Carnitine functions as an enhancer of NRF2 to inhibit osteoclastogenesis via regulating macrophage polarization in osteoporosis.

Osteoporosis, which manifests as reduced bone mass and deteriorated bone quality, is common in the elderly population. It is characterized by persistent elevation of macrophage-associated inflammation and active osteoclast bone resorption. Currently, the roles of intracellular metabolism in regulating these processes remain unclear. In this study, we initially performed bioinformatics analysis and observed a significant increase in the proportion of M1 macrophages in bone marrow with aging. Further metabolomics analysis demonstrated a notable reduction in the expression of carnitine metabolites in aged macrophages, while carnitine was not detected in osteoclasts. During the differentiation process, osteoclasts took up carnitine synthesized by macrophages to regulate their own activity. Mechanistically, carnitine enhanced the function of Nrf2 by inhibiting the Keap1-Nrf2 interaction, reducing the proteasome-dependent ubiquitination and degradation of Nrf2. In silico molecular ligand docking analysis of the interaction between carnitine and Keap1 showed that carnitine binds to Keap1 to stabilize Nrf2 and enhance its function. In this study, we found that the decrease in carnitine levels in aging macrophages causes overactivation of osteoclasts, ultimately leading to osteoporosis. A decrease in serum carnitine levels in patients with osteoporosis was found to have good diagnostic and predictive value. Moreover, supplementation with carnitine was shown to be effective in the treatment of osteoporosis.

Effect of phytate on hypercalciuria secondary to bone resorption in patients with urinary stones: pilot study.

The objective is to evaluate the effect of phytate supplements on calciuria in patients with urinary stones and elevated bone resorption. The secondary objective is to analyze the therapeutic effect of phytate based on measurements of serum markers of bone resorption. This is a controlled randomized study included patients according to predefined inclusion and exclusion criteria, and randomized them into two groups. Patients in the phytate group received a 380 mg capsule of calcium-magnesium InsP6 (Salvat Laboratories®) every 24 h for 3 months and patients in the control group received no treatment. All included patients were male or female, 18-65 years old, had hypercalciuria (> 250 mg/24 h), had a ß-Crosslaps level greater than 0.4 ng/mL, and had bone densitometry results indicative of osteopenia or osteoporosis in the femur and/or spine. At study onset, calciuria was 321 ± 52 mg/24 h in the phytate group and 305 ± 57 mg/24 h in the control group (p > 0.05). At 3 months, calciuria was significantly lower in the phytate group than the control group (226 ± 45 mg/24 h vs. 304 ± 58 mg/24 h, p < 0.05). At study onset, the mean ß-CrossLaps level was 1.25 ± 0.72 ng/mL in the phytate group and 0.57 ± 0.13 ng/mL in the control group (p < 0.05). However, at 3 months, the ß-CrossLaps level was significantly lower in the phytate group than in the control group (0.57 ± 0.13 ng/mL vs. 0.77 ± 0.42 ng/mL, p < 0.05). Phytate reduced calciuria in patients with hypercalciuria secondary to bone resorption. The ß-CrossLaps assay was effective for evaluating the efficacy of phytate on hypercalciuria during follow-up.

Pharmacological Therapies for the Management of Inflammatory Bone Resorption in Periodontal Disease: A Review of Preclinical Studies.

