Efficacy of denosumab co-administered with vitamin D and Ca by baseline vitamin D status.

INTRODUCTION: In anti-osteoporosis drug trials, vitamin D and calcium (Ca) are common supplements; however, the optimal dose of each is unclear. Using data from the randomized, double-blind, placebo-controlled DIRECT trial, we assessed whether baseline serum 25-hydroxy vitamin D (25[OH]D) level influences the efficacy of denosumab co-administered with vitamin D and Ca. MATERIALS AND METHODS: In this prespecified sub-analysis, subjects with primary osteoporosis who received denosumab or placebo, plus vitamin D (≥ 400 IU/day) and Ca (≥ 600 mg/day), were classified as 25(OH)D deficient (< 20 ng/mL), insufficient (≥ 20 to < 30 ng/mL), and sufficient (≥ 30 ng/mL). Study endpoints included absolute serum 25(OH)D level at baseline, 12 months, and 24 months; change in serum 25(OH)D and bone mineral density (BMD) status from baseline; and incidence of new vertebral fractures at 24 months. RESULTS: In 475 denosumab-treated and 481 placebo-treated subjects, proportions with deficient/insufficient/sufficient 25(OH)D at baseline were 53.1%/37.1%/9.9% and 50.9%/42.0%/7.1%, respectively. Supplementation significantly increased mean serum 25(OH)D levels; at 24 months, mean levels were > 30 ng/mL (sufficient) in both treatment groups. Increase in BMD over time was higher in the denosumab group vs. placebo group in all three vitamin D status groups. At month 24, denosumab-treated subjects with deficient/insufficient baseline 25(OH)D had a significantly lower risk of new vertebral fracture vs. placebo-treated subjects. CONCLUSION: Among DIRECT trial subjects supplemented with ≥ 400 IU/day of vitamin D and ≥ 600 mg/day of Ca, baseline 25(OH)D sufficiency may not influence the efficacy of denosumab in increasing BMD or preventing vertebral fractures.

Efficacy and safety of once-monthly risedronate in osteoporosis subjects with mild-to-moderate chronic kidney disease: a post hoc subgroup analysis of a phase III trial in Japan.

Limited data are available on the safety and efficacy of anti-resorptive agents, particularly once-monthly bisphosphonates, for use in osteoporotic patients with chronic kidney disease (CKD). We conducted a post hoc analysis of data from a 12-month, randomized, double-blind, phase III study to evaluate the safety and efficacy of once-monthly risedronate (RIS-OM) 75 mg tablets in Japanese osteoporosis patients with mild-to-moderate CKD. Patients who received RIS-OM 75 mg were stratified by baseline estimated glomerular filtration rate (eGFR; ≥ 90, ≥ 60 to < 90, or ≥ 30 to < 60 mL/min/1.73 m2). Safety endpoints were incidence of adverse events (AEs) and percent change from baseline in eGFR, serum creatinine, calcium, and phosphorus. Efficacy endpoints were percent change from baseline in lumbar spine bone mineral density (BMD) and bone turnover markers (BTMs). In 420 patients included (age 67.7 ± 6.7 years, women 98.8%), the incidence of all AEs, gastrointestinal disorders, acute phase reaction, non-vertebral fractures, and renal and urinary disorders was not significantly different among subgroups. Interaction between subgroups and time was significant for eGFR (p = 0.010) and serum creatinine (p = 0.001) but considered to be regression to the mean and clinically insignificant. BMD significantly increased while BTMs significantly decreased from baseline with a similar degree of change among the subgroups. In conclusion, RIS-OM 75 mg showed consistent safety and efficacy in suppressing bone turnover and increasing BMD in Japanese primary osteoporosis patients with mild-to-moderate CKD. These results should, however, be interpreted with caution because the number of patients with moderate CKD was limited.

Osteoblast AMP-activated protein kinase regulates glucose metabolism and bone mass in adult mice.

