Several cytokines, steroids, hormones and prostaglandins are involved in regulating the differentiation, proliferation, activation and survival of osteoclasts (2-4). These include macrophage colony-stimulating factor (M-CSF), receptor activator of nuclear factor- B ligand (RANKL) and tumour necrosis factor- (TNF - ) (5-8). Osteoclast precursor cells express cell surface receptors which are specific for these factors. In-vitro studies have demonstrated that M-CSF binds to the M-CSF (c-fms) receptor and induces the expression of genes in the osteoclast lineage leading to the differentiation and development of mature osteoclasts and cell survival (3, 9). TNF- is an inflammatory cytokine that binds to the TNF receptors-1 and -2, stimulates osteoclastogenesis and regulates cell apoptosis (10). In addition osteoclast precursor cell s express 2 integrins such as CD11b/Cd18 ) adhesion molecules that are necessary for cell trafficking and differentiation (11). It been demonstrated in CD11b- deficient mice where there was a decrease in bone mass and increase in osteoclast number (12).
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OPG has been used as a neutralizing agent to achieve pharmacologic blockade of RANKL in various models. Destruction of mineralized tissue was effectively blocked by this form of RANKL inhibition in models including adjuvant- induced arthritis, TNF-mediated arthritis, and collagen- induced arthritis [33-36]. In contrast, OPG did not ameliorate the inflammatory signs of disease, and histologic examination of inflamed joints from these models revealed full-blown development of synovial inflammation, suggesting that the interaction between RANKL and RANK is not critically involved in joint inflammation. Despite the presence of inflammation, however, structural damage did not occur to a significant degree in these models, indicating that RANKL- mediated osteoclast formation is a key player in structural damage in arthritis. Treatment with OPG completely blocked osteoclast formation in inflammatory tissue and prevented the formation of osteoclast precursor cells (Figure 4). This arrest of osteoclast differentiation elicited by OPG, together with the effect on function of mature osteoclasts, is crucial to appreciating the effective bone-sparing properties of RANKL blockade. Given the abundance of monocytes as potential osteoclast precursors in the inflammatory tissue and the large number of cells that actually differentiate from this precursor pool to the osteoclast lineage, this dual inhibitory effect on both differentiation and activation of osteoclasts can be considered an important feature of RANKL blockade. This may provide an explanation as to why relatively high amounts of bisphosphonates are necessary to block inflammatory bone erosions in animal models of arthritis . Also, the uptake and deposition of bisphosphonates at cortical bone sites affected by arthritis might be insufficient to allow complete protection of bone.
important role in regulating cell motility by deacetylat- ing a-tubulin and cortactin [51, 52]. HDAC6 also mod- ulates Hsp90-dependent activation of the glucocorticoid receptor through deacetylating Hsp90 [53, 54]. Loss of HDAC6 increased the formation of os- sified bone in TDII embryos, but it did not lead to sig- nificantly different bone mineral densities at later ages [55, 56]. Specific inhibition of HDAC6 with Tubastatin or AC1215 increased osteoblast formation and osteo- clast inhibition . Taking into account previous pub- lished results, we have shown that HDAC6 knockdown promotes dental MSCs differentiation by using shRNA. By employing a small interfering RNA technique, we also knocked down the expression of HDAC6 in mur- ine osteoclast precursor cells line RAW 264.7. The knockdown of HDAC6 in RAW 264.7 cells inhibited osteoclasts formation under RANKL stimulation. How- ever, it is still not clear whether HDAC6 could regulate osteoclast precursor proliferation. Although further studies are required to understand the detailed molecu- lar mechanisms by which HDAC6 regulates the expres- sion of osteoclast marker genes, our results shed light on the role of HDAC6 in dental MSCs and osteoclasts differentiation. Together with previous findings, our study suggests that HDAC6 is a potential regulator for bone growth and maintenance.
Excessive osteoclastic bone resorption plays a central role in the pathogenesis of age-related bone loss and microstructural deterio- ration, leading to fragility fractures (1). Mutinucleated osteoclasts are generated from hematopoietic precursor cells through the action of M-CSF and receptor activator of NF-κB ligand (RANKL) (2–4). These cytokines are produced by osteoclastogenesis-sup- porting marrow stromal cells and act on osteoclast precursor cells that express their receptors, c-fms and receptor activator of NF-κB (RANK), respectively. These cell-surface receptors transmit osteo- clastogenic signals through intracellular kinase cascades that culminate in the activation of transcription factors c-Fos/AP-1 and NF-κB in the nucleus. Accordingly, mice deficient in c-Fos, NF-κB, RANK, RANKL, or M-CSF cannot generate osteoclasts and exhibit osteopetrosis (2–4).
