Ly49Q, an ITIM-bearing NK receptor, positively regulates osteoclast differentiation

International Nuclear Information System (INIS)

Hayashi, Mikihito; Nakashima, Tomoki; Kodama, Tatsuhiko; Makrigiannis, Andrew P.; Toyama-Sorimachi, Noriko; Takayanagi, Hiroshi

2010-01-01

Osteoclasts, multinucleated cells that resorb bone, play a key role in bone remodeling. Although immunoreceptor tyrosine-based activation motif (ITAM)-mediated signaling is critical for osteoclast differentiation, the significance of immunoreceptor tyrosine-based inhibitory motif (ITIM) has not been well understood. Here we report the function of Ly49Q, an Ly49 family member possessing an ITIM motif, in osteoclastogenesis. Ly49Q is selectively induced by receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) stimulation in bone marrow-derived monocyte/macrophage precursor cells (BMMs) among the Ly49 family of NK receptors. The knockdown of Ly49Q resulted in a significant reduction in the RANKL-induced formation of tartrate-resistance acid phosphatase (TRAP)-positive multinucleated cells, accompanied by a decreased expression of osteoclast-specific genes such as Nfatc1, Tm7sf4, Oscar, Ctsk, and Acp5. Osteoclastogenesis was also significantly impaired in Ly49Q-deficient cells in vitro. The inhibitory effect of Ly49Q-deficiency may be explained by the finding that Ly49Q competed for the association of Src-homology domain-2 phosphatase-1 (SHP-1) with paired immunoglobulin-like receptor-B (PIR-B), an ITIM-bearing receptor which negatively regulates osteoclast differentiation. Unexpectedly, Ly49Q deficiency did not lead to impaired osteoclast formation in vivo, suggesting the existence of a compensatory mechanism. This study provides an example in which an ITIM-bearing receptor functions as a positive regulator of osteoclast differentiation.

Siglec-15, a member of the sialic acid-binding lectin, is a novel regulator for osteoclast differentiation

International Nuclear Information System (INIS)

Hiruma, Yoshiharu; Hirai, Takehiro; Tsuda, Eisuke

2011-01-01

Highlights: → Siglec-15 was identified as a gene overexpressed in giant cell tumor. → Siglec-15 mRNA expression increased in association with osteoclast differentiation. → Polyclonal antibody to Siglec-15 inhibited osteoclast differentiation in vitro. -- Abstract: Osteoclasts are tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells derived from monocyte/macrophage-lineage precursors and are critically responsible for bone resorption. In giant cell tumor of bone (GCT), numerous TRAP-positive multinucleated giant cells emerge and severe osteolytic bone destruction occurs, implying that the emerged giant cells are biologically similar to osteoclasts. To identify novel genes involved in osteoclastogenesis, we searched genes whose expression pattern was significantly different in GCT from normal and other bone tumor tissues. By screening a human gene expression database, we identified sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) as one of the genes markedly overexpressed in GCT. The mRNA expression level of Siglec-15 increased in association with osteoclast differentiation in cultures of mouse primary unfractionated bone marrow cells (UBMC), RAW264.7 cells of the mouse macrophage cell line and human osteoclast precursors (OCP). Treatment with polyclonal antibody to mouse Siglec-15 markedly inhibited osteoclast differentiation in primary mouse bone marrow monocyte/macrophage (BMM) cells stimulated with receptor activator of nuclear factor κB ligand (RANKL) or tumor necrosis factor (TNF)-α. The antibody also inhibited osteoclast differentiation in cultures of mouse UBMC and RAW264.7 cells stimulated with active vitamin D 3 and RANKL, respectively. Finally, treatment with polyclonal antibody to human Siglec-15 inhibited RANKL-induced TRAP-positive multinuclear cell formation in a human OCP culture. These results suggest that Siglec-15 plays an important role in osteoclast differentiation.

Siglec-15, a member of the sialic acid-binding lectin, is a novel regulator for osteoclast differentiation

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Hiruma, Yoshiharu, E-mail: hiruma.yoshiharu.hy@daiichisankyo.co.jp [Biological Research Laboratories, Daiichi Sankyo Co. Ltd., Tokyo 134-8630 (Japan); Hirai, Takehiro [Translational Medicine and Clinical Pharmacology Department, Daiichi Sankyo Co. Ltd., Tokyo 134-8630 (Japan); Tsuda, Eisuke [Biological Research Laboratories, Daiichi Sankyo Co. Ltd., Tokyo 134-8630 (Japan)

2011-06-10

Highlights: {yields} Siglec-15 was identified as a gene overexpressed in giant cell tumor. {yields} Siglec-15 mRNA expression increased in association with osteoclast differentiation. {yields} Polyclonal antibody to Siglec-15 inhibited osteoclast differentiation in vitro. -- Abstract: Osteoclasts are tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells derived from monocyte/macrophage-lineage precursors and are critically responsible for bone resorption. In giant cell tumor of bone (GCT), numerous TRAP-positive multinucleated giant cells emerge and severe osteolytic bone destruction occurs, implying that the emerged giant cells are biologically similar to osteoclasts. To identify novel genes involved in osteoclastogenesis, we searched genes whose expression pattern was significantly different in GCT from normal and other bone tumor tissues. By screening a human gene expression database, we identified sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) as one of the genes markedly overexpressed in GCT. The mRNA expression level of Siglec-15 increased in association with osteoclast differentiation in cultures of mouse primary unfractionated bone marrow cells (UBMC), RAW264.7 cells of the mouse macrophage cell line and human osteoclast precursors (OCP). Treatment with polyclonal antibody to mouse Siglec-15 markedly inhibited osteoclast differentiation in primary mouse bone marrow monocyte/macrophage (BMM) cells stimulated with receptor activator of nuclear factor {kappa}B ligand (RANKL) or tumor necrosis factor (TNF)-{alpha}. The antibody also inhibited osteoclast differentiation in cultures of mouse UBMC and RAW264.7 cells stimulated with active vitamin D{sub 3} and RANKL, respectively. Finally, treatment with polyclonal antibody to human Siglec-15 inhibited RANKL-induced TRAP-positive multinuclear cell formation in a human OCP culture. These results suggest that Siglec-15 plays an important role in osteoclast differentiation.

Osteoclasts derive from hematopoietic stem cells according to marker, giant lysosomes of beige mice

International Nuclear Information System (INIS)

Ash, P.; Loutit, J.F.; Townsend, K.M.

1981-01-01

To ascertain the origin of multinucleated osteoclasts from hematopoietic stem cells, giant lysosomes peculiar to cells of beige mice (bg bg) were used as marker cells of that provenance. Radiation chimeras were established reciprocally between bg bg mice and osteopetrotic mi mi mice with defective osteoclasts. As a result, all the derivative cells of the hematopoietic stem cell would depend on the donor's cell line, whereas osteogenesis would remain the province of the host. It was affirmed in the chimeras mi mi/bg bg that the osteopetrosis was cured within six weeks. Thereafter the definitive osteoclasts of the chimeras contained giant lysosomes attributable to the beige cell line. However, the cure was well advanced before donor osteoclasts were prominent, for which several reasons are offered. In the mouse chimeras, bg bg/mi mi, there was a delay of some six weeks before osteopetrosis became evident, histologically before radiologically, at the major metaphyseal growth centers. During the period one to two months after establishment, osteoclasts appeared to be a mixture of two cell lines according to quantitative assessments for giant lysosomes. Assessments consisted of measurements of the percentage area of osteoclasts occupied by lysosomes over 1 micrometer diameter. The means were 0.018% +/- 0.008% for nonbeige stock and 2.09% +/- 0.58% for beige stock

Production and Functional Characterization of Murine Osteoclasts Differentiated from ER-Hoxb8-Immortalized Myeloid Progenitor Cells.

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Frank Zach

Full Text Available In vitro differentiation into functional osteoclasts is routinely achieved by incubation of embryonic stem cells, induced pluripotent stem cells, or primary as well as cryopreserved spleen and bone marrow-derived cells with soluble receptor activator of nuclear factor kappa-B ligand and macrophage colony-stimulating factor. Additionally, osteoclasts can be derived from co-cultures with osteoblasts or by direct administration of soluble receptor activator of nuclear factor kappa-B ligand to RAW 264.7 macrophage lineage cells. However, despite their benefits for osteoclast-associated research, these different methods have several drawbacks with respect to differentiation yields, time and animal consumption, storage life of progenitor cells or the limited potential for genetic manipulation of osteoclast precursors. In the present study, we therefore established a novel protocol for the differentiation of osteoclasts from murine ER-Hoxb8-immortalized myeloid stem cells. We isolated and immortalized bone marrow cells from wild type and genetically manipulated mouse lines, optimized protocols for osteoclast differentiation and compared these cells to osteoclasts derived from conventional sources. In vitro generated ER-Hoxb8 osteoclasts displayed typical osteoclast characteristics such as multi-nucleation, tartrate-resistant acid phosphatase staining of supernatants and cells, F-actin ring formation and bone resorption activity. Furthermore, the osteoclast differentiation time course was traced on a gene expression level. Increased expression of osteoclast-specific genes and decreased expression of stem cell marker genes during differentiation of osteoclasts from ER-Hoxb8-immortalized myeloid progenitor cells were detected by gene array and confirmed by semi-quantitative and quantitative RT-PCR approaches. In summary, we established a novel method for the quantitative production of murine bona fide osteoclasts from ER-Hoxb8 stem cells generated from

[Observation of osteoclasts on the root surface during human deciduous teeth resorption].

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Bao, Xiang-jun; Liang, Xing; Chen, Ming; Wang, Hang; Xie, Zhi-gang; Yang, Xiao-yu

2004-08-01

To observe osteoclasts on the resorbing surface of human deciduous teeth. After fixing the collected deciduous teeth, we prepared the tooth slices without decalcification, treated them with HE and TRAP dyestuff, and observed the osteoclasts under light and scanning electron microscope. There were large quantity of various forms of overlapping and huge osteoclasts with many nuclei and silk-like protuberances on the resorbing surface of deciduous teeth. The multinucleated osteoclasts align on the surface of coarse dentin. On the resorbing surface of human deciduous teeth there are large amount of osteoclasts which can be used as a source of studying human osteoclast.

Microarray Profile of Gene Expression during Osteoclast Differentiation in Modeled Microgravity

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National Aeronautics and Space Administration — Microgravity leads to a 10-15% loss of bone mass in astronauts during space flight. Osteoclast is the multinucleated bone resorbing cell. In this study we used NASA...

Effects of cadmium on osteoclast formation and activity in vitro

International Nuclear Information System (INIS)

Wilson, A.K.; Cerny, E.A.; Smith, B.D.; Wagh, A.

1996-01-01

Chronic exposure to cadmium has been linked to bone loss, low bone mass, and increased incidence of fracture. To determine if Cd could directly increase the formation of cells responsible for bone resorption, we cultured normal canine bone marrow cells containing the progenitor cells for osteoclasts. Cultures were evaluated for the number of multinucleate osteoclast-like cells (MNOCs) formed. Exposure to Cd (10-100 nM) increased the number of MNOCs formed over control values when cultured in the presence but not in the absence of a bone wafer. The MNOCs formed were functional, evidenced by pits excavated on the bone wafers included in the cultures. By 12 days, MNOCs formed in the presence of 50 nM Cd excavated significantly more pits and a greater pit area than did untreated MNOCs. By 14 days, the control values were similar to those of the Cd-exposed MNOCs, but pit formation was enhanced by Cd in that the ratio of pit complexes to single pits was increased twofold over that for untreated cultures. Mature osteoclasts, isolated from the long bones of rat neonates and cultured for 1-3 days on bone slices, provided a direct method to assess the effect of Cd on osteoclast activity. Exposure of osteoclast cultures to 100 nm Cd increased the number of nM osteoclasts present over that for untreated osteoclasts by a factor of 1.7 ± 0.1, the number of pits excavated by 2.8 ± 0.6, the area excavated by 3.2 ± 0.8, and the area excavated per osteoclast by 1.8 ± 0.4 (mean SE; n = six experiments). These data suggest that Cd accelerates the differentiation of new osteoclasts from their progenitor cells and activates or increases the activity of mature osteoclasts. 48 refs., 4 figs., 3 tabs

Regulation of human umbilical cord blood-derived multi-potent stem cells by autogenic osteoclast-based niche-like structure

International Nuclear Information System (INIS)

Sun, Bo; Jeong, Yun-Hyeok; Jung, Ji-Won; Seo, Kwangwon; Lee, Yong-Soon; Kang, Kyung-Sun

2007-01-01

Stem cell niches provide the micro-environment for the development of stem cells. Under our culturing regimen, a kind of osteoclast-centralized structure supports the proliferation of MSCs, derived from human cord blood, once they reside on osteoclasts. MSCs in this structure expressed Oct4 which is a marker of embryonic stem cells. Floating daughter cells of MSCs colony showed abilities to differentiate into osteocyte, adipocyte, and neuronal progenitor cells. Compared with the easy senescence of MSCs without this niche-like structure in vitro, these results suggested that osteoclasts might play an important role the development and maintenance of Umbilical cord blood (UCB)-derived MSCs and might provide a means to expand UCB-MSCs in vitro, more easily, through a stem cell niche-like structure

Low-magnitude high-frequency vibration inhibits RANKL-induced osteoclast differentiation of RAW264.7 cells.

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Wu, Song-Hui; Zhong, Zhao-Ming; Chen, Jian-Ting

2012-01-01

Osteoclasts are the key participants in regulation of bone mass. Low-magnitude high-frequency vibration (LMHFV) has been found to be anabolic to bone in vivo. This study aimed to investigate the effect of LMHFV on osteoclast differentiation in vitro. Murine monocyte cell line RAW264.7 cells in the presence of receptor activator of nuclear factor-kappaB ligand (RANKL) were treated with or without LMHFV at 45 Hz (0.3 g) for 15 min day(-1). Tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) and actin ring formation were evaluated. Expression of the osteoclast-specific genes, such as cathepsin K, matrix metallopeptidase-9 (MMP-9) and TRAP, were analyzed using real time-PCR. c-Fos, an osteoclast-specific transcription factor, was determined using Western blot. We found that LMHFV significantly decreased the number of RANKL-induced TRAP-positive MNCs (P<0.01), and inhibited the actin ring formation. The mRNA expression of the cathepsin K, MMP-9 and TRAP were down-regulated by LMHFV intervention (all P<0.001). Furthermore, LMHFV also inhibited the expression of c-Fos protein in the RANKL-treated RAW264.7 cells (P<0.05). Our results suggest that LMHFV can inhibit the RANKL-induced osteoclast differentiation of RAW264.7 cells, which give some new insight into the anabolic effects of LMHFV on bone.

Diclofenac sodium inhibits NFkappaB transcription in osteoclasts.

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Karakawa, A; Fukawa, Y; Okazaki, M; Takahashi, K; Sano, T; Amano, H; Yamamoto, M; Yamada, S

2009-11-01

A non-steroidal anti-inflammatory drug, diclofenac, acts efficiently against inflammation; however, down-regulation of diclofenac on bone remodeling has raised concerns. The inhibitory mechanisms of diclofenac are poorly understood. We hypothesized that diclofenac down-regulates osteoclast differentiation and activation via inhibition of the translocation of phosphorylated nuclear factor kappa B (NFkappaB). When osteoclasts prepared from mouse hematopoietic stem cells were treated with diclofenac, tartrateresistant acid phosphatase-positive multinucleated cells decreased in a concentration-dependent manner. Pit formation assay revealed the abolition of osteoclastic bone resorption; levels of cathepsin K transcripts, an osteoclastic resorption marker, were down-regulated time-dependently. Diclofenac induced the accumulation of the inhibitor of kappa B in cytosol, which led to suppression of the nuclear translocation of NFkappaB and phosphorylated NFkappaB. These results suggest that the novel mechanism of diclofenac for bone remodeling is associated with phosphorylated NFkappaB reduction, which regulates osteoclast differentiation and activation.

Distinctive and selective route of PI3K/PKCα-PKCδ/RhoA-Rac1 signaling in osteoclastic cell migration.

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Kim, Jin-Man; Kim, Mi Yeong; Lee, Kyunghee; Jeong, Daewon

2016-12-05

Cell migration during specialized stages of osteoclast precursors, mononuclear preosteoclasts, and multinucleated mature osteoclasts remain uncertain. M-CSF- and osteopontin-induced osteoclastic cell migration was inhibited by function-blocking monoclonal antibodies specific to the integrin αv and β3 subunits, suggesting that integrin αvβ3 mediates migratory signaling induced by M-CSF and osteopontin. M-CSF and osteopontin stimulation was shown to regulate two branched signaling processes, PI3K/PKCα/RhoA axis and PI3K/PKCδ/Rac1 axis. Interestingly, inactivation of RhoA or Rac1 blocked preosteoclast and mature osteoclast migration but not osteoclast precursor migration in a transwell-based cell migration assay. Moreover, the inhibitory effect on preosteoclast and mature osteoclast migration induced by Rac1 inactivation was more effective than that by RhoA inactivation. Collectively, our findings suggest that osteoclast precursor migration depends on PI3K/PKCα-PKCδ signaling mediated via integrin αvβ3 bypassing RhoA and Rac1, whereas preosteoclast and mature osteoclast migration relies on PI3K/PKCα-PKCδ/RhoA-Rac1 axis signaling mediated via integrin αvβ3 with increased dependency on PKCδ/Rac1 signaling route as differentiation progresses. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Comparison of direct and indirect radiation effects on osteoclast formation from progenitor cells derived from different hemopoietic sources.

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Scheven, B A; Wassenaar, A M; Kawilarang-de Haas, E W; Nijweide, P J

1987-07-01

Hemopoietic stem and progenitor cells from different sources differ in radiosensitivity. Recently, we have demonstrated that the multinucleated cell responsible for bone resorption and marrow cavity formation, the osteoclast, is in fact of hemopoietic lineage. In this investigation we have studied the radiosensitivity of osteoclast formation from two different hemopoietic tissues: fetal liver and adult bone marrow. Development of osteoclasts from hemopoietic progenitors was induced by coculture of hemopoietic cell populations with fetal mouse long bones depleted of their own osteoclast precursor pool. During culture, osteoclasts developed from the exogenous cell population and invaded the calcified hypertrophic cartilage of the long bone model, thereby giving rise to the formation of a primitive marrow cavity. To analyze the radiosensitivity of osteoclast formation, either the hemopoietic cells or the bone rudiments were irradiated before coculture. Fetal liver cells were found to be less radiosensitive than bone marrow cells. The D0, Dq values and extrapolation numbers were 1.69 Gy, 5.30 Gy, and 24.40 for fetal liver cells and 1.01 Gy, 1.85 Gy, and 6.02 for bone marrow cells. Irradiation of the (pre)osteoclast-free long bone rudiments instead of the hemopoietic sources resulted in a significant inhibition of osteoclast formation at doses of 4 Gy or more. This indirect effect appeared to be more prominent in the cocultures with fetal than with adult hemopoietic cells. Furthermore, radiation doses of 8.0-10.0 Gy indirectly affected the appearance of other cell types (e.g., granulocytes) in the newly formed but underdeveloped marrow cavity. The results indicate that osteoclast progenitors from different hemopoietic sources exhibit a distinct sensitivity to ionizing irradiation. Radiation injury to long bone rudiments disturbs the osteoclast-forming capacity as well as the hemopoietic microenvironment.

The proliferative human monocyte subpopulation contains osteoclast precursors

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Lari, Roya; Kitchener, Peter D; Hamilton, John A

2009-01-01

Introduction Immediate precursors of bone-resorbing osteoclasts are cells of the monocyte/macrophage lineage. Particularly during clinical conditions showing bone loss, it would appear that osteoclast precursors are mobilized from bone marrow into the circulation prior to entering tissues undergoing such loss. The observed heterogeneity of peripheral blood monocytes has led to the notion that different monocyte subpopulations may have special or restricted functions, including as osteoclast precursors. Methods Human peripheral blood monocytes were sorted based upon their degree of proliferation and cultured in macrophage colony-stimulating factor (M-CSF or CSF-1) and receptor activator of nuclear factor-kappa-B ligand (RANKL). Results The monocyte subpopulation that is capable of proliferation gave rise to significantly more multinucleated, bone-resorbing osteoclasts than the bulk of the monocytes. Conclusions Human peripheral blood osteoclast precursors reside in the proliferative monocyte subpopulation. PMID:19222861

Inhibition of osteoclast differentiation by overexpression of NDRG2 in monocytes

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Kang, Kyeongah; Nam, Sorim; Kim, Bomi; Lim, Ji Hyun; Yang, Young; Lee, Myeong-Sok; Lim, Jong-Seok, E-mail: jslim@sookmyung.ac.kr

2015-12-25

N-Myc downstream-regulated gene 2 (NDRG2), a member of the NDRG family of differentiation-related genes, has been characterized as a regulator of dendritic cell differentiation from monocytes, CD34{sup +} progenitor cells, and myelomonocytic leukemic cells. In this study, we show that NDRG2 overexpression inhibits the differentiation of U937 cells into osteoclasts in response to stimulation with a combination of macrophage colony-stimulating factor (M-CSF) and soluble receptor activator of NF-κB ligand (RANKL). U937 cells stably expressing NDRG2 are unable to differentiate into multinucleated osteoclast-like cells and display reduced tartrate-resistant acid phosphatase (TRAP) activity and resorption pit formation. Furthermore, NDRG2 expression significantly suppresses the expression of genes that are crucial for the proliferation, survival, differentiation, and function of osteoclasts, including c-Fos, Atp6v0d2, RANK, and OSCAR. The activation of ERK1/2 and p38 is also inhibited by NDRG2 expression during osteoclastogenesis, and the inhibition of osteoclastogenesis by NDRG2 correlates with the down-regulation of the expression of the transcription factor PU.1. Taken together, our results suggest that the expression of NDRG2 potentially inhibits osteoclast differentiation and plays a role in modulating the signal transduction pathway responsible for osteoclastogenesis. - Highlights: • The expression of NDRG2 significantly impairs osteoclast differentiation. • PU.1 and p38 MAPK inhibitions by NDRG2 are critical for the inhibition of osteoclastogenesis. • Knockdown of NDRG2 rescues the ability of monocytes to differentiate into osteoclasts. • NDRG2 expression in BM and primary macrophages also impairs osteoclast differentiation. • This study implies the potential of NDRG2 expression in the inhibition of osteoclastogenesis.

MECHANICAL VIBRATION INHIBITS OSTEOCLAST FORMATION BY REDUCING DC-STAMP RECEPTOR EXPRESSION IN OSTEOCLAST PRECURSOR CELLS

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Kulkarni, R.N.; Voglewede, P.A.; Liu, D.

2014-01-01

It is well known that physical inactivity leads to loss of muscle mass, but it also causes bone loss. Mechanistically, osteoclastogenesis and bone resorption have recently been shown to be regulated by vibration. However, the underlying mechanism behind the inhibition of osteoclast formation is yet unknown. Therefore, we investigated whether mechanical vibration of osteoclast precursor cells affects osteoclast formation by the involvement of fusion-related molecules such as dendritic cell-specific transmembrane protein (DC-STAMP), and P2X7 receptor (P2X7R). RAW264.7 (a murine osteoclastic-like cell line) cells were treated with 20 ng/ml receptor activator of NF-κB ligand (RANKL). For 3 consecutive days, the cells were subjected to 1 hour of mechanical vibration with 20 µm displacement at a frequency of 4 Hz and compared to the control cells that were treated under the same condition but without the vibration. After 5 days of culture, osteoclast formation was determined. Gene expression of DC-STAMP and P2X7R by RAW264.7 cells were determined after 1 hour mechanical vibration, while protein production of the DC-STAMP was determined after 6 hours of post incubation after vibration. As a result, mechanical vibration of RAW264.7 cells inhibited the formation of osteoclasts. Vibration down-regulated DC-STAMP gene expression by 1.6-fold in the presence of RANKL and by 1.4-fold in the absence of RANKL. Additionally, DC-STAMP protein production was also down-regulated by 1.4-fold in the presence of RANKL and by 1.2-fold in the absence of RANKL in RAW264.7 cells in response to mechanical vibration. However, vibration did not affect P2X7R gene expression. Mouse anti-DC-STAMP antibody inhibited osteoclast formation in the absence of vibration. Our results suggest that mechanical vibration of osteoclast precursor cells reduce DC-STAMP expression in osteoclast precursor cells leading to the inhibition of osteoclast formation. PMID:23994170

Mechanical vibration inhibits osteoclast formation by reducing DC-STAMP receptor expression in osteoclast precursor cells.

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Kulkarni, Rishikesh N; Voglewede, Philip A; Liu, Dawei

2013-12-01

It is well known that physical inactivity leads to loss of muscle mass, but it also causes bone loss. Mechanistically, osteoclastogenesis and bone resorption have recently been shown to be regulated by vibration. However, the underlying mechanism behind the inhibition of osteoclast formation is yet unknown. Therefore, we investigated whether mechanical vibration of osteoclast precursor cells affects osteoclast formation by the involvement of fusion-related molecules such as dendritic cell-specific transmembrane protein (DC-STAMP) and P2X7 receptor (P2X7R). RAW264.7 (a murine osteoclastic-like cell line) cells were treated with 20ng/ml receptor activator of NF-κB ligand (RANKL). For 3 consecutive days, the cells were subjected to 1h of mechanical vibration with 20μm displacement at a frequency of 4Hz and compared to the control cells that were treated under the same condition but without the vibration. After 5days of culture, osteoclast formation was determined. Gene expression of DC-STAMP and P2X7R by RAW264.7 cells was determined after 1h of mechanical vibration, while protein production of the DC-STAMP was determined after 6h of postincubation after vibration. As a result, mechanical vibration of RAW264.7 cells inhibited the formation of osteoclasts. Vibration down-regulated DC-STAMP gene expression by 1.6-fold in the presence of RANKL and by 1.4-fold in the absence of RANKL. Additionally, DC-STAMP protein production was also down-regulated by 1.4-fold in the presence of RANKL and by 1.2-fold in the absence of RANKL in RAW264.7 cells in response to mechanical vibration. However, vibration did not affect P2X7R gene expression. Mouse anti-DC-STAMP antibody inhibited osteoclast formation in the absence of vibration. Our results suggest that mechanical vibration of osteoclast precursor cells reduces DC-STAMP expression in osteoclast precursor cells leading to the inhibition of osteoclast formation. © 2013 Elsevier Inc. All rights reserved.

Class A scavenger receptor promotes osteoclast differentiation via the enhanced expression of receptor activator of NF-{kappa}B (RANK)

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Takemura, Kenichi [Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556 (Japan); Department of Orthopaedic and Neuro-Musculoskeletal Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto (Japan); Sakashita, Naomi; Fujiwara, Yukio; Komohara, Yoshihiro; Lei, XiaoFeng; Ohnishi, Koji [Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556 (Japan); Suzuki, Hiroshi [National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido (Japan); Kodama, Tatsuhiko [Department of Molecular Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo (Japan); Mizuta, Hiroshi [Department of Orthopaedic and Neuro-Musculoskeletal Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto (Japan); Takeya, Motohiro, E-mail: takeya@kumamoto-u.ac.jp [Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556 (Japan)

2010-01-22

Osteoclasts originate from bone marrow monocyte/macrophage lineage cells, and their differentiation depends on macrophage colony-stimulating factor (M-CSF) and receptor activator nuclear factor kappa B (RANK) ligand. Class A scavenger receptor (SR-A) is one of the principal functional molecules of macrophages, and its level of expression declines during osteoclast differentiation. To investigate the role of SR-A in osteoclastogenesis, we examined pathological changes in femoral bone and the expression levels of osteoclastogenesis-related molecules in SR-A{sup -/-} mice. The femoral osseous density of SR-A{sup -/-} mice was higher than that of SR-A{sup +/+} mice, and the number of multinucleated osteoclasts was significantly decreased. An in vitro differentiation assay revealed that the differentiation of multinucleated osteoclasts from bone marrow-derived progenitor cells is impaired in SR-A{sup -/-} mice. Elimination of SR-A did not alter the expression level of the M-CSF receptor, c-fms; however, the expression levels of RANK and RANK-related osteoclast-differentiation molecules such as nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) and microphthalmia-associated transcription factor (MITF) significantly decreased. Furthermore, acetylated low-density lipoprotein (AcLDL), an SR-A ligand, significantly increased the expression level of RANK and MITF during osteoclast differentiation. These data indicate that SR-A promotes osteoclastogenesis via augmentation of the expression level of RANK and its related molecules.

Involvement of Receptor Activator of Nuclear Factor-κB Ligand (RANKL)-induced Incomplete Cytokinesis in the Polyploidization of Osteoclasts*

Science.gov (United States)

Takegahara, Noriko; Kim, Hyunsoo; Mizuno, Hiroki; Sakaue-Sawano, Asako; Miyawaki, Atsushi; Tomura, Michio; Kanagawa, Osami; Ishii, Masaru; Choi, Yongwon

2016-01-01

Osteoclasts are specialized polyploid cells that resorb bone. Upon stimulation with receptor activator of nuclear factor-κB ligand (RANKL), myeloid precursors commit to becoming polyploid, largely via cell fusion. Polyploidization of osteoclasts is necessary for their bone-resorbing activity, but the mechanisms by which polyploidization is controlled remain to be determined. Here, we demonstrated that in addition to cell fusion, incomplete cytokinesis also plays a role in osteoclast polyploidization. In in vitro cultured osteoclasts derived from mice expressing the fluorescent ubiquitin-based cell cycle indicator (Fucci), RANKL induced polyploidy by incomplete cytokinesis as well as cell fusion. Polyploid cells generated by incomplete cytokinesis had the potential to subsequently undergo cell fusion. Nuclear polyploidy was also observed in osteoclasts in vivo, suggesting the involvement of incomplete cytokinesis in physiological polyploidization. Furthermore, RANKL-induced incomplete cytokinesis was reduced by inhibition of Akt, resulting in impaired multinucleated osteoclast formation. Taken together, these results reveal that RANKL-induced incomplete cytokinesis contributes to polyploidization of osteoclasts via Akt activation. PMID:26670608

Dasatinib inhibits both osteoclast activation and prostate cancer PC-3-cell-induced osteoclast formation.

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Araujo, John C; Poblenz, Ann; Corn, Paul; Parikh, Nila U; Starbuck, Michael W; Thompson, Jerry T; Lee, Francis; Logothetis, Christopher J; Darnay, Bryant G

2009-11-01

Therapies to target prostate cancer bone metastases have only limited effects. New treatments are focused on the interaction between cancer cells, bone marrow cells and the bone matrix. Osteoclasts play an important role in the development of bone tumors caused by prostate cancer. Since Src kinase has been shown to be necessary for osteoclast function, we hypothesized that dasatinib, a Src family kinase inhibitor, would reduce osteoclast activity and prostate cancer (PC-3) cell-induced osteoclast formation. Dasatinib inhibited RANKL-induced osteoclast differentiation of bone marrow-derived monocytes with an EC(50) of 7.5 nM. PC-3 cells, a human prostate cancer cell line, were able to differentiate RAW 264.7 cells, a murine monocytic cell line, into osteoclasts, and dasatinib inhibited this differentiation. In addition, conditioned medium from PC-3 cell cultures was able to differentiate RAW 264.7 cells into osteoclasts and this too, was inhibited by dasatinib. Even the lowest concentration of dasatinib, 1.25 nmol, inhibited osteoclast differentiation by 29%. Moreover, dasatinib inhibited osteoclast activity by 58% as measured by collagen 1 release. We performed in vitro experiments utilizing the Src family kinase inhibitor dasatinib to target osteoclast activation as a means of inhibiting prostate cancer bone metastases. Dasatinib inhibits osteoclast differentiation of mouse primary bone marrow-derived monocytes and PC-3 cell-induced osteoclast differentiation. Dasatinib also inhibits osteoclast degradation activity. Inhibiting osteoclast differentiation and activity may be an effective targeted therapy in patients with prostate cancer bone metastases.

Dasatinib inhibits both osteoclast activation and prostate cancer PC-3 cell-induced osteoclast formation

Science.gov (United States)

Araujo, John C.; Poblenz, Ann; Corn, Paul G.; Parikh, Nila U.; Starbuck, Michael W.; Thompson, Jerry T.; Lee, Francis; Logothetis, Christopher J.; Darnay, Bryant G.

2013-01-01

Purpose Therapies to target prostate cancer bone metastases have only limited effects. New treatments are focused on the interaction between cancer cells, bone marrow cells and the bone matrix. Osteoclasts play an important role in the development of bone tumors caused by prostate cancer. Since Src kinase has been shown to be necessary for osteoclast function, we hypothesized that dasatinib, a Src family kinase inhibitor, would reduce osteoclast activity and prostate cancer (PC-3) cell-induced osteoclast formation. Results Dasatinib inhibited RANKL-induced osteoclast differentiation of bone marrow-derived monocytes with an EC50 of 7.5 nM. PC-3 cells, a human prostate cancer cell line, were able to differentiate RAW 264.7 cells, a murine monocytic cell line, into osteoclasts and dasatinib inhibited this differentiation. In addition, conditioned medium from PC-3 cell cultures was able to differentiate RAW 264.7 cells into osteoclasts and this too, was inhibited by dasatinib. Even the lowest concentration of dasatinib, 1.25 nmol, inhibited osteoclast differentiation by 29%. Moreover, dasatinib inhibited osteoclast activity by 58% as measured by collagen 1 release. Experimental design We performed in vitro experiments utilizing the Src family kinase inhibitor dasatinib to target osteoclast activation as a means of inhibiting prostate cancer bone metastases. Conclusion Dasatinib inhibits osteoclast differentiation of mouse primary bone marrow-derived monocytes and PC-3 cell-induced osteoclast differentiation. Dasatinib also inhibits osteoclast degradation activity. Inhibiting osteoclast differentiation and activity may be an effective targeted therapy in patients with prostate cancer bone metastases. PMID:19855158

Nanosilica induced dose-dependent cytotoxicity and cell type-dependent multinucleation in HepG2 and L-02 cells

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Yu, Yongbo [Capital Medical University, Beijing Key Laboratory for Pediatric Diseases of Otolaryngology, Head and Neck Surgery, Beijing Pediatric Research Institute, Beijing Children’s Hospital (China); Duan, Junchao; Li, Yang; Yu, Yang; Hu, Hejing; Wu, Jing; Zhang, Yannan; Li, Yanbo; CaixiaGuo; Zhou, Xianqing; Sun, Zhiwei, E-mail: zwsun@ccmu.edu.cn [Capital Medical University, School of Public Health (China)

2016-11-15

The prevalent exposure to nanosilica gained concerns about health effects of these particles on human beings. Although nanosilica-induced multinucleation has been confirmed previously, the underlying mechanism was still not clear; this study was to investigate the origination of multinucleated cells caused by nanosilica (62 nm) in both HepG2 and L-02 cells. Cell viability and cellular uptake was determined by MTT assay and transmission electron microscope (TEM), respectively. Giemsa staining was applied to detect multinucleation. To clarify the origination of multinucleated cells, fluorescent probes, PKH26 and PKH67, time-lapse observation were further conducted by confocal microscopy. Results indicated that nanosilica particles were internalized into cells and induced cytotoxicity in a dose-dependent manner. Quantification analysis showed that nanosilica significantly increased the rates of binucleated and multinucleated cells, which suggested mitotic catastrophe induction. Moreover, dynamic visualization verified that multinucleation resulted from cell fusion in HepG2 cells not in L-02 cells after nanosilica exposure, suggesting cell type-dependent multinucleation formation. Both multinucleation and cell fusion were involved in genetic instability, which emphasized the significance to explore the multinucleation induced by nanosilica via environmental, occupational and consumer product exposure.

Involvement of Receptor Activator of Nuclear Factor-κB Ligand (RANKL)-induced Incomplete Cytokinesis in the Polyploidization of Osteoclasts.

Science.gov (United States)

Takegahara, Noriko; Kim, Hyunsoo; Mizuno, Hiroki; Sakaue-Sawano, Asako; Miyawaki, Atsushi; Tomura, Michio; Kanagawa, Osami; Ishii, Masaru; Choi, Yongwon

2016-02-12

Osteoclasts are specialized polyploid cells that resorb bone. Upon stimulation with receptor activator of nuclear factor-κB ligand (RANKL), myeloid precursors commit to becoming polyploid, largely via cell fusion. Polyploidization of osteoclasts is necessary for their bone-resorbing activity, but the mechanisms by which polyploidization is controlled remain to be determined. Here, we demonstrated that in addition to cell fusion, incomplete cytokinesis also plays a role in osteoclast polyploidization. In in vitro cultured osteoclasts derived from mice expressing the fluorescent ubiquitin-based cell cycle indicator (Fucci), RANKL induced polyploidy by incomplete cytokinesis as well as cell fusion. Polyploid cells generated by incomplete cytokinesis had the potential to subsequently undergo cell fusion. Nuclear polyploidy was also observed in osteoclasts in vivo, suggesting the involvement of incomplete cytokinesis in physiological polyploidization. Furthermore, RANKL-induced incomplete cytokinesis was reduced by inhibition of Akt, resulting in impaired multinucleated osteoclast formation. Taken together, these results reveal that RANKL-induced incomplete cytokinesis contributes to polyploidization of osteoclasts via Akt activation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Circulating microparticles in acute diabetic Charcot foot exhibit a high content of inflammatory cytokines, and support monocyte-to-osteoclast cell induction.

Science.gov (United States)

Pasquier, Jennifer; Thomas, Binitha; Hoarau-Véchot, Jessica; Odeh, Tala; Robay, Amal; Chidiac, Omar; Dargham, Soha R; Turjoman, Rebal; Halama, Anna; Fakhro, Khalid; Menzies, Robert; Jayyousi, Amin; Zirie, Mahmoud; Al Suwaidi, Jassim; Rafii, Arash; Malik, Rayaz A; Talal, Talal; Abi Khalil, Charbel

2017-11-27

Circulating microparticles (MPs) are major mediators in cardiovascular complications of type 2 diabetes (T2D); however, their contribution to Charcot foot (CF) disease is not known. Here, we purified and assessed the origin, concentration and content of circulating MPs from 33 individuals: 11 with T2D and acute CF, 11 T2D patients with equivalent neuropathy and 11 non-diabetic controls. First, we demonstrated that there were no differences in the distribution of MPs of endothelial, platelet origin among the 3 groups. However, MPs from leukocytes and monocytes origin were increased in CF patients. Moreover, we demonstrated that monocytes-derived MPs originated more frequently from intermediate and non-classical monocytes in CF patients. Five cytokines (G-CSF, GM-CSF, IL-1-ra, IL-2 and IL-16) were significantly increased in MPs from acute CF patients. Applying ingenuity pathways analysis, we found that those cytokines interacted well and induced the activation of pathways that are involved in osteoclast formation. Further, we treated THP-1 monocytes and monocytes sorted from healthy patients with CF-derived MPs during their differentiation into osteoclasts, which increased their differentiation into multinucleated osteoclast-like cells. Altogether, our study suggests that circulating MPs in CF disease have a high content of inflammatory cytokines and could increase osteoclast differentiation in vitro.

Switch-like reprogramming of gene expression after fusion of multinucleate plasmodial cells of two Physarum polycephalum sporulation mutants

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Walter, Pauline; Hoffmann, Xenia-Katharina; Ebeling, Britta; Haas, Markus; Marwan, Wolfgang, E-mail: wolfgang.marwan@ovgu.de

2013-05-24

Highlights: •We investigate reprogramming of gene expression in multinucleate single cells. •Cells of two differentiation control mutants are fused. •Fused cells proceed to alternative gene expression patterns. •The population of nuclei damps stochastic fluctuations in gene expression. •Dynamic processes of cellular reprogramming can be observed by repeated sampling of a cell. -- Abstract: Nonlinear dynamic processes involving the differential regulation of transcription factors are considered to impact the reprogramming of stem cells, germ cells, and somatic cells. Here, we fused two multinucleate plasmodial cells of Physarum polycephalum mutants defective in different sporulation control genes while being in different physiological states. The resulting heterokaryons established one of two significantly different expression patterns of marker genes while the plasmodial halves that were fused to each other synchronized spontaneously. Spontaneous synchronization suggests that switch-like control mechanisms spread over and finally control the entire plasmodium as a result of cytoplasmic mixing. Regulatory molecules due to the large volume of the vigorously streaming cytoplasm will define concentrations in acting on the population of nuclei and in the global setting of switches. Mixing of a large cytoplasmic volume is expected to damp stochasticity when individual nuclei deliver certain RNAs at low copy number into the cytoplasm. We conclude that spontaneous synchronization, the damping of molecular noise in gene expression by the large cytoplasmic volume, and the option to take multiple macroscopic samples from the same plasmodium provide unique options for studying the dynamics of cellular reprogramming at the single cell level.

Switch-like reprogramming of gene expression after fusion of multinucleate plasmodial cells of two Physarum polycephalum sporulation mutants

International Nuclear Information System (INIS)

Walter, Pauline; Hoffmann, Xenia-Katharina; Ebeling, Britta; Haas, Markus; Marwan, Wolfgang

2013-01-01

Highlights: •We investigate reprogramming of gene expression in multinucleate single cells. •Cells of two differentiation control mutants are fused. •Fused cells proceed to alternative gene expression patterns. •The population of nuclei damps stochastic fluctuations in gene expression. •Dynamic processes of cellular reprogramming can be observed by repeated sampling of a cell. -- Abstract: Nonlinear dynamic processes involving the differential regulation of transcription factors are considered to impact the reprogramming of stem cells, germ cells, and somatic cells. Here, we fused two multinucleate plasmodial cells of Physarum polycephalum mutants defective in different sporulation control genes while being in different physiological states. The resulting heterokaryons established one of two significantly different expression patterns of marker genes while the plasmodial halves that were fused to each other synchronized spontaneously. Spontaneous synchronization suggests that switch-like control mechanisms spread over and finally control the entire plasmodium as a result of cytoplasmic mixing. Regulatory molecules due to the large volume of the vigorously streaming cytoplasm will define concentrations in acting on the population of nuclei and in the global setting of switches. Mixing of a large cytoplasmic volume is expected to damp stochasticity when individual nuclei deliver certain RNAs at low copy number into the cytoplasm. We conclude that spontaneous synchronization, the damping of molecular noise in gene expression by the large cytoplasmic volume, and the option to take multiple macroscopic samples from the same plasmodium provide unique options for studying the dynamics of cellular reprogramming at the single cell level

Suppression of T cell-induced osteoclast formation

Energy Technology Data Exchange (ETDEWEB)

Karieb, Sahar; Fox, Simon W., E-mail: Simon.fox@plymouth.ac.uk

2013-07-12

Highlights: •Genistein and coumestrol prevent activated T cell induced osteoclast formation. •Anti-TNF neutralising antibodies prevent the pro-osteoclastic effect of activated T cells. •Phytoestrogens inhibit T cell derived TNF alpha and inflammatory cytokine production. •Phytoestrogens have a broader range of anti-osteoclastic actions than other anti-resorptives. -- Abstract: Inhibition of T cell derived cytokine production could help suppress osteoclast differentiation in inflammatory skeletal disorders. Bisphosphonates are typically prescribed to prevent inflammatory bone loss but are not tolerated by all patients and are associated with an increased risk of osteonecrosis of the jaw. In light of this other anti-resorptives such as phytoestrogens are being considered. However the effect of phytoestrogens on T cell-induced osteoclast formation is unclear. The effect of genistein and coumestrol on activated T cell-induced osteoclastogenesis and cytokine production was therefore examined. Concentrations of genistein and coumestrol (10{sup −7} M) previously shown to directly inhibit osteoclast formation also suppressed the formation of TRAP positive osteoclast induced by con A activated T cells, which was dependent on inhibition of T cell derived TNF-α. While both reduced osteoclast formation their mechanism of action differed. The anti-osteoclastic effect of coumestrol was associated with a dual effect on con A induced T cell proliferation and activation; 10{sup −7} M coumestrol significantly reducing T cell number (0.36) and TNF-α (0.47), IL-1β (0.23) and IL-6 (0.35) expression, whereas genistein (10{sup −7} M) had no effect on T cell number but a more pronounced effect on T cell differentiation reducing expression of TNF-α (0.49), IL-1β (0.52), IL-6 (0.71) and RANKL (0.71). Phytoestrogens therefore prevent the pro-osteoclastic action of T cells suggesting they may have a role in the control of inflammatory bone loss.

Inhibitory effects of methyl-3,5-di-O-caffeoyl-epi-quinate on RANKL-induced osteoclast differentiation.

Science.gov (United States)

Kim, Tae Hoon; Ihn, Hye Jung; Kim, Kiryeong; Cho, Hye-Sung; Shin, Hong-In; Bae, Yong Chul; Park, Eui Kyun

2018-04-09

In this study, we have shown that methyl-3,5-di-O-caffeoyl-epi-quinate, a naturally occurring compound isolated from Ainsliaea acerifolia, inhibits receptor activator of nuclear factor-κB ligand (RANKL)-induced formation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts and the expression of osteoclast marker genes. Methyl-3,5-di-O-caffeoyl-epi-quinate also inhibited RANKL-induced activation of p38, Akt and extracellular signal-regulated kinase (ERK) as well as the expression of nuclear factor of activated T-cell (NFATc1), the key regulator of osteoclast differentiation. Negative regulators for osteoclast differentiation was upregulated by methyl-3,5-di-O-caffeoyl-epi-quinate. Collectively, our results suggested that methyl-3,5-di-O-caffeoyl-epi-quinate suppresses osteoclast differentiation via downregulation of RANK signaling pathways and NFATc1. Copyright © 2018 Elsevier Ltd. All rights reserved.

Osteoclast formation is strongly reduced both in vivo and in vitro in the absence of CD47/SIRPα-interaction

International Nuclear Information System (INIS)

Lundberg, Pernilla; Koskinen, Cecilia; Baldock, Paul A.; Loethgren, Hanna; Stenberg, Asa; Lerner, Ulf H.; Oldenborg, Per-Arne

2007-01-01

Physical interaction between the cell surface receptors CD47 and signal regulatory protein alpha (SIRPα) was reported to regulate cell migration, phagocytosis, cytokine production, and macrophage fusion. However, it is unclear if the CD47/SIRPα-interaction can also regulate macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-κB ligand (RANKL)-stimulated formation of osteoclasts. Here, we show that functional blocking antibodies to either CD47 or SIRPα strongly reduced formation of multinucleated tartrate-resistant acid phosphatase (TRAP) + osteoclasts in cultures of murine hematopoietic cells, stimulated in vitro by M-CSF and RANKL. In addition, the numbers of osteoclasts formed in M-CSF/RANKL-stimulated bone marrow macrophage cultures from CD47 -/- mice were strongly reduced, and bones of CD47 -/- mice exhibited significantly reduced osteoclast numbers, as compared with wild-type controls. We conclude that the CD47/SIRPα interaction is important for M-CSF/RANKL-stimulated osteoclast formation both in vivo and in vitro, and that absence of CD47 results in decreased numbers of osteoclasts in CD47 -/- mice

Assessing cell fusion and cytokinesis failure as mechanisms of clone 9 hepatocyte multinucleation in vitro.

Science.gov (United States)

Simic, Damir; Euler, Catherine; Thurby, Christina; Peden, Mike; Tannehill-Gregg, Sarah; Bunch, Todd; Sanderson, Thomas; Van Vleet, Terry

2012-08-01

In this in vitro model of hepatocyte multinucleation, separate cultures of rat Clone 9 cells are labeled with either red or green cell tracker dyes (Red Cell Tracker CMPTX or Vybrant CFDA SE Cell Tracer), plated together in mixed-color colonies, and treated with positive or negative control agents for 4 days. The fluorescent dyes become cell-impermeant after entering cells and are not transferred to adjacent cells in a population, but are inherited by daughter cells after fusion. The mixed-color cultures are then evaluated microscopically for multinucleation and analysis of the underlying mechanism (cell fusion/cytokinesis). Multinucleated cells containing only one dye have undergone cytokinesis failure, whereas dual-labeled multinucleated cells have resulted from fusion. © 2012 by John Wiley & Sons, Inc.

Fucoidan, a Sulfated Polysaccharide, Inhibits Osteoclast Differentiation and Function by Modulating RANKL Signaling

Directory of Open Access Journals (Sweden)

Young Woo Kim

2014-10-01

Full Text Available Multinucleated osteoclasts differentiate from hematopoietic progenitors of the monocyte/macrophage lineage. Because of its pivotal role in bone resorption, regulation of osteoclast differentiation is a potential therapeutic approach to the treatment of erosive bone disease. In this study, we have found that fucoidan, a sulfated polysaccharide extracted from brown seaweed, inhibited osteoclast differentiation. In particular, addition of fucoidan into the early stage osteoclast cultures significantly inhibited receptor activator of nuclear factor kappa B (NF-κB ligand (RANKL-induced osteoclast formation, thus suggesting that fucoidan affects osteoclast progenitors. Furthermore, fucoidan significantly inhibited the activation of RANKL-dependent mitogen-activated protein kinases (MAPKs such as JNK, ERK, and p38, and also c-Fos and NFATc1, which are crucial transcription factors for osteoclastogenesis. In addition, the activation of NF-κB, which is an upstream transcription factor modulating NFATc1 expression, was alleviated in the fucoidan-treated cells. These results collectively suggest that fucoidan inhibits osteoclastogenesis from bone marrow macrophages by inhibiting RANKL-induced p38, JNK, ERK and NF-κB activation, and by downregulating the expression of genes that partake in both osteoclast differentiation and resorption.

Multinucleated Giant Cancer Cells Produced in Response to Ionizing Radiation Retain Viability and Replicate Their Genome

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Razmik Mirzayans

2017-02-01

Full Text Available Loss of wild-type p53 function is widely accepted to be permissive for the development of multinucleated giant cells. However, whether therapy-induced multinucleation is associated with cancer cell death or survival remains controversial. Herein, we demonstrate that exposure of p53-deficient or p21WAF1 (p21-deficient solid tumor-derived cell lines to ionizing radiation (between 2 and 8 Gy results in the development of multinucleated giant cells that remain adherent to the culture dish for long times post-irradiation. Somewhat surprisingly, single-cell observations revealed that virtually all multinucleated giant cells that remain adherent for the duration of the experiments (up to three weeks post-irradiation retain viability and metabolize 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl-tetrazolium bromide (MTT, and the majority (>60% exhibit DNA synthesis. We further report that treatment of multinucleated giant cells with pharmacological activators of apoptosis (e.g., sodium salicylate triggers their demise. Our observations reinforce the notion that radiation-induced multinucleation may reflect a survival mechanism for p53/p21-deficient cancer cells. With respect to evaluating radiosensitivity, our observations underscore the importance of single-cell experimental approaches (e.g., single-cell MTT as the creation of viable multinucleated giant cells complicates the interpretation of the experimental data obtained by commonly-used multi-well plate colorimetric assays.

Lipocalin-2 inhibits osteoclast formation by suppressing the proliferation and differentiation of osteoclast lineage cells

Energy Technology Data Exchange (ETDEWEB)

Kim, Hyun-Ju, E-mail: biohjk@knu.ac.kr [Department of Molecular Medicine, Cell and Matrix Research Institute, Clinical Trial Center, BK21 Plus KNU Biomedical Convergence Program, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Yoon, Hye-Jin [Department of Molecular Medicine, Cell and Matrix Research Institute, Clinical Trial Center, BK21 Plus KNU Biomedical Convergence Program, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Yoon, Kyung-Ae [Department of Orthopedic Surgery, Skeletal Diseases Genome Research Center, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Gwon, Mi-Ri; Jin Seong, Sook [Department of Molecular Medicine, Cell and Matrix Research Institute, Clinical Trial Center, BK21 Plus KNU Biomedical Convergence Program, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Suk, Kyoungho [Department of Pharmacology, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Kim, Shin-Yoon [Department of Orthopedic Surgery, Skeletal Diseases Genome Research Center, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Yoon, Young-Ran, E-mail: yry@knu.ac.kr [Department of Molecular Medicine, Cell and Matrix Research Institute, Clinical Trial Center, BK21 Plus KNU Biomedical Convergence Program, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of)

2015-06-10

Lipocalin-2 (LCN2) is a member of the lipocalin superfamily and plays a critical role in the regulation of various physiological processes, such as inflammation and obesity. In this study, we report that LCN2 negatively modulates the proliferation and differentiation of osteoclast precursors, resulting in impaired osteoclast formation. The overexpression of LCN2 in bone marrow-derived macrophages or the addition of recombinant LCN2 protein inhibits the formation of multinuclear osteoclasts. LCN2 suppresses macrophage colony-stimulating factor (M-CSF)-induced proliferation of osteoclast precursor cells without affecting their apoptotic cell death. Interestingly, LCN2 decreases the expression of the M-CSF receptor, c-Fms, and subsequently blocks its downstream signaling cascades. In addition, LCN2 inhibits RANKL-induced osteoclast differentiation and attenuates the expression of c-Fos and nuclear factor of activated T cells c1 (NFATc1), which are important modulators in osteoclastogenesis. Mechanistically, LCN2 inhibits NF-κB signaling pathways, as demonstrated by the suppression of IκBα phosphorylation, nuclear translocation of p65, and NF-κB transcriptional activity. Thus, LCN2 is an anti-osteoclastogenic molecule that exerts its effects by retarding the proliferation and differentiation of osteoclast lineage cells. - Highlights: • LCN2 expression is regulated during osteoclast development. • LCN2 suppresses M-CSF-mediated osteoclast precursor proliferation. • LCN2 inhibits RANKL-induced osteoclast differentiation.

Tumor associated osteoclast-like giant cells promote tumor growth and lymphangiogenesis by secreting vascular endothelial growth factor-C

International Nuclear Information System (INIS)

Hatano, Yu; Nakahama, Ken-ichi; Isobe, Mitsuaki; Morita, Ikuo

2014-01-01

Highlights: • M-CSF and RANKL expressing HeLa cells induced osteoclastogenesis in vitro. • We established OGC-containing tumor model in vivo. • OGC-containing tumor became larger independent of M-CSF or RANKL effect. • VEGF-C secreted from OGCs was a one of candidates for OGC-containing tumor growth. - Abstract: Tumors with osteoclast-like giant cells (OGCs) have been reported in a variety of organs and exert an invasive and prometastatic phenotype, but the functional role of OGCs in the tumor environment has not been fully clarified. We established tumors containing OGCs to clarify the role of OGCs in tumor phenotype. A mixture of HeLa cells expressing macrophage colony-stimulating factor (M-CSF, HeLa-M) and receptor activator of nuclear factor-κB ligand (RANKL, HeLa-R) effectively supported the differentiation of osteoclast-like cells from bone marrow macrophages in vitro. Moreover, a xenograft study showed OGC formation in a tumor composed of HeLa-M and HeLa-R. Surprisingly, the tumors containing OGCs were significantly larger than the tumors without OGCs, although the growth rates were not different in vitro. Histological analysis showed that lymphangiogenesis and macrophage infiltration in the tumor containing OGCs, but not in other tumors were accelerated. According to quantitative PCR analysis, vascular endothelial growth factor (VEGF)-C mRNA expression increased with differentiation of osteoclast-like cells. To investigate whether VEGF-C expression is responsible for tumor growth and macrophage infiltration, HeLa cells overexpressing VEGF-C (HeLa-VC) were established and transplanted into mice. Tumors composed of HeLa-VC mimicked the phenotype of the tumors containing OGCs. Furthermore, the vascular permeability of tumor microvessels also increased in tumors containing OGCs and to some extent in VEGF-C-expressing tumors. These results suggest that macrophage infiltration and vascular permeability are possible mediators in these tumors. These

Osteoclast differentiation inhibitors: a patent review (2008 - 2012).

Science.gov (United States)

Kim, Seong Hwan; Moon, Seong-Hee

2013-12-01

Mononuclear macrophage/monocyte-lineage hematopoietic precursors differentiate into multinucleated osteoclasts. Abnormally increased numbers and/or overactivation of osteoclasts can lead to bone loss. Therefore, pharmaceutical inhibition of osteoclast differentiation is one therapeutic strategy for mitigating the occurrence of bone loss-associated disorders and related fractures. This review surveys the patents and patent applications from 2008 to 2012 that are related to inventions of therapeutics and/or methods for inhibiting osteoclast differentiation. Over the past 20 years, the identification and validation of signaling molecules involved in osteoclast differentiation has led to a better understanding of the molecular mechanism, and to the development of new therapeutic agents for treating bone loss-associated disorders. Since 2008, 34 WO patents or patent applications have been filed that relate to inventions of therapeutics and/or methods for chemical-based, natural product-based, or biological-based inhibitors of osteoclast differentiation. Here, analysis of these patents and patent applications is presented, and summarize the disclosed osteoclast differentiation-inhibiting target molecules. This report can support further advances in the development of anti-osteoclastogenic therapeutics for bone loss-associated disorders, including osteoporosis, rheumatoid arthritis, Paget's disease, periodontal disease, osteosarcoma, and cancer bone metastasis.

MECHANICAL VIBRATION INHIBITS OSTEOCLAST FORMATION BY REDUCING DC-STAMP RECEPTOR EXPRESSION IN OSTEOCLAST PRECURSOR CELLS

OpenAIRE

Kulkarni, R.N.; Voglewede, P.A.; Liu, D.

2013-01-01

It is well known that physical inactivity leads to loss of muscle mass, but it also causes bone loss. Mechanistically, osteoclastogenesis and bone resorption have recently been shown to be regulated by vibration. However, the underlying mechanism behind the inhibition of osteoclast formation is yet unknown. Therefore, we investigated whether mechanical vibration of osteoclast precursor cells affects osteoclast formation by the involvement of fusion-related molecules such as dendritic cell-spe...

Furosin, an ellagitannin, suppresses RANKL-induced osteoclast differentiation and function through inhibition of MAP kinase activation and actin ring formation

International Nuclear Information System (INIS)

Park, Eui Kyun; Kim, Myung Sunny; Lee, Seung Ho; Kim, Kyung Hee; Park, Ju-Young; Kim, Tae-Ho; Lee, In-Seon; Woo, Je-Tae; Jung, Jae-Chang; Shin, Hong-In; Choi, Je-Yong; Kim, Shin-Yoon

2004-01-01

Phenolic compounds including tannins and flavonoids have been implicated in suppression of osteoclast differentiation/function and prevention of bone diseases. However, the effects of hydrolysable tannins on bone metabolism remain to be elucidated. In this study, we found that furosin, a hydrolysable tannin, markedly decreased the differentiation of both murine bone marrow mononuclear cells and Raw264.7 cells into osteoclasts, as revealed by the reduced number of tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells and decreased TRAP activity. Furosin appears to target at the early stage of osteoclastic differentiation while having no cytotoxic effect on osteoclast precursors. Analysis of the inhibitory mechanisms of furosin revealed that it inhibited the receptor activator of nuclear factor-κB ligand (RANKL)-induced activation of p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK)/activating protein-1 (AP-1). Furthermore, furosin reduced resorption pit formation in osteoclasts, which was accompanied by disruption of the actin rings. Taken together, these results demonstrate that naturally occurring furosin has an inhibitory activity on both osteoclast differentiation and function through mechanisms involving inhibition of the RANKL-induced p38MAPK and JNK/AP-1 activation as well as actin ring formation

Inactivation of the small GTP binding protein Rho induces multinucleate cell formation and apoptosis in murine T lymphoma EL4.

Science.gov (United States)

Moorman, J P; Bobak, D A; Hahn, C S

1996-06-01

The small G-protein Rho regulates the actin microfilament-dependent cytoskeleton. Exoenzyme C3 of Clostridium botulinum ADP-ribosylates Rho at Asn41, a modification that functionally inactivates Rho. Using a Sindbis virus-based transient gene expression system, we studied the role of Rho in murine EL4 T lymphoma cells. We generated a double subgenomic infectious Sindbis virus (dsSIN:C3) recombinant which expressed C3 in >95% of EL4 cells. This intracellular C3 resulted in modification and inactivation of virtually all endogenous Rho. dsSIN:C3 infection led to the formation of multinucleate cells, likely by inhibiting the actin microfilament-dependent step of cytokinesis. Intriguingly, in spite of the inhibition of cytokinesis, karyokinesis continued, with the result that cells containing a nuclear DNA content as high as 16N (eight nuclei) were observed. In addition, dsSIN:C3-mediated inactivation of Rho was a potent activator of apoptosis in EL4 cells. To discern whether the formation of multinucleate cells was responsible for the activation of apoptosis, 5-fluorouracil (5-FUra) was used to induce cell cycle arrest. As expected, EL4 cells treated with 5-FUra were prevented from forming multinucleate cells upon infection with dsSIN:C3. dsSIN:C3 infection, however, still caused marked apoptosis in 5-FUra-treated cells, indicating that this activation of apoptosis was independent of multinucleate cell formation.

Cell adhesion signaling regulates RANK expression in osteoclast precursors.

