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Abstract Background Isoorientin (ISO) is a glycosylated flavonoid with antitumor, anti-inflammatory, and antioxidant properties. However, its effects on bone metabolism remain largely unknown. Methods In this study, we aimed to investigate the effects of ISO on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation in vitro and bone loss in post-ovariectomy (OVX) rats, as well as to elucidate the underlying mechanism. First, network pharmacology analysis indicated that MAPK1 and AKT1 may be potential therapeutic targets of ISO and that ISO has potential regulatory effects on the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathways, as well as oxidative stress. ISO was added to RAW264.7 cells stimulated by RANKL, and its effects on osteoclast differentiation were evaluated using tartrate‐resistant acid phosphatase (TRAP) staining, TRAP activity measurement, and F-actin ring analysis. Reactive oxygen species (ROS) production in osteoclasts was detected using a ROS assay kit. The effects of ISO on RANKL-triggered molecular cascade response were further investigated by Western blotting, quantitative real-time polymerase chain reaction, and immunofluorescence staining. In addition, the therapeutic effects of ISO were evaluated in vivo. Results ISO inhibited osteoclastogenesis in a time- and concentration-dependent manner. Mechanistically, ISO downregulated the expression of the main transcription factor for osteoclast differentiation by inhibiting MAPK and PI3K/AKT1 signaling pathways. Moreover, ISO exhibited protective effects in OVX-induced bone loss rats. This was consistent with the results derived from network pharmacology. Conclusion Our findings suggest a potential therapeutic utility of ISO in the management of osteoclast-associated bone diseases, including osteoporosis.

The rhizome of Drynaria fortunei (Kunze ex Mett.) J. Sm., which is known as "Golsebo" in Korea, traditionally has been used to heal various inflammatory conditions, including bone metabolism disorders. It relieves blood extravasation, stops bleeding, repairs broken bone tissue, treats bone fractures, and kills bacteria. In this study, we evaluated the modulatory effects of DFP on the differentiation of bone-marrow-derived macrophages into osteoclasts. We performed tartrate-resistant acid phosphatase assays using DFP (at different concentrations and molecular weights) to evaluate the degree of bone resorption in the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis of bone-marrow-derived macrophages. TRAP activity increased with increasing DFP concentrations (0–200 µg/mL). Additionally, DFP significantly inhibited RANKL-induced osteoclastogenesis and controlled RANKL-mediated overexpression of c-Fos and nuclear factor of activated T cells 1, thereby downregulating osteoclast-specific gene (Atp6v0d2, cathepsin K, and DC-STAMP) expression. DFP thus has potential as a nutraceutical candidate for treating bone loss diseases, including osteoporosis in postmenopausal women. [ABSTRACT FROM AUTHOR]

The rhizome of Drynaria fortunei (Kunze ex Mett.) J. Sm., which is known as “Golsebo” in Korea, traditionally has been used to heal various inflammatory conditions, including bone metabolism disorders. It relieves blood extravasation, stops bleeding, repairs broken bone tissue, treats bone fractures, and kills bacteria. In this study, we evaluated the modulatory effects of DFP on the differentiation of bone-marrow-derived macrophages into osteoclasts. We performed tartrate-resistant acid phosphatase assays using DFP (at different concentrations and molecular weights) to evaluate the degree of bone resorption in the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis of bone-marrow-derived macrophages. TRAP activity increased with increasing DFP concentrations (0–200 µg/mL). Additionally, DFP significantly inhibited RANKL-induced osteoclastogenesis and controlled RANKL-mediated overexpression of c-Fos and nuclear factor of activated T cells 1, thereby downregulating osteoclast-specific gene (Atp6v0d2, cathepsin K, and DC-STAMP) expression. DFP thus has potential as a nutraceutical candidate for treating bone loss diseases, including osteoporosis in postmenopausal women.

Osteoporosis is among the major contributors of pathologic fracture in postmenopausal women, which is caused by the bone metabolic disorder owing to the over-activation of osteoclasts. Inhibition of osteoclast differentiation and maturation has become a mainstream research interest in the prevention of osteoporosis. Isoliensinine (Iso) is a dibenzyl isoquinoline alkaloid with antioxidant, anti-inflammatory, and anti-cancer activities. However, whether it can be used as a potential treatment for osteoporosis remains undiscovered. Here, we investigated whether Iso might suppress the differentiation of osteoclasts in vitro and in vivo to play an anti-osteoporosis role. Our results showed that Iso inhibits the formation of mature multinuclear osteoclasts induced by RANKL, the bone resorption, and the osteoclast-specific genes expression by blocking the nuclear translocation of NF-κB p65, and the effect was in a dosage-dependent way. Furthermore, we investigated the therapeutic effect of Iso on osteoporosis in ovariectomized (OVX) mice. We found that Iso attenuated bone loss in the OVX mice and significantly promoted BS, Conn. DN, Tb.Th, TB.N, and BV/TV Index. All in all, Iso showed a prominent effect of osteoclast inhibition, with great promise for treating osteoporosis. [ABSTRACT FROM AUTHOR]

It is a viable strategy to inhibit osteoclast differentiation for the treatment of osteolytic diseases such as osteoporosis, rheumatoid arthritis and tumor bone metastases. Here we assessed the effects of insulicolide A, a natural nitrobenzoyl sesquiterpenoid derived from marine fungus, on receptor activator of nuclear factor-κB ligand (RANKL)-stimulated osteoclastogenesis in vitro and its protective effects on LPS-induced osteolysis mice model in vivo. The results demonstrated that insulicolide A inhibited osteoclastogenesis from 1 μM in vitro. Insulicolide A could prevent c-Fos and nuclear factor of activated T-cell cytoplasmic 1 (NFATc1) nuclear translocation and attenuate the expression levels of osteoclast-related genes and DC-STAMP during RANKL-stimulated osteoclastogenesis but have no effects on NF-κB and MAPKs. Insulicolide A can also protect the mice from LPS-induced osteolysis. Our research provides the first evidence that insulicolide A may inhibit osteoclastogenesis both in vitro and in vivo, and indicates that it may have potential for the treatment of osteoclast-related diseases.

