Risedronate Reduces Osteoclast Precursors and Cytokine Production in Postmenopausal Osteoporotic Women

This paper studies the effect of oral risedronate on osteoclast precursors, osteoclast formation, and cytokine production in 25 osteoporotic women. Risedronate is effective in reducing the number of osteoclast precursors, their formation, vitality, and activity and the level of RANKL and TNF‐α in cultures. Introduction: Bisphosphonates inhibit bone resorption by acting against osteoclasts. Some in vitro studies suggest that they induce osteoclast apoptosis; others suggest that they exert an effect on the production of pro‐osteoclastogenic cytokines. The effect of risedronate on osteoclastogenesis by peripheral blood mononuclear cells (PBMCs) in postmenopausal osteoporosis has not been previously studied. This paper examined the influence of risedronate on the formation of osteoclast precursors and cytokine production within the compass of osteoclastogenesis in osteoporosis. Materials and Methods: This study was conducted on 38 osteoporotic women; 25 patients were treated with risedronate 5 mg/d, whereas 13 were treated with calcium 1 g/d and vitamin D 800 UI/d. The following parameters were assessed: changes in bone turnover, circulating osteoclast precursors, formation of osteoclasts in PBMC cultures, their activity and vitality, and variations in the production of pro‐osteoclastogenic cytokines before and after therapy. Results: After 3 mo of risedronate, there was a significant reduction in the number and degree of differentiation of osteoclast precursors, osteoclast formation, vitality and activity, and in the level of RANKL and TNF in cultures and of TNF and osteoprotegerin (OPG) in serum, whereas in the group treated with calcium and vitamin D, there were no significant changes. Conclusions: Our data show that risedronate is effective in lowering the number of circulating osteoclast precursors, their formation, vitality, and activity in cultures, and in reducing the level of pro‐osteoclastogenic cytokines in culture supernatants and in serum.     

Macrophage Migration Inhibitory Factor (MIF) Supports Homing of Osteoclast Precursors to Peripheral Osteolytic Lesions

By binding to its chemokine receptor CXCR4 on osteoclast precursor cells (OCPs), it is well known that stromal cell‐derived factor‐1 (SDF‐1) promotes the chemotactic recruitment of circulating OCPs to the homeostatic bone remodeling site. However, the engagement of circulating OCPs in pathogenic bone resorption remains to be elucidated. The present study investigated a possible chemoattractant role of macrophage migration inhibitory factor (MIF), another ligand for C‐X‐C chemokine receptor type 4 (CXCR4), in the recruitment of circulating OCPs to the bone lytic lesion. To accomplish this, we used Csf1r‐eGFP‐knock‐in (KI) mice to establish an animal model of polymethylmethacrylate (PMMA) particle‐induced calvarial osteolysis. In the circulating Csf1r‐eGFP+ cells of healthy Csf1r‐eGFP‐KI mice, Csf1r+/CD11b+ cells showed a greater degree of RANKL‐induced osteoclastogenesis compared to a subset of Csf1r+/RANK+ cells in vitro. Therefore, Csf1r‐eGFP+/CD11b+ cells were targeted as functionally relevant OCPs in the present study. Although expression of the two cognate receptors for MIF, CXCR2 and CXCR4, was elevated on Csf1r+/CD11b+ cells, transmigration of OCPs toward recombinant MIF in vitro was facilitated by ligation with CXCR4, but not CXCR2. Meanwhile, the level of PMMA‐induced bone resorption in calvaria was markedly greater in wild‐type (WT) mice compared to that detected in MIF‐knockout (KO) mice. Interestingly, in contrast to the elevated MIF, diminished SDF‐1 was detected in a particle‐induced bone lytic lesion of WT mice in conjunction with an increased number of infiltrating CXCR4+ OCPs. However, such diminished SDF‐1 was not found in the PMMA‐injected calvaria of MIF‐KO mice. Furthermore, stimulation of osteoblasts with MIF in vitro suppressed their production of SDF‐1, suggesting that MIF can downmodulate SDF‐1 production in bone tissue. Systemically administered anti‐MIF neutralizing monoclonal antibody (mAb) inhibited the homing of CXCR4+ OCPs, as well as bone resorption, in the PMMA‐injected calvaria, while increasing locally produced SDF‐1. Collectively, these data suggest that locally produced MIF in the inflammatory bone lytic site is engaged in the chemoattraction of circulating CXCR4+ OCPs. © 2016 American Society for Bone and Mineral Research.     