Directory of Open Access Journals (Sweden)

Ayako Mochizuki

Full Text Available Cells with monocyte/macrophage lineage expressing receptor activator of NF-κB (RANK differentiate into osteoclasts following stimulation with the RANK ligand (RANKL. Cell adhesion signaling is also required for osteoclast differentiation from precursors. However, details of the mechanism by which cell adhesion signals induce osteoclast differentiation have not been fully elucidated. To investigate the participation of cell adhesion signaling in osteoclast differentiation, mouse bone marrow-derived macrophages (BMMs were used as osteoclast precursors, and cultured on either plastic cell culture dishes (adherent condition or the top surface of semisolid methylcellulose gel loaded in culture tubes (non-adherent condition. BMMs cultured under the adherent condition differentiated into osteoclasts in response to RANKL stimulation. However, under the non-adherent condition, the efficiency of osteoclast differentiation was markedly reduced even in the presence of RANKL. These BMMs retained macrophage characteristics including phagocytic function and gene expression profile. Lipopolysaccharide (LPS and tumor necrosis factor -αTNF-α activated the NF-κB-mediated signaling pathways under both the adherent and non-adherent conditions, while RANKL activated the pathways only under the adherent condition. BMMs highly expressed RANK mRNA and protein under the adherent condition as compared to the non-adherent condition. Also, BMMs transferred from the adherent to non-adherent condition showed downregulated RANK expression within 24 hours. In contrast, transferring those from the non-adherent to adherent condition significantly increased the level of RANK expression. Moreover, interruption of cell adhesion signaling by echistatin, an RGD-containing disintegrin, decreased RANK expression in BMMs, while forced expression of either RANK or TNFR-associated factor 6 (TRAF6 in BMMs induced their differentiation into osteoclasts even under the non

Retinal pigment epithelial cell multinucleation in the aging eye - a mechanism to repair damage and maintain homoeostasis.

Science.gov (United States)

Chen, Mei; Rajapakse, Dinusha; Fraczek, Monika; Luo, Chang; Forrester, John V; Xu, Heping

2016-06-01

Retinal pigment epithelial (RPE) cells are central to retinal health and homoeostasis. Dysfunction or death of RPE cells underlies many age-related retinal degenerative disorders particularly age-related macular degeneration. During aging RPE cells decline in number, suggesting an age-dependent cell loss. RPE cells are considered to be postmitotic, and how they repair damage during aging remains poorly defined. We show that RPE cells increase in size and become multinucleate during aging in C57BL/6J mice. Multinucleation appeared not to be due to cell fusion, but to incomplete cell division, that is failure of cytokinesis. Interestingly, the phagocytic activity of multinucleate RPE cells was not different from that of mononuclear RPE cells. Furthermore, exposure of RPE cells in vitro to photoreceptor outer segment (POS), particularly oxidized POS, dose-dependently promoted multinucleation and suppressed cell proliferation. Both failure of cytokinesis and suppression of proliferation required contact with POS. Exposure to POS also induced reactive oxygen species and DNA oxidation in RPE cells. We propose that RPE cells have the potential to proliferate in vivo and to repair defects in the monolayer. We further propose that the conventionally accepted 'postmitotic' status of RPE cells is due to a modified form of contact inhibition mediated by POS and that RPE cells are released from this state when contact with POS is lost. This is seen in long-standing rhegmatogenous retinal detachment as overtly proliferating RPE cells (proliferative vitreoretinopathy) and more subtly as multinucleation during normal aging. Age-related oxidative stress may promote failure of cytokinesis and multinucleation in RPE cells. © 2016 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Effects of Cinnamoyloxy-mammeisin from Geopropolis on Osteoclast Differentiation and Porphyromonas gingivalis-Induced Periodontitis.

Science.gov (United States)

da Cunha, Marcos Guilherme; Ramos-Junior, Erivan Schnaider; Franchin, Marcelo; Taira, Thaise Mayumi; Beutler, John A; Franco, Gilson Cesar Nobre; Ikegaki, Masaharu; de Alencar, Severino Matias; Fukada, Sandra Yasuyo; Rosalen, Pedro Luiz

2017-06-23

Bone-loss-related diseases such as rheumatoid arthritis, osteomyelitis, osteoporosis, and periodontitis are associated with high rates of morbidity worldwide. These disorders are characterized by an imbalance between the formation and activity of osteoblasts and osteoclasts, leading to bone loss. In this context, we evaluated the effect of cinnamoyloxy-mammeisin (CNM), an anti-inflammatory coumarin found in Melipona scutellaris geopropolis, on key targets related to bone remodeling. In the present study we investigated the in vitro effects of CNM on osteoclast differentiation and M-CSF+RANKL-induced osteoclastogenic marker expression. Additionally, the interference of CNM treatment on osteoclast activity was evaluated by zymography and resorption area. Finally, we assessed the capacity of the compound to mitigate alveolar bone loss in vivo in experimental murine periodontitis induced by Porphyromonas gingivalis. We observed that treatment with CNM impaired osteoclast differentiation, as evidenced by a reduced number of tartrate-resistant acid-phosphatase-positive multinucleated cells (TRAP+) as well as the expression of osteoclastogenic markers upon M-CSF+RANKL-induced stimulation. Similarly, we observed reduced gelatinolytic and resorption capacity in M-CSF+RANKL-induced cells in vitro. Lastly, CNM attenuated alveolar bone loss in an experimental murine periodontitis model. These findings indicate that CNM may be considered a promising treatment for bone loss diseases.

Conditional deletion of CD98hc inhibits osteoclast development

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Hideki Tsumura

2016-03-01

Full Text Available The CD98 heavy chain (CD98hc regulates virus-induced cell fusion and monocyte fusion, and is involved in amino acid transportation. Here, we examined the role that CD98hc plays in the formation of osteoclasts using CD98hcflox/floxLysM-cre peritoneal macrophages (CD98hc-defect macrophages. Peritoneal macrophages were stimulated with co-cultured with osteoblasts in the presence of 1,25(OH2 vitamin D3, and thereafter stained with tartrate-resistant acid phosphatase staining solution. The multinucleated osteoclast formation was severely impaired in the peritoneal macrophages isolated from the CD98hc-defect mice compared with those from wild-type mice. CD98hc mediates integrin signaling and amino acid transport through the CD98 light chain (CD98lc. In integrin signaling, suppression of the M-CSF-RANKL-induced phosphorylation of ERK, Akt, JNK and p130Cas were observed at the triggering phase in the CD98h-defect peritoneal macrophages. Moreover, we showed that the general control non-derepressible (GCN pathway, which was activated by amino acid starvation, was induced by the CD98hc-defect peritoneal macrophages stimulated with RANKL. These results indicate that CD98 plays two important roles in osteoclast formation through integrin signaling and amino acid transport.

Arctigenin suppresses receptor activator of nuclear factor κB ligand (RANKL)-mediated osteoclast differentiation in bone marrow-derived macrophages.

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Kim, A-Ram; Kim, Hyuk Soon; Lee, Jeong Min; Choi, Jung Ho; Kim, Se Na; Kim, Do Kyun; Kim, Ji Hyung; Mun, Se Hwan; Kim, Jie Wan; Jeon, Hyun Soo; Kim, Young Mi; Choi, Wahn Soo

2012-05-05

Osteoclasts, multinucleated bone-resorbing cells, are closely associated with bone diseases such as rheumatoid arthritis and osteoporosis. Osteoclasts are derived from hematopoietic precursor cells, and their differentiation is mediated by two cytokines, including macrophage colony stimulating factor and receptor activator of nuclear factor κB ligand (RANKL). Previous studies have shown that arctigenin exhibits an anti-inflammatory effect. However, the effect of arctigenin on osteoclast differentiation is yet to be elucidated. In this study, we found that arctigenin inhibited RANKL-mediated osteoclast differentiation in bone marrow macrophages in a dose-dependent manner and suppressed RANKL-mediated bone resorption. Additionally, the expression of typical marker proteins, such as NFATc1, c-Fos, TRAF6, c-Src, and cathepsin K, were significantly inhibited. Arctigenin inhibited the phosphorylation of Erk1/2, but not p38 and JNK, in a dose-dependent manner. Arctigenin also dramatically suppressed immunoreceptor tyrosine-based activation motif-mediated costimulatory signaling molecules, including Syk and PLCγ2, and Gab2. Notably, arctigenin inhibited the activation of Syk through RANKL stimulation. Furthermore, arctigenin prevented osteoclast differentiation in the calvarial bone of mice following stimulation with lipopolysaccharide. Our results show that arctigenin inhibits osteoclast differentiation in vitro and in vivo. Therefore, arctigenin may be useful for treating rheumatoid arthritis and osteoporosis. Copyright © 2012 Elsevier B.V. All rights reserved.

Involvement of multiple myeloma cell-derived exosomes in osteoclast differentiation

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Raimondi, Lavinia; De Luca, Angela; Amodio, Nicola; Manno, Mauro; Raccosta, Samuele; Taverna, Simona; Bellavia, Daniele; Naselli, Flores; Fontana, Simona; Schillaci, Odessa; Giardino, Roberto; Fini, Milena; Tassone, Pierfrancesco; Santoro, Alessandra; De Leo, Giacomo; Giavaresi, Gianluca; Alessandro, Riccardo

2015-01-01

Bone disease is the most frequent complication in multiple myeloma (MM) resulting in osteolytic lesions, bone pain, hypercalcemia and renal failure. In MM bone disease the perfect balance between bone-resorbing osteoclasts (OCs) and bone-forming osteoblasts (OBs) activity is lost in favour of OCs, thus resulting in skeletal disorders. Since exosomes have been described for their functional role in cancer progression, we here investigate whether MM cell-derived exosomes may be involved in OCs differentiation. We show that MM cells produce exosomes which are actively internalized by Raw264.7 cell line, a cellular model of osteoclast formation. MM cell-derived exosomes positively modulate pre-osteoclast migration, through the increasing of CXCR4 expression and trigger a survival pathway. MM cell-derived exosomes play a significant pro-differentiative role in murine Raw264.7 cells and human primary osteoclasts, inducing the expression of osteoclast markers such as Cathepsin K (CTSK), Matrix Metalloproteinases 9 (MMP9) and Tartrate-resistant Acid Phosphatase (TRAP). Pre-osteoclast treated with MM cell-derived exosomes differentiate in multinuclear OCs able to excavate authentic resorption lacunae. Similar results were obtained with exosomes derived from MM patient's sera. Our data indicate that MM-exosomes modulate OCs function and differentiation. Further studies are needed to identify the OCs activating factors transported by MM cell-derived exosomes. PMID:25944696

Arctigenin inhibits osteoclast differentiation and function by suppressing both calcineurin-dependent and osteoblastic cell-dependent NFATc1 pathways.

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Yamashita, Teruhito; Uehara, Shunsuke; Udagawa, Nobuyuki; Li, Feng; Kadota, Shigetoshi; Esumi, Hiroyasu; Kobayashi, Yasuhiro; Takahashi, Naoyuki

2014-01-01

Arctigenin, a lignan-derived compound, is a constituent of the seeds of Arctium lappa. Arctigenin was previously shown to inhibit osteoclastogenesis; however, this inhibitory mechanism has yet to be elucidated. Here, we showed that arctigenin inhibited the action of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a key transcription factor for osteoclastogenesis. NFATc1 in osteoclast precursors was activated through two distinct pathways: the calcineurin-dependent and osteoblastic cell-dependent pathways. Among the several lignan-derived compounds examined, arctigenin most strongly inhibited receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast-like cell formation in mouse bone marrow macrophage (BMM) cultures, in which the calcineurin-dependent NFATc1 pathway was activated. Arctigenin suppressed neither the activation of nuclear factor κB and mitogen-activated protein kinases nor the up-regulation of c-Fos expression in BMMs treated with RANKL. However, arctigenin suppressed RANKL-induced NFATc1 expression. Interestingly, the treatment of osteoclast-like cells with arctigenin converted NFATc1 into a lower molecular weight species, which was translocated into the nucleus even in the absence of RANKL. Nevertheless, arctigenin as well as cyclosporin A (CsA), a calcineurin inhibitor, suppressed the NFAT-luciferase reporter activity induced by ionomycin and phorbol 12-myristate 13-acetate in BMMs. Chromatin immunoprecipitation analysis confirmed that arctigenin inhibited the recruitment of NFATc1 to the promoter region of the NFATc1 target gene. Arctigenin, but not CsA suppressed osteoclast-like cell formation in co-cultures of osteoblastic cells and bone marrow cells, in which the osteoblastic cell-dependent NFATc1 pathway was activated. The forced expression of constitutively active NFATc1 rescued osteoclastogenesis in BMM cultures treated with CsA, but not that treated with arctigenin. Arctigenin also suppressed the pit

Arctigenin Inhibits Osteoclast Differentiation and Function by Suppressing Both Calcineurin-Dependent and Osteoblastic Cell-Dependent NFATc1 Pathways

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Yamashita, Teruhito; Uehara, Shunsuke; Udagawa, Nobuyuki; Li, Feng; Kadota, Shigetoshi; Esumi, Hiroyasu; Kobayashi, Yasuhiro; Takahashi, Naoyuki

2014-01-01

Arctigenin, a lignan-derived compound, is a constituent of the seeds of Arctium lappa. Arctigenin was previously shown to inhibit osteoclastogenesis; however, this inhibitory mechanism has yet to be elucidated. Here, we showed that arctigenin inhibited the action of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a key transcription factor for osteoclastogenesis. NFATc1 in osteoclast precursors was activated through two distinct pathways: the calcineurin-dependent and osteoblastic cell-dependent pathways. Among the several lignan-derived compounds examined, arctigenin most strongly inhibited receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast-like cell formation in mouse bone marrow macrophage (BMM) cultures, in which the calcineurin-dependent NFATc1 pathway was activated. Arctigenin suppressed neither the activation of nuclear factor κB and mitogen-activated protein kinases nor the up-regulation of c-Fos expression in BMMs treated with RANKL. However, arctigenin suppressed RANKL-induced NFATc1 expression. Interestingly, the treatment of osteoclast-like cells with arctigenin converted NFATc1 into a lower molecular weight species, which was translocated into the nucleus even in the absence of RANKL. Nevertheless, arctigenin as well as cyclosporin A (CsA), a calcineurin inhibitor, suppressed the NFAT-luciferase reporter activity induced by ionomycin and phorbol 12-myristate 13-acetate in BMMs. Chromatin immunoprecipitation analysis confirmed that arctigenin inhibited the recruitment of NFATc1 to the promoter region of the NFATc1 target gene. Arctigenin, but not CsA suppressed osteoclast-like cell formation in co-cultures of osteoblastic cells and bone marrow cells, in which the osteoblastic cell-dependent NFATc1 pathway was activated. The forced expression of constitutively active NFATc1 rescued osteoclastogenesis in BMM cultures treated with CsA, but not that treated with arctigenin. Arctigenin also suppressed the pit

Arctigenin inhibits osteoclast differentiation and function by suppressing both calcineurin-dependent and osteoblastic cell-dependent NFATc1 pathways.

Directory of Open Access Journals (Sweden)

Teruhito Yamashita

Full Text Available Arctigenin, a lignan-derived compound, is a constituent of the seeds of Arctium lappa. Arctigenin was previously shown to inhibit osteoclastogenesis; however, this inhibitory mechanism has yet to be elucidated. Here, we showed that arctigenin inhibited the action of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1, a key transcription factor for osteoclastogenesis. NFATc1 in osteoclast precursors was activated through two distinct pathways: the calcineurin-dependent and osteoblastic cell-dependent pathways. Among the several lignan-derived compounds examined, arctigenin most strongly inhibited receptor activator of nuclear factor κB ligand (RANKL-induced osteoclast-like cell formation in mouse bone marrow macrophage (BMM cultures, in which the calcineurin-dependent NFATc1 pathway was activated. Arctigenin suppressed neither the activation of nuclear factor κB and mitogen-activated protein kinases nor the up-regulation of c-Fos expression in BMMs treated with RANKL. However, arctigenin suppressed RANKL-induced NFATc1 expression. Interestingly, the treatment of osteoclast-like cells with arctigenin converted NFATc1 into a lower molecular weight species, which was translocated into the nucleus even in the absence of RANKL. Nevertheless, arctigenin as well as cyclosporin A (CsA, a calcineurin inhibitor, suppressed the NFAT-luciferase reporter activity induced by ionomycin and phorbol 12-myristate 13-acetate in BMMs. Chromatin immunoprecipitation analysis confirmed that arctigenin inhibited the recruitment of NFATc1 to the promoter region of the NFATc1 target gene. Arctigenin, but not CsA suppressed osteoclast-like cell formation in co-cultures of osteoblastic cells and bone marrow cells, in which the osteoblastic cell-dependent NFATc1 pathway was activated. The forced expression of constitutively active NFATc1 rescued osteoclastogenesis in BMM cultures treated with CsA, but not that treated with arctigenin. Arctigenin also suppressed the

Tumor cell anaplasia and multinucleation are predictors of disease recurrence in oropharyngeal squamous cell carcinoma, including among just the human papillomavirus-related cancers.

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Lewis, James S; Scantlebury, Juliette B; Luo, Jingqin; Thorstad, Wade L

2012-07-01

Oropharyngeal squamous cell carcinoma (SCC) is frequently related to high risk human papillomavirus. This tumor expresses p16, frequently has a nonkeratinizing morphology, and has improved outcomes. Despite having a good prognosis, tumors can have focal or diffuse nuclear anaplasia or multinucleation, the significance of which is unknown. From a database of 270 oropharyngeal SCCs with known histologic typing (using our established system) and p16 immunohistochemistry, all surgically resected cases (149) were reviewed. Anaplasia was defined as any × 40 field with ≥ 3 tumor nuclei with diameters ≥ 5 lymphocyte nuclei (~25 μm), and multinucleation was defined as any × 40 field with ≥ 3 tumor cells with multiple nuclei. p16 was positive in 128 cases (85.9%), 64 cases (43.0%) showed anaplasia, and 71 (47.7%) showed multinucleation. Anaplasia and multinucleation were highly related (Panaplasia or multinucleation had worse overall, disease-specific, and disease-free survival (Panaplasia and multinucleation both predicted worse disease-specific survival (hazard ratio 9.9, P=0.04; and hazard ratio 11.9, P=0.02, respectively) independent of the other variables. In summary, among surgically resectable oropharyngeal SCC (including among just the p16-positive cohort), tumor cell anaplasia and multinucleation independently correlated with disease recurrence and poorer survival.

Absence of p53 in Clara cells favours multinucleation and loss of cell cycle arrest

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Clarke Alan R

2002-11-01

Full Text Available Abstract Background The p53 oncosuppressor protein is a critical mediator of the response to injury in mammalian cells and is mutationally inactivated in the majority of lung malignancies. In this analysis, the effects of p53-deficiency were investigated in short-term primary cultures of murine bronchiolar Clara cells. Clara cells, isolated from gene-targeted p53-deficient mice, were compared to cells derived from wild type littermates. Results p53 null cultures displayed abnormal morphology; specifically, a high incidence of multinucleation, which increased with time in culture. Multinucleated cells were proficient in S phase DNA synthesis, as determined by BrdU incorporation. However, multinucleation did not reflect altered rates of S phase synthesis, which were similar between wild type and p53-/- cultures. Nucleation defects in p53-/- Clara cells associated with increased centrosome number, as determined by confocal microscopy of pericentrin-stained cultures, and may highlight a novel role of p53 in preserving genomic integrity in lung epithelial cells. Effects of p53-deficiency were also studied following exposure to DNA damage. A p53-dependent reduction in the BrdU index was observed in Clara cells following ionizing radiation. The reduction in BrdU index in wild type cells displayed serum-dependency, and occurred only in the absence of serum. Taken together, these findings demonstrate that in murine primary Clara cell culture, cell cycle arrest is a p53-mediated response to DNA damage, and that extracellular factors, such as serum, influence this response. Conclusion These findings highlight functions of wild type p53 protein in bipolar spindle formation, centrosome regulation, and growth control in bronchiolar Clara cells.

Identification of a subpopulation of marrow MSC-derived medullary adipocytes that express osteoclast-regulating molecules: marrow adipocytes express osteoclast mediators.

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Vance Holt

Full Text Available Increased marrow medullary adipogenesis and an associated decrease in bone mineral density, usually observed in elderly individuals, is a common characteristic in senile osteoporosis. In this study we investigated whether cells of the medullary adipocyte lineage have the potential to directly support the formation of osteoclasts, whose activity in bone leads to bone degradation. An in vitro mesenchymal stem cell (MSC-derived medullary adipocyte lineage culture model was used to study the expression of the important osteoclast mediators RANKL, M-CSF, SDF-1, and OPG. We further assessed whether adipocytes at a specific developmental stage were capable of supporting osteoclast-like cell formation in culture. In vitro MSC-derived medullary adipocytes showed an mRNA and protein expression profile of M-CSF, RANKL, and OPG that was dependent on its developmental/metabolic stage. Furthermore, RANKL expression was observed in MSC-derived adipocytes that were at a distinct lineage stage and these cells were also capable of supporting osteoclast-like cell formation in co-cultures with peripheral blood mononuclear cells. These results suggest a connection between medullary adipocytes and osteoclast formation in vivo and may have major significance in regards to the mechanisms of decreased bone density in senile osteoporosis.

The two inverse pathways: the apoptosis and the multinucleated cell switched by the magnesium/zinc balance in irradiated Sarcoma in vivo

International Nuclear Information System (INIS)

Harada, S.; Sera, K.; Futatsugawa, S.; Yanagisawa, T.; Takahashi, T.; Tamakawa, Y.

1999-01-01

The Mg dependence on apoptosis, and the Zn dependence on the multinucleated cell were evaluated in irradiated Sarcoma-180 in vivo in BALB/c mice. The kinetics of total concentration of Mg strongly correlated with the frequency of apoptosis from 6 to 24 h after irradiation and that of Zn strongly correlated with the frequency of the multinucleated cell from 12 to 48 h after irradiation under the 10 or 20 Gy irradiation. There was an inverse proportion of frequency of apoptosis to that of the multinucleated cell. The administration of Mg significantly increased the frequency of apoptosis and significantly decreased the frequency of the multinucleated cell, from 6 to 12 h after the 20 Gy irradiation. In contrast, the administration of Zn significantly increased the frequency of the multinucleated cell and significantly decreased the frequency of apoptosis from 6 to 12 h under the 20 Gy irradiation. The combined administration of Mg and Zn did not change the frequency of apoptosis and the multinucleated cell. The administration of these elements did not change those correlation coefficients. The two inverse pathways were considered: the one for apoptosis; and another for the multinuclear cell, switched by the Mg/Zn balance

A novel phthalimide derivative, TC11, has preclinical effects on high-risk myeloma cells and osteoclasts.

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Maiko Matsushita

Full Text Available Despite the recent advances in the treatment of multiple myeloma (MM, MM patients with high-risk cytogenetic changes such as t(4;14 translocation or deletion of chromosome 17 still have extremely poor prognoses. With the goal of helping these high-risk MM patients, we previously developed a novel phthalimide derivative, TC11. Here we report the further characterization of TC11 including anti-myeloma effects in vitro and in vivo, a pharmacokinetic study in mice, and anti-osteoclastogenic activity. Intraperitoneal injections of TC11 significantly delayed the growth of subcutaneous tumors in human myeloma-bearing SCID mice. Immunohistochemical analyses showed that TC11 induced apoptosis of MM cells in vivo. In the pharmacokinetic analyses, the Cmax was 2.1 μM at 1 h after the injection of TC11, with 1.2 h as the half-life. TC11 significantly inhibited the differentiation and function of tartrate-resistant acid phosphatase (TRAP-positive multinucleated osteoclasts in mouse osteoclast cultures using M-CSF and RANKL. We also revealed that TC11 induced the apoptosis of myeloma cells accompanied by α-tubulin fragmentation. In addition, TC11 and lenalidomide, another phthalimide derivative, directly bound to nucleophosmin 1 (NPM1, whose role in MM is unknown. Thus, through multiple molecular interactions, TC11 is a potentially effective drug for high-risk MM patients with bone lesions. The present results suggest the possibility of the further development of novel thalidomide derivatives by drug designing.

Bropirimine inhibits osteoclast differentiation through production of interferon-β

International Nuclear Information System (INIS)

Suzuki, Hiroaki; Mochizuki, Ayako; Yoshimura, Kentaro; Miyamoto, Yoichi; Kaneko, Kotaro; Inoue, Tomio; Chikazu, Daichi; Takami, Masamichi; Kamijo, Ryutaro

2015-01-01

Bropirimine is a synthetic agonist for toll-like receptor 7 (TLR7). In this study, we investigated the effects of bropirimine on differentiation and bone-resorbing activity of osteoclasts in vitro. Bropirimine inhibited osteoclast differentiation of mouse bone marrow-derived macrophages (BMMs) induced by receptor activator of nuclear factor κB ligand (RANKL) in a concentration-dependent manner. Furthermore, it suppressed the mRNA expression of nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1), a master transcription factor for osteoclast differentiation, without affecting BMM viability. Bropirimine also inhibited osteoclast differentiation induced in co-cultures of mouse bone marrow cells (BMCs) and mouse osteoblastic UAMS-32 cells in the presence of activated vitamin D_3. Bropirimine partially suppressed the expression of RANKL mRNA in UAMS-32 cells induced by activated vitamin D_3. Finally, the anti-interferon-β (IFN-β) antibody restored RANKL-dependent differentiation of BMMs into osteoclasts suppressed by bropirimine. These results suggest that bropirimine inhibits differentiation of osteoclast precursor cells into osteoclasts via TLR7-mediated production of IFN-β.

Bropirimine inhibits osteoclast differentiation through production of interferon-β

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Suzuki, Hiroaki [Department of Biochemistry, Showa University School of Dentistry, Tokyo 142-8555 (Japan); Mochizuki, Ayako [Department of Oral Physiology, Showa University School of Dentistry, Tokyo 142-8555 (Japan); Yoshimura, Kentaro; Miyamoto, Yoichi [Department of Biochemistry, Showa University School of Dentistry, Tokyo 142-8555 (Japan); Kaneko, Kotaro [Department of Biochemistry, Showa University School of Dentistry, Tokyo 142-8555 (Japan); Department of Oral and Maxillofacial Surgery, Tokyo Medical University, Tokyo 160-0023 (Japan); Inoue, Tomio [Department of Oral Physiology, Showa University School of Dentistry, Tokyo 142-8555 (Japan); Chikazu, Daichi [Department of Oral and Maxillofacial Surgery, Tokyo Medical University, Tokyo 160-0023 (Japan); Takami, Masamichi [Department of Pharmacology, Showa University School of Dentistry, Tokyo 142-8555 (Japan); Kamijo, Ryutaro, E-mail: kamijor@dent.showa-u.ac.jp [Department of Biochemistry, Showa University School of Dentistry, Tokyo 142-8555 (Japan)

2015-11-06

Bropirimine is a synthetic agonist for toll-like receptor 7 (TLR7). In this study, we investigated the effects of bropirimine on differentiation and bone-resorbing activity of osteoclasts in vitro. Bropirimine inhibited osteoclast differentiation of mouse bone marrow-derived macrophages (BMMs) induced by receptor activator of nuclear factor κB ligand (RANKL) in a concentration-dependent manner. Furthermore, it suppressed the mRNA expression of nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1), a master transcription factor for osteoclast differentiation, without affecting BMM viability. Bropirimine also inhibited osteoclast differentiation induced in co-cultures of mouse bone marrow cells (BMCs) and mouse osteoblastic UAMS-32 cells in the presence of activated vitamin D{sub 3}. Bropirimine partially suppressed the expression of RANKL mRNA in UAMS-32 cells induced by activated vitamin D{sub 3}. Finally, the anti-interferon-β (IFN-β) antibody restored RANKL-dependent differentiation of BMMs into osteoclasts suppressed by bropirimine. These results suggest that bropirimine inhibits differentiation of osteoclast precursor cells into osteoclasts via TLR7-mediated production of IFN-β.

"Ruffled border" formation on a CaP-free substrate: A first step towards osteoclast-recruiting bone-grafts materials able to re-establish bone turn-over.

Science.gov (United States)

Merolli, Antonio; Fung, Stephanie; Murthy, N Sanjeeva; Pashuck, E Thomas; Mao, Yong; Wu, Xiaohuan; Steele, Joseph A M; Martin, Daniel; Moghe, Prabhas V; Bromage, Timothy; Kohn, Joachim

2018-03-21

Osteoclasts are large multinucleated giant cells that actively resorb bone during the physiological bone turnover (BTO), which is the continuous cycle of bone resorption (by osteoclasts) followed by new bone formation (by osteoblasts). Osteoclasts secrete chemotactic signals to recruit cells for regeneration of vasculature and bone. We hypothesize that a biomaterial that attracts osteoclasts and re-establishes BTO will induce a better healing response than currently used bone graft materials. While the majority of bone regeneration efforts have focused on maximizing bone deposition, the novelty in this approach is the focus on stimulating osteoclastic resorption as the starter for BTO and its concurrent new vascularized bone formation. A biodegradable tyrosine-derived polycarbonate, E1001(1k), was chosen as the polymer base due to its ability to support bone regeneration in vivo. The polymer was functionalized with a RGD peptide or collagen I, or blended with β-tricalcium phosphate. Osteoclast attachment and early stages of active resorption were observed on all substrates. The transparency of E1001(1k) in combination with high resolution confocal imaging enabled visualization of morphological features of osteoclast activation such as the formation of the "actin ring" and the "ruffled border", which previously required destructive forms of imaging such as transmission electron microscopy. The significance of these results is twofold: (1) E1001(1k) is suitable for osteoclast attachment and supports osteoclast maturation, making it a base polymer that can be further modified to optimize stimulation of BTO and (2) the transparency of this polymer makes it a suitable analytical tool for studying osteoclast behavior.

Influence of surface microstructure and chemistry on osteoinduction and osteoclastogenesis by biphasic calcium phosphate discs

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NL Davison

2015-06-01

Full Text Available It has been reported that surface microstructural dimensions can influence the osteoinductivity of calcium phosphates (CaPs, and osteoclasts may play a role in this process. We hypothesised that surface structural dimensions of ≤ 1 μm trigger osteoinduction and osteoclast formation irrespective of macrostructure (e.g., concavities, interconnected macropores, interparticle space or surface chemistry. To test this, planar discs made of biphasic calcium phosphate (BCP: 80 % hydroxyapatite, 20 % tricalcium phosphate were prepared with different surface structural dimensions – either ~ 1 μm (BCP1150 or ~ 2-4 μm (BCP1300 – and no macropores or concavities. A third material was made by sputter coating BCP1150 with titanium (BCP1150Ti, thereby changing its surface chemistry but preserving its surface structure and chemical reactivity. After intramuscular implantation in 5 dogs for 12 weeks, BCP1150 formed ectopic bone in 4 out of 5 samples, BCP1150Ti formed ectopic bone in 3 out of 5 samples, and BCP1300 formed no ectopic bone in any of the 5 samples. In vivo, large multinucleated osteoclast-like cells densely colonised BCP1150, smaller osteoclast-like cells formed on BCP1150Ti, and osteoclast-like cells scarcely formed on BCP1300. In vitro, RAW264.7 cells cultured on the surface of BCP1150 and BCP1150Ti in the presence of osteoclast differentiation factor RANKL (receptor activator for NF-κB ligand proliferated then differentiated into multinucleated osteoclast-like cells with positive tartrate resistant acid phosphatase (TRAP activity. However, cell proliferation, fusion, and TRAP activity were all significantly inhibited on BCP1300. These results indicate that of the material parameters tested – namely, surface microstructure, macrostructure, and surface chemistry – microstructural dimensions are critical in promoting osteoclastogenesis and triggering ectopic bone formation.

Osteoclastic giant cell tumor of the pancreas: an immunohistochemical study

DEFF Research Database (Denmark)

Dizon, M A; Multhaupt, H A; Paskin, D L

1996-01-01

A case of an osteoclastic giant cell tumor of the pancreas is presented. Immunohistochemical studies were performed, which showed keratin (CAM, AE1) and epithelial membrane antigen positivity in the tumor cells. The findings support an epithelial origin for this tumor.......A case of an osteoclastic giant cell tumor of the pancreas is presented. Immunohistochemical studies were performed, which showed keratin (CAM, AE1) and epithelial membrane antigen positivity in the tumor cells. The findings support an epithelial origin for this tumor....

The Effects of Aronia melanocarpa 'Viking' Extracts in Attenuating RANKL-Induced Osteoclastic Differentiation by Inhibiting ROS Generation and c-FOS/NFATc1 Signaling.

Science.gov (United States)

Ghosh, Mithun; Kim, In Sook; Lee, Young Min; Hong, Seong Min; Lee, Taek Hwan; Lim, Ji Hong; Debnath, Trishna; Lim, Beong Ou

2018-03-08

This study aimed to determine the anti-osteoclastogenic effects of extracts from Aronia melanocarpa 'Viking' (AM) and identify the underlying mechanisms in vitro. Reactive oxygen species (ROS) are signal mediators in osteoclast differentiation. AM extracts inhibited ROS production in RAW 264.7 cells in a dose-dependent manner and exhibited strong radical scavenging activity. The extracts also attenuated the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts. To attain molecular insights, the effect of the extracts on the signaling pathways induced by receptor activator of nuclear factor kappa B ligand (RANKL) were also investigated. RANKL triggers many transcription factors through the activation of mitogen-activated protein kinase (MAPK) and ROS, leading to the induction of osteoclast-specific genes. The extracts significantly suppressed RANKL-induced activation of MAPKs, such as extracellular signal-regulated kinase (ERK), c-Jun- N -terminal kinase (JNK) and p38 and consequently led to the downregulation of c-Fos and nuclear factor of activated T cells 1 (NFATc1) protein expression which ultimately suppress the activation of the osteoclast-specific genes, cathepsin K, TRAP, calcitonin receptor and integrin β₃. In conclusion, our findings suggest that AM extracts inhibited RANKL-induced osteoclast differentiation by downregulating ROS generation and inactivating JNK/ERK/p38, nuclear factor kappa B (NF-κB)-mediated c-Fos and NFATc1 signaling pathway.

Mitotic Stress Is an Integral Part of the Oncogene-Induced Senescence Program that Promotes Multinucleation and Cell Cycle Arrest

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Dina Dikovskaya

2015-09-01

Full Text Available Oncogene-induced senescence (OIS is a tumor suppression mechanism that blocks cell proliferation in response to oncogenic signaling. OIS is frequently accompanied by multinucleation; however, the origin of this is unknown. Here, we show that multinucleate OIS cells originate mostly from failed mitosis. Prior to senescence, mutant H-RasV12 activation in primary human fibroblasts compromised mitosis, concordant with abnormal expression of mitotic genes functionally linked to the observed mitotic spindle and chromatin defects. Simultaneously, H-RasV12 activation enhanced survival of cells with damaged mitoses, culminating in extended mitotic arrest and aberrant exit from mitosis via mitotic slippage. ERK-dependent transcriptional upregulation of Mcl1 was, at least in part, responsible for enhanced survival and slippage of cells with mitotic defects. Importantly, mitotic slippage and oncogene signaling cooperatively induced senescence and key senescence effectors p21 and p16. In summary, activated Ras coordinately triggers mitotic disruption and enhanced cell survival to promote formation of multinucleate senescent cells.

The Effects of Aronia melanocarpa ‘Viking’ Extracts in Attenuating RANKL-Induced Osteoclastic Differentiation by Inhibiting ROS Generation and c-FOS/NFATc1 Signaling

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Mithun Ghosh

2018-03-01

Full Text Available This study aimed to determine the anti-osteoclastogenic effects of extracts from Aronia melanocarpa ‘Viking’ (AM and identify the underlying mechanisms in vitro. Reactive oxygen species (ROS are signal mediators in osteoclast differentiation. AM extracts inhibited ROS production in RAW 264.7 cells in a dose-dependent manner and exhibited strong radical scavenging activity. The extracts also attenuated the number of tartrate-resistant acid phosphatase (TRAP-positive multinucleated osteoclasts. To attain molecular insights, the effect of the extracts on the signaling pathways induced by receptor activator of nuclear factor kappa B ligand (RANKL were also investigated. RANKL triggers many transcription factors through the activation of mitogen-activated protein kinase (MAPK and ROS, leading to the induction of osteoclast-specific genes. The extracts significantly suppressed RANKL-induced activation of MAPKs, such as extracellular signal-regulated kinase (ERK, c-Jun-N-terminal kinase (JNK and p38 and consequently led to the downregulation of c-Fos and nuclear factor of activated T cells 1 (NFATc1 protein expression which ultimately suppress the activation of the osteoclast-specific genes, cathepsin K, TRAP, calcitonin receptor and integrin β3. In conclusion, our findings suggest that AM extracts inhibited RANKL-induced osteoclast differentiation by downregulating ROS generation and inactivating JNK/ERK/p38, nuclear factor kappa B (NF-κB-mediated c-Fos and NFATc1 signaling pathway.

Nanocrystallinity effects on osteoblast and osteoclast response to silicon substituted hydroxyapatite.

Science.gov (United States)

Casarrubios, Laura; Matesanz, María Concepción; Sánchez-Salcedo, Sandra; Arcos, Daniel; Vallet-Regí, María; Portolés, María Teresa

2016-11-15

Silicon substituted hydroxyapatites (SiHA) are highly crystalline bioceramics treated at high temperatures (about 1200°C) which have been approved for clinical use with spinal, orthopedic, periodontal, oral and craniomaxillofacial applications. The preparation of SiHA with lower temperature methods (about 700°C) provides nanocrystalline SiHA (nano-SiHA) with enhanced bioreactivity due to higher surface area and smaller crystal size. The aim of this study has been to know the nanocrystallinity effects on the response of both osteoblasts and osteoclasts (the two main cell types involved in bone remodelling) to silicon substituted hydroxyapatite. Saos-2 osteoblasts and osteoclast-like cells (differentiated from RAW-264.7 macrophages) have been cultured on the surface of nano-SiHA and SiHA disks and different cell parameters have been evaluated: cell adhesion, proliferation, viability, intracellular content of reactive oxygen species, cell cycle phases, apoptosis, cell morphology, osteoclast-like cell differentiation and resorptive activity. This comparative in vitro study evidences that nanocrystallinity of SiHA affects the cell/biomaterial interface inducing bone cell apoptosis by loss of cell anchorage (anoikis), delaying osteoclast-like cell differentiation and decreasing the resorptive activity of this cell type. These results suggest the potential use of nano-SiHA biomaterial for preventing bone resorption in treatment of osteoporotic bone. Copyright © 2016 Elsevier Inc. All rights reserved.

Tetraspanin CD9 regulates osteoclastogenesis via regulation of p44/42 MAPK activity

International Nuclear Information System (INIS)

Yi, TacGhee; Kim, Hye-Jin; Cho, Je-Yoel; Woo, Kyung Mi; Ryoo, Hyun-Mo; Kim, Gwan-Shik; Baek, Jeong-Hwa

2006-01-01

Tetraspanin CD9 has been shown to regulate cell-cell fusion in sperm-egg fusion and myotube formation. However, the role of CD9 in osteoclast, another multinucleated cell type, is not still clear. Therefore, we investigated the role of CD9 in osteoclast differentiation. CD9 was expressed in osteoclast lineage cells and its expression level increased during the progression of RANKL-induced osteoclastogenesis. KMC8, a neutralizing antibody specific to CD9, significantly suppressed RANKL-induced multinucleated osteoclast formation and the mRNA expression of osteoclast differentiation marker genes. To define CD9-regulated osteoclastogenic signaling pathway, MAPK pathways were examined. KMC8 induced long-term phosphorylation of p44/42 MAPK, but not of p38 MAPK. Constitutive activation of p44/42 MAPK by overexpressing constitutive-active mutant of MEK1 almost completely blocked osteoclast differentiation. Taken together, these results suggest that CD9 expressed on osteoclast lineage cells might positively regulate osteoclastogenesis via the regulation of p44/42 MAPK activity

A diagnostic dilemma in breast pathology – benign fibroadenoma with multinucleated stromal giant cells

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Tobbia Igdam

2008-08-01

Full Text Available Abstract Fibroadenomas are common benign breast tumours that display a characteristic pathological morphology, although several epithelial and stromal variations exist. A very rare histological finding is the presence of multinucleated giant cells throughout the stroma of a benign fibroadenoma. Cells of this type, which are more commonly found incidentally within the interlobular stroma of breast tissue, are benign and should not be mistaken for malignant cells on microscopic examination. Unfortunately a lack of awareness of this pathological entity can lead to diagnostic confusion amongst pathologists resulting in the multinucleate giant cells being mistaken for highly mitotic cells and consequently the fibroadenoma being mistaken for a malignant lesion. This may have serious implications for the subsequent management of the patient. The presence of this unusual cell type in the stroma does not alter the prognosis of otherwise benign lesion. We encountered two such cases at our institution in a six month period recently. We present their histories along with relevant radiological, microscopic and immunohistochemical features, followed by a discussion of this unusual pathological entity.

Differences in responses to X-ray exposure between osteoclast and osteoblast cells

International Nuclear Information System (INIS)

Zhang, Jian; Wang, Ziyang; Wu, Anqing; Nie, Jing; Pei, Hailong; Hu, Wentao; Wang, Bing; Shang, Peng; Li, Bingyan; Zhou, Guangming

2017-01-01

Radiation-induced bone loss is a potential health concern for cancer patients undergoing radiotherapy. Enhanced bone resorption by osteoclasts and decreased bone formation by osteoblasts were thought to be the main reasons. In this study, we showed that both pre-differentiating and differentiating osteoclasts were relatively sensitive to X-rays compared with osteoblasts. X-rays decreased cell viability to a greater degree in RAW264.7 cells and in differentiating cells than than in osteoblastic MC3T3-E1 cells. X-rays at up to 8 Gy had little effects on osteoblast mineralization. In contrast, X-rays at 1 Gy induced enhanced osteoclastogenesis by enhanced cell fusion, but had no effects on bone resorption. A higher dose of X-rays at 8 Gy, however, had an inhibitory effect on bone resorption. In addition, actin ring formation was disrupted by 8 Gy of X-rays and reorganized into clusters. An increased activity of Caspase 3 was found after X-ray exposure. Actin disorganization and increased apoptosis may be the potential effects of X-rays at high doses, by inhibiting osteoclast differentiation. Taken together, our data indicate high radiosensitivity of osteoclasts. X-ray irradiation at relatively low doses can activate osteoclastogenesis, but not osteogenic differentiation. The radiosensitive osteoclasts are the potentially responsive cells for X-ray-induced bone loss.

Osteoclast cell-surface specializations and nuclear kinetics during egg-laying in Japanese quail

International Nuclear Information System (INIS)

Miller, S.C.

1981-01-01

Medullary bone deposits serve as a reservoir of labile calcium for egg-shell calcification in birds. Quantitative transmission-electron-microscope methods and light-microscope autoradiographic cell-population-kinetic analyses were used to determine changes in cell-surface specializations and population dynamics of medullary bone osteoclasts during egg-laying in Japanese quail. Prior to egg-shell formation, from 0 to about 8 hours after the previous oviposition, very few osteoclast profiles had ruffled borders. The appearance of ruffled borders coincided with the beginning of egg-shell calcification, about 9-10 hours after the previous oviposition. During egg-shell calcification, about 10-21 hours after the previous oviposition, most osteoclast profiles had ruffled borders. Ruffled borders disappeared at the completion of egg-shell calcification and commencement of egg-shell pigmentation. Thus, functional activities of medullary bone osteoclasts appear to be closely synchronized with egg-shell calcification during egg-laying. From 1 to 48 hours after a single injection of 3H-thymidine (3H-TdR), very few labeled osteoclast nuclei were seen during egg-laying. Following multiple injections of 3H-TdR, the percentage of labeled nuclei reached a peak at about 170 hours after the first injection. At this peak-labeling time, relatively few of the osteoclast profiles that had labeled nuclei had two or more; although the average number of nuclei per osteoclast profile was about 3.6. These kinetic data suggest that the medullary bone osteoclast population has a prolonged rate of turnover compared to rapid changes in cell activities associated with each 24-hour egg-laying cycle; and collectively they would suggest that rapid changes in osteoclast functions occur independently of changes in cell-population dynamics

Melittin inhibits osteoclast formation through the downregulation of the RANKL-RANK signaling pathway and the inhibition of interleukin-1β in murine macrophages.

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Choe, Jung-Yoon; Kim, Seong-Kyu

2017-03-01

Melittin is a major toxic component of bee venom (Apis mellifera). It is not known whether melittin is involved in bone metabolism and osteoclastogenesis. The aim of this study was to determine the role of melittin in the regulation of osteoclastogenesis. In vitro osteoclastogenesis assays were performed using mouse RAW 264.7 cells and bone marrow-derived macrophages (BMMs) treated with receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Morphologic and functional analyses for osteoclast-like multinucleated cells (MNCs) were performed by tartrate-resistant acid phosphatase (TRAP) staining, F-actin staining and pit formation methods. The gene expression of TRAP, cathepsin K, matrix metalloproteinase-9 (MMP-9) and carbonic anhydrase II was measured by reverse transcription-quantitative PCR. The protein expression levels of mitogen-activated protein kinases (MAPKs), the p65 subunit of nuclear factor-κB (NF-κB), c-Fos, c-Jun, nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), TNF receptor-associated factor-6 (TRAF6), and interleukin-1β (IL-1β) were assessed by western blot analysis. Melittin inhibited the mRNA expression of TRAP, cathepsin K, MMP-9 and carbonic anhydrase II in RANKL-stimulated RAW 264.7 cells. The increased protein expression of TRAF6, p-extracellular signal-regulated kinase (ERK), p-JNK, p-p65, p-c-Fos and NFATc1 induced by RANKL was significantly suppressed in the RAW 264.7 cells treated with melittin. A synergistic effect of IL-1β on the formation of RANKL-induced osteoclast-like MNCs was found in two experimental cells. The increased expression of IL-1β following the stimulation of RAW 264.7 cells with RANKL activated TRAF6, p-ERK, p-JNK, p-p65, p-c-Fos and NFATc1. These effects were attenuated by the downregulation of IL-1β using siRNA against IL-1β, and also by treatment with melittin. On the whole, the findings of this study demonstrate that melittin

Silica nanoparticles induce multinucleation through activation of PI3K/Akt/GSK-3β pathway and downregulation of chromosomal passenger proteins in L-02 cells

Energy Technology Data Exchange (ETDEWEB)

Geng, Weijia; Li, Yang; Yu, Yongbo; Yu, Yang; Duan, Junchao; Jiang, Lizhen; Li, Qiuling; Sun, Zhiwei, E-mail: zwsun@ccmu.edu.cn, E-mail: zwsun@hotmail.com [Capital Medical University, School of Public Health (China)

2016-04-15

Silica nanoparticles (SNPs) are applicable in various fields due to their unique physicochemical characteristics. However, concerns over their potential adverse effects have been raised. In our previous studies, we reported that SNPs could induce abnormal high incidence of multinucleation. The aim of this study is to further investigate the mechanisms of multinucleation induced by SNPs (68 nm) in human normal liver L-02 cells (L-02 cells). In order to determine the cytotoxicity of SNPs, MTT assay was performed, and the cell viability was decreased in a dose-dependent manner. The intracellular reactive oxygen species (ROS) detected by flow cytometry and multinucleation observed by Giemsa stain showed that ROS generation and rate of multinucleated cells increased after SNPs exposure. N-acetyl-cysteine (NAC), a glutathione precursor against SNP-induced toxicity, was used as a ROS inhibitor to elucidate the relationship between ROS and multinucleation. The presence of NAC resulted in inhibition of both ROS generation and rate of multinucleation. Moreover, Western blot analysis showed that the protein levels of Cdc20, Aurora B, and Survivin were down-regulated, and the PI3K/Akt/GSK-3β pathway was activated by SNPs. In conclusion, our findings strongly suggested that multinucleation induced by SNPs was related to PI3K/Akt/GSK-3β signal pathway activation and downregulation of G2/M phase-related protein and chromosomal passenger proteins.

Silica nanoparticles induce multinucleation through activation of PI3K/Akt/GSK-3β pathway and downregulation of chromosomal passenger proteins in L-02 cells

Science.gov (United States)

Geng, Weijia; Li, Yang; Yu, Yongbo; Yu, Yang; Duan, Junchao; Jiang, Lizhen; Li, Qiuling; Sun, Zhiwei

2016-04-01

Silica nanoparticles (SNPs) are applicable in various fields due to their unique physicochemical characteristics. However, concerns over their potential adverse effects have been raised. In our previous studies, we reported that SNPs could induce abnormal high incidence of multinucleation. The aim of this study is to further investigate the mechanisms of multinucleation induced by SNPs (68 nm) in human normal liver L-02 cells (L-02 cells). In order to determine the cytotoxicity of SNPs, MTT assay was performed, and the cell viability was decreased in a dose-dependent manner. The intracellular reactive oxygen species (ROS) detected by flow cytometry and multinucleation observed by Giemsa stain showed that ROS generation and rate of multinucleated cells increased after SNPs exposure. N-acetyl-cysteine (NAC), a glutathione precursor against SNP-induced toxicity, was used as a ROS inhibitor to elucidate the relationship between ROS and multinucleation. The presence of NAC resulted in inhibition of both ROS generation and rate of multinucleation. Moreover, Western blot analysis showed that the protein levels of Cdc20, Aurora B, and Survivin were down-regulated, and the PI3K/Akt/GSK-3β pathway was activated by SNPs. In conclusion, our findings strongly suggested that multinucleation induced by SNPs was related to PI3K/Akt/GSK-3β signal pathway activation and downregulation of G2/M phase-related protein and chromosomal passenger proteins.

Giant cell tumor of soft tissues of low malignant potential: A rare diagnosis on fine needle aspiration cytology

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Maithili M Kulkarni

2016-01-01

Full Text Available Primary giant cell tumors of soft tissues (GCT-ST are extremely rare soft tissue tumors, located in both superficial and deep soft tissues. They resemble osseous giant cell tumors morphologically and immunohistochemically. The tumor exhibits strong positive immunoreactivity for cluster of differentiation 68 (CD68 within multinucleated osteoclast-like giant cells and focal staining of mononuclear cells. Case reports describing the cytohistological features of this entity are very few. We report a case of GCT-ST of low malignant potential diagnosed on fine needle aspiration (FNA and confirmed on histological and immunohistochemical studies.

In vitro formation of osteoclasts from long-term cultures of bone marrow mononuclear phagocytes

International Nuclear Information System (INIS)

Burger, E.H.; Van der Meer, J.W.; van de Gevel, J.S.; Gribnau, J.C.; Thesingh, G.W.; van Furth, R.

1982-01-01

The origin of osteoclasts was studied in an in vitro model using organ cultures of periosteum-free embryonic mouse long-bone primordia, which were co-cultured with various cell populations. The bone rudiments were freed of their periosteum-perichondrium by collagenase treatment in a stage before cartilage erosion and osteoclast formation, and co-cultured for 7 d with either embryonic liver or mononuclear phagocytes from various sources. Light and electron microscopic examination of the cultures showed that mineralized matrix-resorbing osteoclasts developed only in bones co-cultured with embryonic liver or with cultured bone marrow mononuclear phagocytes but not when co-cultured with blood monocytes or resident or exudate peritoneal macrophages. Osteoclasts developed from the weakly adherent, but not from the strongly adherent cells of bone marrow cultures, whereas 1,000 rad irradiation destroyed the capacity of such cultures to form osteoclasts. In bone cultures to which no other cells were added, osteoclasts were virtually absent. Bone-resorbing activity of in vitro formed osteoclasts was demonstrated by 45 Ca release studies. These studies demonstrate that osteoclasts develop from cells present in cultures of proliferating mononuclear phagocytes and that, at least in our system, monocytes and macrophages are unable to form osteoclasts. The most likely candidates for osteoclast precursor cells seem to be monoblasts and promonocytes

A comparison of osteoclast-rich and osteoclast-poor osteopetrosis in adult mice sheds light on the role of the osteoclast in coupling bone resorption and bone formation

DEFF Research Database (Denmark)

Thudium, Christian S; Moscatelli, Ilana; Flores, Carmen

2014-01-01

that osteoclasts are important for regulating osteoblast activity. To illuminate the role of the osteoclast in controlling bone remodeling, we transplanted irradiated skeletally mature 3-month old wild-type mice with hematopoietic stem cells (HSCs) to generate either an osteoclast-rich or osteoclast-poor adult......Osteopetrosis due to lack of acid secretion by osteoclasts is characterized by abolished bone resorption, increased osteoclast numbers, but normal or even increased bone formation. In contrast, osteoclast-poor osteopetrosis appears to have less osteoblasts and reduced bone formation, indicating...... osteopetrosis model. We used fetal liver HSCs from (1) oc/oc mice, (2) RANK KO mice, and (3) compared these to wt control cells. TRAP5b activity, a marker of osteoclast number and size, was increased in the oc/oc recipients, while a significant reduction was seen in the RANK KO recipients. In contrast, the bone...

Bone substitute material composition and morphology differentially modulate calcium and phosphate release through osteoclast-like cells.

Science.gov (United States)

Konermann, A; Staubwasser, M; Dirk, C; Keilig, L; Bourauel, C; Götz, W; Jäger, A; Reichert, C

2014-04-01

The aim of this study was to determine the material composition and cell-mediated remodelling of different calcium phosphate-based bone substitutes. Osteoclasts were cultivated on bone substitutes (Cerabone, Maxresorb, and NanoBone) for up to 5 days. Bafilomycin A1 addition served as the control. To determine cellular activity, the supernatant content of calcium and phosphate was measured by inductively coupled plasma optical emission spectrometry. Cells were visualized on the materials by scanning electron microscopy. Material composition and surface characteristics were assessed by energy-dispersive X-ray spectroscopy. Osteoclast-induced calcium and phosphate release was material-specific. Maxresorb exhibited the highest ion release to the medium (P = 0.034; calcium 40.25mg/l day 5, phosphate 102.08 mg/l day 5) and NanoBone the lowest (P = 0.021; calcium 8.43 mg/l day 5, phosphate 15.15 mg/l day 5); Cerabone was intermediate (P = 0.034; calcium 16.34 mg/l day 5, phosphate 30.6 mg/l day 5). All investigated materials showed unique resorption behaviours. The presented methodology provides a new perspective on the investigation of bone substitute biodegradation, maintaining the material-specific micro- and macrostructure. Copyright © 2013 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

Osteoclasts in multiple myeloma are derived from Gr-1+CD11b+myeloid-derived suppressor cells.

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Junling Zhuang

Full Text Available Osteoclasts play a key role in the development of cancer-associated osteolytic lesions. The number and activity of osteoclasts are often enhanced by tumors. However, the origin of osteoclasts is unknown. Myeloid-derived suppressor cells (MDSCs are one of the pre-metastatic niche components that are induced to expand by tumor cells. Here we show that the MDSCs can differentiate into mature and functional osteoclasts in vitro and in vivo. Inoculation of 5TGM1-GFP myeloma cells into C57BL6/KaLwRij mice led to a significant expansion of MDSCs in blood, spleen, and bone marrow over time. When grown in osteoclastogenic media in vitro, MDSCs from tumor-challenged mice displayed 14 times greater potential to differentiate into mature and functional osteoclasts than those from non-tumor controls. Importantly, MDSCs from tumor-challenged LacZ transgenic mice differentiated into LacZ+osteoclasts in vivo. Furthermore, a significant increase in tumor burden and bone loss accompanied by increased number of osteoclasts was observed in mice co-inoculated with tumor-challenged MDSCs and 5TGM1 cells compared to the control animals received 5TGM1 cells alone. Finally, treatment of MDSCs from myeloma-challenged mice with Zoledronic acid (ZA, a potent inhibitor of bone resorption, inhibited the number of osteoclasts formed in MDSC cultures and the expansion of MDSCs and bone lesions in mice. Collectively, these data provide in vitro and in vivo evidence that tumor-induced MDSCs exacerbate cancer-associated bone destruction by directly serving as osteoclast precursors.

Identification of an osteoclast transcription factor that binds to the human T cell leukemia virus type I-long terminal repeat enhancer element.

Science.gov (United States)

Inoue, D; Santiago, P; Horne, W C; Baron, R

1997-10-03

Transgenic mice expressing human T cell leukemia virus type I (HTLV-I)-tax under the control of HTLV-I-long terminal repeat (LTR) promoter develop skeletal abnormalities with high bone turnover and myelofibrosis. In these animals, Tax is highly expressed in bone with a pattern of expression restricted to osteoclasts and spindle-shaped cells within the endosteal myelofibrosis. To test the hypothesis that lineage-specific transcription factors promote transgene expression from the HTLV-I-LTR in osteoclasts, we first examined tax expression in transgenic bone marrow cultures. Expression was dependent on 1alpha,25-dihydroxycholecalciferol and coincided with tartrate-resistant acid phosphatase (TRAP) expression, a marker of osteoclast differentiation. Furthermore, Tax was expressed in vitronectin receptor-positive mononuclear precursors as well as in mature osteoclast-like cells (OCLs). Consistent with our hypothesis, electrophoretic mobility shift assays revealed the presence of an OCL nuclear factor (NFOC-1) that binds to the LTR 21-base pair direct repeat, a region critical for the promoter activity. This binding is further enhanced by Tax. Since NFOC-1 is absent in macrophages and conserved in osteoclasts among species including human, such a factor may play a role in lineage determination and/or in expression of the differentiated osteoclast phenotype.

Myeloma cell-induced disruption of bone remodelling compartments leads to osteolytic lesions and generation of osteoclast-myeloma hybrid cells

DEFF Research Database (Denmark)

Andersen, Thomas L; Søe, Kent; Søndergaard, Teis Esben

2010-01-01

on the physical organisation of the myeloma cell microenvironment. The proximity between myeloma cells and osteoclasts or osteoblasts was shown to be conditioned by the recently discovered layer of flat cells that separates the osteoclasts and osteoblasts from the bone marrow, by forming a canopy over bone...

Stem cell factor stimulates chicken osteoclast activity in vitro

NARCIS (Netherlands)

van't Hof, R. J.; von Lindern, M.; Nijweide, P. J.; Beug, H.

1997-01-01

Stem cell factor (SCF) is a polypeptide growth factor active on multiple cell types, mainly of hematopoietic origin. We studied the effects of avian SCF on the differentiation of chicken osteoclasts from their putative progenitors as well as on the bone-resorbing activity of terminally

Cell fusion in osteoclasts plays a critical role in controlling bone mass and osteoblastic activity

International Nuclear Information System (INIS)

Iwasaki, Ryotaro; Ninomiya, Ken; Miyamoto, Kana; Suzuki, Toru; Sato, Yuiko

2008-01-01

The balance between osteoclast and osteoblast activity is central for maintaining the integrity of bone homeostasis. Here we show that mice lacking dendritic cell specific transmembrane protein (DC-STAMP), an essential molecule for osteoclast cell-cell fusion, exhibited impaired bone resorption and upregulation of bone formation by osteoblasts, which do not express DC-STAMP, which led to increased bone mass. On the contrary, DC-STAMP over-expressing transgenic (DC-STAMP-Tg) mice under the control of an actin promoter showed significantly accelerated cell-cell fusion of osteoclasts and bone resorption, with decreased osteoblastic activity and bone mass. Bone resorption and formation are known to be regulated in a coupled manner, whereas DC-STAMP regulates bone homeostasis in an un-coupled manner. Thus our results indicate that inhibition of a single molecule provides both decreased osteoclast activity and increased bone formation by osteoblasts, thereby increasing bone mass in an un-coupled and a tissue specific manner.

Ebselen Is a Potential Anti-Osteoporosis Agent by Suppressing Receptor Activator of Nuclear Factor Kappa-B Ligand-Induced Osteoclast Differentiation In vitro and Lipopolysaccharide-Induced Inflammatory Bone Destruction In vivo.

Science.gov (United States)

Baek, Jong Min; Kim, Ju-Young; Yoon, Kwon-Ha; Oh, Jaemin; Lee, Myeung Su

2016-01-01

Ebselen is a non-toxic seleno-organic drug with anti-inflammatory and antioxidant properties that is currently being examined in clinical trials to prevent and treat various diseases, including atherosclerosis, stroke, and cancer. However, no reports are available for verifying the pharmacological effects of ebselen on major metabolic bone diseases such as osteoporosis. In this study, we observed that ebselen suppressed the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in an osteoblast/osteoclast co-culture by regulating the ratio of receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin secreted by osteoblasts. In addition, ebselen treatment in the early stage of osteoclast differentiation inhibited RANKL-dependent osteoclastogenesis by decreasing the phosphorylation of IκB, PI3K, and Akt in early signaling pathways and by subsequently inducing c-Fos and nuclear factor of activated T-cells c1. Further, ebselen induced apoptosis of osteoclasts in the late stage of osteoclast differentiation. In addition, ebselen treatment suppressed filamentous actin ring formation and bone resorption activity of mature osteoclasts. Reflecting these in vitro effects, administration of ebselen recovered bone loss and its µ-CT parameters in lipopolysaccharide-mediated mouse model. Histological analysis confirmed that ebselen prevented trabecular bone matrix degradation and osteoclast formation in the bone tissues. Finally, it was proved that the anti-osteoclastogenic action of ebselen is achieved through targeting N-methyl-D-aspartate (NMDA) receptor. These results indicate that ebselen is a potentially safe drug for treating metabolic bone diseases such as osteoporosis.

