Nucleic acid recognition by Toll-like receptors is coupled to stepwise processing by cathepsins and asparagine endopeptidase

TLR3, TLR7, and TLR9 are cleaved in the same step-wise manner in all immune cell types examined

Directed differentiation of hematopoietic precursors and functional osteoclasts from human ES and iPS cells

This article has been made available through the Brunel Open Access Publishing Fund and is available from the specified link - 2010 by The American Society of HematologyThe directed differentiation of human pluripotent stem cells offers the unique opportunity to generate a broad spectrum of human cell types and tissues for transplantation, drug discovery, and studying disease mechanisms. Here, we report the stepwise generation of bone-resorbing osteoclasts from human embryonic and induced pluripotent stem cells. Generation of a primitive streak-like population in embryoid bodies, followed by specification to hematopoiesis and myelopoiesis by vascular endothelial growth factor and hematopoietic cytokines in serum-free media, yielded a precursor population enriched for cells expressing the monocyte-macrophage lineage markers CD14, CD18, CD11b, and CD115. When plated in monolayer culture in the presence of macrophage colony-stimulating factor and receptor activator of nuclear factor-kappa B ligand (RANKL), these precursors formed large, multinucleated osteoclasts that expressed tartrate-resistant acid phosphatase and were capable of resorption. No tartrate-resistant acid phosphatase-positive multinucleated cells or resorption pits were observed in the absence of RANKL. Molecular analyses confirmed the expression of the osteoclast marker genes NFATc1, cathepsin K, and calcitonin receptor in a RANKL-dependent manner, and confocal microscopy demonstrated the coexpression of the alpha v beta 3 integrin, cathepsin K and F-actin rings characteristic of active osteoclasts. Generating hematopoietic and osteoclast populations from human embryonic and induced pluripotent stem cells will be invaluable for understanding embryonic bone development and postnatal bone disease. (Blood. 2010; 115(14): 2769-2776)This study was supported (in part) by research funding from the Nuffield Foundation-Oliver Bird Rheumatism Program to A.E.G., Arthritis Research Campaign (ARC ref 18197) to G.S., National Institutes of Health grants R01 HL080627 and P20 GM075019 to G.M.K., and Austrian Science Fund (FWF) and Anabonos EU-FP7 to E.F.W

Transgenic Mice for a Tamoxifen-Induced, Conditional Expression of the Cre Recombinase in Osteoclasts

Background: Studies on osteoclasts, the bone resorbing cells, have remained limited due to the lack of transgenic mice allowing the conditional knockout of genes in osteoclasts at any time during development or adulthood. Methodology/Principal Finding: We report here on the generation of transgenic mice which specifically express a tamoxifen-inducible Cre recombinase in osteoclasts. These mice, generated on C57BL/6 and FVB background, express a fusion Cre recombinase-ERT2 protein whose expression is driven by the promoter of cathepsin K (CtsK), a gene highly expressed in osteoclasts. We tested the cellular specificity of Cre activity in CtsKCreERT2 strains by breeding with Rosa26LacZ reporter mice. PCR and histological analyses of the CtsKCreERT2LacZ positive adult mice and E17.5 embryos show that Cre activity is restricted largely to bone tissue. In vitro, primary osteoclasts derived from the bone marrow of CtsKCreERT2+/2LacZ+/2 adult mice show a Cre-dependent b-galactosidase activity after tamoxifen stimulation

Nicotinic acetylcholine receptors modulate osteoclastogenesis

Background: Our aim was to investigate the role of nicotinic acetylcholine receptors (nAChRs) in in-vitro osteoclastogenesis and in in-vivo bone homeostasis. Methods: The presence of nAChR subunits as well as the in-vitro effects of nAChR agonists were investigated by ex vivo osteoclastogenesis assays, real-time polymerase chain reaction, Western blot and flow cytometry in murine bone marrow-derived macrophages differentiated in the presence of recombinant receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). The bone phenotype of mice lacking various nAChR subunits was investigated by peripheral quantitative computed tomography and histomorphometric analysis. Oscillations in the intracellular calcium concentration were detected by measuring the Fura-2 fluorescence intensity. Results: We could demonstrate the presence of several nAChR subunits in bone marrow-derived macrophages stimulated with RANKL and M-CSF, and showed that they are capable of producing acetylcholine. nAChR ligands reduced the number of osteoclasts as well as the number of tartrate-resistant acidic phosphatase-positive mononuclear cells in a dose-dependent manner. In vitro RANKL-mediated osteoclastogenesis was reduced in mice lacking α7 homomeric nAChR or β2-containing heteromeric nAChRs, while bone histomorphometry revealed increased bone volume as well as impaired osteoclastogenesis in male mice lacking the α7 nAChR. nAChR ligands inhibited RANKL-induced calcium oscillation, a well-established phenomenon of osteoclastogenesis. This inhibitory effect on Ca2+ oscillation subsequently led to the inhibition of RANKL-induced NFATc1 and c-fos expression after long-term treatment with nicotine. Conclusions: We have shown that the activity of nAChRs conveys a marked effect on osteoclastogenesis in mice. Agonists of these receptors inhibited calcium oscillations in osteoclasts and blocked the RANKL-induced activation of c-fos and NFATc1. RANKL-mediated in-vitro osteoclastogenesis was reduced in α7 knockout mice, which was paralleled by increased tibial bone volume in male mice in vivo. © 2016 Mandl et al