Previous studies have shown that AMP-activated protein kinase (AMPK), a crucial regulator of energy homeostasis, plays important roles in osteoblast differentiation and mineralization. However, little is known about in vivo roles of osteoblastic AMPK in glucose metabolism and bone mass regulation in adult mice. Here, we used the inducible Cre system to control the onset of Ampk disruption after birth by removing doxycycline supplementation. We conditionally inactivated Ampk in osterix (Osx)-expressing cells in 3-week-old Ampk-/- mice. After 6 months of Ampk inactivation, the Ampk-/- mice displayed lower serum osteocalcin levels as well as glucose intolerance and insulin resistance, as indicated by glucose tolerance and insulin tolerance tests, respectively, when compared with wild-type mice. After 18 months of Ampk inactivation, micro computed tomography showed significant reductions in trabecular bone volume and cortical bone thickness in the femur of Ampk-/- mice when compared with wild-type mice. Moreover, bone stiffness was significantly lower in Ampk-/- mice than in wild-type mice. This is the first study to show that osteoblast AMPK plays an important roles in glucose metabolism and in maintaining trabecular bone volume, cortical thickness, and bone strength in adult mice.

[Update on recent progress in vitamin D research. Vitamin D and metabolic disease.]

Numerous epidemiological studies and meta-analyses have indicated that there is a link between Vitamin D insufficiency/deficiency and metabolic disorders such as type 1 and type 2 diabetes mellitus as well as metabolic syndrome. However, vitamin D supplementation has not demonstrated improvement effects in obesity, disorders of glucose and lipid metabolism in any of these illnesses;therefore, the details of the causal relationship remain unclear. Improvement in glucose metabolism was observed in a study in which only vitamin D deficient patients with 25-hydroxyvitamin D[25(OH)D]levels of less than 20 ng/mL were given native vitamin D supplementation. Further studies are needed to determine the 25(OH)D level at which intervention is needed along with the required amount and duration of such supplementation.

Nationwide survey of fibroblast growth factor 23 (FGF23)-related hypophosphatemic diseases in Japan: prevalence, biochemical data and treatment.

A nationwide epidemiologic survey of fibroblast growth factor 23 (FGF23)-related hypophosphatemic diseases was conducted in 2010 to clarify the prevalence and the clinical presentations of the disorders. A questionnaire inquiring the experience of patients with these diseases was sent to randomly selected hospitals throughout Japan. The estimated annual incidence of the diseases was 117 cases (95% CI 75 - 160), 55 males (95% CI 30 - 81) and 62 females (95% CI 40 - 84). Tumor-induced osteomalacia (TIO) and X-linked hypophosphatemic rickets (XLH) were the most prevalent causes of acquired and genetic FGF23-related hypophosphatemic diseases, respectively. The estimated incidence of XLH was about 1 in 20,000. We have also collected clinical data of the patients by a secondary survey. These patients showed FGF23 levels of above 30 pg/mL by intact assay in the presence of hypophosphatemia. While complete resection of responsible tumors improved biochemical abnormalities in patients with TIO, treatment with phosphate and/or active vitamin D3 did not normalize serum phosphate and tubular maximum transport of phosphate in patients with XLH. Our results suggest that there is no racial difference in the incidence of XLH. While FGF23 measurement is useful for the diagnosis of FGF23-related hypophosphatemic diseases, the better management is necessary especially for patients with genetic hypophosphatemic rickets caused by excessive actions of FGF23.

Clinical Trials Express: fracture risk reduction with denosumab in Japanese postmenopausal women and men with osteoporosis: denosumab fracture intervention randomized placebo controlled trial (DIRECT).

CONTEXT: Denosumab 60 mg sc injection every 6 months for 36 months was well tolerated and effective in reducing the incidence of vertebral, nonvertebral, and hip fracture in predominantly Caucasian postmenopausal women with osteoporosis. OBJECTIVE: The objective of this phase 3 fracture study was to examine the antifracture efficacy and safety of denosumab 60 mg in Japanese women and men with osteoporosis compared with placebo. DESIGN AND SETTING: A randomized, double-blind, placebo-controlled trial with an open-label active comparator as a referential arm was conducted. PATIENTS: Subjects were 1262 Japanese patients with osteoporosis aged 50 years or older, who had one to four prevalent vertebral fractures. INTERVENTION: Subjects were randomly assigned to receive denosumab 60 mg sc every 6 months (n = 500), placebo for denosumab (n = 511), or oral alendronate 35 mg weekly (n = 251). All subjects received daily supplements of calcium and vitamin D. MAIN OUTCOME MEASURE: The primary endpoint was the 24-month incidence of new or worsening vertebral fracture for denosumab vs placebo. RESULTS: Denosumab significantly reduced the risk of new or worsening vertebral fracture by 65.7%, with incidences of 3.6% in denosumab and 10.3% in placebo at 24 months (hazard ratio 0.343; 95% confidence interval 0.194-0.606, P = .0001). No apparent difference in adverse events was found between denosumab and placebo during the first 24 months of the study. CONCLUSION: These results provide evidence of the efficacy and safety of denosumab 60 mg sc every 6 months in Japanese subjects with osteoporosis.

