Effects of Alendronate and Dexamethasone on Osteoclast Gene Expression and Bone Resorption in Mouse Marrow Cultures

2021 ◽  
pp. 002215542110635
Author(s):  
Lorraine Perciliano de Faria ◽  
Giuliana Sueyoshi ◽  
Taís Carvalho de Oliveira ◽  
L. Shannon Holliday ◽  
Victor E. Arana-Chavez
Keyword(s):  
Gene Expression ◽  
Bone Matrix ◽  
Giant Cells ◽  
Inhibitory Effect ◽  
Tartrate Resistant Acid Phosphatase ◽  
Main Function ◽  
Dihydroxyvitamin D3 ◽  
Primary Mouse ◽  
Receptor Activator ◽  
Marrow Cultures

Osteoclasts are cells whose main function is the resorption of bone matrix. However, several factors, including medications, can interfere with the resorption process. Alendronate (ALN), a nitrogen-containing type of bisphosphonate, and dexamethasone (DEX), a glucocorticoid, are drugs that may affect the resorption activity. The aim of this study is to investigate the effects of ALN, and/or DEX on osteoclast gene expression and resorption activity in primary mouse marrow cultures stimulated with 1,25-dihydroxyvitamin D3, a model for the bone microenvironment. Cultures were treated only with ALN (10−5 M), DEX (10−6 M), and with a combination of both agents. Viability assays performed at days 5, 7, and 9 showed the highest number of viable cells at day 7. All the following assays were then performed at day 7 of cell culture: tartrate resistant acid phosphatase (TRAP) histochemistry, receptor activator of nuclear factor kappa B ligand (RANKL) immunofluorescence, osteoprotegerin (OPG), and RANKL gene expression by qPCR and resorption analysis by scanning electron microscopy. Treatment with ALN, DEX, and the combination of both did not promote significant changes in the number of TRAP+ cells, although larger giant cells were detected in groups treated with DEX. DEX treatment increased the gene expression of RANKL and reduced OPG. The treatment with ALN reduced the depth of the resorption pits, but their inhibitory effect was less effective when administered with DEX:

scholarly journals RANKL-induced DC-STAMP Is Essential for Osteoclastogenesis

2004 ◽  
Vol 200 (7) ◽  
pp. 941-946 ◽  
Author(s):  
Toshio Kukita ◽  
Naohisa Wada ◽  
Akiko Kukita ◽  
Takashi Kakimoto ◽  
Ferry Sandra ◽  
...  
Keyword(s):  
Transmembrane Protein ◽  
Nuclear Factor Κb ◽  
Giant Cells ◽  
Precursor Cells ◽  
Tartrate Resistant Acid Phosphatase ◽  
Small Interfering Rnas ◽  
Surface Molecules ◽  
Receptor Activator ◽  
Targeted Inhibition ◽  
First Time

Osteoclasts are bone-resorbing, multinucleated giant cells that are essential for bone remodeling and are formed through cell fusion of mononuclear precursor cells. Although receptor activator of nuclear factor–κB ligand (RANKL) has been demonstrated to be an important osteoclastogenic cytokine, the cell surface molecules involved in osteoclastogenesis are mostly unknown. Here, we report that the seven-transmembrane receptor-like molecule, dendritic cell–specific transmembrane protein (DC-STAMP) is involved in osteoclastogenesis. Expression of DC-STAMP is rapidly induced in osteoclast precursor cells by RANKL and other osteoclastogenic stimulations. Targeted inhibition of DC-STAMP by small interfering RNAs and specific antibody markedly suppressed the formation of multinucleated osteoclast-like cells. Overexpression of DC-STAMP enhanced osteoclastogenesis in the presence of RANKL. Furthermore, DC-STAMP directly induced the expression of the osteoclast marker tartrate-resistant acid phosphatase. These data demonstrate for the first time that DC-STAMP has an essential role in osteoclastogenesis.

scholarly journals Influence of Cu2+ on Osteoclast Formation and Activity In Vitro