TRAF-6 Dependent Signaling Pathway Is Essential for TNF-Related Apoptosis-Inducing Ligand (TRAIL) Induces Osteoclast Differentiation

Human osteoclast formation from mononuclear phagocyte precursors involves interactions between tumor necrosis factor (TNF) ligand superfamily members and their receptors. Recent evidence indicates that in addition to triggering apoptosis, the TNF-related apoptosis-inducing ligand (TRAIL) induces osteoclast differentiation. To understand TRAIL-mediated signal transduction mechanism in osteoclastogenesis, we demonstrated that TRAIL induces osteoclast differentiation via a Tumor necrosis factor receptor-associated factor 6 (TRAF-6)-dependent signaling pathway. TRAIL-induced osteoclast differentiation was significantly inhibited by treatment with TRAF-6 siRNA and TRAF6 decoy peptides in both human monocytes and murine RAW264.7 macrophage cell lines, as evaluated in terms of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and bone resorption activity. Moreover, TRAIL-induced osteoclast differentiation was also abolished in TRAF6 knockout bone marrow macrophages. In addition to induction of NFATc1, treatment of TRAIL also induced ubiquitination of TRAF6 in osteoclast differentiation. Thus, our data demonstrate that TRAIL induces osteoclastic differentiation via a TRAF-6 dependent signaling pathway. This study suggests TRAF6-dependent signaling may be a central pathway in osteoclast differentiation, and that TNF superfamily molecules other than RANKL may modify RANK signaling by interaction with TRAF6-associated signaling

Bone turnover markers for early detection of fracture healing disturbances: A review of the scientific literature

Imaging techniques are the standard method for assessment of fracture healing processes. However, these methods are perhaps not entirely reliable for early detection of complications, the most frequent of these being delayed union and non-union. A prompt diagnosis of such disorders could prevent prolonged patient distress and disability. Efforts should be directed towards the development of new technologies for improving accuracy in diagnosing complications following bone fractures. The variation in the levels of bone turnover markers (BTMs) have been assessed with regard to there ability to predict impaired fracture healing at an early stage, nevertheless the conclusions of some studies are not consensual. In this article the authors have revised the potential of BTMs as early predictors of prognosis in adult patients presenting traumatic bone fractures but who did not suffer from osteopenia or postmenopausal osteoporosis. The available information from the different studies performed in this field was systematized in order to highlight the most promising BTMs for the assessment of fracture healing outcome.As técnicas imagiológicas são o método convencional para a avaliação dos processos de cicatrização das fraturas. No entanto, estes métodos não são talvez totalmente confiáveis para a deteção precoce de complicações, as mais frequentes destas sendo o atraso da união e a não-união. Um diagnóstico eficaz destas desordens poderia prevenir a dor e a incapacidade prolongada do paciente. Esforços devem ser dirigidos no sentido do desenvolvimento de novas tecnologias para melhorar a exatidão no diagnóstico de complicações após fraturas ósseas. A variação nos níveis dos marcadores do turnover ósseo (BTMs) têm sido avaliados com vista à sua capacidade para prever o comprometimento da cicatrização das fraturas numa fase inicial, no entanto, as conclusões de alguns estudos não são consensuais. Neste artigo os autores fizeram uma revisão do potencial dos BTMs como fatores de previsibilidade precoce do prognóstico em doentes adultos que apresentavam fraturas ósseas traumáticas mas que não sofriam de osteopenia ou osteoporose pós-menopausa. A informação disponível nos diferentes estudos realizados neste campo foi sistematizada com vista a evidenciar-se os BTMs mais promissores para a avaliação da evolução da cicatrização das fraturas.SFRH/BD/45018/200

Cathepsin L Inhibition Prevents Murine Autoimmune Diabetes via Suppression of CD8+ T Cell Activity