Periodontitis, a highly prevalent multicausal chronic inflammatory and destructive disease, develops as a result of complex host-parasite interactions. Dysbiotic bacterial biofilm in contact with the gingival tissues initiates a cascade of inflammatory events, mediated and modulated by the host's immune response, which is characterized by increased expression of several inflammatory mediators such as cytokines and chemokines in the connective tissue. If periodontal disease (PD) is left untreated, it results in the destruction of the supporting tissues around the teeth, including periodontal ligament, cementum, and alveolar bone, which lead to a wide range of disabilities and poor quality of life, thus imposing significant burdens. This process depends on the differentiation and activity of osteoclasts, the cells responsible for reabsorbing the bone tissue. Therefore, the inhibition of differentiation or activity of these cells is a promising strategy for controlling bone resorption. Several pharmacological drugs that target osteoclasts and inflammatory cells with immunomodulatory and anti-inflammatory effects, such as bisphosphonates, anti-RANK-L antibody, strontium ranelate, cathepsin inhibitors, curcumin, flavonoids, specialized proresolving mediators, and probiotics, were already described to manage inflammatory bone resorption during experimental PD progression in preclinical studies. Meantime, a growing number of studies have described the beneficial effects of herbal products in inhibiting bone resorption in experimental PD. Therefore, this review summarizes the role of several pharmacological drugs used for PD prevention and treatment and highlights the targeted action of all those drugs with antiresorptive properties. In addition, our review provides a timely and critical appraisal for the scientific rationale use of the antiresorptive and immunomodulatory medications in preclinical studies, which will help to understand the basis for its clinical application.

Testosterone supplementation and bone parameters: a systematic review and meta-analysis study.

BACKGROUND: The role of testosterone (T) replacement therapy (TRT) in subjects with late onset hypogonadism is still the object of an intense debate. METHODS: All observational studies and placebo-controlled or -uncontrolled randomized trials (RCTs) comparing the effect of TRT on different bone parameters were considered. RESULTS: Out of 349 articles, 36 were considered, including 3103 individuals with a mean trial duration of 66.6 weeks. TRT improves areal bone mineral density (aBMD) at the spine and femoral neck levels in observational studies, whereas placebo-controlled RTCs showed a positive effect of TRT only at lumber spine and when trials included only hypogonadal patients at baseline (total testosterone < 12 nM). The effects on aBMD were more evident in subjects with lower T levels at baseline and increased as a function of trial duration and a higher prevalence of diabetic subjects. Either T or estradiol increase at endpoint contributed to aBMD improvement. TRT was associated with a significant reduction of bone resorption markers in observational but not in controlled studies. CONCLUSION: TRT is able to inhibit bone resorption and increase bone mass, particularly at the lumbar spine level and when the duration is long enough to allow the anabolic effect of T and estrogens on bone metabolism to take place.

Carnosol suppresses RANKL-induced osteoclastogenesis and attenuates titanium particles-induced osteolysis.

Osteolysis is a common medical condition characterized by excessive activity of osteoclasts and bone resorption, leading to severe poor quality of life. It is essential to identify the medications that can effectively suppress the excessive differentiation and function of osteoclasts to prevent and reduce the osteolytic conditions. It has been reported that Carnosol (Car), isolated from rosemary and salvia, has anti-inflammatory, antioxidative, and anticancer effects, but its activity on osteolysis has not been determined. In this study, we found that Car has a strong inhibitory effect on the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation dose-dependently without any observable cytotoxicity. Moreover, Car can inhibit the RANKL-induced osteoclastogenesis and resorptive function via suppressing NFATc1, which is a result of affecting MAPK, NF-κB and Ca2+ signaling pathways. Moreover, the particle-induced osteolysis mouse model confirmed that Car could be effective for the treatment of bone loss in vivo. Taken together, by suppressing the formation and function of RANKL-induced osteoclast, Car, may be a therapeutic supplementary in the prevention or the treatment of osteolysis.

Panax Notoginseng Saponins Prevent Bone Loss by Promoting Angiogenesis in an Osteoporotic Mouse Model.