It is a viable strategy to inhibit osteoclast differentiation for the treatment of osteolytic diseases such as osteoporosis, rheumatoid arthritis and tumor bone metastases. Here we assessed the effects of insulicolide A, a natural nitrobenzoyl sesquiterpenoid derived from marine fungus, on receptor activator of nuclear factor-κB ligand (RANKL)-stimulated osteoclastogenesis in vitro and its protective effects on LPS-induced osteolysis mice model in vivo. The results demonstrated that insulicolide A inhibited osteoclastogenesis from 1 μM in vitro. Insulicolide A could prevent c-Fos and nuclear factor of activated T-cell cytoplasmic 1 (NFATc1) nuclear translocation and attenuate the expression levels of osteoclast-related genes and DC-STAMP during RANKL-stimulated osteoclastogenesis but have no effects on NF-κB and MAPKs. Insulicolide A can also protect the mice from LPS-induced osteolysis. Our research provides the first evidence that insulicolide A may inhibit osteoclastogenesis both in vitro and in vivo , and indicates that it may have potential for the treatment of osteoclast-related diseases. [ABSTRACT FROM AUTHOR]

Objectives: The present study was designed to investigate the effects of microRNA‐21 (miR‐21) on orthodontic tooth movement. Methods: The orthodontic tooth movement model was established in C57BL/6 and miR‐21−/− mice with or without implantation of activated T cells. Histological and histomorphometrical analyses were performed by hematoxylin–eosin staining. Tartrate‐resistant acid phosphate staining was used to analyze the osteoclast numbers during tooth movement. Enzyme‐linked immunosorbent assay, reverse transcription polymerase chain reaction, and immunohistochemistry analysis were used to examine the expression of RANKL and OPG. Results: In miR‐21−/− mice, the distance of tooth movement was retarded, the osteoclast number was decreased, and serum RANKL expression was strongly reduced. MiR‐21 promoted the secretion of RANKL from activated T cells. Furthermore, activated T cells could partially rescue the decreased orthodontic tooth movement distance in miR‐21−/− mice. MiR‐21 was shown to promote orthodontic tooth movement by modulating the RANKL/OPG balance in T cells. Conclusions: The impact of miR‐21 on tooth movement was better elucidated, furthering our understanding of its role and clinical applications in orthodontics. [ABSTRACT FROM AUTHOR]

Osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) are regulators of bone remodeling, but are also considered to play important roles in coronary artery disease (CAD). This study evaluated potential associations of soluble (s) RANKL and OPG with atherosclerosis-relevant cytokines. Blood was collected from 414 individuals who presented to our hospital with intermediate likelihood for CAD for further examination. Plasma concentrations of total sRANKL, OPG, and 20 cytokines were measured using sandwich-type enzyme-linked immunoassays (ELISAs; OPG and sRANKL) and Luminex laser-based fluorescence analysis and correlated with each other. The plasma levels of interferon-γ (IFN-γ) and the T-helper cell 2 cytokines interleukin-4 (IL-4) and IL-13 showed a positive correlation with sRANKL. The association with sRANKL levels was negative for IFN-γ-induced protein-10 (IP-10) and monocyte chemotactic protein-1 (MCP-1). The strongest independent association with sRANKL in multivariable analyses was found for IFN-γ (positive) and IP-10 (negative), while IL-13 showed a positive and independent association with OPG plasma levels. OPG and sRANKL plasma levels correlate strongly and independently with specific circulating atherosclerosis-related cytokines in patients with intermediate cardiovascular risk. [ABSTRACT FROM AUTHOR]

Bone remodeling, a physiological process characterized by bone formation by osteoblasts and bone resorption by osteoclasts, is important for the maintenance of healthy bone in adult humans. Osteoclasts play a critical role in bone erosion and osteoporosis and are bone-specific multinucleated cells generated through differentiation of monocyte/macrophage lineage precursors. Receptor activator of NF-κB ligand (RANKL) has been reported to induce osteoclast differentiation. In this study, we explored whether Gracilaria verrucosa extracts (GE) could affect RANKL-mediated osteoclast differentiation. GE significantly inhibited RANKL-activated osteoclast differentiation by inhibiting protein expression of c-fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), vital factors in RANKL-mediated osteoclastogenesis. In addition, GE attenuated ovariectomy-induced bone loss in mice. In summary, GE can prevent osteoclastogenesis and hormone-related bone loss via blockage of c-fos-NFATc1 signaling. Our results suggest that GE may have therapeutic potential in the treatment of postmenopausal osteoporosis. [ABSTRACT FROM AUTHOR]

Background Osteoarthritis (OA) is a major health problem in the increasingly elderly population. Therefore, it is crucial to prevent and treat OA at an early stage. The present study investigated whether pamidronate disodium (PAM), a bone-loss inhibitor, can significantly prevent or reverse the progression of early anterior cruciate ligament transection (ACLT)- induced OA. Whether therapeutic intervention is associated with regulation of the expression of osteoprotegerin (OPG), receptor activator of nuclear factor-κB ligand (RANKL), metalloproteinase-9 (MMP-9) or Toll-like receptor-4 (TLR-4) in cartilage and/or subchondral bone was also investigated. Methods 60 New Zealand rabbits were randomized into four groups: Sham-operated (n =20); ACLT (n =20); short-term treatment with PAM (PAM-S, n =10) and long-term treatment with PAM (PAM-L, n =10). For cartilage and subchondral bone testing, rabbits from Sham and ACLT groups were harvested at 2, 4, 6, and 14 weeks. Rabbits were given PAM from the 4th week after ACLT operation in PAM-S and PAM-L group, and were harvested at 6 and 14 weeks, respectively. Trabecular characteristics and cartilage changes were detected using Micro-CT, safranin O and rapid green staining, respectively. Immunohistochemical staining for OPG and RANKL were also performed. OPG, RANKL, MMP-9 and TLR-4 expression was evaluated by western blot analysis. Results Micro-CT and histology analyses indicated that PAM treatment for 2 or 10 weeks could completely prevent or reverse osteoarthritic subchondral bone loss and cartilage surface erosion. Immunohistochemistry and western blot analysis indicated that expression of OPG and RANKL increased, although RANKL expression increased more significantly than that of OPG. Therefore the ratio of OPG to RANKL was lower in the ACLT group. However, the ratio of OPG to RANKL in the PAM group was significantly higher than that in the ACLT group. Additionally, expression of MMP-9 and TLR-4 were upregulated in the ACLT group and downregulated in the PAM treated groups. Conclusions PAM can significantly inhibit and even reverse early osteoarthritic subchondral bone loss, thus alleviating the process of cartilaginous degeneration. The mechanisms involved may be associated with the upregulation of OPG expression, and downregulation of RANKL, MMP- 9 and TLR-4 expression. [ABSTRACT FROM AUTHOR]

Abstract: RELAXIN (RLN) is a polypeptide hormone of the insulin-like hormone family; it facilitates birth by softening and widening the pubic symphysis and cervix in many mammals, including humans. The role of RLN in bone metabolism was recently suggested by its ability to induce osteoclastogenesis and activate osteoclast function. RLN binds to RELAXIN/INSULIN-LIKE FAMILY PEPTIDE 1 (RXFP1) and 2 (RXFP2), with varying species-specific affinities. Young men with mutated RXFP2 are at high risk for osteoporosis, as RXFP2 influences osteoblast metabolism by binding to INSULIN-LIKE PEPTIDE 3 (INSL3). However, there have been no reports on RLN function in osteoblast differentiation and mineralization or on the functionally dominant receptors for RLN in osteoblasts. We previously described Rxfp1 and 2 expression patterns in developing mouse oral components, including the maxillary and mandibular bones, Meckel's cartilage, tongue, and tooth primordia. We hypothesized that Rln/Rxfp signaling is a key mediator of skeletal development and metabolism. Here, we present the gene expression patterns of Rxfp1 and 2 in developing mouse calvarial frontal bones as determined by in situ hybridization. In addition, RLN enhanced osteoblastic differentiation and caused abnormal mineralization and extracellular matrix metabolism through Rxfp2, which was predominant over Rxfp1 in MC3T3-E1 mouse calvarial osteoblasts. Our data suggest a novel role for Rln in craniofacial skeletal development and metabolism through Rxfp2. [Copyright &y& Elsevier]