2021 ◽  
Vol 22 (5) ◽  
pp. 2451
Author(s):  
Anne Bernhardt ◽  
Jana Bacova ◽  
Uwe Gbureck ◽  
Michael Gelinsky
Keyword(s):  
Gene Expression ◽  
Enzyme Activities ◽  
Bone Matrix ◽  
Antimicrobial Properties ◽  
Osteoclast Formation ◽  
Tartrate Resistant Acid Phosphatase ◽  
Specific Enzyme ◽  
Cytotoxic Effects ◽  
Resorptive Activity

Background: Copper-containing biomaterials are increasingly applied for bone regeneration due to their pro-angiogenetic, pro-osteogenetic and antimicrobial properties. Therefore, the effect of Cu2+ on osteoclasts, which play a major role in bone remodeling was studied in detail. Methods: Human primary osteoclasts, differentiated from human monocytes were differentiated or cultivated in the presence of Cu2+. Osteoclast formation and activity were analyzed by measurement of osteoclast-specific enzyme activities, gene expression analysis and resorption assays. Furthermore, the glutathione levels of the cells were checked to evaluate oxidative stress induced by Cu2+. Results: Up to 8 µM Cu2+ did not induce cytotoxic effects. Activity of tartrate-resistant acid phosphatase (TRAP) was significantly increased, while other osteoclast specific enzyme activities were not affected. However, gene expression of TRAP was not upregulated. Resorptive activity of osteoclasts towards dentin was not changed in the presence of 8 µM Cu2+ but decreased in the presence of extracellular bone matrix. When Cu2+ was added to mature osteoclasts TRAP activity was not increased and resorption decreased only moderately. The glutathione level of both differentiating and mature osteoclasts was significantly decreased in the presence of Cu2+. Conclusions: Differentiating and mature osteoclasts react differently to Cu2+. High TRAP activities are not necessarily related to high resorption.

scholarly journals Calcitonin inhibits osteoclast formation in mouse haematopoetic cells independently of transcriptional regulation by receptor activator of NF-κB and c-Fms

2007 ◽  
Vol 195 (3) ◽  
pp. 415-427 ◽  
Author(s):  
Susanne Granholm ◽  
Pernilla Lundberg ◽  
Ulf H Lerner
Keyword(s):  
Transmembrane Protein ◽  
Osteoclast Differentiation ◽  
Inhibitory Effect ◽  
Osteoclast Formation ◽  
Tartrate Resistant Acid Phosphatase ◽  
Mouse Bone Marrow ◽  
Osteoclast Progenitor ◽  
Receptor Activator ◽  
Macrophage Colony ◽  
A Disintegrin And Metalloproteinase

The effects of calcitonin (CT) on osteoclast formation and gene expression have been studied in cultured mouse spleen cells and mouse bone marrow macrophages (BMMs). CT inhibited the formation of multinucleated osteoclasts and resorption pits in spleen cell cultures and BMM as well as in CD115+ CD3− CD45R−sorted BMM cultures, incubated in the presence of macrophage colony-stimulating factor and receptor activator of NF-κB ligand (RANKL). No effect on apoptosis by CT was observed. CT did not affect the mRNA expressions of RANK and c-Fms, or the mRNA expressions of a wide variety of transcription factors and genes important for osteoclast differentiation and activity. CT induced inhibition of tartrate-resistant acid phosphatase (TRAP), positive multinucleated osteoclast formation was not associated with any decrease of total TRAP activity, resulting in a large number of TRAP+ mononucleated cells in CT-treated cultures. CT did not affect the mRNA expression of dendritic cell-specific transmembrane protein, d2 isoform of vacuolar (H+) ATPase vo domain, a disintegrin and metalloproteinase domain 8 (ADAM8), ADAM12, DNAX-activating protein or Fc receptor common γ chain suggested to be involved in fusion of mononucleated osteoclast progenitor cells. The inhibitory effect by CT was mimicked not only by compounds activating cAMP and protein kinase A (PKA) but also by a cAMP analogue activating the exchange protein directly activated by cAMP (Epac) pathway. It is concluded that CT, through cAMP/PKA/Epac cascades, inhibits osteoclast formation and that this effect is not associated with decreased transcription of genes known to be important for osteoclast progenitor cell differentiation, fusion or function.