With the aging of the population and the extension of life expectancy, osteoporosis is becoming a global epidemic. Although there are several drugs used to treat osteoporosis in clinical practice, such as parathyroid hormone or bisphosphonates, they all have some serious side effects. Therefore, a safer drug is called for osteoporosis, especially for the prevention in the early stage of the disease, not only the treatment in the later stage. Panax notoginseng saponin (PNS), a traditional Chinese herb, has been used as anti-ischemic drug due to its function on improving vascular circulation. In order to verify whether Panax notoginseng saponins (PNS) could be used to prevent osteoporosis, ovariectomy (OVX) was induced in female C57BL/C6J mice, followed by orally administration with 40 mg/kg/d, 80 mg/kg/d, and 160 mg/kg/d of three different dosages of PNS for 9 weeks. Serum biochemical analysis, micro-CT, histological evaluation, and immunostaining of markers of osteogenesis and angiogenesis were performed in the sham, osteoporotic (OVX), and treatment (OVX+PNS) groups. Micro-CT and histological evaluation showed that compared to sham group, the bone mass of OVX group reduced significantly, while it was significantly restored in the moderate-dose PNS (40 mg/kg and 80 mg/kg) treatment groups. The expression of CD31 and osteocalcin (OCN) in the bone tissue of treatment group also increased, suggesting that PNS activated osteogenesis and angiogenesis, which subsequently increased the bone mass. These results confirmed the potential function of PNS on the prevention of osteoporosis. However, in the high dose of PNS (160 mg/kg) group, the antiosteoportic effect had been eliminated, which also suggested the importance of proper dose of PNS for the prevention and treatment of osteoporosis in postmenopausal women.

Acute hypercalcemia and excessive bone resorption following anti-RANKL withdrawal: Case report and brief literature review.

Denosumab is an anti-RANKL antibody that is commonly used for the treatment of osteoporosis; in oncology, bisphosphonates and denosumab have become the standard therapies for the treatment and prevention of skeletal complications in patients with myeloma and solid tumors. In recent years, excessive bone remodeling following the discontinuation of denosumab has raised concerns. Several cases of hypercalcemia have been reported after the discontinuation of high-dose denosumab (120 mg every 4 weeks), mainly in children. In this study, we report a new case of severe refractory hypercalcemia in a 54-year-old woman who received high-dose denosumab for 5 years as an adjuvant therapy for breast cancer. She is currently in remission and undergoing treatment with anastrazole, an aromatase inhibitor. The peculiarities of this case are the presence of associated bone pain with subperiosteal bone resorption on hand X-rays, and diffuse, long bone diaphyseal uptake on a bone scan. Hyperparathyroidism has been ruled out, and existing evidence suggests that this high-level of bone remodeling could be due to a rebound hyperactivation of the RANKL pathway. In addition to rehydration, repeated use of i.v. bisphosphonates was required to control recurrent hypercalcemia. As hypercalcemia is a serious metabolic complication, a gradual dose reduction should be considered when interruption of high dose denosumab therapy is planned.

Exploring the potential of tocotrienol from Bixa orellana as a single agent targeting metabolic syndrome and bone loss.

Metabolic syndrome (MetS) is associated with osteoporosis due to the underlying inflammatory and hormonal changes. Annatto tocotrienol has been shown to improve medical complications associated with MetS or bone loss in animal studies. This study aimed to investigate the effects of annatto tocotrienol as a single treatment for MetS and osteoporosis in high-carbohydrate high-fat (HCHF) diet-induced MetS animals. Three-month-old male Wistar rats were randomly divided into five groups. The baseline group was euthanized at the onset of the study. The normal group received standard rat chow and tap water. The remaining groups received HCHF diet and treated with three different regimens orally daily: (a) tocopherol-stripped corn oil (the vehicle of tocotrienol), (b) 60 mg/kg annatto tocotrienol, and (c) 100 mg/kg annatto tocotrienol. At the end of the study, measurements of MetS parameters, body compositions, and bone mineral density were performed in animals before sacrifice. Upon euthanasia, blood and femur of the rats were harvested for the evaluations of bone microstructure, biomechanical strength, remodelling activities, hormonal changes, and inflammatory response. Treatment with annatto tocotrienol improved all MetS parameters (except abdominal obesity), trabecular bone microstructure, bone strength, increased osteoclast number, normalized hormonal changes and inflammatory response in the HCHF animals. In conclusion, annatto tocotrienol is a potential agent for managing MetS and osteoporosis concurrently. The beneficial effects of annatto tocotrienol may be attributed to its ability to prevent the hormonal changes and pro-inflammatory state in animals with MetS.