Vertebral fracture risk after once-weekly teriparatide injections: follow-up study of Teriparatide Once-Weekly Efficacy Research (TOWER) trial.

OBJECTIVE: To evaluate fracture risk and bone mineral density (BMD) in patients with primary osteoporosis, 1 year after completing 72 weeks of weekly teriparatide injections. RESEARCH DESIGN AND METHODS: After 72 weeks of teriparatide injections or placebo (original trial), treatment was unblinded and subjects were subsequently treated with bisphosphonates or other therapeutic regimens at the discretion of their physicians and followed for 1 year. Spine radiographs and BMD measurements at the lumbar spine, femoral neck, and total hip by dual energy X-ray absorptiometry were performed. MAIN OUTCOME MEASURE: Incident vertebral fracture rate. RESULTS: A total of 465 patients were enrolled and 447 (96.1%) completed the study. In the 1 year follow-up period, new morphometric vertebral fractures occurred in 7/203 (3.4%) in the post-teriparatide group and 33/241 (13.7%) in the post-placebo group (relative risk [RR]: 0.23, 95% confidence interval [CI]: 0.10 to 0.52, P < 0.05). The cumulative incidences from the start of the original trial were 4.9% and 22.8%, respectively (RR: 0.18, 95% CI: 0.09 to 0.36, P < 0.05). There were no significant differences in incidences of vertebral fractures between subsequent therapeutic regimens in the post-teriparatide group. In subjects treated with bisphosphonates, mean BMD values further significantly increased by 9.6%, 2.9%, and 4.1% at the lumbar spine, femoral neck, and total hip, respectively (P < 0.05). CONCLUSIONS: The reduced risk of vertebral fracture was sustained for 1 year after completion of 72 weeks of weekly teriparatide injections. The effects did not differ between subsequent therapeutic regimens. BMD gains continued with sequential bisphosphonate treatment, but not with the other sequential therapeutic regimens. Bisphosphonates seem to be a useful choice as a subsequent treatment to weekly teriparatide. LIMITATION: This study was an observational follow-up study and the regimens of subsequent medication after discontinuation of the original TOWER trial were not randomly allocated.

Bone metabolism and fracture risk in type 2 diabetes mellitus.

Osteoporosis and type 2 diabetes mellitus (T2DM), both prevalent in aging and westernized societies, adversely affect the health of elderly people by causing fractures and vascular complications, respectively. Recent experimental and clinical studies show that the disorders are etiologically related through the actions of osteocalcin and adiponectin. Meta-analyses of multiple clinical studies show that the hip fracture risk of T2DM patients is increased 1.4-1.7-fold compared with non-DM controls, even though the patients' bone mineral density (BMD) is not diminished. Vertebral fracture risk of the T2DM patients is also increased, and BMD measurement is not sensitive enough to assess this risk. These findings suggest that bone fragility in T2DM patients depends on bone quality deterioration rather than bone mass reduction. Surrogate markers are therefore needed to supplement the partial effectiveness of BMD testing in assessing the fracture risk of the T2DM patients. Markers related to advanced glycation end products may be candidates. These substances modulate bone quality in DM. Until research establishes the usefulness of surrogate markers, physicians should assess fracture risk in T2DM patients not only by measuring the BMD, but also by taking a fracture history and evaluating prior vertebral fractures using spinal X-rays.

Vitamin D insufficiency defined by serum 25-hydroxyvitamin D and parathyroid hormone before and after oral vitamin D3 load in Japanese subjects.