Abstract: This study aimed to generate a monoclonal antibody (mAb) targeting both tumor necrosis factor-α (TNF-α) and receptor activator of NF-κB ligand (RANKL) and to evaluate the therapeutic effects of this antibody on acute inflammation and osteoporosis. We used hybridoma techniques to generate potential mAbs and enzyme-linked immunosorbent assay (ELISA) to determine their specificity. Crystal violet staining was performed to measure the effective dose of the candidate mAbs. The neutralizing effect of the mAbs was evaluated by TNF-α-mediated cytotoxicity and RANKL-induced osteoclastogenesis assays. We further assessed the therapeutic effect of the mAbs in BALB/c mice with carrageenan-induced acute inflammation and ovariectomy-induced osteoporosis. We successfully generated an IgG1 isotype mAb that recognizes human TNF-α and RANKL, which we named 8G12. The 50% effective dose of 8G12 was approximately 1μg/mL. L929 cells treated with 8G12 exhibited decreased levels of apoptosis (20.04% compared to 63.28% in the positive controls). In addition, treatment with 8G12 inhibited osteoclastogenesis in a dose-dependent manner in vitro. Carrageenan-induced paw edema was significantly reduced in the 8G12-treated mice compared to the positive controls. Treatment with 8G12 also reduced the number of infiltrating leukocytes by more than 50%. The 8G12 treatment not only prevented bone loss but also increased the number, thickness and volume of trabeculae and reduced trabecular separation in ovariectomized mice. Our data suggest that the 8G12 effectively neutralizes the bioactivity of TNF-α and RANKL, ameliorating osteoporosis and inflammation. We therefore propose that 8G12 could be a candidate for generating therapeutic antibodies for treating inflammatory bone diseases. [Copyright &y& Elsevier]

Highlights: [•] LC/MS method for total antigen protein after dosing of mAb drug with LLOQ 3.13ng/mL. [•] Immunoaffinity enrichment by anti-human Fc region antibody to capture immune complex. [•] No need to prepare antigen specific antibody that is different epitope from mAb drug. [•] Applicable to multivalent antigen protein forming bulky immune complex with mAb drug. [•] LC/MS method without interference by coexistent mAb unlike ELISA. [Copyright &y& Elsevier]

Abstract: Osteoclastogenesis is comprised of several stage s including progenitor survival, differentiation to mononuclear preosteoclasts, cell fusion to multinuclear mature osteoclasts, and activation to osteoclasts with bone resorbing activity. Botanical antioxidants are now being increasingly investigated for their health-promoting effects on bone. This study investigated that fisetin, a flavonol found naturally in many fruits and vegetables, suppressed osteoclastogenesis by disturbing receptor activator of nuclear factor (NF)-κB ligand (RANKL)-mediated signaling pathway and demoting osteoclastogenic protein induction. Nontoxic fisetin at ≤10μM inhibited the induction of RANK, tumor necrosis factor receptor associated factor 6 (TRAF6) and the activation of NF-κB in RANKL-stimulated RAW 264.7 macrophages. In RANKL-differentiated osteoclasts cell fusion protein of E-cadherin was induced, which was dampened by fisetin. The formation of tartrate-resistance acid phosphatase-positive multinucleated osteoclasts was suppressed by adding fisetin to RANKL-exposed macrophages. It was also found that fisetin reduced actin ring formation and gelsolin induction of osteclasts enhanced by RANKL through disturbing c-Src-proline-rich tyrosine kinase 2 signaling. Fisetin deterred preosteoclasts from the cell-cell fusion and the organization of the cytoskeleton to seal the resorbing area and to secret protons for bone resorption. Consistently, the 5 day-treatment of fisetin diminished RANKL-induced cellular expression of carbonic anhydrase II and integrin β3 concurrently with a reduction of osteoclast bone-resorbing activity. Therefore, fisetin was a natural therapeutic agent retarding osteoclast fusion and cytoskeletal organization such as actin rings and ruffled boarder, which is a property of mature osteoclasts and is required for osteoclasts to resorb bone. [Copyright &y& Elsevier]

Osterix/Sp7 is a zinc finger transcription factor and critical regulator of osteoblast differentiation, maturation and activity. Osterix expression has also been described in non-skeletal tissues but functional analyses are lacking. In the present study, we show that in the teleost model medaka, osterix is present as two alternatively spliced transcripts, osx_tv1 and osx_tv2. Knock-down of osx_tv1 and/or osx_tv2 results in mineralization loss in early intramembranous bones while cartilage formation is mostly unaffected. Formation of the parasphenoid, the earliest mineralized bone in the medaka skeleton, is impaired and fails to recover at later stages. Ossification of later bones, such as the operculum and cleithrum, is delayed but recovers during further development. In the axial skeleton, formation of the neural arches and centra is strongly delayed. In vivo analyses using osterix:nlGFP and osteocalcin:GFP transgenic medaka and whole mount in situ hybridization suggest that bone defects observed after knock-down of osterix are caused by a delay of osteoblast maturation and activity. Furthermore, we analyzed expression profile and function of osterix during ear and otolith formation. We show that osterix is expressed in otic placodes at the otic vesicle stage and that its knock-down results in a loss of otoliths. Taken together, we show that osterix is required for bone formation in a teleost fish and that its important regulatory functions are conserved between teleosts and mammals. Furthermore, we provide the first functional evidence for a role of Osterix in a non-skeletal tissue, i.e. the otoliths. [ABSTRACT FROM AUTHOR]

Abstract: In this study we determined the molecular mechanisms of how homocysteine differentially affects receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) synthesis in the bone. The results showed that oxidative stress induced by homocysteine deranges insulin-sensitive FOXO1 and MAP kinase signaling cascades to decrease OPG and increase RANKL synthesis in osteoblast cultures. We observed that downregulation of insulin/FOXO1 and p38 MAP kinase signaling mechanisms due to phosphorylation of protein phosphatase 2A (PP2A) was the key event that inhibited OPG synthesis in homocysteine-treated osteoblast cultures. siRNA knockdown experiments confirmed that FOXO1 is integral to OPG and p38 synthesis. Conversely homocysteine increased RANKL synthesis in osteoblasts through c-Jun/JNK MAP kinase signaling mechanisms independent of FOXO1. In the rat bone milieu, high-methionine diet-induced hyperhomocysteinemia lowered FOXO1 and OPG expression and increased synthesis of proresorptive and inflammatory cytokines such as RANKL, M-CSF, IL-1α, IL-1β, G-CSF, GM-CSF, MIP-1α, IFN-γ, IL-17, and TNF-α. Such pathophysiological conditions were exacerbated by ovariectomy. Lowering the serum homocysteine level by a simultaneous supplementation with N-acetylcysteine improved OPG and FOXO1 expression and partially antagonized RANKL and proresorptive cytokine synthesis in the bone milieu. These results emphasize that hyperhomocysteinemia alters the redox regulatory mechanism in the osteoblast by activating PP2A and deranging FOXO1 and MAPK signaling cascades, eventually shifting the OPG:RANKL ratio toward increased osteoclast activity and decreased bone quality. [Copyright &y& Elsevier]