α-Linolenic Acid Inhibits Receptor Activator of NF-κB Ligand Induced (RANKL-Induced) Osteoclastogenesis and Prevents Inflammatory Bone Loss via Downregulation of Nuclear Factor-KappaB-Inducible Nitric Oxide Synthases (NF-κB-iNOS) Signaling Pathways.

BACKGROUND Inflammation is a major cellular strain causing increased risk of osteo-degenerative diseases. Omega-3 fatty acids have been great source in suppressing inflammation. We investigated the effect of α-linolenic acid (ALA) on RANKL-stimulated osteoclast differentiation, LPS-induced and ovariectomized bone loss in mice models. MATERIAL AND METHODS The bone marrow macrophages (BMMs) were isolated from femurs of ICR mice, stimulated with RANKL, and treated with ALA (100, 200, 300 µM). Major analytical methods include histological analysis, osteoclasts viability assay, serum cytokines and chemokines ELISA, and gene expression by qPCR. RESULTS ALA intervention inhibited RANKL-induced osteoclasts proliferation and differentiation. ALA inhibited bone resorption activity as measured by materialization of F-actin ring structures as well. ALA suppressed the RANKL-induced osteoclast markers c-Fos, c-Jun and NFATc1 together with transcription factor proteins TRAP, OSCAR, cathepsin K and ß3-integrin. ALA also suppressed the RANKL-stimulated phosphorylation of JNK, ERK, and AKT as well as NF-κB and BCL-2 proteins. ALA intervention (100 and 300 mg/kg) to LPS-challenged mice showed annulled morphometric changes induced by LPS by suppressing the levels of proinflammatory cytokines and chemokines. ALA (100 and 300 mg/kg) intervention to estrogen-deficiency induced bone loss mice (ovariectomized) showed reductions in TRAP+ osteoclasts count, CTX-I expression, levels of IL-1ß, IL-2, IL-6, IL10, TNF-α and MCP-1 and iNOS and COX-2. CONCLUSIONS ALA suppresses RANKL-induced osteoclast differentiation and prevents inflammatory bone loss via downregulation of NF-κB-iNOS-COX-2 signaling. ALA is suggested to be a preventive herbal medicine against inflammatory bone disorders.

Inflammatory arthritis and systemic bone loss are attenuated by gastrointestinal helminth parasites.

Infections with different helminth species have been observed to ameliorate a variety of chronic inflammatory diseases. Herein, we show that the natural murine helminth species, Heligmosomoides polygyrus bakeri (Hp) is capable of attenuating disease severity in two different inflammatory arthritis models. Furthermore, we show that excretory-secretory (ES) products from Hp directly suppress osteoclast differentiation in vitro. Taken together, these results demonstrate that helminth infections can dampen autoimmune diseases and highlight a previously unrecognized and important role for ES products, by directly impacting on bone destruction.

Pharmacologically Inactive Bisphosphonates as an Alternative Strategy for Targeting Osteoclasts: In Vivo Assessment of 5-Fluorodeoxyuridine-Alendronate in a Preclinical Model of Breast Cancer Bone Metastases.