We have previously reported that depletion of LIS1, a key regulator of microtubules and cytoplasmic dynein motor complex, in osteoclast precursor cells by shRNAs attenuates osteoclastogenesis in vitro. However, the underlying mechanisms remain unclear. In this study, we show that conditional deletion of LIS1 in osteoclast progenitors in mice led to increased bone mass and decreased osteoclast number on trabecular bone. In vitro mechanistic studies revealed that loss of LIS1 had little effects on cell cycle progression but accelerated apoptosis of osteoclast precursor cells. Furthermore, deletion of LIS1 prevented prolonged activation of ERK by M-CSF and aberrantly enhanced prolonged JNK activation stimulated by RANKL. Finally, lack of LIS1 abrogated M-CSF and RANKL induced CDC42 activation and retroviral transduction of a constitutively active form of CDC42 partially rescued osteoclastogenesis in LIS1-deficient macrophages. Therefore, these data identify a key role of LIS1 in regulation of cell survival of osteoclast progenitors by modulating M-CSF and RANKL induced signaling pathways and CDC42 activation.
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Therefore, we examined the mRNA expression levels of SIRT1, FOXO3a and Bim on RANKL-induced osteoc- last differentiation after EGCG treatment using RT-PCR. Upregulated FOXO3a, Bim, and reduced SIRT1 indi- cated that the apoptosis of RAW264 cells might be en- hanced by EGCG treatment. EGCG-induced apoptosis through downstream activation of caspase is well cha- racterized in various cancer cells. However, it is reported thatcatechin, a component of green tea, causes caspase- independent necrosis-like cell death in chronic myelo- genous leukemia . Whether caspase activation was involved in the death of murine osteoclasts by EGCG remains to be clarified. Western blotting results showed increased Caspase-3 activation. Furthermore, the apopto- sis-inducing effect of EGCG on osteoclast precursor cells was confirmed by Hoechst staining, while the MTT assay revealed that up to 50 µM, EGCG treatment had no ef- fect on cell viability, which might to be explained as the result of increased mitochondrial activity in RAW264 cells due to treatment with EGCG. These results suggest that the pro-apoptotic function of Caspase-3-Bim might play an important role in EGCG-induced apoptosis in RAW264 cells.
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Regulation of osteoclast differentiation is an aspect central to the understanding of the pathogenesis and the treatment of bone diseases such as autoimmune arthritis and osteoporosis. In fact, excessive signaling by RANKL (receptor activator of nuclear factor κ B ligand), a member of the tumor necrosis factor (TNF) family essential for osteoclastogenesis, may contribute to such pathological conditions. Here we summarize our current work on the negative regulation of osteoclastogenesis by unique signaling crosstalk between RANKL and interferons (IFNs). First, activated T cells maintain bone homeostasis by counterbalancing the action of RANKL through production of IFN- γ . This cytokine induces rapid degradation of the RANK (receptor activator of nuclear factor κ B) adapter protein TRAF6 (TNF-receptor-associated factor 6), resulting in strong inhibition of the RANKL-induced activation of NF-κB and JNK (c-Jun N-terminal kinase). Second, RANKL induces the IFN-β gene but not IFN- α genes, in osteoclast precursor cells, and that IFN- β strongly inhibits the osteoclast differentiation by interfering with the RANKL-induced expression of c-Fos. The series of in vivo experiments revealed that these two distinct IFN-mediated regulatory mechanisms are both important to maintain homeostasis of bone resorption. Collectively, these studies revealed novel aspects of the two types of IFN, beyond their original roles in the immune response, and may offer a molecular basis for the treatment of bone diseases.
In 1889, Paget  postulated that cancer cells (seeds) metastasize towards a favorable microenvironment (soil), and recent studies have provided a molecular explanation of his theory. Bone-homing tumor cells overexpress chemokine receptors, such as C–X–C motif chemokine- receptor-4 (CXCR-4), whose ligand C–X–C motif chemokine-ligand-12 (CXCL-12) is secreted by stromal cells, including BMSC. Other chemo- kine axes, namely CXCR-6/CXCL-16 and CXCR-3/CXCL-10, are in- volved in this process [18,19], while the calcium sensing receptor is implicated in BC cell migration towards calcium-rich sites . From these metastatic niches, cancer cells may spread to other organs; in the meantime, they enter a state of dormancy, promoted by BMPs and growth-arrest specific-6 (GAS6) protein, secreted by mesenchymal cells [21,22]. This quiescent state, together with the acquisition of osteoblast and/or osteoclast markers (the so-called “osteomimicry”) permit tumor cell escape from anti-cancer drugs and immune response .