SH3BP2 is a Critical Regulator of Macrophage and Osteoclast Response to M-CSF and RANKL Stimulation

The protein Src homology 3 domain-binding protein 2 (SH3BP2) is a regulator of tumor necrosis factor (TNF)-alpha generation in macrophages and of osteoclast differentiation. SH3BP2 regulates bone marrow monocyte responses to macrophage and osteoclast differentiation signals downstream of the receptors for macrophage colony-stimulating factor and receptor activator of nuclear factor kappaB ligand. SH3BP2 is a potential target for the development of novel anti-TNF-alpha therapeutic interventions as well as a target for suppression of osteoclastogenesis. SH3BP2 is a critical regulator of macrophage and osteoclast response to M-CSF and RANKL stimulation.     

Osteoclast differentiation by RANKL requires NF-kappaB-mediated downregulation of cyclin-dependent kinase 6 (Cdk6).

This study investigated the involvement of cell cycle factors in RANKL-induced osteoclast differentiation. Among the G1 cell cycle factors, Cdk6 was found to be a key molecule in determining the differentiation rate of osteoclasts as a downstream effector of the NF-kappaB signaling. INTRODUCTION: A temporal arrest in the G1 phase of the cell cycle is a prerequisite for cell differentiation, making it possible that cell cycle factors regulate not only the proliferation but also the differentiation of cells. This study investigated cell cycle factors that critically influence differentiation of the murine monocytic RAW264.7 cells to osteoclasts induced by RANKL. MATERIALS AND METHODS: Growth-arrested RAW cells were stimulated with serum in the presence or absence of soluble RANKL (100 ng/ml). Expressions of the G1 cell cycle factors cyclin D1, D2, D3, E, cyclin-dependent kinase (Cdk) 2, 4, 6, and Cdk inhibitors (p18 and p27) were determined by Western blot analysis. Involvement of NF-kappaB and c-jun N-terminal kinase (JNK) pathways was examined by overexpressing dominant negative mutants of the IkappaB kinase 2 (IKK(DN)) gene and mitogen-activated protein kinase kinase 7 (MKK7(DN)) gene, respectively, using the adenovirus vectors. To determine the direct effect of Cdk6 on osteoclast differentiation, stable clones of RAW cells transfected with Cdk6 cDNA were established. Osteoclast differentiation was determined by TRACP staining, and cell cycle regulation was determined by BrdU uptake and flow cytometric analysis. RESULTS AND CONCLUSION: Among the cell cycle factors examined, the Cdk6 level was downregulated by RANKL synchronously with the appearance of multinucleated osteoclasts. Inhibition of the NF-kappaB pathway by IKK(DN) overexpression, but not that of the JNK pathway by MKK7(DN) overexpression, caused the decreases in both Cdk6 downregulation and osteoclastogenesis by RANKL. RAW cells overexpressing Cdk6 resist RANKL-induced osteoclastogenesis; however, cell cycle regulation was not affected by the levels of Cdk6 overexpression, suggesting that the inhibitory effect of Cdk6 on osteoclast differentiation was not exerted through cell cycle regulation. These results indicate that Cdk6 is a critical regulator of RANKL-induced osteoclast differentiation and that its NF-kappaB-mediated downregulation is essential for efficient osteoclast differentiation.     

Osteoclast differentiation from circulating mononuclear precursors in Paget's disease is hypersensitive to 1,25-dihydroxyvitamin D(3) and RANKL