Abstract: Background: Major depressive disorder (MDD) has been linked with accelerated bone loss leading to the development of low bone mineral density (BMD). Several mechanisms have been discussed as causative factors, e.g. lifestyle, selective serotonin reuptake inhibitor (SSRI) intake, or the influence of proinflammatory cytokines. Methods: In a cross-sectional study of in-patients with a current episode of MDD, without somatic comorbidities, we determined various parameters of bone metabolism, inflammatory parameters and parameters of depression. BMD was measured by dual x-ray absorptiometry. Results: Of 50 patients, only one had low BMD in any of the measure sites. Body mass index (BMI) correlated positively with Z-scores. 83.3% of the examined patients had elevated osteoprotegerin (OPG) levels. SSRI intake did not have an effect on BMD. BMD in the femoral neck was significantly lower in smokers. We also found a positive correlation between the level of physical activity and osteocalcin levels. Conclusions: In our sample, young to middle-aged, somatically healthy, and acutely depressed patients with a history of MDD showed no reduction of BMD. This could be due to compensatory mechanisms, as suggested by elevated OPG levels. Physical activity and high BMI could also have served as protective factors. Still, as patients with MDD often suffer from comorbidities or take medication with a negative effect on bone, this population should be appreciated as a high-risk group for the development of osteopenia and osteoporosis. [Copyright &y& Elsevier]

Abstract: Background: The interactions between metastatic breast cancer cells and host cells of osteoclastic lineage in bone microenvironment are essential for osteolysis. In vitro studies to evaluate pharmacological agents are mainly limited to their direct effects on cell lines. To mimic the communication between breast cancer cells and human osteoclasts, a simple and reproducible cellular model was established to evaluate the effects of zoledronate (zoledronic acid, ZOL), a bisphosphonate which exerts antiresorptive properties. Methods: Human precursor osteoclasts were cultured on bone-like surfaces in the presence of stimuli (sRANKL, M-CSF) to ensure their activation. Furthermore, immature as well as activated osteoclasts were co-cultured with MDA-MB-231 breast cancer cells. TRAP5b and type I collagen N-terminal telopeptide (NTx) were used as markers. Osteoclasts’ adhesion to bone surface and subsequent bone breakdown were evaluated by studying the expression of cell surface receptors and certain functional matrix macromolecules in the presence of ZOL. Results: ZOL significantly suppresses the precursor osteoclast maturation, even when the activation stimuli (sRANKL and M-SCF) are present. Moreover, it significantly decreases bone osteolysis and activity of MMPs as well as precursor osteoclast maturation by breast cancer cells. Additionally, ZOL inhibits the osteolytic activity of mature osteoclasts and the expression of integrin β3, matrix metalloproteinases and cathepsin K, all implicated in adhesion and bone resorption. Conclusions: ZOL exhibits a beneficial inhibitory effect by restricting activation of osteoclasts, bone particle decomposition and the MMP-related breast cancer osteolysis. General significance: The proposed cellular model can be reliably used for enhancing preclinical evaluation of pharmacological agents in metastatic bone disease. [Copyright &y& Elsevier]

Abstract: Autophagy and apoptosis are crucial cellular housekeeping and tissue survival mechanisms. There is emerging evidence of important crosstalk between apoptosis and autophagy which can be linked to inflammasome activation. Beclin 1 is a platform protein which assembles an interactome consisting of diverse proteins which control the initiation of autophagocytosis and distinct phases in endocytosis. Recent studies have demonstrated that the anti-apoptotic Bcl-2 family members can interact with Beclin 1 and inhibit autophagy. Consequently, impaired autophagy can trigger inflammasome activation. Interestingly, the hallmarks of the ageing process include a decline in autophagy, increased resistance to apoptosis and a low-grade inflammatory phenotype. Age-related stresses, e.g. genotoxic, metabolic and environmental insults, enhance the expression of NF-κB-driven anti-apoptotic Bcl-2 proteins which repress the Beclin 1-dependent autophagy. Suppression of autophagocytosis provokes inflammation including NF-κB activation which further potentiates anti-apoptotic defence. In a context-dependent manner, this feedback defence mechanism can enhance the aging process or provoke tumorigenesis or cellular senescence. We will review the role of Beclin 1 interactome in the crosstalk between apoptosis, autophagy and inflammasomes emphasizing that disturbances in Beclin 1-dependent autophagy can have a crucial impact on the aging process. [Copyright &y& Elsevier]

Osteolytic bone disease is the most common complication of multiple myeloma, resulting in skeletal-related events (SREs) that cause significant morbidity. Bone destruction in myeloma is due to an increased activity of osteoclasts coupled with suppressed bone formation by osteoblasts. Currently, bisphosphonates are the mainstay of the treatment of myeloma bone disease. Zoledronic acid and pamidronate have shown similar efficacy in reducing SREs in a randomized study in the conventional chemotherapy era. However, in a recent study (the Myeloma-IX trial of the UK Medical Research Council, MRC), zoledronic acid was found to be superior to clodronate in reducing SREs, but also it produced a survival advantage of approximately 10 months in patients with bone disease at baseline. During recent years, novel agents targeting bone have been used in myeloma. This review focuses on the established therapy of myeloma bone disease and also on recent advances in treatment that take advantage of the better understanding of the pathophysiology of bone disease. [ABSTRACT FROM AUTHOR]

Abstract: Differentiation into osteoclasts is induced by a macrophage colony-stimulating factor and receptor activator of nuclear-factor κB ligand. The macrophage-like cell line, C7 has the potential to differentiate into osteoclasts when it is cultured with both factors for 6days. Although C7 is an established cell line, the frequency of differentiation into this lineage was less than 10%, and the ratio was maintained at a constant level, even after repeated cloning. In this study, to increase the differentiation of C7 cells to osteoclasts, C7 derivative treatments with several activators and/or inhibitors were performed for 3days prior to setting osteoclast induction analysis; however, a reagent to significantly up-regulate the frequency of differentiation was not found. Only extended cultures for osteoclastogenesis exponentially increased the frequency of osteoclast precursors. It is likely that C7 cell differentiation into committed osteoclast precursors is on ‘autopilot’ rather than requiring specific signals to drive this process. [Copyright &y& Elsevier]