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On entering the peritoneum, we found a large amount of bloody ascites. Immediately after the delivery of the baby, uncontrolled haemorrhage was encountered posterior to the uterus from what appeared to be a solid mass in the retroperitoneum. The mass was large, haemorrhagic and necrotic. It invaded the uterus, right ovary and appendix. The right ureter was also involved by the tumour, resulting in severe hydronephrosis. In order to stop the haemorrhage, an en-block resection of the tumour together with total hysterectomy, right salpingo-oopho- rectomy, appendectomy and partial right ureterectomy was performed. Estimated blood loss was 12,860 g, and the patient received a massive transfusion of 24 units of packed red blood cells, 20 units of fresh frozen plasma and 20 units of platelet concentrate. The postoperative course of the patient was uneventful and the patient was discharged on the ninth postoperative day.
To evaluate the cytotoxicity induced by silica particle treatment of the mouse macrophage cell line RAW264.7 cells, a WST-8 cell proliferation assay was carried out. The results showed that 30 μ g/ml of nSP300 and mSP1000 treatment for 5 days did not induce cytotoxi- city in RAW264.7 cells. In contrast, nSP70 treatment induced higher cytotoxicity (about 40% viability of non- treated cells) at 30 μ g/ml, although 10 μ g/ml of nSP70 treatment produced only marginal cytotoxicity (Figure 1). These results indicated that decreasing the silica par- ticle size to below 100 nm increased the cytotoxicity sig- nificantly. We have confirmed that the number of silica particles ingested by cells increases as the particle size decreases, and only nSP70 invaded into the nuclei of dendritic cells which, along with macrophages, have a phagocytic capacity . We also found that only nSP70 invaded into the nucleus, in other words the intracellu- lar localization of nSP70 differed from that of nSP300 and mSP1000 . From these results, it was suspected that differences in the number of ingested silica particles and/or in their intracellular localization were significant factors in their observed cytotoxicity in RAW264.7 cells. Results of this experiment confirmed that not all silica particles induced cytotoxicity at 10 μg/ml in RAW264.7 cells, and therefore subsequent studies were carried out at 10 μg/ml silica particle treatment.
Although Paget's disease is the most flagrant example of a primary osteoclast disorder, little is known of osteoclast biology in this disease. In this report we have studied the formation of cells with the osteoclast phenotype in long-term cultures of marrow mononuclear cells derived from patients with Paget's disease, and compared these with similar cells formed in long-term marrow cultures from normal individuals, and with osteoclasts present in pagetic bone. Osteoclasts formed in pagetic marrow cultures resembled osteoclasts present in pagetic bone, but were distinctly different from osteoclasts formed in normal marrow cultures. Osteoclast formation was 10-20-fold greater in pagetic marrow cultures than in normal cultures. The multinucleated cells formed in cultures of pagetic marrow were much larger in size, were hyperresponsive to 1,25(OH)2 vitamin D, had more nuclei per cell, had increased levels of tartrate-resistant acid phosphatase activity and had ultrastructural features which were not seen in multinucleated cells formed from normal marrow mononuclear cells. These pagetic marrow-derived multinucleated cells formed large resorption lacunae on calcified matrices and cross-reacted with monoclonal antibodies which preferentially bind to osteoclasts. The multinucleated cells formed from marrow obtained from uninvolved sites in Paget's patients also displayed these abnormal features.