Osteoclast nuclei of myeloma patients show chromosome translocations specific for the myeloma cell clone: a new type of cancer-host partnership?

DEFF Research Database (Denmark)

Levin Andersen, Thomas; Boissy, Patrice; Sondergaard, T E

2007-01-01

through fusion between myeloma cells and osteoclasts. In conclusion, malignant cells contribute significantly to the formation of bone-resorbing osteoclasts in multiple myeloma. Osteoclast-myeloma clone hybrids reflect a previously unrecognized mechanism of bone destruction in which malignant cells...

[Separation of osteoclasts by lectin affinity chromatography].

Science.gov (United States)

Itokazu, M; Tan, A; Tanaka, S

1991-09-01

Newborn rat calvaria bone cells obtained by digestion were fractionated on columns of wheat-germ agglutinin (WGA) sepharose 6MB for osteoclast isolation. The initial nonspecific binding cells which were passed through the WGA sepharose column by a buffer acquired a high enzyme activity of alkaline phosphatase, but not that of acid phosphatase. However, elution of cells using a buffer with the addition of N-acetyl-D-glucosamine resulted in a high acid phosphatase activity but no alkaline phosphatase activity. The former WGA binding negative fraction enriched osteoblasts averaging 30 microns in size. The latter WGA binding positive fraction enriched osteoclasts ranging from 20 microns to 60 microns in size. The electron-microscope clearly demonstrated the cellular details of osteoclasts. Isolated cell counts showed a ratio of six to four. These results indicate that our method of osteoclast isolation is simple and useful in lectin affinity chromatography because all cells have sugar moieties on their surface and the binding of osteoclasts can be reversed by the addition of specific lectin-binding sugars to the eluting buffer.

Resveratrol inhibits myeloma cell growth, prevents osteoclast formation, and promotes osteoblast differentiation

DEFF Research Database (Denmark)

Boissy, Patrice; Andersen, Thomas L; Abdallah, Basem M

2005-01-01

, a challenge for treating multiple myeloma is discovering drugs targeting not only myeloma cells but also osteoclasts and osteoblasts. Because resveratrol (trans-3,4',5-trihydroxystilbene) is reported to display antitumor activities on a variety of human cancer cells, we investigated the effects...... of this natural compound on myeloma and bone cells. We found that resveratrol reduces dose-dependently the growth of myeloma cell lines (RPMI 8226 and OPM-2) by a mechanism involving cell apoptosis. In cultures of human primary monocytes, resveratrol inhibits dose-dependently receptor activator of nuclear factor......RNA and cell surface protein levels and a decrease of NFATc1 stimulation and NF-kappaB nuclear translocation, whereas the gene expression of c-fms, CD14, and CD11a is up-regulated. Finally, resveratrol promotes dose-dependently the expression of osteoblast markers like osteocalcin and osteopontin in human bone...

Scanning electron microscopical observation of an osteoblast/osteoclast co-culture on micropatterned orthopaedic ceramics.

Science.gov (United States)

Halai, Mansur; Ker, Andrew; Meek, Rm Dominic; Nadeem, Danish; Sjostrom, Terje; Su, Bo; McNamara, Laura E; Dalby, Matthew J; Young, Peter S

2014-01-01

In biomaterial engineering, the surface of an implant can influence cell differentiation, adhesion and affinity towards the implant. On contact with an implant, bone marrow-derived mesenchymal stromal cells demonstrate differentiation towards bone forming osteoblasts, which can improve osteointegration. The process of micropatterning has been shown to improve osteointegration in polymers, but there are few reports surrounding ceramics. The purpose of this study was to establish a co-culture of bone marrow-derived mesenchymal stromal cells with osteoclast progenitor cells and to observe the response to micropatterned zirconia toughened alumina ceramics with 30 µm diameter pits. The aim was to establish whether the pits were specifically bioactive towards osteogenesis or were generally bioactive and would also stimulate osteoclastogenesis that could potentially lead to osteolysis. We demonstrate specific bioactivity of micropatterns towards osteogenesis, with more nodule formation and less osteoclastogenesis compared to planar controls. In addition, we found that that macrophage and osteoclast-like cells did not interact with the pits and formed fewer full-size osteoclast-like cells on the pitted surfaces. This may have a role when designing ceramic orthopaedic implants.

The elementary fusion modalities of osteoclasts

DEFF Research Database (Denmark)

Søe, Kent; Hobolt-Pedersen, Anne Sofie; Delaisse, Jean Marie

2015-01-01

, are not known for the osteoclast. Here we show that osteoclast fusion partners are characterized by differences in mobility, nuclearity, and differentiation level. Our demonstration was based on time-laps videos of human osteoclast preparations from three donors where 656 fusion events were analyzed. Fusions......The last step of the osteoclast differentiation process is cell fusion. Most efforts to understand the fusion mechanism have focused on the identification of molecules involved in the fusion process. Surprisingly, the basic fusion modalities, which are well known for fusion of other cell types...... between a mobile and an immobile partner were most frequent (62%), while fusion between two mobile (26%) or two immobile partners (12%) was less frequent (p fusion partner contained more nuclei than the mobile one (p

Sr-substituted bone cements direct mesenchymal stem cells, osteoblasts and osteoclasts fate.

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Monica Montesi

Full Text Available Strontium-substituted apatitic bone cements enriched with sodium alginate were developed as a potential modulator of bone cells fate. The biological impact of the bone cement were investigated in vitro through the study of the effect of the nanostructured apatitic composition and the doping of strontium on mesenchymal stem cells, pre-osteoblasts and osteoclasts behaviours. Up to 14 days of culture the bone cells viability, proliferation, morphology and gene expression profiles were evaluated. The results showed that different concentrations of strontium were able to evoke a cell-specific response, in fact an inductive effect on mesenchymal stem cells differentiation and pre-osteoblasts proliferation and an inhibitory effect on osteoclasts activity were observed. Moreover, the apatitic structure of the cements provided a biomimetic environment suitable for bone cells growth. Therefore, the combination of biological features of this bone cement makes it as promising biomaterials for tissue regeneration.

Rare sugar D-allose strongly induces thioredoxin-interacting protein and inhibits osteoclast differentiation in Raw264 cells.

Science.gov (United States)

Yamada, Kana; Noguchi, Chisato; Kamitori, Kazuyo; Dong, Youyi; Hirata, Yuko; Hossain, Mohammad A; Tsukamoto, Ikuko; Tokuda, Masaaki; Yamaguchi, Fuminori

2012-02-01

Oxidative stress modulates the osteoclast differentiation via redox systems, and thioredoxin 1 (Trx) promotes the osteoclast formation by regulating the activity of transcription factors. The function of Trx is known to be regulated by its binding partner, thioredoxin-interacting protein (TXNIP). We previously reported that the expression of TXNIP gene is strongly induced by a rare sugar D-allose. In this study, we tested the hypothesis that D-allose could inhibit the osteoclast differentiation by regulating the Trx function. We used a murine Raw264 cell line that differentiates to the osteoclast by the receptor activator of nuclear factor-κB ligand (RANKL) treatment. The effect of sugars was evaluated by tartrate-resistant acid phosphatase staining. The expression and localization of TXNIP and Trx protein were examined by Western blotting and immunohistochemisty. The activity of the nuclear factor-κB, nuclear factor of activated T cells, and activator protein 1 transcription factors was measured by the luciferase reporter assay. The addition of D-allose (25 mmol/L) inhibited the osteoclast differentiation down to 9.53% ± 1.27% of a receptor activator of nuclear factor-κB ligand-only treatment. During the osteoclast differentiation, a significant increase of TNXIP was observed by D-allose treatment. The immunohistochemical analysis showed that both Trx and TXNIP existed in the nucleus in preosteoclasts and osteoclasts. Overexpression of TXNIP by plasmid transfection also inhibited the osteoclast formation, indicating the functional importance of TXNIP for the osteoclast differentiation. Transcriptional activity of the activator protein 1, nuclear factor-κB, and nuclear factor of activated T cells, known to be modulated by Trx, were inhibited by D-allose. In conclusion, our data indicate that D-allose is a strong inhibitor of the osteoclast differentiation, and this effect could be caused by TXNIP induction and a resulting inhibition of the Trx function

Multinucleation during C. trachomatis infections is caused by the contribution of two effector pathways.

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Heather M Brown

Full Text Available Chlamydia trachomatis is an obligate intracellular bacterial pathogen and the second leading cause of sexually transmitted infections in the US. Infections cause significant morbidity and can lead to serious reproductive sequelae, including an epidemiological link to increased rates of reproductive cancers. One of the overt changes that infected cells exhibit is the development of genomic instability leading to multinucleation. Here we demonstrate that the induction of multinucleation is not conserved equally across chlamydial species; C. trachomatis L2 caused high levels of multinucleation, C. muridarum intermediate levels, and C. caviae had very modest effects on multinucleation. Our data show that at least two effector pathways together cause genomic instability during infection leading to multinucleation. We find that the highly conserved chlamydial protease CPAF is a key effector for one of these pathways. CPAF secretion is required for the loss of centrosome duplication regulation as well as inducing early mitotic exit. The second effector pathway involves the induction of centrosome position errors. This function is not conserved in three chlamydial species tested. Together these two pathways contribute to the induction of high levels of genomic instability and multinucleation seen in C. trachomatis infections.

MCP-1 expressed by osteoclasts stimulates osteoclastogenesis in an autocrine/paracrine manner

International Nuclear Information System (INIS)

Miyamoto, Kana; Ninomiya, Ken; Sonoda, Koh-Hei; Miyauchi, Yoshiteru; Hoshi, Hiroko; Iwasaki, Ryotaro; Miyamoto, Hiroya

2009-01-01

Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that plays a critical role in the recruitment and activation of leukocytes. Here, we describe that multinuclear osteoclast formation was significantly inhibited in cells derived from MCP-1-deficient mice. MCP-1 has been implicated in the regulation of osteoclast cell-cell fusion; however defects of multinuclear osteoclast formation in the cells from mice deficient in DC-STAMP, a seven transmembrane receptor essential for osteoclast cell-cell fusion, was not rescued by recombinant MCP-1. The lack of MCP-1 in osteoclasts resulted in a down-regulation of DC-STAMP, NFATc1, and cathepsin K, all of which were highly expressed in normal osteoclasts, suggesting that osteoclast differentiation was inhibited in MCP-1-deficient cells. MCP-1 alone did not induce osteoclastogenesis, however, the inhibition of osteoclastogenesis in MCP-1-deficient cells was restored by addition of recombinant MCP-1, indicating that osteoclastogenesis was regulated in an autocrine/paracrine manner by MCP-1 under the stimulation of RANKL in osteoclasts.

Neuropeptide substance P stimulates the formation of osteoclasts via synovial fibroblastic cells

International Nuclear Information System (INIS)

Matayoshi, Takaaki; Goto, Tetsuya; Fukuhara, Eiji; Takano, Hiroshi; Kobayashi, Shigeru; Takahashi, Tetsu

2005-01-01

The present study was designed to evaluate the effects of neuropeptide substance P (Sp) on the formation of osteoclasts via synovial fibroblastic cells. Synovial fibroblastic cells derived from rat knee joint expressed the Sp receptor, neurokinin-1 receptor (NK 1 -R). The addition of Sp stimulated the proliferation of synovial fibroblastic cells and this effect was inhibited by Sp or NK 1 -R antagonists. Increased expression of the receptor activator of nuclear factor κB ligand (Rankle) in synovial fibroblastic cells after the addition of Sp was demonstrated by reverse transcriptase-polymerase chain reaction and immunofluorescence staining. Osteoprotegerin expression in synovial fibroblastic cells was decreased after incubation with SP. In co-cultures of synovial fibroblastic cells and rat peripheral blood monocytes, SP stimulated osteoclastogenesis. These results suggest that SP in the joint cavity may cause both hypertrophy of the synovium and induction of increased osteoclast formation through the increased expression of RANKL in the synovium

Genetic ablation of CD68 results in mice with increased bone and dysfunctional osteoclasts.

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Jason W Ashley

Full Text Available CD68 is a member of the lysosome associated membrane protein (LAMP family that is restricted in its expression to cells of the monocyte/macrophage lineage. This lineage restriction includes osteoclasts, and, while previous studies of CD68 in macrophages and dendritic cells have proposed roles in lipid metabolism, phagocytosis, and antigen presentation, the expression and function of CD68 in osteoclasts have not been explored. In this study, we investigated the expression and localization of CD68 in macrophages and osteoclasts in response to the monocyte/macrophage-colony stimulating factor (M-CSF and the receptor activator of NF-κB ligand (RANKL. We found that M-CSF stimulates CD68 expression and RANKL alters the apparent molecular weight of CD68 as measured by Western immunoblotting. In addition, we explored the significance of CD68 expression in osteoclasts by generating mice that lack expression of CD68. These mice have increased trabecular bone, and in vitro assessment of CD68(-/- osteoclasts revealed that, in the absence of CD68, osteoclasts demonstrate an accumulation of intracellular vesicle-like structures, and do not efficiently resorb bone. These findings demonstrate a role for CD68 in the function of osteoclasts, and future studies will determine the mechanistic nature of the defects seen in CD68(-/- osteoclasts.

Osteoclastic Giant Cell Rich Squamous Cell Carcinoma of the Uterine Cervix: A Case Report and Review of the Literature

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Lucía Alemán-Meza

2014-01-01

Full Text Available Cervical carcinoma is the most common malignancy of the female genital tract and represents the second most common malignancy in women worldwide. Histologically 85 to 90% of cervical cancers are squamous cell carcinoma. Osteoclastic giant cell rich squamous cell carcinoma is an unusual histological variant of which only 4 cases have been reported. We present the case of a 49-year-old woman with a 6-month history of irregular vaginal bleeding. Examination revealed a 2.7 cm polypoid mass in the anterior lip of the uterine cervix. The patient underwent hysterectomy with bilateral salpingo-oophorectomy. Microscopically the tumor was composed of infiltrative nests of poorly differentiated nonkeratinizing squamous cell carcinoma. Interspersed in between these tumor cells were numerous osteoclastic giant cells with abundant eosinophilic cytoplasm devoid of nuclear atypia, hyperchromatism, or mitotic activity. Immunohistochemistry was performed; CK and P63 were strongly positive in the squamous component and negative in the osteoclastic giant cells, while CD68 and Vimentin were strongly positive in the giant cell population and negative in the squamous component. The patient received chemo- and radiotherapy for recurrent disease identified 3 months later on a follow-up CT scan; 7 months after the surgical procedure the patient is clinically and radiologically disease-free.

Dlx homeobox gene family expression in osteoclasts.

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Lézot, F; Thomas, B L; Blin-Wakkach, C; Castaneda, B; Bolanos, A; Hotton, D; Sharpe, P T; Heymann, D; Carles, G F; Grigoriadis, A E; Berdal, A

2010-06-01

Skeletal growth and homeostasis require the finely orchestrated secretion of mineralized tissue matrices by highly specialized cells, balanced with their degradation by osteoclasts. Time- and site-specific expression of Dlx and Msx homeobox genes in the cells secreting these matrices have been identified as important elements in the regulation of skeletal morphology. Such specific expression patterns have also been reported in osteoclasts for Msx genes. The aim of the present study was to establish the expression patterns of Dlx genes in osteoclasts and identify their function in regulating skeletal morphology. The expression patterns of all Dlx genes were examined during the whole osteoclastogenesis using different in vitro models. The results revealed that Dlx1 and Dlx2 are the only Dlx family members with a possible function in osteoclastogenesis as well as in mature osteoclasts. Dlx5 and Dlx6 were detected in the cultures but appear to be markers of monocytes and their derivatives. In vivo, Dlx2 expression in osteoclasts was examined using a Dlx2/LacZ transgenic mouse. Dlx2 is expressed in a subpopulation of osteoclasts in association with tooth, brain, nerve, and bone marrow volumetric growths. Altogether the present data suggest a role for Dlx2 in regulation of skeletal morphogenesis via functions within osteoclasts. (c) 2010 Wiley-Liss, Inc.

Key Triggers of Osteoclast-Related Diseases and Available Strategies for Targeted Therapies: A Review

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Haidi Bi

2017-12-01

Full Text Available Osteoclasts, the only cells with bone resorption functions in vivo, maintain the balance of bone metabolism by cooperating with osteoblasts, which are responsible for bone formation. Excessive activity of osteoclasts causes many diseases such as osteoporosis, periprosthetic osteolysis, bone tumors, and Paget’s disease. In contrast, osteopetrosis results from osteoclast deficiency. Available strategies for combating over-activated osteoclasts and the subsequently induced diseases can be categorized into three approaches: facilitating osteoclast apoptosis, inhibiting osteoclastogenesis, and impairing bone resorption. Bisphosphonates are representative molecules that function by triggering osteoclast apoptosis. New drugs, such as tumor necrosis factor and receptor activator of nuclear factor kappa-B ligand (RANKL inhibitors (e.g., denosumab have been developed for targeting the receptor activator of nuclear factor kappa-B /RANKL/osteoprotegerin system or CSF-1/CSF-1R axis, which play critical roles in osteoclast formation. Furthermore, vacuolar (H+-ATPase inhibitors, cathepsin K inhibitors, and glucagon-like peptide 2 impair different stages of the bone resorption process. Recently, significant achievements have been made in this field. The aim of this review is to provide an updated summary of the current progress in research involving osteoclast-related diseases and of the development of targeted inhibitors of osteoclast formation.

Apatite-mediated actin dynamics in resorbing osteoclasts.

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Saltel, Frédéric; Destaing, Olivier; Bard, Frédéric; Eichert, Diane; Jurdic, Pierre

2004-12-01

The actin cytoskeleton is essential for osteoclasts main function, bone resorption. Two different organizations of actin have been described in osteoclasts, the podosomes belt corresponding to numerous F-actin columns arranged at the cell periphery, and the sealing zone defined as a unique large band of actin. To compare the role of these two different actin organizations, we imaged osteoclasts on various substrata: glass, dentin, and apatite. Using primary osteoclasts expressing GFP-actin, we found that podosome belts and sealing zones, both very dynamic actin structures, were present in mature osteoclasts; podosome belts were observed only in spread osteoclasts adhering onto glass, whereas sealing zone were seen in apico-basal polarized osteoclasts adherent on mineralized matrix. Dynamic observations of several resorption cycles of osteoclasts seeded on apatite revealed that 1) podosomes do not fuse together to form the sealing zone; 2) osteoclasts alternate successive stationary polarized resorption phases with a sealing zone and migration, nonresorption phases without any specific actin structure; and 3) apatite itself promotes sealing zone formation though c-src and Rho signaling. Finally, our work suggests that apatite-mediated sealing zone formation is dependent on both c-src and Rho whereas apico-basal polarization requires only Rho.

Nucleoli in large (giant bi- and multinucleate cells after apoptosis-inducing photodynamic treatment

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K Smetana

2009-06-01

Full Text Available The present experimental study was undertaken to provide information on nucleolar changes accompanying the apoptotic process in large or giant binucleate and multinucleate cells (LBMNCs. Such cells were present in a small but constant percentage in cultures of HL-60 cells. The apoptotic process was induced by photodynamic treatment (PDT by means of 5-aminolaevulinic acid (ALA as the precursor of the photosensitizer protoporphyrin IX and irradiation with broad spectrum blue light (BL. Nucleolar changes in LBMNCs were characterized by marked reduction or disappearance of silver stained particles representing AgNORs in nucleoli including the large ones. In addition, PDT also significantly reduced the number of nucleoli regardless of their size. These changes apparently reflected the decrease or cessation of nucleolar biosynthetic activities and resembled those which were previously observed in naturally maturing bone marrow megakaryocytes (Janoutová et al., 2001.

Fisetin antagonizes cell fusion, cytoskeletal organization and bone resorption in RANKL-differentiated murine macrophages.

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Kim, Yun-Ho; Kim, Jung-Lye; Lee, Eun-Jung; Park, Sin-Hye; Han, Seon-Young; Kang, Soon Ah; Kang, Young-Hee

2014-03-01

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 ≤10 μM 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 © 2014 Elsevier Inc. All rights reserved.

Erk1 positively regulates osteoclast differentiation and bone resorptive activity.

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Yongzheng He

Full Text Available The extracellular signal-regulated kinases (ERK1 and 2 are widely-expressed and they modulate proliferation, survival, differentiation, and protein synthesis in multiple cell lineages. Altered ERK1/2 signaling is found in several genetic diseases with skeletal phenotypes, including Noonan syndrome, Neurofibromatosis type 1, and Cardio-facio-cutaneous syndrome, suggesting that MEK-ERK signals regulate human skeletal development. Here, we examine the consequence of Erk1 and Erk2 disruption in multiple functions of osteoclasts, specialized macrophage/monocyte lineage-derived cells that resorb bone. We demonstrate that Erk1 positively regulates osteoclast development and bone resorptive activity, as genetic disruption of Erk1 reduced osteoclast progenitor cell numbers, compromised pit formation, and diminished M-CSF-mediated adhesion and migration. Moreover, WT mice reconstituted long-term with Erk1(-/- bone marrow mononuclear cells (BMMNCs demonstrated increased bone mineral density as compared to recipients transplanted with WT and Erk2(-/- BMMNCs, implicating marrow autonomous, Erk1-dependent osteoclast function. These data demonstrate Erk1 plays an important role in osteoclast functions while providing rationale for the development of Erk1-specific inhibitors for experimental investigation and/or therapeutic modulation of aberrant osteoclast function.

Influence of Bisphosphonate Treatment on Medullary Macrophages and Osteoclasts: An Experimental Study

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Natalia Daniela Escudero

2012-01-01

Full Text Available Nitrogen-containing bisphosphonates are widely used for treating diverse bone pathologies. They are anticatabolic drugs that act on osteoclasts inhibiting bone resorption. It remains unknown whether the mechanism of action is by decreasing osteoclast number, impairing osteoclast function, or whether they continue to effectively inhibit bone resorption despite the increase in osteoclast number. There is increasing evidence that bisphosphonates also act on bone marrow cells like macrophages and monocytes. The present work sought to evaluate the dynamics of preosteoclast fusion and possible changes in medullary macrophage number in bisphosphonate-treated animals. Healthy female Wistar rats received olpadronate, alendronate, or vehicle during 5 weeks, and 5-bromo-2-deoxyuridine (BrdU on day 7, 28, or 34 of the experiment. Histomorphometric studies were performed to study femurs and evaluate: number of nuclei per osteoclast (N.Nu/Oc; number of BrdU-positive nuclei (N.Nu BrdU+/Oc; percentage of BrdU-positive nuclei per osteoclast (%Nu.BrdU+/Oc; medullary macrophage number (mac/mm2 and correlation between N.Nu/Oc and mac/mm2. Results showed bisphosphonate-treated animals exhibited increased N.Nu/Oc, caused by an increase in preosteoclast fusion rate and evidenced by higher N.Nu BrdU+/Oc, and significantly decreased mac/mm2. Considering the common origin of osteoclasts and macrophages, the increased demand for precursors of the osteoclast lineage may occur at the expense of macrophage lineage precursors.

Interleukin-15-activated natural killer cells kill autologous osteoclasts via LFA-1, DNAM-1 and TRAIL, and inhibit osteoclast-mediated bone erosion in vitro

DEFF Research Database (Denmark)

Feng, Shan; Madsen, Suzi H; Viller, Natasja N

2015-01-01

Osteoclasts reside on bone and are the main bone resorbing cells playing an important role in bone homeostasis, while natural killer (NK) cells are bone-marrow-derived cells known to play a crucial role in immune defence against viral infections. Although mature NK cells traffic through bone marrow...

A scrutiny of matrix metalloproteinases in osteoclasts: evidence for heterogeneity and for the presence of MMPs synthesized by other cells

DEFF Research Database (Denmark)

Andersen, Thomas L; del Carmen Ovejero, Maria; Kirkegaard, Tove

2004-01-01

osteoclasts appears insensitive to MMP inhibitors. Our study shows that the confusion about osteoclastic MMPs in the literature reflects the remarkable ability of osteoclasts to adapt to their environment, as required by the structural or functional diversity of bone tissue. Our observations provide basic...... information needed for understanding the emerging role of MMPs in controlling cell signaling and bone resorption....

INI Expressing Epithelioid Sarcoma with Osteoclastic Giant Cells in a Child: A Case Report with Summary of Prior Published Cases.

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Bhattacharyya, Riju; Ghosh, Ranajoy; Saha, Koushik; Chatterjee, Uttara

2017-08-01

Epithelioid sarcoma is a heterogeneous tumor with 2 subtypes, classic and proximal. The proximal variant is more aggressive and occurs in proximal location in young adults. We present a proximal epithelioid sarcoma in the leg of an 8 year old girl with rhabdoid morphology and scattered osteoclastic giant cells. Nuclear INI-1 was retained. Despite wide local excision, local recurrence occurred at 8 months. Following re-excision, she developed a chest wall metastasis after 9 months. Epithelioid sarcoma, proximal type with osteoclastic giant cells in the pediatric age group has not been reported previously and should be considered in the differential diagnoses of tumors with epithelioid cell morphology and scattered osteoclastic giant cells. Retained INI expression helped to differentiate this tumor from malignant rhabdoid tumor.

[Osteoclasts and early bone remodeling after orthodontic micro-implant placement].

Science.gov (United States)

Zhang, Wei; Guo, Jia-jia; Zhu, Wen-qian; Tang, Guo-hua

2013-08-01

To observe the incidence of osteoclasts during early bone remodeling after orthodontic micro-implant placement. Twenty New Zealand rabbits were randomly allotted into 4 groups. One micro-implant was implanted proximal to the epiphyseal plate of the tibia. Animals were sacrificed on day 3, 7, 14 and 28 (n=5). The sequence of histological changes around the micro-implants were evaluated by hematoxylin and eosin (HE) staining. Osteoclasts were identified by TRAP staining. The differences of the number of the osteoclasts among each time point were analyzed by one way ANOVA with SPSS 19.0 software package. After 3 days of implantation, a large number of erythrocytes, inflammatory cells, mesenchymal cells and bone debris were seen at the implant bone interfaces. Few osteoclasts were observed. On day 7, granular woven bone was formed and some osteoclasts were found in the Howship's lacunae. New bone formation and mineralization were apparent on day 14. Meanwhile, large amounts of osteoclasts were found in the latticed woven bone. On day 28, woven trabeculae with lamellate structures connected to lamellar bone and fewer osteoclasts were identified. Semi-quantitative analysis showed that the number of the osteoclasts was at peak on day 14. There were significant differences among each time point (Pmicro-implant insertion.

Steering the osteoclast through the demineralization-collagenolysis balance

DEFF Research Database (Denmark)

Søe, Kent; Merrild, Ditte Marie Horslev; Delaissé, Jean-Marie

2013-01-01

are generated when collagen degradation is slower than demineralization, and trenches when collagen degradation is as fast as demineralization. Next we treated the osteoclasts with a low dose of a carbonic anhydrase inhibitor to slightly decrease the rate of demineralization, thereby allowing collagen......, forming a pit, and continues parallel to the bone surface, forming a trench. Importantly, we show that the progress of the osteoclast along this route depends on the balance between the rate of collagenolysis and demineralization. We propose that the osteocytes and bone lining cells surrounding...... the osteoclast may act on this balance to steer the osteoclast resorptive activity in order to give the excavations a specific shape....

Coexistence of reduced function of natural killer cells and osteoclasts in two distinct osteopetrotic mutations in the rat

International Nuclear Information System (INIS)

Popoff, S.N.; Jackson, M.E.; Koevary, S.B.; Marks, S.C. Jr.

1991-01-01

Recent evidence suggesting that immune cells and their products (cytokines) play an important role in the regulation of skeletal development and function, particularly of the osteoclast, implies that immune cell dysfunction may be involved in the pathogenesis of certain skeletal disorders. The mammalian osteopetroses are a pathogenetically heterogeneous group of skeletal disorders characterized by skeletal sclerosis resulting from reduced osteoclast-mediated bone resorption. Using a 51 Cr release microcytotoxicity assay we demonstrated that splenic natural killer (NK) cell activity was significantly reduced in two distinctly different osteopetrotic mutations in the rat, osteopetrosis (op) and toothless (tl). To determine whether this reduction in NK cell-mediated cytotoxicity is caused by decreased cell number and/or function in these osteopetrotic mutants, we quantitated NK cells by analyzing mononuclear cell suspensions labeled for two-color fluorescence with OX8 and OX19 monoclonal antibodies in a fluorescence-activated cell sorter. Flow cytometry of these double-labeled cells revealed that the percentage of NK cells (OX8+/OX19- subset) in op and tl spleens was not significantly different from that of normal spleens. These results suggest that NK cells in these osteopetrotic mutants are functionally defective. Thus aberrations in osteoclast and NK cell function coexist in these mutations, and their developmental relationships deserve further study

Inhibition of osteoclastogenesis by osteoblast-like cells genetically engineered to produce interleukin-10.

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Fujioka, Kazuki; Kishida, Tsunao; Ejima, Akika; Yamamoto, Kenta; Fujii, Wataru; Murakami, Ken; Seno, Takahiro; Yamamoto, Aihiro; Kohno, Masataka; Oda, Ryo; Yamamoto, Toshiro; Fujiwara, Hiroyoshi; Kawahito, Yutaka; Mazda, Osam

2015-01-16

Bone destruction at inflamed joints is an important complication associated with rheumatoid arthritis (RA). Interleukin-10 (IL-10) may suppress not only inflammation but also induction of osteoclasts that play key roles in the bone destruction. If IL-10-producing osteoblast-like cells are induced from patient somatic cells and transplanted back into the destructive bone lesion, such therapy may promote bone remodeling by the cooperative effects of IL-10 and osteoblasts. We transduced mouse fibroblasts with genes for IL-10 and Runx2 that is a crucial transcription factor for osteoblast differentiation. The IL-10-producing induced osteoblast-like cells (IL-10-iOBs) strongly expressed osteoblast-specific genes and massively produced bone matrix that were mineralized by calcium phosphate in vitro and in vivo. Culture supernatant of IL-10-iOBs significantly suppressed induction of osteoclast from RANKL-stimulated Raw264.7 cells as well as LPS-induced production of inflammatory cytokine by macrophages. The IL-10-iOBs may be applicable to novel cell-based therapy against bone destruction associated with RA. Copyright © 2014 Elsevier Inc. All rights reserved.

Facilitation of bone resorption activities in synovial lavage fluid patients with mandibular condyle fractures.

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Takano, H; Takahashi, T; Nakata, A; Nogami, S; Yusa, K; Kuwajima, S; Yamazaki, M; Fukuda, M

2016-05-01

The aim of this study was to investigate the bone resorption effect of the mediators delivered in joint cavity of patients with mandibular condyle fractures by detecting osteoclast markers using cellular biochemistry methods, and by analysing bone resorption activities via inducing osteoclast differentiation of the infiltrated cells from arthrocentesis. Sixteen joints in 10 patients with mandibular condyle fractures were evaluated. The control group consisted of synovial fluid (SF) samples from seven joints of four volunteers who had no clinical signs or symptoms involving the temporomandibular joint (TMJ) or disc displacement. We collected SF cells from all patients during therapeutic arthrocentesis. The infiltrating cells from TMJ SF were cultured, differentiated into tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like cells and examined bone resorption activities. We also investigated factors related to osteoclast induction of SF, using ELISA procedures. Osteoclast-like cells were induced from the SF cells obtained from all patients with condylar fractures. These multinucleated giant cells were positive for TRAP and actin, and had the ability to absorb dentin slices. The levels of macrophage colony-stimulating factor (M-CSF), prostaglandin E2 (PGE2), soluble form of receptor activator of nuclear factor kappa-B ligand (sRANKL) and osteoprotegerin (OPG), in SF samples from the patients, were significantly higher than in the controls. These findings indicate that bone resorption activities in SF from patients with mandibular condyle fractures were upregulated and may participate in the pathogenesis and wound healing. © 2016 The Authors. Journal of Oral Rehabilitation Published by John Wiley & Sons Ltd.

Involvement of multiple myeloma cell-derived exosomes in osteoclast differentiation

OpenAIRE

Raimondi, Lavinia; De Luca, Angela; Amodio, Nicola; Manno, Mauro; Raccosta, Samuele; Taverna, Simona; Bellavia, Daniele; Naselli, Flores; Fontana, Simona; Schillaci, Odessa; Giardino, Roberto; Fini, Milena; Tassone, Pierfrancesco; Santoro, Alessandra; De Leo, Giacomo

2015-01-01

Bone disease is the most frequent complication in multiple myeloma (MM) resulting in osteolytic lesions, bone pain, hypercalcemia and renal failure. In MM bone disease the perfect balance between bone-resorbing osteoclasts (OCs) and bone-forming osteoblasts (OBs) activity is lost in favour of OCs, thus resulting in skeletal disorders. Since exosomes have been described for their functional role in cancer progression, we here investigate whether MM cell-derived exosomes may be involved in OCs ...

Tumor Cell Anaplasia and Multinucleation Are Predictors of Disease Recurrence in Oropharyngeal Squamous Cell Carcinoma, Including Among Just the Human Papillomavirus-Related Cancers

OpenAIRE

Lewis, James S.; Scantlebury, Juliette B.; Luo, Jingqin; Thorstad, Wade L.

2012-01-01

Oropharyngeal squamous cell carcinoma (SCC) is frequently related to high risk human papillomavirus. This tumor expresses p16, frequently has a nonkeratinizing morphology, and has improved outcomes. Despite having a good prognosis, tumors can have focal or diffuse nuclear anaplasia or multinucleation, the significance of which is unknown. From a database of 270 oropharyngeal SCCs with known histologic typing (using our established system) and p16 immunohistochemistry, all su...

Fibroadenoma and phyllodes tumors of anogenital mammary-like glands: a series of 13 neoplasms in 12 cases, including mammary-type juvenile fibroadenoma, fibroadenoma with lactation changes, and neurofibromatosis-associated pseudoangiomatous stromal hyperplasia with multinucleated giant cells.

Science.gov (United States)

Kazakov, Dmitry V; Spagnolo, Dominic V; Stewart, Colin J; Thompson, Jane; Agaimy, Abbas; Magro, Gaetano; Bisceglia, Michele; Vazmitel, Marina; Kacerovska, Denisa; Kutzner, Heinz; Mukensnabl, Petr; Michal, Michal

2010-01-01

The authors present a series of 13 fibroepithelial neoplasms involving anogenital mammary-like glands, all occurring in 12 female patients, whose age at diagnosis ranged from 30 to 51 years (mean, 38 y; median, 42 y). All women presented with a solitary asymptomatic nodule in the vulva (n=8), perineum (n=2), or near the anus (n=2) ranging in size from 1.5 to 4.5 cm. Microscopically, 8 lesions were classified as fibroadenoma, and 5, including 1 recurrent tumor, as phyllodes tumor, of which 1 was benign and 4 low-grade malignant. In addition to conventional findings, we describe several hitherto unreported features including juvenile fibroadenoma-like proliferation, fibroadenoma with lactation change, and pseudoangiomatous stromal hyperplasia with multinucleated stromal giant cells in a patient with neurofibromatosis, type 1 all constituting potential diagnostic pitfalls, which are best averted by using the same approach to diagnosis as for their analogous mammary counterparts.

Hydroxyapatite nanocrystals functionalized with alendronate as bioactive components for bone implant coatings to decrease osteoclastic activity

Science.gov (United States)

Bosco, Ruggero; Iafisco, Michele; Tampieri, Anna; Jansen, John A.; Leeuwenburgh, Sander C. G.; van den Beucken, Jeroen J. J. P.

2015-02-01

The integration of bone implants within native bone tissue depends on periprosthetic bone quality, which is severely decreased in osteoporotic patients. In this work, we have synthesized bone-like hydroxyapatite nanocrystals (nHA) using an acid-base neutralization reaction and analysed their physicochemical properties. Subsequently, we have functionalized the nHA with alendronate (nHAALE), a well-known bisphosphonate drug used for the treatment of osteoporosis. An in vitro osteoclastogenesis test was carried out to evaluate the effect of nHAALE on the formation of osteoclast-like cells from monocytic precursor cells (i.e. RAW264.7 cell line) showing that nHAALE significantly promoted apoptosis of osteoclast-like cells. Subsequently, nHA and nHAALE were deposited on titanium disks using electrospray deposition (ESD), for which characterisation of the deposited coatings confirmed the presence of alendronate in nHAALE coatings with nanoscale thickness of about 700 nm. These results indicate that alendronate linked to hydroxyapatite nanocrystals has therapeutic potential and nHAALE can be considered as an appealing coating constituent material for orthopaedic and oral implants for application in osteoporotic patients.

Biochemical and morphological changes associated with macrophages and osteoclasts when challenged with infection - biomed 2011.

Science.gov (United States)

Wiggers, Erin Callie; Johnson, William; Tucci, Michelle; Benghuzzi, Hamed

2011-01-01

Osteomyelitis is a bacterial infection of the bone that occurs frequently as a complication of open fractures and various kinds of orthopedic surgery. This infection can often lead to more extensive surgeries and even death of the patient. In animal models of osteomyelitis, the site of infection by Staphylococcus aureus was observed to have high numbers of both macrophages and osteoclasts, both of which may contribute to large amounts of osteolysis and tissue damage. In order to evaluate the immune response in both types of cells, two cells lines, a macrophage cell line and a macrophage cell line stimulated to become osteoclasts by the addition of receptor activator of nuclear-factor B (RANKL), were exposed to lipopolysaccharides, opsonized S. aureus, and unopsonized S. aureus. The results showed that both cell types activated a biochemical cascade that included the release of cytokines and nitric oxide associated with cell damage and death in response to infection. However, macrophages and osteoclasts differed in response magnitude, most likely due to differences in cell-membrane receptors. This data supports the growing body of research that links the immune and skeletal systems. Further understanding of biochemical pathways shared by the two systems could lead to significant advances in the treatment of osteomyelitis and the success of prostheses.

Monosodium Urate in the Presence of RANKL Promotes Osteoclast Formation through Activation of c-Jun N-Terminal Kinase

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Jung-Yoon Choe

2015-01-01

Full Text Available The aim of this study was to clarify the role of monosodium urate (MSU crystals in receptor activator of nuclear factor kB ligand- (RANKL- RANK-induced osteoclast formation. RAW 264.7 murine macrophage cells were incubated with MSU crystals or RANKL and differentiated into osteoclast-like cells as confirmed by staining for tartrate-resistant acid phosphatase (TRAP and actin ring, pit formation assay, and TRAP activity assay. MSU crystals in the presence of RANKL augmented osteoclast differentiation, with enhanced mRNA expression of NFATc1, cathepsin K, carbonic anhydrase II, and matrix metalloproteinase-9 (MMP-9, in comparison to RAW 264.7 macrophages incubated in the presence of RANKL alone. Treatment with both MSU crystals and RANKL induced osteoclast differentiation by activating downstream molecules in the RANKL-RANK pathway including tumor necrosis factor receptor-associated factor 6 (TRAF-6, JNK, c-Jun, and NFATc1. IL-1b produced in response to treatment with both MSU and RANKL is involved in osteoclast differentiation in part through the induction of TRAF-6 downstream of the IL-1b pathway. This study revealed that MSU crystals contribute to enhanced osteoclast formation through activation of RANKL-mediated pathways and recruitment of IL-1b. These findings suggest that MSU crystals might be a pathologic causative agent of bone destruction in gout.

The Use of Patient-Specific Induced Pluripotent Stem Cells (iPSCs to Identify Osteoclast Defects in Rare Genetic Bone Disorders

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I-Ping Chen

2014-12-01

Full Text Available More than 500 rare genetic bone disorders have been described, but for many of them only limited treatment options are available. Challenges for studying these bone diseases come from a lack of suitable animal models and unavailability of skeletal tissues for studies. Effectors for skeletal abnormalities of bone disorders may be abnormal bone formation directed by osteoblasts or anomalous bone resorption by osteoclasts, or both. Patient-specific induced pluripotent stem cells (iPSCs can be generated from somatic cells of various tissue sources and in theory can be differentiated into any desired cell type. However, successful differentiation of hiPSCs into functional bone cells is still a challenge. Our group focuses on the use of human iPSCs (hiPSCs to identify osteoclast defects in craniometaphyseal dysplasia. In this review, we describe the impact of stem cell technology on research for better treatment of such disorders, the generation of hiPSCs from patients with rare genetic bone disorders and current protocols for differentiating hiPSCs into osteoclasts.

Deletion of Adseverin in Osteoclasts Affects Cell Structure But Not Bone Metabolism.

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Cao, Yixuan; Wang, Yongqiang; Sprangers, Sara; Picavet, Daisy I; Glogauer, Michael; McCulloch, Christopher A; Everts, Vincent

2017-08-01

Adseverin is an actin-severing/capping protein that may contribute to osteoclast differentiation in vitro but its role in bone remodeling of healthy animals is not defined. We analyzed bone and osteoclast structure in adseverin conditional null mice at alveolar and long bone sites. In wild-type and adseverin null mice, as measured by dual-energy X-ray absorptiometry, there were no differences of bone mineral content or bone mineral density, indicating no change of bone metabolism. In tibiae, TRAcP + osteoclasts were formed in comparable numbers in adseverin null and wild-type mice. Ultrastructural analysis showed normal and similar abundance of ruffled borders, sealing zones, and mitochondria, and with no difference of osteoclast nuclear numbers. In contrast, analyses of long bone showed that in the absence of adseverin osteoclasts were smaller (120 ± 13 vs. 274 ± 19 µm 2 ; p structure but not to bone metabolism in vivo.

Characterization of osteoclasts derived from CD14+ monocytes isolated from peripheral blood

DEFF Research Database (Denmark)

Sørensen, Mette Grøndahl; Henriksen, Kim; Schaller, Sophie

2007-01-01

Bone resorption is solely mediated by osteoclasts. Therefore, a pure osteoclast population is of high interest for the investigation of biological aspects of the osteoclasts, such as the direct effect of growth factors and hormones, as well as for testing and characterizing inhibitors of bone...... resorption. We have established a pure, stable, and reproducible system for purification of human osteoclasts from peripheral blood. We isolated CD14-positive (CD14+) monocytes using anti-CD14-coated beads. After isolation, the monocytes are differentiated into mature osteoclasts by stimulation...... of osteoclast precursors. No expression of osteoclast markers was observed in the absence of RANKL, whereas RANKL dose-dependently induced the expression of cathepsin K, tartrate-resistant acid phosphatase (TRACP), and matrix metallo proteinase (MMP)-9. Furthermore, morphological characterization of the cells...

The Inhibitory Effect of Alisol A 24-Acetate from Alisma canaliculatum on Osteoclastogenesis

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Kwang-Jin Kim

2015-01-01

Full Text Available Osteoporosis is a disease that decreases bone mass. The number of patients with osteoporosis has been increasing, including an increase in patients with bone fractures, which lead to higher medical costs. Osteoporosis treatment is all-important in preventing bone loss. One strategy for osteoporosis treatment is to inhibit osteoclastogenesis. Osteoclasts are bone-resorbing multinucleated cells, and overactive osteoclasts and/or their increased number are observed in bone disorders including osteoporosis and rheumatoid arthritis. Bioactivity-guided fractionations led to the isolation of alisol A 24-acetate from the dried tuber of Alisma canaliculatum. Alisol A 24-acetate inhibited RANKL-mediated osteoclast differentiation by downregulating NFATc1, which plays an essential role in osteoclast differentiation. Furthermore, it inhibited the expression of DC-STAMP and cathepsin K, which are related to cell-cell fusion of osteoclasts and bone resorption, respectively. Therefore, alisol A 24-acetate could be developed as a new structural scaffold for inhibitors of osteoclast differentiation in order to develop new drugs against osteoporosis.

A specific subtype of osteoclasts secretes factors inducing nodule formation by osteoblasts

DEFF Research Database (Denmark)

Henriksen, Kim; Andreassen, Kim V; Thudium, Christian S

2012-01-01

Osteoclasts are known to be important for the coupling process between bone resorption and formation. The aim of this study was to address when osteoclasts are anabolically active. Human monocytes were differentiated into mature osteoclasts by treatment with M-CSF and RANKL. Conditioned medium wa...... dependent and independent of their resorptive activity, secrete factors stimulating osteoblastic bone formation.......Osteoclasts are known to be important for the coupling process between bone resorption and formation. The aim of this study was to address when osteoclasts are anabolically active. Human monocytes were differentiated into mature osteoclasts by treatment with M-CSF and RANKL. Conditioned medium...... release. The osteoblastic cell line 2T3 was treated with 50% of CM or non-CM for 12days. Bone formation was assessed by Alizarin Red extraction. CM from mature osteoclasts induced bone formation, while CM from macrophages did not. Non-resorbing osteoclasts generated from osteopetrosis patients showed...

Interleukin-3 plays dual roles in osteoclastogenesis by promoting the development of osteoclast progenitors but inhibiting the osteoclastogenic process

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Hong, Huixian [Department of Hematology, Guangzhou First People’s Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510180 (China); Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Shi, Zhenqi [Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Qiao, Ping [Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Department of Pharmacology, Norman Bethune Medical College, Jilin University, Changchun, Jilin 130021 (China); Li, Hui [Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); McCoy, Erin M. [Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Mao, Ping [Department of Hematology, Guangzhou First People’s Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510180 (China); Xu, Hui [Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Feng, Xu [Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Wang, Shunqing, E-mail: shqwang_cn@yahoo.com [Department of Hematology, Guangzhou First People’s Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510180 (China)

2013-11-01

Highlights: •IL-3 treatment of bone marrow cells generates a population of hematopoietic cells. •IL-3-dependent hematopoietic cells are capable of differentiating into osteoclasts. •Osteoclasts derived from IL-3-dependent hematopoietic cells are functional. •IL-3 promotes the development of osteoclast progenitors. •IL-3 inhibits the osteoclastogenic process. -- Abstract: Interleukin (IL)-3, a multilineage hematopoietic growth factor, is implicated in the regulation of osteoclastogenesis. However, the role of IL-3 in osteoclastogenesis remains controversial; whereas early studies showed that IL-3 stimulates osteoclastogenesis, recent investigations demonstrated that IL-3 inhibits osteoclast formation. The objective of this work is to further address the role of IL-3 in osteoclastogenesis. We found that IL-3 treatment of bone marrow cells generated a population of cells capable of differentiating into osteoclasts in tissue culture dishes in response to the stimulation of the monocyte/macrophage-colony stimulating factor (M-CSF) and the receptor activator of nuclear factor kappa B ligand (RANKL). The IL-3-dependent hematopoietic cells were able to further proliferate and differentiate in response to M-CSF stimulation and the resulting cells were also capable of forming osteoclasts with M-CSF and RANKL treatment. Interestingly, IL-3 inhibits M-CSF-/RANKL-induced differentiation of the IL-3-dependent hematopoietic cells into osteoclasts. The flow cytometry analysis indicates that while IL-3 treatment of bone marrow cells slightly affected the percentage of osteoclast precursors in the surviving populations, it considerably increased the percentage of osteoclast precursors in the populations after subsequent M-CSF treatment. Moreover, osteoclasts derived from IL-3-dependent hematopoietic cells were fully functional. Thus, we conclude that IL-3 plays dual roles in osteoclastogenesis by promoting the development of osteoclast progenitors but inhibiting the

Esculetin attenuates receptor activator of nuclear factor kappa-B ligand-mediated osteoclast differentiation through c-Fos/nuclear factor of activated T-cells c1 signaling pathway

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Baek, Jong Min; Park, Sun-Hyang; Cheon, Yoon-Hee; Ahn, Sung-Jun [Department of Anatomy, School of Medicine, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Lee, Myeung Su [Division of Rheumatology, Department of Internal Medicine, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Imaging Science-based Lung and Bone Diseases Research Center, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Institute for Skeletal Disease, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Oh, Jaemin, E-mail: jmoh@wku.ac.kr [Department of Anatomy, School of Medicine, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Imaging Science-based Lung and Bone Diseases Research Center, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Institute for Skeletal Disease, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Kim, Ju-Young, E-mail: kimjy1014@gmail.com [Imaging Science-based Lung and Bone Diseases Research Center, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of)

2015-05-29

Esculetin exerts various biological effects on anti-oxidation, anti-tumors, and anti-inflammation. However, the involvement of esculetin in the bone metabolism process, particularly osteoclast differentiation has not yet been investigated. In the present study, we first confirmed the inhibitory effect of esculetin on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation. We then revealed the relationship between esculetin and the expression of osteoclast-specific molecules to elucidate its underlying mechanisms. Esculetin interfered with the expression of c-Fos and nuclear factor of activated T cell c1 (NFATc1) both at the mRNA and protein level with no involvement in osteoclast-associated early signaling pathways, suppressing the expression of various transcription factors exclusively expressed in osteoclasts such as tartrate-resistant acid phosphatase (Trap), osteoclast-associated receptor (Oscar), dendritic cell-specific transmembrane protein (Dcstamp), osteoclast stimulatory transmembrane protein (Ocstamp), cathepsin K, αvβ3 integrin, and calcitonin receptor (Ctr). Additionally, esculetin inhibited the formation of filamentous actin (F-actin) ring-positive osteoclasts during osteoclast differentiation. However, the development of F-actin structures and subsequent bone resorbing activity of mature osteoclasts, which are observed in osteoclast/osteoblast co-culture systems were not affected by esculetin. Taken together, our results indicate for the first time that esculetin inhibits RANKL-mediated osteoclastogenesis via direct suppression of c-Fos and NFATc1 expression and exerts an inhibitory effect on actin ring formation during osteoclastogenesis. - Highlights: • We first investigated the effects of esculetin on osteoclast differentiation and function. • Our data demonstrate for the first time that esculetin can suppress osteoclastogenesis in vitro. • Esculetin acts as an inhibitor of c-Fos and NFATc1 activation. �

Osteoclasts Are Required for Hematopoietic Stem and Progenitor Cell Mobilization but Not for Stress Erythropoiesis in Plasmodium chabaudi adami Murine Malaria

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Hugo Roméro

2016-01-01

Full Text Available The anemia and inflammation concurrent with blood stage malaria trigger stress haematopoiesis and erythropoiesis. The activity of osteoclasts seems required for the mobilization of hematopoietic stem and progenitor cells (HSPC from the bone marrow to the periphery. Knowing that BALB/c mice with acute Plasmodium chabaudi adami malaria have profound alterations in bone remodelling cells, we evaluated the extent to which osteoclasts influence their hematopoietic response to infection. For this, mice were treated with osteoclast inhibiting hormone calcitonin prior to parasite inoculation, and infection as well as hematological parameters was studied. In agreement with osteoclast-dependent HSPC mobilization, administration of calcitonin led to milder splenomegaly, reduced numbers of HSPC in the spleen, and their retention in the bone marrow. Although C-terminal telopeptide (CTX levels, indicative of bone resorption, were lower in calcitonin-treated infected mice, they remained comparable in naive and control infected mice. Calcitonin-treated infected mice conveniently responded to anemia but generated less numbers of splenic macrophages and suffered from exacerbated infection; interestingly, calcitonin also decreased the number of macrophages generated in vitro. Globally, our results indicate that although osteoclast-dependent HSC mobilization from bone marrow to spleen is triggered in murine blood stage malaria, this activity is not essential for stress erythropoiesis.

CD147 promotes the formation of functional osteoclasts through NFATc1 signalling

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Nishioku, Tsuyoshi; Terasawa, Mariko; Baba, Misaki; Yamauchi, Atsushi; Kataoka, Yasufumi

2016-01-01

CD147, a membrane glycoprotein of the immunoglobulin superfamily, is highly upregulated during dynamic cellular events including tissue remodelling. Elevated CD147 expression is present in the joint of rheumatoid arthritis patients. However, the role of CD147 in bone destruction remains unclear. To determine whether CD147 is involved in osteoclastogenesis, we studied its expression in mouse osteoclasts and its role in osteoclast differentiation and function. CD147 expression was markedly upregulated during osteoclast differentiation. To investigate the role of CD147 in receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis and bone resorption activity, osteoclast precursor cells were transfected with CD147 siRNA. Decreased CD147 expression inhibited osteoclast formation and bone resorption, inhibited RANKL-induced nuclear translocation of the nuclear factor of activated T cells (NFAT) c1 and decreased the expression of the d2 isoform of vacuolar ATPase Vo domain and cathepsin K. Therefore, CD147 plays a critical role in the differentiation and function of osteoclasts by upregulating NFATc1 through the autoamplification of its expression in osteoclastogenesis. - Highlights: • CD147 expression was markedly upregulated during osteoclast differentiation. • Downregulation of CD147 expression inhibited osteoclastgenesis and bone resorption. • Decreased CD147 expression inhibited RANKL-induced nuclear translocation of NFATc1.

CD147 promotes the formation of functional osteoclasts through NFATc1 signalling

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Nishioku, Tsuyoshi, E-mail: nishiokut@niu.ac.jp [Department of Clinical Pharmacology, Faculty of Pharmaceutical Sciences, Nagasaki International University, 2825-7 Huis Ten Bosch, Sasebo, Nagasaki 859-3298 (Japan); Department of Pharmaceutical Care and Health Sciences, Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, Fukuoka 814-0180 (Japan); Terasawa, Mariko; Baba, Misaki; Yamauchi, Atsushi; Kataoka, Yasufumi [Department of Pharmaceutical Care and Health Sciences, Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, Fukuoka 814-0180 (Japan)

2016-04-29

CD147, a membrane glycoprotein of the immunoglobulin superfamily, is highly upregulated during dynamic cellular events including tissue remodelling. Elevated CD147 expression is present in the joint of rheumatoid arthritis patients. However, the role of CD147 in bone destruction remains unclear. To determine whether CD147 is involved in osteoclastogenesis, we studied its expression in mouse osteoclasts and its role in osteoclast differentiation and function. CD147 expression was markedly upregulated during osteoclast differentiation. To investigate the role of CD147 in receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis and bone resorption activity, osteoclast precursor cells were transfected with CD147 siRNA. Decreased CD147 expression inhibited osteoclast formation and bone resorption, inhibited RANKL-induced nuclear translocation of the nuclear factor of activated T cells (NFAT) c1 and decreased the expression of the d2 isoform of vacuolar ATPase Vo domain and cathepsin K. Therefore, CD147 plays a critical role in the differentiation and function of osteoclasts by upregulating NFATc1 through the autoamplification of its expression in osteoclastogenesis. - Highlights: • CD147 expression was markedly upregulated during osteoclast differentiation. • Downregulation of CD147 expression inhibited osteoclastgenesis and bone resorption. • Decreased CD147 expression inhibited RANKL-induced nuclear translocation of NFATc1.

Tetraspanin 7 regulates sealing zone formation and the bone-resorbing activity of osteoclasts

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Kwon, Jun-Oh; Lee, Yong Deok; Kim, Haemin; Kim, Min Kyung; Song, Min-Kyoung; Lee, Zang Hee; Kim, Hong-Hee, E-mail: hhbkim@snu.ac.kr

2016-09-02

Tetraspanin family proteins regulate morphology, motility, fusion, and signaling in various cell types. We investigated the role of the tetraspanin 7 (Tspan7) isoform in the differentiation and function of osteoclasts. Tspan7 was up-regulated during osteoclastogenesis. When Tspan7 expression was reduced in primary precursor cells by siRNA-mediated gene knock-down, the generation of multinuclear osteoclasts was not affected. However, a striking cytoskeletal abnormality was observed: the formation of the podosome belt structure was inhibited and the microtubular network were disrupted by Tspan7 knock-down. Decreases in acetylated microtubules and levels of phosphorylated Src and Pyk2 in Tspan7 knock-down cells supported the involvement of Tspan7 in cytoskeletal rearrangement signaling in osteoclasts. This cytoskeletal defect interfered with sealing zone formation and subsequently the bone-resorbing activity of mature osteoclasts on dentin surfaces. Our results suggest that Tspan7 plays an important role in cytoskeletal organization required for the bone-resorbing function of osteoclasts by regulating signaling to Src, Pyk2, and microtubules. - Highlights: • Tspan7 expression is up-regulated during osteoclastogenesis. • Tspan7 regulates podosome belt organization in osteoclasts. • Tspan7 is crucial for sealing zone formation and bone-resorption by osteoclasts. • Src and Pyk2 phosphorylation and microtubule acetylation mediate Tspan7 function.

Effects of transforming growth factor beta 1 on the regulation of osteoclastic development and function

International Nuclear Information System (INIS)

Hattersley, G.; Chambers, T.J.

1991-01-01

Transforming growth factor (TGF) beta 1 is a multifunctional cytokine with powerful effects on osteoblastic cells. Its role in the regulation of osteoclast generation and function, however, is unclear. It has been reported both to stimulate and to inhibit resorption in organ culture and to inhibit multinuclear cell formation in bone marrow cultures. We tested the effects of TGF-beta 1 on bone resorption by osteoclasts isolated from neonatal rat long bones. We found potent stimulation of osteoclastic bone resorption, mediated by osteoblastic cells, with an EC50 of 10 pg/ml, considerably lower than that of well-documented osteotropic hormones. Stimulation was not mediated by Swiss mouse 3T3 cells, a nonosteoblastic cell line. TGF-beta 1 strongly inhibited the generation of calcitonin receptor (CTR)-positive cells in mouse bone marrow cultures, but as for isolated osteoclasts, bone resorption per CTR-positive cell was increased. The inhibition of CTR-positive cell formation was associated with suppression of maturation of other bone marrow derivatives and may be related more to the known ability of TGF-beta 1 to suppress the proliferation of primitive hematopoietic cells than to a specific role of TGF-beta 1 in osteoclast generation

Live imaging of osteoclast inhibition by bisphosphonates in a medaka osteoporosis model

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Tingsheng Yu

2016-02-01

Full Text Available Osteoclasts are bone-resorbing cells derived from the monocyte/macrophage lineage. Excess osteoclast activity leads to reduced bone mineral density, a hallmark of diseases such as osteoporosis. Processes that regulate osteoclast activity are therefore targeted in current osteoporosis therapies. To identify and characterize drugs for treatment of bone diseases, suitable in vivo models are needed to complement cell-culture assays. We have previously reported transgenic medaka lines expressing the osteoclast-inducing factor receptor activator of nuclear factor κB ligand (Rankl under control of a heat shock-inducible promoter. Forced Rankl expression resulted in ectopic osteoclast formation, as visualized by live imaging in fluorescent reporter lines. This led to increased bone resorption and a dramatic reduction of mineralized matrix similar to the situation in humans with osteoporosis. In an attempt to establish the medaka as an in vivo model for osteoporosis drug screening, we treated Rankl-expressing larvae with etidronate and alendronate, two bisphosphonates commonly used in human osteoporosis therapy. Using live imaging, we observed an efficient, dose-dependent inhibition of osteoclast activity, which resulted in the maintenance of bone integrity despite an excess of osteoclast formation. Strikingly, we also found that bone recovery was efficiently promoted after inhibition of osteoclast activity and that osteoblast distribution was altered, suggesting effects on osteoblast-osteoclast coupling. Our data show that transgenic medaka lines are suitable in vivo models for the characterization of antiresorptive or bone-anabolic compounds by live imaging and for screening of novel osteoporosis drugs.

Osteocytes, not Osteoblasts or Lining Cells, are the Main Source of the RANKL Required for Osteoclast Formation in Remodeling Bone.

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Jinhu Xiong

Full Text Available The cytokine receptor activator of nuclear factor kappa B ligand (RANKL, encoded by the Tnfsf11 gene, is essential for osteoclastogenesis and previous studies have shown that deletion of the Tnfsf11 gene using a Dmp1-Cre transgene reduces osteoclast formation in cancellous bone by more than 70%. However, the Dmp1-Cre transgene used in those studies leads to recombination in osteocytes, osteoblasts, and lining cells making it unclear whether one or more of these cell types produce the RANKL required for osteoclast formation in cancellous bone. Because osteoblasts, osteocytes, and lining cells have distinct locations and functions, distinguishing which of these cell types are sources of RANKL is essential for understanding the orchestration of bone remodeling. To distinguish between these possibilities, we have now created transgenic mice expressing the Cre recombinase under the control of regulatory elements of the Sost gene, which is expressed in osteocytes but not osteoblasts or lining cells in murine bone. Activity of the Sost-Cre transgene in osteocytes, but not osteoblast or lining cells, was confirmed by crossing Sost-Cre transgenic mice with tdTomato and R26R Cre-reporter mice, which express tdTomato fluorescent protein or LacZ, respectively, only in cells expressing the Cre recombinase or their descendants. Deletion of the Tnfsf11 gene in Sost-Cre mice led to a threefold decrease in osteoclast number in cancellous bone and increased cancellous bone mass, mimicking the skeletal phenotype of mice in which the Tnfsf11 gene was deleted using the Dmp1-Cre transgene. These results demonstrate that osteocytes, not osteoblasts or lining cells, are the main source of the RANKL required for osteoclast formation in remodeling cancellous bone.