PERIARTICULAR GENE EXPRESSION OF OSTEOPONTIN AND PROGRESSION IN OSTEOPENIA FOLLOWING JOINT IMMOBILIZATION

2001 ◽  
Vol 05 (04) ◽  
pp. 225-234
Author(s):  
TAKAHIDE MIYAMA ◽  
NORIMASA NAKAMURA ◽  
TAKANOBU NAKASE ◽  
YUKIYOSHI TORITSUKA ◽  
AKIRA MYOUI ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Matrix ◽  
Knee Joints ◽  
Tartrate Resistant Acid Phosphatase ◽  
Positive Staining ◽  
Multinucleated Cells ◽  
Molecular Events ◽  
Bone Matrix Proteins ◽  
Temporal And Spatial

In order to elucidate the molecular events involved in periarticular osteopenia following joint immobilization, we investigated the temporal and spatial gene expression of one of the major non-collagenous bone matrix proteins, osteopontin (OPN), in periarticular regions, after immobilizing rat knee joints. Significant decreases in total BMD was observed in the immobilized limbs after 2 weeks of immobilization. Histologically, multinucleated cells with positive staining for tartrate resistant acid phosphatase (TRAP) were significantly increased in the metaphyseal regions, 1 to 4 weeks following immobilization. In situ hybridization studies revealed that OPN mRNA was present in a significantly larger number of cells in the metaphysial regions of joints immobilized for 1 to 3 weeks; compared with those levels detected in the control limbs, OPN mRNA was mainly detected in osteoblast/osteocyte-lineage cells of the spongiosa. Such temporal and spatial similarities in the expression of TRAP-positive multinucleated cells and OPN transcripts suggest the possible involvement of OPN in the periarticular osteoclastogenesis, leading to the activation of regional bone resorption following joint immobilization.

scholarly journals ATP downregulates P2X7 and inhibits osteoclast formation in RAW cells

2004 ◽  
Vol 287 (2) ◽  
pp. C403-C412 ◽  
Author(s):  
Jeffrey F. Hiken ◽  
Thomas H. Steinberg
Keyword(s):  
Prolonged Exposure ◽  
Nuclear Factor Κb ◽  
Giant Cells ◽  
Osteoclast Formation ◽  
Marker Genes ◽  
Tartrate Resistant Acid Phosphatase ◽  
Marker Enzyme ◽  
Multinucleated Giant Cells ◽  
Membrane Pores ◽  
Receptor Activator

Multinucleated giant cells derive from fusion of precursor cells of the macrophage lineage. It has been proposed that the purinoreceptor P2X7 is involved in this fusion process. Prolonged exposure of macrophages to ATP, the ligand for P2X7, induces the formation of plasma membrane pores and eventual cell death. We took advantage of this cytolytic property to select RAW 264.7 (RAW) cells that lacked P2X7 function by maintaining them in ATP (RAW ATP-R cells). RAW ATP-R cells failed to fuse to form multinucleated osteoclasts in response to receptor activator nuclear factor-κB ligand, although they did become positive for the osteoclast marker enzyme tartrate-resistant acid phosphatase, and upregulated expression of other osteoclast marker genes. RAW ATP-R cells and wild-type RAW cells expressed similar amounts of P2X7 protein, but little P2X7 was present on the surface of RAW ATP-R cells. After ATP was removed from the medium of RAW ATP-R cells, the cells reexpressed P2X7 on the cell surface, regained sensitivity to ATP, and formed multinucleated osteoclasts. These results suggest that P2X7 or another protein that is downregulated in concert with P2X7 is involved either in the mechanics of cell fusion to form osteoclasts or in a signaling pathway proximal to this event. These results also suggest that P2X7 may be regulated by ligand-mediated internalization and that extracellular ATP may regulate the formation of osteoclasts and other multinucleated giant cells.