Bisphosphonates have effects that are antiresorptive, antitumor, and antiapoptotic to osteoblasts and osteocytes, but an effective means of eliciting these multiple activities in the treatment of bone metastases has not been identified. Antimetabolite-bisphosphonate conjugates have potential for improved performance as a class of bone-specific antineoplastic drugs. The primary objective of the study was to determine whether an antimetabolite-bisphosphonate conjugate will preserve bone formation concomitant with antiresorptive and antitumor activity. 5-FdU-ale, a highly stable conjugate between the antimetabolite 5-fluoro-2'-deoxyuridine and the bisphosphonate alendronate, was tested for its therapeutic efficacy in a mouse model of MDA-MB231 breast cancer bone metastases. In vitro testing revealed osteoclasts to be highly sensitive to 5-FdU-ale. In contrast, osteoblasts had significantly reduced sensitivity. Tumor cells were resistant in vitro but in vivo tumor burden was nevertheless significantly reduced compared with untreated mice. Sensitivity to 5-FdU-ale was not mediated through inhibition of farnesyl diphosphate synthase activity, but cell cycle arrest was observed. Although serum tartrate-resistant acid phosphatase (TRAP) levels were greatly reduced by both drugs, there was no significant decrease in the serum bone formation marker osteocalcin with 5-FdU-ale treatment. In contrast, there was more than a fivefold decrease in serum osteocalcin levels with alendronate treatment (p < 0.001). This finding is supported by time-lapse micro-computed tomography analyses, which revealed bone formation volume to be on average 1.6-fold higher with 5-FdU-ale treatment compared with alendronate (p < 0.001). We conclude that 5-FdU-ale, which is a poor prenylation inhibitor but maintains potent antiresorptive activity, does not reduce bone formation and has cytostatic antitumor efficacy. These results document that conjugation of an antimetabolite with bisphosphonates offers flexibility in creating potent bone-targeting drugs with cytostatic, bone protection properties that show limited nephrotoxicity. This unique class of drugs may offer distinct advantages in the setting of targeted adjuvant therapy and chemoprevention of bone diseases. © 2016 American Society for Bone and Mineral Research.

Aqueous extract of pomegranate seed attenuates glucocorticoid-induced bone loss and hypercalciuria in mice: A comparative study with alendronate.

The present study was performed in order to examine bone loss and calcium homeostasis in mice with glucocorticoid (GC)-induced osteoporosis (GIOP) following treatment with the aqueous extract of pomegranate seed (AE-PS). In addition, a comparative study with alendronate was performed. Biomarkers in the serum and the urine were measured. The tibias, kidney and duodenum were removed in order to measure the levels of bone calcium, protein expression as well as to perform histomorphological analysis of the bone. GC treatment facilitated the induction of hypercalciuria in the mice, and the AE-PS­treated mice exhibited a greater increase in serum calcium and a decrease in urine calcium. The AE-PS reversed the deleterious effects on the trabecular bone induced by DXM and stimulated bone remodeling, including an increase in bone calcium and alkaline phosphatase­b (ALP-b) and a decrease in a the critical bone resorption markers C-terminal telopeptide of type I collagen (CTX) and tartrate­resistant acid phosphatase-5b (TRAP-5b). Hematoxylin and eosin (H&E) staining revealed the increased disconnections and separation between the growth plate and the trabecular bone network as well as the reduction in the trabecular bone mass of the primary and secondary spongiosa throughout the proximal metaphysis of the tibia in the DXM group. Moreover, the decreased protein expression of transient receptor potential vanilloid (TRPV)5, TRPV6 and calbindin­D9k (CaBP­9k) was reversed by the AE-PS or alendronate supplementation in the kidneys and the duodenum as well as plasma membrane Ca2+­ATPase1 (PMCA1) expression in the kidneys of mice with GIOP. There was no marked difference in pharmacological effectiveness between alendronate and the AE-PS. Taken together, these findings suggest that the AE-PS may be an alternative therapy suitable for use in the management of secondary osteoporosis.

Anti-osteoporosis activity of red yeast rice extract on ovariectomy-induced bone loss in rats.