Carbon and nitrogen depletion-induced nucleophagy and selective autophagic sequestration of a whole nucleus in multinucleate cells of the filamentous fungus Aspergillus oryzae.

Science.gov (United States)

Kikuma, Takashi; Mitani, Takahiro; Kohara, Takahiro; Maruyama, Jun-Ichi; Kitamoto, Katsuhiko

2017-05-12

Autophagy is a conserved cellular degradation process in eukaryotes, in which cytoplasmic components and organelles are digested in vacuoles/lysosomes. Recently, autophagic degradation of nuclear materials, termed "nucleophagy", has been reported. In the multinucleate filamentous fungus Aspergillus oryzae, a whole nucleus is degraded by nucleophagy after prolonged culture. While developing an H2B-EGFP processing assay for the evaluation of nucleophagy in A. oryzae, we found that nucleophagy is efficiently induced by carbon or nitrogen depletion. Microscopic observations in a carbon depletion condition clearly demonstrated that autophagosomes selectively sequester a particular nucleus, despite the presence of multiple nuclei in the same cell. Furthermore, AoNsp1, the A. oryzae homolog of the yeast nucleoporin Nsp1p, mainly localized at the nuclear periphery, but its localization was restricted to the opposite side of the autophagosome being formed around a nucleus. In contrast, the perinuclear ER visualized with the calnexin AoClxA was not morphologically affected by nucleophagy. The findings of nucleophagy-inducing conditions enabled us to characterize the morphological process of autophagic degradation of a whole nucleus in multinucleate cells.

IL-1β Suppresses the Formation of Osteoclasts by Increasing OPG Production via an Autocrine Mechanism Involving Celecoxib-Related Prostaglandins in Chondrocytes

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Yusuke Watanabe

2009-01-01

Full Text Available Elevated interleukin (IL-1 concentrations in synovial fluid have been implicated in joint bone and cartilage destruction. Previously, we showed that IL-1β stimulated the expression of prostaglandin (PG receptor EP4 via increased PGE2 production. However, the effect of IL-1β on osteoclast formation via chondrocytes is unclear. Therefore, we examined the effect of IL-1β and/or celecoxib on the expression of macrophage colony-stimulating factor (M-CSF, receptor activator of NF-κB ligand (RANKL, and osteoprotegerin (OPG in human chondrocytes, and the indirect effect of IL-1β on osteoclast-like cell formation using RAW264.7 cells. OPG and RANKL expression increased with IL-1β; whereas M-CSF expression decreased. Celecoxib blocked the stimulatory effect of IL-1β. Conditioned medium from IL-1β-treated chondrocytes decreased TRAP staining in RAW264.7 cells. These results suggest that IL-1β suppresses the formation of osteoclast-like cells via increased OPG production and decreased M-CSF production in chondrocytes, and OPG production may increase through an autocrine mechanism involving celecoxib-related PGs.

CD147 promotes the formation of functional osteoclasts through NFATc1 signalling.

Science.gov (United States)

Nishioku, Tsuyoshi; Terasawa, Mariko; Baba, Misaki; Yamauchi, Atsushi; Kataoka, Yasufumi

2016-04-29

CD147, a membrane glycoprotein of the immunoglobulin superfamily, is highly upregulated during dynamic cellular events including tissue remodelling. Elevated CD147 expression is present in the joint of rheumatoid arthritis patients. However, the role of CD147 in bone destruction remains unclear. To determine whether CD147 is involved in osteoclastogenesis, we studied its expression in mouse osteoclasts and its role in osteoclast differentiation and function. CD147 expression was markedly upregulated during osteoclast differentiation. To investigate the role of CD147 in receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis and bone resorption activity, osteoclast precursor cells were transfected with CD147 siRNA. Decreased CD147 expression inhibited osteoclast formation and bone resorption, inhibited RANKL-induced nuclear translocation of the nuclear factor of activated T cells (NFAT) c1 and decreased the expression of the d2 isoform of vacuolar ATPase Vo domain and cathepsin K. Therefore, CD147 plays a critical role in the differentiation and function of osteoclasts by upregulating NFATc1 through the autoamplification of its expression in osteoclastogenesis. Copyright © 2016 Elsevier Inc. All rights reserved.

Water extract of Acer tegmentosum reduces bone destruction by inhibiting osteoclast differentiation and function.

Science.gov (United States)

Ha, Hyunil; Shim, Ki-Shuk; Kim, Taesoo; An, Hyosun; Lee, Chung-Jo; Lee, Kwang Jin; Ma, Jin Yeul

2014-04-01

The stem of Acer tegmentosum has been widely used in Korea for the treatment of hepatic disorders. In this study, we investigated the bone protective effect of water extract of the stem of Acer tegmentosum (WEAT). We found that WEAT inhibits osteoclast differentiation induced by receptor activator of nuclear factor-κB ligand (RANKL), an essential cytokine for osteoclast differentiation. In osteoclast precursor cells, WEAT inhibited RANKL-induced activation of JNK, NF-κB, and cAMP response element-binding protein, leading to suppression of the induction of c-Fos and nuclear factor of activated T cells cytoplasmic 1, key transcription factors for osteoclast differentiation. In addition, WEAT inhibited bone resorbing activity of mature osteoclasts. Furthermore, the oral administration of WEAT reduced RANKL-induced bone resorption and trabecular bone loss in mice. Taken together, our study demonstrates that WEAT possesses a protective effect on bone destruction by inhibiting osteoclast differentiation and function.

TRAF family member-associated NF-κB activator (TANK) induced by RANKL negatively regulates osteoclasts survival and function.

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Wu, Mengrui; Wang, Yiping; Deng, Lianfu; Chen, Wei; Li, Yi-Ping

2012-01-01

Osteoclasts are the principle bone-resorbing cells. Precise control of balanced osteoclast activity is indispensable for bone homeostasis. Osteoclast activation mediated by RANK-TRAF6 axis has been clearly identified. However, a negative regulation-machinery in osteoclast remains unclear. TRAF family member-associated NF-κB activator (TANK) is induced by about 10 folds during osteoclastogenesis, according to a genome-wide analysis of gene expression before and after osteoclast maturation, and confirmed by western blot and quantitative RT-PCR. Bone marrow macrophages (BMMs) transduced with lentivirus carrying tank-shRNA were induced to form osteoclast in the presence of RANKL and M-CSF. Tank expression was downregulated by 90% by Tank-shRNA, which is confirmed by western blot. Compared with wild-type (WT) cells, osteoclastogenesis of Tank-silenced BMMs was increased, according to tartrate-resistant acid phosphatase (TRAP) stain on day 5 and day 7. Number of bone resorption pits by Tank-silenced osteoclasts was increased by 176% compared with WT cells, as shown by wheat germ agglutinin (WGA) stain and scanning electronic microscope (SEM) analysis. Survival rate of Tank-silenced mature osteoclast is also increased. However, acid production of Tank-knockdown cells was not changed compared with control cells. IκBα phosphorylation is increased in tank-silenced cells, indicating that TANK may negatively regulate NF-κB activity in osteoclast. In conclusion, Tank, whose expression is increased during osteoclastogenesis, inhibits osteoclast formation, activity and survival, by regulating NF-κB activity and c-FLIP expression. Tank enrolls itself in a negative feedback loop in bone resorption. These results may provide means for therapeutic intervention in diseases of excessive bone resorption.

Id1 represses osteoclast-dependent transcription and affects bone formation and hematopoiesis.

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April S Chan

2009-11-01

Full Text Available The bone-bone marrow interface is an area of the bone marrow microenvironment in which both bone remodeling cells, osteoblasts and osteoclasts, and hematopoietic cells are anatomically juxtaposed. The close proximity of these cells naturally suggests that they interact with one another, but these interactions are just beginning to be characterized.An Id1(-/- mouse model was used to assess the role of Id1 in the bone marrow microenvironment. Micro-computed tomography and fracture tests showed that Id1(-/- mice have reduced bone mass and increased bone fragility, consistent with an osteoporotic phenotype. Osteoclastogenesis and pit formation assays revealed that loss of Id1 increased osteoclast differentiation and resorption activity, both in vivo and in vitro, suggesting a cell autonomous role for Id1 as a negative regulator of osteoclast differentiation. Examination by flow cytometry of the hematopoietic compartment of Id1(-/- mice showed an increase in myeloid differentiation. Additionally, we found increased expression of osteoclast genes, TRAP, Oscar, and CTSK in the Id1(-/- bone marrow microenvironment. Lastly, transplantation of wild-type bone marrow into Id1(-/- mice repressed TRAP, Oscar, and CTSK expression and activity and rescued the hematopoietic and bone phenotype in these mice.In conclusion, we demonstrate an osteoporotic phenotype in Id1(-/- mice and a mechanism for Id1 transcriptional control of osteoclast-associated genes. Our results identify Id1 as a principal player responsible for the dynamic cross-talk between bone and bone marrow hematopoietic cells.

Chondroitin Sulfate-E Binds to Both Osteoactivin and Integrin αVβ3 and Inhibits Osteoclast Differentiation.

Science.gov (United States)

Miyazaki, Tatsuya; Miyauchi, Satoshi; Anada, Takahisa; Tawada, Akira; Suzuki, Osamu

2015-10-01

Integrins and their ligands have been suggested to be associated with osteoclast-mediated bone resorption. The present study was designed to investigate whether chondroitin sulfate E (CS-E), which is one of the sulfated glycosaminoglycans (GAGs), is involved in osteoactivin (OA) activity, and osteoclast differentiation. The binding affinity of sulfated GAGs to integrin and its ligand was measured using biotin-labeled CS-E, and the osteoclast differentiation was evaluated by tartrate-resistant acid phosphatase staining and a pit formation assay. CS-E as well as CS-B, synthetic chondroitin polysulfate, and heparin inhibited osteoclast differentiation of bone marrow-derived macrophages. Pre-coating of OA to synthetic calcium phosphate-coated plates enhanced the osteoclastic differentiation of RAW264 cells, and addition of a neutralizing antibody to OA inhibited its differentiation. CS-E bound not only to OA, fibronectin, and vitronectin, but also to its receptor integrin αVβ3, and inhibited the direct binding of OA to integrin αVβ3. Furthermore, CS-E blocked the binding of OA to cells and inhibited OA-induced osteoclastic differentiation. On the other hand, heparinase treatment of RAW264 cells inhibited osteoclastic differentiation. Since binding of OA to the cells was inhibited by the presence of heparan sulfate or heparinase treatment of cells, heparan sulfate proteoglycan (HSPG) was also considered to be an OA receptor. Taken together, the present results suggest that CS-E is capable of inhibiting OA-induced osteoclast differentiation by blocking the interaction of OA to integrin αVβ3 and HSPG. © 2015 Wiley Periodicals, Inc.

Excavatolide B Attenuates Rheumatoid Arthritis through the Inhibition of Osteoclastogenesis.

Science.gov (United States)

Lin, Yen-You; Jean, Yen-Hsuan; Lee, Hsin-Pai; Lin, Sung-Chun; Pan, Chieh-Yu; Chen, Wu-Fu; Wu, Shu-Fen; Su, Jui-Hsin; Tsui, Kuan-Hao; Sheu, Jyh-Horng; Sung, Ping-Jyun; Wen, Zhi-Hong

2017-01-06

Osteoclasts are multinucleated giant cells of macrophage/monocyte lineage, and cell differentiation with the upregulation of osteoclast-related proteins is believed to play a major role in the destruction of the joints in the course of rheumatoid arthritis (RA). Pro-inflammatory cytokines, such as interleukin-17A (IL-17A) and macrophage colony-stimulating factor (M-CSF), can be overexpressed in RA and lead to osteoclastogenesis. In a previous study, we found that cultured-type soft coral-derived excavatolide B (Exc-B) exhibited anti-inflammatory properties. In the present study, we thus aimed to evaluate the anti-arthritic activity of Exc-B in in vitro and in vivo models. The results demonstrated that Exc-B inhibits LPS-induced multinucleated cell and actin ring formation, as well as TRAP, MMP-9, and cathepsin K expression. Additionally, Exc-B significantly attenuated the characteristics of RA in adjuvant (AIA) and type II collagen-induced arthritis (CIA) in rats. Moreover, Exc-B improved histopathological features, and reduced the number of TRAP-positive multinucleated cells in the in vivo AIA and CIA models. Immunohistochemical analysis showed that Exc-B attenuated the protein expression of cathepsin K, MMP-2, MMP-9, CD11b, and NFATc1 in ankle tissues of AIA and CIA rats. Level of interleukin-17A and macrophage colony-stimulating factor were also decreased by Exc-B. These findings strongly suggest that Exc-B could be of potential use as a therapeutic agent by inhibiting osteoclast differentiation in arthritis. Moreover, this study also illustrates the use of the anti-inflammatory marine compound, Exc-B, as a potential therapeutic strategy for RA.

Excavatolide B Attenuates Rheumatoid Arthritis through the Inhibition of Osteoclastogenesis

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Yen-You Lin

2017-01-01

Full Text Available Osteoclasts are multinucleated giant cells of macrophage/monocyte lineage, and cell differentiation with the upregulation of osteoclast-related proteins is believed to play a major role in the destruction of the joints in the course of rheumatoid arthritis (RA. Pro-inflammatory cytokines, such as interleukin-17A (IL-17A and macrophage colony-stimulating factor (M-CSF, can be overexpressed in RA and lead to osteoclastogenesis. In a previous study, we found that cultured-type soft coral-derived excavatolide B (Exc-B exhibited anti-inflammatory properties. In the present study, we thus aimed to evaluate the anti-arthritic activity of Exc-B in in vitro and in vivo models. The results demonstrated that Exc-B inhibits LPS-induced multinucleated cell and actin ring formation, as well as TRAP, MMP-9, and cathepsin K expression. Additionally, Exc-B significantly attenuated the characteristics of RA in adjuvant (AIA and type II collagen-induced arthritis (CIA in rats. Moreover, Exc-B improved histopathological features, and reduced the number of TRAP-positive multinucleated cells in the in vivo AIA and CIA models. Immunohistochemical analysis showed that Exc-B attenuated the protein expression of cathepsin K, MMP-2, MMP-9, CD11b, and NFATc1 in ankle tissues of AIA and CIA rats. Level of interleukin-17A and macrophage colony-stimulating factor were also decreased by Exc-B. These findings strongly suggest that Exc-B could be of potential use as a therapeutic agent by inhibiting osteoclast differentiation in arthritis. Moreover, this study also illustrates the use of the anti-inflammatory marine compound, Exc-B, as a potential therapeutic strategy for RA.

Deletion of Adseverin in Osteoclasts Affects Cell Structure But Not Bone Metabolism

NARCIS (Netherlands)

Cao, Yixuan; Wang, Yongqiang; Sprangers, Sara; Picavet, Daisy I.; Glogauer, Michael; McCulloch, Christopher A.; Everts, Vincent

2017-01-01

Adseverin is an actin-severing/capping protein that may contribute to osteoclast differentiation in vitro but its role in bone remodeling of healthy animals is not defined. We analyzed bone and osteoclast structure in adseverin conditional null mice at alveolar and long bone sites. In wild-type and

(-)-Epigallocatechin gallate inhibition of osteoclastic differentiation via NF-κB

International Nuclear Information System (INIS)

Lin, R.-W.; Chen, C.-H.; Wang, Y.-H.; Ho, M.-L.; Hung, S.-H.; Chen, I.-S.; Wang, G.-J.

2009-01-01

People who regularly drink tea have been found to have a higher bone mineral density (BMD) and to be at less risk of hip fractures than those who do not drink it. Green tea catechins such as (-)-epigallocatechin gallate (EGCG) have been reported to increase osteogenic functioning in mesenchymal stem cells. However, its effect on osteoclastogenesis remains unclear. In this study, we investigated the effect of EGCG on RANKL-activation osteoclastogenesis and NF-κB in RAW 264.7, a murine preosteoclast cell line. EGCG (10-100 μM) significantly suppressed the RANKL-induced differentiation of osteoclasts and the formation of pits in murine RAW 264.7 cells and bone marrow macrophages (BMMs). EGCG appeared to target osteoclastic differentiation at an early stage but had no cytotoxic effect on osteoclast precursors. In addition, it significantly inhibited RANKL-induced NF-κB transcriptional activity and nuclear translocation. We conclude that EGCG inhibits osteoclastogenesis through its activation of NF-κB.

Follicle-Stimulating Hormone Increases the Risk of Postmenopausal Osteoporosis by Stimulating Osteoclast Differentiation.

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Jie Wang

Full Text Available The objectives of this study were to observe the changes in follicle-stimulating hormone (FSH and bone mineral density (BMD in postmenopausal women, to research the relationship between FSH and postmenopausal osteoporosis, and to observe the effects of FSH on osteoclast differentiation in RAW264.7 cells.We analyzed 248 postmenopausal women with normal bone metabolism. A radioimmunoassay (RIA was used to detect serum FSH, luteinizing hormone (LH, and estradiol (E2. Dual-energy X-ray absorptiometry was used to measure forearm BMD. Then, we analyzed the age-related changes in serum FSH, LH and E2. Additionally, FSH serum concentrations were compared between a group of postmenopausal women with osteoporosis and a control group. Osteoclasts were induced from RAW264.7 cells in vitro by receptor activator of nuclear factor kappa B ligand (RANKL, and these cells were treated with 0, 5, 10, and 20 ng/ml FSH. After the osteoclasts matured, tartrate-resistant acid phosphatase (TRAP staining was used to identify osteoclasts, and the mRNA expression levels of genes involved in osteoclastic phenotypes and function, such as receptor activator of NF-κB (Rank, Trap, matrix metalloproteinase-9 (Mmp-9 and Cathepsin K, were detected in different groups using real-time PCR (polymerase chain reaction.1. FSH serum concentrations in postmenopausal women with osteoporosis increased notably compared with the control group. 2. RANKL induced RAW264.7 cell differentiation into mature osteoclasts in vitro. 3. FSH increased mRNA expression of genes involved in osteoclastic phenotypes and function, such as Rank, Trap, Mmp-9 and Cathepsin K, in a dose-dependent manner.The circulating concentration of FSH may play an important role in the acceleration of bone loss in postmenopausal women. FSH increases osteoclastogenesis in vitro.

A-Type Cranberry Proanthocyanidins Inhibit the RANKL-Dependent Differentiation and Function of Human Osteoclasts

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Amy B. Howell

2011-03-01

Full Text Available This study investigated the effect of A-type cranberry proanthocyanidins (AC-PACs on osteoclast formation and bone resorption activity. The differentiation of human pre-osteoclastic cells was assessed by tartrate-resistant acid phosphatase (TRAP staining, while the secretion of interleukin-8 (IL-8 and matrix metalloproteinases (MMPs was measured by ELISA. Bone resorption activity was investigated by using a human bone plate coupled with an immunoassay that detected the release of collagen helical peptides. AC-PACs up to 100 µg/mL were atoxic for osteoclastic cells. TRAP staining evidenced a dose-dependent inhibition of osteoclastogenesis. More specifically, AC-PACs at 50 µg/mL caused a 95% inhibition of RANKL-dependent osteoclast differentiation. This concentration of AC-PACs also significantly increased the secretion of IL-8 (6-fold and inhibited the secretion of both MMP-2 and MMP-9. Lastly, AC-PACs (10, 25, 50 and 100 µg/ml affected bone degradation mediated by mature osteoclasts by significantly decreasing the release of collagen helical peptides. This study suggests that AC-PACs can interfere with osteoclastic cell maturation and physiology as well as prevent bone resorption. These compounds may be considered as therapeutic agents for the prevention and treatment of periodontitis.

Noncanonical Wnt signaling promotes osteoclast differentiation and is facilitated by the human immunodeficiency virus protease inhibitor ritonavir

International Nuclear Information System (INIS)

Santiago, Francisco; Oguma, Junya; Brown, Anthony M.C.; Laurence, Jeffrey

2012-01-01

Highlights: ► First demonstration of direct role for noncanonical Wnt in osteoclast differentiation. ► Demonstration of Ryk as a Wnt5a/b receptor in inhibition of canonical Wnt signaling. ► Modulation of noncanonical Wnt signaling by a clinically important drug, ritonavir. ► Establishes a mechanism for an important clinical problem: HIV-associated bone loss. -- Abstract: Wnt proteins that signal via the canonical Wnt/β-catenin pathway directly regulate osteoblast differentiation. In contrast, most studies of Wnt-related effects on osteoclasts involve indirect changes. While investigating bone mineral density loss in the setting of human immunodeficiency virus (HIV) infection and its treatment with the protease inhibitor ritonavir (RTV), we observed that RTV decreased nuclear localization of β-catenin, critical to canonical Wnt signaling, in primary human and murine osteoclast precursors. This occurred in parallel with upregulation of Wnt5a and Wnt5b transcripts. These Wnts typically stimulate noncanonical Wnt signaling, and this can antagonize the canonical Wnt pathway in many cell types, dependent upon Wnt receptor usage. We now document RTV-mediated upregulation of Wnt5a/b protein in osteoclast precursors. Recombinant Wnt5b and retrovirus-mediated expression of Wnt5a enhanced osteoclast differentiation from human and murine monocytic precursors, processes facilitated by RTV. In contrast, canonical Wnt signaling mediated by Wnt3a suppressed osteoclastogenesis. Both RTV and Wnt5b inhibited canonical, β-catenin/T cell factor-based Wnt reporter activation in osteoclast precursors. RTV- and Wnt5-induced osteoclast differentiation were dependent upon the receptor-like tyrosine kinase Ryk, suggesting that Ryk may act as a Wnt5a/b receptor in this context. This is the first demonstration of a direct role for Wnt signaling pathways and Ryk in regulation of osteoclast differentiation, and its modulation by a clinically important drug, ritonavir. These studies

GIANT CELL-RICH LESIONS OF BONE AND JOINTS: A ONE YEAR PROSPECTIVE STUDY

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Sri Nithisa H

2016-07-01

Full Text Available BACKGROUND Giant cell-rich lesions constitute a group of biologically and morphologically diverse bone and joint tumours. The common feature is presence of numerous multinucleated osteoclast-like giant cells. However, they differ from each other by in terms of clinical and radiographic features and in many cases by their distinct morphological features. METHODS All the bone and joint specimens with giant cell-rich lesions received in the period of one year were studied along with clinical and radiological data available. Gross and microscopic findings were noted. RESULTS In a period of one year, 10 cases of giant cell-rich lesions of bone and joints have been studied, which were and correlated with clinical and radiological findings. Five were lesions from bone and two were from joints, which are chondroblastoma, chondromyxoid fibroma, osteoclastoma, aneurysmal bone cyst, pigmented villonodular synovitis, giant cell lesion of tendon sheath, and tendinous xanthoma. CONCLUSION In the present study, variety of giant cell lesions of bone and joints are studied. Of which, the mean age in young patients being 20 years and in elderly patients being 50 years. The common site being lower end of femur.

Potential of Resveratrol Analogues as Antagonists of Osteoclasts and Promoters of Osteoblasts

DEFF Research Database (Denmark)

Kupisiewicz, Katarzyna; Boissy, Patrice; Abdallah, Basem M

2010-01-01

The plant phytoalexin resveratrol was previously demonstrated to inhibit the differentiation and bone resorbing activity of osteoclasts, to promote the formation of osteoblasts from mesenchymal precursors in cultures, and inhibit myeloma cell proliferation, when used at high concentrations....... In the current study, we screened five structurally modified resveratrol analogues for their ability to modify the differentiation of osteoclasts and osteoblasts and proliferation of myeloma cells. Compared to resveratrol, analogues showed an up to 5,000-fold increased potency to inhibit osteoclast...... differentiation. To a lesser extent, resveratrol analogues also promoted osteoblast maturation. However, they did not antagonize the proliferation of myeloma cells. The potency of the best-performing candidate in vitro was tested in vivo in an ovariectomy-induced model of osteoporosis, but an effect on bone loss...

Biglycan deficiency increases osteoclast differentiation and activity due to defective osteoblasts

DEFF Research Database (Denmark)

Bi, Yanming; Nielsen, Karina L; Kilts, Tina M

2006-01-01

to be independent of the differential production of soluble RANKL and OPG and, instead, due to a decrease in osteoblast maturation accompanied by increase in osteoblastic proliferation. In addition to the imbalance between differentiation and proliferation, there was a differential decrease in secretory leukocyte......Bone mass is maintained by a fine balance between bone formation by osteoblasts and bone resorption by osteoclasts. Although osteoblasts and osteoclasts have different developmental origins, it is generally believed that the differentiation, function, and survival of osteoclasts are regulated...... by osteogenic cells. We have previously shown that the extracellular matrix protein, biglycan (Bgn), plays an important role in the differentiation of osteoblast precursors. In this paper, we showed that Bgn is involved in regulating osteoclast differentiation through its effect on osteoblasts...

Pharmacological blocking of the osteoclastic biocorrosion of surgical stainless steel in vitro.

Science.gov (United States)

Lionetto, S; Little, A; Moriceau, G; Heymann, D; Decurtins, M; Plecko, M; Filgueira, L; Cadosch, D

2013-04-01

In vitro studies suggest that human osteoclasts (OC) are able to corrode surgical stainless steel 316L (SS). The aim of this study was to investigate whether osteoclastic biocorrosion can be blocked pharmacologically. Human OCs were generated in vitro from peripheral blood monocytic cells (PBMCs) in the presence of OC differentiation cytokines. The osteoclastic viability, differentiation, and resorptive function (on both bone and SS) were assessed using standard colorimetric cell viability assay 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenil)-2H-tetrazolium, inner salt (MTS), fluorescence microscopy, tartrate-resistant acid phosphatase expression (flow cytometry), and scanning electron microscopy. OCs cultured on SS were exposed to nontoxic concentrations of bafilomycin A1, amiloride hydrochloride, or zoledronic acid. The extent of biocorrosion was quantified using atomic emission spectrometry (to measure the concentration of metal ions released into the supernatant) and scanning electron microscopy. PBMCs differentiated into mature and functional OC in the presence of all the drugs used. Osteoclastic resorption of SS was noted with differences in the resorption pattern for all drug treatments. Under the drug treatments, single areas of osteoclastic resorption were larger in size but less abundant when compared with positive controls. None of the drugs used were able to inhibit osteoclastic biocorrosion of SS. Copyright © 2012 Wiley Periodicals, Inc.

Global epigenomic analysis indicates protocadherin-7 activates osteoclastogenesis by promoting cell–cell fusion

Energy Technology Data Exchange (ETDEWEB)

Nakamura, Haruhiko [Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033 (Japan); Department of Cell Signaling, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8549 (Japan); Nakashima, Tomoki [Department of Cell Signaling, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8549 (Japan); Japan Science and Technology Agency, PRESTO, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8549 (Japan); Hayashi, Mikihito [Department of Cell Signaling, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8549 (Japan); Japan Science and Technology Agency, ERATO, Takayanagi Osteonetwork Project, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Izawa, Naohiro; Yasui, Tetsuro [Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033 (Japan); Aburatani, Hiroyuki [Genome Science Division, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1, Komaba, Meguro-ku, Tokyo 153-8904 (Japan); Tanaka, Sakae [Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033 (Japan); Takayanagi, Hiroshi, E-mail: takayana@m.u-tokyo.ac.jp [Japan Science and Technology Agency, ERATO, Takayanagi Osteonetwork Project, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Department of Immunology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan)

2014-12-12

Highlights: • Identification of epigenetically regulated genes during osteoclastogenesis. • Pcdh7 is regulated by H3K4me3 and H3K27me3 during osteoclastogenesis. • Pcdh7 expression is increased by RANKL during osteoclastogenesis. • Establishment of novel cell fusion analysis for osteoclasts by imaging cytometer. • Pcdh7 regulates osteoclastogenesis by promoting cell fusion related gene expressions. - Abstract: Gene expression is dependent not only on genomic sequences, but also epigenetic control, in which the regulation of chromatin by histone modification plays a crucial role. Histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 trimethylation (H3K27me3) are related to transcriptionally activated and silenced sequences, respectively. Osteoclasts, the multinucleated cells that resorb bone, are generated by the fusion of precursor cells of monocyte/macrophage lineage. To elucidate the molecular and epigenetic regulation of osteoclast differentiation, we performed a chromatin immunoprecipitation sequencing (ChIP-seq) analysis for H3K4me3 and H3K27me3 in combination with RNA sequencing. We focused on the histone modification change from H3K4me3(+)H3K27me3(+) to H3K4me3(+)H3K27me3(–) and identified the protocadherin-7 gene (Pcdh7) to be among the genes epigenetically regulated during osteoclastogenesis. Pcdh7 was induced by RANKL stimulation in an NFAT-dependent manner. The knockdown of Pcdh7 inhibited RANKL-induced osteoclast differentiation due to the impairment of cell–cell fusion, accompanied by a decreased expression of the fusion-related genes Dcstamp, Ocstamp and Atp6v0d2. This study demonstrates that Pcdh7 plays a key role in osteoclastogenesis by promoting cell–cell fusion.

Global epigenomic analysis indicates protocadherin-7 activates osteoclastogenesis by promoting cell–cell fusion

International Nuclear Information System (INIS)

Nakamura, Haruhiko; Nakashima, Tomoki; Hayashi, Mikihito; Izawa, Naohiro; Yasui, Tetsuro; Aburatani, Hiroyuki; Tanaka, Sakae; Takayanagi, Hiroshi

2014-01-01

Highlights: • Identification of epigenetically regulated genes during osteoclastogenesis. • Pcdh7 is regulated by H3K4me3 and H3K27me3 during osteoclastogenesis. • Pcdh7 expression is increased by RANKL during osteoclastogenesis. • Establishment of novel cell fusion analysis for osteoclasts by imaging cytometer. • Pcdh7 regulates osteoclastogenesis by promoting cell fusion related gene expressions. - Abstract: Gene expression is dependent not only on genomic sequences, but also epigenetic control, in which the regulation of chromatin by histone modification plays a crucial role. Histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 trimethylation (H3K27me3) are related to transcriptionally activated and silenced sequences, respectively. Osteoclasts, the multinucleated cells that resorb bone, are generated by the fusion of precursor cells of monocyte/macrophage lineage. To elucidate the molecular and epigenetic regulation of osteoclast differentiation, we performed a chromatin immunoprecipitation sequencing (ChIP-seq) analysis for H3K4me3 and H3K27me3 in combination with RNA sequencing. We focused on the histone modification change from H3K4me3(+)H3K27me3(+) to H3K4me3(+)H3K27me3(–) and identified the protocadherin-7 gene (Pcdh7) to be among the genes epigenetically regulated during osteoclastogenesis. Pcdh7 was induced by RANKL stimulation in an NFAT-dependent manner. The knockdown of Pcdh7 inhibited RANKL-induced osteoclast differentiation due to the impairment of cell–cell fusion, accompanied by a decreased expression of the fusion-related genes Dcstamp, Ocstamp and Atp6v0d2. This study demonstrates that Pcdh7 plays a key role in osteoclastogenesis by promoting cell–cell fusion

Recombinant Human Endostatin Suppresses Mouse Osteoclast Formation by Inhibiting the NF-κB and MAPKs Signaling Pathways

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Non eChen

2016-06-01

Full Text Available Rheumatoid arthritis is an autoimmune disease characterized by synovial hyperplasia and progressive joint destruction. As reported previously, recombinant human endostatin (rhEndostatin is associated with inhibition of joint bone destruction present in rat adjuvant-induced arthritis; however, the effect of rhEndostatin on bone destruction is not known. This study was designed to assess the inhibitory effect and mechanisms of rhEndostatin on formation and function of osteoclasts in vitro, and to gain insight into the mechanism underlying the inhibitory effect of bone destruction. Bone marrow-derived macrophages isolated from BALB/c mice were stimulated with receptor activator of NF-κB ligand (RANKL and macrophage colony-stimulating factor to establish osteoclast formation. Osteoclast formation was determined by TRAP staining. Cell viability of BMMs affected by rhEndostatin was determined using a MTT assay. Bone resorption was examined with a bone resorption pits assay. The expression of osteoclast-specific markers was analyzed using quantitative real-time PCR. The related signaling pathways were examined using a Luciferase reporter assay and western blot analysis. Indeed, rhEndostatin showed a significant reduction in the number of osteoclast-like cells and early-stage bone resorption. Moreover, molecular analysis demonstrated that rhEndostatin attenuated RANKL-induced NF-κB signaling by inhibiting the phosphorylation of IκBα and NF-κB p65 nuclear translocation. Furthermore, rhEndostatin significantly inhibited the activation of RANKL-dependent mitogen-activated protein kinases (MAPKs, such as ERK1/2, JNK, and p38. Hence, we demonstrated for the first time that preventing the formation and function of osteoclasts is an important anti-bone destruction mechanism of rhEndostatin, which might be useful in the prevention and treatment of bone destruction in RA.

MicroRNAs: Potential Biomarkers and Therapeutic Targets for Alveolar Bone Loss in Periodontal Disease

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Tadayoshi Kagiya

2016-08-01

Full Text Available Periodontal disease is an inflammatory disease caused by bacterial infection of tooth-supporting structures, which results in the destruction of alveolar bone. Osteoclasts play a central role in bone destruction. Osteoclasts are tartrate-resistant acid phosphatase (TRAP-positive multinucleated giant cells derived from hematopoietic stem cells. Recently, we and other researchers revealed that microRNAs are involved in osteoclast differentiation. MicroRNAs are novel, single-stranded, non-coding, small (20–22 nucleotides RNAs that act in a sequence-specific manner to regulate gene expression at the post-transcriptional level through cleavage or translational repression of their target mRNAs. They regulate various biological activities such as cellular differentiation, apoptosis, cancer development, and inflammatory responses. In this review, the roles of microRNAs in osteoclast differentiation and function during alveolar bone destruction in periodontal disease are described.

Nucleic adaptability of heterokaryons to fungicides in a multinucleate fungus, Sclerotinia homoeocarpa.

Science.gov (United States)

Kessler, Dylan; Sang, Hyunkyu; Bousquet, Amanda; Hulvey, Jonathan P; Garcia, Dawlyn; Rhee, Siyeon; Hoshino, Yoichiro; Yamada, Toshihiko; Jung, Geunhwa

2018-06-01

Sclerotinia homoeocarpa is the causal organism of dollar spot in turfgrasses and is a multinucleate fungus with a history of resistance to multiple fungicide classes. Heterokaryosis gives rise to the coexistence of genetically distinct nuclei within a cell, which contributes to genotypic and phenotypic plasticity in multinucleate fungi. We demonstrate that field isolates, resistant to either a demethylation inhibitor or methyl benzimidazole carbamate fungicide, can form heterokaryons with resistance to each fungicide and adaptability to serial combinations of different fungicide concentrations. Field isolates and putative heterokaryons were assayed on fungicide-amended media for in vitro sensitivity. Shifts in fungicide sensitivity and microsatellite genotypes indicated that heterokaryons could adapt to changes in fungicide pressure. Presence of both nuclei in heterokaryons was confirmed by detection of a single nucleotide polymorphism in the β-tubulin gene, the presence of microsatellite alleles of both field isolates, and the live-cell imaging of two different fluorescently tagged nuclei using laser scanning confocal microscopy. Nucleic adaptability of heterokaryons to fungicides was strongly supported by the visualization of changes in fluorescently labeled nuclei to fungicide pressure. Results from this study suggest that heterokaryosis is a mechanism by which the pathogen adapts to multiple fungicide pressures in the field. Copyright © 2018 Elsevier Inc. All rights reserved.

TRAF Family Member-Associated NF-κB Activator (TANK) Induced by RANKL Negatively Regulates Osteoclasts Survival and Function

OpenAIRE

Mengrui Wu, Yiping Wang, Lianfu Deng, Wei Chen, Yi-Ping Li

2012-01-01

Osteoclasts are the principle bone-resorbing cells. Precise control of balanced osteoclast activity is indispensable for bone homeostasis. Osteoclast activation mediated by RANK-TRAF6 axis has been clearly identified. However, a negative regulation-machinery in osteoclast remains unclear. TRAF family member-associated NF-κB activator (TANK) is induced by about 10 folds during osteoclastogenesis, according to a genome-wide analysis of gene expression before and after osteoclast maturation...

TRAF Family Member-Associated NF-κB Activator (TANK) Induced by RANKL Negatively Regulates Osteoclasts Survival and Function

OpenAIRE

Wu, Mengrui; Wang, Yiping; Deng, Lianfu; Chen, Wei; Li, Yi-Ping

2012-01-01

Osteoclasts are the principle bone-resorbing cells. Precise control of balanced osteoclast activity is indispensable for bone homeostasis. Osteoclast activation mediated by RANK-TRAF6 axis has been clearly identified. However, a negative regulation-machinery in osteoclast remains unclear. TRAF family member-associated NF-κB activator (TANK) is induced by about 10 folds during osteoclastogenesis, according to a genome-wide analysis of gene expression before and after osteoclast maturation, and...

Molecular Stress-inducing Compounds Increase Osteoclast Formation in a Heat Shock Factor 1 Protein-dependent Manner*

Science.gov (United States)

Chai, Ryan C.; Kouspou, Michelle M.; Lang, Benjamin J.; Nguyen, Chau H.; van der Kraan, A. Gabrielle J.; Vieusseux, Jessica L.; Lim, Reece C.; Gillespie, Matthew T.; Benjamin, Ivor J.; Quinn, Julian M. W.; Price, John T.

2014-01-01

Many anticancer therapeutic agents cause bone loss, which increases the risk of fractures that severely reduce quality of life. Thus, in drug development, it is critical to identify and understand such effects. Anticancer therapeutic and HSP90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) causes bone loss by increasing osteoclast formation, but the mechanism underlying this is not understood. 17-AAG activates heat shock factor 1 (Hsf1), the master transcriptional regulator of heat shock/cell stress responses, which may be involved in this negative action of 17-AAG upon bone. Using mouse bone marrow and RAW264.7 osteoclast differentiation models we found that HSP90 inhibitors that induced a heat shock response also enhanced osteoclast formation, whereas HSP90 inhibitors that did not (including coumermycin A1 and novobiocin) did not affect osteoclast formation. Pharmacological inhibition or shRNAmir knockdown of Hsf1 in RAW264.7 cells as well as the use of Hsf1 null mouse bone marrow cells demonstrated that 17-AAG-enhanced osteoclast formation was Hsf1-dependent. Moreover, ectopic overexpression of Hsf1 enhanced 17-AAG effects upon osteoclast formation. Consistent with these findings, protein levels of the essential osteoclast transcription factor microphthalmia-associated transcription factor were increased by 17-AAG in an Hsf1-dependent manner. In addition to HSP90 inhibitors, we also identified that other agents that induced cellular stress, such as ethanol, doxorubicin, and methotrexate, also directly increased osteoclast formation, potentially in an Hsf1-dependent manner. These results, therefore, indicate that cellular stress can enhance osteoclast differentiation via Hsf1-dependent mechanisms and may significantly contribute to pathological and therapeutic related bone loss. PMID:24692538

Molecular stress-inducing compounds increase osteoclast formation in a heat shock factor 1 protein-dependent manner.

Science.gov (United States)

Chai, Ryan C; Kouspou, Michelle M; Lang, Benjamin J; Nguyen, Chau H; van der Kraan, A Gabrielle J; Vieusseux, Jessica L; Lim, Reece C; Gillespie, Matthew T; Benjamin, Ivor J; Quinn, Julian M W; Price, John T

2014-05-09

Many anticancer therapeutic agents cause bone loss, which increases the risk of fractures that severely reduce quality of life. Thus, in drug development, it is critical to identify and understand such effects. Anticancer therapeutic and HSP90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) causes bone loss by increasing osteoclast formation, but the mechanism underlying this is not understood. 17-AAG activates heat shock factor 1 (Hsf1), the master transcriptional regulator of heat shock/cell stress responses, which may be involved in this negative action of 17-AAG upon bone. Using mouse bone marrow and RAW264.7 osteoclast differentiation models we found that HSP90 inhibitors that induced a heat shock response also enhanced osteoclast formation, whereas HSP90 inhibitors that did not (including coumermycin A1 and novobiocin) did not affect osteoclast formation. Pharmacological inhibition or shRNAmir knockdown of Hsf1 in RAW264.7 cells as well as the use of Hsf1 null mouse bone marrow cells demonstrated that 17-AAG-enhanced osteoclast formation was Hsf1-dependent. Moreover, ectopic overexpression of Hsf1 enhanced 17-AAG effects upon osteoclast formation. Consistent with these findings, protein levels of the essential osteoclast transcription factor microphthalmia-associated transcription factor were increased by 17-AAG in an Hsf1-dependent manner. In addition to HSP90 inhibitors, we also identified that other agents that induced cellular stress, such as ethanol, doxorubicin, and methotrexate, also directly increased osteoclast formation, potentially in an Hsf1-dependent manner. These results, therefore, indicate that cellular stress can enhance osteoclast differentiation via Hsf1-dependent mechanisms and may significantly contribute to pathological and therapeutic related bone loss.

Human mesenchymal stem cells inhibit osteoclastogenesis through osteoprotegerin production.

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Oshita, Koichi; Yamaoka, Kunihiro; Udagawa, Nobuyuki; Fukuyo, Shunsuke; Sonomoto, Koshiro; Maeshima, Keisuke; Kurihara, Ryuji; Nakano, Kazuhisa; Saito, Kazuyoshi; Okada, Yosuke; Chiba, Kenji; Tanaka, Yoshiya

2011-06-01

Mesenchymal stem cells (MSCs) have been proposed to be a useful tool for treatment of rheumatoid arthritis (RA), not only because of their multipotency but also because of their immunosuppressive effect on lymphocytes, dendritic cells, and other proinflammatory cells. Since bone destruction caused by activated osteoclasts occurs in RA, we undertook the present study to investigate the effect of MSCs on osteoclast function and differentiation in order to evaluate their potential use in RA therapy. Human MSCs and peripheral blood mononuclear cells were cultured under cell-cell contact-free conditions with osteoclast induction medium. Differentiation into osteoclast-like cells was determined by tartrate-resistant acid phosphatase staining and expression of osteoclast differentiation markers. The number of osteoclast-like cells was decreased and expression of cathepsin K and nuclear factor of activated T cells c1 (NF-ATc1) was down-regulated by the addition of either MSCs or a conditioned medium obtained from MSCs. Osteoprotegerin (OPG) was constitutively produced by MSCs and inhibited osteoclastogenesis. However, osteoclast differentiation was not fully recovered upon treatment with either anti-OPG antibody or OPG small interfering RNA, suggesting that OPG had only a partial role in the inhibitory effect of MSCs. Moreover, bone-resorbing activity of osteoclast-like cells was partially recovered by addition of anti-OPG antibody into the conditioned medium. The present results indicate that human MSCs constitutively produce OPG, resulting in inhibition of osteoclastogenesis and expression of NF-ATc1 and cathepsin K in the absence of cell-cell contact. Therefore, we conclude that human MSCs exert a suppressive effect on osteoclastogenesis, which may be beneficial in inhibition of joint damage in RA. Copyright © 2011 by the American College of Rheumatology.

Avenanthramides Prevent Osteoblast and Osteocyte Apoptosis and Induce Osteoclast Apoptosis in Vitro in an Nrf2-Independent Manner

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Gretel G. Pellegrini

2016-07-01

Full Text Available Oats contain unique bioactive compounds known as avenanthramides (AVAs with antioxidant properties. AVAs might enhance the endogenous antioxidant cellular response by activation of the transcription factor Nrf2. Accumulation of reactive oxygen species plays a critical role in many chronic and degenerative diseases, including osteoporosis. In this disease, there is an imbalance between bone formation by osteoblasts and bone resorption by osteoclasts, which is accompanied by increased osteoblast/osteocyte apoptosis and decreased osteoclast apoptosis. We investigated the ability of the synthethic AVAs 2c, 2f and 2p, to 1-regulate gene expression in bone cells, 2-affect the viability of osteoblasts, osteocytes and osteoclasts, and the generation of osteoclasts from their precursors, and 3-examine the potential involvement of the transcription factor Nrf2 in these actions. All doses of AVA 2c and 1 and 5 µM dose of 2p up-regulated collagen 1A expression. Lower doses of AVAs up-regulated OPG (osteoprotegerin in OB-6 osteoblastic cells, whereas 100 μM dose of 2f and all concentrations of 2c down-regulated RANKL gene expression in MLO-Y4 osteocytic cells. AVAs did not affect apoptosis of OB-6 osteoblastic cells or MLO-Y4 osteocytic cells; however, they prevented apoptosis induced by the DNA topoisomerase inhibitor etoposide, the glucocorticoid dexamethasone, and hydrogen peroxide. AVAs prevented apoptosis of both wild type (WT and Nrf2 Knockout (KO osteoblasts, demonstrating that AVAs-induced survival does not require Nrf2 expression. Further, KO osteoclast precursors produced more mature osteoclasts than WT; and KO cultures exhibited less apoptotic osteoclasts than WT cultures. Although AVAs did not affect WT osteoclasts, AVA 2p reversed the low apoptosis of KO osteoclasts. These in vitro results demonstrate that AVAs regulate, in part, the function of osteoblasts and osteocytes and prevent osteoblast/osteocyte apoptosis and increase osteoclast

Diphlorethohydroxycarmalol from Ishige okamurae Suppresses Osteoclast Differentiation by Downregulating the NF-κB Signaling Pathway

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Hye Jung Ihn

2017-12-01

Full Text Available Marine algae possess a variety of beneficial effects on human health. In this study, we investigated whether diphlorethohydroxycarmalol (DPHC, isolated from Ishige okamurae, a brown alga, suppresses receptor activator of nuclear factor-κB ligand (RANKL-induced osteoclast differentiation. DPHC significantly suppressed RANKL-induced osteoclast differentiation and macrophage-colony stimulating factor (M-CSF expression in a dose-dependent manner. In addition, it significantly inhibited actin ring formation, the expression of osteoclast marker genes, such as tartrate-resistant acid phosphatase (TRAP, nuclear factor of activated T-cells cytoplasmic 1 (Nfatc1, cathepsin K (Ctsk, and dendritic cell-specific transmembrane protein (Dcstamp, and osteoclast-induced bone resorption. Analysis of the RANKL-mediated signaling pathway showed that the phosphorylation of both IκB and p65 was specifically inhibited by DPHC. These results suggest that DPHC substantially suppresses osteoclastogenesis by downregulating the RANK-NF-κB signaling pathway. Thus, it holds significant potential for the treatment of skeletal diseases associated with an enhanced osteoclast activity.

Biocorrosion and uptake of titanium by human osteoclasts.

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Cadosch, Dieter; Al-Mushaiqri, Mohamed S; Gautschi, Oliver P; Meagher, James; Simmen, Hans-Peter; Filgueira, Luis

2010-12-15

All metals in contact with a biological system undergo corrosion through an electrochemical redox reaction. This study investigated whether human osteoclasts (OC) are able to grow on titanium and aluminum, and directly corrode the metals leading to the release of corresponding metal ions, which are believed to cause inflammatory reactions and activate osteoclastic differentiation. Scanning electron microscopy analysis demonstrated long-term viable OC cultures on the surface of titanium and aluminum foils. Atomic emission spectrometry investigations showed significantly increased levels of aluminum in the supernatant of OC cultured on aluminum; however, all measurements in the supernatants of cell cultures on titanium were below detection limits. Despite this, confocal microscopy analysis with Newport Green DCF diacetate ester staining depicted intense fluorescence throughout the cytoplasm and nucleolus of OC cultured on titanium foils. Comparable fluorescence intensities were not observed in monocytes and control cells cultured on glass. The present study demonstrated that human osteoclast precursors are able to grow and differentiate toward mature OC on titanium and aluminum. Furthermore, it established that the mature cells are able to directly corrode the metal surface and take up corresponding metal ions, which subsequently may be released and thereby induce the formation of osteolytic lesions in the periprosthetic bone, contributing to the loosening of the implant. Copyright © 2010 Wiley Periodicals, Inc.

The polyphenol fisetin protects bone by repressing NF-κB and MKP-1-dependent signaling pathways in osteoclasts.

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Laurent Léotoing

Full Text Available Osteoporosis is a bone pathology leading to increase fractures risk and challenging quality of life. Since current treatments could exhibit deleterious side effects, the use of food compounds derived from plants represents a promising innovative alternative due to their potential therapeutic and preventive activities against human diseases. In this study, we investigated the ability of the polyphenol fisetin to counter osteoporosis and analyzed the cellular and molecular mechanisms involved. In vivo, fisetin consumption significantly prevented bone loss in estrogen deficiency and inflammation mice osteoporosis models. Indeed, bone mineral density, micro-architecture parameters and bone markers were positively modulated by fisetin. Consistent with in vivo results, we showed that fisetin represses RANKL-induced osteoclast differentiation and activity as demonstrated by an inhibition of multinucleated cells formation, TRAP activity and differentiation genes expression. The signaling pathways NF-κB, p38 MAPK, JNK and the key transcription factors c-Fos and NFATc1 expressions induced by RANKL, were negatively regulated by fisetin. We further showed that fisetin inhibits the constitutive proteasomal degradation of MKP-1, the phosphatase that deactivates p38 and JNK. Consistently, using shRNA stable cell lines, we demonstrated that impairment of MKP-1 decreases fisetin potency. Taken together, these results strongly support that fisetin should be further considered as a bone protective agent.

The Polyphenol Fisetin Protects Bone by Repressing NF-κB and MKP-1-Dependent Signaling Pathways in Osteoclasts

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Léotoing, Laurent; Wauquier, Fabien; Guicheux, Jérôme; Miot-Noirault, Elisabeth; Wittrant, Yohann; Coxam, Véronique

2013-01-01

Osteoporosis is a bone pathology leading to increase fractures risk and challenging quality of life. Since current treatments could exhibit deleterious side effects, the use of food compounds derived from plants represents a promising innovative alternative due to their potential therapeutic and preventive activities against human diseases. In this study, we investigated the ability of the polyphenol fisetin to counter osteoporosis and analyzed the cellular and molecular mechanisms involved. In vivo, fisetin consumption significantly prevented bone loss in estrogen deficiency and inflammation mice osteoporosis models. Indeed, bone mineral density, micro-architecture parameters and bone markers were positively modulated by fisetin. Consistent with in vivo results, we showed that fisetin represses RANKL-induced osteoclast differentiation and activity as demonstrated by an inhibition of multinucleated cells formation, TRAP activity and differentiation genes expression. The signaling pathways NF-κB, p38 MAPK, JNK and the key transcription factors c-Fos and NFATc1 expressions induced by RANKL, were negatively regulated by fisetin. We further showed that fisetin inhibits the constitutive proteasomal degradation of MKP-1, the phosphatase that deactivates p38 and JNK. Consistently, using shRNA stable cell lines, we demonstrated that impairment of MKP-1 decreases fisetin potency. Taken together, these results strongly support that fisetin should be further considered as a bone protective agent. PMID:23861901

Antioxidant and bone repair properties of quercetin-functionalized hydroxyapatite: An in vitro osteoblast-osteoclast-endothelial cell co-culture study.

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Forte, Lucia; Torricelli, Paola; Boanini, Elisa; Gazzano, Massimo; Rubini, Katia; Fini, Milena; Bigi, Adriana

2016-03-01

Quercetin (3,3',4',5,7-pentahydroxy-flavone) is a flavonoid known for its pharmacological activities, which include antioxidant and anti-inflammatory properties, as well as possible beneficial action on diseases involving bone loss. In this work, we explored the possibility to functionalize hydroxyapatite (HA) with quercetin in order to obtain new materials for bone repair through local administration of the flavonoid. HA was synthesized in presence of different concentrations of quercetin according to two different procedures: direct synthesis and phase transition from monetite. Direct synthesis lead to composite nanocrystals containing up to 3.1 wt% quercetin, which provokes a reduction of the crystals mean dimensions and of the length of the coherently scattering domains. Synthesis conditions provoke a partial oxidation of quercetin and, as a consequence, a significant reduction of its radical scavenging activity (RSA). On the other hand, synthesis through phase transition yields samples containing up to 1.3 wt% of quercetin incorporated into hydroxyapatite, with minor structural modifications, which exhibit relevant anti-oxidant activities, as testified by their high RSA levels, (slightly lower than that of pure quercetin). The biological response to these materials was tested using an innovative triculture model involving osteoblast, osteoclast and endothelial cells, in order to mimic bone microenvironment. The results show that the presence of quercetin in the composite materials enhances human osteoblast-like MG63 proliferation and differentiation, whereas it downregulates osteoclastogenesis of osteoclast precursors 2T-110, and supports proliferation and differentiation of human umbilical vein endothelial cells (HUVEC). The pharmacological activities of the flavonoid quercetin include anti-oxidant and antiinflammatory properties, as well as capability to prevent bone loss. In this paper, we demonstrate that it is possible to synthesize hydroxyapatite

DC-STAMP Is an Osteoclast Fusogen Engaged in Periodontal Bone Resorption.

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Wisitrasameewong, W; Kajiya, M; Movila, A; Rittling, S; Ishii, T; Suzuki, M; Matsuda, S; Mazda, Y; Torruella, M R; Azuma, M M; Egashira, K; Freire, M O; Sasaki, H; Wang, C Y; Han, X; Taubman, M A; Kawai, T

2017-06-01

Dendritic cell-specific transmembrane protein (DC-STAMP) plays a key role in the induction of osteoclast (OC) cell fusion, as well as DC-mediated immune regulation. While DC-STAMP gene expression is upregulated in the gingival tissue with periodontitis, its pathophysiological roles in periodontitis remain unclear. To evaluate the effects of DC-STAMP in periodontitis, anti-DC-STAMP-monoclonal antibody (mAb) was tested in a mouse model of ligature-induced periodontitis ( n = 6-7/group) where Pasteurella pneumotropica ( Pp)-reactive immune response activated T cells to produce receptor activator of nuclear factor kappa-B ligand (RANKL), which, in turn, promotes the periodontal bone loss via upregulation of osteoclastogenesis. DC-STAMP was expressed on the cell surface of mature multinuclear OCs, as well as immature mononuclear OCs, in primary cultures of RANKL-stimulated bone marrow cells. Anti-DC-STAMP-mAb suppressed the emergence of large, but not small, multinuclear OCs, suggesting that DC-STAMP is engaged in the late stage of cell fusion. Anti-DC-STAMP-mAb also inhibited pit formation caused by RANKL-stimulated bone marrow cells. Attachment of ligature to a second maxillary molar induced DC-STAMP messenger RNA and protein, along with elevated tartrate-resistant acid phosphatase-positive (TRAP+) OCs and alveolar bone loss. As we expected, systemic administration of anti-DC-STAMP-mAb downregulated the ligature-induced alveolar bone loss. Importantly, local injection of anti-DC-STAMP-mAb also suppressed alveolar bone loss and reduced the total number of multinucleated TRAP+ cells in mice that received ligature attachment. Attachment of ligature induced significantly elevated tumor necrosis factor-α, interleukin-1β, and RANKL in the gingival tissue compared with the control site without ligature ( P < 0.05), which was unaffected by local injection with either anti-DC-STAMP-mAb or control-mAb. Neither in vivo anti- Pp IgG antibody nor in vitro anti- Pp T-cell

Distinctive subdomains in the resorbing surface of osteoclasts.

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Kinga A Szewczyk

Full Text Available We employed a novel technique to inspect the substrate-apposed surface of activated osteoclasts, the cells that resorb bone, in the scanning electron microscope. The surface revealed unexpected complexity. At the periphery of the cells were circles and crescents of individual or confluent nodules. These corresponded to the podosomes and actin rings that form a 'sealing zone', encircling the resorptive hemivacuole into which protons and enzymes are secreted. Inside these rings and crescents the osteoclast surface was covered with strips and patches of membrane folds, which were flattened against the substrate surface and surrounded by fold-free membrane in which many orifices could be seen. Corresponding regions of folded and fold-free membrane were found by transmission electron microscopy in osteoclasts incubated on bone. We correlated these patterns with the distribution of several proteins crucial to resorption. The strips and patches of membrane folds corresponded in distribution to vacuolar H+-ATPase, and frequently co-localized with F-actin. Cathepsin K localized to F-actin-free foci towards the center of cells with circular actin rings, and at the retreating pole of cells with actin crescents. The chloride/proton antiporter ClC-7 formed a sharply-defined band immediately inside the actin ring, peripheral to vacuolar H+-ATPase. The sealing zone of osteoclasts is permeable to molecules with molecular mass up to 10,000. Therefore, ClC-7 might be distributed at the periphery of the resorptive hemivacuole in order to prevent protons from escaping laterally from the hemivacuole into the sealing zone, where they would dissolve the bone mineral. Since the activation of resorption is attributable to recognition of the αVβ3 ligands bound to bone mineral, such leakage would, by dissolving bone mineral, release the ligands and so terminate resorption. Therefore, ClC-7 might serve not only to provide the counter-ions that enable proton pumping, but

Effects of extracellular magnesium on the differentiation and function of human osteoclasts.

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Wu, Lili; Luthringer, Bérengère J C; Feyerabend, Frank; Schilling, Arndt F; Willumeit, Regine

2014-06-01

Magnesium-based implants have been shown to influence the surrounding bone structure. In an attempt to partially reveal the cellular mechanisms involved in the remodelling of magnesium-based implants, the influence of increased extracellular magnesium content on human osteoclasts was studied. Peripheral blood mononuclear cells were driven towards an osteoclastogenesis pathway via stimulation with receptor activator of nuclear factor kappa-B ligand and macrophage colony-stimulating factor for 28 days. Concomitantly, the cultures were exposed to variable magnesium concentrations (from either magnesium chloride or magnesium extracts). Osteoclast proliferation and differentiation were evaluated based on cell metabolic activity, total protein content, tartrate-resistant acid phosphatase activity, cathepsin K and calcitonin receptor immunocytochemistry, and cellular ability to form resorption pits. While magnesium chloride first enhanced and then opposed cell proliferation and differentiation in a concentration-dependent manner (peaking between 10 and 15mM magnesium chloride), magnesium extracts (with lower magnesium contents) appeared to decrease cell metabolic activity (≈50% decrease at day 28) while increasing osteoclast activity at a lower concentration (twofold higher). Together, the results indicated that (i) variations in the in vitro extracellular magnesium concentration affect osteoclast metabolism and (ii) magnesium extracts should be used preferentially in vitro to more closely mimic the in vivo environment. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Peri-Implant Crestal Bone Loss: A Putative Mechanism

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Yuko Ujiie

2012-01-01

Full Text Available Purpose. The immunological mechanisms of peri-implant crestal bone loss have, hitherto, not been elucidated. We hypothesized that bacterial products from the microgap cause upregulation of cytokines in otherwise healthy peri-implant cells, which results in osteoclast formation and, ultimately, in bone resorption. Materials and Methods. We used RT-PCR and ELISA to assay mediators of osteoclastogenesis in rat and human macrophages (r-and hMO; bone marrow derived stromal cells (r-and hBMCs; and human gingival fibroblasts (hGF—with or without stimulation by LPS. TRAP positive multinucleate cells were assessed for their resorptive ability. Results. We show that IL-1α, IL-1β, and IL-6 were expressed by all examined cell types, and TNF-α was upregulated in hGF. Secretion of IL-1α and IL-1β proteins was stimulated in hMO by LPS, and IL-6 protein secretion was highly stimulated in hBMCs and hGF. Both LPS and RANKL stimulated macrophages to form osteoclast-like TRAP positive cells, which resorbed calcium phosphate substrates. Conclusion. Taken together, the results of our study support the hypothesis that bacterial endotoxins upregulate enhanced mediators of osteoclastogenesis in resident cells found in the healthy peri-implant compartment and that the local synergistic action of cytokines secreted by such cells results in the genesis of resorptively active osteoclasts.

Transgenic mice for a tamoxifen-induced, conditional expression of the Cre recombinase in osteoclasts.

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Maria Arantzazu Sanchez-Fernandez

Full Text Available 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+/-LacZ+/- adult mice show a Cre-dependent β-galactosidase activity after tamoxifen stimulation. CONCLUSIONS/SIGNIFICANCE: We have generated transgenic lines that enable the tamoxifen-induced, conditional deletion of loxP-flanked genes in osteoclasts, thus circumventing embryonic and postnatal gene lethality and avoiding gene deletion in other cell types. Such CtsKCreERT2 mice provide a convenient tool to study in vivo the different facets of osteoclast function in bone physiology during different developmental stages and adulthood of mice.

Conditional abrogation of Atm in osteoclasts extends osteoclast lifespan and results in reduced bone mass.