A characteristic feature of Paget's disease is an increase in the number of osteoclasts in bone. Osteoclasts are formed from mononuclear phagocyte precursors that circulate in the monocyte fraction of peripheral blood. These cells require the presence of RANK ligand (RANKL)-expressing osteoblastic cells and human macrophage colony-stimulating factor (M-CSF) to form osteoclasts in vitro. To determine the role of osteoclast differentiation from circulating precursors in Paget's disease, we cultured monocytes from Paget's patients and gender- and age-matched normal controls with no evidence of bone disease for up to 21 days in the presence of UMR 106 cells and various concentrations of M-CSF (1-25 ng/mL) and 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] (10(-10) to 10(-7) mol/L). Relative to controls, there was a significant increase in the extent of osteoclast differentiation from pagetic monocytes as assessed by expression of tartrate-resistant acid phosphatase (TRAP), vitronectin receptor (VNR), and lacunar bone resorption. Serial dilution experiments (2 x 10(5) to 2 x 10(2) cells/well) showed no difference in the concentration of osteoclast precursors in the peripheral blood. In Paget's patients with high serum alkaline phosphatase (sAP) levels, increased sensitivity to the osteoclastogenic effect of 1,25(OH)(2)D(3) was noted. Osteoclast differentiation did not occur when M-CSF was substituted by interleukin-6 (IL-6) and soluble IL-6 receptor (sIL-6R), and these factors did not stimulate osteoclast differentiation in the presence of M-CSF. In this in vitro coculture system, osteoclast formation was inhibited by osteoprotegerin in a dose-dependent manner. In the presence of RANKL (5-30 ng/mL) and M-CSF (25 ng/mL), osteoclast formation and bone resorption were significantly increased in cultures of monocytes from patients with high and low sAP levels as compared with normal controls. Our findings suggest that the increase in osteoclast numbers seen in Paget's disease results not from an increase in the number of circulating precursors in peripheral blood but rather from an increased sensitivity of osteoclast precursors to the humoral factors, 1,25(OH)(2)D(3) and RANKL, which regulate osteoclast formation.     

ErbB Receptor Negative Regulatory Mechanisms: Implications in Cancer

Activation of ErbB receptor tyrosine kinases (RTKs) must be precisely regulated to ensure the fidelity of developmental and homeostatic processes mediated by growth factors. Insufficient receptor stimulation will lead to defects in tissue development, while excessive stimulation can lead to hyperplastic events associated with cancer and other diseases. A coordinated balance of the intensity and timing of receptor signaling, achieved through both receptor activation and negative regulatory mechanisms, is required for signaling fidelity. While considerable effort has gone into understanding mechanisms by which ErbB receptors are activated, our understanding of the suppression of growth factor receptor activity remains limited. While ligand-stimulated receptor degradation is the most thoroughly examined mechanism for preventing hyper-signaling by ErbBs, recent studies indicate that several other mechanisms act directly on receptors to suppress receptor levels, or the magnitude or duration of receptor signaling. ErbB receptor overexpression or aberrant activation contributes to the progression of numerous solid tumor types. Hence, tumor cells must overcome these endogenous receptor negative regulatory mechanisms before they can exploit ErbB receptors to achieve uncontrolled growth. Here we will discuss several proteins that directly interact with ErbB receptors to suppress signaling, highlighting the potential impact of their loss on tumor progression.     

Menatetrenone (Vitamin K<Subscript>2</Subscript>) Acts Directly on Circulating Human Osteoclast Precursors

It is still not certain what the direct effect of menatetrenone is on osteoclast precursors. In the present study, we investigated whether menatetrenone has a direct effect on circulating osteoclast precursors to influence osteoclast differentiation. Monocytes isolated from human peripheral blood were cultured with receptor-activated NF-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Menatetrenone or vitamin K₁ was then added to the cultures. Geranylgeraniol or phytol (the respective side chain) was also added to the cultures instead of menatetrenone or vitamin K₁, respectively. After 7 and 14 days incubation, cultures were evaluated for cytochemical and functional evidence of osteoclast formation. The number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) and the percentage area of lacunar resorption induced by RANKL and M-CSF were decreased when menatetrenone or geranylgeraniol was added to the cultures. Dose-dependent inhibition of osteoclast formation and lacunar resorption was seen when the cultures were treated with menatetrenone or geranylgeraniol. In contrast, vitamin K₁ or phytol did not affect the number of TRAP-positive MNCs nor the percentage area of lacunar resorption. These results indicate that menatetrenone not only influences osteoclast formation via bone stromal cells but also acts directly on circulating osteoclast precursors to influence osteoclast differentiation. These findings also suggest that geranylgeraniol, the side chain of menatetrenone, plays an important role in this inhibitory effect.     