Reparação óssea no implante dental

2020 ◽  
Vol 12 (45) ◽  
pp. 63-66
Author(s):  
Halim Nagem Filho ◽  
Reinaldo Francisco Maia ◽  
Reinaldo Missaka ◽  
Nasser Hussein Fares
Keyword(s):  
Gene Expression ◽  
Titanium Surface ◽  
Close Contact ◽  
The Other ◽  
Main Function ◽  
Indirect Interaction ◽  
M2 Macrophages ◽  
Activated Macrophages ◽  
M1 Macrophages ◽  
High Production

The osseointegration is the stable and functional union between the bone and a titanium surface. A new bone can be found on the surface of the implant about 1 week after its installation; the bone remodeling begins between 6 and 12 weeks and continues throughout life. After the implant insertion, depending on the energy of the surface, the plasma fluid immediately adheres, in close contact with the surface, promoting the adsorption of proteins and inducing the indirect interaction of the cells with the material. Macrophages are cells found in the tissues and originated from bone marrow monocytes. The M1 macrophages orchestrate the phagocytic phase in the inflammatory region and also produce inflammatory cytokines involved with the chronic inflammation and the cleaning of the wound and damaged tissues from bacteria. On the other hand, alternative-activated macrophages (M2) are activated by IL-10, the immune complex. Its main function consists on regulating negatively the inflammation through the secretion of the immunosuppressant IL-10. The M2 macrophages present involvement with the immunosuppression, besides having a low capacity for presenting antigens and high production of cytokines; these can be further divided into M2a, M2b, and M2c, based on the gene expression profile.

Efficacy and safety of denosumab treatment in a prepubertal patient with cherubism

2020 ◽  
Vol 33 (7) ◽  
pp. 963-966
Author(s):  
Haruka Kawamura ◽  
Satoshi Watanabe ◽  
Takashi I ◽  
Izumi Asahina ◽  
Hiroyuki Moriuchi ◽  
...  
Keyword(s):  
Autosomal Dominant ◽  
Therapeutic Potential ◽  
Giant Cells ◽  
Efficacy And Safety ◽  
Autosomal Dominant Disorder ◽  
Osteolytic Lesions ◽  
Case Presentation ◽  
Childhood Growth ◽  
Receptor Activator ◽  
Denosumab Treatment

AbstractBackgroundDenosumab is an inhibitor of receptor activator of nuclear factor kappa-B ligand, which strongly suppresses osteoclasts. Cherubism is a rare autosomal dominant disorder characterized by symmetrical swelling of the jaws, in which the bone is replaced by a fibrous granuloma containing osteoclast-like giant cells.Case presentationWe report the efficacy and safety of denosumab treatment in a prepubertal boy with progressive cherubism. The treatment consisting of eight subcutaneous denosumab injections (120 mg/dose) in 6 months not only suppressed the expansion of the osteolytic lesions but also dramatically ossified them. However, a transiently decreased growth rate and rebounded asymptomatic hypercalcemia were associated with the treatment.ConclusionsThe present case demonstrated the therapeutic potential of denosumab for treatment of cherubism, although adverse effects, especially those on childhood growth, remain obscure. Further studies are needed to establish a safe and effective protocol for denosumab treatment of children.

scholarly journals ‘Educated’ Osteoblasts Reduce Osteoclastogenesis in a Bone-Tumor Mimetic Microenvironment

Cancers ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 263
Author(s):  
Alexus D. Kolb ◽  
Jinlu Dai ◽  
Evan T. Keller ◽  
Karen M. Bussard
Keyword(s):  
Bone Resorption ◽  
Bone Tumor ◽  
Bone Matrix ◽  
Necrosis Factor Alpha ◽  
Tumor Bearing ◽  
Receptor Activator ◽  
Factor Alpha ◽  
Osteoclast Resorption ◽  
Necrosis Factor