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Hirozane, Toru; Tohmonda, Takahide; Yoda, Masaki; Shimoda, Masayuki; Kanai, Yae; Matsumoto, Morio; Morioka, Hideo; Nakamura, Masaya; Horiuchi, Keisuke

2016-09-28

Ataxia-telangiectasia mutated (ATM) kinase is a central component involved in the signal transduction of the DNA damage response (DDR) and thus plays a critical role in the maintenance of genomic integrity. Although the primary functions of ATM are associated with the DDR, emerging data suggest that ATM has many additional roles that are not directly related to the DDR, including the regulation of oxidative stress signaling, insulin sensitivity, mitochondrial homeostasis, and lymphocyte development. Patients and mice lacking ATM exhibit growth retardation and lower bone mass; however, the mechanisms underlying the skeletal defects are not fully understood. In the present study, we generated mutant mice in which ATM is specifically inactivated in osteoclasts. The mutant mice did not exhibit apparent developmental defects but showed reduced bone mass due to increased osteoclastic bone resorption. Osteoclasts lacking ATM were more resistant to apoptosis and showed a prolonged lifespan compared to the controls. Notably, the inactivation of ATM in osteoclasts resulted in enhanced NF-κB signaling and an increase in the expression of NF-κB-targeted genes. The present study reveals a novel function for ATM in regulating bone metabolism by suppressing the lifespan of osteoclasts and osteoclast-mediated bone resorption.

Osteoclasts prefer aged bone

DEFF Research Database (Denmark)

Henriksen, K; Leeming, Diana Julie; Byrjalsen, I

2007-01-01

We investigated whether the age of the bones endogenously exerts control over the bone resorption ability of the osteoclasts, and found that osteoclasts preferentially develop and resorb bone on aged bone. These findings indicate that the bone matrix itself plays a role in targeted remodeling...... of aged bones....

Fluoride Stimulates the Proliferation of Osteoclasts in vitro by Upregulating MCM3

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Shengbin Bai

2016-06-01

Full Text Available We have previously shown that the expression of the minichromosome maintenance protein 3 (MCM3 gene was upregulated in lymphocytes of patients with skeletal fluorosis. We speculated that increased MCM3 expression may be contribute to osteopathy in patients with skeletal fluorosis. Here, we investigated the effect of fluoride on the proliferation of osteoclasts derived from RAW264.7 cells and the involvement of MCM3. Our MTT assays showed that 0.25 mM NaF markedly stimulated the proliferation of RAW264.7 cells. The RT-PCR and immunoblotting assays revealed that 0.25 mM NaF upregulated MCM3 expression in RAW264.7 cells. The MTT assays additionally demonstrated that stimulation with MCM3 potentiated the effect of fluorine on the proliferation of RAW264.7 cells. These results demonstrated that fluoride at clinical relevant concentration upregulates MCM3 expression in osteoclasts in vitro. We are currently conducting a series of experiments to examine whether increased MCM3 in osteoclasts indeed contributes to osteopathy in skeletal fluorosis.

IL-33 inhibits RANKL-induced osteoclast formation through the regulation of Blimp-1 and IRF-8 expression

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Kiyomiya, Hiroyasu [Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580 (Japan); Division of Oral and Maxillofacial Surgery, Department of Science of Physical Functions, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580 (Japan); Ariyoshi, Wataru; Okinaga, Toshinori [Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580 (Japan); Kaneuji, Takeshi [Division of Oral Medicine, Department of Science of Physical Functions, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580 (Japan); Mitsugi, Sho [Division of Oral and Maxillofacial Surgery, Department of Science of Physical Functions, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580 (Japan); Sakurai, Takuma [Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580 (Japan); Division of Oral and Maxillofacial Surgery, Department of Science of Physical Functions, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580 (Japan); Habu, Manabu [Division of Oral and Maxillofacial Surgery, Department of Science of Physical Functions, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580 (Japan); Yoshioka, Izumi [Division of Oral Medicine, Department of Science of Physical Functions, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580 (Japan); Tominaga, Kazuhiro [Division of Oral and Maxillofacial Surgery, Department of Science of Physical Functions, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580 (Japan); and others

2015-05-01

Interleukin (IL)-33 is a recently discovered proinflammatory cytokine that belongs to the IL-1 family. Several studies have reported that IL-33 inhibits osteoclast differentiation. However, the mechanism of IL-33 regulation of osteoclastogenesis remains unclear. In the present study, we examined the effect of IL-33 on osteoclast formation in vitro. IL-33 suppressed osteoclast formation in both mouse bone marrow cells and monocyte/macrophage cell line RAW264.7 cells induced by receptor activator of NF-κB ligand (RANKL) and/or macrophage stimulating factor (M-CSF). IL-33 also inhibited the expression of RANKL-induced nuclear factor of activated T-cell cytoplasmic 1 (NFATc1), thereby decreasing the expression of osteoclastogenesis-related marker genes, including Cathepsin K, Osteoclast stimulatory transmembrane protein (Oc-stamp) and Tartrate-resistant acid phosphatase (Trap). Blockage of IL-33-ST2 binding suppressed the IL-33-mediated inhibition of NFATc1. RANKL-induced B-lymphocyte-induced maturation protein-1 (Blimp-1) expression was also suppressed by IL-33, which was followed by the stimulation of anti-osteoclastic genes such as interferon regulatory factor-8 (IRF-8). These results suggest that IL-33-ST2 interactions down-regulate both RANKL-induced NFATc1 activation and osteoclast differentiation via the regulation of Blimp-1 and IRF-8 expression. - Highlights: • IL-33 inhibits RANKL-induced osteoclast formation. • IL-33 has inhibitory effect on the RANKL-induced NFATc1 expression. • IL-33-induced NFATc1 suppression depends on the regulation of Blimp-1 and IRF-8.

IL-33 inhibits RANKL-induced osteoclast formation through the regulation of Blimp-1 and IRF-8 expression

International Nuclear Information System (INIS)

Kiyomiya, Hiroyasu; Ariyoshi, Wataru; Okinaga, Toshinori; Kaneuji, Takeshi; Mitsugi, Sho; Sakurai, Takuma; Habu, Manabu; Yoshioka, Izumi; Tominaga, Kazuhiro

2015-01-01

Interleukin (IL)-33 is a recently discovered proinflammatory cytokine that belongs to the IL-1 family. Several studies have reported that IL-33 inhibits osteoclast differentiation. However, the mechanism of IL-33 regulation of osteoclastogenesis remains unclear. In the present study, we examined the effect of IL-33 on osteoclast formation in vitro. IL-33 suppressed osteoclast formation in both mouse bone marrow cells and monocyte/macrophage cell line RAW264.7 cells induced by receptor activator of NF-κB ligand (RANKL) and/or macrophage stimulating factor (M-CSF). IL-33 also inhibited the expression of RANKL-induced nuclear factor of activated T-cell cytoplasmic 1 (NFATc1), thereby decreasing the expression of osteoclastogenesis-related marker genes, including Cathepsin K, Osteoclast stimulatory transmembrane protein (Oc-stamp) and Tartrate-resistant acid phosphatase (Trap). Blockage of IL-33-ST2 binding suppressed the IL-33-mediated inhibition of NFATc1. RANKL-induced B-lymphocyte-induced maturation protein-1 (Blimp-1) expression was also suppressed by IL-33, which was followed by the stimulation of anti-osteoclastic genes such as interferon regulatory factor-8 (IRF-8). These results suggest that IL-33-ST2 interactions down-regulate both RANKL-induced NFATc1 activation and osteoclast differentiation via the regulation of Blimp-1 and IRF-8 expression. - Highlights: • IL-33 inhibits RANKL-induced osteoclast formation. • IL-33 has inhibitory effect on the RANKL-induced NFATc1 expression. • IL-33-induced NFATc1 suppression depends on the regulation of Blimp-1 and IRF-8

[The polyploidization characteristics of the hepatocytes of the mouse-like hamster Calomyscus mystax].

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Anatskaia, O V; Malikov, V G; Meĭer, M N; Kudriavtsev, B N

1995-01-01

A cytophotometric measurement of DNA content in hepatocytes of maturing mouse-like hamsters was made. Cells belonging to ordinary mammalian ploidy classes 2c, 2c x 2, 4c, and 4c x 2 made about 90% of the hepatocyte population. The share of binucleated cells wa high (about 80%), the majority of these cells being 2c X 2 hepatocytes. Binucleated cells with tetraploid and diploid nuclei occur in almost every animal. An average hepatocyte ploidy level in mouse-like hamster is 4.6c. The main peculiarity of parenchymal liver cell populations is that up 5% of hepatocytes contain 3--11 nuclei of different ploidy classes. Multinucleated cells increase in number from 1.5% to 4% within the period from one year (the age of maturation) to two years. Later on their percentage does not change. It is found that in binucleated and multinucleated hepatocytes DNA synthesis can proceed asynchronously. Asynchrony in DNA synthesis elevates as the number of nuclei increases. Among the 2c x 2 and 2c x 3 cells an uneven distribution of 3H-thymidine label can occur, respectively, in 5 and in 50% cases, whereas all the cells with more than 3 nuclei display an uneven an uneven 3H-thymidin label distribution. The formation of multinucleated cells is supposed to be associated with asynchrony in DNA-synthesis in binucleated cells and with the restitution of mitosis.

Estrogen directly attenuates human osteoclastogenesis, but has no effect on resorption by mature osteoclasts

DEFF Research Database (Denmark)

Sørensen, M G; Henriksen, K; Dziegiel, Morten Hanefeld

2006-01-01

+ monocytes were cultured in the presence of M-CSF and RANKL to induce osteoclast differentiation. Addition of 0.1-10 nM 17beta-estradiol to differentiating osteoclasts resulted in a dose-dependent reduction in tartrate resistant acid phosphatase (TRACP) activity reaching 60% at 0.1 nM. In addition, 17beta-estradiol...... inhibited bone resorption, as measured by the release of the C-terminal crosslinked telopeptide (CTX), by 60% at 0.1 nM, but had no effect on the overall cell viability. In contrast to the results obtained with differentiating osteoclasts, addition of 17beta-estradiol (0.001-10 nM) to mature osteoclasts did...

The inhibitory effect of vitamin K on RANKL-induced osteoclast differentiation and bone resorption.

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Wu, Wei-Jie; Kim, Min Seuk; Ahn, Byung-Yong

2015-10-01

To further understand the correlation between vitamin K and bone metabolism, the effects of vitamins K1, menaquinone-4 (MK-4), and menaquinone-7 (MK-7) on RANKL-induced osteoclast differentiation and bone resorption were comparatively investigated. Vitamin K2 groups (MK-4 and MK-7) were found to significantly inhibit RANKL-medicated osteoclast cell formation of bone marrow macrophages (BMMs) in a dose-dependent manner, without any evidence of cytotoxicity. The mRNA expression of specific osteoclast differentiation markers, such as c-Fos, NFATc1, OSCAR, and TRAP, as well as NFATc1 protein expression and TRAP activity in RANKL-treated BMMs were inhibited by vitamin K2, although MK-4 exhibited a significantly greater efficiency compared to MK-7. In contrast, the same dose of vitamin K1 had no inhibitory effect on RANKL-induced osteoclast cell formation, but increased the expression of major osteoclastogenic genes. Interestingly, vitamins K1, MK-4 and MK-7 all strongly inhibited osteoclastic bone resorption (p vitamins K1, MK-4 and MK-7 have anti-osteoporotic properties, while their regulation effects on osteoclastogenesis are somewhat different.

The inhibitors of cyclin-dependent kinases and GSK-3β enhance osteoclastogenesis

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Yosuke Akiba

2016-03-01

Full Text Available Osteoclasts are multinucleated cells with bone resorption activity that is crucial for bone remodeling. RANK‐RANKL (receptor activator of nuclear factor κB ligand signaling has been shown as a main signal pathway for osteoclast differentiation. However, the molecular mechanism and the factors regulating osteoclastogenesis remain to be fully understood. In this study, we performed a chemical genetic screen, and identified a Cdks/GSK-3β (cyclin-dependent kinases/glycogen synthase kinase 3β inhibitor, kenpaullone, and two Cdks inhibitors, olomoucine and roscovitine, all of which significantly enhance osteoclastogenesis of RAW264.7 cells by upregulating NFATc1 (nuclear factor of activated T cells, cytoplasmic 1 levels. We also determined that the all three compounds increase the number of osteoclast differentiated from murine bone marrow cells. Furthermore, the three inhibitors, especially kenpaullone, promoted maturation of cathepsin K, suggesting that the resorption activity of the resultant osteoclasts is also activated. Our findings indicate that inhibition of GSK-3β and/or Cdks enhance osteoclastogenesis by modulating the RANK–RANKL signaling pathway.

Distribution of Inositol 1,4,5-Trisphosphate Receptors in Rat Osteoclasts

International Nuclear Information System (INIS)

Morikawa, Kazumasa; Goto, Tetsuya; Tanimura, Akihiko; Kobayashi, Shigeru; Maki, Kenshi

2008-01-01

Inositol 1,4,5-trisphosphate (IP 3 ) receptors (IP 3 Rs) are Ca 2+ channels that localize to intracellular Ca 2+ stores such as the endoplasmic reticulum (ER). Recently, IP 3 Rs were found to participate in the formation of the cytoskeleton and cellular adhesions. In this study, we examined the cellular localization of type I, II, and III IP 3 Rs to assess their role in cellular adhesion in rat osteoclasts. Rat bone marrow cells were cultured in α-MEM with 10% fetal bovine serum, M-CSF, RANKL, and 1,25(OH) 2 D 3 for 1 week to promote osteoclast formation. Type I, II, and III IP 3 R expression in the osteoclasts was then examined by RT-PCR. Double-staining was performed using antibodies against type I, II, and III IP 3 Rs and DiOC 6 , an ER marker, or TRITC-phalloidin, an actin filament marker. Expression of all three IP 3 Rs was detected in the newly formed osteoclasts; however, the localization of the type I and II IP 3 Rs was predominantly close to nuclear, and possibly colocalized with the ER, while the type III IP 3 Rs were localized to the ER and podosomes, actin-rich adhesion structures in osteoclasts. These findings suggest that type III IP 3 Rs are associated with osteoclast adhesion

Technetium-99 conjugated with methylene diphosphonate inhibits receptor activator of nuclear factor-κB ligand-induced osteoclastogenesis.

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Gong, Wei; Dou, Huan; Liu, Xianqin; Sun, Lingyun; Hou, Yayi

2012-10-01

1. In the present study, we investigated the effects of technetium-99 conjugated with methylene diphosphonate ((99)Tc-MDP), an agent used in radionuclide therapy, on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis and explored the underlying mechanisms. 2. The murine macrophage cell line RAW264.7 and bone marrow-derived-macrophages from C57BL/6 mice (BMM) were used as models for osteoclastogenesis in vitro. The expression of some key factors in RANKL (50 ng/mL)-induced osteoclastogenesis in RAW264.7 cells was investigated by flow cytometry and real-time reverse transcription-polymerase chain reaction (RT-PCR). To detect multinucleated osteoclast formation, RAW264.7 cells were induced with RANKL for 4 days, whereas BMM were induced by 50 ng/mL RANKL and 20 ng/mL macrophage colony-stimulating factor for 7 days, before being stained with tartrate-resistant acid phosphatase. 3. Osteoclastogenesis was evaluated using the osteoclast markers CD51, matrix metalloproteinase (MMP)-9 and cathepsin K. At 0.01 μg/mL, (99)Tc-MDP significantly inhibited RANKL-induced osteoclastogenesis without any cytotoxicity. In addition, (99)Tc-MDP abolished the appearance of multinucleated osteoclasts. 4. Real-time RT-PCR analysis of transcription factor expression revealed that (99)Tc-MDP inhibited the expression of c-Fos and nuclear factor of activated T cells. In addition, (99)Tc-MDP inhibited the expression of the inflammatory factors interleukin (IL)-6, tumour necrosis factor-α and IL-1β. Finally, (99)Tc-MDP inhibited the activation of mitogen-activated protein kinases in RAW264.7 cells following RANKL stimulation. 5. In conclusion, (99)Tc-MDP possesses anti-osteoclastogenic activity against RANKL-induced osteoclast formation. © 2012 The Authors Clinical and Experimental Pharmacology and Physiology © 2012 Wiley Publishing Asia Pty Ltd.

The polymethoxy flavonoid sudachitin suppresses inflammatory bone destruction by directly inhibiting osteoclastogenesis due to reduced ROS production and MAPK activation in osteoclast precursors.

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Yoko Ohyama

Full Text Available Inflammatory bone diseases, including rheumatoid arthritis, periodontitis and peri-implantitis, are associated not only with the production of inflammatory cytokines but also with local oxidative status, which is defined by intracellular reactive oxygen species (ROS. Osteoclast differentiation has been reported to be related to increased intracellular ROS levels in osteoclast lineage cells. Sudachitin, which is a polymethoxyflavone derived from Citrus sudachi, possesses antioxidant properties and regulates various functions in mammalian cells. However, the effects of sudachitin on inflammatory bone destruction and osteoclastogenesis remain unknown. In calvaria inflamed by a local lipopolysaccharide (LPS injection, inflammation-induced bone destruction and the accompanying elevated expression of osteoclastogenesis-related genes were reduced by the co-administration of sudachitin and LPS. Moreover, sudachitin inhibited osteoclast formation in cultures of isolated osteoblasts and osteoclast precursors. However, sudachitin rather increased the expression of receptor activator of NF-κB ligand (RANKL, which is an important molecule triggering osteoclast differentiation, and the mRNA ratio of RANKL/osteoprotegerin that is a decoy receptor for RANKL, in the isolated osteoblasts, suggesting the presence of additional target cells. When osteoclast formation was induced from osteoclast precursors derived from bone marrow cells in the presence of soluble RANKL and macrophage colony-stimulating factor, sudachitin inhibited osteoclastogenesis without influencing cell viability. Consistently, the expression of osteoclast differentiation-related molecules including c-fos, NFATc1, cathepsin K and osteoclast fusion proteins such as DC-STAMP and Atp6v0d2 was reduced by sudachitin. In addition, sudachitin decreased activation of MAPKs such as Erk and JNK and the ROS production evoked by RANKL in osteoclast lineage cells. Our findings suggest that sudachitin is a

Effects of transforming growth factor-beta on long-term human cord blood monocyte cultures

International Nuclear Information System (INIS)

Orcel, P.; Bielakoff, J.; De Vernejoul, M.C.

1990-01-01

Transforming growth factor-beta (TGF-beta) modulates growth and differentiation in many cell types and is abundant in bone matrix. We recently showed that human cord blood monocytes cultured in the presence of 1,25(OH)2D3 acquire some features of osteoclast precursors. Since TGF-beta has been shown to influence bone resorption in organ culture, we have studied the effect of TGF-beta (1-1,000 pg/ml) on cord blood monocyte cultures. These cells were cultured on plastic substrate during 3 weeks in the presence of 20% horse serum and 10(-9) M 1,25(OH)2D3. TGF-beta, from a concentration of 10 pg/ml in the culture medium, decreased in a dose dependent manner the formation of multinucleated cells. At a concentration of TGF-beta of 1 ng/ml, the multinucleated cells were reduced to 2.1% +/- 0.3%, compared to 19.3% +/- 1.5% in control cultures. TGF-beta inhibited in a dose-dependent manner the proliferation of cord blood monocytes as assessed by 3H-thymidine incorporation at 7 and 14 days of culture. The fusion index was also decreased by 3 weeks of treatment with TGF-beta. Indomethacin did not reverse the inhibitory effects of TGF-beta. The expression of the osteoclastic phenotype was assessed using two different antibodies: 23C6, a monoclonal antibody directed against the vitronectin receptor, which is highly expressed by osteoclasts but not by adult monocytes, and an antibody to HLA-DR, which is not present on osteoclast. TGF-beta decreased the expression of HLA-DR and increased in a dose-dependent manner the proportion of 23C6-labeled cells; these results suggest that TGF-beta could modulate a differentiation effect to the osteoclastic phenotype. However, when cord blood monocytes were cultured on devitalized rat calvariae prelabeled with 45Ca, TGF-beta did not induce any 45Ca release from bone cultured with monocytes

Interaction of osteoblast-like cells with serum and fibronectin: effects on cell motility and proliferation in vitro

International Nuclear Information System (INIS)

Zuk, A.

1986-01-01

Osteoblast migration and proliferation are believed to occur during bone remodelling, in particular after osteoclastic bone resorption and prior to osteoblastic bone formation. In order to study migration and proliferation in vitro, the model of Alessandri et al. (1983) was modified. The model entailed seeding osteoblast-like cells into wells cut in agar and quantifying migration and proliferation peripheral to the well. Cell morphology also was described. The data indicated that on growth surfaces enriched with varying concentrations of fetal calf serum (FSC), the quantification of migration and proliferation was related both to percent cell attachment and to FCS-concentration. Because few osteoblast-like cells incorporated ( 3 H-TdR), it was concluded that the appearance of cells peripheral to the well was due to migration, and not to proliferation. Cell morphology and myosin distribution and organization indicated that osteoblast-like cells at the periphery of the cell culture (i.e. leading edge) may have been directionally migrating whereas cells behind the leading edge may have been engaged in non-directional migration. The migration, proliferation, and morphology of osteoblast-like cells cultured on fibronectin (FN) enriched growth surfaces also was examined. The quantification of migration and proliferation was related to the FN-concentration applied to the growth surface. Because few osteoblast-like cells incorporated 3 H-TdR and cell morphology indicated migration, it was concluded that osteoblast-like cells on FN-enriched growth surfaces are specialized, in part, for migration

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice.

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Maridas, David E; Rendina-Ruedy, Elizabeth; Le, Phuong T; Rosen, Clifford J

2018-01-06

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

Regulation of ITAM adaptor molecules and their receptors by inhibition of calcineurin-NFAT signalling during late stage osteoclast differentiation

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Zawawi, M.S.F. [Universiti Sains Malaysia (USM) (Malaysia); Discipline of Anatomy and Pathology, School of Medical Sciences, University of Adelaide, Adelaide, SA 5005 (Australia); Dharmapatni, A.A.S.S.K.; Cantley, M.D. [Discipline of Anatomy and Pathology, School of Medical Sciences, University of Adelaide, Adelaide, SA 5005 (Australia); McHugh, K.P. [University of Florida, College of Dentistry, Fl (United States); Haynes, D.R. [Discipline of Anatomy and Pathology, School of Medical Sciences, University of Adelaide, Adelaide, SA 5005 (Australia); Crotti, T.N., E-mail: tania.crotti@adelaide.edu.au [Discipline of Anatomy and Pathology, School of Medical Sciences, University of Adelaide, Adelaide, SA 5005 (Australia)

2012-10-19

Highlights: Black-Right-Pointing-Pointer Calcineurin/NFAT inhibitors FK506 and VIVIT treated human PBMC derived osteoclasts in vitro. Black-Right-Pointing-Pointer Differential regulation of ITAM receptors and adaptor molecules by calcineurin/NFAT inhibitors. Black-Right-Pointing-Pointer FK506 and VIVIT suppress ITAM factors during late phase osteoclast differentiation. -- Abstract: Osteoclasts are specialised bone resorptive cells responsible for both physiological and pathological bone loss. Osteoclast differentiation and activity is dependent upon receptor activator NF-kappa-B ligand (RANKL) interacting with its receptor RANK to induce the transcription factor, nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1). The immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathway has been identified as a co-stimulatory pathway in osteoclasts. Osteoclast-associated receptor (OSCAR) and triggering receptor expressed in myeloid cells (TREM2) are essential receptors that pair with adaptor molecules Fc receptor common gamma chain (FcR{gamma}) and DNAX-activating protein 12 kDa (DAP12) respectively to induce calcium signalling. Treatment with calcineurin-NFAT inhibitors, Tacrolimus (FK506) and the 11R-VIVIT (VIVIT) peptide, reduces NFATc1 expression consistent with a reduction in osteoclast differentiation and activity. This study aimed to investigate the effects of inhibiting calcineurin-NFAT signalling on the expression of ITAM factors and late stage osteoclast genes including cathepsin K (CathK), Beta 3 integrin ({beta}3) and Annexin VIII (AnnVIII). Human peripheral blood mononuclear cells (PBMCs) were differentiated with RANKL and macrophage-colony stimulating factor (M-CSF) over 10 days in the presence or absence of FK506 or VIVIT. Osteoclast formation (as assessed by tartrate resistant acid phosphatase (TRAP)) and activity (assessed by dentine pit resorption) were significantly reduced with treatment. Quantitative real

Intercellular calcium signaling occurs between human osteoblasts and osteoclasts and requires activation of osteoclast P2X7 receptors

DEFF Research Database (Denmark)

Jørgensen, Niklas R; Henriksen, Zanne; Sørensen, Ole

2002-01-01

that human osteoclasts expressed functional P2Y1 receptors, but, unexpectedly, desensitization of P2Y1 did not block calcium signaling to osteoclasts. We also found that osteoclasts expressed functional P2X7 receptors and showed that pharmacological inhibition of these receptors blocked calcium signaling...

Matrix metalloproteinase-12 (MMP-12) in osteoclasts

DEFF Research Database (Denmark)

Hou, Peng; Troen, Tine; Ovejero, Maria C

2004-01-01

Osteoclasts require matrix metalloproteinase (MMP) activity and cathepsin K to resorb bone, but the critical MMP has not been identified. Osteoclasts express MMP-9 and MMP-14, which do not appear limiting for resorption, and the expression of additional MMPs is not clear. MMP-12, also called...... bone show MMP-12 expression in osteoclasts in calvariae and long bones. We also demonstrate that recombinant MMP-12 cleaves the putative functional domains of osteopontin and bone sialoprotein, two bone matrix proteins that strongly influence osteoclast activities, such as attachment, spreading...

The Cdc42 guanine nucleotide exchange factor FGD6 coordinates cell polarity and endosomal membrane recycling in osteoclasts.

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Steenblock, Charlotte; Heckel, Tobias; Czupalla, Cornelia; Espírito Santo, Ana Isabel; Niehage, Christian; Sztacho, Martin; Hoflack, Bernard

2014-06-27

The initial step of bone digestion is the adhesion of osteoclasts onto bone surfaces and the assembly of podosomal belts that segregate the bone-facing ruffled membrane from other membrane domains. During bone digestion, membrane components of the ruffled border also need to be recycled after macropinocytosis of digested bone materials. How osteoclast polarity and membrane recycling are coordinated remains unknown. Here, we show that the Cdc42-guanine nucleotide exchange factor FGD6 coordinates these events through its Src-dependent interaction with different actin-based protein networks. At the plasma membrane, FGD6 couples cell adhesion and actin dynamics by regulating podosome formation through the assembly of complexes comprising the Cdc42-interactor IQGAP1, the Rho GTPase-activating protein ARHGAP10, and the integrin interactors Talin-1/2 or Filamin A. On endosomes and transcytotic vesicles, FGD6 regulates retromer-dependent membrane recycling through its interaction with the actin nucleation-promoting factor WASH. These results provide a mechanism by which a single Cdc42-exchange factor controlling different actin-based processes coordinates cell adhesion, cell polarity, and membrane recycling during bone degradation. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Curcumol suppresses RANKL-induced osteoclast formation by attenuating the JNK signaling pathway

International Nuclear Information System (INIS)

Yu, Mingxiang; Chen, Xianying; Lv, Chaoyang; Yi, Xilu; Zhang, Yao; Xue, Mengjuan; He, Shunmei; Zhu, Guoying; Wang, Hongfu

2014-01-01

Highlights: • Curcumol suppresses osteoclasts differentiation in vitro. • Curcumol impairs JNK/AP-1 signaling pathway. • Curcumol may be used for treating osteoclast related diseases. - Abstract: Osteoclasts, derived from hemopoietic progenitors of the monocyte/macrophage lineage, have a unique role in bone resorption, and are considered a potential therapeutic target in the treatment of such pathologic bone diseases as osteoporosis, rheumatoid arthritis, and periodontitis. In the present study, we demonstrate that curcumol, one of the major components of the essential oil of Rhizoma Curcumae, exhibits an inhibitory effect on receptor activator of nuclear factor kappaB ligand (RANKL)-induced osteoclast differentiation with both bone marrow-derived macrophages and RAW264.7 cells in a dose-dependent manner. In addition, RANKL-induced mRNA expression of osteoclast-specific genes, such as tartrate-resistant acid phosphatase, calcitonin receptor, and cathepsin K, is prominently reduced in the presence of curcumol. Furthermore, the molecular mechanism of action was investigated, and curcumol inhibited osteoclastogenesis by specifically impairing RANKL-induced c-Jun N-terminal kinase (JNK)/activator protein-1 (AP-1) signaling, which was further identified in rescue studies by means of anisomycin, a JNK signaling-specific activator. Taken together, these findings suggest that curcumol suppresses RANKL-induced osteoclast differentiation through the JNK/AP-1 signaling pathway, and may be useful as a therapeutic treatment for bone resorption-associated diseases

Curcumol suppresses RANKL-induced osteoclast formation by attenuating the JNK signaling pathway

Energy Technology Data Exchange (ETDEWEB)

Yu, Mingxiang, E-mail: yu.mingxiang@zs-hospital.sh.cn [Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan University, Shanghai (China); Chen, Xianying [Department of Endocrinology and Metabolism, Hainan Provincial Nong Ken Hospital, Hainan (China); Lv, Chaoyang [Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan University, Shanghai (China); Yi, Xilu [Department of Endocrinology and Metabolism, Shanghai Songjiang District Central Hospital, Shanghai (China); Zhang, Yao; Xue, Mengjuan; He, Shunmei [Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan University, Shanghai (China); Zhu, Guoying [Institute of Radiation Medicine, Fudan University, Shanghai (China); Wang, Hongfu, E-mail: hfwang@shmu.edu.cn [Institute of Radiation Medicine, Fudan University, Shanghai (China)

2014-05-02

Highlights: • Curcumol suppresses osteoclasts differentiation in vitro. • Curcumol impairs JNK/AP-1 signaling pathway. • Curcumol may be used for treating osteoclast related diseases. - Abstract: Osteoclasts, derived from hemopoietic progenitors of the monocyte/macrophage lineage, have a unique role in bone resorption, and are considered a potential therapeutic target in the treatment of such pathologic bone diseases as osteoporosis, rheumatoid arthritis, and periodontitis. In the present study, we demonstrate that curcumol, one of the major components of the essential oil of Rhizoma Curcumae, exhibits an inhibitory effect on receptor activator of nuclear factor kappaB ligand (RANKL)-induced osteoclast differentiation with both bone marrow-derived macrophages and RAW264.7 cells in a dose-dependent manner. In addition, RANKL-induced mRNA expression of osteoclast-specific genes, such as tartrate-resistant acid phosphatase, calcitonin receptor, and cathepsin K, is prominently reduced in the presence of curcumol. Furthermore, the molecular mechanism of action was investigated, and curcumol inhibited osteoclastogenesis by specifically impairing RANKL-induced c-Jun N-terminal kinase (JNK)/activator protein-1 (AP-1) signaling, which was further identified in rescue studies by means of anisomycin, a JNK signaling-specific activator. Taken together, these findings suggest that curcumol suppresses RANKL-induced osteoclast differentiation through the JNK/AP-1 signaling pathway, and may be useful as a therapeutic treatment for bone resorption-associated diseases.

Effects of local and whole body irradiation on appearance of osteoclasts during wound healing of tooth extraction sockets in rats

International Nuclear Information System (INIS)

Hosokawa, Yoichiro; Sakakura, Yasunori; Tanaka, Likinobu; Okumura, Kazuhiko; Yajima, Toshihiko; Kaneko, Masayuki

2007-01-01

We examined effects of local and whole body irradiation before tooth extraction on appearance and differentiation of osteoclasts in the alveolar bone of rat maxillary first molars. Wistar rats weighting 100 g were divided into three groups: non-irradiation group, local irradiation group, and whole body irradiation group. In the local irradiation group, a field made with lead blocks was placed over the maxillary left first molar tooth. In the whole body irradiation group, the animals were irradiated in cages. Both groups were irradiated at 8 Gy. The number of osteoclasts around the interradicular alveolar bone showed chronological changes common to non-irradiated and irradiated animals. Several osteoclasts appeared one day after tooth extraction, and the maximal peak was observed 3 days after extraction. Local irradiation had no difference from non-irradiated controls. In animals receiving whole body irradiation, tooth extraction one day after irradiation caused smaller number of osteoclasts than that 7 day after irradiation during the experimental period. Whole body-irradiated rats had small osteoclasts with only a few nuclei and narrow resorption lacunae, indicating deficiency of radioresistant osteoclast precursor cells. Injection of intact bone marrow cells to whole body-irradiated animals immediately after tooth extraction recovered to some content the number of osteoclasts. These findings suggest that bone resorption in the wound healing of alveolar socket requires radioresistant, postmitotic osteoclast precursor cells from hematopoietic organs, but not from local sources around the alveolar socket, at the initial phase of wound healing. (author)

Effects of local and whole body irradiation on appearance of osteoclasts during wound healing of tooth extraction sockets in rats.

Science.gov (United States)

Hosokawa, Yoichiro; Sakakura, Yasunori; Tanaka, Likinobu; Okumura, Kazuhiko; Yajima, Toshihiko; Kaneko, Masayuki

2007-07-01

We examined effects of local and whole body irradiation before tooth extraction on appearance and differentiation of osteoclasts in the alveolar bone of rat maxillary first molars. Wistar rats weighting 100 g were divided into three groups: non-irradiation group, local irradiation group, and whole body irradiation group. In the local irradiation group, a field made with lead blocks was placed over the maxillary left first molar tooth. In the whole body irradiation group, the animals were irradiated in cages. Both groups were irradiated at 8 Gy. The number of osteoclasts around the interradicular alveolar bone showed chronological changes common to non-irradiated and irradiated animals. Several osteoclasts appeared one day after tooth extraction, and the maximal peak was observed 3 days after extraction. Local irradiation had no difference from non-irradiated controls. In animals receiving whole body irradiation, tooth extraction one day after irradiation caused smaller number of osteoclasts than that 7 day after irradiation during the experimental period. Whole body-irradiated rats had small osteoclasts with only a few nuclei and narrow resorption lacunae, indicating deficiency of radioresistant osteoclast precursor cells. Injection of intact bone marrow cells to whole body-irradiated animals immediately after tooth extraction recovered to some content the number of osteoclasts. These findings suggest that bone resorption in the wound healing of alveolar socket requires radioresistant, postmitotic osteoclast precursor cells from hematopoietic organs, but not from local sources around the alveolar socket, at the initial phase of wound healing.

Adenosine A1 receptors (A1Rs) play a critical role in osteoclast formation and function

Science.gov (United States)

Kara, Firas M.; Chitu, Violeta; Sloane, Jennifer; Axelrod, Matthew; Fredholm, Bertil B.; Stanley, E. Richard; Cronstein, Bruce N.

2010-01-01

Adenosine regulates a wide variety of physiological processes via interaction with one or more G-protein-coupled receptors (A1R, A2AR, A2BR, and A3R). Because A1R occupancy promotes fusion of human monocytes to form giant cells in vitro, we determined whether A1R occupancy similarly promotes osteoclast function and formation. Bone marrow cells (BMCs) were harvested from C57Bl/6 female mice or A1R-knockout mice and their wild-type (WT) littermates and differentiated into osteoclasts in the presence of colony stimulating factor-1 and receptor activator of NF-κB ligand in the presence or absence of the A1R antagonist 1,3-dipropyl-8-cyclopentyl xanthine (DPCPX). Osteoclast morphology was analyzed in tartrate-resistant acid phosphatase or F-actin-stained samples, and bone resorption was evaluated by toluidine blue staining of dentin. BMCs from A1R-knockout mice form fewer osteoclasts than BMCs from WT mice, and the A1R antagonist DPCPX inhibits osteoclast formation (IC50=1 nM), with altered morphology and reduced ability to resorb bone. A1R blockade increased ubiquitination and degradation of TRAF6 in RAW264.7 cells induced to differentiate into osteoclasts. These studies suggest a critical role for adenosine in bone homeostasis via interaction with adenosine A1R and further suggest that A1R may be a novel pharmacologic target to prevent the bone loss associated with inflammatory diseases and menopause.—Kara, F. M., Chitu, V., Sloane, J., Axelrod, M., Fredholm, B. B., Stanley, R., Cronstein, B. N. Adenosine A1 receptors (A1Rs) play a critical role in osteoclast formation and function. PMID:20181934

Matrix metalloproteinases (MMP) and cathepsin K contribute differently to osteoclastic activities

DEFF Research Database (Denmark)

Delaissé, Jean-Marie; Andersen, Thomas L; Engsig, Michael T

2003-01-01

The best established proteolytic event of osteoclasts is bone matrix solubilization by the cysteine proteinase cathepsin K. Here, however, we draw the attention on osteoclastic activities depending on matrix metalloproteinases (MMPs). We discuss the observations supporting that MMPs contribute...... significantly to bone matrix solubilization in specific areas of the skeleton and in some developmental and pathological situations. Our discussion takes into account (1) the characteristics of the bone remodeling persisting in the absence of cathepsin K, (2) the ultrastructure of the resorption zone...... in response to inactivation of MMPs and of cathepsin K in different bone types, (3) bone resorption levels in MMP knockout mice compared to wild-type mice, (4) the identification of MMPs in osteoclasts and surrounding cells, and (5) the effect of different bone pathologies on the serum concentrations...

RANK ligand signaling modulates the matrix metalloproteinase-9 gene expression during osteoclast differentiation

International Nuclear Information System (INIS)

Sundaram, Kumaran; Nishimura, Riko; Senn, Joseph; Youssef, Rimon F.; London, Steven D.; Reddy, Sakamuri V.

2007-01-01

Osteoclast differentiation is tightly regulated by receptor activator of NF-κB ligand (RANKL) signaling. Matrix metalloproteinase-9 (MMP-9), a type IV collagenase is highly expressed in osteoclast cells and plays an important role in degradation of extracellular matrix; however, the molecular mechanisms that regulate MMP-9 gene expression are unknown. In this study, we demonstrate that RANKL signaling induces MMP-9 gene expression in osteoclast precursor cells. We further show that RANKL regulates MMP-9 gene expression through TRAF6 but not TRAF2. Interestingly, blockade of p38 MAPK activity by pharmacological inhibitor, SB203580 increases MMP-9 activity whereas ERK1/2 inhibitor, PD98059 decreases RANKL induced MMP-9 activity in RAW264.7 cells. These data suggest that RANKL differentially regulates MMP-9 expression through p38 and ERK signaling pathways during osteoclast differentiation. Transient expression of MMP-9 gene (+ 1 to - 1174 bp relative to ATG start codon) promoter-luciferase reporter plasmids in RAW264.7 cells and RANKL stimulation showed significant increase (20-fold) of MMP-9 gene promoter activity; however, there is no significant change with respect to + 1 bp to - 446 bp promoter region and empty vector transfected cells. These results indicated that MMP-9 promoter sequence from - 446 bp to - 1174 bp relative to start codon is responsive to RANKL stimulation. Sequence analysis of the mouse MMP-9 gene promoter region further identified the presence of binding motif (- 1123 bp to - 1153 bp) for the nuclear factor of activated T cells 1 (NFATc1) transcription factor. Inhibition of NFATc1 using siRNA and VIVIT peptide inhibitor significantly decreased RANKL stimulation of MMP-9 activity. We further confirm by oligonucleotide pull-down assay that RANKL stimuli enhanced NFATc1 binding to MMP-9 gene promoter element. In addition, over-expression of constitutively active NFAT in RAW264.7 cells markedly increased (5-fold) MMP-9 gene promoter activity in

A metabolomics study of the inhibitory effect of 17-beta-estradiol on osteoclast proliferation and differentiation.

Science.gov (United States)

Liu, Xiaoyan; Liu, Yanqiu; Cheng, Mengchun; Zhang, Xiaozhe; Xiao, Hongbin

2015-02-01

Estradiol is a major drug used clinically to alleviate osteoporosis, partly through inhibition of the activity of osteoclasts, which play a crucial role in bone resorption. So far, little is known about the effects of estradiol on osteoclast metabolism. In this study, ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC/MS)-based metabolomics strategy was used to investigate the metabolite response to 17β-estradiol in mouse osteoclast RAW264.7, a commonly used cell model for studying osteoporosis. Our results showed that the application of estradiol altered the levels of 27 intracellular metabolites, including lysophosphatidylcholines (LysoPCs), other lipids and amino acid derivants. The changes of all the 27 metabolites were observed in the study of estradiol induced osteoclast proliferation inhibition (1 μM estradiol applied), while the changes of only 18 metabolites were observed in the study of differentiation inhibition (0.1 μM estradiol applied). Further pathway impact analysis determined glycerophospholipid metabolism as the main potential target pathway of estradiol, which was further confirmed by LCAT (phosphatidylcholine-sterol acyltransferase) activity changes and lipid peroxidative product (MDA, methane dicarboxylic aldehyde) changes caused by estradiol. Additionally, we found that estradiol significantly decreased intracellular oxidative stress during cell proliferation but not during cell differentiation. Our study suggested that estradiol generated a highly condition-dependent influence on osteoclast metabolism.

Visualization of structural organization of ventral membranes of sheared-open resorbing osteoclasts attached to apatite pellets.

Science.gov (United States)

Akisaka, Toshitaka; Yoshida, Atsushi

2015-05-01

Osteoclasts are highly polarized cells from both morphological and functional points of view. Using quick-freeze, rotary-replication methods combined with cell-shearing, we clarified the variability of cytoplasmic surface of the polarized membranes of osteoclasts seeded on apatite. As to the organization of actin filaments and clathrin sheets, we confirmed almost the same ventral membrane specializations of osteoclasts on apatite as seen on glass plates. The organized actin filaments and membrane-associated particles supported the ruffled border membranes. Inside the actin sealing zone, membrane specializations were not always occupied with the ruffled border but also with other types of membranes. Some osteoclasts formed an actin ring but lacked the ruffled border projections. We report a unique and distinctive membrane modification of apatite-attached osteoclasts, i.e., the presence of dense aggregates of membrane-associated particles and related structures not found in the osteoclasts seeded on glass plates. Actin filament polarity in the podosomes was determined by decoration with myosin S1. The actin filament polarity within podosome appears to be oriented predominantly with its barbed ends toward the core, whereas the interconnecting F-actin appears to be mixed oriented. Two different types of clathrin plaques displayed different distributions: clathrin-dependent endocytosis was observed in the ruffled border regions, whereas flat clathrin sheets were found in the leading edge of lamellipodia and near podosomes. The clathrin sheets adhered to the apatite surface tightly on the ventral membranes overlaying the resorption lacunae. All these membrane specializations as mentioned above may indicate the functional variability of osteoclasts seeded on apatite.

Multi-nucleate retinal pigment epithelium cells of the human macula exhibit a characteristic and highly specific distribution.

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Starnes, Austin C; Huisingh, Carrie; McGwin, Gerald; Sloan, Kenneth R; Ablonczy, Zsolt; Smith, R Theodore; Curcio, Christine A; Ach, Thomas

2016-01-01

The human retinal pigment epithelium (RPE) is reportedly 3% bi-nucleated. The importance to human vision of multi-nucleated (MN)-RPE cells could be clarified with more data about their distribution in central retina. Nineteen human RPE-flatmounts (9 ≤ 51 years, 10 > 80 years) were imaged at 12 locations: 3 eccentricities (fovea, perifovea, near periphery) in 4 quadrants (superior, inferior, temporal, nasal). Image stacks of lipofuscin-attributable autofluorescence and phalloidin labeled F-actin cytoskeleton were obtained using a confocal fluorescence microscope. Nuclei were devoid of autofluorescence and were marked using morphometric software. Cell areas were approximated by Voronoi regions. Mean number of nuclei per cell among eccentricity/quadrant groups and by age were compared using Poisson and binominal regression models. A total of 11,403 RPE cells at 200 locations were analyzed: 94.66% mono-, 5.31% bi-, 0.02% tri-nucleate, and 0.01% with 5 nuclei. Age had no effect on number of nuclei. There were significant regional differences: highest frequencies of MN-cells were found at the perifovea (9.9%) and near periphery (6.8%). The fovea lacked MN-cells almost entirely. The nasal quadrant had significantly more MN-cells compared to other quadrants, at all eccentricities. This study demonstrates MN-RPE cells in human macula. MN-cells may arise due to endoreplication, cell fusion, or incomplete cell division. The topography of MN-RPE cells follows the topography of photoreceptors; with near-absence at the fovea (cones only) and high frequency at perifovea (highest rod density). This distribution might reflect specific requirements of retinal metabolism or other mechanisms addressable in further studies.

Screening of protein kinase inhibitors identifies PKC inhibitors as inhibitors of osteoclastic acid secretion and bone resorption

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Boutin Jean A

2010-10-01

Full Text Available Abstract Background Bone resorption is initiated by osteoclastic acidification of the resorption lacunae. This process is mediated by secretion of protons through the V-ATPase and chloride through the chloride antiporter ClC-7. To shed light on the intracellular signalling controlling extracellular acidification, we screened a protein kinase inhibitor library in human osteoclasts. Methods Human osteoclasts were generated from CD14+ monocytes. The effect of different kinase inhibitors on lysosomal acidification in human osteoclasts was investigated using acridine orange for different incubation times (45 minutes, 4 and 24 hours. The inhibitors were tested in an acid influx assay using microsomes isolated from human osteoclasts. Bone resorption by human osteoclasts on bone slices was measured by calcium release. Cell viability was measured using AlamarBlue. Results Of the 51 compounds investigated only few inhibitors were positive in both acidification and resorption assays. Rottlerin, GF109203X, Hypericin and Ro31-8220 inhibited acid influx in microsomes and bone resorption, while Sphingosine and Palmitoyl-DL-carnitine-Cl showed low levels of inhibition. Rottlerin inhibited lysosomal acidification in human osteoclasts potently. Conclusions In conclusion, a group of inhibitors all indicated to inhibit PKC reduced acidification in human osteoclasts, and thereby bone resorption, indicating that acid secretion by osteoclasts may be specifically regulated by PKC in osteoclasts.

Real-time intravital imaging of pH variation associated with osteoclast activity.

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Maeda, Hiroki; Kowada, Toshiyuki; Kikuta, Junichi; Furuya, Masayuki; Shirazaki, Mai; Mizukami, Shin; Ishii, Masaru; Kikuchi, Kazuya

2016-08-01

Intravital imaging by two-photon excitation microscopy (TPEM) has been widely used to visualize cell functions. However, small molecular probes (SMPs), commonly used for cell imaging, cannot be simply applied to intravital imaging because of the challenge of delivering them into target tissues, as well as their undesirable physicochemical properties for TPEM imaging. Here, we designed and developed a functional SMP with an active-targeting moiety, higher photostability, and a fluorescence switch and then imaged target cell activity by injecting the SMP into living mice. The combination of the rationally designed SMP with a fluorescent protein as a reporter of cell localization enabled quantitation of osteoclast activity and time-lapse imaging of its in vivo function associated with changes in cell deformation and membrane fluctuations. Real-time imaging revealed heterogenic behaviors of osteoclasts in vivo and provided insights into the mechanism of bone resorption.

Ethanol Extract of Atractylodes macrocephala Protects Bone Loss by Inhibiting Osteoclast Differentiation

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Youn-Hwan Hwang

2013-06-01

Full Text Available The rhizome of Atractylodes macrocephala has been used mainly in Traditional Chinese Medicine for invigorating the functions of the stomach and spleen. In the present study, we investigated the inhibitory effect of the 70% ethanol extract of the rhizome of Atractylodes macrocephala (AMEE on osteoclast differentiation. We found that AMEE inhibits osteoclast differentiation from its precursors induced by receptor activator of nuclear factor-κB ligand (RANKL, an essential cytokine required for osteoclast differentiation. AMEE attenuated RANKL-induced activation of NF-κB signaling pathway, subsequently inhibiting the induction of osteoclastogenic transcription factors, c-Fos and nuclear factor of activated T cells cytoplasmic 1. Consistent with the in vitro results, administration of AMEE protected RANKL-induced bone loss in mice. We also identified atractylenolide I and II as active constituents contributing to the anti-osteoclastogenic effect of AMEE. Taken together, our results demonstrate that AMEE has a protective effect on bone loss via inhibiting osteoclast differentiation and suggest that AMEE may be useful in preventing and treating various bone diseases associated with excessive bone resorption.

Fisetin Inhibits Osteoclast Differentiation via Downregulation of p38 and c-Fos-NFATc1 Signaling Pathways

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Sik-Won Choi

2012-01-01

Full Text Available The prevention or therapeutic treatment of loss of bone mass is an important means of improving the quality of life for patients with disorders related to osteoclast-mediated bone loss. Fisetin, a flavonoid dietary ingredient found in the smoke tree (Continus coggygria, exhibits various biological activities, but its effect on osteoclast differentiation is unknown. In this study, fisetin dose-dependently inhibited the RANKL-induced osteoclast differentiation with downregulation of the activity or expression of p38, c-Fos, and NFATc1 signaling molecules. The p38/c-Fos/NFATc1-regulated expression of genes required for cell fusion and bone resorption, such as DC-STAMP and cathepsin K, was also inhibited by fisetin. Considering the rescue of fisetin's inhibitory action by NFATc1 over-expression, the cascade of p38-c-Fos-NFATc1 could be strongly involved in the inhibitory effect of fisetin on osteoclast differentiation. Furthermore, fisetin inhibited the bone-resorbing activity of mature osteoclasts. In conclusion, fisetin may be of use in the treatment of osteoclast-related disorders, including osteoporosis.

Down-regulation of Irf8 by Lyz2-cre/loxP accelerates osteoclast differentiation in vitro.

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Saito, Emi; Suzuki, Dai; Kurotaki, Daisuke; Mochizuki, Ayako; Manome, Yoko; Suzawa, Tetsuo; Toyoshima, Yoichi; Ichikawa, Takahiro; Funatsu, Takahiro; Inoue, Tomio; Takami, Masamichi; Tamura, Tomohiko; Inagaki, Katsunori; Kamijo, Ryutaro

2017-06-01

Interferon regulatory factor 8 (Irf8) is a transcription factor that negatively regulates osteoclast differentiation and Irf8 global knockout (Irf8 -/- ) mice have been shown to have reduced bone volume resulting from increased osteoclast numbers. However, detailed analysis of the functions of Irf8 in osteoclast precursors with a monocyte/macrophage linage is difficult, because the population and properties of hematopoietic cells in Irf8 -/- mice are severely altered. Therefore, to clearly elucidate the functions of Irf8 during osteoclastogenesis, we established myeloid cell-specific Irf8 conditional knockout (Irf8 fl/fl ;Lyz2 cre/+ ) mice. We found that trabecular bone volume in the Irf8 fl/fl ;Lyz2 cre/+ mice was not significantly affected, while exposure to M-CSF and RANKL significantly increased TRAP activity in vitro in osteoclasts that underwent osteoclastogenesis from bone marrow-derived macrophages (BMMs) induced from bone marrow cells (BMCs) of those mice by addition of M-CSF. Our results also showed that expression of Irf8 mRNA and protein in BMMs obtained from Irf8 fl/fl ;Lyz2 cre/+ mice and cultured with M-CSF was reduced. These findings predicted that Lyz2/Lyz2-cre expression is induced when BMCs differentiate into BMMs in cultures with M-CSF. In osteoclast differentiation cultures, Lyz2 was gradually increased by M-CSF during the first 3 days of culture, then rapidly decreased by the addition of RANKL with M-CSF during the next 3 days. Furthermore, BMCs differentiated into osteoclasts while maintaining a low level of Lyz2 expression when cultured simultaneously with both M-CSF and RANKL from the initiation of culture. These findings suggest that Lyz2-cre expression is induced along with differentiation to BMMs by BMCs obtained from Irf8 fl/fl ;Lyz2 cre/+ mice and cultured with M-CSF. In addition, Irf8 was down-regulated by activation of the cre/loxP recombination system in BMMs and osteoclastogenesis was accelerated. Based on our results, we propose

Role of notch signaling in osteoimmunology-from the standpoint of osteoclast differentiation

NARCIS (Netherlands)

Duan, Li; Ren, Yijin

The Notch signaling pathway is a highly conserved cell signaling system present in most multicellular organisms. Osteoimmunology comprises the interplay between the immune system and bone metabolism. Osteoclasts, cells that resorb bone, play a crucial role in bone metabolism. In this review, we

Normalizing the bone marrow microenvironment with p38 inhibitor reduces multiple myeloma cell proliferation and adhesion and suppresses osteoclast formation

International Nuclear Information System (INIS)

Nguyen, Aaron N.; Stebbins, Elizabeth G.; Henson, Margaret; O'Young, Gilbert; Choi, Sun J.; Quon, Diana; Damm, Debby; Reddy, Mamatha; Ma, Jing Y.; Haghnazari, Edwin; Kapoun, Ann M.; Medicherla, Satyanarayana; Protter, Andy; Schreiner, George F.; Kurihara, Noriyoshi; Anderson, Judy; Roodman, G. David; Navas, Tony A.; Higgins, Linda S.

2006-01-01

The multiple myeloma (MM) bone marrow (BM) microenvironment plays a critical role in supporting tumor growth and survival as well as in promoting formation of osteolytic lesions. Recent results suggest that the p38 mitogen-activated protein kinase (MAPK) is an important factor in maintaining this activated environment. In this report, we demonstrate that the p38α MAPK inhibitor, SCIO-469, suppresses secretion of the tumor-supportive factors IL-6 and VEGF from BM stromal cells (BMSCs) as well as cocultures of BMSCs with MM cells, resulting in reduction in MM cell proliferation. Additionally, we show that SCIO-469 prevents TNFα-induced adhesion of MM cells to BMSCs through an ICAM-1- and VCAM-1-independent mechanism. Microarray analysis revealed a novel set of TNFα-induced chemokines in BMSCs that is strongly inhibited by SCIO-469. Furthermore, reintroduction of chemokines CXCL10 and CCL8 to BMSCs overcomes the inhibitory effect of SCIO-469 on TNFα-induced MM adhesion. Lastly, we show that SCIO-469 inhibits secretion and expression of the osteoclast-activating factors IL-11, RANKL, and MIP-1α as well as prevents human osteoclast formation in vitro. Collectively, these results suggest that SCIO-469 treatment can suppress factors in the bone marrow microenvironment to inhibit MM cell proliferation and adhesion and also to alleviate osteolytic activation in MM

Validation of an automated counting procedure for phthalate-induced testicular multinucleated germ cells.

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Spade, Daniel J; Bai, Cathy Yue; Lambright, Christy; Conley, Justin M; Boekelheide, Kim; Gray, L Earl

2018-06-15

In utero exposure to certain phthalate esters results in testicular toxicity, characterized at the tissue level by induction of multinucleated germ cells (MNGs) in rat, mouse, and human fetal testis. Phthalate exposures also result in a decrease in testicular testosterone in rats. The anti-androgenic effects of phthalates have been more thoroughly quantified than testicular pathology due to the significant time requirement associated with manual counting of MNGs on histological sections. An automated counting method was developed in ImageJ to quantify MNGs in digital images of hematoxylin-stained rat fetal testis tissue sections. Timed pregnant Sprague Dawley rats were exposed by daily oral gavage from gestation day 17 to 21 with one of eight phthalate test compounds or corn oil vehicle. Both the manual counting method and the automated image analysis method identified di-n-butyl phthalate, butyl benzyl phthalate, dipentyl phthalate, and di-(2-ethylhexyl) phthalate as positive for induction of MNGs. Dimethyl phthalate, diethyl phthalate, the brominated phthalate di-(2-ethylhexyl) tetrabromophthalate, and dioctyl terephthalate were negative. The correlation between automated and manual scoring metrics was high (r = 0.923). Results of MNG analysis were consistent with these compounds' anti-androgenic activities, which were confirmed in an ex vivo testosterone production assay. In conclusion, we have developed a reliable image analysis method that can be used to facilitate dose-response studies for the reproducible induction of MNGs by in utero phthalate exposure. Copyright © 2018 Elsevier B.V. All rights reserved.

Arctigenin Inhibits Osteoclast Differentiation and Function by Suppressing Both Calcineurin-Dependent and Osteoblastic Cell-Dependent NFATc1 Pathways

OpenAIRE

Yamashita, Teruhito; Uehara, Shunsuke; Udagawa, Nobuyuki; Li, Feng; Kadota, Shigetoshi; Esumi, Hiroyasu; Kobayashi, Yasuhiro; Takahashi, Naoyuki

2014-01-01

Arctigenin, a lignan-derived compound, is a constituent of the seeds of Arctium lappa. Arctigenin was previously shown to inhibit osteoclastogenesis; however, this inhibitory mechanism has yet to be elucidated. Here, we showed that arctigenin inhibited the action of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a key transcription factor for osteoclastogenesis. NFATc1 in osteoclast precursors was activated through two distinct pathways: the calcineurin-dependent and osteoblasti...

Central Giant Cell Granuloma of Posterior Maxilla: First Expression of Primary Hyperparathyroidism

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Deepanshu Gulati

2015-01-01

Full Text Available A case of 19-year-old male patient reported with the chief complaint of slowly growing diffuse painless swelling over the right part of the face from last 6 months. Intraoral examination revealed a swelling on right side of palate in relation to molar region with buccal cortical plate expansion. Radiographic examination (orthopantograph and 3DCT showed large multilocular radiolucency in right maxilla with generalized loss of lamina dura. Incisional biopsy was done and specimen was sent for histopathological examination which showed multinucleated giant cells containing 15–30 nuclei. Based on clinical, radiological, and histopathological findings provisional diagnosis of central giant cell granuloma was made. Blood tests after histopathology demonstrated elevated serum calcium level and alkaline phosphatase level. Immunoassay of parathyroid hormone (PTH level was found to be highly elevated. Radiographic examination of long bones like humerus and femur, mandible, and skull was also done which showed osteoclastic lesions. Considering the clinical, radiographic, histopathological, and blood investigation findings, final diagnosis of brown tumour of maxilla was made. The patient underwent partial parathyroidectomy under general anaesthesia to control primary hyperparathyroidism. Surgical removal of the bony lesion was done by curettage. The patient has been followed up for 1 year with no postoperative complications and the lesion healed uneventfully.

Adenosine A1 receptors (A1Rs) play a critical role in osteoclast formation and function

OpenAIRE

Kara, Firas M.; Chitu, Violeta; Sloane, Jennifer; Axelrod, Matthew; Fredholm, Bertil B.; Stanley, E. Richard; Cronstein, Bruce N.

2010-01-01

Adenosine regulates a wide variety of physiological processes via interaction with one or more G-protein-coupled receptors (A1R, A2AR, A2BR, and A3R). Because A1R occupancy promotes fusion of human monocytes to form giant cells in vitro, we determined whether A1R occupancy similarly promotes osteoclast function and formation. Bone marrow cells (BMCs) were harvested from C57Bl/6 female mice or A1R-knockout mice and their wild-type (WT) littermates and differentiated into osteoclasts in the pre...

Omental leiomyosarcoma with unusual giant cells in a Beagle dog - Short communication.

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Sasaki, Jun; Toyoshima, Megumi; Okamura, Yasuhiko; Goryo, Masanobu

2016-06-01

A 10-year-old castrated male Beagle dog was presented with a 2-month history of intermittent vomiting and abdominal pain. The dog was referred to the Veterinary Teaching Hospital at Iwate University for further evaluation, and a splenic tumour was suspected on the basis of ultrasonography and computed tomography. Surgery identified a large, solid, light-pink mass on the greater omentum with blood-coloured ascites in the abdominal cavity, and resection was performed. Microscopically, the mass comprised spindle-shaped tumour cells and scattered osteoclast-like giant cells. Most spindle-shaped cells were positive for vimentin, desmin, and smooth muscle actin (α-SMA), whereas osteoclast-like giant cells were positive only for vimentin. On the basis of histopathological and immunohistochemical findings, a diagnosis of leiomyosarcoma was made. To the best of our knowledge, this represents the first report of leiomyosarcoma associated with osteoclast-like giant cells developing from the greater omentum in a dog.

Immunological Reaction in TNF-α-Mediated Osteoclast Formation and Bone Resorption In Vitro and In Vivo

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Hideki Kitaura

2013-01-01

Full Text Available Tumor necrosis factor-α (TNF-α is a cytokine produced by monocytes, macrophages, and T cells and is induced by pathogens, endotoxins, or related substances. TNF-α may play a key role in bone metabolism and is important in inflammatory bone diseases such as rheumatoid arthritis. Cells directly involved in osteoclastogenesis include macrophages, which are osteoclast precursor cells, osteoblasts, or stromal cells. These cells express receptor activator of NF-κB ligand (RANKL to induce osteoclastogenesis, and T cells, which secrete RANKL, promote osteoclastogenesis during inflammation. Elucidating the detailed effects of TNF-α on bone metabolism may enable the identification of therapeutic targets that can efficiently suppress bone destruction in inflammatory bone diseases. TNF-α is considered to act by directly increasing RANK expression in macrophages and by increasing RANKL in stromal cells. Inflammatory cytokines such as interleukin- (IL- 12, IL-18, and interferon-γ (IFN-γ strongly inhibit osteoclast formation. IL-12, IL-18, and IFN-γ induce apoptosis in bone marrow cells treated with TNF-α  in vitro, and osteoclastogenesis is inhibited by the interactions of TNF-α-induced Fas and Fas ligand induced by IL-12, IL-18, and IFN-γ. This review describes and discusses the role of cells concerned with osteoclast formation and immunological reactions in TNF-α-mediated osteoclastogenesis in vitro and in vivo.