Osteoclast formation is related to bone matrix age

Summary Little is known about the relationship between the age of the skeleton and the development of multinucleated bone-resorbing cells, osteoclasts. It has been shown that mineralized bone implanted onto the chick chorioallantoic membrane (CAM) is effective in the recruitment and differentiation of osteoclast precursors. In studies reported here we used the CAM system to examine the influence of bone matrix age on osteoclast formation. Devitalized mineralized bone particles (75–250 μm) were prepared from rats of various ages (2, 4, 9, 12, and 16 months). The particles were implanted onto the chick chorioallantoic membrane and 8 days later implants were harvested and processed for morphometric or immunohistochemical analysis. Osteoclast number, cell area, nucleocytoplasmic ratio, and the presence of a distinctive osteoclast antigen, defined by the 121F monoclonal antibody, were determined. Bone particles of each age group resulted in the formation of osteoclast-like giant cells. Compared with multinucleated cells that formed in response to bone particles obtained from 2-month-old rats, matrix from the oldest age group (16 months) elicited significantly fewer and smaller cells which contained a smaller number of nuclei. These data suggest that with aging, bone undergoes qualitative and/or quantitative changes that affect the recruitment and differentiation of osteoclast precursor cells.     

Phosphoproteomic Analysis Reveals Regulatory Mechanisms at the Kidney Filtration Barrier

Diseases of the kidney filtration barrier are a leading cause of ESRD. Most disorders affect the podocytes, polarized cells with a limited capacity for self-renewal that require tightly controlled signaling to maintain their integrity, viability, and function. Here, we provide an atlas of in vivo phosphorylated, glomerulus-expressed proteins, including podocyte-specific gene products, identified in an unbiased tandem mass spectrometry–based approach. We discovered 2449 phosphorylated proteins corresponding to 4079 identified high-confidence phosphorylated residues and performed a systematic bioinformatics analysis of this dataset. We discovered 146 phosphorylation sites on proteins abundantly expressed in podocytes. The prohibitin homology domain of the slit diaphragm protein podocin contained one such site, threonine 234 (T234), located within a phosphorylation motif that is mutated in human genetic forms of proteinuria. The T234 site resides at the interface of podocin dimers. Free energy calculation through molecular dynamic simulations revealed a role for T234 in regulating podocin dimerization. We show that phosphorylation critically regulates formation of high molecular weight complexes and that this may represent a general principle for the assembly of proteins containing prohibitin homology domains.The kidney filter consists of three layers: fenestrated endothelial cells, the glomerular basement membrane, and podocytes.¹ Damage to any of these compartments becomes clinically evident as proteinuria and the development of kidney disease.² Of particular importance for the regulation of podocyte biology through signaling is the slit diaphragm, a specialized intercellular junction that bridges the 40-nm gap in between foot processes of neighboring podocytes. It also serves as a signaling platform regulating podocyte function. Mutations in genes encoding for components of the slit diaphragm, such as nephrin,³ podocin,⁴ CD2AP,⁵ and TRPC6,^6,7 are important causes of genetic forms of proteinuria. Alteration of these proteins results in defective signaling causing podocyte dysfunction, progressive glomerulosclerosis, and kidney failure. The slit diaphragm protein complex is a lipid-multiprotein supercomplex.⁸ Of central importance to the integrity and function of the protein complex is the prohibitin homology (PHB) domain protein podocin,⁹ which forms multimeric complexes and is required to control signal transduction through associated transmembrane proteins.^10,11Signaling processes governing podocyte function, integrity, and survival largely depend on signaling processes involving phosphorylation.^12,13 Comprehensive analyses of the signaling events in podocytes in vivo have been hampered by the fact that interference with these signaling cascades by genetic deletion often results in massively disrupted and dysfunctional podocytes. One of the primary aims of this study was to use phosphoproteomics to analyze thousands of phosphorylation sites in native murine glomeruli within single samples. Within this study, we show that this approach allows the introduction of new concepts into signaling processes at the kidney filtration barrier.     