Osteoporosis is the most common bone disease, affecting millions of people worldwide and leading to significant morbidity and high costs. Monacolin K, an extract of red yeast rice (RYR, Hongqu), plays important roles in the management of dyslipidemia, coronary heart disease, and diabetes. Our study aimed to investigate the protective effect of monacolin K on ovariectomy-induced bone loss in rats. Fifty female Sprague-Dawley rats were randomly divided into a sham-operated and five ovariectomized (OVX) groups: OVX with vehicle, OVX with fluvastatin, and OVX with RYR extract of three graded doses. Bone mineral density (BMD), biochemical markers, and cell viability were analyzed by dual energy X-ray absorptiometry, enzyme-linked immunosorbent assay, and 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays. Gene expression was evaluated by real-time polymerase chain reaction amplification and western blot. Our results showed that administration of RYR extract markedly increased the bone mineral density in OVX rats. Moreover, RYR extract decreased the levels of bone turnover markers, including osteocalcin and tartrate resistant acid phosphatase activity. The MMT assay revealed that RYR extract treatment significantly improved the osteoblast viabilities in a dose-dependent manner (P < 0.05). At the molecular level, we further demonstrated that RYR extract enhanced the expression of Bmp2 and Bmp4 both at the mRNA and protein levels. Collectively, these data suggested RYR extract could protect against osteoporosis in ovariectomized rats, most likely through activation of BMP2/4 expression.

Effects of Curculigoside on Memory Impairment and Bone Loss via Anti-Oxidative Character in APP/PS1 Mutated Transgenic Mice.

Alzheimer's disease (AD) and osteoporosis are two closely related multifactorial progressively degenerative diseases that predominantly affect aged people. These two diseases share many common risk factors, including old age, being female, smoking, excessive drinking, low estrogen, and vitamin D3 levels. Additionally, oxidative damage and the dysfunction of the antioxidant system play important roles in the pathogenesis of osteoporosis and AD. Aß not only leads to impaired memory but also plays a crucial role in the demineralization process of bone tissues of older people and women with menopause. Curculigoside can promote calcium deposition and increase the levels of ALP and Runx2 in osteoblasts under oxidative stress via anti-oxidative character. Therefore, we investigated the effects of CUR on the spatial learning and memory by the Morris water maze and brain immunohistochemistry, and bone microstructure and material properties of femurs by micro-computed tomography and mechanical testing in APP/PS1 mutated transgenic mice. Oral administration of CUR can significantly enhance learning performance and ameliorate bone loss in APP/PS1 mutated transgenic mice, and the mechanism may be related to its antioxidant effect. Based on these results, CUR has real potential as a new natural resource for developing medicines or dietary supplements for the prevention and treatment of the two closely linked multifactorial progressive degenerative disorders, AD and osteoporosis.

The effects of thyrotropin-suppressing therapy on bone metabolism in patients with well-differentiated thyroid carcinoma.

Studies on the effects of levothyroxine (LT4) therapy on bone and bone metabolism have yielded conflicting results. This 1-year prospective study examined whether LT4 in patients with well-differentiated thyroid carcinoma (DTC) is a risk factor for bone mass loss and the subsequent development of osteoporosis. We examined 93 patients with DTC over 12months after initiating LT4 therapy (early postoperative period). We examined another 33 patients on long-term LT4 therapy for DTC (late postoperative period). Dual energy X-ray absorptiometry was performed at baseline and after 1year. The mean bone losses during the early postoperative period in the lumbar spine, femoral neck, and total hip, calculated as the percentage change between levels at baseline and 12months, were -0.88, -1.3 and -0.81%, respectively. Bone loss was more evident in postmenopausal women (lumbar spine -2.1%, femoral neck -2.2%, and hip -2.1%; all P<0.05). We compared the changes in annual bone mineral density (BMD) in postmenopausal women according to calcium/vitamin D supplementation. Bone loss tended to be higher in the postmenopausal women receiving no supplementation. There was no decrease in BMD among patients during the late postoperative period. The mean bone loss was generally greater in the early than in the late postoperative group, and this was significant at the lumbar spine (P=0.041) and femoral neck (P=0.010). TSH-suppressive levothyroxine therapy accelerates bone loss, predominantly in postmenopausal women and exclusively during the early post-thyroidectomy period.

Effect of calcium supplementation on bone resorption in pregnancy and the early postpartum: a randomized controlled trial in Mexican women.