Tumor Necrosis Factor α Stimulates Osteoclast Differentiation by a Mechanism Independent of the Odf/Rankl–Rank Interaction

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Kobayashi, Kanichiro; Takahashi, Naoyuki; Jimi, Eijiro; Udagawa, Nobuyuki; Takami, Masamichi; Kotake, Shigeru; Nakagawa, Nobuaki; Kinosaki, Masahiko; Yamaguchi, Kyoji; Shima, Nobuyuki; Yasuda, Hisataka; Morinaga, Tomonori; Higashio, Kanji; Martin, T. John; Suda, Tatsuo

2000-01-01

Osteoclast differentiation factor (ODF, also called RANKL/TRANCE/OPGL) stimulates the differentiation of osteoclast progenitors of the monocyte/macrophage lineage into osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF, also called CSF-1). When mouse bone marrow cells were cultured with M-CSF, M-CSF–dependent bone marrow macrophages (M-BMMφ) appeared within 3 d. Tartrate-resistant acid phosphatase–positive osteoclasts were also formed when M-BMMφ were further cultured for 3 d with mouse tumor necrosis factor α (TNF-α) in the presence of M-CSF. Osteoclast formation induced by TNF-α was inhibited by the addition of respective antibodies against TNF receptor 1 (TNFR1) or TNFR2, but not by osteoclastogenesis inhibitory factor (OCIF, also called OPG, a decoy receptor of ODF/RANKL), nor the Fab fragment of anti–RANK (ODF/RANKL receptor) antibody. Experiments using M-BMMφ prepared from TNFR1- or TNFR2-deficient mice showed that both TNFR1- and TNFR2-induced signals were important for osteoclast formation induced by TNF-α. Osteoclasts induced by TNF-α formed resorption pits on dentine slices only in the presence of IL-1α. These results demonstrate that TNF-α stimulates osteoclast differentiation in the presence of M-CSF through a mechanism independent of the ODF/RANKL–RANK system. TNF-α together with IL-1α may play an important role in bone resorption of inflammatory bone diseases. PMID:10637272

Stromal cells and osteoclasts are responsible for exacerbated collagen-induced arthritis in interferon-beta-deficient mice

DEFF Research Database (Denmark)

Treschow, Alexandra P; Teige, Ingrid; Nandakumar, Kutty S

2005-01-01

OBJECTIVE: Clinical trials using interferon-beta (IFNbeta) in the treatment of rheumatoid arthritis have shown conflicting results. We undertook this study to understand the mechanisms of IFNbeta in arthritis at a physiologic level. METHODS: Collagen-induced arthritis (CIA) was induced in IFNbeta....... Differences in osteoclast maturation were determined in situ by histology of arthritic and naive paws and by in vitro maturation studies of naive bone marrow cells. The importance of IFNbeta-producing fibroblasts was determined by transferring fibroblasts into mice at the time of CIA immunization. RESULTS...

Identification of the Common Origins of Osteoclasts, Macrophages, and Dendritic Cells in Human Hematopoiesis

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Yanling Xiao

2015-06-01

Full Text Available Osteoclasts (OCs originate from the myeloid cell lineage, but the successive steps in their lineage commitment are ill-defined, especially in humans. To clarify OC origin, we sorted cell populations from pediatric bone marrow (BM by flow cytometry and assessed their differentiation potential in vitro. Within the CD11b−CD34+c-KIT+ BM cell population, OC-differentiation potential was restricted to FLT3+ cells and enriched in an IL3 receptor (Rαhigh subset that constituted less than 0.5% of total BM. These IL3Rαhigh cells also generated macrophages (MΦs and dendritic cells (DCs but lacked granulocyte (GR-differentiation potential, as demonstrated at the clonal level. The IL3Rαlow subset was re-defined as common progenitor of GR, MΦ, OC, and DC (GMODP and gave rise to the IL3Rαhigh subset that was identified as common progenitor of MΦ, OC, and DC (MODP. Unbiased transcriptome analysis of CD11b−CD34+c-KIT+FLT3+ IL3Rαlow and IL3Rαhigh subsets corroborated our definitions of the GMODP and MODP and their developmental relationship.

Intercellular Communication between Keratinocytes and Fibroblasts Induces Local Osteoclast Differentiation: a Mechanism Underlying Cholesteatoma-Induced Bone Destruction.

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Iwamoto, Yoriko; Nishikawa, Keizo; Imai, Ryusuke; Furuya, Masayuki; Uenaka, Maki; Ohta, Yumi; Morihana, Tetsuo; Itoi-Ochi, Saori; Penninger, Josef M; Katayama, Ichiro; Inohara, Hidenori; Ishii, Masaru

2016-06-01

Bone homeostasis is maintained by a balance in activity between bone-resorbing osteoclasts and bone-forming osteoblasts. Shifting the balance toward bone resorption causes osteolytic bone diseases such as rheumatoid arthritis and periodontitis. Osteoclast differentiation is regulated by receptor activator of nuclear factor κB ligand (RANKL), which, under some pathological conditions, is produced by T and B lymphocytes and synoviocytes. However, the mechanism underlying bone destruction in other diseases is little understood. Bone destruction caused by cholesteatoma, an epidermal cyst in the middle ear resulting from hyperproliferation of keratinizing squamous epithelium, can lead to lethal complications. In this study, we succeeded in generating a model for cholesteatoma, epidermal cyst-like tissue, which has the potential for inducing osteoclastogenesis in mice. Furthermore, an in vitro coculture system composed of keratinocytes, fibroblasts, and osteoclast precursors was used to demonstrate that keratinocytes stimulate osteoclast differentiation through the induction of RANKL in fibroblasts. Thus, this study demonstrates that intercellular communication between keratinocytes and fibroblasts is involved in the differentiation and function of osteoclasts, which may provide the molecular basis of a new therapeutic strategy for cholesteatoma-induced bone destruction. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

NHE10, a novel osteoclast-specific member of the Na+/H+ exchanger family, regulates osteoclast differentiation and survival

International Nuclear Information System (INIS)

Lee, Seoung Hoon; Kim, Taesoo; Park, Eui-Soon; Yang, Sujeong; Jeong, Daewon; Choi, Yongwon; Rho, Jaerang

2008-01-01

Bone homeostasis is tightly regulated by the balanced actions of osteoblasts (OBs) and osteoclasts (OCs). We previously analyzed the gene expression profile of OC differentiation using a cDNA microarray, and identified a novel osteoclastogenic gene candidate, clone OCL-1-E7 [J. Rho, C.R. Altmann, N.D. Socci, L. Merkov, N. Kim, H. So, O. Lee, M. Takami, A.H. Brivanlou, Y. Choi, Gene expression profiling of osteoclast differentiation by combined suppression subtractive hybridization (SSH) and cDNA microarray analysis, DNA Cell Biol. 21 (2002) 541-549]. In this study, we have isolated full-length cDNAs corresponding to this clone from mice and humans to determine the functional roles of this gene in osteoclastogenesis. The full-length cDNA of OCL-1-E7 encodes 12 membrane-spanning domains that are typical of isoforms of the Na + /H + exchangers (NHEs), indicating that this clone is a novel member of the NHE family (hereafter referred to as NHE10). Here, we show that NHE10 is highly expressed in OCs in response to receptor activator of nuclear factor-κB ligand signaling and is required for OC differentiation and survival

Sitagliptin, An Anti-diabetic Drug, Suppresses Estrogen Deficiency-Induced OsteoporosisIn Vivo and Inhibits RANKL-Induced Osteoclast Formation and Bone Resorption In Vitro

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Chuandong Wang

2017-06-01

Full Text Available Postmenopausal osteoporosis is a disease characterized by excessive osteoclastic bone resorption. Some anti-diabetic drugs were demonstrated for anti-osteoclastic bone-loss effects. The present study investigated the skeletal effects of chronic administration of sitagliptin, a dipeptidyl peptidase IV (DPP IV inhibitor that is increasingly used for type 2 diabetes treatments, in an estrogen deficiency-induced osteoporosis and elucidated the associated mechanisms. This study indicated that sitagliptin effectively prevented ovariectomy-induced bone loss and reduced osteoclast numbers in vivo. It was also indicated that sitagliptin suppressed receptor activator of nuclear factor-κB ligand (RANKL-mediated osteoclast differentiation, bone resorption, and F-actin ring formation in a manner of dose-dependence. In addition, sitagliptin significantly reduced the expression of osteoclast-specific markers in mouse bone-marrow-derived macrophages, including calcitonin receptor (Calcr, dendrite cell-specific transmembrane protein (Dc-stamp, c-Fos, and nuclear factor of activated T-cells cytoplasmic 1 (Nfatc1. Further study indicated that sitagliptin inhibited osteoclastogenesis by suppressing AKT and ERK signaling pathways, scavenging ROS activity, and suppressing the Ca2+ oscillation that consequently affects the expression and/or activity of the osteoclast-specific transcription factors, c-Fos and NFATc1. Collectively, these findings suggest that sitagliptin possesses beneficial effects on bone and the suppression of osteoclast number implies that the effect is exerted directly on osteoclastogenesis.

Characterization of Mason--Pfizer monkey virus-induced cell fusion

International Nuclear Information System (INIS)

Chatterjee, S.; Hunter, E.

1979-01-01

The characteristics and requirements of multinucleate cell (syncytium) induction by Mason--Pfizer monkey virus (M-PMV) on human and non-human primate cells have been investigated. Multinucleate cell induction by this D-type retrovirus shows single-hit kinetics on human foreskin and rhesus monkey fetal lung cells. The peak of syncytium-forming activity in an isopycnic sucrose gradient coincides with the peak of M-PMV virions as assessed by electron microscopy and analysis of viral polypeptides. Unlike the paramyxoviruses, M-PMV does not induce early cell fusion when added in high concentrations to the target cells. Furthermore, multinucleate cell formation is maximal 48 hr postinfection and the size of the syncytia remains constant after this time. Ultraviolet irradiation of M-PMV reduces its ability to form syncytia and to replicate with single-hit kinetics, suggesting that a functional viral genome is required for syncytium formation. Proviral DNA synthesis and assembly of virions are not necessary for cell fusion since the addition of cytosine arabinoside at concentrations which block virus replication has little effect on multinucleate cell formation. Moreover both multinucleate cells lacking detectable intracellular virus polypeptides, and groups of individual, nonfused but brightly staining cells can be observed in immunofluorescence assays at times when multinucleate cell formation is maximal. Cell fusion is inhibited by the addition of cycloheximide during the first 12 hr of infection, suggesting that de novo protein synthesis is required for multinucleate cell formation. The possibility that the translation of genomic RNA yields a fusion-inducing product is discussed

Dual Effect of Chrysanthemum indicum Extract to Stimulate Osteoblast Differentiation and Inhibit Osteoclast Formation and Resorption In Vitro

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Jong Min Baek

2014-01-01

Full Text Available The risk of bone-related diseases increases due to the imbalance between bone resorption and bone formation by osteoclasts and osteoblasts, respectively. The goal in the development of antiosteoporotic treatments is an agent that will improve bone through simultaneous osteoblast stimulation and osteoclast inhibition without undesirable side effects. To achieve this goal, numerous studies have been performed to identify novel approaches using natural oriental herbs to treat bone metabolic diseases. In the present study, we investigated the effect of Chrysanthemum indicum extract (CIE on the differentiation of osteoclastic and osteoblastic cells. CIE inhibited the formation of TRAP-positive mature osteoclasts and of filamentous-actin rings and disrupted the bone-resorbing activity of mature osteoclasts in a dose-dependent manner. CIE strongly inhibited Akt, GSK3β, and IκB phosphorylation in RANKL-stimulated bone marrow macrophages and did not show any effects on MAP kinases, including p38, ERK, and JNK. Interestingly, CIE also enhanced primary osteoblast differentiation via upregulation of the expression of alkaline phosphatase and the level of extracellular calcium concentrations during the early and terminal stages of differentiation, respectively. Our results revealed that CIE could have a potential therapeutic role in bone-related disorders through its dual effects on osteoclast and osteoblast differentiation.

Monocytes/Macrophages Upregulate the Hyaluronidase HYAL1 and Adapt Its Subcellular Trafficking to Promote Extracellular Residency upon Differentiation into Osteoclasts.

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Emeline Puissant

Full Text Available Osteoclasts are giant bone-resorbing cells originating from monocytes/macrophages. During their differentiation, they overexpress two lysosomal enzymes, cathepsin K and TRAP, which are secreted into the resorption lacuna, an acidified sealed area in contact with bone matrix where bone degradation takes place. Here we report that the acid hydrolase HYAL1, a hyaluronidase able to degrade the glycosaminoglycans hyaluronic acid (HA and chondroitin sulfate, is also upregulated upon osteoclastogenesis. The mRNA expression and protein level of HYAL1 are markedly increased in osteoclasts differentiated from RAW264.7 mouse macrophages or primary mouse bone marrow monocytes compared to these precursor cells. As a result, the HYAL1-mediated HA hydrolysis ability of osteoclasts is strongly enhanced. Using subcellular fractionation, we demonstrate that HYAL1 proteins are sorted to the osteoclast lysosomes even though, in contrast to cathepsin K and TRAP, HYAL1 is poorly mannose 6-phosphorylated. We reported previously that macrophages secrete HYAL1 proforms by constitutive secretion, and that these are recaptured by the cell surface mannose receptor, processed in endosomes and sorted to lysosomes. Present work highlights that osteoclasts secrete HYAL1 in two ways, through lysosomal exocytosis and constitutive secretion, and that these cells promote the extracellular residency of HYAL1 through downregulation of the mannose receptor. Interestingly, the expression of the other main hyaluronidase, HYAL2, and of lysosomal exoglycosidases involved in HA degradation, does not increase similarly to HYAL1 upon osteoclastogenesis. Taken together, these findings point out the predominant involvement of HYAL1 in bone HA metabolism and perhaps bone remodeling via the resorption lacuna.

Macrophage fusion is controlled by the cytoplasmic protein tyrosine phosphatase PTP-PEST/PTPN12.

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Rhee, Inmoo; Davidson, Dominique; Souza, Cleiton Martins; Vacher, Jean; Veillette, André

2013-06-01

Macrophages can undergo cell-cell fusion, leading to the formation of multinucleated giant cells and osteoclasts. This process is believed to promote the proteolytic activity of macrophages toward pathogens, foreign bodies, and extracellular matrices. Here, we examined the role of PTP-PEST (PTPN12), a cytoplasmic protein tyrosine phosphatase, in macrophage fusion. Using a macrophage-targeted PTP-PEST-deficient mouse, we determined that PTP-PEST was not needed for macrophage differentiation or cytokine production. However, it was necessary for interleukin-4-induced macrophage fusion into multinucleated giant cells in vitro. It was also needed for macrophage fusion following implantation of a foreign body in vivo. Moreover, in the RAW264.7 macrophage cell line, PTP-PEST was required for receptor activator of nuclear factor kappa-B ligand (RANKL)-triggered macrophage fusion into osteoclasts. PTP-PEST had no impact on expression of fusion mediators such as β-integrins, E-cadherin, and CD47, which enable macrophages to become fusion competent. However, it was needed for polarization of macrophages, migration induced by the chemokine CC chemokine ligand 2 (CCL2), and integrin-induced spreading, three key events in the fusion process. PTP-PEST deficiency resulted in specific hyperphosphorylation of the protein tyrosine kinase Pyk2 and the adaptor paxillin. Moreover, a fusion defect was induced upon treatment of normal macrophages with a Pyk2 inhibitor. Together, these data argue that macrophage fusion is critically dependent on PTP-PEST. This function is seemingly due to the ability of PTP-PEST to control phosphorylation of Pyk2 and paxillin, thereby regulating cell polarization, migration, and spreading.

Osteoclast inhibition impairs chondrosarcoma growth and bone destruction.

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Otero, Jesse E; Stevens, Jeff W; Malandra, Allison E; Fredericks, Douglas C; Odgren, Paul R; Buckwalter, Joseph A; Morcuende, Jose

2014-12-01

Because Chondrosarcoma is resistant to available chemotherapy and radiation regimens, wide resection is the mainstay in treatment, which frequently results in high morbidity and which may not prevent local recurrence. There is a clear need for improved adjuvant treatment of this malignancy. We have observed the presence of osteoclasts in the microenvironment of chondrosarcoma in human pathological specimens. We utilized the Swarm rat chondrosarcoma (SRC) model to test the hypothesis that osteoclasts affect chondrosarcoma pathogenesis. We implanted SRC tumors in tibia of Sprague-Dawley rats and analyzed bone histologically and radiographically for bone destruction and tumor growth. At three weeks, tumors invaded local bone causing cortical disruption and trabecular resorption. Bone destruction was accompanied by increased osteoclast number and resorbed bone surface. Treatment of rats with the zoledronic acid prevented cortical destruction, inhibited trabecular resorption, and resulted in decreased tumor volume in bone. To confirm that inhibition of osteoclasts per se, and not off-target effects of drug, was responsible for the prevention of tumor growth and bone destruction, we implanted SRC into osteopetrotic rat tibia. SRC-induced bone destruction and tumor growth were impaired in osteopetrotic bone compared with control bone. The results from our animal model demonstrate that osteoclasts contribute to chondrosarcoma-mediated bone destruction and tumor growth and may represent a therapeutic target in particular chondrosarcoma patients. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Adaptor protein GRB2 promotes Src tyrosine kinase activation and podosomal organization by protein-tyrosine phosphatase ϵ in osteoclasts.

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Levy-Apter, Einat; Finkelshtein, Eynat; Vemulapalli, Vidyasiri; Li, Shawn S-C; Bedford, Mark T; Elson, Ari

2014-12-26

The non-receptor isoform of protein-tyrosine phosphatase ϵ (cyt-PTPe) supports adhesion of bone-resorbing osteoclasts by activating Src downstream of integrins. Loss of cyt-PTPe reduces Src activity in osteoclasts, reduces resorption of mineralized matrix both in vivo and in cell culture, and induces mild osteopetrosis in young female PTPe KO mice. Activation of Src by cyt-PTPe is dependent upon this phosphatase undergoing phosphorylation at its C-terminal Tyr-638 by partially active Src. To understand how cyt-PTPe activates Src, we screened 73 Src homology 2 (SH2) domains for binding to Tyr(P)-638 of cyt-PTPe. The SH2 domain of GRB2 bound Tyr(P)-638 of cyt-PTPe most prominently, whereas the Src SH2 domain did not bind at all, suggesting that GRB2 may link PTPe with downstream molecules. Further studies indicated that GRB2 is required for activation of Src by cyt-PTPe in osteoclast-like cells (OCLs) in culture. Overexpression of GRB2 in OCLs increased activating phosphorylation of Src at Tyr-416 and of cyt-PTPe at Tyr-638; opposite results were obtained when GRB2 expression was reduced by shRNA or by gene inactivation. Phosphorylation of cyt-PTPe at Tyr-683 and its association with GRB2 are integrin-driven processes in OCLs, and cyt-PTPe undergoes autodephosphorylation at Tyr-683, thus limiting Src activation by integrins. Reduced GRB2 expression also reduced the ability of bone marrow precursors to differentiate into OCLs and reduced the fraction of OCLs in which podosomal adhesion structures assume organization typical of active, resorbing cells. We conclude that GRB2 physically links cyt-PTPe with Src and enables cyt-PTPe to activate Src downstream of activated integrins in OCLs. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Osteoclasts and CD8 T cells form a negative feedback loop that contributes to homeostasis of both the skeletal and immune systems.

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Buchwald, Zachary S; Aurora, Rajeev

2013-01-01

There are a number of dynamic regulatory loops that maintain homeostasis of the immune and skeletal systems. In this review, we highlight a number of these regulatory interactions that contribute to maintaining homeostasis. In addition, we review data on a negative regulatory feedback loop between osteoclasts and CD8 T cells that contributes to homeostasis of both the skeletal and immune systems.

Effect of bisphosphonates on NFATc1 and correlators p-NF-κB and p-c-Jun in osteoclast differentiation

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Wei DONG

2015-11-01

Full Text Available Objective　To study the effect of alendronate (ALN on NFATc1 and correlated signaling factors p-NF-κB and p-c-Jun in osteoclast differentiation. Methods　Osteoclasts were inductively cultivated with mouse mononuclear macrophage RAW264.7, and they were divided into 2 groups: group A (control group and group B (ALN-treated group. The protein expressions of NFATc1, p-NF-κB and p-c-Jun were determined with Western blotting at the 2nd day of cultivation; the expression of NFATc1 was assessed by immunofluorescent assay on the 4th day; and the osteoclast formation was examined on the 7th day of cultivation. RAW264.7 cells were inoculated on abrasive dentine disk, and divided into 2 groups and treated as mentioned above. The resorption function of osteoclast was observed on the 9th day of inoculation. Results　TRAP positive multinuclear osteoclasts were observed, and resorption lacunaes formed in the abrasive dentine disks of the 2 groups. More TRAP positive multinuclear cells and resorption lacunaes in large size were found in group A than those in group B. Immunofluorescence assay showed the expression of NFATc1 was higher in group A than in group B. The gene expressions of NFATc1, p-NF-κB and p-c-Jun were lower in group B than in group A(P<0.01 as determined with Western blotting. Conclusion　By down-regulating the expressions of p-NF-κB and p-c-Jun, ALN may strongly inhibit the osteoclast formation and its resorption function, thus inhibiting NFATc1 expression. DOI: 10.11855/j.issn.0577-7402.2015.10.02

Characterization of osteoclasts from patients harboring a G215R mutation in ClC-7 causing autosomal dominant osteopetrosis type II

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Henriksen, Kim; Gram, Jeppe; Schaller, Sophie

2004-01-01

from ADOII patients and healthy age- and sex-matched controls, were used to evaluate osteoclastogenesis, cell fusion, acidification, and resorptive activity. ADOII osteoclasts in vivo have increased number and size. However, in vitro we observed no significant changes in the osteoclast formation rate......, the morphology, and the expression of markers, such as cathepsin K and tartrate-resistant acid phosphatase. When mature ADOII osteoclasts were investigated on mineralized bone, they degraded the bone material, however only to 10 to 20% of the level in controls. We show by acridine orange, that the reduced...

G protein-coupled receptor 84 controls osteoclastogenesis through inhibition of NF-κB and MAPK signaling pathways.

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Park, Ji-Wan; Yoon, Hye-Jin; Kang, Woo Youl; Cho, Seungil; Seong, Sook Jin; Lee, Hae Won; Yoon, Young-Ran; Kim, Hyun-Ju

2018-02-01

GPR84, a member of the G protein-coupled receptor family, is found predominantly in immune cells, such as macrophages, and functions as a pivotal modulator of inflammatory responses. In this study, we investigated the role of GPR84 in receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation. Our microarray data showed that GPR84 was significantly downregulated in osteoclasts compared to in their precursors, macrophages. The overexpression of GPR84 in bone marrow-derived macrophages suppressed the formation of multinucleated osteoclasts without affecting precursor proliferation. In addition, GPR84 overexpression attenuated the induction of c-Fos and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), which are transcription factors that are critical for osteoclastogenesis. Furthermore, knockdown of GPR84 using a small hairpin RNA promoted RANKL-mediated osteoclast differentiation and gene expression of osteoclastogenic markers. Mechanistically, GPR84 overexpression blocked RANKL-stimulated phosphorylation of IκBα and three MAPKs, JNK, ERK, and p38. GPR84 also suppressed NF-κB transcriptional activity mediated by RANKL. Conversely, GPR84 knockdown enhanced RANKL-induced activation of IκBα and the three MAPKs. Collectively, our results revealed that GPR84 functions as a negative regulator of osteoclastogenesis, suggesting that it may be a potential therapeutic target for osteoclast-mediated bone-destructive diseases. © 2017 Wiley Periodicals, Inc.

Tfe3 expression is closely associated to macrophage terminal differentiation of human hematopoietic myeloid precursors

International Nuclear Information System (INIS)

Zanocco-Marani, Tommaso; Vignudelli, Tatiana; Gemelli, Claudia; Pirondi, Sara; Testa, Anna; Montanari, Monica; Parenti, Sandra; Tenedini, Elena; Grande, Alexis; Ferrari, Sergio

2006-01-01

The MItf-Tfe family of basic helix-loop-helix leucine zipper (bHLH-Zip) transcription factors encodes four family members: MItf, Tfe3, TfeB and TfeC. In vitro, each protein of the family binds DNA in a homo- or heterodimeric form with other family members. Tfe3 is involved in chromosomal translocations recurrent in different tumors and it has been demonstrated, by in vivo studies, that it plays, redundantly with MItf, an important role in the process of osteoclast formation, in particular during the transition from mono-nucleated to multi-nucleated osteoclasts. Since mono-nucleated osteoclasts derive from macrophages we investigated whether Tfe3 might play a role upstream during hematopoietic differentiation. Here we show that Tfe3 is able to induce mono-macrophagic differentiation of U937 cells, in association with a decrease of cell proliferation and an increase of apoptosis. We also show that Tfe3 does not act physiologically during commitment of CD34+ hematopoietic stem cells (HSCs), since it is not able to direct HSCs toward a specific lineage as observed by clonogenic assay, but is a strong actor of terminal differentiation since it allows human primary myeloblasts' maturation toward the macrophage lineage

Salicortin inhibits osteoclast differentiation and bone resorption by down-regulating JNK and NF-κB/NFATc1 signaling pathways

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Nie, Shaobo [Department of Orthopaedics, PLA General Hospital, Beijing 100853 (China); Xu, Jiawei [Department of Orthopaedics, Shanghai Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011 (China); Zhang, Chenghua [Department of Orthopaedics, Changle County Hospital of Traditional Chinese Medicine, Weifang 262400 (China); Xu, Chen [Department of Orthopaedics, Shanghai Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011 (China); Liu, Ming, E-mail: ming_li4717@sina.com [Department of Orthopaedics, Shanghai Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011 (China); Yu, Degang, E-mail: ydg163@126.com [Department of Orthopaedics, Shanghai Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011 (China)

2016-01-29

Receptor activator of nuclear factor (NF)-κB ligand (RANKL)-activated signaling is essential for osteoclast differentiation, activation, and survival. Salicortin is a phenolic glycoside that has been isolated from many plants such as Populus and Salix species, and has been shown to have anti-amnesic and anti-adipogenic effects. In this study, we investigated the effect of salicortin on RANKL-induced osteoclasts formation, bone resorption, and activation of osteoclast-related signaling pathways. Salicortin suppressed RANKL-induced osteoclastogenesis in bone marrow macrophage cultures in a dose-dependent manner, and inhibited osteoclastic bone resorption activity without any cytotoxicity. Salicortin inhibited RANKL-induced c-Jun N-terminal kinase and NF-κB activation, concomitant with retarded IκBα phosphorylation and inhibition of p65 nuclear translocation, leading to impaired transcription of nuclear factor of activated T cells c1 (NFATc1) and expression of osteoclastic-specific genes. Taken together, our findings demonstrate that salicortin inhibits NF-κB and NFATc1 activation, leading to attenuation of osteoclastogenesis and bone resorption. Thus, salicortin may be of interest in developments of treatment for osteoclast related diseases. - Highlights: • Salicortin suppresses osteoclastogenesis in vitro. • Salicortin impairs the JNK and NF-κB/NFATc1 signaling pathway. • Salicortin may be of interest in developments of osteoporosis treatment.

Salicortin inhibits osteoclast differentiation and bone resorption by down-regulating JNK and NF-κB/NFATc1 signaling pathways

International Nuclear Information System (INIS)

Nie, Shaobo; Xu, Jiawei; Zhang, Chenghua; Xu, Chen; Liu, Ming; Yu, Degang

2016-01-01

Receptor activator of nuclear factor (NF)-κB ligand (RANKL)-activated signaling is essential for osteoclast differentiation, activation, and survival. Salicortin is a phenolic glycoside that has been isolated from many plants such as Populus and Salix species, and has been shown to have anti-amnesic and anti-adipogenic effects. In this study, we investigated the effect of salicortin on RANKL-induced osteoclasts formation, bone resorption, and activation of osteoclast-related signaling pathways. Salicortin suppressed RANKL-induced osteoclastogenesis in bone marrow macrophage cultures in a dose-dependent manner, and inhibited osteoclastic bone resorption activity without any cytotoxicity. Salicortin inhibited RANKL-induced c-Jun N-terminal kinase and NF-κB activation, concomitant with retarded IκBα phosphorylation and inhibition of p65 nuclear translocation, leading to impaired transcription of nuclear factor of activated T cells c1 (NFATc1) and expression of osteoclastic-specific genes. Taken together, our findings demonstrate that salicortin inhibits NF-κB and NFATc1 activation, leading to attenuation of osteoclastogenesis and bone resorption. Thus, salicortin may be of interest in developments of treatment for osteoclast related diseases. - Highlights: • Salicortin suppresses osteoclastogenesis in vitro. • Salicortin impairs the JNK and NF-κB/NFATc1 signaling pathway. • Salicortin may be of interest in developments of osteoporosis treatment.

Allium cepa L. and Quercetin Inhibit RANKL/Porphyromonas gingivalis LPS-Induced Osteoclastogenesis by Downregulating NF-κB Signaling Pathway

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Tatiane Oliveira

2015-01-01

Full Text Available Objectives. We evaluated the in vitro modulatory effects of Allium cepa L. extract (AcE and quercetin (Qt on osteoclastogenesis under inflammatory conditions (LPS-induced. Methods. RAW 264.7 cells were differentiated with 30 ng/mL of RANKL, costimulated with PgLPS (1 µg/mL, and treated with AcE (50–1000 µg/mL or Qt (1.25, 2.5, or 5 µM. Cell viability was determined by alamarBlue and protein assays. Nuclei morphology was analysed by DAPI staining. TRAP assays were performed as follows: p-nitrophenyl phosphate was used to determine the acid phosphatase activity of the osteoclasts and TRAP staining was used to evaluate the number and size of TRAP-positive multinucleated osteoclast cells. Von Kossa staining was used to measure osteoclast resorptive activity. Cytokine levels were measured on osteoclast precursor cell culture supernatants. Using western blot analysis, p-IκBα and IκBα degradation, inhibitor of NF-kappaB, were evaluated. Results. Both AcE and Qt did not affect cell viability and significantly reduced osteoclastogenesis compared to control. We observed lower production of IL-6 and IL-1α and an increased production of IL-3 and IL-4. AcE and Qt downregulated NF-κB pathway. Conclusion. AcE and Qt may be inhibitors of osteoclastogenesis under inflammatory conditions (LPS-induced via attenuation of RANKL/PgLPS-induced NF-κB activation.

Renal medullary AA amyloidosis, hepatocyte dissociation and multinucleated hepatocytes in a 14-year-old free-ranging lioness (Panthera leo

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J.H. Williams

2005-06-01

Full Text Available A 14-year-old lioness, originating from Etosha in Namibia, and a member of a pride in Pilanesberg National Park since translocation in 1994, was euthanased due to fight-related vertebral fracture and spinal injury, incurred approximately 6-8 weeks previously. Blood specimens collected at the time of death showed mild anaemia and a leukogram reflecting stress and chronic infection. Necropsy conducted within 2 hours of death was on a dehydrated, emaciated animal with hindquarter wasting and chronic traumatic friction injuries from dragging her hindlegs. There was cellulitis in the region of bite-wounds adjacent to the thoraco-lumbar vertebral fracture, at which site there was spinal cord compression, and there was marked intestinal helminthiasis. The outer renal medullae appeared pale and waxy and the liver was macroscopically unremarkable. Histopathology and electron microscopy of the kidneys revealed multifocal to coalescing deposits of proximal medullary interstitial amyloid, which fluoresced strongly with thioflavine T, and was sensitive to potassium permanganate treatment prior to Congo Red staining, thus indicating inflammatory (AA origin. There was diffuse hepatocyte dissociation, as well as numerous binucleated and scattered multinucleated (up to 8 nuclei/cell hepatocytes, with swollen hepatocyte mitochondria, in liver examined light microscopically. Ultrastructurally, the mono-, bi- and multinucleated hepatocytes contained multifocal irregular membrane-bound accumulations of finely-granular, amorphous material both intra-cytoplasmically and intra-nuclearly, as well as evidence of irreversible mitochondrial injury. The incidence and relevance in cats and other species of amyloidosis, particularly with renal medullary distribution, as well as of hepatocyte dissociation and multinucleation, as reported in selected literature, is briefly overviewed and their occurrence in this lioness is discussed.

Stress induced by premature chromatin condensation triggers chromosome shattering and chromothripsis at DNA sites still replicating in micronuclei or multinucleate cells when primary nuclei enter mitosis.

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Terzoudi, Georgia I; Karakosta, Maria; Pantelias, Antonio; Hatzi, Vasiliki I; Karachristou, Ioanna; Pantelias, Gabriel

2015-11-01

Combination of next-generation DNA sequencing, single nucleotide polymorphism array analyses and bioinformatics has revealed the striking phenomenon of chromothripsis, described as complex genomic rearrangements acquired in a single catastrophic event affecting one or a few chromosomes. Via an unproven mechanism, it is postulated that mechanical stress causes chromosome shattering into small lengths of DNA, which are then randomly reassembled by DNA repair machinery. Chromothripsis is currently examined as an alternative mechanism of oncogenesis, in contrast to the present paradigm that considers a stepwise development of cancer. While evidence for the mechanism(s) underlying chromosome shattering during cancer development remains elusive, a number of hypotheses have been proposed to explain chromothripsis, including ionizing radiation, DNA replication stress, breakage-fusion-bridge cycles, micronuclei formation and premature chromosome compaction. In the present work, we provide experimental evidence on the mechanistic basis of chromothripsis and on how chromosomes can get locally shattered in a single catastrophic event. Considering the dynamic nature of chromatin nucleoprotein complex, capable of rapid unfolding, disassembling, assembling and refolding, we first show that chromatin condensation at repairing or replicating DNA sites induces the mechanical stress needed for chromosome shattering to ensue. Premature chromosome condensation is then used to visualize the dynamic nature of interphase chromatin and demonstrate that such mechanical stress and chromosome shattering can also occur in chromosomes within micronuclei or asynchronous multinucleate cells when primary nuclei enter mitosis. Following an aberrant mitosis, chromosomes could find themselves in the wrong place at the wrong time so that they may undergo massive DNA breakage and rearrangement in a single catastrophic event. Specifically, our results support the hypothesis that premature chromosome

Formation of multinucleated giant cells and microglial degeneration in rats expressing a mutant Cu/Zn superoxide dismutase gene

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Streit Wolfgang J

2007-02-01

Full Text Available Abstract Background Microglial neuroinflammation is thought to play a role in the pathogenesis of amyotrophic lateral sclerosis (ALS. The purpose of this study was to provide a histopathological evaluation of the microglial neuroinflammatory response in a rodent model of ALS, the SOD1G93A transgenic rat. Methods Multiple levels of the CNS from spinal cord to cerebral cortex were studied in SOD1G93A transgenic rats during three stages of natural disease progression, including presymptomatic, early symptomatic (onset, and late symptomatic (end stage, using immuno- and lectin histochemical markers for microglia, such as OX-42, OX-6, and Griffonia simplicifolia isolectin B4. Results Our studies revealed abnormal aggregates of microglia forming in the spinal cord as early as the presymptomatic stage. During the symptomatic stages there was prominent formation of multinucleated giant cells through fusion of microglial cells in the spinal cord, brainstem, and red nucleus of the midbrain. Other brain regions, including substantia nigra, cranial nerve nuclei, hippocampus and cortex showed normal appearing microglia. In animals during end stage disease at 4–5 months of age virtually all microglia in the spinal cord gray matter showed extensive fragmentation of their cytoplasm (cytorrhexis, indicative of widespread microglial degeneration. Few microglia exhibiting nuclear fragmentation (karyorrhexis indicative of apoptosis were identified at any stage. Conclusion The current findings demonstrate the occurrence of severe abnormalities in microglia, such as cell fusions and cytorrhexis, which may be the result of expression of mutant SOD1 in these cells. The microglial changes observed are different from those that accompany normal microglial activation, and they demonstrate that aberrant activation and degeneration of microglia is part of the pathogenesis of motor neuron disease.

Early reversal cells in adult human bone remodeling

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Abdelgawad, Mohamed Essameldin; Delaissé, Jean-Marie; Hinge, Maja

2016-01-01

The mechanism coupling bone resorption and formation is a burning question that remains incompletely answered through the current investigations on osteoclasts and osteoblasts. An attractive hypothesis is that the reversal cells are likely mediators of this coupling. Their nature is a big matter...... of debate. The present study performed on human cancellous bone is the first one combining in situ hybridization and immunohistochemistry to demonstrate their osteoblastic nature. It shows that the Runx2 and CD56 immunoreactive reversal cells appear to take up TRAcP released by neighboring osteoclasts....... Earlier preclinical studies indicate that reversal cells degrade the organic matrix left behind by the osteoclasts and that this degradation is crucial for the initiation of the subsequent bone formation. To our knowledge, this study is the first addressing these catabolic activities in adult human bone...

The orally available Btk inhibitor ibrutinib (PCI-32765) protects against osteoclast-mediated bone loss.

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Shinohara, Masahiro; Chang, Betty Y; Buggy, Joseph J; Nagai, Yusuke; Kodama, Tatsuhiko; Asahara, Hiroshi; Takayanagi, Hiroshi

2014-03-01

Bone-resorbing osteoclasts play an essential role in normal bone homeostasis, as well as in various bone disorders such as osteoporosis and rheumatoid arthritis. Previously we showed that the Tec family of tyrosine kinases is essential for the differentiation of osteoclasts and the inhibition of Btk is a promising strategy for the prevention of the bone loss in osteoclast-associated bone disorders. Here we demonstrate that an orally available Btk inhibitor, ibrutinib (PCI-32765), suppresses osteoclastic bone resorption by inhibiting both osteoclast differentiation and function. Ibrutinib downregulated the expression of NFATc1, the key transcription factor for osteoclastogenesis, and disrupted the formation of the actin ring in mature osteoclasts. In addition, genome-wide screening revealed that Btk regulates the expression of the genes involved in osteoclast differentiation and function in both an NFATc1-dependent and -independent manner. Finally, we showed that ibrutinib administration ameliorated the bone loss that developed in a RANKL-induced osteoporosis mouse model. Thus, this study suggests ibrutinib to be a promising therapeutic agent for osteoclast-associated bone diseases. Copyright © 2013 Elsevier Inc. All rights reserved.

Dioscin inhibits osteoclast differentiation and bone resorption though down-regulating the Akt signaling cascades

International Nuclear Information System (INIS)

Qu, Xinhua; Zhai, Zanjing; Liu, Xuqiang; Li, Haowei; Ouyang, Zhengxiao; Wu, Chuanlong; Liu, Guangwang; Fan, Qiming; Tang, Tingting; Qin, An; Dai, Kerong

2014-01-01

Highlights: •A natural-derived compound, dioscin, suppresses osteoclast formation and bone resorption. •Dioscin inhibits osteolytic bone loss in vivo. •Dioscin impairs the Akt signaling cascades pathways during osteoclastogenesis. •Dioscin have therapeutic value in treating osteoclast-related diseases. -- Abstract: Bone resorption is the unique function of osteoclasts (OCs) and is critical for both bone homeostasis and pathologic bone diseases including osteoporosis, rheumatoid arthritis and tumor bone metastasis. Thus, searching for natural compounds that may suppress osteoclast formation and/or function is promising for the treatment of osteoclast-related diseases. In this study, we for the first time demonstrated that dioscin suppressed RANKL-mediated osteoclast differentiation and bone resorption in vitro in a dose-dependent manner. The suppressive effect of dioscin is supported by the reduced expression of osteoclast-specific markers. Further molecular analysis revealed that dioscin abrogated AKT phosphorylation, which subsequently impaired RANKL-induced nuclear factor-kappaB (NF-κB) signaling pathway and inhibited NFATc1 transcriptional activity. Moreover, in vivo studies further verified the bone protection activity of dioscin in osteolytic animal model. Together our data demonstrate that dioscin suppressed RANKL-induced osteoclast formation and function through Akt signaling cascades. Therefore, dioscin is a potential natural agent for the treatment of osteoclast-related diseases

Dioscin inhibits osteoclast differentiation and bone resorption though down-regulating the Akt signaling cascades

Energy Technology Data Exchange (ETDEWEB)

Qu, Xinhua; Zhai, Zanjing; Liu, Xuqiang; Li, Haowei [Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopaedics, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai (China); Ouyang, Zhengxiao [Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopaedics, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai (China); Department of Orthopaedics, Hunan Provincial Tumor Hospital and Tumor Hospital of Xiangya School of Medicine, Central South University, Changsha (China); Wu, Chuanlong [Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopaedics, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai (China); Liu, Guangwang [Department of Orthopaedic Surgery, The Central Hospital of Xuzhou, Affiliated Hospital of Medical Collage of Southeast University, Xuzhou (China); Fan, Qiming; Tang, Tingting [Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopaedics, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai (China); Qin, An, E-mail: dr.qinan@gmail.com [Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopaedics, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai (China); Dai, Kerong, E-mail: krdai@163.com [Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopaedics, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai (China)

2014-01-10

Highlights: •A natural-derived compound, dioscin, suppresses osteoclast formation and bone resorption. •Dioscin inhibits osteolytic bone loss in vivo. •Dioscin impairs the Akt signaling cascades pathways during osteoclastogenesis. •Dioscin have therapeutic value in treating osteoclast-related diseases. -- Abstract: Bone resorption is the unique function of osteoclasts (OCs) and is critical for both bone homeostasis and pathologic bone diseases including osteoporosis, rheumatoid arthritis and tumor bone metastasis. Thus, searching for natural compounds that may suppress osteoclast formation and/or function is promising for the treatment of osteoclast-related diseases. In this study, we for the first time demonstrated that dioscin suppressed RANKL-mediated osteoclast differentiation and bone resorption in vitro in a dose-dependent manner. The suppressive effect of dioscin is supported by the reduced expression of osteoclast-specific markers. Further molecular analysis revealed that dioscin abrogated AKT phosphorylation, which subsequently impaired RANKL-induced nuclear factor-kappaB (NF-κB) signaling pathway and inhibited NFATc1 transcriptional activity. Moreover, in vivo studies further verified the bone protection activity of dioscin in osteolytic animal model. Together our data demonstrate that dioscin suppressed RANKL-induced osteoclast formation and function through Akt signaling cascades. Therefore, dioscin is a potential natural agent for the treatment of osteoclast-related diseases.

Osteoclasts secrete non-bone derived signals that induce bone formation

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Karsdal, Morten A; Neutzsky-Wulff, Anita V; Dziegiel, Morten Hanefeld

2008-01-01

Bone turnover is a highly regulated process, where bone resorption in the normal healthy individual always is followed by bone formation in a manner referred to as coupling. Patients with osteopetrosis caused by defective acidification of the resorption lacuna have severely decreased resorption......) from human osteoclasts cultured on either bone or plastic, and tested their effects on bone nodule formation by osteoblasts. Both types of CM were shown to dose-dependently induce bone nodule formation, whereas non-conditioned osteoclast culture medium had no effects. These data show that osteoclasts...

Expression and Function of Transmembrane-4 Superfamily (Tetraspanin Proteins in Osteoclasts: Reciprocal Roles of Tspan-5 and NET-6 during Osteoclastogenesis

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Kaori Iwai

2007-01-01

Conclusions: These data indicate that a diversity of tetraspanins is expressed in osteoclast precursors, and that cell fusion during osteoclastogenesis is regulated by cooperation of distinct tetraspanin family proteins such as Tspan-5 and NET-6. This study indicates that functional alterations of tetraspanin family proteins may have therapeutic potential in diseases where osteoclasts play a major role, such as rheumatoid arthritis and osteoporosis.

The Effects of Kaempferol-Inhibited Autophagy on Osteoclast Formation.

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Kim, Chang-Ju; Shin, Sang-Hun; Kim, Bok-Joo; Kim, Chul-Hoon; Kim, Jung-Han; Kang, Hae-Mi; Park, Bong-Soo; Kim, In-Ryoung

2018-01-02

Kaempferol, a flavonoid compound, is derived from the rhizome of Kaempferia galanga L ., which is used in traditional medicine in Asia. Autophagy has pleiotropic functions that are involved in cell growth, survival, nutrient supply under starvation, defense against pathogens, and antigen presentation. There are many studies dealing with the inhibitory effects of natural flavonoids in bone resorption. However, no studies have explained the relationship between the autophagic and inhibitory processes of osteoclastogenesis by natural flavonoids. The present study was undertaken to investigate the inhibitory effects of osteoclastogenesis through the autophagy inhibition process stimulated by kaempferol in murin macrophage (RAW 264.7) cells. The cytotoxic effect of Kaempferol was investigated by MTT assay. The osteoclast differentiation and autophagic process were confirmed via tartrate-resistant acid phosphatase (TRAP) staining, pit formation assay, western blot, and real-time PCR. Kaempferol controlled the expression of autophagy-related factors and in particular, it strongly inhibited the expression of p62/SQSTM1. In the western blot and real time-PCR analysis, when autophagy was suppressed with the application of 3-Methyladenine (3-MA) only, osteoclast and apoptosis related factors were not significantly affected. However, we found that after cells were treated with kaempferol, these factors inhibited autophagy and activated apoptosis. Therefore, we presume that kaempferol-inhibited autophagy activated apoptosis by degradation of p62/SQSTM1. Further study of the p62/SQSTM1 gene as a target in the autophagy mechanism, may help to delineate the potential role of kaempferol in the treatment of bone metabolism disorders.

Pseudoangiomatous stromal hyperplasia with multinucleated stromal giant cells is neither exceptional in gynecomastia nor characteristic of neurofibromatosis type 1.

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Pižem, Jože; Velikonja, Mojca; Matjašič, Alenka; Jerše, Maja; Glavač, Damjan

2015-04-01

Six cases of gynecomastia with pseudoangiomatous stromal hyperplasia (PASH) and multinucleated stromal giant cells (MSGC) associated with neurofibromatosis type 1 (NF1) have been reported, and finding MSGC within PASH in gynecomastia has been suggested as being a characteristic of NF1. The frequency of PASH with MSGC in gynecomastia and its specificity for NF1 have not, however, been systematically studied. A total of 337 gynecomastia specimens from 215 patients, aged from 8 to 78 years (median, 22 years) were reevaluated for the presence of PASH with MSGC. Breast tissue samples of 25 patients were analyzed for the presence of an NF1 gene mutation using next generation sequencing. Rare MSGC, usually in the background of PASH, were noted at least unilaterally in 27 (13 %) patients; and prominent MSGC, always in the background of PASH, were noted in 8 (4 %) patients. The NF1 gene was mutated in only 1 (an 8-year-old boy with known NF1 and prominent MSGC) of the 25 tested patients, including 6 patients with prominent MSGC and 19 patients with rare MSGC. MSGC, usually in the background of PASH, are not characteristic of NF1.

Characterization of two types of osteoclasts from human peripheral blood monocytes

International Nuclear Information System (INIS)

Yuasa, Kimitaka; Mori, Kouki; Ishikawa, Hitoshi; Sudo, Akihiro; Uchida, Atsumasa; Ito, Yasuhiko

2007-01-01

The two osteoclastogenesis pathways, receptor activator nuclear factor (NF)-κB ligand (RANKL)-mediated and fusion regulatory protein-1 (FRP-1)-mediated osteoclastogenesis, have recently been reported. There were significant differences in differentiation and activation mechanisms between the two pathways. When monocytes were cultured with FRP-1 without adding M-CSF, essential for the RANKL system, TRAP-positive polykaryocyte formation occurred. FRP-1-mediated osteoclasts formed larger pits on mineralized calcium phosphate plates than RANKL+M-CSF-mediated osteoclasts did. Lacunae on dentin surfaces induced by FRP-1-mediated osteoclasts were inclined to be single and isolated. However, osteoclasts induced by RANKL+M-CSF made many connected pits on dentin surfaces as if they crawled on there. Interestingly, FRP-1 osteoclastogenesis was enhanced by M-CSF/IL-1α, while chemotactic behavior to the dentin slices was not effected. There were differences in pH and concentration of HCO3- at culture endpoint and in adherent feature to dentin surfaces. Our findings indicate there are two types of osteoclasts with distinct properties

c-Fms signaling mediates neurofibromatosis Type-1 osteoclast gain-in-functions.

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Yongzheng He

Full Text Available Skeletal abnormalities including osteoporosis and osteopenia occur frequently in both pediatric and adult neurofibromatosis type 1 (NF1 patients. NF1 (Nf1 haploinsufficient osteoclasts and osteoclast progenitors derived from both NF1 patients and Nf1(+/- mice exhibit increased differentiation, migration, and bone resorptive capacity in vitro, mediated by hyperactivation of p21(Ras in response to limiting concentrations of macrophage-colony stimulating factor (M-CSF. Here, we show that M-CSF binding to its receptor, c-Fms, results in increased c-Fms activation in Nf1(+/ (- osteoclast progenitors, mediating multiple gain-in-functions through the downstream effectors Erk1/2 and p90RSK. PLX3397, a potent and selective c-Fms inhibitor, attenuated M-CSF mediated Nf1(+/- osteoclast migration by 50%, adhesion by 70%, and pit formation by 60%. In vivo, we administered PLX3397 to Nf1(+/- osteoporotic mice induced by ovariectomy (OVX and evaluated changes in bone mass and skeletal architecture. We found that PLX3397 prevented bone loss in Nf1(+/--OVX mice by reducing osteoclast differentiation and bone resorptive activity in vivo. Collectively, these results implicate the M-CSF/c-Fms signaling axis as a critical pathway underlying the aberrant functioning of Nf1 haploinsufficient osteoclasts and may provide a potential therapeutic target for treating NF1 associated osteoporosis and osteopenia.

Osteoclastic finger arthrosis - a subtype of polyarthrosis of the hand

International Nuclear Information System (INIS)

Dihlmann, W.; Dihlmann, A.

1998-01-01

Aim: Description of a subtype of arthrosis deformans of the hand which is characterised as osteoclastic arthrosis. Patients and methods: Retrospective analysis of radiographs of the hands of 150 women and 100 men with radiological findings of arthrosis deformans. Results: 5% of women and 2% of men showed at least one digital joint with subchondral osteolysis of one or both articulating bones involving at least a third of the phalanx. This subchondral osteolysis far exceeds the cysts which are situated in the epiphyseal part of the articular region. It may develop within a year. Conclusion: Osteoclastic arthrosis of the finger is a subtype of polyarthrosis of the hand. Serial observations suggest that an osteoclast stimulating substance is produced by the cysts or arises directly from the synovial fluid; this enters the subchondral part of the bone through clefts which may or may not be visible radiologically and that this produces osteoclastic activity. The most important differential diagnoses are chronic tophacious gout and a benign tumor. (orig.) [de

Role of Muramyl Dipeptide in Lipopolysaccharide-Mediated Biological Activity and Osteoclast Activity

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Hideki Kitaura

2018-01-01

Full Text Available Lipopolysaccharide (LPS is an endotoxin and bacterial cell wall component that is capable of inducing inflammation and immunological activity. Muramyl dipeptide (MDP, the minimal essential structural unit responsible for the immunological activity of peptidoglycans, is another inflammation-inducing molecule that is ubiquitously expressed by bacteria. Several studies have shown that inflammation-related biological activities were synergistically induced by interactions between LPS and MDP. MDP synergistically enhances production of proinflammatory cytokines that are induced by LPS exposure. Injection of MDP induces lethal shock in mice challenged with LPS. LPS also induces osteoclast formation and pathological bone resorption; MDP enhances LPS induction of both processes. Furthermore, MDP enhances the LPS-induced receptor activator of NF-κB ligand (RANKL expression and toll-like receptor 4 (TLR4 expression both in vivo and in vitro. Additionally, MDP enhances LPS-induced mitogen-activated protein kinase (MAPK signaling in stromal cells. Taken together, these findings suggest that MDP plays an important role in LPS-induced biological activities. This review discusses the role of MDP in LPS-mediated biological activities, primarily in relation to osteoclastogenesis.

Leucine-rich repeat kinase-1 regulates osteoclast function by modulating RAC1/Cdc42 Small GTPase phosphorylation and activation.

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Zeng, Canjun; Goodluck, Helen; Qin, Xuezhong; Liu, Bo; Mohan, Subburaman; Xing, Weirong

2016-10-01

Leucine-rich repeat kinase-1 (Lrrk1) consists of ankyrin repeats (ANK), leucine-rich repeats (LRR), a GTPase-like domain of Roc (ROC), a COR domain, a serine/threonine kinase domain (KD), and WD40 repeats (WD40). Previous studies have revealed that knockout (KO) of Lrrk1 in mice causes severe osteopetrosis, and a human mutation of Lrrk1 leads to osteosclerotic metaphysial dysplasia. The molecular mechanism by which Lrrk1 regulates osteoclast function is unknown. In this study, we generated a series of Lrrk1 mutants and evaluated their ability to rescue defective bone resorption in Lrrk1-deficient osteoclasts by use of pit formation assays. Overexpression of Lrrk1 or LRR-truncated Lrrk1, but not ANK-truncated Lrrk1, WD40-truncated Lrrk1, Lrrk1-KD, or K651A mutant Lrrk1, rescued bone resorption function of Lrrk1 KO osteoclasts. We next examined whether RAC1/Cdc42 small GTPases are direct substrates of Lrrk1 in osteoclasts. Western blot and pull-down assays revealed that Lrrk1 deficiency in osteoclasts resulted in reduced phosphorylation and activation of RAC1/Cdc42. In vitro kinase assays confirmed that recombinant Lrrk1 phosphorylated RAC1-GST protein, and immunoprecipitation showed that the interaction of Lrrk1 with RAC1 occurred within 10 min after RANKL treatment. Overexpression of constitutively active Q61L RAC1 partially rescued the resorptive function of Lrrk1-deficient osteoclasts. Furthermore, lack of Lrrk1 in osteoclasts led to reduced autophosphorylation of p21 protein-activated kinase-1 at Ser 144 , catalyzed by RAC1/Cdc42 binding and activation. Our data indicate that Lrrk1 regulates osteoclast function by directly modulating phosphorylation and activation of small GTPase RAC1/Cdc42 and that its function depends on ANK, ROC, WD40, and kinase domains. Copyright © 2016 the American Physiological Society.

Osteoblast and osteoclast behaviors in the turnover of attachment bones during medaka tooth replacement.

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Mantoku, Akiko; Chatani, Masahiro; Aono, Kazushi; Inohaya, Keiji; Kudo, Akira

2016-01-15

Tooth replacement in polyphyodont is a well-organized system for maintenance of homeostasis of teeth, containing the dynamic structural change in skeletal tissues such as the attachment bone, which is the supporting element of teeth. Histological analyses have revealed the character of tooth replacement, however, the cellular mechanism of how skeletal tissues are modified during tooth replacement is largely unknown. Here, we showed the important role of osteoblasts for controlling osteoclasts to modify the attachment bone during tooth replacement in medaka pharyngeal teeth, coupled with an osterix-DsRed/TRAP-GFP transgenic line to visualize osteoblasts and osteoclasts. In the turnover of the row of attachment bones, these bones were resorbed at the posterior side where most developed functional teeth were located, and generated at the anterior side where teeth were newly erupted, which caused continuous tooth replacement. In the cellular analysis, osteoclasts and osteoblasts were located at attachment bones separately, since mature osteoclasts were localized at the resorbing side and osteoblasts gathered at the generating side. To demonstrate the role of osteoclasts in tooth replacement, we established medaka made deficient in c-fms-a by TALEN. c-fms-a deficient medaka showed hyperplasia of attachment bones along with reduced bone resorption accompanied by a low number of TRAP-positive osteoclasts, indicating an important role of osteoclasts in the turnover of attachment bones. Furthermore, nitroreductase-mediated osteoblast-specific ablation induced disappearance of osteoclasts, indicating that osteoblasts were essential for maintenance of osteoclasts for the proper turnover. Taken together, our results suggested that the medaka attachment bone provides the model to understand the cellular mechanism for tooth replacement, and that osteoblasts act in the coordination of bone morphology by supporting osteoclasts. Copyright © 2015 Elsevier Inc. All rights reserved.

Sympathetic Neurotransmitters Modulate Osteoclastogenesis and Osteoclast Activity in the Context of Collagen-Induced Arthritis

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Muschter, Dominique; Schäfer, Nicole; Stangl, Hubert; Straub, Rainer H.; Grässel, Susanne

2015-01-01

Excessive synovial osteoclastogenesis is a hallmark of rheumatoid arthritis (RA). Concomitantly, local synovial changes comprise neuronal components of the peripheral sympathetic nervous system. Here, we wanted to analyze if collagen-induced arthritis (CIA) alters bone marrow-derived macrophage (BMM) osteoclastogenesis and osteoclast activity, and how sympathetic neurotransmitters participate in this process. Therefore, BMMs from Dark Agouti rats at different CIA stages were differentiated into osteoclasts in vitro and osteoclast number, cathepsin K activity, matrix resorption and apoptosis were analyzed in the presence of acetylcholine (ACh), noradrenaline (NA) vasoactive intestinal peptide (VIP) and assay-dependent, adenylyl cyclase activator NKH477. We observed modulation of neurotransmitter receptor mRNA expression in CIA osteoclasts without affecting protein level. CIA stage-dependently altered marker gene expression associated with osteoclast differentiation and activity without affecting osteoclast number or activity. Neurotransmitter stimulation modulated osteoclast differentiation, apoptosis and activity. VIP, NA and adenylyl cyclase activator NKH477 inhibited cathepsin K activity and osteoclastogenesis (NKH477, 10-6M NA) whereas ACh mostly acted pro-osteoclastogenic. We conclude that CIA alone does not affect metabolism of in vitro generated osteoclasts whereas stimulation with NA, VIP plus specific activation of adenylyl cyclase induced anti-resorptive effects probably mediated via cAMP signaling. Contrary, we suggest pro-osteoclastogenic and pro-resorptive properties of ACh mediated via muscarinic receptors. PMID:26431344

Probiotic consumption decreases the number of osteoclasts during orthodontic movement in mice.

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Pazzini, Camila Alessandra; Pereira, Luciano José; da Silva, Tarcília Aparecida; Montalvany-Antonucci, Carina Cristina; Macari, Soraia; Marques, Leandro Silva; de Paiva, Saul Martins

2017-07-01

The aim of the present study was to investigate the effect of probiotic (Bacillus Subtilis) supplementation on bone remodelling induced by mechanical loading. C57BL/6 mice were divided in two groups: (1) Probiotic and (2) Vehicle (water). The probiotic (1.5×10 8 CFU/mL) was administered orally for 14 days, starting two days before the induction of orthodontic tooth movement (OTM). OTM was determined by histomorphometric analysis by comparing the right to the left side of the maxilla. The number of osteoclasts was determined by counting TRAP-positive cells. Osteoblasts were counted on Masson's trichrome-stained slides. OTM was similar between groups (with and without probiotic supplementation) (p=0.46). The number of TRAP-positive cells increased (pprobiotic group, in comparison to the vehicle group. There was an increase in the number of osteoblasts (p˂0.05) in both the Vehicle and Probiotic groups on the side under OTM, independent of probiotic supplementation. Oral Supplementation with a probiotic influenced the number of osteoclasts adjacent to the tooth root during orthodontic movement in mice. Copyright © 2017 Elsevier Ltd. All rights reserved.

The interleukin-20 receptor axis in early rheumatoid arthritis: novel links between disease-associated autoantibodies and radiographic progression

DEFF Research Database (Denmark)

Kragstrup, Tue Wenzel; Greisen, Stinne Ravn; Nielsen, Morten Aagaard

2016-01-01

BACKGROUND: Rheumatoid arthritis (RA) is often characterized by the presence of rheumatoid factor, anti-citrullinated protein antibodies, and bone erosions. Current therapies can compromise immunity, leading to risk of infection. The interleukin-20 receptor (IL-20R) axis comprising IL-19, IL-20...... the production of the IL-20R cytokines by monocytes/macrophages. Increased baseline plasma concentrations of IL-20 and IL-24 were associated with Sharp-van der Heijde score progression after 24 months (Spearman's rho = 0.19 and 0.26, both P ... by osteoclast precursors and in multinucleated osteoclasts. IL-20 and IL-24 increased the secretion of monocyte chemoattractant protein 1 by these cells. CONCLUSIONS: This study suggests that IL-20 and IL-24 link RA-associated autoantibodies with radiographic progression via the IL-22R1. Modulation of this axis...

Growth differentiation factor-15 secreted by prostate cancer cells inhibits differentiation of osteoclasts

Czech Academy of Sciences Publication Activity Database

Vaňhara, P.; Lincová, Eva; Souček, Karel; Šmarda, J.

2009-01-01

Roč. 276, č. 1 (2009), s. 226 ISSN 1742-464X. [34th FEBS Congress. 04.07.2009-09.07.2009, Prague] R&D Projects: GA ČR(CZ) GA204/07/0834 Grant - others:GA ČR(CZ) GA301/09/1115 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : growth-differentiation factor-15 * osteoclasts * differentiation Subject RIV: BO - Biophysics

Requirement of the inducible nitric oxide synthase pathway for IL-1-induced osteoclastic bone resorption.