Booij-Vrieling HE, Ferbus D, Tryfonidou MA, Riemers FM, Penning LC, Berdal A, Everts V, Hazewinkel HAW. Increased vitamin D-driven signalling and expression of the vitamin D receptor, MSX2, and RANKL in tooth resorption in cats. Eur J Oral Sci 2010; 118: 39–46. © 2010 The Authors. Journal compilation© 2010 Eur J Oral Sci Tooth resorption occurs in 20–75% of cats ( Felis catus). The aetiology is not known, but vitamin D is suggested to be involved. Vitamin D acts through a nuclear receptor (VDR) and increases the expression of receptor activator of nuclear factor-κB ligand ( rankl) and muscle segment homeobox 2 ( msx2) genes. Mice lacking the muscle segment homeobox 2 ( msx2) gene show decreased levels of rankl, suggesting an interaction among VDR, MSX2, and RANKL. Here, we investigated the expression of VDR, MSX2, and RANKL proteins, and the activity of the VDR-mediated signalling pathway (using the quantitative polymerase chain reaction on VDR target genes), in tooth resorption, and measured the serum levels of vitamin D metabolites in cats. Tooth resorption was categorized into either resorptive or reparative stages. In the resorptive stage, odontoclasts expressed MSX2 and RANKL (100% and 88%, respectively) and fibroblasts expressed VDR and MSX2 (both at 100%), whereas fibroblasts expressed RANKL in only 29% of the sites analysed. In the reparative stage, cementoblasts expressed VDR, MSX2, and RANKL, whereas fibroblasts expressed VDR and MSX2, but not RANKL. The vitamin D status did not differ between the groups, based on the serum levels of 25-hydroxycholecalciferol. However, increased expression of VDR protein, and the relative gene expression levels of 1α-hydroxylase and the VDR-target gene, 24-hydroxylase, indicated the involvement of an active vitamin D signalling in the pathophysiology of tooth resorption in cats. [ABSTRACT FROM AUTHOR]

Abstract: Loosening of total hip arthroplasty (THA) caused by periprosthetic osteolysis induced by ultra-high molecular weight polyethylene (UHMWPE) particles is a major clinical problem. We investigated whether there are differences between loosened THA patients and primary THA patients in (1) receptor activator of nuclear factor-κB ligand (RANKL) expression on periprosthetic bone marrow cells; (2) RANKL levels, osteoprotegerin (OPG)/RANKL ratios, the levels of inflammatory cytokines and chemokines in synovial fluid. We used flow cytometric analysis to detect RANKL expression on periprosthetic bone marrow cells. We used enzyme-linked immunoassay and multiplex microsphere-based immunoassay to measure RANKL, OPG, cytokines, and chemokines in synovial fluid. We found loosened THA patients had higher RANKL expression on osteoblastic stromal cells, higher levels of RANKL, interleukin (IL)-6, IL-8, IL-10, interferon-γ-inducible protein (IP)-10, monocyte chemoattractant protein (MCP)-1, monokine induced by interferon-γ (MIG), and lower OPG/RANKL ratios in synovial fluid than primary THA patients. There was positive correlation between the levels of IL-6, IL-8, IL-10, IP-10, MCP-1, or MIG and RANKL levels in synovial fluid or RANKL expression on osteoblastic stromal cells. These suggest that UHMWPE particles induce over-expression of RANKL, IL-6, IL-8, IP-10, MCP-1, and MIG in human periprosthetic microenvironment. This results in periprosthetic osteolysis and loosening of THA. [Copyright &y& Elsevier]

Background and Objective: Cigarette smoking is a major risk factor in the development and further progression of periodontal diseases. However, little is known about how nicotine influences the expression of osteolytic mediators in cigarette smoking-associated periodontal diseases. The aim of this study was to investigate the expression of interleukin-1, interleukin-8, receptor activator of nuclear factor-κB ligand (RANKL), gelatinases and tissue-type plasminogen activator in U2OS cells (from the human osteosarcoma cell line) stimulated with nicotine. Material and Methods: Differences in the expression of interleukin-1, interleukin-8 and RANKL mRNAs, in response to exposure to various concentrations of nicotine (0, 0.125, 0.25, 0.5 and 1 mm) were evaluated in U2OS cells using the reverse transcripttion–polymerase chain reaction.In addition, the levels of interleukin-1, interleukin-8 and RANKL proteins were determined using enzyme-linked immunosorbent assays. The gelatinolytic and caseinolytic activities in nicotine treated-U2OS cells were demonstrated using gelatin and casein zymography, respectively. Results: Nicotine was found to increase the expression of interleukin-1, interleukin-8 and RANKL mRNA and protein in U2OS cells ( p < 0.05). The gelatin zymograms revealed that matrix metalloproteinase (MMP)-2 and MMP-9 were secreted by U2OS cells. The secretion of MMP-2 and MMP-9 occurred in a dose-dependent manner that was dependent on the concentration of nicotine ( p < 0.05). Casein zymography exhibited a caseinolytic band with a molecular weight of 70 kDa, indicative of the presence of tissue-type plasminogen activator. Tissue-type plasminogen activator was also found to be up-regulated by nicotine in a dose-dependent manner ( p < 0.05). Conclusion: Taken together, the results of the present study indicated that smoking modulation of bone destruction in periodontal disease may involve various osteolytic mediators, such as interleukin-1, interleukin-8, RANKL, MMP-2, MMP-9, and tissue-type plasminogen activator. [ABSTRACT FROM AUTHOR]

Background and Objective: Inflammatory immune reactions that occur in response to periodontopathogens are thought to protect the host against infection, but may trigger periodontal destruction. However, the molecular and genetic mechanisms underlying host susceptibility to periodontal infection and to periodontitis development have still not been established in detail. Material and Methods: In this study, we examined the mechanisms that modulate the outcome of Aggregatibacter (Actinobacillus) actinomycetemcomitans-induced periodontal disease in mice mouse strains selected for maximal (AIRmax) or minimal (AIRmin) inflammatory reactions. Results: Our results showed that AIRmax mice developed a more severe periodontitis than AIRmin mice in response to A. actinomycetemcomitans infection, and this periodontitis was characterized by increased alveolar bone loss and inflammatory cell migration to periodontal tissues. In addition, enzyme-linked immunosorbent assays demonstrated that the levels of the cytokines interleukin-1β, tumor necrosis factor-α and interleukin-17 were higher in AIRmax mice, as were the levels of matrix metalloproteinase (MMP)-2, MMP-13 and receptor activator of nuclear factor-κB ligand (RANKL) mRNA levels. However, the more intense inflammatory immune reaction raised by the AIRmax strain, in spite of the higher levels of antimicrobial mediators myeloperoxidase and inducible nitric oxide synthase, did not enhance the protective immunity to A. actinomycetemcomitans infection, because both AIRmax and AIRmin strains presented similar bacterial loads in periodontal tissues. In addition, the AIRmax strain presented a trend towards higher levels of serum C-reactive protein during the course of disease. Conclusion: Our results demonstrate that the intensity of the inflammatory immune reaction is associated with the severity of experimental periodontitis, but not with the control of A. actinomycetemcomitans periodontal infection, suggesting that the occurrence of hyperinflammatory genotypes may not be an evolutionary advantage in the complex host–pathogen interaction observed in periodontal diseases. [ABSTRACT FROM AUTHOR]