Activation of mTORC1 in B Lymphocytes Promotes Osteoclast Formation via Regulation of β‐Catenin and RANKL/OPG

The cytokine receptor activator of nuclear factor‐κB ligand (RANKL) induces osteoclast formation from monocyte/macrophage lineage cells. However, the mechanisms by which RANKL expression is controlled in cells that support osteoclast differentiation are still unclear. We show that deletion of TSC1 (tuberous sclerosis complex 1) in murine B cells causes constitutive activation of mechanistic target of rapamycin complex 1 (mTORC1) and stimulates RANKL but represses osteoprotegerin (OPG) expression and subsequently promotes osteoclast formation and causes osteoporosis in mice. Furthermore, the regulation of RANKL/OPG and stimulation of osteoclastogenesis by mTORC1 was confirmed in a variety of RANKL‐expressing cells and in vivo. Mechanistically, mTORC1 controls RANKL/OPG expression through negative feedback inactivation of Akt, destabilization of β‐catenin mRNA, and downregulation of β‐catenin. Our findings demonstrate that mTORC1 activation‐stimulated RANKL expression in B cells is sufficient to induce bone loss and osteoporosis. The study also established a link between mTORC1 and the RANKL/OPG axis via negative regulation of β‐catenin. © 2016 American Society for Bone and Mineral Research.     

LOX Fails to Substitute for RANKL in Osteoclastogenesis

Osteoclasts are the exclusive bone‐resorbing cells that have a central role in bone homeostasis as well as bone destruction in cancer and autoimmune disease. Both mouse and human genetic studies have clearly proven that receptor activator of NF‐κB ligand (RANKL; encoded by the Tnfsf11 gene) and its receptor RANK are essential for osteoclastogenesis. Although there have been several reports on RANKL‐independent osteoclastogenesis, previous studies have never provided in vivo evidence showing RANKL can be substituted by other molecules using RANKL‐ or RANK‐deficient genetic backgrounds. Thus, to date, there is no clear evidence of RANKL‐independent osteoclastogenesis and no molecule has ever been proven capable of inducing osteoclast differentiation more efficiently than RANKL. Recently, lysyl oxidase (LOX), the enzyme that mediates collagen cross‐linking, has been shown to induce human osteoclasts in the absence of RANKL and has a stronger osteoclastogenic activity than RANKL. Here, we investigated the effect of LOX on osteoclast differentiation using RANKL‐ and RANK‐deficient cells to strictly explore RANKL‐independent osteoclastogenesis. CD14⁺ human peripheral blood cells as well as osteoclast precursor cells derived from wild‐type, RANKL‐ and RANK‐deficient mice were treated with RANKL and/or LOX in short‐term (3 days) or long‐term (3 weeks) experimental settings. LOX treatment alone did not result in the formation of tartrate‐resistant acid phosphatase (TRAP)⁺ cells or resorption pits in either short‐term or long‐term culture. In combination with RANKL, long‐term treatment with LOX synergistically promoted osteoclastogenesis in cells derived from wild‐type mice; however, this was abrogated in RANKL‐deficient cells. Long‐term treatment with LOX stimulated RANKL expression in mouse bone marrow stromal cells via the production of reactive oxygen species (ROS). Furthermore, LOX injection failed to rescue the phenotype of RANKL‐deficient mice. These results suggest that LOX has the ability to induce RANKL expression on stromal cells; however, it fails to substitute for RANKL in osteoclastogenesis. © 2016 American Society for Bone and Mineral Research.     

负载丹酚酸B的破骨前体细胞膜纳米颗粒对成骨细胞和破骨细胞分化的影响

目的 通过制备破骨前体细胞膜纳米颗粒（nanoparticles,NPs）负载丹酚酸B（salvianolic acid,SalB）,构建载药纳米颗粒SalB-NPs,观察其对破骨细胞及成骨细胞分化的影响。方法 采用超声裂解、挤膜的方法制备NPs,并将NPs与SalB共孵育后,使用200 nm聚碳酸酯膜挤出,获得SalB-NPs。在诱导小鼠原代破骨细胞分化和成骨前体细胞（MC3T3-E1）成骨分化的过程中,按照处理方式不同,分为对照组、SalB组、SalB-NPs组。采用透射电镜、纳米粒度及ZETA电位仪和Western Blot对材料进行表征,采用噻唑蓝检测试剂盒检测材料对RAW 264.7和MC3T3-E1细胞活力的影响,采用高效液相色谱法检测SalB的释放率和装载率。采用抗酒石酸酸性磷酸酶（TRAP）染色评价破骨细胞分化能力,通过碱性磷酸酶（ALP）染色和茜素红染色评估成骨分化能力,采用Real time-PCR检测破骨细胞分化及成骨分化相关基因表达水平。结果 制备的纳米颗粒直径在200 nm左右,同时表达RANK蛋白。TRAP染色显示SalB-NPs显著抑制破骨细胞的形成,下调破骨分化相关基因水平,与对照组和SalB组相比,差异有统计学意义（P＜0.05）。使用成骨诱导培养基诱导MC3T3-E1细胞成骨分化,14 d ALP染色和21 d茜素红染色均显示SalB-NPs组ALP活性和钙盐沉积量较SalB组明显增加,差异有统计学意义（P＜0.05）。结论 SalB-NPs体外发挥促进成骨分化、抑制破骨细胞形成双重功效,作为骨质疏松的治疗药物开发,有很好的应用前景,未来仍需在骨质疏松模型动物进一步验证其治疗效果。     