Vitamin D insufficiency is a risk for both skeletal and nonskeletal health. However, some ambiguity remains about threshold serum 25(OH)D for vitamin D insufficiency. To determine the threshold serum 25(OH)D to maintain normal calcium availability without elevation in serum parathyroid hormone (PTH) among Japanese subjects with various calcium intakes, we conducted a multicenter prospective open-labeled study. We recruited 107 ambulatory subjects without disorders affecting vitamin D metabolism to whom oral vitamin D3 800 IU/day for 4 weeks or 1,200 IU/day for 8 weeks was given. Serum 25(OH)D, PTH, calcium, phosphate, and magnesium were measured before and after vitamin D3 supplementation. Calcium intake was assessed by questionnaires. When all the data were combined, serum 25(OH)D was negatively correlated with PTH. The cubic spline curve between serum 25(OH)D and PTH indicated PTH reached its plateau between 35 and 40 pg/ml at 25(OH)D between 25 and 30 ng/ml. Vitamin D3 supplementation increased serum 25(OH)D and decreased PTH. Change in PTH correlated positively with baseline serum 25(OH)D. From the regression analyses, baseline serum 25(OH)D above 28 ng/ml corresponded to the threshold level without reduction in PTH after vitamin D3 supplementation. In multivariate regression analyses, age but not calcium intake was a significant determinant of PTH. We concluded that a serum 25(OH)D level of 28 ng/ml was identified as a threshold for vitamin D insufficiency necessary to stabilize PTH to optimal levels.

Fasudil hydrochloride induces osteoblastic differentiation of stromal cell lines, C3H10T1/2 and ST2, via bone morphogenetic protein-2 expression.

Rho-kinase (ROK), downstream of the mevalonate pathway, is detrimental to vessels, and suppressing its activity is a target for the treatment of human disease such as coronary artery disease and pulmonary hypertension. Recent studies have shown that ROK has a crucial role in bone metabolism. However, the role of ROK in stromal cells is still unclear. The present study was undertaken to investigate the effect of a ROK inhibitor, fasudil hydrochloride, on stromal cell lines, C3H10T1/2 and ST2. In both cells, Fasudil significantly stimulated alkaline phosphatase activity and enhanced cell mineralization. Moreover, fasudil significantly increased the mRNA expression of collagen-I, osteocalcin, and bone morphogenetic protein-2 (BMP-2). Supplementation of noggin, a BMP-2 antagonist, significantly reversed the fasudil-induced collagen-I and osteocalcin mRNA expression in both cells. These findings suggest that fasudil induces the osteoblastic differentiation of stromal cells via enhancing BMP-2 expression, and that this drug might be beneficial for not only atherosclerosis but also osteoporosis by promoting bone formation.

Bone metabolism after cinacalcet administration in patients with secondary hyperparathyroidism.

Cinacalcet, an allosteric modulator of a calcium (Ca)-sensing receptor, significantly suppresses parathyroid hormone (PTH) secretion and bone turnover rate in chronic hemodialysis (HD) patients with secondary hyperparathyroidism (SHPT). In this study, bone metabolism after cinacalcet treatment was examined, because hungry bone syndrome is sometimes experienced after parathyroidectomy in severe SHPT. We conducted a prospective observational study in 17 HD patients with SHPT. Cinacalcet was started at 25 mg/day, and the dose was increased step by step based on serum calcium level. A significant decrease in serum Ca and intact PTH concentration was found within 2 weeks. Tartrate-resistant acid phosphatase 5b, a good bone resorption marker, was significantly decreased at week 2 of the study. Serum bone alkaline phosphatase, a marker of bone formation, was increased at week 2 compared with the basal level. It became, however, gradually decreased until week 14. Only one patient whose bone turnover was considerably high had a mild numbness feeling. These results suggest that cinacalcet treatment might transiently accelerate bone formation with rapid suppression of bone resorption. This uncoupling could be involved in a mechanism by which cinacalcet decreases serum Ca level.

[Clinical aspect of recent progress in phosphate metabolism. Distribution of phosphorus and its physiological roles in the body: the form, distribution, and physiological function].