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van't Hof, R J; Armour, K J; Smith, L M; Armour, K E; Wei, X Q; Liew, F Y; Ralston, S H

2000-07-05

Nitric oxide has been suggested to be involved in the regulation of bone turnover, especially in pathological conditions characterized by release of bone-resorbing cytokines. The cytokine IL-1 is thought to act as a mediator of periarticular bone loss and tissue damage in inflammatory diseases such as rheumatoid arthritis. IL-1 is a potent stimulator of both osteoclastic bone resorption and expression of inducible nitric oxide synthase (iNOS) in bone cells and other cell types. In this study, we investigated the role that the iNOS pathway plays in mediating the bone-resorbing effects of IL-1 by studying mice with targeted disruption of the iNOS gene. Studies in vitro and in vivo showed that iNOS-deficient mice exhibited profound defects of IL-1-induced osteoclastic bone resorption but responded normally to calciotropic hormones such as 1,25 dihydroxyvitamin D3 and parathyroid hormone. Immunohistochemical studies and electrophoretic mobility shift assays performed on bone marrow cocultures from iNOS-deficient mice showed abnormalities in IL-1-induced nuclear translocation of the p65 component of NFkappaB and in NFkappaB-DNA binding, which were reversed by treatment with the NO donor S-nitroso-acetyl penicillamine. These results show that the iNOS pathway is essential for IL-1-induced bone resorption and suggest that the effects of NO may be mediated by modulating IL-1-induced nuclear activation of NFkappaB in osteoclast precursors.

Requirement of the inducible nitric oxide synthase pathway for IL-1-induced osteoclastic bone resorption

Science.gov (United States)

van't Hof, R. J.; Armour, K. J.; Smith, L. M.; Armour, K. E.; Wei, X. Q.; Liew, F. Y.; Ralston, S. H.

2000-01-01

Nitric oxide has been suggested to be involved in the regulation of bone turnover, especially in pathological conditions characterized by release of bone-resorbing cytokines. The cytokine IL-1 is thought to act as a mediator of periarticular bone loss and tissue damage in inflammatory diseases such as rheumatoid arthritis. IL-1 is a potent stimulator of both osteoclastic bone resorption and expression of inducible nitric oxide synthase (iNOS) in bone cells and other cell types. In this study, we investigated the role that the iNOS pathway plays in mediating the bone-resorbing effects of IL-1 by studying mice with targeted disruption of the iNOS gene. Studies in vitro and in vivo showed that iNOS-deficient mice exhibited profound defects of IL-1-induced osteoclastic bone resorption but responded normally to calciotropic hormones such as 1,25 dihydroxyvitamin D3 and parathyroid hormone. Immunohistochemical studies and electrophoretic mobility shift assays performed on bone marrow cocultures from iNOS-deficient mice showed abnormalities in IL-1-induced nuclear translocation of the p65 component of NFκB and in NFκB-DNA binding, which were reversed by treatment with the NO donor S-nitroso-acetyl penicillamine. These results show that the iNOS pathway is essential for IL-1-induced bone resorption and suggest that the effects of NO may be mediated by modulating IL-1-induced nuclear activation of NFκB in osteoclast precursors. PMID:10869429

Tributyltin and triphenyltin inhibit osteoclast differentiation through a retinoic acid receptor-dependent signaling pathway

International Nuclear Information System (INIS)

Yonezawa, Takayuki; Hasegawa, Shin-ichi; Ahn, Jae-Yong; Cha, Byung-Yoon; Teruya, Toshiaki; Hagiwara, Hiromi; Nagai, Kazuo; Woo, Je-Tae

2007-01-01

Organotin compounds, such as tributyltin (TBT) and triphenyltin (TPT), have been widely used in agriculture and industry. Although these compounds are known to have many toxic effects, including endocrine-disrupting effects, their effects on bone resorption are unknown. In this study, we investigated the effects of organotin compounds, such as monobutyltin (MBT), dibutyltin (DBT), TBT, and TPT, on osteoclast differentiation using mouse monocytic RAW264.7 cells. MBT and DBT had no effects, whereas TBT and TPT dose-dependently inhibited osteoclast differentiation at concentrations of 3-30 nM. Treatment with a retinoic acid receptor (RAR)-specific antagonist, Ro41-5253, restored the inhibition of osteoclastogenesis by TBT and TPT. TBT and TPT reduced receptor activator of nuclear factor-κB ligand (RANKL) induced nuclear factor of activated T cells (NFAT) c1 expression, and the reduction in NFATc1 expression was recovered by Ro41-5253. Our results suggest that TBT and TPT suppress osteoclastogenesis by inhibiting RANKL-induced NFATc1 expression via an RAR-dependent signaling pathway

Picrasidine I from Picrasma Quassioides Suppresses Osteoclastogenesis via Inhibition of RANKL Induced Signaling Pathways and Attenuation of ROS Production

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Lingbo Kong

2017-10-01

Full Text Available Background/Aims: Osteoporosis is a metabolic bone disorder that tortures about millions of people worldwide. Recent study demonstrated agents derived from picrasma quassioides is a promising drug for targets multiple signaling pathways. However its potential in treatment of bone loss has not been fully understood. Methods: The bone marrow macrophages (BMMs were cultured and induced with M-CSF and RANKL followed by picrasidine I (PI treatment. Then the effects of PI on osteoclast formation were evaluated by counting tartrate-resistant acid phosphatase (TRAP-positive multinucleated cells. Moreover, effects of PI on bone resorption activity of mature osteoclast were studied through bone resorption pit counting and actin ring structure analysis. Further, the involved potential signaling pathways cross-talking were investigated by performed Western blotting and quantitative real-time PCR examination. Results: Results demonstrated PI strongly inhibited RANKL induced osteoclast formation from its precursors. Mechanistically, the inhibitory effect of PI on osteoclast differentiation was due to the suppression of osteoclastogenic transcription factors, c-Fos and NFATc1. Moreover, PI markedly blocked the RANKL-induced osteoclastogenesis by attenuating MAPKs and NF-κB signaling pathways. In addition, PI decreased the ROS generation in osteoclast and osteoblast. Conclusion: Taken together our data demonstrate that PI has antiosteoclastogenic effect by inhibiting inflammation induced activation of MAPKs, NF-κB and ROS generation followed by suppressing the gene expression of c-Fos and NFATc1 in osteoclast precursors.

Dendrobium moniliforme Exerts Inhibitory Effects on Both Receptor Activator of Nuclear Factor Kappa-B Ligand-Mediated Osteoclast Differentiation in Vitro and Lipopolysaccharide-Induced Bone Erosion in Vivo.

Science.gov (United States)

Baek, Jong Min; Kim, Ju-Young; Ahn, Sung-Jun; Cheon, Yoon-Hee; Yang, Miyoung; Oh, Jaemin; Choi, Min Kyu

2016-03-01

Dendrobium moniliforme (DM) is a well-known plant-derived extract that is widely used in Oriental medicine. DM and its chemical constituents have been reported to have a variety of pharmacological effects, including anti-oxidative, anti-inflammatory, and anti-tumor activities; however, no reports discuss the beneficial effects of DM on bone diseases such as osteoporosis. Thus, we investigated the relationship between DM and osteoclasts, cells that function in bone resorption. We found that DM significantly reduced receptor activator of nuclear factor kappa-B ligand (RANKL)-induced tartrate-resistant acid phosphatase (TRAP)-positive osteoclast formation; DM directly induced the down-regulation of c-Fos and nuclear factor of activated T cells c1 (NFATc1) without affecting other RANKL-dependent transduction pathways. In the later stages of osteoclast maturation, DM negatively regulated the organization of filamentous actin (F-actin), resulting in impaired bone-resorbing activity by the mature osteoclasts. In addition, micro-computed tomography (μ-CT) analysis of the murine model revealed that DM had a beneficial effect on lipopolysaccharide (LPS)-mediated bone erosion. Histological analysis showed that DM attenuated the degradation of trabecular bone matrix and formation of TRAP-positive osteoclasts in bone tissues. These results suggest that DM is a potential candidate for the treatment of metabolic bone disorders such as osteoporosis.

Estrogen inhibits RANKL-stimulated osteoclastic differentiation of human monocytes through estrogen and RANKL-regulated interaction of estrogen receptor-α with BCAR1 and Traf6

International Nuclear Information System (INIS)

Robinson, Lisa J.; Yaroslavskiy, Beatrice B.; Griswold, Reed D.; Zadorozny, Eva V.; Guo, Lida; Tourkova, Irina L.; Blair, Harry C.

2009-01-01

The effects of estrogen on osteoclast survival and differentiation were studied using CD14-selected mononuclear osteoclast precursors from peripheral blood. Estradiol at ∼ 1 nM reduced RANKL-dependent osteoclast differentiation by 40-50%. Osteoclast differentiation was suppressed 14 days after addition of RANKL even when estradiol was withdrawn after 18 h. In CD14+ cells apoptosis was rare and was not augmented by RANKL or by 17-β-estradiol. Estrogen receptor-α (ERα) expression was strongly down-regulated by RANKL, whether or not estradiol was present. Mature human osteoclasts thus cannot respond to estrogen via ERα. However, ERα was present in CD14+ osteoclast progenitors, and a scaffolding protein, BCAR1, which binds ERα in the presence of estrogen, was abundant. Immunoprecipitation showed rapid (∼ 5 min) estrogen-dependent formation of ERα-BCAR1 complexes, which were increased by RANKL co-treatment. The RANKL-signaling intermediate Traf6, which regulates NF-κB activity, precipitated with this complex. Reduction of NF-κB nuclear localization occurred within 30 min of RANKL stimulation, and estradiol inhibited the phosphorylation of IκB in response to RANKL. Inhibition by estradiol was abolished by siRNA knockdown of BCAR1. We conclude that estrogen directly, but only partially, curtails human osteoclast formation. This effect requires BCAR1 and involves a non-genomic interaction with ERα.

Estrogen inhibits RANKL-stimulated osteoclastic differentiation of human monocytes through estrogen and RANKL-regulated interaction of estrogen receptor-{alpha} with BCAR1 and Traf6

Energy Technology Data Exchange (ETDEWEB)

Robinson, Lisa J., E-mail: robinsonlj@msx.upmc.edu [Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Yaroslavskiy, Beatrice B.; Griswold, Reed D.; Zadorozny, Eva V.; Guo, Lida; Tourkova, Irina L. [Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Blair, Harry C. [Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Veteran' s Affairs Medical Center, Pittsburgh, PA 15243 (United States)

2009-04-15

The effects of estrogen on osteoclast survival and differentiation were studied using CD14-selected mononuclear osteoclast precursors from peripheral blood. Estradiol at {approx} 1 nM reduced RANKL-dependent osteoclast differentiation by 40-50%. Osteoclast differentiation was suppressed 14 days after addition of RANKL even when estradiol was withdrawn after 18 h. In CD14+ cells apoptosis was rare and was not augmented by RANKL or by 17-{beta}-estradiol. Estrogen receptor-{alpha} (ER{alpha}) expression was strongly down-regulated by RANKL, whether or not estradiol was present. Mature human osteoclasts thus cannot respond to estrogen via ER{alpha}. However, ER{alpha} was present in CD14+ osteoclast progenitors, and a scaffolding protein, BCAR1, which binds ER{alpha} in the presence of estrogen, was abundant. Immunoprecipitation showed rapid ({approx} 5 min) estrogen-dependent formation of ER{alpha}-BCAR1 complexes, which were increased by RANKL co-treatment. The RANKL-signaling intermediate Traf6, which regulates NF-{kappa}B activity, precipitated with this complex. Reduction of NF-{kappa}B nuclear localization occurred within 30 min of RANKL stimulation, and estradiol inhibited the phosphorylation of I{kappa}B in response to RANKL. Inhibition by estradiol was abolished by siRNA knockdown of BCAR1. We conclude that estrogen directly, but only partially, curtails human osteoclast formation. This effect requires BCAR1 and involves a non-genomic interaction with ER{alpha}.

Optimal Zn-Modified Ca–Si-Based Ceramic Nanocoating with Zn Ion Release for Osteoblast Promotion and Osteoclast Inhibition in Bone Tissue Engineering

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Jiangming Yu

2017-01-01

Full Text Available We investigated the slow release of Zn ion (Zn2+ from nanocoatings and compared the in vitro response of osteoblasts (MC3T3-E1 and proosteoclasts (RAW 264.7 cultured on Ca2ZnSi2O7 nanocoated with different Zn/Ca molar ratios on a Ti-6Al-4V (i.e., Ti substrate to optimize cell behaviors and molecule levels. Significant morphology differences were observed among samples. By comparing with pure Ti and CaSiO3 nanocoating, the morphology of Ca2ZnSi2O7 ceramic nanocoatings was rough and contained small nanoparticles or aggregations. Slow Zn2+ release from nanocoatings was observed and Zn2+ concentration was regulated by varying the Zn/Ca ratios. The cell-response results showed Ca2ZnSi2O7 nanocoating at different Zn/Ca molar ratios for osteoblasts and osteoclasts. Compared to other nanocoatings and Ti, sample Zn/Ca (0.3 showed the highest cell viability and upregulated expression of the osteogenic differentiation genes ALP, COL-1, and OCN. Additionally, sample Zn/Ca (0.3 showed the greatest inhibition of RAW 264.7 cell growth and decreased the mRNA levels of osteoclast-related genes OAR, TRAP, and HYA1. Therefore, the optimal Zn-Ca ratio of 0.3 in Ca2ZnSi2O7 ceramic nanocoating on Ti had a dual osteoblast-promoting and osteoclast-inhibiting effect to dynamically balance osteoblasts/osteoclasts. These optimal Zn-Ca ratios are valuable for Ca2ZnSi2O7 ceramic nanocoating on Ti-coated implants for potential applications in bone tissue regeneration.

Similar healthy osteoclast and osteoblast activity on nanocrystalline hydroxyapatite and nanoparticles of tri-calcium phosphate compared to natural bone

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MacMillan AK

2014-12-01

Full Text Available Adam K MacMillan,1 Francis V Lamberti,1 Julia N Moulton,2 Benjamin M Geilich,2 Thomas J Webster2,3 1RTI Surgical, Alachua, FL, USA; 2Department of Chemical Engineering, Northeastern University, Boston, MA, USA; 3Center of Excellence for Advanced Materials Research, King Abdulaziz University, Jeddah, Saudi Arabia Abstract: While there have been numerous studies to determine osteoblast (bone forming cell functions on nanocrystalline compared to micron crystalline ceramics, there have been few studies which have examined osteoclast activity (including tartrate-resistant acid phosphatase, formation of resorption pits, size of resorption pits, and receptor activator of nuclear factor κB [RANK]. This is despite the fact that osteoclasts are an important part of maintaining healthy bone since they resorb bone during the bone remodeling process. Moreover, while it is now well documented that bone formation is enhanced on nanoceramics compared to micron ceramics, some have pondered whether osteoblast functions (such as osteoprotegerin and RANK ligand [RANKL] are normal (ie, non-diseased on such materials compared to natural bone. For these reasons, the objective of the present in vitro study was to determine various functions of osteoclasts and osteoblasts on nanocrystalline and micron crystalline hydroxyapatite as well as tri-calcium phosphate materials and compare such results to cortical and cancellous bone. Results showed for the first time similar osteoclast activity (including tartrate-resistant acid phosphatase, formation of resorption pits, size of resorption pits, and RANK and osteoblast activity (osteoprotegerin and RANKL on nanocrystalline hydroxyapatite compared to natural bone, whereas osteoclast and osteoblast functions on micron crystalline versions of these ceramics were much different than natural bone. In this manner, this study provides additional evidence that nanocrystalline calcium phosphates can serve as suitable synthetic

Osteoclast TGF-β Receptor Signaling Induces Wnt1 Secretion and Couples Bone Resorption to Bone Formation

Science.gov (United States)

Weivoda, Megan M; Ruan, Ming; Pederson, Larry; Hachfeld, Christine; Davey, Rachel A; Zajac, Jeffrey D; Westendorf, Jennifer J; Khosla, Sundeep; Oursler, Merry Jo

2016-01-01

Osteoblast-mediated bone formation is coupled to osteoclast-mediated bone resorption. These processes become uncoupled with age, leading to increased risk for debilitating fractures. Therefore, understanding how osteoblasts are recruited to sites of resorption is vital to treating age-related bone loss. Osteoclasts release and activate TGF-β from the bone matrix. Here we show that osteoclastspecific inhibition of TGF-β receptor signaling in mice results in osteopenia due to reduced osteoblast numbers with no significant impact on osteoclast numbers or activity. TGF-β induced osteoclast expression of Wnt1, a protein crucial to normal bone formation, and this response was blocked by impaired TGF-β receptor signaling. Osteoclasts in aged murine bones had lower TGF-β signaling and Wnt1 expression in vivo. Ex vivo stimulation of osteoclasts derived from young or old mouse bone marrow macrophages showed no difference in TGF-β–induced Wnt1 expression. However, young osteoclasts expressed reduced Wnt1 when cultured on aged mouse bone chips compared to young mouse bone chips, consistent with decreased skeletal TGF-β availability with age. Therefore, osteoclast responses to TGF-β are essential for coupling bone resorption to bone formation, and modulating this pathway may provide opportunities to treat age-related bone loss. PMID:26108893

Dynamin and PTP-PEST cooperatively regulate Pyk2 dephosphorylation in osteoclasts

Science.gov (United States)

Eleniste, Pierre P.; Du, Liping; Shivanna, Mahesh; Bruzzaniti, Angela

2012-01-01

Bone loss is caused by the dysregulated activity of osteoclasts which degrade the extracellular bone matrix. The tyrosine kinase Pyk2 is highly expressed in osteoclasts, and mice lacking Pyk2 exhibit an increase in bone mass, in part due to impairment of osteoclast function. Pyk2 is activated by phosphorylation at Y402 following integrin activation, but the mechanisms leading to Pyk2 dephosphorylation are poorly understood. In the current study, we examined the mechanism of action of the dynamin GTPase on Pyk2 dephosphorylation. Our studies reveal a novel mechanism for the interaction of Pyk2 with dynamin, which involves the binding of Pyk2’s FERM domain with dynamin’s plextrin homology domain. In addition, we demonstrate that the dephosphorylation of Pyk2 requires dynamin’s GTPase activity and is mediated by the tyrosine phosphatase PTP-PEST. The dephosphorylation of Pyk2 by dynamin and PTP-PEST may be critical for terminating outside-in integrin signaling, and for stabilizing cytoskeletal reorganization during osteoclast bone resorption. PMID:22342188

Different effects of inorganic and dimethylated arsenic compounds on cell morphology, cytoskeletal organization, and DNA synthesis in cultured Chinese hamster V79 cells

Energy Technology Data Exchange (ETDEWEB)

Ochi, Takafumi; Nakajima, Fumie [Department of Environmental Toxicology, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa (Japan); Fukumori, Nobutaka [Department of Toxicology, Tokyo Metropolitan Research Laboratory of Public Health, Hyakuninchou, Shinjyuku (Japan)

1998-09-01

Changes in cytoskeletal organization of cultured V79 cells exposed to arsenite and dimethylarsinic acid (DMAA), a methylated derivative of inorganic arsenics, and related changes, such as mitotic arrest and induction of multinucleated cells, were investigated in comparison with their effects on DNA synthesis. DMAA caused mitotic arrest and induction of multinucleated cells with a delay of 12 h relative to the mitotic arrest. By contrast, arsenite at equitoxic concentrations to DMAA was less effective than DMAA in causing mitotic arrest and in inducing multinucleated cells. Post-mitotic incubation of cells arrested in metaphase by 6 h incubation with 10 mM DMAA showed that the incidence of multinucleated cells increased conversely with a rapid decrease in metaphase cells. This suggests that metaphase-arrested cells can escape from metaphase, resulting in the appearance of multinucleated cells. The mitotic arrest caused by DMAA was accompanied by disruption of the microtubule network. By contrast, both arsenite and DMAA did not cause disorganization of actin stress fibers even when incubated at concentrations that caused a marked retardation of cell growth. Cells exposed to arsenite for 6 h showed marked inhibition of DNA synthesis, whereas inhibition by DMAA was not observed. When incubation was prolonged by 18 h, the arsenite-induced inhibition of DNA synthesis was mitigated. By contrast, inhibition of DNA synthesis by DMAA occurred in parallel with an increase in the population of mitotic cells. These results suggest that DMAA caused growth retardation and morphological changes via disruption of the microtubule network, and that arsenite-induced retardation of cell growth and inhibition of DNA synthesis were not attributable to the cytoskeletal changes. (orig.) (orig.) With 7 figs., 31 refs.

Disulfiram attenuates osteoclast differentiation in vitro: a potential antiresorptive agent.

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Hua Ying

Full Text Available Disulfiram (DSF, a cysteine modifying compound, has long been clinically employed for the treatment of alcohol addiction. Mechanistically, DSF acts as a modulator of MAPK and NF-κB pathways signaling pathways. While these pathways are crucial for osteoclast (OC differentiation, the potential influence of DSF on OC formation and function has not been directly assessed. Here, we explore the pharmacological effects of DSF on OC differentiation, activity and the modulation of osteoclastogenic signaling cascades. We first analyzed cytotoxicity of DSF on bone marrow monocytes isolated from C57BL/6J mice. Upon the establishment of optimal dosage, we conducted osteoclastogenesis and bone resorption assays in the presence or absence of DSF treatment. Luciferase assays in RAW264.7 cells were used to examine the effects of DSF on major transcription factors activation. Western blot, reverse transcription polymerase chain reaction, intracellular acidification and proton influx assays were employed to further dissect the underlying mechanism. DSF treatment dose-dependently inhibited both mouse and human osteoclastogenesis, especially at early stages of differentiation. This inhibition correlated with a decrease in the expression of key osteoclastic marker genes including CtsK, TRAP, DC-STAMP and Atp6v0d2 as well as a reduction in bone resorption in vitro. Suppression of OC differentiation was found to be due, at least in part, to the blockade of several key receptor activators of nuclear factor kappa-B ligand (RANKL-signaling pathways including ERK, NF-κB and NFATc1. On the other hand, DSF failed to suppress intracellular acidification and proton influx in mouse and human osteoclasts using acridine orange quenching and microsome-based proton transport assays. Our findings indicate that DSF attenuates OC differentiation via the collective suppression of several key RANKL-mediated signaling cascades, thus making it an attractive agent for the treatment of OC

Multifunctional role of osteopontin in directing intrafibrillar mineralization of collagen and activation of osteoclasts

Science.gov (United States)

Rodriguez, Douglas E.; Thula-Mata, Taili; Toro, Edgardo J.; Yeh, Ya-Wen; Holt, Carl; Holliday, L. Shannon; Gower, Laurie B.

2013-01-01

Mineralized collagen composites are of interest because they have the potential to provide a bone-like scaffold that stimulates the natural processes of resorption and remodeling. Working toward this goal, our group has previously shown that the nanostructure of bone can be reproduced using a polymer-induced liquid-precursor (PILP) process, which enables intrafibrillar mineralization of collagen with hydroxyapatite (HA) to be achieved. This prior work used polyaspartic acid (pASP), a simple mimic for acidic non-collagenous proteins (NCPs), to generate nanodroplets/nanoparticles of an amorphous mineral precursor which can infiltrate the interstices of type-I collagen fibrils. In this study we show that osteopontin (OPN) can similarly serve as a process-directing agent for the intrafibrillar mineralization of collagen, even though OPN is generally considered a mineralization inhibitor. We also found that inclusion of OPN in the mineralization process promotes the interaction of mouse marrow-derived osteoclasts with PILP-remineralized bone that was previously demineralized, as measured by actin ring formation. While osteoclast activation occurred when pASP was used as the process-directing agent, using OPN resulted in a dramatic effect on osteoclast activation, presumably because of the inherent arginine-glycine-aspartate acid (RGD) ligands of OPN. By capitalizing on the multifunctionality of OPN, these studies may lead the way to producing biomimetic bone substitutes with the capability of tailorable bioresorption rates. PMID:24140612

Schisantherin A suppresses osteoclast formation and wear particle-induced osteolysis via modulating RANKL signaling pathways

Energy Technology Data Exchange (ETDEWEB)

He, Yi; Zhang, Qing; Shen, Yi; Chen, Xia; Zhou, Feng; Peng, Dan, E-mail: xyeypd@163.com

2014-07-04

Highlights: • Schisantherin A suppresses osteoclasts formation and function in vitro. • Schisantherin A impairs RANKL signaling pathway. • Schisantherin A suppresses osteolysis in vivo. • Schisantherin A may be used for treating osteoclast related diseases. - Abstract: Receptor activator of NF-κB ligand (RANKL) plays critical role in osteoclastogenesis. Targeting RANKL signaling pathways has been a promising strategy for treating osteoclast related bone diseases such as osteoporosis and aseptic prosthetic loosening. Schisantherin A (SA), a dibenzocyclooctadiene lignan isolated from the fruit of Schisandra sphenanthera, has been used as an antitussive, tonic, and sedative agent, but its effect on osteoclasts has been hitherto unknown. In the present study, SA was found to inhibit RANKL-induced osteoclast formation and bone resorption. The osteoclastic specific marker genes induced by RANKL including c-Src, SA inhibited OSCAR, cathepsin K and TRAP in a dose dependent manner. Further signal transduction studies revealed that SA down-regulate RANKL-induced nuclear factor-kappaB (NF-κB) signaling activation by suppressing the phosphorylation and degradation of IκBα, and subsequently preventing the NF-κB transcriptional activity. Moreover, SA also decreased the RANKL-induced MAPKs signaling pathway, including JNK and ERK1/2 posphorylation while had no obvious effects on p38 activation. Finally, SA suppressed the NF-κB and MAPKs subsequent gene expression of NFATc1 and c-Fos. In vivo studies, SA inhibited osteoclast function and exhibited bone protection effect in wear-particle-induced bone erosion model. Taken together, SA could attenuate osteoclast formation and wear particle-induced osteolysis by mediating RANKL signaling pathways. These data indicated that SA is a promising therapeutic natural compound for the treatment of osteoclast-related prosthesis loosening.

Granulocyte macrophage colony-stimulating factor enhances the modulatory effect of cytokines on monocyte-derived multinucleated giant cell formation and fungicidal activity against Paracoccidioides brasiliensis

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Magda Paula Pereira do Nascimento

2011-09-01

Full Text Available Multinucleated giant cells (MGC are cells present in characteristic granulomatous inflammation induced by intracellular infectious agents or foreign materials. The present study evaluated the modulatory effect of granulocyte macrophage colony-stimulating factor (GM-CSF in association with other cytokines such as interferon-gamma (IFN-γ, tumour necrosis factor-alpha, interleukin (IL-10 or transforming growth factor beta (TGF-β1 on the formation of MGC from human peripheral blood monocytes stimulated with Paracoccidioides brasiliensis antigen (PbAg. The generation of MGC was determined by fusion index (FI and the fungicidal activity of these cells was evaluated after 4 h of MGC co-cultured with viable yeast cells of P. brasiliensis strain 18 (Pb18. The results showed that monocytes incubated with PbAg and GM-CSF plus IFN-γ had a significantly higher FI than in all the other cultures, while the addition of IL-10 or TGF-β1 had a suppressive effect on MGC generation. Monocytes incubated with both pro and anti-inflammatory cytokines had a higher induction of foreign body-type MGC rather than Langhans-type MGC. MGC stimulated with PbAg and GM-CSF in association with the other cytokines had increased fungicidal activity and the presence of GM-CSF also partially inhibited the suppressive effects of IL-10 and TGF-β1. Together, these results suggest that GM-CSF is a positive modulator of PbAg-stimulated MGC generation and on the fungicidal activity against Pb18.

Effects of extracellular magnesium extract on the proliferation and differentiation of human osteoblasts and osteoclasts in coculture.

Science.gov (United States)

Wu, Lili; Feyerabend, Frank; Schilling, Arndt F; Willumeit-Römer, Regine; Luthringer, Bérengère J C

2015-11-01

Coculture of osteoblasts and osteoclasts is a subject of interest in the understanding of how magnesium (Mg)-based implants influence the bone metabolism and remodeling upon degradation. Human telomerase reverse transcriptase (hTERT) transduced mesenchymal stem cells (SCP-1) were first differentiated into osteoblasts with osteogenic supplements and then further cocultured with peripheral blood mononucleated cells (PBMC) without the addition of osteoclastogenesis promoting factors. Concomitantly, the cultures were exposed to variable Mg extract dilutions (0, 30×, 10×, 5×, 3×, 2× and 1×). Phenotype characterization documented that while 2× dilution of Mg extract was extremely toxic to osteoclast monoculture, monocytes in coculture with osteoblasts exhibited a greater tolerance to higher Mg extract concentration. The dense growth of osteoblasts in cultures with 1× dilution of Mg extract suggested that high concentration of Mg extract promoted osteoblast proliferation/differentiation behavior. The results of intracellular alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) activities as well as protein and gene expressions of receptor activator of nuclear factor kappa-B ligand (RANKL), macrophage colony-stimulating factor (M-CSF), and osteoclast-associated receptor (OSCAR) revealed significantly enhanced formation of osteoblasts whereas decreased osteoclastogenesis in the cultures with high concentrations of Mg extract (2× and 1× dilutions). In conclusion, while an increased osteoinductivity has been demonstrated, the impact of potentially decreased osteoclastogenesis around the Mg-based implants should be also taken into account. Cocultures containing both bone-forming osteoblasts and bone-resorbing osteoclasts should be preferentially performed for in vitro cytocompatibility assessment of Mg-based implants as they more closely mimic the in vivo environment. An attractive human osteoblasts and osteoclasts cocultivation regime was

Scoparone attenuates RANKL-induced osteoclastic differentiation through controlling reactive oxygen species production and scavenging

Energy Technology Data Exchange (ETDEWEB)

Lee, Sang-Hyun; Jang, Hae-Dong, E-mail: haedong@hnu.kr

2015-02-15

Scoparone, one of the bioactive components of Artemisia capillaris Thunb, has various biological properties including immunosuppressive, hepatoprotective, anti-allergic, anti-inflammatory, and antioxidant effects. This study aims at evaluating the anti-osteoporotic effect of scoparone and its underlying mechanism in vitro. Scoparone demonstrated potent cellular antioxidant capacity. It was also found that scoparone inhibited the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and suppressed cathepsin K and tartrate-resistant acid phosphatase (TRAP) expression via c-jun N-terminal kinase (JNK)/extracellular signal-regulated kinase (ERK)/p38-mediated c-Fos–nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) signaling pathway. During osteoclast differentiation, the production of general reactive oxygen species (ROS) and superoxide anions was dose-dependently attenuated by scoparone. In addition, scoparone diminished NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 1 (Nox1) expression and activation via the tumor necrosis factor receptor-associated factor 6 (TRAF6)–cSrc–phosphatidylinositol 3-kinase (PI3k) signaling pathway and prevented the disruption of mitochondrial electron transport chain system. Furthermore, scoparone augmented the expression of superoxide dismutase 1 (SOD1) and catalase (CAT). The overall results indicate that the inhibitory effect of scoparone on RANKL-induced osteoclast differentiation is attributed to the suppressive effect on ROS and superoxide anion production by inhibiting Nox1 expression and activation and protecting the mitochondrial electron transport chain system and the scavenging effect of ROS resulting from elevated SOD1 and CAT expression. - Highlights: • Scoparone dose-dependently inhibited RANKL-induced osteoclast differentiation. • Scoparone diminished general ROS and superoxide anions in a dose-dependent manner. • Scoparone inhibited Nox1 expression and

Interleukin-2 stimulates osteoclastic activity: Increased acid production and radioactive calcium release

International Nuclear Information System (INIS)

Ries, W.L.; Seeds, M.C.; Key, L.L.

1989-01-01

Recombinant human interleukin-2 (IL-2) was studied to determine effects on acid production by individual osteoclasts in situ on mouse calvarial bones. This analysis was performed using a microspectrofluorimetric technique to quantify acid production in individual cells. Radioactive calcium release was determined using calvarial bones in a standard tissue culture system. This allowed us to correlate changes in acid production with a measure of bone resorption. IL-2 stimulated acid production and bone resorbing activity. Both effects were inhibited by calcitonin. No stimulation of bone resorption occurred when IL-2-containing test media was incubated with a specific anti-IL-2 antibody and ultrafiltered. Our data demonstrated a correlation between acid production and bone resorbing activity in mouse calvaria exposed to parathyroid hormone (PTH). The data obtained from cultured mouse calvaria exposed to IL-2 demonstrated similar stimulatory effects to those seen during PTH exposure. These data suggest that calvaria exposed to IL-2 in vitro have increased osteoclastic acid production corresponding with increased bone resorption. (author)

Neutrophil-derived MRP-14 is up-regulated in infectious osteomyelitis and stimulates osteoclast generation.

Science.gov (United States)

Dapunt, Ulrike; Giese, Thomas; Maurer, Susanne; Stegmaier, Sabine; Prior, Birgit; Hänsch, G Maria; Gaida, Matthias M

2015-10-01

Bone infections of patients with joint replacement by endoprosthesis (so called "periprosthetic joint infection") pose a severe problem in the field of orthopedic surgery. The diagnosis is often difficult, and treatment is, in most cases, complicated and prolonged. Patients often require an implant exchange surgery, as the persistent infection and the accompanying inflammation lead to tissue damage with bone degradation and consequently, to a loosening of the implant. To gain insight into the local inflammatory process, expression of the proinflammatory cytokine MRP-14, a major content of neutrophils, and its link to subsequent bone degradation was evaluated. We found MRP-14 prominently expressed in the affected tissue of patients with implant-associated infection, in close association with the chemokine CXCL8 and a dense infiltrate of neutrophils and macrophages. In addition, the number of MRP-14-positive cells correlated with the presence of bone-resorbing osteoclasts. MRP-14 plasma concentrations were significantly higher in patients with implant-associated infection compared with patients with sterile inflammation or healthy individuals, advocating MRP-14 as a novel diagnostic marker. A further biologic activity of MRP-14 was detected: rMRP-14 directly induced the differentiation of monocytes to osteoclasts, thus linking the inflammatory response in implant infections with osteoclast generation, bone degradation, and implant loosening. © Society for Leukocyte Biology.

Acidification of the osteoclastic resorption compartment provides insight into the coupling of bone formation to bone resorption

DEFF Research Database (Denmark)

Karsdal, M.A.; Henriksen, K.; Sorensen, M.G.

2005-01-01

Patients with defective osteoclastic acidification have increased numbers of osteoclasts, with decreased resorption, but bone formation that remains unchanged. We demonstrate that osteoclast survival is increased when acidification is impaired, and that impairment of acidification results in inhi...

Modified salicylanilide and 3-phenyl-2H-benzo[e][1,3]oxazine-2,4(3H)-dione derivatives as novel inhibitors of osteoclast differentiation and bone resorption.

Science.gov (United States)

Chen, Chun-Liang; Liu, Fei-Lan; Lee, Chia-Chung; Chen, Tsung-Chih; Ahmed Ali, Ahmed Atef; Sytwu, Huey-Kang; Chang, Deh-Ming; Huang, Hsu-Shan

2014-10-09

Inhibition of osteoclast formation is a potential strategy to prevent inflammatory bone resorption and to treat bone diseases. In the present work, the purpose was to discover modified salicylanilides and 3-phenyl-2H-benzo[e][1,3]oxazine-2,4(3H)-dione derivatives as potential antiosteoclastogenic agents. Their inhibitory effects on RANKL-induced osteoclastogenesis from RAW264.7 cells were evaluated by TRAP stain assay. The most potent compounds, 1d and 5d, suppressed RANKL-induced osteoclast formation and TRAP activity dose-dependently. The cytotoxicity assay on RAW264.7 cells suggested that the inhibition of osteoclastic bone resorption by these compounds did not result from their cytotoxicity. Moreover, both compounds downregulated RANKL-induced NF-κB and NFATc1 in the nucleus, suppressed the expression of osteoclastogenesis-related marker genes during osteoclastogenesis, and prevented osteoclastic bone resorption but did not impair osteoblast differentiation in MC3T3-E1. Therefore, these modified salicylanilides and 3-phenyl-2H-benzo[e][1,3]oxazine-2,4(3H)-diones could be potential lead compounds for the development of a new class of antiresorptive agents.

Grafting effectiveness of Anadara granosa shell combined with sardinella longiseps gel on the number of osteoblast-osteoclast cells

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Eddy Hermanto

2017-09-01

Full Text Available Background: Bone grafts derived from Anadara granosa shells contain calcium carbonate that possesses bone-healing properties. The combination of Sardinella Longiceps fish oil, containing EPA and DHA, and Anadara granosa shells was assumed to regulate the number of osteoblasts-osteoclasts during the bone-healing process. Purpose: This study aimed to determine the effectiveness of Anadara granosa shell grafts, combined with Sardinella Longiceps fish oil, in the bone-healing process by observing the ratio of osteoblasts-osteoclasts in Rattus novergicus rats. Methods: The Wistar rat subjects (n = 25 were divided into five groups, namely: one untreated group (control, one group treated with bone grafts derived from Anadara granosa shells (P1, and the other three groups treated with a combination of Anadara granosa shells and Sardinella longiceps fish oil at concentrations of 10%, 20%, and 30% (P2, P3, and P4. Then, a wound equivalent in size to half the diameter of a round bur (±1.5mm was intentionally inflicted on the right femur of all the subjects. The rats were subsequently sacrificed on day 14, their femur in the transversal side being cut before HE staining was completed. Thereafter, the ratio of osteoblasts to osteoclasts was measured by means of a light microscopy. The data was subsequently analyzed using one-way ANOVA. Results: The average number of osteoblasts in all research groups increased, viz: 9.420±0.8044 for control group (K, 12.080±0.79811 for group P1, 20.020±0.7190 for group P2, 25.940±0.7197 for group P3, and 36.280±0.9985 for group P4. Similarly, the number of osteoclasts in all groups subject to analysis also increased, namely: 1.73±0.098 for group K, 2.19±0.305 for group P1, 1.60±0.088 for group P2, 1.60±0.724 for group P3, and 1.80±1.302 for group P4. Moreover, the results of the One-way Anova test confirmed that there were no significant differences in osteoclasts between all research groups (p>0.05. The results

Evidence for an amoeba-like infectious stage of ichthyophonus sp. and description of a circulating blood stage: a probable mechanism for dispersal within the fish host

Science.gov (United States)

Kocan, Richard; LaPatra, Scott; Hershberger, Paul

2013-01-01

Small amoeboid cells, believed to be the infectious stage of Ichthyophonus sp., were observed in the bolus (stomach contents) and tunica propria (stomach wall) of Pacific staghorn sculpins and rainbow trout shortly after they ingested Ichthyophonus sp.–infected tissues. By 24–48 hr post-exposure (PE) the parasite morphed from the classically reported multinucleate thick walled schizonts to 2 distinct cell types, i.e., a larger multinucleate amoeboid cell surrounded by a narrow translucent zone and a smaller spherical cell surrounded by a “halo” and resembling a small schizont. Both cell types also appeared in the tunica propria, indicating that they had recently penetrated the columnar epithelium of the stomach. No Ichthyophonus sp. pseudo-hyphae (“germination tubes”) were observed in the bolus or penetrating the stomach wall. Simultaneously, Ichthyophonus sp. was isolated in vitro from aortic blood, which was consistently positive from 6 to 144 hr PE, then only intermittently for the next 4 wk. Small PAS-positive cells observed in blood cultures grew into colonies consisting of non-septate tubules (pseudo-hyphae) terminating in multinucleated knob-like apices similar to those seen in organ explant cultures. Organ explants were culture positive every day; however, typical Ichthyophonus sp. schizonts were not observed histologically until 20–25 days PE. From 20 to 60 days PE, schizont diameter increased from ≤25 μm to ≥82 μm. Based on the data presented herein, we are confident that we have resolved the life cycle of Ichthyophonus sp. within the piscivorous host.

Evidence for an amoeba-like infectious stage of Ichthyophonus sp. and description of a circulating blood stage: a probable mechanism for dispersal within the fish host.

Science.gov (United States)

Kocan, Richard; LaPatra, Scott; Hershberger, Paul

2013-04-01

Small amoeboid cells, believed to be the infectious stage of Ichthyophonus sp., were observed in the bolus (stomach contents) and tunica propria (stomach wall) of Pacific staghorn sculpins and rainbow trout shortly after they ingested Ichthyophonus sp.-infected tissues. By 24-48 hr post-exposure (PE) the parasite morphed from the classically reported multinucleate thick walled schizonts to 2 distinct cell types, i.e., a larger multinucleate amoeboid cell surrounded by a narrow translucent zone and a smaller spherical cell surrounded by a "halo" and resembling a small schizont. Both cell types also appeared in the tunica propria, indicating that they had recently penetrated the columnar epithelium of the stomach. No Ichthyophonus sp. pseudo-hyphae ("germination tubes") were observed in the bolus or penetrating the stomach wall. Simultaneously, Ichthyophonus sp. was isolated in vitro from aortic blood, which was consistently positive from 6 to 144 hr PE, then only intermittently for the next 4 wk. Small PAS-positive cells observed in blood cultures grew into colonies consisting of non-septate tubules (pseudo-hyphae) terminating in multinucleated knob-like apices similar to those seen in organ explant cultures. Organ explants were culture positive every day; however, typical Ichthyophonus sp. schizonts were not observed histologically until 20-25 days PE. From 20 to 60 days PE, schizont diameter increased from ≤ 25 μm to ≥ 82 μm. Based on the data presented herein, we are confident that we have resolved the life cycle of Ichthyophonus sp. within the piscivorous host.

Undifferentiated pleomorphic sarcoma with osteoclast-like giant cells of the female breast

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Balbi Giancarlo

2013-01-01

Full Text Available Abstract The authors describe a case of undifferentiated pleomorphic sarcoma of the breast occurring in a 50-year-old woman who presented with a palpable mass in her right breast. She first noticed the mass one month previously. Core needle biopsy showed connective tissue including epithelioid and spindle cells. The patient underwent total mastectomy without axillary lymph node dissection. Based on examination of the excised tumor, the initial pathologic diagnosis was atypical spindle-shaped and ovoid cells with uncertain malignant potential. Histological findings with immunomarkers led to the final diagnosis of undifferentiated pleomorphic sarcoma. This case highlights a rare and interesting variant of primary breast sarcoma and the important role of immunohistochemistry in defining histological type and differential diagnosis. Hence, undifferentiated pleomorphic sarcoma has been a diagnosis of exclusion performed through sampling and critical use of ancillary diagnostic techniques.

Activation of Src kinase by protein-tyrosine phosphatase-PEST in osteoclasts: comparative analysis of the effects of bisphosphonate and protein-tyrosine phosphatase inhibitor on Src activation in vitro.

Science.gov (United States)

Chellaiah, Meenakshi A; Schaller, Michael D

2009-08-01

PTP-PEST is involved in the regulation of sealing ring formation in osteoclasts. In this article, we have shown a regulatory role for PTP-PEST on dephosphorylation of c-Src at Y527 and phosphorylation at Y418 in the catalytic site. Activation of Src in osteoclasts by over-expression of PTP-PEST resulted in the phosphorylation of cortactin at Y421 and WASP at Y294. Also enhanced as a result, is the interaction of Src, cortactin, and Arp2 with WASP. Moreover, the number of osteoclasts displaying sealing ring and bone resorbing activity was increased in response to PTP-PEST over-expression as compared with control osteoclasts. Cells expressing constitutively active-Src (527YDeltaF) simulate the effects mediated by PTP-PEST. Treatment of osteoclasts with a bisphosphonate alendronate or a potent PTP inhibitor PAO decreased the activity and phosphorylation of Src at Y418 due to reduced dephosphorylation state at Y527. Therefore, Src-mediated phosphorylation of cortactin and WASP as well as the formation of WASP.cortactin.Arp2 complex and sealing ring were reduced in these osteoclasts. Similar effects were observed in osteoclasts treated with an Src inhibitor PP2. We have shown that bisphosphonates could modulate the function of osteoclasts by inhibiting downstream signaling mediated by PTP-PEST/Src, in addition to its effect on the inhibition of the post-translational modification of small GTP-binding proteins such as Rab, Rho, and Rac as shown by others. The promising effects of the inhibitors PP2 and PAO on osteoclast function suggest a therapeutic approach for patients with bone metastases and osteoporosis as an alternative to bisphosphonates.

Imprint cytology of clear cell sarcoma-like tumor of the gastrointestinal tract in the small intestine: A case report.

Science.gov (United States)

Kato, Takashi; Ichihara, Shin; Gotoda, Hiroko; Muraoka, Shunji; Kubo, Terufumi; Sugita, Shintaro; Hasegawa, Tadashi

2017-12-01

Clear cell sarcoma-like tumor of the gastrointestinal tract (CCSLGT) is an extremely rare malignant neoplasm in the digestive tract. Its cytomorphologic features have never previously been reported. Here, we describe a case of CCSLGT, including its cytologic examination findings. A 47-year-old woman presented with a mass in the small intestine, which was resected and sent for imprint cytology. Imprint smears revealed tumor cells with light eosinophilic or clear cytoplasm in a necrotic background. Many of the tumor cells were arranged in a perivascular growth with a pseudopapillary formation, and there were some non-neoplastic osteoclast-like giant cells. Histological examination revealed solid nests and a pseudopapillary pattern of the tumor cells with clear or pale eosinophilic cytoplasm and large nuclei with small nucleoli. Immunohistochemistry showed positive for vimentin, S-100, and SOX-10, and negative for SMA, c-KIT, cytokeratin, HMB-45, and MelanA. The EWSR1 gene split signal was detected by reverse transcriptase fluorescence in situ hybridization, and EWSR1-CREB1 gene fusion was indicated by reverse transcriptase polymerase chain reaction analysis. From these findings, we diagnosed the tumor as CCSLGT. To best of our knowledge, this is the first description of the imprint cytology features of CCSLGT. © 2017 Wiley Periodicals, Inc.

Requirement of the inducible nitric oxide synthase pathway for IL-1-induced osteoclastic bone resorption

OpenAIRE

van't Hof, R. J.; Armour, K. J.; Smith, L. M.; Armour, K. E.; Wei, X. Q.; Liew, F. Y.; Ralston, S. H.

2000-01-01

Nitric oxide has been suggested to be involved in the regulation of bone turnover, especially in pathological conditions characterized by release of bone-resorbing cytokines. The cytokine IL-1 is thought to act as a mediator of periarticular bone loss and tissue damage in inflammatory diseases such as rheumatoid arthritis. IL-1 is a potent stimulator of both osteoclastic bone resorption and expression of inducible nitric oxide synthase (iNOS) in bone cells and other cell types. In this study,...

Giant Cell Angiofibroma in Unusual Localization: A Case Report

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Emel Ebru Pala

2012-01-01

Full Text Available Giant cell angiofibroma (GCA was initially described as a potentially recurrent tumor in the orbit of adults. However, it is now recognized that it can also present in other locations. The morphological hallmark is a richly vascularized patternless spindle cell proliferation containing pseudovascular spaces and floret like multinucleate giant cells. Our case was a 32-years-old female complaining of painless solitary nodule arising on the occipital region of the scalp, which was diagnosed as giant cell angiofibroma. We report the case because of its extremely rare localization.

Donepezil prevents RANK-induced bone loss via inhibition of osteoclast differentiation by downregulating acetylcholinesterase

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Tsuyoshi Sato

2015-09-01

Conclusions: AChE promotes osteoclast differentiation in vitro. Donepezil inhibits osteoclast function in vitro and prevents bone loss by suppressing bone resorption in vivo, suggesting the possibility that donepezil reduces fracture risk in patients with Alzheimer's disease.

Effect of heparin and alendronate coating on titanium surfaces on inhibition of osteoclast and enhancement of osteoblast function

Energy Technology Data Exchange (ETDEWEB)

Moon, Ho-Jin; Yun, Young-Pil [Department of Maxillofacial Biomedical Engineering, School of Dentistry, Kyung Hee University, Seoul 130-701 (Korea, Republic of); Han, Choong-Wan; Kim, Min Sung [Department of Oral and Maxillofacial Radiology, School of Dentistry, Kyung Hee University, Seoul 130-701 (Korea, Republic of); Institute of Oral Biology, Kyung Hee University, Seoul 130-701 (Korea, Republic of); Kim, Sung Eun; Bae, Min Soo [Department of Maxillofacial Biomedical Engineering, School of Dentistry, Kyung Hee University, Seoul 130-701 (Korea, Republic of); Kim, Gyu-Tae; Choi, Yong-Suk; Hwang, Eui-Hwan [Department of Oral and Maxillofacial Radiology, School of Dentistry, Kyung Hee University, Seoul 130-701 (Korea, Republic of); Institute of Oral Biology, Kyung Hee University, Seoul 130-701 (Korea, Republic of); Lee, Joon Woo [Department of Technology Commercialization Information, Korea Institute of Science and Technology Information (KISTI), 66, Hoegi-ro, Dongdaemun-gu, Seoul 130-741 (Korea, Republic of); Lee, Jin-Moo; Lee, Chang-Hoon [Department of Oriental Gynecology, College of Oriental Medicine, Kyung Hee University, Seoul 130-701 (Korea, Republic of); Kim, Duck-Su [Department of Conservative Dentistry, School of Dentistry, Kyung Hee University, Seoul 130-701 (Korea, Republic of); Kwon, Il Keun, E-mail: kwoni@khu.ac.kr [Department of Maxillofacial Biomedical Engineering, School of Dentistry, Kyung Hee University, Seoul 130-701 (Korea, Republic of); Institute of Oral Biology, Kyung Hee University, Seoul 130-701 (Korea, Republic of)

2011-09-23

Highlights: {yields} We examine bone metabolism of engineered alendronate attached to Ti surfaces. {yields} Alendronate-immobilized Ti enhances activation of osteoblast differentiation. {yields} Alendronate-immobilized Ti inhibits osteoclast differentiation. {yields} Alendronate-immobilized Ti may be a bioactive implant with dual functions. -- Abstract: The failure of orthopedic and dental implants has been attributed mainly to loosening of the implant from host bone, which may be due to weak bonding of the implant material to bone tissue. Titanium (Ti) is used in the field of orthopedic and dental implants because of its excellent biocompatibility and outstanding mechanical properties. Therefore, in the field of materials science and tissue engineering, there has been extensive research to immobilize bioactive molecules on the surface of implant materials in order to provide the implants with improved adhesion to the host bone tissue. In this study, chemically active functional groups were introduced on the surface of Ti by a grafting reaction with heparin and then the Ti was functionalized by immobilizing alendronate onto the heparin-grafted surface. In the MC3T3-E1 cell osteogenic differentiation study, the alendronate-immobilized Ti substrates significantly enhanced alkaline phosphatase activity (ALP) and calcium content. Additionally, nuclear factor kappa B ligand (RANKL)-induced osteoclast differentiation of RAW264.7 cells was inhibited with the alendronate-immobilized Ti as confirmed by TRAP analysis. Real time PCR analysis showed that mRNA expressions of osteocalcin and osteopontin, which are markers for osteogenesis, were upregulated in MC3T3-E1 cells cultured on alendronate-immobilized Ti. The mRNA expressions of TRAP and Cathepsin K, markers for osteoclastogenesis, in RAW264.7 cells cultured on alendronate-immobilized Ti were down-regulated. Our study suggests that alendronate-immobilized Ti may be a bioactive implant with dual functions to enhance

The Study of Mechanisms of Protective Effect of Rg1 against Arthritis by Inhibiting Osteoclast Differentiation and Maturation in CIA Mice

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Yanqing Gu

2014-01-01

Full Text Available Ginsenoside Rg1 is a natural product extracted from Panax ginseng C.A. Although Rg1 protects tissue structure and functions by inhibiting local inflammatory reaction, the mechanism remains poorly understood. In vitro, Rg1 dose-dependently inhibited TRAP activity in receptor activator of nuclear factor-κB ligand- (RANKL- induced osteoclasts and decreased the number of osteoclasts and osteoclast resorption area. Rg1 also significantly inhibited the RANK signaling pathway, including suppressing the expression of Trap, cathepsin K, matrix metalloproteinase 9 (MMP9, and calcitonin receptor (CTR. In vivo, Rg1 dramatically decreased arthritis scores in CIA mice and effectively controlled symptoms of inflammatory arthritis. Pathologic analysis demonstrated that Rg1 significantly attenuated pathological changes in CIA mice. Pronounced reduction in synovial hyperplasia and inflammatory cell invasion were observed in CIA mice after Rg1 therapy. Alcian blue staining results illustrated that mice treated with Rg1 had significantly reduced destruction in the articular cartilage. TRAP and cathepsin K staining results demonstrated a significant reduction of numbers of OCs in the articular cartilage in proximal interphalangeal joints and ankle joints in Rg1-treated mice. In summary, this study revealed that Rg1 reduced the inflammatory destruction of periarticular bone by inhibiting differentiation and maturation of osteoclasts in CIA mice.

Hydroxyapatite/collagen bone-like nanocomposite.

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Kikuchi, Masanori

2013-01-01

Our group has succeeded to synthesize material with bone-like nanostructure and bone-like inorganic and organic composition via self-organization mechanism between them using simultaneous titration method under controlled pH and temperature. The hydroxyapatite/collagen (HAp/Col) bone-like nanocomposite completely incorporated into bone remodeling process to be substituted by new bone. Cells cultured on the HAp/Col revealed very interesting reactions. Osteoblast-like MG63 cells showed upregulation of alkaline phosphatase >3 times greater than MG63 cells cultured on tissue culture polystyrene (TCPS). MG63 cells 3-dimensionally cultured in a "HAp/Col sponge," a porous HAp/Col having sponge-like viscoelasticity, accumulated calcium phosphate nodules on extracellular matrices they secreted. Bone marrow cells co-cultured with osteoblasts on HAp/Col differentiated to osteoclasts without differentiation supplements. This phenomenon is not found in cells cultured on hydroxyapatite ceramics and TCPS, and rarely in cells cultured on dentin. These results suggest that HAp/Col is a good candidate for tissue engineering of bone as well as bone filler. In a clinical test as a bone filler, the HAp/Col sponge was significantly better than porous β-tricalcium phosphate. The HAp/Col sponge has been approved by the Japanese government and will be used as greatly needed bone filler in patients. In addition to the above, HAp/Col coating on titanium revealed higher osteo-conductivity than HAp-coated titanium and bare titanium and improved direct bonding between titanium and newly formed bone. The HAp/Col coating may be used for metal devices requiring osseointegration.

Bortezomib modulates CHIT1 and YKL40 in monocyte-derived osteoclast and in myeloma cells

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Tibullo eDaniele

2015-10-01

Full Text Available Osteolytic bone disease is a common manifestation of multiple myeloma (MM that leads to progressive skeleton destruction and is the most severe cause of morbidity in MM patients.It results from increased osteolytic activity and decrease osteoblastic function. Activation of mammalian chitinases CHIT1 and YKL40 is associated with osteoclast (OCs differentiation and bone digestion. In the current study, we investigated the effect of two Bortezomib’s concentration (BO (2.5 nM and 5nM on osteoclastogenesis by analyzing regulation of chitinase expression. OCs exposition to BO was able to inhibit the expression of different OCs markers such as RANK, CTSK, TRAP and MMP9. In addition BO-treatment reduced CHIT1 enzymatic activity and both CHIT1 and YKL40 mRNA expression levels and cytoplasmatic and secreted protein. Moreover, immunofluorescence evaluation of mature OCs showed that BO was able to translocate YKL40 into the nucleus, while CHIT1 remained into the cytoplasm. Since MM cell lines such as U266, SKM-M1 and MM1 showed high levels of CHIT1 activity, we analyzed bone resorption ability of U266 using dentin disc assay resorption pits. Silencing chitinase proteins in U266 cell line with specific siRNAs, resulted in pits number reduction on dentine discs. In conclusion, we showed that BO decreases osteoclastogenesis and reduces bone resorption in OCs and U266 cell line by modulating the chitinases CHIT1 and YKL40. These results indicate that chitinases may be a therapeutic target for bone disease in MM patients.

Bortezomib modulates CHIT1 and YKL40 in monocyte-derived osteoclast and in myeloma cells.

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Tibullo, Daniele; Di Rosa, Michelino; Giallongo, Cesarina; La Cava, Piera; Parrinello, Nunziatina L; Romano, Alessandra; Conticello, Concetta; Brundo, Maria V; Saccone, Salvatore; Malaguarnera, Lucia; Di Raimondo, Francesco

2015-01-01

Osteolytic bone disease is a common manifestation of multiple myeloma (MM) that leads to progressive skeleton destruction and is the most severe cause of morbidity in MM patients. It results from increased osteolytic activity and decrease osteoblastic function. Activation of mammalian chitinases chitotriosidase (CHIT1) and YKL40 is associated with osteoclast (OCs) differentiation and bone digestion. In the current study, we investigated the effect of two Bortezomib's concentration (2.5 and 5 nM) on osteoclastogenesis by analyzing regulation of chitinase expression. OCs exposition to bortezomib (BO) was able to inhibit the expression of different OCs markers such as RANK, CTSK, TRAP, and MMP9. In addition BO-treatment reduced CHIT1 enzymatic activity and both CHIT1 and YKL40 mRNA expression levels and cytoplasmatic and secreted protein. Moreover, immunofluorescence evaluation of mature OCs showed that BO was able to translocate YKL40 into the nucleus, while CHIT1 remained into the cytoplasm. Since MM cell lines such as U266, SKM-M1 and MM1 showed high levels of CHIT1 activity, we analyzed bone resorption ability of U266 using dentin disk assay resorption pits. Silencing chitinase proteins in U266 cell line with specific small interfering RNA, resulted in pits number reduction on dentine disks. In conclusion, we showed that BO decreases osteoclastogenesis and reduces bone resorption in OCs and U266 cell line by modulating the chitinases CHIT1 and YKL40. These results indicate that chitinases may be a therapeutic target for bone disease in MM patients.

Involvement of human endogenous retroviral syncytin-1 in human osteoclast fusion

DEFF Research Database (Denmark)

Søe, Kent; Andersen, Thomas Lykke; Hobolt-Pedersen, Anne-Sofie

2011-01-01

fusion of the lipid bilayers of their cell membranes are still unknown. Syncytin-1 is a protein encoded by a human endogenous retroviral gene which was stably integrated into the human ancestor genome more than 24 million years ago. Upon activation, syncytin-1 is able to destabilize the lipid bilayer....... This was documented through Q-PCR, Western blot and immunofluorescence analyses. These in vitro findings were confirmed by immunohistochemical stainings in human iliac crest biopsies. A syncytin-1 inhibitory peptide reduced the number of nuclei per osteoclast by 30%, as well as TRACP activity. From a mechanistic...

Substrate adhesion regulates sealing zone architecture and dynamics in cultured osteoclasts.

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Fabian Anderegg

Full Text Available The bone-degrading activity of osteoclasts depends on the formation of a cytoskeletal-adhesive super-structure known as the sealing zone (SZ. The SZ is a dynamic structure, consisting of a condensed array of podosomes, the elementary adhesion-mediating structures of osteoclasts, interconnected by F-actin filaments. The molecular composition and structure of the SZ were extensively investigated, yet despite its major importance for bone formation and remodelling, the mechanisms underlying its assembly and dynamics are still poorly understood. Here we determine the relations between matrix adhesiveness and the formation, stability and expansion of the SZ. By growing differentiated osteoclasts on micro-patterned glass substrates, where adhesive areas are separated by non-adhesive PLL-g-PEG barriers, we show that SZ growth and fusion strictly depend on the continuity of substrate adhesiveness, at the micrometer scale. We present a possible model for the role of mechanical forces in SZ formation and reorganization, inspired by the current data.

Osteoclasts but not osteoblasts are affected by a calcified surface treated with zoledronic acid in vitro

International Nuclear Information System (INIS)

Schindeler, Aaron; Little, David G.

2005-01-01

Bisphosphonates are potent inhibitors of osteoclast-mediated bone resorption. Recent interest has centered on the effects of bisphosphonates on osteoblasts. Chronic dosing of osteoblasts with solubilized bisphosphonates has been reported to enhance osteogenesis and mineralization in vitro. However, this methodology poorly reflects the in vivo situation, where free bisphosphonate becomes rapidly bound to mineralized bone surfaces. To establish a more clinically relevant cell culture model, we cultured bone cells on calcium phosphate coated quartz discs pre-treated with the potent nitrogen-containing bisphosphonate, zoledronic acid (ZA). Binding studies utilizing [ 14 C]-labeled ZA confirmed that the bisphosphonate bound in a concentration-dependent manner over the 1-50 μM dose range. When grown on ZA-treated discs, the viability of bone-marrow derived osteoclasts was greatly reduced, while the viability and mineralization of the osteoblastic MC3T3-E1 cell line were largely unaffected. This suggests that only bone resorbing cells are affected by bound bisphosphonate. However, this system does not account for transient exposure to unbound bisphosphonate in the hours following a clinical dosing. To model this event, we transiently treated osteoblasts with ZA in the absence of a calcified surface. Osteoblasts proved highly resistant to all transitory treatment regimes, even when utilizing ZA concentrations that prevented mineralization and/or induced cell death when dosed chronically. This study represents a pharmacologically more relevant approach to modeling bisphosphonate treatment on cultured bone cells and implies that bisphosphonate therapies may not directly affect osteoblasts at bone surfaces

Impact of different synthetic bone fillers on healing of extraction sockets: an experimental study in dogs.