Background: Type 1 diabetes (T1D) is an autoimmune disease resulting from defects in central and peripheral tolerance and characterized by T cell-mediated destruction of islet b cells. To determine whether specific lysosomal proteases might influence the outcome of a T cell–mediated autoimmune response, we examined the functional significance of cathepsin inhibition on autoimmune T1D-prone non-obese diabetic (NOD) mice. Methods and Findings: Here it was found that specific inhibition of cathepsin L affords strong protection from cyclophosphamide (CY)-induced insulitis and diabetes of NOD mice at the advanced stage of CD8 + T cell infiltration via inhibiting granzyme activity. It was discovered that cathepsin L inhibition prevents cytotoxic activity of CD8 + T cells in the pancreatic islets through controlling dipeptidyl peptidase I activity. Moreover, the gene targeting for cathepsin L with application of in vivo siRNA administration successfully prevented CY-induced diabetes of NOD mice. Finally, cathepsin L mRNA expression of peripheral CD8 + T cells from NOD mice developing spontaneous T1D was significantly increased compared with that from control mice. Conclusions: Our results identified a novel function of cathepsin L as an enzyme whose activity is essential for the progression of CD8 + T cell-mediated autoimmune diabetes, and inhibition of cathepsin L as a powerful therapeutic strateg

Dasatinib as a Bone-Modifying Agent: Anabolic and Anti-Resorptive Effects

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al.[Background]: Bone loss, in malignant or non-malignant diseases, is caused by increased osteoclast resorption and/or reduced osteoblast bone formation, and is commonly associated with skeletal complications. Thus, there is a need to identify new agents capable of influencing bone remodeling. We aimed to further pre-clinically evaluate the effects of dasatinib (BMS-354825), a multitargeted tyrosine kinase inhibitor, on osteoblast and osteoclast differentiation and function. [Methods]: For studies on osteoblasts, primary human bone marrow mensenchymal stem cells (hMSCs) together with the hMSC-TERT and the MG-63 cell lines were employed. Osteoclasts were generated from peripheral blood mononuclear cells (PBMC) of healthy volunteers. Skeletally-immature CD1 mice were used in the in vivo model. [Results]: Dasatinib inhibited the platelet derived growth factor receptor-β (PDGFR-β), c-Src and c-Kit phosphorylation in hMSC-TERT and MG-63 cell lines, which was associated with decreased cell proliferation and activation of canonical Wnt signaling. Treatment of MSCs from healthy donors, but also from multiple myeloma patients with low doses of dasatinib (2-5 nM), promoted its osteogenic differentiation and matrix mineralization. The bone anabolic effect of dasatinib was also observed in vivo by targeting endogenous osteoprogenitors, as assessed by elevated serum levels of bone formation markers, and increased trabecular microarchitecture and number of osteoblast-like cells. By in vitro exposure of hemopoietic progenitors to a similar range of dasatinib concentrations (1-2 nM), novel biological sequelae relative to inhibition of osteoclast formation and resorptive function were identified, including F-actin ring disruption, reduced levels of c-Fos and of nuclear factor of activated T cells 1 (NFATc1) in the nucleus, together with lowered cathepsin K, αVβ3 integrin and CCR1 expression. [Conclusions]: Low dasatinib concentrations show convergent bone anabolic and reduced bone resorption effects, which suggests its potential use for the treatment of bone diseases such as osteoporosis, osteolytic bone metastasis and myeloma bone disease. © 2012 Garcia-Gomez et al.This work was supported by grants from the Spanish Ministry of Science and Innovation – ISCIII (PI081825); Mutua Madrileña Medical Research Foundation (AP27262008); Centro en Red of Regenerative Medicine and Cellular Therapy from Castilla y León, Consejería de Sanidad JCyL – ISCIII; the Cooperative Research Thematic Network in Cancer (RTICC; RD06/0020/0006 and RD03/0020/0041); and Spanish FIS (PS09/01897). AG-G and CS are supported by the Centro en Red of Regenerative Medicine and Cellular Therapy from Castilla y León Project.Peer Reviewe

Prevention of Wear Particle-Induced Osteolysis by a Novel V-ATPase Inhibitor Saliphenylhalamide through Inhibition of Osteoclast Bone Resorption

Wear particle-induced peri-implant loosening (Aseptic prosthetic loosening) is one of the most common causes of total joint arthroplasty. It is well established that extensive bone destruction (osteolysis) by osteoclasts is responsible for wear particle-induced peri-implant loosening. Thus, inhibition of osteoclastic bone resorption should prevent wear particle induced osteolysis and may serve as a potential therapeutic avenue for prosthetic loosening. Here, we demonstrate for the first time that saliphenylhalamide, a new V-ATPase inhibitor attenuates wear particle-induced osteolysis in a mouse calvarial model. In vitro biochemical and morphological assays revealed that the inhibition of osteolysis is partially attributed to a disruption in osteoclast acidification and polarization, both a prerequisite for osteoclast bone resorption. Interestingly, the V-ATPase inhibitor also impaired osteoclast differentiation via the inhibition of RANKL-induced NF-κB and ERK signaling pathways. In conclusion, we showed that saliphenylhalamide affected multiple physiological processes including osteoclast differentiation, acidification and polarization, leading to inhibition of osteoclast bone resorption in vitro and wear particle-induced osteolysis in vivo. The results of the study provide proof that the new generation V-ATPase inhibitors, such as saliphenylhalamide, are potential anti-resorptive agents for treatment of peri-implant osteolysis