Breast cancer (BC) metastases to bone disrupt the balance between osteoblasts and osteoclasts, leading to excessive bone resorption. We identified a novel subpopulation of osteoblasts with tumor-inhibitory properties, called educated osteoblasts (EOs). Here we sought to examine the effect of EOs on osteoclastogenesis during tumor progression. We hypothesized that EOs affect osteoclast development in the bone-tumor niche, leading to suppressed pre-osteoclast fusion and bone resorption. Conditioned media (CM) was analyzed for protein expression of osteoclast factors receptor activator of nuclear factor kappa-β ligand (RANKL), osteoprotegerin (OPG), and tumor necrosis factor alpha (TNFα) via ELISA. EOs were co-cultured with pre-osteoclasts on a bone mimetic matrix to assess osteoclast resorption. Pre-osteoclasts were tri-cultured with EOs plus metastatic BC cells and assessed for tartrate-resistance acid phosphatase (TRAP)-positive, multinucleated (≥3 nuclei), mature osteoclasts. Tumor-bearing murine tibias were stained for TRAP to determine osteoclast number in-vivo. EO CM expressed reduced amounts of soluble TNFα and OPG compared to naïve osteoblast CM. Osteoclasts formed in the presence of EOs were smaller and less in number. Upon co-culture on a mimetic bone matrix, a 50% reduction in the number of TRAP-positive osteoclasts formed in the presence of EOs was observed. The tibia of mice inoculated with BC cells had less osteoclasts per bone surface in bones with increased numbers of EO cells. These data suggest EOs reduce osteoclastogenesis and bone resorption. The data imply EOs provide a protective effect against bone resorption in bone metastatic BC.

scholarly journals Sex-dependent dynamics of metabolism in primary mouse hepatocytes

2021 ◽  
Author(s):  
Luise Hochmuth ◽  
Christiane Körner ◽  
Fritzi Ott ◽  
Daniela Volke ◽  
Kaja Blagotinšek Cokan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Amino Acid ◽  
Sexual Dimorphism ◽  
New Drugs ◽  
Model Systems ◽  
Specific Gene ◽  
Primary Hepatocytes ◽  
Alcoholic Fatty Liver ◽  
Male And Female ◽  
Primary Mouse

AbstractThe liver is one of the most sexually dimorphic organs. The hepatic metabolic pathways that are subject to sexual dimorphism include xenobiotic, amino acid and lipid metabolism. Non-alcoholic fatty liver disease and hepatocellular carcinoma are among diseases with sex-dependent prevalence, progression and outcome. Although male and female livers differ in their abilities to metabolize foreign compounds, including drugs, sex-dependent treatment and pharmacological dynamics are rarely applied in all relevant cases. Therefore, it is important to consider hepatic sexual dimorphism when developing new treatment strategies and to understand the underlying mechanisms in model systems. We isolated primary hepatocytes from male and female C57BL6/N mice and examined the sex-dependent transcriptome, proteome and extracellular metabolome parameters in the course of culturing them for 96 h. The sex-specific gene expression of the general xenobiotic pathway altered and the female-specific expression of Cyp2b13 and Cyp2b9 was significantly reduced during culture. Sex-dependent differences of several signaling pathways increased, including genes related to serotonin and melatonin degradation. Furthermore, the ratios of male and female gene expression were inversed for other pathways, such as amino acid degradation, beta-oxidation, androgen signaling and hepatic steatosis. Because the primary hepatocytes were cultivated without the influence of known regulators of sexual dimorphism, these results suggest currently unknown modulatory mechanisms of sexual dimorphism in vitro. The large sex-dependent differences in the regulation and dynamics of drug metabolism observed during cultivation can have an immense influence on the evaluation of pharmacodynamic processes when conducting initial preclinical trials to investigate potential new drugs.

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