Phosphorus plays pivotal roles in the survival such as the cellular structure, genomic information, energy metabolism, and cell signaling. Total amount of phosphorus is 500-700 g in human, most of which is stored in the bone in an insoluble form of calcium salt. About 15% of phosphorus is located in the cell membrane and the intracellular fluid in the soft tissues in a form of organic phosphate. Only 0.1% is present in the extracellular fluid. This phosphate pool plays a role in the dynamic equilibrium through the gut, kidney, bone and other tissues. Most of inorganic phosphates in the extracellular fluid are present in a form of ions such as H2PO4- and HPO(4)2-, and the concentration of phosphatic acids is about 1.2 mM. The form, distribution, and physiological function of phosphorus in the body are summarized in this review.

Activation of AMP kinase and inhibition of Rho kinase induce the mineralization of osteoblastic MC3T3-E1 cells through endothelial NOS and BMP-2 expression.

AMP-activated protein kinase (AMPK) and Rho kinase (ROK) are known to modulate the mevalonate pathway. Activation of AMPK suppresses 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase. ROK acts downstream of HMG-CoA reductase, and its inhibition exerts antiatherosclerosis effects. However, whether or not these enzymes are involved in bone metabolism is unclear. The present study was undertaken to investigate the effects of an AMPK activator, 5-aminoimidazole-4-carboxamide1-beta-d-ribonucleoside (AICAR), and a ROK inhibitor, fasudil hydrochrolide, on the mineralization of osteoblastic MC3T3-E1 cells. Real-time PCR and mineralization stainings revealed that both AICAR and fasudil significantly stimulated endothelial nitric oxide synthase (eNOS), bone morphogenetic protein-2 (BMP-2), and osteocalcin mRNA expression as well as mineralization in the cells. Supplementation of either mevalonate or geranyl-geranyl pyrophosphate, the downstream molecules of HMG-CoA reductase, or coincubation with either a nitric oxide synthase inhibitor, N(G)-nitro-l-arginine methyl ester, or a BMP-2 antagonist, noggin, significantly reversed these AICAR-induced reactions. Western blot analysis showed that AICAR activated protein kinase B and extracellular signal-regulated kinase (ERK). ERK inhibitor significantly reversed the AICAR-induced increase in eNOS and BMP-2 mRNA expression. Measurement of ROK activities by enzyme-linked immunosorbent assay revealed that both AICAR and fasudil significantly suppressed the phosphorylation of the myosin-binding subunit of myosin phosphate, a ROK substrate. These findings suggest that the AMPK activator and the ROK inhibitor are able to stimulate the mineralization of osteoblasts through modulating the mevalonate pathway. These agents could be candidate drugs that promote bone formation for the treatment of osteoporosis.

Metformin enhances the differentiation and mineralization of osteoblastic MC3T3-E1 cells via AMP kinase activation as well as eNOS and BMP-2 expression.

It is unclear whether metformin, one of the anti-hyperglycemic agents commonly used for type 2 diabetes, could affect bone formation through activation of AMP-activated protein kinase (AMPK). In order to clarify this issue, we investigated the effects of metformin on the differentiation and mineralization of osteoblastic MC3T3-E1 cells as well as intracellular signal transduction. Metformin (50 microM) significantly increased collagen-I and osteocalcin mRNA expression, stimulated alkaline phosphatase activity, and enhanced cell mineralization. Moreover, metformin significantly activated AMPK in dose- and time-dependent manners, and induced endothelial nitric oxide synthase (eNOS) and bone morphogenetic protein-2 (BMP-2) expressions. Supplementation of Ara-A (0.1mM), a specific AMPK inhibitor, significantly reversed the metformin-induced eNOS and BMP-2 expressions. Our findings suggest that metformin can induce the differentiation and mineralization of osteoblasts via activation of the AMPK signaling pathway, and that this drug might be beneficial for not only diabetes but also osteoporosis by promoting bone formation.

Short-term effects of glucocorticoid therapy on biochemical markers of bone metabolism in Japanese patients: a prospective study.