BACKGROUND: Calcium needs are physiologically upregulated during pregnancy and lactation to meet demands of the developing fetus and breastfeeding infant. Maternal calcium homeostasis is maintained by hormonal adaptive mechanisms, thus, the role of dietary calcium supplementation in altering maternal responses to fetal-infant demand for calcium is thought to be limited. However, increased calcium absorption is directly related to maternal calcium intake and dietary supplementation has been suggested to prevent transient bone loss associated with childbearing. METHODS: In a double-blind, randomized placebo-controlled trial, we randomly assigned 670 women in their first trimester of pregnancy to 1,200 mg/day calcium (N = 334) or placebo (N = 336). Subjects were followed through 1-month postpartum and the effect on urinary cross-linked N-telopeptides (NTx) of type I collagen, a specific marker of bone resorption, was evaluated using an intent-to-treat analysis. Women with a baseline and at least one follow-up measurement (N = 563; 84%) were included. Subsequent analyses were conducted stratifying subjects by compliance assessed using pill counts. In random subsets of participants, bone-specific alkaline phosphatase (BAP) (N = 100) and quantitative ultrasound (QUS) (N = 290) were also measured. RESULTS: Calcium was associated with an overall reduction of 15.8% in urinary NTx relative to placebo (p < 0.001). Among those who consumed ≥50%, ≥67%, and ≥75% of pills, respectively, the effect was associated with 17.3%, 21.3%, and 22.1% reductions in bone resorption (all p < 0.001). There was no significant effect of calcium on bone formation measured by BAP. However, by 1-month postpartum, those in the calcium group had significantly lower NTx/BAP ratios than those in the placebo group (p = 0.04) indicating a net reduction in bone loss in the supplement group by the end of follow-up. Among subjects who consumed ≥50% and ≥75% of pills, respectively, calcium was also associated with an increase of 26.3 m/s (p = 0.03) and 59.0 m/s (p = 0.009) in radial SOS relative to placebo by 1-month postpartum. CONCLUSIONS: Calcium administered during pregnancy and the early postpartum period, to women with intakes around adequacy, was associated with reduced bone resorption and, thus, may constitute a practical intervention to prevent transient skeletal loss associated with childbearing. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT00558623.

Proline/arginine-rich end leucine-rich repeat protein N-terminus is a novel osteoclast antagonist that counteracts bone loss.

(hbd) PRELP is a peptide corresponding to the N-terminal heparin binding domain of the matrix protein proline/arginine-rich end leucine-rich repeat protein (PRELP). (hbd) PRELP inhibits osteoclastogenesis entering pre-fusion osteoclasts through a chondroitin sulfate- and annexin 2-dependent mechanism and reducing the nuclear factor-κB transcription factor activity. In this work, we hypothesized that (hbd) PRELP could have a pharmacological relevance, counteracting bone loss in a variety of in vivo models of bone diseases induced by exacerbated osteoclast activity. In healthy mice, we demonstrated that the peptide targeted the bone and increased trabecular bone mass over basal level. In mice treated with retinoic acid to induce an acute increase of osteoclast formation, the peptide consistently antagonized osteoclastogenesis and prevented the increase of the serum levels of the osteoclast-specific marker tartrate-resistant acid phosphatase. In ovariectomized mice, in which osteoclast activity was chronically enhanced by estrogen deficiency, (hbd) PRELP counteracted exacerbated osteoclast activity and bone loss. In mice carrying osteolytic bone metastases, in which osteoclastogenesis and bone resorption were enhanced by tumor cell-derived factors, (hbd) PRELP reduced the incidence of osteolytic lesions, both preventively and curatively, with mechanisms involving impaired tumor cell homing to bone and tumor growth in the bone microenvironment. Interestingly, in tumor-bearing mice, (hbd) PRELP also inhibited breast tumor growth in orthotopic sites and development of metastatic disease in visceral organs, reducing cachexia and improving survival especially when administered preventively. (hbd) PRELP was retained in the tumor tissue and appeared to affect tumor growth by interacting with the microenvironment rather than by directly affecting the tumor cells. Because safety studies and high-dose treatments revealed no adverse effects, (hbd) PRELP could be employed as a novel biological agent to combat experimentally induced bone loss and breast cancer metastases, with a potential translational impact.