Recent years brought the discovery of new members of TNF receptor superfamily - osteoprotegerin/receptor activator of nuclear factor-κB and its ligand (OPG/RANK/RANKL) system as regulator of bone remodeling. Further studies showed its involvement in control of vascular and immune system. Animal studies' results confirm the OPG/RANK/RANKL role in pathogenesis of vascular calcifications and osteoporosis. Human studies, especially in patients with chronic kidney disease (CKD), have brought many conflicting data. Understanding of exact contribution of each molecule creating this axis may be crucial for diagnosis and treatment of CKD complications involving renal osteodystrophy and vascular calcification. In this review we try to summarize recent knowledge and OPG/RANK/RANKL role in patient with chronic kidney diseases. [ABSTRACT FROM AUTHOR]

Abstract: Receptor of Activated NF-κB Ligand (RANKL) is implicated as one of a number of effector molecules that mediate progesterone and prolactin signaling in the murine mammary epithelium. Using a mouse transgenic approach, we demonstrate that installation of the RANKL signaling axis into the mammary epithelium results in precocious ductal side-branching and alveologenesis in the virgin animal. These morphological changes occur due to RANKL-induced mammary epithelial proliferation, which is accompanied by increases in expression of activated NF-kB and cyclin D1. With age, prolonged RANKL exposure elicits limited mammary epithelial hyperplasia. While these transgenics exhibit RANKL-induced salivary gland adenocarcinomas, palpable mammary tumors are not observed due to RANKL-suppression of its own signaling receptor (RANK) in the mammary epithelium. Together, these studies reveal not only that the RANKL signaling axis can program many of the normal epithelial changes attributed to progesterone and prolactin action in the normal mammary gland during early pregnancy, but underscore the necessity for tight control of this signaling molecule to avoid unwarranted developmental changes that could lead to mammary hyperplasia in later life. [Copyright &y& Elsevier]

RAW 264.7 macrophage cells differentiate into osteoclast-like cells in the presence of RANKL. Participation of M-CSF in RANKL-induced osteoclast formation of RAW 264.7 cells was examined. TRAP-positive osteoclast-like cells appeared in RAW 264.7 cells cultured in the presence of RANKL. RANKL-induced osteoclast formation was markedly inhibited by anti-M-CSF antibody. RANKL augmented M-CSF mRNA expression and M-CSF production in RAW 264.7 cells. Further, anti-M-CSF antibody inhibited the expression of RANK, c-fms, c-fos and TRAP mRNA in RANKL-stimulated RAW 264.7 cells. However, anti-M-CSF antibody did not affect the expression of DC-STAMP in the stimulated cells. Therefore, RANKL was suggested to induce osteoclast formation in RAW 264.7 cells via augmented production of M-CSF. The putative role of M-CSF in RANKL-induced osteoclast formation of RAW 264.7 cells is discussed. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Evaluation of serum and synovial fluid OPG and sRANKL in 37 patients with primary knee osteoarthritis. Design and method: OPG and sRANKL were measured using ELISA. Results: OPG, sRANKL and sRANKL/OPG were increased in osteoarthritis patients'' serum. Synovial OPG was higher than serum OPG, while sRANKL/OPG was higher in the serum; both correlated with disease severity. Discussion: RANKL/OPG pathway is implicated in the pathogenesis of knee osteoarthritis being a suitable target for therapeutic intervention. [Copyright &y& Elsevier]

Abstract: Lipopolysaccharide (LPS) is a potent bone resorbing factor. The effect of LPS on osteoclast formation was examined by using murine RAW 264.7 macrophage cells. LPS-induced the formation of multinucleated giant cells (MGC) in RAW 264.7 cells 3days after the exposure. MGCs were positive for tartrate-resistant acid phosphatase (TRAP) activity. Further, MGC formed resorption pits on calcium-phosphate thin film that is a substrate for osteoclasts. Therefore, LPS was suggested to induce osteoclast formation in RAW 264.7 cells. LPS-induced osteoclast formation was abolished by anti-tumor necrosis factor (TNF)-α antibody, but not antibodies to macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-κB ligand (RANKL). TNF-α might play a critical role in LPS-induced osteoclast formation in RAW 264.7 cells. Inhibitors of NF-κB and stress activated protein kinase (SAPK/JNK) prevented the LPS-induced osteoclast formation. The detailed mechanism of LPS-induced osteoclast formation is discussed. [Copyright &y& Elsevier]

Abstract: In addition to systemic and local factors, mechanical strain plays a crucial role in bone remodeling during growth, development, and fracture healing, and especially in orthodontic tooth movement. Although many papers have been published on the effects of mechanical stress on osteoblasts or osteoblastic cells, little is known about the effects of different magnitudes of mechanical strain on such cells. In the present study, we investigated how different magnitudes of cyclic tensile strain affected osteoblasts. MC3T3-E1 osteoblastic cells were subjected to 0%, 6%, 12% or 18% elongation for 24h using a Flexercell Strain Unit, and then the mRNA and protein expressions of osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) were examined. The results showed that cyclic tensile strain induced a magnitude-dependent increase (0%, 6%, 12%, and 18%) in OPG synthesis and a concomitant decrease in RANKL mRNA expression and sRANKL release from the osteoblasts. Furthermore, the induction of OPG mRNA expression by stretching was inhibited by indomethacin or genistein, and the stretch-induced reduction of RANKL mRNA was inhibited by PD098059. These results indicate that different magnitudes of cyclic tensile strain influence the biological behavior of osteoblasts, which profoundly affects bone remodeling. [Copyright &y& Elsevier]

The aim of this study was the evaluation of the effect of intermediate doses of thalidomide with dexamethasone (Thal/Dex) on disease course and bone disease in patients with refractory/relapsed myeloma who were under zoledronic acid therapy. We studied 35 patients, who received thalidomide at a dose of 200 mg/daily. We measured, pre-, 3 and 6 months post-treatment soluble receptor activator of nuclear factor-κB ligand (sRANKL), osteoprotegerin (OPG), osteopontin (OPN), markers of bone resorption and formation. Before treatment, patients had increased levels of sRANKL/OPG ratio, bone resorption markers and OPN, while they had suppressed bone formation. The pretreatment sRANKL/OPG ratio correlated with the extent of bone disease. Thal/Dex administration resulted in a significant reduction of sRANKL/OPG ratio, and bone resorption. Bone formation, OPG and OPN did not show any alteration. Changes of sRANKL/OPG ratio correlated with changes of bone resorption markers. Thal/Dex was given for a median time of 10 months and the median follow-up period was 22 months. The response rate was 65.7%. The median survival was 19.5 months. β2-microglobulin, type of response and International Staging System predicted for survival. These results suggest that the combination of intermediate dose of Thal/Dex is effective in patients with refractory/relapsed myeloma and improves abnormal bone remodeling through the reduction of sRANKL/OPG ratio.Leukemia (2005) 19, 1969–1976. doi:10.1038/sj.leu.2403890; published online 4 August 2005 [ABSTRACT FROM AUTHOR]