Glucocorticoid (GC) therapy induces rapid bone loss, but the early changes in calcium and bone metabolism in patients treated with GC have not been clarified. To investigate the changes in calcium and bone metabolism during the early stage of GC therapy, we analyzed various biochemical markers of bone metabolism. The serum levels of calcium (Ca), phosphorus, parathyroid hormone (PTH), osteocalcin (OC), bone alkaline phosphatase (BAP), and type I collagen cross-linked N-telopeptide (NTx), as well as the urinary levels of Ca, creatinine, and NTx, were measured on days 0, 3, 7, and 28 of GC therapy. The subjects were divided into the following four groups: 9 patients receiving pulse therapy (P), 18 patients receiving prednisolone (PSL) at doses > or =40 mg/day (H), 9 patients receiving PSL at doses > or =20 mg/day (M), and 11 patients receiving PSL at doses < or =10 mg/day (S). The serum OC level showed a marked decrease on day 3 of GC therapy (-41.2% +/- 6.6%, P < 0.01), while the BAP level decreased gradually. Both serum and urinary NTx levels significantly increased on day 7 of GC therapy (9.9% +/- 4.5%, P < 0.05, and 42.2% +/- 10.6%, P < 0.01, respectively). Urinary Ca excretion was increased on day 3 of GC therapy and continued to increase until 4 weeks, while intact PTH showed an increase on day 3 and then remained constant until 4 weeks. In groups P and H, there were significant early changes in OC, BAP, NTx, and intact PTH levels, as well as urinary Ca excretion. Even a PSL dose of <10 mg/day caused a decrease in the serum OC level. In conclusion, the biochemical markers of Ca and bone metabolism showed different kinetics depending on the dose of GC, and it is important for patients on high-dose GC therapy to receive prophylaxis for bone loss from the start of GC treatment.

Clinical usefulness of measurement of fibroblast growth factor 23 (FGF23) in hypophosphatemic patients: proposal of diagnostic criteria using FGF23 measurement.

Fibroblast growth factor 23 (FGF23) plays important roles in the development of hypophosphatemic diseases such as tumor-induced osteomalacia (TIO) and X-linked hypophosphatemic rickets/osteomalacia (XLH). However, clinical usefulness of measurement of FGF23 has not been established. The objective of this study is to examine the importance of FGF23 measurement in the diagnosis of hypophosphatemic diseases. Biochemical parameters concerning phosphate metabolism were analyzed in a cross-sectional study. 32 patients with TIO, 28 patients with XLH and 16 hypophosphatemic patients with other causes including vitamin D deficiency, Fanconi's syndrome and Cushing's syndrome were studied. In patients with TIO and XLH, FGF23 was above the upper limit of the reference range in most patients irrespective of medical treatment. The lowest FGF23 in these patients was 38.0 pg/ml. FGF23 in hypophosphatemic patients with other causes was undetectable (less than 3 pg/ml) in 12 patients and the highest FGF23 in this group was 23.9 pg/ml. Relationship between phosphate and FGF23 indicated that TIO and XLH are diseases with high FGF23 and hypophosphatemia judged by age-dependent reference ranges for serum phosphate. FGF23 measurement is useful for differential diagnosis of hypophosphatemic diseases caused by excess actions of FGF23 and other etiologies. High FGF23 with low phosphate judged by age-dependent reference ranges for phosphate establishes the diagnosis of diseases caused by excess actions of FGF23.

A case of magnesium deficiency associated with insufficient parathyroid hormone action and severe osteoporosis.

The relationship between osteoporosis and magnesium (Mg) deficiency is still controversial. Here we report a case of an 82-year-old woman with a giant adenomatous goiter and severe osteoporosis with multiple vertebral fractures, whose clinical course indicated that her osteoporosis was probably due to Mg deficiency. She visited our hospital for treatments of tetany. Laboratory data showed the existence of hypomagnesemia, hypocalcemia, hypokalemia, vitamin D deficiency, and slightly elevated intact PTH. Intravenous administration of Mg not only improved these electrolyte abnormalities but also increased serum levels of intact PTH, bone formation markers, 1,25-dihydroxyvitamin D, as well as bone resorption markers in the urine, and lowered urinary phosphate reabsorption. Hypomagnesemia on admission seemed to arise from long-lasting poor food intake and malnutrition, because it improved after the disappearance of dysphagia with a goiter resection. After the operation, BMD values at the lumbar spine and femoral neck obviously increased during 6 months of Mg supplementation without any specific therapies for osteoporosis. Mg deficiency in this case seemed to cause impaired secretion of PTH from the parathyroid and the refractoriness of bone and kidney to the hormone, which led to the suppression of both bone remodeling and renal vitamin D production. These processes were probably linked to her severe osteoporosis, which was reversed by Mg supplementation.

