylate (AU)-rich element binding proteins (AU-rich RNA-binding factor 1 and Hu antigen R).

Our previous results in mouse osteoclasts suggested that calcitonin (CT) alters CT receptor (CTR) mRNA stability. The CTR mRNA transcript contains several adenylate/uridylate (AU)-rich destabilizing elements in the 3' untranslated region (3'UTR). When the 3'UTR of mouse CTR mRNA was labeled by [alpha-(32)P]-uridine 5-triphosphate, interactions were observed between the transcript and several cell extracts, including those from the osteoclast progenitor monocyte/macrophage cell line, RAW 264.7. The molecular masses of the interacting proteins ranged from approximately 35 to 50 kDa, similar to AU-rich RNA-binding factor 1 (AUF1) and Hu antigen R (HuR). Radiolabeled 3'UTR transcripts bound with a 40-kDa protein, which could be extracted from cells transfected with AUF1 p40. To confirm the binding specificity, a pSG5 vector construct, containing the AUF1 p40 with an hemagglutinin tag, was transiently transfected into NIH3T3 cells. The extracts were incubated with poly(A)-added CTR3'UTR. The reaction mixture was immunoprecipitated using an antihemagglutinin antibody and precipitated mRNA species were extracted and reverse transcribed using oligo-dT primers. It was found that PCR primers specific for the 3'UTR of CTR mRNA sequence generated a PCR signal. No signal was observed when mutated AUF1 p40 was transfected. In a manner similar to the AUF1 binding, HuR was also found to bind to the 3'UTR. Specific binding of AUF1 p40 and HuR was also found with RNA extracted from mouse osteoclasts. Treatment of osteoclasts with CT did not significantly affect the expression of AUF1 but decreased the levels of HuR and its mRNA. The role of CTR3'UTR in mRNA stability was further tested by expressing luciferase reporter constructs that did, or did not, contain the CTR3'UTR, under the control of the tetracycline-regulatory system. The results showed that the addition of 3'UTR considerably shortened the mRNA half-life of the luciferase reporter gene. These results suggest that AUF1 p40, HuR, and the 3'UTR of the CTR mRNA transcript could be involved in posttranscriptional regulation of CTR mRNA expression.

Preclinical Cushing's syndrome due to ACTH-independent bilateral macronodular adrenocortical hyperplasia with excessive secretion of 18-hydroxydeoxycorticosterone and corticosterone.

A 64-year-old woman developed hypertension and hypokalemia, due to ACTH-independent bilateral macronodular adrenocortical hyperplasia (AIMAH) with excessive secretion of 18-hydroxydeoxycorticosterone and corticosterone. Plasma cortisol did not show a diurnal rhythm, and was not suppressed by dexamethasone (8 mg). Plasma cortisol responded to ACTH and was increased by hypoglycemia without modifying ACTH levels. Radiological studies demonstrated that adrenal glands were enlarged with macronodules. Although the patient exhibited a low plasma renin activity and aldosterone levels, hypokalemia and hypertension were observed. Hormonal findings would support the hypothesis that the tumor of AIMAH originated from cells of the upper zona fasciculata.