All osteogenic cells (osteoclasts, osteoblasts) contribute individually to bone remodeling. Their cellular interactions control their cellular activities and the bone remodeling intensity. These interactions can be established either through a cell–cell contact, involving molecules of the integrin family, or by the release of many polypeptidic factors and/or their soluble receptor chains. These factors can act directly on osteogenic cells and their precursors to control differentiation, formation and functions (matrix formation, mineralization, resorption …). Here, we present the involvement of three groups of cytokines which seem to be of particular importance in bone physiology: interleukin-6 (IL-6), tumor necrosis factor -alpha(TNF-α) (TNF-α)/IL-1, and the more recently known triad osteoprotegerin (OPG)/receptor activator of NF-κB (RANK)/RANK ligand (RANKL). The interactions between these three groups are presented within the framework of bone resorption pathophysiology such as tumor associated osteolysis. The central role of the OPG/RANK/RANKL triad is pointed out. [Copyright &y& Elsevier]

Background: To clarify the roles of osteoclast regulatory factors in progression of odontogenic tumors, expression of parathyroid hormone-related protein (PTHrP), osteoclast differentiation factor (ODF)/receptor activator of nuclear factor-kappaB ligand (RANKL), and osteoclastogenesis inhibitory factor (OCIF)/osteoprotegerin (OPG) were analyzed in ameloblastomas as well as tooth germs.Methods: Tissue specimens of nine tooth germs and 36 benign and one malignant ameloblastomas were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry for the expression of PTHrP, ODF/RANKL, and OCIF/OPG.Results: Expression of PTHrP, ODF/RANKL, and OCIF/OPG mRNA was detected in all tooth germ and ameloblastoma samples. Immunohistochemical reactivity for PTHrP was recognized in both normal and neoplastic odontogenic epithelial cells. In ameloblastomas, PTHrP reactivity in peripheral columnar or cuboidal cells was stronger than that in central polyhedral cells, and keratinizing cells showed increased PTHrP reactivity. ODF/RANKL and OCIF/OPG were expressed predominantly in mesenchymal cells rather than in odontogenic epithelial cells in both tooth germs and ameloblastomas. Epithelial ODF/RANKL and OCIF/OPG expression was slightly lower in ameloblastomas than in tooth germs. Tumor cells in plexiform ameloblastomas showed slightly higher reactivity for PTHrP and ODF/RANKL than tumor cells in follicular ameloblastomas.Conclusion: Expression of PTHrP, ODF/RANKL and OCIF/OPG in tooth germs and ameloblastomas suggests that these factors might locally regulate bone metabolism and dynamics in tooth development as well as in progression of ameloblastomas. These factors might also be involved in tumor cell differentiation and/or tumor tissue structuring in ameloblastomas. [ABSTRACT FROM AUTHOR]

Nucleotides such as adenosine triphosphate (ATP) and uridine triphosphate (UTP) exist in the extracellular environment where they are agonists at P2 receptors. Both P2Y G-protein-coupled receptors and P2X ligand-gated ion channels are expressed by osteoblasts and osteoclasts, reflected in the diverse nucleotide-induced effects reported to occur in bone. Previous reports have implicated ATP as a proresorptive agent; however, these studies were unable to determine whether ATP mediated its actions directly on osteoclasts, or indirectly via osteoblasts. The development of techniques to generate human osteoclasts in vitro has allowed us to further investigate the intriguing role of extracellular nucleotides with regard to osteoclast activity. This study reports that nearly all P2-receptor-subtype mRNAs were expressed throughout human osteoclast development, and provides evidence for functional P2 receptor expression by these cells. In cultures of human osteoclasts alone, neither ATP nor UTP affected the quantity of resorption by these cells; however, in cocultures of osteoblast-like UMR-106 cells and human osteoclasts, ATP, but not UTP, greatly enhanced resorption, indicating a role for osteoblasts in mediating the proresorptive effects of ATP. Furthermore, ATP, but not UTP, elevated receptor activator of nuclear factor-κB ligand (RANKL) mRNA and protein expression by UMR-106 cells. These data are consistent with observations that UMR-106 cells predominantly express P2Y1 with low expression of P2Y2, thereby explaining the response to ATP and not UTP, and further substantiating the involvement of osteoblasts in ATP-induced effects on osteoclasts. These results significantly advance our understanding of the role of P2 receptors in bone, and indicate that local-acting ATP may play a pivotal role in osteoclast activation at bone-resorbing sites by inducing elevated expression of RANKL. [Copyright &y& Elsevier]

Parathyroid hormone (PTH) stimulates receptor activator of nuclear factor-κB ligand (RANKL) mRNA and inhibits osteoprotegerin (OPG) mRNA expression in murine bone marrow cultures. To understand the mechanisms influencing these responses, we investigated the role of the protein kinase A (PKA) and protein kinase C (PKC) pathways in the regulation of RANKL and OPG mRNA expression in murine bone marrow cultures. Murine bone marrow cells were stimulated with bovine PTH(1-34) and (1-34) amide, which activate both pathways; PTH(3-34), which more selectively activates the PKC and calcium pathways; and human PTH (1-31), which stimulates adenylyl cyclase, but not protein kinase C. We also examined agents that more directly activate either the PKA pathway (forskolin [FSK] and 8-bromo cAMP [8-Br-cAMP]) or the PKC pathway (phorbol 12-myristate 13-acetate [PMA]) in murine bone marrow cultures. After 1 h, RANKL mRNA expression was stimulated to a similar degree by agents that activate either or both the PKA and PKC pathways. However, this effect was sustained for 24 h only with agents that stimulated PKA. OPG mRNA expression was inhibited by all agents that stimulated PKA at 6 h. In contrast, PKC-specific stimulators [PMA and bPTH(3-34)] had no effect on OPG regulation in this culture system. To determine the involvement of the PKC signaling pathway in responses of RANKL, bone marrow cells were pretreated with PMA for 24 h and then treated with PTH(1-34) or FSK for 2 h. PMA pretreatment did not alter the ability of PTH or FSK to stimulate RANKL or inhibit OPG mRNA expression. Treatment of cells with H-89, a PKA inhibitor, significantly reduced the ability of PTH and FSK to induce RANKL and inhibit OPG mRNA expression. Calphostin C, a PKC inhibitor, significantly reduced PMA-stimulated RANKL mRNA expression without altering PTH- or FSK-mediated effects on RANKL or OPG mRNA. Cycloheximide, an inhibitor for protein synthesis, inhibited PTH-stimulated RANKL mRNA expression by 60% without altering the effect of PTH on OPG mRNA expression. To examine the involvement of prostaglandin in PMA-mediated responses, cells were treated with indomethacin, a nonspecific prostaglandin G/H synthase (PGHS) inhibitor, or NS-398, a selective inhibitor of PGHS-2. Neither PGHS inhibitor altered PMA-induced effects on RANKL and OPG mRNA expression. These results demonstrate that the PKA pathway is predominantly involved in the effects of PTH on RANKL mRNA expression in murine bone marrow cultures, but there is also a PKC-mediated response, which is not sustained. Inhibition of OPG by PTH appears to be a selective PKA response. [Copyright &y& Elsevier]