vitronectin, osteopontin, or fibronectin but not to laminin or collagen; ( b ) coimmunoprecipitation of PYK2 and c-Src from OCLs; ( c ) PYK2 binding to the SH2 domains of Src; ( d ) marked reduction in tyrosine phosphorylation and ki- nase activity of PYK2 in OCLs derived from Src ( 2 / 2 ) mice, which do not form actin rings and do not resorb bone; ( e ) PYK2 phosphorylation by exogeneous c-Src; ( f ) translo- cation of PYK2 to the Triton X-100 insoluble cytoskeletal fraction upon adhesion; ( g ) localization of PYK2 in podo- somes and the ring-like structures in OCLs plated on glass and in the sealing zone in OCLs plated on bone; and ( h ) ac- tivation of PYK2, in the presence of MB1.8 cells, parallels the formation of sealing zones and pit resorption in vitro and is reduced by echistatin or calcitonin and cytochalasin D. Taken together, these findings suggest that Src-depen- dent tyrosine phosphorylation of PYK2 is involved in the adhesion-induced formation of the sealing zone, required for osteoclastic bone resorption. ( J. Clin. Invest. 1998. 102: 881–892.) Key words: tyrosine kinase • podosome • clear zone • bone resorption • vitronectin receptor
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In addition to chemokines, various cytokines have also been reported to be expressed following TBI, including TNF-α associated with activated microglia and astro- cytes that may initiate the inflammatory process . IL- 6 in the injured brain has been associated with reactive astrogliosis, neuronal injury, and infiltration of periph- eral cells [78-81]. TGF-β expression in the astrocytes and microglia after injury has been implicated in the pathology and dysfunction of the CNS and IL-1, IL-6, IL-8, IL-10, granulocyte colony-stimulating factor, TNF- α, FAS ligand and monocyte chemo-attractant protein 1 [18,82-84] are thought to account for the progressive in- jury. In a rat fluid percussion injury model a biphasic production of TGF-β, mainly of TGF-β 2, was detected in the ipsilateral cortex, with a first peak at 30 minutes and a second peak at 48 hours after the lesion. This re- sponse was accompanied by transient production of TNF-α and IL-6 occurring between five and eighteen hours after trauma. From this temporal pattern, Rimaniol et al. suggested an alternative pro- and anti- inflammatory role of TGF-β in the regulation of the brain cytokine network providing an endogenous mech- anism for the control of the inflammatory reaction in traumatic brain injury .
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In the final part of the study, the authors tried to explore the effects of OC-STAMP and P2X7 receptors during the LPA-stimulated osteoclast fusion, using siRNA to silence OC- STAMP and P2X7 receptors in a different model of cell fusion using RAW 264.7 cells. Although OC-STAMP and P2X7 receptors were only partly knocked down according to the RT-PCR, western blotting and quantitative real-time RT-PCR results, the siRNA transfected RAW 264.7 cells were shown, qualitatively, to have relatively narrower diameters and reduced pit-forming ability after 5 days differentiation under LPA treatment and RANKL- limiting conditions, compared to control cells. Fifteen days after differentiated under the same conditions, the function markers of osteoclast resorption matrix metalloproteinase 9 (MMP-9) and cathepsin K were also found to be reduced in culture medium of OC-STAMP and P2X7 silenced cells. Furthermore, quantitative real-time RT-PCR results demonstrated that following OC-STAMP and P2X7 receptor knockdown, NF-ATc1, cathepsin K, c-Jun, c-Fos, meltrin-a, and c-Src mRNA levels decreased during LPA-stimulated osteoclast fusion. The authors suggest that these data show that OC-STAMP and P2X7 receptors are involved in not only cell-cell fusion but also mature osteoclast bone resorption activities in RANKL-limiting, LPA induced osteoclast fusion. In addition, the authors also showed that NF-ATc1 nuclear translocation was inhibited by cyclosporine (CsA) treatment as was OC-STAMP and P2X7 receptor mRNA suggesting that NF-ATc1 directly regulates OC-STAMP and P2X7 receptors in this process.
Sample of whole blood was layered over Ficoll-Hypaque density gradient and direct immunostaining for Peripheral blood mononuclear cells (PBMC) was performed. Mononuclear cells were stained with monoclonal antihuman RANK- PE (9A725, Thermofisher, scientific) and caspase-3 NucView 488 antibodies. Cells were incubated for 30 minutes at room temperature, followed by washing using phosphate buffer saline (PBS) with 2% fetal bovine serum (FBS). The cells were finally acquired using flow cytometer (BD FACS CANTO- II, Becton Dickinson, CA, USA) and data were analyzed using FACSDiva software. Single color tubes were used for compensation. Cells were first gated on the basis of forward and side scatter (P1), followed by gating of RANK positive cells (P2). caspase-3 activity was assessed in these RANK positive precursor osteoclast cells. The frequency of caspase-3 expressing precursor osteoclasts and median fluorescence intensity (MFI) of caspase-3 were compared among subject groups. The OI patient’s demography, biochemistry, radiology & ZOL dose are summarized in Table 1.