A new active vitamin D, ED-71, increases bone mass in osteoporotic patients under vitamin D supplementation: a randomized, double-blind, placebo-controlled clinical trial.

CONTEXT: ED-71 has been shown to increase lumbar bone mineral density (BMD) in osteoporotic subjects. However, vitamin D insufficiency might have influenced the effect of ED-71 on BMD. OBJECTIVE: Our objective was to examine whether ED-71 can increase BMD in osteoporotic patients under vitamin D supplementation. DESIGN, SETTING, AND PATIENTS: We conducted a randomized, double-blind, placebo-controlled clinical trial of 219 osteoporotic patients (49-87 yr of age). INTERVENTIONS: Subjects were randomly assigned to receive placebo or 0.5, 0.75, or 1.0 microg/d ED-71 for 12 months. All the subjects received 200 or 400 IU/d vitamin D(3). MAIN OUTCOME MEASURES: We assessed changes in lumbar and hip BMD and bone turnover markers from baseline. RESULTS: Lumbar BMD increased with ED-71 treatment for 12 months (2.2, 2.6, and 3.1% from baseline and 2.9, 3.4, and 3.8% vs. placebo group in subjects receiving 0.5, 0.75, and 1.0 microg ED-71, respectively). Total hip BMD also increased with 0.75 and 1.0 microg ED-71 (-0.8, 0.6, and 0.9% from baseline and 0.1, 1.5, and 1.8% vs. placebo group in the 0.5, 0.75, and 1.0 microg ED-71 groups, respectively). Bone formation and resorption markers were suppressed by approximately 20% after 12 months of 0.75 and 1.0 microg ED-71 treatment. Transient hypercalcemia over 2.6 mmol/liter occurred in 7, 5, and 23% of subjects in the 0.5, 0.75, and 1.0 microg ED-71 groups, respectively, but none of them developed sustained hypercalcemia. CONCLUSIONS: These results demonstrate that ED-71 treatment at around 0.75 microg/d can effectively and safely increase lumbar and hip BMD in osteoporotic patients with vitamin D supplementation.

Menin suppresses osteoblast differentiation by antagonizing the AP-1 factor, JunD.

Mice null for menin, the product of the multiple endocrine neoplasia type 1 (MEN1) gene, exhibit cranial and facial hypoplasia suggesting a role for menin in bone formation. We have shown previously that menin is required for the commitment of multipotential mesenchymal stem cells into the osteoblast lineage in part by interacting with the bone morphogenetic protein (BMP)-2 signaling molecules Smad1/5, and the key osteoblast transcriptional regulator, Runx2 (Sowa H., Kaji, H., Hendy, G. N., Canaff, L., Komori, T., Sugimoto, T., and Chihara, K. (2004) J. Biol. Chem. 279, 40267-40275). However, menin inhibits the later differentiation of committed osteoblasts. The activator protein-1 (AP-1) transcription factor, JunD, is expressed in osteoblasts and has been shown to interact with menin in other cell types. Here, we examined the consequences of menin-JunD interaction on osteoblast differentiation in mouse osteoblastic MC3T3-E1 cells. JunD expression, assessed by immunoblot, gradually increased during osteoblast differentiation. Stable expression of JunD enhanced expression of the differentiation markers, Runx2, type 1 collagen (COL1), and osteocalcin (OCN) and alkaline phosphatase (ALP) activity and mineralization. Hence, JunD promotes osteoblast differentiation. In MC3T3-E1 cells in which menin expression was reduced by stable menin antisense DNA transfection, JunD levels were increased. When JunD and menin were co-transfected in MC3T3-E1 cells, they co-immunoprecipitated. JunD overexpression increased the transcriptional activity of an AP-1 luciferase reporter construct, and this activity was reduced by co-transfection of menin. Therefore, JunD and menin interact both physically and functionally in osteoblasts. Furthermore, menin overexpression inhibited the ALP activity induced by JunD. In conclusion, the data suggest that menin suppresses osteoblast maturation, in part, by inhibiting the differentiation actions of JunD.