Interleukin-11 (IL-11) is a stromal cell-derived cytokine that can enhance osteoclast formation and stimulate bone resorption. In the present study, the characteristics of the resorptive effect of IL-11 in mouse calvarial bones were investigated. Both recombinant mouse IL-11 and human IL-11 caused concentration- and time-dependent stimulations of 45Ca release from prelabeled mouse calvariae. Half-maximal responses were obtained at 0.7 ng/mL (&ap;40 pmol/L). Mouse and human IL-11 also stimulated release of 3H from [3H]-proline-labeled bones. The magnitude of the 45Ca and 3H release (1.4–1.6-fold) caused by a maximally effective concentration of IL-11 was less than the stimulation (2.5–4.0-fold) elicited by a maximum concentration of parathyroid hormone (PTH). Release of 45Ca by IL-11 was unaffected by the mitotic inhibitors, hydroxyurea and aphidicolin. In addition to resorption of bone, IL-11 caused a small (1.5–2.0-fold) enhancement of prostaglandin E2 (PGE2) biosynthesis in calvariae, but had no effect on the mRNA expression of cyclooxygenase-1 and -2, or cytosolic phospholipase A2. Indomethacin and flurbiprofen abolished the formation of PGE2 and partially reduced 45Ca release stimulated by IL-11. When either mouse interleukin-4 (IL-4) or interleukin-13 (IL-13) was added to calvariae treated with IL-11, 45Ca release was inhibited. Resorption caused by IL-11 was also inhibited by both anti-mouse glycoprotein 130 (gp130) and an antibody neutralizing IL-11, but these agents had no effect on 45Ca release caused by PTH or 1,25(OH)2vitamin D3 (D3). Real-time, quantitative polymerase chain reaction (PCR) analysis (TaqMan PCR) and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that IL-11 caused concentration-dependent enhancements of receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) mRNA, without affecting the mRNA expression of RANK. Mouse RANKL stimulated 45Ca release in the calvarial bones. The stimulatory effects of RANKL and IL-11 were inhibited by mouse OPG. These data demonstrate that IL-11 stimulates osteoclastic resorption in mouse calvariae by mechanisms that are independent of cell proliferation; partially dependent on prostaglandin biosynthesis; sensitive to inhibition by IL-4, IL-13, and OPG; and associated with enhanced expression of RANKL and OPG. In addition, IL-11 was not found to play an essential role in resorption stimulated by other calciotropic agents in calvariae. [Copyright &y& Elsevier]

Recent studies have shown that stimulation of osteoclastogenesis in cocultures of osteoblasts and spleen cells in response to prostaglandin E2 (PGE2) is markedly decreased when the osteoblasts are derived from cells lacking either the EP2 or the EP4 receptor. Induction of osteoclast formation requires upregulation of receptor activator of nuclear factor-κB ligand (RANKL) on cells of the osteoblastic lineage, which then binds to the RANK receptor on cells of the osteoclast lineage. Osteoprotegerin (OPG) is a decoy receptor for RANKL that can block its interaction with RANK. In addition, macrophage-colony stimulating factor (M-CSF) is essential for osteoclast formation. Finally, PGE2 can increase interleukin-6 (IL-6), which may further enhance osteoclastogenesis. To study the relative influence of the EP2 and EP4 receptors on response of these factors to PGE2, we examined mRNA levels for RANKL, OPG, M-CSF, and IL-6 in primary osteoblastic cell cultures derived from two lines of EP2 knockout mice (EP2−/−) and one line of EP4 knockout mice (EP4−/−) and the relevant wild-type controls (EP2+/+ and EP4+/+). The responses of cells from wild-type animals of all three lines were similar. After PGE2 treatment, RANKL mRNA levels were increased at 2 h, and this was sustained over 72 h. Basal RANKL expression was moderately reduced in EP2−/− cells and markedly reduced in EP4−/− cells. PGE2 increased RANKL mRNA in EP2−/− cells and EP4−/− cells, but the levels were significantly reduced compared with wild-type cells. There were no consistent changes in expression of M-CSF or OPG in the different genotypes or with PGE2 treatment. IL-6 mRNA was variably increased by PGE2 in both wild-type and knockout cells, although the absolute levels were somewhat lower in both EP2−/− and EP4 −/− cultures. Parathyroid hormone (PTH) increased RANKL and IL-6 and decreased OPG mRNA levels similarly in both wild-type and EP2−/− or EP4−/− cells. The major defect in the response to PGE2 in animals lacking either EP2 or EP4 receptors is a reduction in basal and stimulated RANKL levels. Loss of EP4 receptor appears to have a greater effect on basal RANKL expression than EP2. [ABSTRACT FROM AUTHOR]

Thyroid hormones enhance osteoclast formation and their excess is an important cause of secondary osteoporosis. 3,5,3′-Triiodo-l-thyronine (T3) induced the mRNA expression of receptor activator of nuclear factor-κB ligand (RANKL), which is a key molecule in osteoclast formation, in primary osteoblastic cells (POB). This effect was amplified in the copresence of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). Although T3 alone did not induce octeoclasts in coculture of bone marrow cells with POB, T3 enhanced 1,25(OH)2D3-induced osteoclast formation. Thyroxine (T4) also enhanced 1,25(OH)2D3-induced osteoclast formation. These data suggested that T4 was locally metabolized to T3 for its action, since T4 is a prohormone with little hormonal activity. The mRNA expression of type-2 iodothyronine deiodinase (D2), which is responsible for maintaining local T3 concentration, was induced by 1,25(OH)2D3 dose- and time-dependently. Our data would facilitate our understanding of the mechanism of osteoclast formation by thyroid hormones and suggest a novel interaction between thyroid hormones and 1,25(OH)2D3. [Copyright &y& Elsevier]

Denosumab–Prolia®, Xgeva® (Amgen) is a fully human antibody to the receptor activator of the nuclear factor-K ligand (RANKL). Hepatotoxicity is extremely rare, with only one reported case of immune origin. We present a second case of hepatotoxicity resulting from an immune reaction to denosumab. A 43-year-old female was referred to the Endocrinology, Diabetes & Metabolism Department for treatment of low bone mineral density (BMD) following endocrine therapy with letrozole and lucrin because of breast cancer. She developed premature menopause at the age of 36 years when she underwent a left lumpectomy due to an infiltrating duct carcinoma of the breast (T1 NO MO) and was subsequently started on endocrine therapy. Denosumab was started to prevent osteoporosis. On the third year after starting on denosumab and one month after she received the last injection, she became ill. The routine biochemical analysis showed that the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) rose appreciatively to 10 times the upper limit of normal (ULN). The gamma-glutamyl transferase (GGT) level was elevated slightly to 67 U/L (0–38 U/L). The serum gamma-globulin level was elevated to 1.72 g/dL (0.7–1.6 gr/dl), while the total bilirubin (TB) and serum albumin levels were normal. A liver biopsy revealed a moderate to severe chronic inflammatory infiltrate containing MUM-1 positive plasma cells. In addition, numerous CD-3 positive small T lymphocytes and few CD-20 positive B lymphocytes and eosinophils were seen in the portal tracts. Moderate to severe interface hepatitis, bile duct proliferation and mild portal fibrosis were also identified. The results could be consistent with the diagnosis of drug-induced liver injury (DILI). [ABSTRACT FROM AUTHOR]