Anchoring of heterologous Tat substrates in the inner membrane has previously occurred via the addition of extra amino acid residues to the protein sequence (Karlsson et al., 2012). However, this study has shown that only a single substitution mutation in the signal peptidase cleavage site of a TorA signal peptide, fused to hGH, resulted in the precursor protein being “ stalled ” at the inner membrane, with no alterations to the mature protein. The previous studies by (Karlsson et al., 2012) and (Ren et al., 2013) showed that cleavage by leader peptidase occurs only after transfer of the mature protein to the periplasm, and given that hGH is exported by Tat in an extremely efficient manner, it is to be expected that the mature hGH protein will be similarly positioned on the periplasmic face of the inner membrane. This could be confirmed using a structural biology approach whereby E. coli spheroblasts (overexpressing TorA ‐ hGH or TorA ‐ A39L ‐ hGH) are immunogold labeled specifically for hGH and subsequently visualized by SEM. This would allow visualization of the periplasmic side of the inner membrane with the hGH ‐ bound immunogold, and confirm the topology of the mature hGH as periplasmically exposed.
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Despite the high cellularity in the case presented here, there was no evidence of epithelioid or dysplastic fea- tures, or growth pattern suggestive of malignancy. The Ki-67 immunostaining disclosed a relatively low to mo- derate cell proliferation index and the tumor cells did not show any immunoreactivity for the p53 protein. Oliveira et al. investigated the cell proliferation marker PCNA, as well as the p53 immunohistochemical expression and p53 gene mutations in classical, atypical osteoblastomas in comparison to osteosarcomas . Atypical osteoblas- tomas, osteosarcomas and tumor recurrence were statis- tically correlated with a high PCNA labelling index and p53 immunoexpression .
RANKL/RANK/NF kB pathway is the classical pathway regulates osteoclast differentiation, activation and apoptosis. After RANKL bound to the RANK receptor in the osteoclast surface, the Motif (including Motif 1, 2, 3) in the cyto- plasmic end of RANK receptor combined with TRAF-6, which can activate NF kB, Akt/PKB and JNK, ERK, and p383 mitogen activated protein kinase (MAPK) signal pathway. Under the stimulation of RANKL, TGF-βactivated kinase 1 (TAK1) can be combined with TRAF-6 and activated with the help of its connexin 2 and 3 (TAB2, TAB3) . The activated TAK1 further kindled the NF-kB and JNK signaling pathway. The NF kB is the key transcription reg- ulatory factor and involved in the expression regulation of many specific genes in osteo- blasts, and it’s crucial to the differentiation, cell fusion, adhesion, acidification and bone absorption. The NF κB-knockout mice present- ed as bone sclerosis; its geneticly defective embryonic stem cell was unable to differentiate into mature osteoclasts [17, 18].
antibody (E11). Western blot analysis using a monoclonal antibody to pp60c-src (327) revealed that protooncogene c-src expression by the platelets of the CMD patient was comparable to the normal control. These data suggest that: (a) the hyperostosis and the metaphyseal long bone deformity in the present CMD patient might be explained by osteoclast dysfunction due to impaired expression […]
Mesenchymal precursor cells found in the blood (BMPCs) of normal persons adhere to plastic and glass and proliferate logarithmically in DMEM–20% fetal calf serum (FCS) without growth factors. They form cells with fibroblast-like and stromal morphology, which is not affected by eliminating CD34, CD3, or CD14 cells. Osteogenic supplements (dexamethasone, ascorbic acid, and β -glycerophosphate) added to the culture inhibited fibroblast formation, and BMPCs assumed the cuboidal shape of osteoblasts. After 5 days in supplemented medium, the elutriated cells displayed alkaline phosphatase (AP), and the addition of bone morphogenetic protein (BMP)2 (1 ng) doubled AP production (P< 0.04). Two weeks later, 30% of the cells were very large and reacted with anti-osteocalcin antibody. The same cultures also contained sudanophlic adipocytes and multinucleated giant cells that stained for tartrate-resistant acid phosphatase (TRAP) and vitronectin receptors. Cultured BMPCs immunostain with antibodies to vimentin, type I collagen, and BMP receptors, heterodimeric structures expressed on mesenchymal lineage cells. In addition, BMPCs stain with anti-CD105 (endoglin), a putative marker for bone-marrow mesenchymal stem cells (MSCs).
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