Alternative NF‐κB Regulates RANKL‐Induced Osteoclast Differentiation and Mitochondrial Biogenesis via Independent Mechanisms

Mitochondrial biogenesis, the generation of new mitochondrial DNA and proteins, has been linked to osteoclast (OC) differentiation and function. In this study we used mice with mutations in key alternative NF‐κB pathway proteins, RelB and NF‐κB–inducing kinase (NIK), to dissect the complex relationship between mitochondrial biogenesis and osteoclastogenesis. In OC precursors lacking either NIK or RelB, receptor activator of NF‐κB ligand (RANKL) was unable to increase mitochondrial DNA or oxidative phosphorylation (OxPhos) protein expression, which was associated with lower oxygen consumption rates. Transgenic OC precursors expressing constitutively active NIK showed normal RANKL‐induced mitochondrial biogenesis (OxPhos expression and mitochondria copy number) compared to controls, but larger mitochondrial dimensions and increased oxygen consumption rates, suggesting increased mitochondrial function. To deduce the mechanism for mitochondrial biogenesis defects in NIK‐deficient and RelB‐deficient precursors, we examined expression of genes known to control this process. PGC‐1β (Ppargc1b) expression, but not PGC‐1α, PPRC1, or ERRα, was significantly reduced in RelB^–/– and NIK^–/– OCs. Because PGC‐1β has been reported to positively regulate both mitochondrial biogenesis and differentiation in OCs, we retrovirally overexpressed PGC‐1β in RelB^–/– cells, but surprisingly found that it did not affect differentiation, nor did it restore RANKL‐induced mitochondrial biogenesis. To determine whether the blockade in osteoclastogenesis in RelB‐deficient cells precludes mitochondrial biogenesis, we rescued RelB^–/– differentiation via overexpression of NFATc1. Mitochondrial parameters in neither WT nor RelB‐deficient cultures were affected by NFATc1 overexpression, and bone resorption in RelB^–/– was not restored. Furthermore, NFATc1 co‐overexpression with PGC‐1β, although allowing OC differentiation, did not rescue mitochondrial biogenesis or bone resorption in RelB^–/– OCs, by CTX‐I levels. Thus, our results indicate that the alternative NF‐κB pathway plays dual, but distinct, roles in controlling the independent processes of OC differentiation and OC mitochondrial biogenesis. Furthermore, the inability of PGC‐1β to drive mitochondrial biogenesis in OCs without RelB indicates a cell‐type specificity in mitochondria regulation. © 2015 American Society for Bone and Mineral Research.     

Presentation of abdominal tuberculosis to general surgeons.

Abdominal tuberculosis (TB) continues to give rise to diagnostic and therapeutic challenges. A total of 24 patients with abdominal TB who presented to general surgeons over a 9-year period have been reviewed. Most (92 per cent) of these patients were Asian; only one had a past history of pulmonary TB. The most common presenting complaint was abdominal pain in 21 patients (88 per cent) with the associated symptoms of weight loss in 18 (75 per cent), anorexia in 15 (62 per cent) and night sweats in 13 (54 per cent). A tissue diagnosis was obtained in 18 patients (75 per cent) and 17 patients (71 per cent) underwent laparotomy. These results show that the diagnosis of abdominal TB is still difficult to establish, and that many patients undergo laparotomy despite the existence of less invasive diagnostic procedures.