Effect of calcitriol on bone turnover and osteocalcin in recent-onset type 1 diabetes.

BACKGROUND: Vitamin D supplementation in childhood improves the achievement of peak bone mass. We investigated the effect of supplementation with calcitriol on bone turnover in recent-onset type 1 diabetes (T1D). Moreover, the association between osteocalcin and parameters of ß-cell function and metabolic control was examined. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a post-hoc analysis of a double-blind, placebo-controlled study of calcitriol supplementation to preserve ß-cell function. 27 recent-onset T1D subjects, mean age 22 years, were randomized to 0.25 µg calcitriol per day or placebo (1:1) and followed up for one year. Changes in bone formation (osteoclacin) and resorption (beta-CrossLaps) markers, and differences between placebo and calcitriol-treated group were evaluated. At baseline, osteocalcin levels were significantly lower in female than in male patients (P<0.01) while no other metabolic parameters as HbA1c and C-peptide differed between gender. No significant correlations were found in relation to HbA1c, insulin requirement and C-peptide. At 1 year follow-up, no significant differences were observed between calcitriol and placebo groups for osteocalcin and ß-CrossLaps. In the placebo group osteocalcin levels were unrelated with parameters of metabolic control, such as C-peptide, insulin requirement or HbA1c. Changes of C-peptide, insulin requirement and HbA1c were not related to osteocalcin levels. CONCLUSIONS: Supplementation with 0.25 µg calcitriol per day to patients with new-onset T1D does not affect circulating markers of bone turnover. OC levels were unrelated to ß-cell function and other metabolic parameters suggesting that OC is ineffective to control pancreatic function in presence of aggressive autoimmune destruction.

Bisphosphonates in preclinical bone oncology.

Bisphosphonates, especially nitrogen-containing bisphosphonates, are widely used to block bone destruction in cancer patients with bone metastasis because they are effective inhibitors of osteoclast-mediated bone resorption. In addition to their antiresorptive effects, preclinical evidence strongly suggests that nitrogen-containing bisphosphonates exert direct and indirect anticancer activities through inhibition of tumor cell functions, enhancement of the cytotoxic activity of chemotherapy agents, inhibition of tumor angiogenesis, and stimulation of antitumor immune reactions. This review examines the current evidence and provides insights into ongoing preclinical research on anticancer activities of these bisphosphonates in animal models of tumorigenesis and metastasis.

Elevated cytokine production restores bone resorption by human Btk-deficient osteoclasts.

Mutations in Bruton's tyrosine kinase (Btk) cause the B-cell disorder X-linked agammaglobulinaemia (XLA) in humans, but the effect of Btk deficiency in human bone health has not been investigated previously. In this study, we show that human Btk-deficient osteoclasts are defective at resorption activity in vitro owing to a dysregulation of the actin cytoskeletal function. Contrary to expectation, XLA patients did not exhibit increased bone density or alterations in serum markers of bone turnover, indicating that a potential compensation mechanism normalizes bone homeostasis. In contrast to the bone turnover markers, the levels of inflammatory cytokines interleukin 6 (IL-6), IL-1ß, and tumor necrosis factor α (TNF-α) were significantly elevated in XLA patients' serum compared with control individuals. Supplementation of osteoclast cultures from normal and XLA subjects with serum from XLA patients or recombinant inflammatory cytokines IL-6, IL-1ß, and TNF-α resulted in a stimulation of osteoclast activity in vitro, whereas the addition of cytokine-neutralizing antibodies inhibited this stimulatory effect, confirming that elevated inflammatory cytokines in XLA serum heightened osteoclast activity in vitro. This study provides novel evidence that Btk signaling is crucial for optimal actin cytoskeletal organization and lacunar resorption in isolated osteoclasts. In XLA patients, however, these inherent osteoclast defects are corrected by increased inflammatory cytokine levels, restoring osteoclast activity and leading to the normalization of bone density.