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Hong, Ji-Youn; Lee, Jung-Seok; Pang, Eun-Kyoung; Jung, Ui-Won; Choi, Seong-Ho; Kim, Chong-Kwan

2014-02-01

The objective of this study was to elucidate the socket healing process and biodegradation of incorporating synthetic bone fillers followed by grafting of the fresh extraction socket. Third premolars in four quadrants of eight beagle dogs were extracted and randomly treated with either one of hydroxyapatite (HA), biphasic calcium phosphate (BCP), β-tricalcium phosphate (β-TCP), or no graft (C). Histologic observations and histomorphometric analysis at three zones (apical, middle, and coronal) of the socket were performed. Socket area (S) and the proportions of newly formed bone (%NB), residual biomaterials (%RB), and fibrovascular connective tissue (%FCT) at 2, 4, and 8 weeks were measured. The numbers of osteoclast-like multinucleated cells (No.OC) were also determined at the three zones. %NB was significantly higher in control group compared with the grafted groups at all healing periods. %NB of HA and BCP increased with time, whereas %RB showed different patterns that decreased in BCP, unlike the minimal change observed in HA. %NB of β-TCP showed smallest portion compared with other grafted groups at 2 and 4 weeks, however, significantly increased at 8 weeks. %RB of β-TCP was less than HA and BCP at all healing periods. Numbers of multinucleated cells were greater in BCP and β-TCP, followed by HA and smallest in control group. Within the limit of this study, bone formation of the extraction socket was delayed in the sockets grafted with synthetic bone fillers and showed different healing process according to the biodegradation patterns. © 2012 John Wiley & Sons A/S.

Effect of Cytokines on Osteoclast Formation and Bone Resorption during Mechanical Force Loading of the Periodontal Membrane

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Hideki Kitaura

2014-01-01

Full Text Available Mechanical force loading exerts important effects on the skeleton by controlling bone mass and strength. Several in vivo experimental models evaluating the effects of mechanical loading on bone metabolism have been reported. Orthodontic tooth movement is a useful model for understanding the mechanism of bone remodeling induced by mechanical loading. In a mouse model of orthodontic tooth movement, TNF-α was expressed and osteoclasts appeared on the compressed side of the periodontal ligament. In TNF-receptor-deficient mice, there was less tooth movement and osteoclast numbers were lower than in wild-type mice. These results suggest that osteoclast formation and bone resorption caused by loading forces on the periodontal ligament depend on TNF-α. Several cytokines are expressed in the periodontal ligament during orthodontic tooth movement. Studies have found that inflammatory cytokines such as IL-12 and IFN-γ strongly inhibit osteoclast formation and tooth movement. Blocking macrophage colony-stimulating factor by using anti-c-Fms antibody also inhibited osteoclast formation and tooth movement. In this review we describe and discuss the effect of cytokines in the periodontal ligament on osteoclast formation and bone resorption during mechanical force loading.

Bone impairment in phenylketonuria is characterized by circulating osteoclast precursors and activated T cell increase.

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Ilaria Roato

Full Text Available BACKGROUND: Phenylketonuria (PKU is a rare inborn error of metabolism often complicated by a progressive bone impairment of uncertain etiology, as documented by both ionizing and non- ionizing techniques. METHODOLOGY: Peripheral blood mononuclear cell (PBMC cultures were performed to study osteoclastogenesis, in the presence or absence of recombinant human monocyte-colony stimulating factor (M-CSF and receptor activator of NFκB ligand (RANKL. Flow cytometry was utilized to analyze osteoclast precursors (OCPs and T cell phenotype. Tumour necrosis factor α (TNF-α, RANKL and osteoprotegerin (OPG were quantified in cell culture supernatants by ELISA. The effects of RANKFc and anti-TNF-α antibodies were also investigated to determine their ability to inhibit osteoclastogenesis. In addition, bone conditions and phenylalanine levels in PKU patients were clinically evaluated. PRINCIPAL FINDINGS: Several in vitro studies in PKU patients' cells identified a potential mechanism of bone formation inhibition commonly associated with this disorder. First, PKU patients disclosed an increased osteoclastogenesis compared to healthy controls, both in unstimulated and M-CSF/RANKL stimulated PBMC cultures. OCPs and the measured RANKL/OPG ratio were higher in PKU patients compared to healthy controls. The addition of specific antagonist RANKFc caused osteoclastogenesis inhibition, whereas anti-TNF-α failed to have this effect. Among PBMCs isolated from PKU patients, activated T cells, expressing CD69, CD25 and RANKL were identified. Confirmatory in vivo studies support this proposed model. These in vivo studies included the analysis of osteoclastogenesis in PKU patients, which demonstrated an inverse relation to bone condition assessed by phalangeal Quantitative Ultrasound (QUS. This was also directly related to non-compliance to therapeutic diet reflected by hyperphenylalaninemia. CONCLUSIONS: Our results indicate that PKU spontaneous osteoclastogenesis

Changes in osteocyte density correspond with changes in osteoblast and osteoclast activity in an osteoporotic sheep model.

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Zarrinkalam, M R; Mulaibrahimovic, A; Atkins, G J; Moore, R J

2012-04-01

Histomorphometric assessment of trabecular bone in osteoporotic sheep showed that bone volume, osteoid surface area, bone formation rate, and osteocyte density were reduced. In contrast, eroded surface area and empty lacunae density were increased. Changes in osteocyte density correlated with changes in osteoblast and osteoclast activity. Osteocytes contribute to the regulation of the activity of osteoclasts and osteoblasts that together control bone mass. Osteocytes therefore likely play a role in the loss of bone mass associated with osteoporosis. The purpose of this study was to investigate the relationships between osteocyte lacunar density and other bone histomorphometric parameters in the iliac crest (IC) and lumbar spine (LS) of osteoporotic sheep. Osteoporosis was induced in ten mature ewes by an established protocol involving a combination of ovariectomy, dexamethasone injection, and low calcium diet for 6 months. Five ewes were used as controls. Post-mortem IC and LS biopsies were collected and processed for further histomorphometric assessment. Bone volume, osteoid surface, and bone formation rate in the IC and LS of osteoporotic sheep were reduced compared to those of the controls. In contrast, eroded surface area was increased in osteoporotic sheep. In the osteoporotic group, osteocyte density was reduced in the LS region and to a greater extent in the IC region. The empty osteocyte lacunae were increased 1.7-fold in LS and 2.1-fold in IC in the osteoporotic group. The osteocyte density correlated positively with markers of osteoblast activity and negatively with those of osteoclast activity. Depletion of osteocytes and an increase in the empty lacunae could be important factors contributing to bone loss in this model since they may adversely affect intercellular communication between osteoblasts and osteoclasts. The regional differences in histology suggest that there may be different pathological mechanisms operating at different anatomical sites.

Static and dynamic hypergravity responses of osteoblasts and osteoclasts in medaka scales

OpenAIRE

Yano, Sachiko; Kitamura, Kei-ichiro; Satoh, Yusuke; Nakano, Masaki; Hattori, Atsuhiko; Sekiguchi, Toshio; Ikegame, Mika; Nakashima, Hiroshi; Omori, Katsunori; Hayakawa, Kazuichi; Chiba, Atsuhiko; Sasayama, Yuichi; Ejiri, Sadakazu; Mikuni-Takagaki, Yuko; Mishima, Hiroyuki

2013-01-01

Fish scales are a form of calcified tissue similar to that found in human bone. In medaka scales, we detected both osteoblasts and osteoclasts and subsequently developed a new scale assay system. Using this system, we analyzed the osteoblastic and osteoclastic responses under 2-, 3-, and 4-gravity (G) loading by both centrifugation and vibration. After loading for 10 min, the scales from centrifugal and vibration loading were incubated for 6 and 24 hrs, respectively, after which the osteoblas...

Suppression Effect of Astaxanthin on Osteoclast Formation In Vitro and Bone Loss In Vivo

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Yun-Ho Hwang

2018-03-01

Full Text Available Osteoporosis is characterized by a reduction of the bone mineral density (BMD and microarchitectural deterioration of the bone, which lead to bone fragility and susceptibility to fracture. Astaxanthin (AST has a variety of biological activities, such as a protective effect against asthma or neuroinflammation, antioxidant effect, and decrease of the osteoclast number in the right mandibles in the periodontitis model. Although treatment with AST is known to have an effect on inflammation, no studies on the effect of AST exposure on bone loss have been performed. Thus, in the present study, we examined the antiosteoporotic effect of AST on bone mass in ovariectomized (OVX mice and its possible mechanism of action. The administration of AST (5, 10 mg/kg for 6 weeks suppressed the enhancement of serum calcium, inorganic phosphorus, alkaline phosphatase, total cholesterol, and tartrate-resistant acid phosphatase (TRAP activity. The bone mineral density (BMD and bone microarchitecture of the trabecular bone in the tibia and femur were recovered by AST exposure. Moreover, in the in vitro experiment, we demonstrated that AST inhibits osteoclast formation through the expression of the nuclear factor of activated T cells (NFAT c1, dendritic cell-specific transmembrane protein (DC-STAMP, TRAP, and cathepsin K without any cytotoxic effects on bone marrow-derived macrophages (BMMs. Therefore, we suggest that AST may have therapeutic potential for the treatment of postmenopausal osteoporosis.

Murine osteoblastic and osteoclastic differentiation on strontium releasing hydroxyapatite forming cements

Energy Technology Data Exchange (ETDEWEB)

Singh, Satish S., E-mail: sss42@pitt.edu [Department of Chemical & Petroleum Engineering, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Roy, Abhijit, E-mail: abr20@pitt.edu [Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Lee, Boeun, E-mail: bol11@pitt.edu [Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Parekh, Shrey, E-mail: smp116@pitt.edu [Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Kumta, Prashant N., E-mail: pkumta@pitt.edu [Department of Chemical & Petroleum Engineering, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Center for Craniofacial Regeneration, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Department of Mechanical Engineering and Materials Science, University of Pittsburgh, PA 15261 (United States); Center for Complex Engineered Multifunctional Materials, University of Pittsburgh, PA 15261 (United States)

2016-06-01

Ionic substitutions in hydroxyapatite (HA) scaffolds and self-setting cements containing Sr{sup 2+} ions incorporated are particularly of interest in bone regeneration. To date, the approach widely used to incorporate Sr{sup 2+} ions into HA cements has been the addition of Sr{sup 2+} containing salts, such as SrCO{sub 3}, SrCl{sub 2} ∙ 6H{sub 2}O, or SrHPO{sub 4}. However, this approach is dependent upon the relative solubility of Sr{sup 2+} containing salts with respect to calcium phosphate (CaP) precursors. Therefore, in the current study Sr{sup 2+} substituted dicalcium phosphate dihydrate (DCPD) was first synthesized and directly reacted with tetracalcium phosphate (TTCP) to form Sr{sup 2+} substituted HA forming cements. Rietveld refinement indicated that after one week of aging in phosphate buffered saline, cements prepared with and without Sr{sup 2+} were composed of 75% HA and 25% unreacted TTCP by weight. Cements prepared with 10% Sr{sup 2+} DCPD exhibited increased compressive strengths in comparison to unsubstituted cements. Increased MC3T3-E1 proliferation and differentiation were also observed on the cements prepared with increasing Sr{sup 2+} content. It was concluded that both the scaffold microstructure and Sr{sup 2+} ion release supported osteogenic differentiation. With respect to osteoclastic differentiation, no statistically significant differences in TRAP activity or cell morphology were observed. This suggests that the amount of Sr{sup 2+} released may have been too low to influence osteoclast formation in comparison to unsubstituted cements. The results obtained herein demonstrate that the use of Sr{sup 2+} substituted DCPD precursors rather than individually separate Sr{sup 2+} containing salts may be a useful approach to prepare Sr{sup 2+} containing HA cements. - Highlights: • Sr{sup 2+} containing HA cement was prepared by reacting TTCP with Sr{sup 2+} substituted DCPD. • Cements with increased Sr{sup 2+} supported increased

The controlled release of simvastatin from TiO{sub 2} nanotubes to promote osteoblast differentiation and inhibit osteoclast resorption

Energy Technology Data Exchange (ETDEWEB)

Lai, Min, E-mail: minlai@jsnu.edu.cn [School of Life Science, Jiangsu Normal University, Xuzhou, Jiangsu 221116 (China); Jin, Ziyang; Yang, Xinyi; Wang, Huaying [School of Life Science, Jiangsu Normal University, Xuzhou, Jiangsu 221116 (China); Xu, Kui [Biomedical Engineering Center, School of Medicine, Ningbo University, Ningbo, Zhejiang 315211 (China)

2017-02-28

Highlights: • The TiO{sub 2} nanotube substrates filled with simvastatin were successfully coated using chitosan/gelatin multilayers. • The bio-functionalized substrates display controlled release of simvastatin in a sustained manner. • The bio-functionalized substrates have great potential for improving osteoblast differentiation. • The bio-functionalized substrates effectively inhibit osteoclast differentiation. - Abstract: The aim of this study was to fabricate a novel drug-releasing bioactive platform that has excellent potential for improving osteoblast differentiation and inhibiting osteoclast resorption. TiO{sub 2} nanotubes (TNTs) with an outer diameter of around 70 nm were prepared by an anodization method. TNTs were filled with simvastatin (SV) and then coated using chitosan/gelatin multilayers (TNT-SV-LBL). The successful fabrication of TNT-SV-LBL substrates was confirmed by field emission scanning electron microscopy (FE-SEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and contact angle measurement, respectively. The in vitro release behavior of simvastatin from TNT-SV-LBL substrates showed a sustained release as compared to the uncoated group. Osteoblasts adhering to TNT-SV-LBL substrates attached well and displayed significantly higher (p < 0.01) cell viability compared with the other substrates. More importantly, osteoblasts grown on TNT-SV-LBL substrates displayed a statistically significant (p < 0.01 or p < 0.05) increase in protein production levels of alkaline phosphatase (ALP), osteocalcin (OC) and mRNA expression of runt related transcription factor 2 (Runx2), ALP, collagen type I (Col I), osteopontin (OPN), OC and osteoprotegerin (OPG) compared to the other groups after 4, 7 and 14 days of culture, respectively. Additionally, multinuclear osteoclastic differentiation of RAW264.7 cells grown on TNT-SV-LBL substrates was inhibited as confirmed by tartrate-resistant acid phosphatase (TRAP) analysis. These

Adhesion structures and their cytoskeleton-membrane interactions at podosomes of osteoclasts in culture.

Science.gov (United States)

Akisaka, Toshitaka; Yoshida, Hisaho; Suzuki, Reiko; Takama, Keiko

2008-03-01

The organization of the cytoskeleton in the podosomes of osteoclasts was studied by use of cell shearing, rotary replication, and fluorescence cytochemical techniques. After shearing, clathrin plaques and particles associated with the cytoskeleton were left behind on the exposed cytoplasmic side of the membrane. The cytoskeleton of the podosomes was characterized by two types of actin filaments: relatively long filaments in the portion surrounding the podosome core, and highly branched short filaments in the core. Individual actin filaments radiating from the podosomes interacted with several membrane particles along the length of the filaments. Many lateral contacts with the membrane surface by the particles were made along the length of individual actin filaments. The polarity of actin filaments in podosomes became oriented such that their barbed ends were directed toward the core of podosomes. The actin cytoskeletons terminated or branched at the podosomes, where the membrane tightly adhered to the substratum. Microtubules were not usually present in the podosome structures; however, certain microtubules appeared to be morphologically in direct contact with the podosome core. Most of the larger clathrin plaques consisted of flat sheets of clathrin lattices that interconnected neighboring clathrin lattices to form an extensive clathrin area. However, the small deeply invaginated clathrin plaques and the podosomal cytoskeleton were located close together. Thus, the clathrin plaques on the ventral membrane of osteoclasts might be involved in both cell adhesion and the formation of receptor-ligand complexes, i.e., endocytosis.

Prevention of wear particle-induced osteolysis by a novel V-ATPase inhibitor saliphenylhalamide through inhibition of osteoclast bone resorption.

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An Qin

Full Text Available 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.

Characterization of acid flux in osteoclasts from patients harboring a G215R mutation in ClC-7

DEFF Research Database (Denmark)

Henriksen, Kim; Gram, Jeppe; Neutzsky-Wulff, Anita Vibsig

2008-01-01

-I, calcium release, and pit area when comparing to age and sex matched controls. In addition, the ADOII osteoclasts showed no differences in actin ring formation. Finally, V-ATPase and chloride channel inhibitors completely abrogated the H(+) and Cl(-) driven acidification. Finally, the acid influx...... mutation in ClC-7 (G215R) were isolated, and used these to investigate bone resorption measured by CTX-I, calcium release and pit scoring. The actin cytoskeleton of the osteoclasts was also investigated. ClC-7 enriched membranes from the osteoclasts were isolated, and used to test acidification rates...... in the presence of a V-ATPase and a chloride channel inhibitor, using a H(+) and Cl(-) driven approach. Finally, acidification rates in ClC-7 enriched membranes from ADOII osteoclasts and their corresponding controls were compared. Resorption by the G215R osteoclasts was reduced by 60% when measured by both CTX...

F4/80 inhibits osteoclast differentiation via downregulation of nuclear factor of activated T cells, cytoplasmic 1.

Science.gov (United States)

Kang, Ju-Hee; Sim, Jung-Sun; Zheng, Ting; Yim, Mijung

2017-04-01

Osteoclastogenesis is an essential process in bone metabolism, which can be induced by RANKL stimulation. The F4/80 glycoprotein is a member of the EGF-transmembrane 7 (TM7) family and has been established as a specific cell-surface marker for murine macrophages. This study aimed to identify the role of F4/80 in osteoclastogenesis. Using mouse bone marrow-derived macrophages (BMMs), we observed that the mRNA level of F4/80 was dramatically reduced as these cells differentiated into osteoclasts. Furthermore, osteoclastogenesis was decreased in F4/80 high BMMs compared to F4/80 -/low BMMs. The inhibitory effect of F4/80 was associated with decreased expression of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1). Ectopic overexpression of a constitutively active form of NFATc1 rescued the anti-osteoclastogenic effect of F4/80 completely, suggesting that the anti-osteoclastogenic effect of F4/80 was mainly due to reduction in NFATc1 expression. As an underlying mechanism, we demonstrated that the presence of F4/80 abrogated the effect of RANKL on the phosphorylation of CREB and activated the expression of IFN-β, which are restored by cyclic AMP. Collectively, our results demonstrate that the presence of F4/80 suppresses RANKL-induced osteoclastogenesis by impairing the expression of NFATc1 via CREB and IFN-β. Therefore, F4/80 may hold therapeutic potential for bone destructive diseases.

Dietary emu oil supplementation suppresses 5-fluorouracil chemotherapy-induced inflammation, osteoclast formation, and bone loss.

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Raghu Nadhanan, Rethi; Abimosleh, Suzanne M; Su, Yu-Wen; Scherer, Michaela A; Howarth, Gordon S; Xian, Cory J

2012-06-01

Cancer chemotherapy can cause osteopenia or osteoporosis, and yet the underlying mechanisms remain unclear, and currently, no preventative treatments are available. This study investigated damaging effects of 5-fluorouracil (5-FU) on histological, cellular, and molecular changes in the tibial metaphysis and potential protective benefits of emu oil (EO), which is known to possess a potent anti-inflammatory property. Female dark agouti rats were gavaged orally with EO or water (1 ml·day(-1)·rat(-1)) for 1 wk before a single ip injection of 5-FU (150 mg/kg) or saline (Sal) was given. The treatment groups were H(2)O + Sal, H(2)O + 5-FU, EO + 5-FU, and EO + Sal. Oral gavage was given throughout the whole period up to 1 day before euthanasia (days 3, 4, and 5 post-5-FU). Histological analysis showed that H(2)O + 5-FU significantly reduced heights of primary spongiosa on days 3 and 5 and trabecular bone volume of secondary spongiosa on days 3 and 4. It reduced density of osteoblasts slightly and caused an increase in the density of osteoclasts on trabecular bone surface on day 4. EO supplementation prevented reduction of osteoblasts and induction of osteoclasts and bone loss caused by 5-FU. Gene expression studies confirmed an inhibitory effect of EO on osteoclasts since it suppressed 5-FU-induced expression of proinflammatory and osteoclastogenic cytokine TNFα, osteoclast marker receptor activator of nuclear factor-κB, and osteoclast-associated receptor. Therefore, this study demonstrated that EO can counter 5-FU chemotherapy-induced inflammation in bone, preserve osteoblasts, suppress osteoclast formation, and potentially be useful in preventing 5-FU chemotherapy-induced bone loss.

Approach to novel functional foods for stress control 4. Regulation of serotonin transporter by food factors.

Science.gov (United States)

Ito, Mikiko; Haito, Sakiko; Furumoto, Mari; Kawai, Yoshichika; Terao, Junji; Miyamoto, Ken-ichi

2005-11-01

Serotonin transporters (SERTs) are pre-synaptic proteins specialized for the clearance of serotonin following vesicular release at central nervous system (CNS) and enteric nervous system synapses. SERTs are high affinity targets in vivo for antidepressants such as serotonin selective reuptake inhibitors (SSRIs). These include 'medical' psychopharmacological agents such as analgesics and antihistamines, a plant extract called St John's Wort (Hypericum). Osteoclasts are the primary cells responsible for bone resorption. They arise by the differentiation of osteoclast precursors of the monocyte/macrophage lineage. The expression of SERTs was increased in RANKL-induced osteoclast-like cells. Using RANKL stimulation of RAW264.7 cells as a model system for osteoclast differentiation, we studied the direct effects of food factor on serotonin uptake. The SSRIs (fluoxetine and fluvoxamine) inhibited markedly (approximately 95%) in serotonin transport in differentiated osteoclast cells. The major components of St. John's Wort, hyperforin and hypericine were significantly decreased in serotonin transport activity. Thus, a new in vitro model using RANKL-induced osteoclast-like cells may be useful to analyze the regulation of SERT by food factors and SSRIs.

P2X7 receptor regulates osteoclast function and bone loss in a mouse model of osteoporosis

DEFF Research Database (Denmark)

Wang, Ning; Agrawal, Ankita; Jørgensen, Niklas Rye

2018-01-01

Post-menopausal osteoporosis is a condition that affects millions worldwide and places a huge socio-economic burden on society. Previous research has shown an association of loss of function SNPs in the gene for the purinergic receptor P2X7R with low bone mineral density, increased rates of bone...... loss and vertebral fractures in post-menopausal women. In this study we use a mouse model of oestrogen deficiency-induced bone loss and the BALB/cJ P2X7R-/- to show that absence of the P2X7R resulted in increased bone loss. Osteoclast precursors were isolated from both BALB/cJ P2X7R-/- and BALB/cJ P2X7......R+/+ mice and then cultured in vitro to form mature resorbing osteoclasts. The BALB/cJ P2X7R-/- derived precursors generated slightly more osteoclasts but with a significant reduction in the amount of resorption per osteoclast. Furthermore, when using modified culture conditions osteoclast activity...

Long-Term Endurance Exercise in Humans Stimulates Cell Fusion of Myoblasts along with Fusogenic Endogenous Retroviral Genes In Vivo.

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Sebastian Frese

Full Text Available Myogenesis is defined as growth, differentiation and repair of muscles where cell fusion of myoblasts to multinucleated myofibers is one major characteristic. Other cell fusion events in humans are found with bone resorbing osteoclasts and placental syncytiotrophoblasts. No unifying gene regulation for natural cell fusions has been found. We analyzed skeletal muscle biopsies of competitive cyclists for muscle-specific attributes and expression of human endogenous retrovirus (ERV envelope genes due to their involvement in cell fusion of osteoclasts and syncytiotrophoblasts. Comparing muscle biopsies from post- with the pre-competitive seasons a significant 2.25-fold increase of myonuclei/mm fiber, a 2.38-fold decrease of fiber area/nucleus and a 3.1-fold decrease of satellite cells (SCs occurred. We propose that during the pre-competitive season SC proliferation occurred following with increased cell fusion during the competitive season. Expression of twenty-two envelope genes of muscle biopsies demonstrated a significant increase of putative muscle-cell fusogenic genes Syncytin-1 and Syncytin-3, but also for the non-fusogenic erv3. Immunohistochemistry analyses showed that Syncytin-1 mainly localized to the sarcolemma of myofibers positive for myosin heavy-chain isotypes. Cellular receptors SLC1A4 and SLC1A5 of Syncytin-1 showed significant decrease of expression in post-competitive muscles compared with the pre-competitive season, but only SLC1A4 protein expression localized throughout the myofiber. Erv3 protein was strongly expressed throughout the myofiber, whereas envK1-7 localized to SC nuclei and myonuclei. Syncytin-1 transcription factors, PPARγ and RXRα, showed no protein expression in the myofiber, whereas the pCREB-Ser133 activator of Syncytin-1 was enriched to SC nuclei and myonuclei. Syncytin-1, Syncytin-3, SLC1A4 and PAX7 gene regulations along with MyoD1 and myogenin were verified during proliferating or actively-fusing human

The plant limonoid 7-oxo-deacetoxygedunin inhibits RANKL-induced osteoclastogenesis by suppressing activation of the NF-{kappa}B and MAPK pathways

Energy Technology Data Exchange (ETDEWEB)

Wisutsitthiwong, Chonnaree; Buranaruk, Chayanit [Graduate Program in Industrial Microbiology, Department of Microbiology, Faculty of Science, Chulalongkorn University, Phayathai Road, Bangkok 10330 (Thailand); Pudhom, Khanitha [Department of Chemistry, Faculty of Science and Center for Petroleum, Petrochemicals and Advanced Materials, Chulalongkorn University, Phayathai Road, Bangkok 10330 (Thailand); Palaga, Tanapat, E-mail: tanapat.p@chula.ac.th [Graduate Program in Industrial Microbiology, Department of Microbiology, Faculty of Science, Chulalongkorn University, Phayathai Road, Bangkok 10330 (Thailand)

2011-11-18

Highlights: Black-Right-Pointing-Pointer A gedunin type limonoid from seeds of mangroves, 7-oxo-7-deacetoxygedunin, exhibits strong anti-osteoclastogenic activity. Black-Right-Pointing-Pointer Treatment with this limonoid results in significant decrease in expression of NFATc1 and osteoclast-related genes. Black-Right-Pointing-Pointer The mode of action of this limonoid is by inhibiting activation of the NF-{kappa}B and MAPK pathways which are activated by RANKL. -- Abstract: Osteoclasts together with osteoblasts play pivotal roles in bone remodeling. Aberrations in osteoclast differentiation and activity contribute to osteopenic disease. Osteoclasts differentiate from monocyte/macrophage progenitors, a process that is initiated by the interaction between receptor activator of NF-{kappa}B (RANK) and its ligand, RANKL. In this study, we identified 7-oxo-7-deacetoxygedunin (7-OG), a gedunin type limonoid from seeds of the mangrove Xylocarpus moluccensis, as a potent inhibitor of osteoclastogenesis. Additionally, 7-OG showed strong anti-osteoclastogenic activity with low cytotoxicity against the monocyte/macrophage progenitor cell line, RAW264.7. The IC50 for anti-osteoclastogenic activity was 4.14 {mu}M. Treatment with 7-OG completely abolished the appearance of multinucleated giant cells with tartrate-resistant acid phosphatase activity in RAW264.7 cells stimulated with RANKL. When the expression of genes related to osteoclastogenesis was investigated, a complete downregulation of NFATc1 and cathepsin K and a delayed downregulation of irf8 were observed upon 7-OG treatment in the presence of RANKL. Furthermore, treatment with this limonoid suppressed RANKL-induced activation of p38, MAPK and Erk and nuclear localization of NF-{kappa}B p65. Taken together, we present evidence indicating a plant limonoid as a novel osteoclastogenic inhibitor that could be used for osteoporosis and related conditions.

The plant limonoid 7-oxo-deacetoxygedunin inhibits RANKL-induced osteoclastogenesis by suppressing activation of the NF-κB and MAPK pathways

International Nuclear Information System (INIS)

Wisutsitthiwong, Chonnaree; Buranaruk, Chayanit; Pudhom, Khanitha; Palaga, Tanapat

2011-01-01

Highlights: ► A gedunin type limonoid from seeds of mangroves, 7-oxo-7-deacetoxygedunin, exhibits strong anti-osteoclastogenic activity. ► Treatment with this limonoid results in significant decrease in expression of NFATc1 and osteoclast-related genes. ► The mode of action of this limonoid is by inhibiting activation of the NF-κB and MAPK pathways which are activated by RANKL. -- Abstract: Osteoclasts together with osteoblasts play pivotal roles in bone remodeling. Aberrations in osteoclast differentiation and activity contribute to osteopenic disease. Osteoclasts differentiate from monocyte/macrophage progenitors, a process that is initiated by the interaction between receptor activator of NF-κB (RANK) and its ligand, RANKL. In this study, we identified 7-oxo-7-deacetoxygedunin (7-OG), a gedunin type limonoid from seeds of the mangrove Xylocarpus moluccensis, as a potent inhibitor of osteoclastogenesis. Additionally, 7-OG showed strong anti-osteoclastogenic activity with low cytotoxicity against the monocyte/macrophage progenitor cell line, RAW264.7. The IC50 for anti-osteoclastogenic activity was 4.14 μM. Treatment with 7-OG completely abolished the appearance of multinucleated giant cells with tartrate-resistant acid phosphatase activity in RAW264.7 cells stimulated with RANKL. When the expression of genes related to osteoclastogenesis was investigated, a complete downregulation of NFATc1 and cathepsin K and a delayed downregulation of irf8 were observed upon 7-OG treatment in the presence of RANKL. Furthermore, treatment with this limonoid suppressed RANKL-induced activation of p38, MAPK and Erk and nuclear localization of NF-κB p65. Taken together, we present evidence indicating a plant limonoid as a novel osteoclastogenic inhibitor that could be used for osteoporosis and related conditions.

Effect of Zn and Mg in tricalcium phosphate and in culture medium on apoptosis and actin ring formation of mature osteoclasts

International Nuclear Information System (INIS)

Li Xia; Ito, Atsuo; Sogo, Yu; Senda, Koji; Yamazaki, Atsushi

2008-01-01

This study investigated the resorptive activity of osteoclasts on tricalcium phosphate (TCP), zinc-containing tricalcium phosphate (ZnTCP) and magnesium-containing tricalcium phosphate (MgTCP) ceramics in different Zn- or Mg-containing culture media. On the TCP ceramic, an increase in Zn ions in the culture medium within the range between 0.3 and 6.8 ppm significantly induced an increase in osteoclast apoptosis and a decrease in actin ring formation. However, even a high level of Mg ions up to 100 ppm in the culture medium was unlikely to induce an increase in osteoclast apoptosis. Mg ions in the MgTCP ceramics have no effect on osteoclast apoptosis and actin ring formation. There was almost no significant difference in osteoclast apoptosis and actin ring formation between ZnTCP and MgTCP ceramics which have the same solubility and dissolution rates. It is indicated that only an increase in Zn level outside resorption lacuna has an inhibitory effect on osteoclast resorption and that an increase in Zn level inside resorption lacuna could not influence the osteoclast activity.

Targeting BCL2 Family in Human Myeloid Dendritic Cells: A Challenge to Cure Diseases with Chronic Inflammations Associated with Bone Loss

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Selma Olsson Åkefeldt

2013-01-01

Full Text Available Rheumatoid arthritis (RA and Langerhans cell histiocytosis (LCH are common and rare diseases, respectively. They associate myeloid cell recruitment and survival in inflammatory conditions with tissue destruction and bone resorption. Manipulating dendritic cell (DC, and, especially, regulating their half-life and fusion, is a challenge. Indeed, these myeloid cells display pathogenic roles in both diseases and may be an important source of precursors for differentiation of osteoclasts, the bone-resorbing multinucleated giant cells. We have recently documented that the proinflammatory cytokine IL-17A regulates long-term survival of DC by inducing BCL2A1 expression, in addition to the constitutive MCL1 expression. We summarize bibliography of the BCL2 family members and their therapeutic targeting, with a special emphasis on MCL1 and BCL2A1, discussing their potential impact on RA and LCH. Our recent knowledge in the survival pathway, which is activated to perform DC fusion in the presence of IL-17A, suggests that targeting MCL1 and BCL2A1 in infiltrating DC may affect the clinical outcomes in RA and LCH. The development of new therapies, interfering with MCL1 and BCL2A1 expression, to target long-term surviving inflammatory DC should be translated into preclinical studies with the aim to increase the well-being of patients with RA and LCH.

Effects of interleukin-7/interleukin-7 receptor on RANKL-mediated osteoclast differentiation and ovariectomy-induced bone loss by regulating c-Fos/c-Jun pathway.

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Zhao, Ji-Jun; Wu, Zhao-Feng; Yu, Ying-Hao; Wang, Ling; Cheng, Li

2018-09-01

To explore the effects of IL-7/IL-7R on the RANKL-mediated osteoclast differentiation in vitro and OVX-induced bone loss in vivo. BMMs and RAW264.7 were transfected with IL-7, IL-7R siRNA, c-Fos siRNA, and c-jun siRNA and later stimulated by RANKL. TRAP and toluidine blue staining were used to observe osteoclast formation and bone resorption, respectively. HE and TRAP staining were used to detect trabecular bone microstructure and osteoclasts of mice, respectively. qRT-PCR and Western blot analysis were used to examine expression. IL-7 unregulated the expression of CTSK, NFATc1, MMP9, and the phosphorylation of p38 and Akt by activating the c-Fos/c-Jun pathway, which increased osteoclast numbers and bone resorption in RANKL-stimulated macrophages. While IL-7R siRNA and c-Fos siRNA decreased the expression, as well as and the phosphorylation of p38 and Akt.IL-7 decreased the BMD and OPG expression in OVX-induced mice and increased the TRAP positive cells, the mRNA expression of c-fos, c-jun, and RANKL, which was contradictory to IL-7R siRNA, and c-Fos siRNA. Furthermore, IL-7R siRNA and c-Fos siRNA caused thicker trabeculae, increased trabecular number, and decreased osteolysis in OVX mice. IL-7/IL-7R can promote RANKL-mediated osteoclast formation and bone resorption by activating the c-Fos/c-Jun pathway, as well as inducing bone loss in OVX mice. © 2018 Wiley Periodicals, Inc.

Methyl Gallate Inhibits Osteoclast Formation and Function by Suppressing Akt and Btk-PLCγ2-Ca2+ Signaling and Prevents Lipopolysaccharide-Induced Bone Loss.

Science.gov (United States)

Baek, Jong Min; Kim, Ju-Young; Lee, Chang Hoon; Yoon, Kwon-Ha; Lee, Myeung Su

2017-03-07

In the field of bone research, various natural derivatives have emerged as candidates for osteoporosis treatment by targeting abnormally elevated osteoclastic activity. Methyl gallate, a plant-derived phenolic compound, is known to have numerous pharmacological effects against inflammation, oxidation, and cancer. Our purpose was to explore the relation between methyl gallate and bone metabolism. Herein, we performed screening using methyl gallate by tartrate resistant acid phosphatase (TRAP) staining and revealed intracellular mechanisms responsible for methyl gallate-mediated regulation of osteoclastogenesis by Western blotting and quantitative reverse transcription polymerase chain reaction (RT-PCR). Furthermore, we assessed the effects of methyl gallate on the characteristics of mature osteoclasts. We found that methyl gallate significantly suppressed osteoclast formation through Akt and Btk-PLCγ2-Ca 2+ signaling. The blockade of these pathways was confirmed through transduction of cells with a CA-Akt retrovirus and evaluation of Ca 2+ influx intensity (staining with Fluo-3/AM). Indeed, methyl gallate downregulated the formation of actin ring-positive osteoclasts and resorption pit areas. In agreement with in vitro results, we found that administration of methyl gallate restored osteoporotic phenotype stimulated by acute systemic injection of lipopolysaccharide in vivo according to micro-computed tomography and histological analysis. Our data strongly indicate that methyl gallate may be useful for the development of a plant-based antiosteoporotic agent.

Methyl Gallate Inhibits Osteoclast Formation and Function by Suppressing Akt and Btk-PLCγ2-Ca2+ Signaling and Prevents Lipopolysaccharide-Induced Bone Loss

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Jong Min Baek

2017-03-01

Full Text Available In the field of bone research, various natural derivatives have emerged as candidates for osteoporosis treatment by targeting abnormally elevated osteoclastic activity. Methyl gallate, a plant-derived phenolic compound, is known to have numerous pharmacological effects against inflammation, oxidation, and cancer. Our purpose was to explore the relation between methyl gallate and bone metabolism. Herein, we performed screening using methyl gallate by tartrate resistant acid phosphatase (TRAP staining and revealed intracellular mechanisms responsible for methyl gallate-mediated regulation of osteoclastogenesis by Western blotting and quantitative reverse transcription polymerase chain reaction (RT-PCR. Furthermore, we assessed the effects of methyl gallate on the characteristics of mature osteoclasts. We found that methyl gallate significantly suppressed osteoclast formation through Akt and Btk-PLCγ2-Ca2+ signaling. The blockade of these pathways was confirmed through transduction of cells with a CA-Akt retrovirus and evaluation of Ca2+ influx intensity (staining with Fluo-3/AM. Indeed, methyl gallate downregulated the formation of actin ring-positive osteoclasts and resorption pit areas. In agreement with in vitro results, we found that administration of methyl gallate restored osteoporotic phenotype stimulated by acute systemic injection of lipopolysaccharide in vivo according to micro-computed tomography and histological analysis. Our data strongly indicate that methyl gallate may be useful for the development of a plant-based antiosteoporotic agent.

Naringin prevents ovariectomy-induced osteoporosis and promotes osteoclasts apoptosis through the mitochondria-mediated apoptosis pathway

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Li, Fengbo [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin TJ 300050 (China); Graduate School of Tianjin Medical University, No. 22, Qixiangtai Street, Heping District, Tianjin 300070 (China); Sun, Xiaolei; Ma, Jianxiong [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin TJ 300050 (China); Ma, Xinlong, E-mail: gengxiao502@163.com [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin TJ 300050 (China); Zhao, Bin; Zhang, Yang; Tian, Peng [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin TJ 300050 (China); Li, Yanjun [Graduate School of Tianjin Medical University, No. 22, Qixiangtai Street, Heping District, Tianjin 300070 (China); Han, Zhe [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin TJ 300050 (China)

2014-09-26

Highlights: • Naringin possesses many pharmacological activities, promotes the proliferation of osteoblast. • Undecalcified histological obtain dynamic parameters of callus formation and remodeling. • Naringin regulate osteoclast apoptosis by mitochondrial pathway. - Abstract: Naringin, the primary active compound of the traditional Chinese medicine Rhizoma drynariae, possesses many pharmacological activities. The present study is an effort to explore the anti-osteoporosis potential of naringin in vivo and in vitro. In vivo, we used ovariectomized rats to clarify the mechanisms by which naringin anti-osteoporosis. In vitro, we used osteoclasts to investigate naringin promotes osteoclasts apoptosis. Naringin was effective at enhancing BMD, trabecular thickness, bone mineralization, and mechanical strength in a dose-dependent manner. The result of RT-PCR analysis revealed that naringin down-regulated the mRNA expression levels of BCL-2 and up-regulated BAX, caspase-3 and cytochrome C. In addition, naringin significantly reduced the bone resorption area in vitro. These findings suggest that naringin promotes the apoptosis of osteoclasts by regulating the activity of the mitochondrial apoptosis pathway and prevents OVX-induced osteoporosis in rats.

Naringin prevents ovariectomy-induced osteoporosis and promotes osteoclasts apoptosis through the mitochondria-mediated apoptosis pathway

International Nuclear Information System (INIS)

Li, Fengbo; Sun, Xiaolei; Ma, Jianxiong; Ma, Xinlong; Zhao, Bin; Zhang, Yang; Tian, Peng; Li, Yanjun; Han, Zhe

2014-01-01

Highlights: • Naringin possesses many pharmacological activities, promotes the proliferation of osteoblast. • Undecalcified histological obtain dynamic parameters of callus formation and remodeling. • Naringin regulate osteoclast apoptosis by mitochondrial pathway. - Abstract: Naringin, the primary active compound of the traditional Chinese medicine Rhizoma drynariae, possesses many pharmacological activities. The present study is an effort to explore the anti-osteoporosis potential of naringin in vivo and in vitro. In vivo, we used ovariectomized rats to clarify the mechanisms by which naringin anti-osteoporosis. In vitro, we used osteoclasts to investigate naringin promotes osteoclasts apoptosis. Naringin was effective at enhancing BMD, trabecular thickness, bone mineralization, and mechanical strength in a dose-dependent manner. The result of RT-PCR analysis revealed that naringin down-regulated the mRNA expression levels of BCL-2 and up-regulated BAX, caspase-3 and cytochrome C. In addition, naringin significantly reduced the bone resorption area in vitro. These findings suggest that naringin promotes the apoptosis of osteoclasts by regulating the activity of the mitochondrial apoptosis pathway and prevents OVX-induced osteoporosis in rats

Osteopetrorickets due to Snx10 deficiency in mice results from both failed osteoclast activity and loss of gastric acid-dependent calcium absorption.

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Liang Ye

2015-03-01

Full Text Available Mutations in sorting nexin 10 (Snx10 have recently been found to account for roughly 4% of all human malignant osteopetrosis, some of them fatal. To study the disease pathogenesis, we investigated the expression of Snx10 and created mouse models in which Snx10 was knocked down globally or knocked out in osteoclasts. Endocytosis is severely defective in Snx10-deficient osteoclasts, as is extracellular acidification, ruffled border formation, and bone resorption. We also discovered that Snx10 is highly expressed in stomach epithelium, with mutations leading to high stomach pH and low calcium solubilization. Global Snx10-deficiency in mice results in a combined phenotype: osteopetrosis (due to osteoclast defect and rickets (due to high stomach pH and low calcium availability, resulting in impaired bone mineralization. Osteopetrorickets, the paradoxical association of insufficient mineralization in the context of a positive total body calcium balance, is thought to occur due to the inability of the osteoclasts to maintain normal calcium-phosphorus homeostasis. However, osteoclast-specific Snx10 knockout had no effect on calcium balance, and therefore led to severe osteopetrosis without rickets. Moreover, supplementation with calcium gluconate rescued mice from the rachitic phenotype and dramatically extended life span in global Snx10-deficient mice, suggesting that this may be a life-saving component of the clinical approach to Snx10-dependent human osteopetrosis that has previously gone unrecognized. We conclude that tissue-specific effects of Snx10 mutation need to be considered in clinical approaches to this disease entity. Reliance solely on hematopoietic stem cell transplantation can leave hypocalcemia uncorrected with sometimes fatal consequences. These studies established an essential role for Snx10 in bone homeostasis and underscore the importance of gastric acidification in calcium uptake.

Effect of vibration on osteoblastic and osteoclastic activities: Analysis of bone metabolism using goldfish scale as a model for bone

Science.gov (United States)

Suzuki, N.; Kitamura, K.; Nemoto, T.; Shimizu, N.; Wada, S.; Kondo, T.; Tabata, M. J.; Sodeyama, F.; Ijiri, K.; Hattori, A.

In osteoclastic activity during space flight as well as hind limb unloading by tail suspension, inconsistent results have been reported in an in vivo study. The bone matrix plays an important role in the response to physical stress. However, there is no suitable in vitro co-culture system of osteoblasts and osteoclasts including bone matrix. On the other hand, fish scale is a calcified tissue that contains osteoblasts, osteoclasts, and bone matrix, all of which are similar to those found in human bones. Recently, we developed a new in vitro model system using goldfish scale. This system can detect the activities of osteoclasts and osteoblasts with tartrate-resistant acid phosphatase and alkaline phosphatase as the respective markers and precisely analyze the co-relationship between osteoblasts and osteoclasts. Using this system, we analyzed the bone metabolism under various degrees of acceleration (0.5-, 1-, 2-, 4-, and 6-G) by vibration with a G-load apparatus. After loading for 5 and 10 min, the scales were incubated for 6 and 24 h. The osteoblastic and osteoclastic activities were then measured. The osteoblastic activities gradually increased corresponding to 1-G to 6-G acceleration. In addition, ER mRNA expression was the highest under 6-G acceleration. On the other hand, the osteoclastic activity decreased at 24 h of incubation under low acceleration (0.5- and 1-G). This change coincided with TRAP mRNA expression. Under 2-G acceleration, the strength of suppression in osteoclastic activity was the highest. The strength of the inhibitory action under 4- and 6-G acceleration was lower than that under 2-G acceleration. In our co-culture system, osteoblasts and osteoclasts in the scale sensitively responded to several degrees of acceleration. Therefore, we strongly believe that our in vitro co-culture system is useful for the analysis of bone metabolism under loading or unloading.

Combined treatment with Dendrobium candidum and black tea extract promotes osteoprotective activity in ovariectomized estrogen deficient rats and osteoclast formation.

Science.gov (United States)

Wang, Ming-Yue; Shen, Chang; An, Meng-Fei; Xie, Chuan-Qi; Wu, Xin; Zhu, Qiang-Qiang; Sun, Bin; Huang, Yan-Ping; Zhao, Yun-Li; Wang, Xuan-Jun; Sheng, Jun

2018-05-01

Dendrobium candidum (DC) and black tea, are traditional chinese drinks, which contain multiple active ingredients. However, whether or not the combination of these two ingredients can improve osteoporosis remains unknown. This study therefore aimed to examine the effects of the combination of DC and black tea extract (BTE) on osteoporosis. Ovariectomy (OVX)-induced osteoporosis in vivo as well as receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis in vitro was selected. Results showed that OVX rats that were treated orally with a DC and BTE combination for 12 weeks maintained their calcium (Ca) and phosphorus (P) homeostasis and exhibited significantly enhanced estradiol (E 2 ) and OPG levels. This combination treatment also simultaneously reduced levels of interleukin (IL)-1β, IL-6 and improved the organ coefficients of the uterus and femur as well as BMD and BMC in OVX rats. In addition, this DC and BTE combination suppressed osteoclast differentiation in the RANKL-stimulated osteoclastogenesis of RAW 264.7 cells and effectively inhibited the expression of osteoclast-associated genes and proteins. The results of this study further highlight the fact that a combination of DC and BTE improved ovariectomy-induced osteoporosis in rats and suppressed RANKL-stimulated osteoclastogenesis in RAW 264.7 cells. This combination also significantly alleviated osteoporosis when compared to the alternative sole treatments above, due to synergistic effects among components. One partial mechanism of this combination might be the inhibition of osteoclast proliferation and the regulation of NFATC1/c-Fos expression. Copyright © 2018 Elsevier Inc. All rights reserved.

Selected Alkylating Agents Can Overcome Drug Tolerance of G0-like Tumor Cells and Eradicate BRCA1-Deficient Mammary Tumors in Mice.

Science.gov (United States)

Pajic, Marina; Blatter, Sohvi; Guyader, Charlotte; Gonggrijp, Maaike; Kersbergen, Ariena; Küçükosmanoğlu, Aslι; Sol, Wendy; Drost, Rinske; Jonkers, Jos; Borst, Piet; Rottenberg, Sven

2017-11-15

Purpose: We aimed to characterize and target drug-tolerant BRCA1-deficient tumor cells that cause residual disease and subsequent tumor relapse. Experimental Design: We studied responses to various mono- and bifunctional alkylating agents in a genetically engineered mouse model for BRCA1/p53 -mutant breast cancer. Because of the large intragenic deletion of the Brca1 gene, no restoration of BRCA1 function is possible, and therefore, no BRCA1-dependent acquired resistance occurs. To characterize the cell-cycle stage from which Brca1 -/- ;p53 -/- mammary tumors arise after cisplatin treatment, we introduced the fluorescent ubiquitination-based cell-cycle indicator (FUCCI) construct into the tumor cells. Results: Despite repeated sensitivity to the MTD of platinum drugs, the Brca1 -mutated mammary tumors are not eradicated, not even by a frequent dosing schedule. We show that relapse comes from single-nucleated cells delaying entry into the S-phase. Such slowly cycling cells, which are present within the drug-naïve tumors, are enriched in tumor remnants. Using the FUCCI construct, we identified nonfluorescent G 0 -like cells as the population most tolerant to platinum drugs. Intriguingly, these cells are more sensitive to the DNA-crosslinking agent nimustine, resulting in an increased number of multinucleated cells that lack clonogenicity. This is consistent with our in vivo finding that the nimustine MTD, among several alkylating agents, is the most effective in eradicating Brca1 -mutated mouse mammary tumors. Conclusions: Our data show that targeting G 0 -like cells is crucial for the eradication of BRCA1/p53-deficient tumor cells. This can be achieved with selected alkylating agents such as nimustine. Clin Cancer Res; 23(22); 7020-33. ©2017 AACR . ©2017 American Association for Cancer Research.

Time-lapse reveals that osteoclasts can move across the bone surface while resorbing

DEFF Research Database (Denmark)

Søe, Kent; Delaissé, Jean-Marie

2017-01-01

, clear real-time observations are still lacking. Herein, we used specific markers and time-lapse to monitor live the spatiotemporal generation of resorption events by osteoclasts cultured on bone slices. In accordance with the current view, we found alternating episodes of resorption and migration...... trenches. Compared to pit events, trench events show properties enabling higher aggressiveness: long duration (days), high erosion speed (two times faster) and long-distance erosion (several 100 µm). Simultaneous resorption and migration reflect a unique situation where epithelial/secretory and mesenchymal....../migratory characteristics are integrated into just one cell phenotype, and deserves attention in future research....

Urokinase plasminogen activator receptor affects bone homeostasis by regulating osteoblast and osteoclast function

DEFF Research Database (Denmark)

Furlan, Federico; Galbiati, Clara; Jørgensen, Niklas R

2007-01-01

PAR and produce urokinase (uPA). The purpose of this study was to investigate the role of uPAR in bone remodeling. MATERIALS AND METHODS: In vivo studies were performed in uPAR knockout (KO) and wildtype (WT) mice on a C57Bl6/SV129 (75:25) background. Bone mass was analyzed by pQCT. Excised tibias were subjected......The uPAR and its ligand uPA are expressed by both osteoblasts and osteoclasts. Their function in bone remodeling is unknown. We report that uPAR-lacking mice display increased BMD, increased osteogenic potential of osteoblasts, decreased osteoclasts formation, and altered cytoskeletal...... of macrophage-colony stimulating factor (M-CSF) and RANKL. Phalloidin staining in osteoclasts served to study actin ring and podosome formation. RESULTS: pQCT revealed increased bone mass in uPAR-null mice. Mechanical tests showed reduced load-sustaining capability in uPAR KO tibias. uPAR KO osteoblasts showed...

Psoralidin, a prenylated coumestan, as a novel anti-osteoporosis candidate to enhance bone formation of osteoblasts and decrease bone resorption of osteoclasts

DEFF Research Database (Denmark)

Zhai, Yuankun; Li, Yingying; Wang, Yanping

2017-01-01

Traditional Chinese medicines (TCM) have been proven to prevent osteoporosis, but their clinical applications are not widely recognized due to their complicated ingredients. Psoralidin, a prenylated coumestan, has been reported to prevent bone loss of ovariectomized rats, but detailed mechanisms...... and osteoclastic bone resorption, as demonstrated by the lower tartrate-resistant acid phosphatase activity and smaller area, with fewer resorption pits formed. Interestingly, psoralidin showed much stronger effects than coumestrol at enhancing osteoblast proliferation/differentiation or inhibiting osteoclast...... differentiation and bone resorption. Moreover, we found that both psoralidin and coumestrol suppressed COX-2 and ROS production in rat osteoblastic calvarias cells, and psoralidin showed stronger effects than coumestrol. Furthermore, we detected that by blocking estrogen receptors with ICI 182.780 (an estrogen...

Age-related effects on osteoclastic activities after orthodontic tooth movement.

Science.gov (United States)

Li, X; Li, M; Lu, J; Hu, Y; Cui, L; Zhang, D; Yang, Y

2016-10-01

To elucidate the effects of age on the expression levels of the receptor activator of the nuclear factor-κB ligand (RANKL) and osteoclasts in the periodontal ligament during orthodontic mechanical loading and post-orthodontic retention. The study included 20 male Sprague-Dawley rats, ten in the young group (aged four to five weeks) and ten in the adult group (aged 18 to 20 weeks). In each rat, the upper-left first molar was subjected to a seven-day orthodontic force loading followed by a seven-day retention period. The upper-right first molar served as a control. The amount of orthodontic tooth movement was measured after seven-day force application and seven-day post-orthodontic retention. The expression levels of RANKL and the tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts were evaluated on day 7 (end of mechanical force loading) and day 14 (after seven days of post-orthodontic retention). Statistical analysis was performed using the t-test, and significance was set at p 0.05) after the seven-day force application. On the compression side, the expression of RANKL and TRAP-positive osteoclasts in both the young and the adult groups increased after the application of force for seven days, and then decreased at the end of the seven-day retention period. However, by the end of the period, the expression of RANKL on the compression side dropped to the control level in the young group (p > 0.05), while it was still higher than that on the control side in the adult group (p 0.05), but it was significantly higher in the adult group than that in the young group after seven-day post-orthodontic retention (p Cite this article: X. Li, M. Li, J. Lu, Y. Hu, L. Cui, D. Zhang, Y. Yang. Age-related effects on osteoclastic activities after orthodontic tooth movement. Bone Joint Res 2016;5:492-499. DOI: 10.1302/2046-3758.510.BJR-2016-0004.R2. © 2016 Li et al.

P2X7 receptor regulates osteoclast function and bone loss in a mouse model of osteoporosis.

Science.gov (United States)

Wang, Ning; Agrawal, Ankita; Jørgensen, Niklas Rye; Gartland, Alison

2018-02-22

Post-menopausal osteoporosis is a condition that affects millions worldwide and places a huge socio-economic burden on society. Previous research has shown an association of loss of function SNPs in the gene for the purinergic receptor P2X7R with low bone mineral density, increased rates of bone loss and vertebral fractures in post-menopausal women. In this study we use a mouse model of oestrogen deficiency-induced bone loss and the BALB/cJ P2X7R -/- to show that absence of the P2X7R resulted in increased bone loss. Osteoclast precursors were isolated from both BALB/cJ P2X7R -/- and BALB/cJ P2X7R +/+ mice and then cultured in vitro to form mature resorbing osteoclasts. The BALB/cJ P2X7R -/- derived precursors generated slightly more osteoclasts but with a significant reduction in the amount of resorption per osteoclast. Furthermore, when using modified culture conditions osteoclast activity was additionally increased in the absence of the P2X7R suggest that P2X7R may regulate the lifespan and activity of osteoclasts. Finally using mechanical loading as an anabolic stimulus for bone formation, we demonstrated that the increased oestrogen-deficient bone loss could be rescued, even in the absence of P2X7R. This study paves the way for clinical intervention for women with post-menopausal osteoporosis and P2XR7 loss of function polymorphisms.

Ion transporters involved in acidification of the resorption lacuna in osteoclasts

DEFF Research Database (Denmark)

Henriksen, K.; Sorensen, M.G.; Jensen, V.K.

2008-01-01

Osteoclasts possess a large amount of ion transporters, which participate in bone resorption; of these, the vacuolar-adenosine trisphosphatase (V-ATPase) and the chloride-proton antiporter ClC-7 acidify the resorption lacuna. However, whether other ion transporters participate in this process is ......, including carbonic anhydrase II, the NHEs, and potassium-chloride cotransporters, are all involved in resorption but do not seem to directly be involved in acidification of the lysosomes Udgivelsesdato: 2008/9......Osteoclasts possess a large amount of ion transporters, which participate in bone resorption; of these, the vacuolar-adenosine trisphosphatase (V-ATPase) and the chloride-proton antiporter ClC-7 acidify the resorption lacuna. However, whether other ion transporters participate in this process...

Growth/differentiation factor-15 inhibits differentiation into osteoclasts - A novel factor involved in control of osteoclast differentiation

Czech Academy of Sciences Publication Activity Database

Vaňhara, P.; Lincová, Eva; Kozubík, Alois; Jurdic, P.; Souček, Karel; Šmarda, J.

2009-01-01

Roč. 78, č. 4 (2009), s. 213-222 ISSN 0301-4681 R&D Projects: GA ČR(CZ) GA204/07/0834 Grant - others:GA ČR(CZ) GA301/06/0036; GA ČR(CZ) GD204/08/H054; GA ČR(CZ) GA310/07/0961 Program:GA Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : osteoclast differentiation * GDF-15 * prostate cancer Subject RIV: BO - Biophysics Impact factor: 3.311, year: 2009

Therapeutic effects of anti-CD115 monoclonal antibody in mouse cancer models through dual inhibition of tumor-associated macrophages and osteoclasts.

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Laetitia Fend

Full Text Available Tumor progression is promoted by Tumor-Associated Macrophages (TAMs and metastasis-induced bone destruction by osteoclasts. Both myeloid cell types depend on the CD115-CSF-1 pathway for their differentiation and function. We used 3 different mouse cancer models to study the effects of targeting cancer host myeloid cells with a monoclonal antibody (mAb capable of blocking CSF-1 binding to murine CD115. In mice bearing sub-cutaneous EL4 tumors, which are CD115-negative, the anti-CD115 mAb depleted F4/80(+ CD163(+ M2-type TAMs and reduced tumor growth, resulting in prolonged survival. In the MMTV-PyMT mouse model, the spontaneous appearance of palpable mammary tumors was delayed when the anti-CD115 mAb was administered before malignant transition and tumors became palpable only after termination of the immunotherapy. When administered to mice already bearing established PyMT tumors, anti-CD115 treatment prolonged their survival and potentiated the effect of chemotherapy with Paclitaxel. As shown by immunohistochemistry, this therapeutic effect correlated with the depletion of F4/80(+CD163(+ M2-polarized TAMs. In a breast cancer model of bone metastasis, the anti-CD115 mAb potently blocked the differentiation of osteoclasts and their bone destruction activity. This resulted in the inhibition of cancer-induced weight loss. CD115 thus represents a promising target for cancer immunotherapy, since a specific blocking antibody may not only inhibit the growth of a primary tumor through TAM depletion, but also metastasis-induced bone destruction through osteoclast inhibition.

Degradation of the organic phase of bone by osteoclasts

DEFF Research Database (Denmark)

Henriksen, Kim; Sørensen, Mette G; Nielsen, Rasmus H

2006-01-01

Osteoclasts degrade bone matrix by secretion of hydrochloric acid and proteases. We studied the processes involved in the degradation of the organic matrix of bone in detail and found that lysosomal acidification is involved in this process and that MMPs are capable of degrading the organic matrix...

Safflower bud inhibits RANKL-induced osteoclast differentiation and prevents bone loss in ovariectomized mice.

Science.gov (United States)

Choi, Joo-Hee; Lim, Seul-Ki; Kim, Dong-Il; Park, Min-Jung; Kim, Young-Kuk; Lee, An-Chul; Kim, Young-Min; Yang, Soo-Jin; Park, Jong-Hwan

2017-10-15

The powder and extract of safflower seeds are known to be effective in the prevention of bone loss in ovariectomized animals. However, the inhibitory effect and molecular mechanisms of safflower bud (SB), the germinated safflower, on bone destruction is unclear. The present study was designed to investigate the inhibitory effect and molecular mechanism of SB on osteoclastic differentiation and on bone loss in ovarietomized (OVX) mice. Osteoclastogenesis was determined by TRAP staining, F-actin ring formation, and bone resorption assay. NF-κB and MAPKs activation was analyzed by transfection assay and Western blot, respectively. Real-time PCR was performed to examine the expression of osteoclastogenesis-related genes. Histological changes, increases in TRAP-positive cells, and cathepsin K expression were examined in the metaphysis of OVX mice. Density of bone marrow was evaluated by µCT. SB inhibited the RANKL-induced differentiation of BMDMs into osteoclasts in a dose-dependent manner. F-actin ring formation and bone resorption were also reduced by SB in RANKL-treated BMDMs. In addition, SB decreased the activation of NF-κB and MAPKs and the expression of osteoclastogenesis-related genes in BMDMs treated with RANKL. Feeding of SB-included diet prevented bone loss in OVX mice. The number of TRAP-positive cells and level of protein expression of cathepsin K was reduced and bone mineral density was increased in the metaphysis of mice fed SB compared with OVX mice. These findings suggest that SB can be a preventive and therapeutic candidate for destructive bone diseases. Copyright © 2017. Published by Elsevier GmbH.

Structure-based design of an osteoclast-selective, nonpeptide Src homology 2 inhibitor with in vivo antiresorptive activity

Science.gov (United States)

Shakespeare, William; Yang, Michael; Bohacek, Regine; Cerasoli, Franklin; Stebbins, Karin; Sundaramoorthi, Raji; Azimioara, Mihai; Vu, Chi; Pradeepan, Selvi; Metcalf, Chester; Haraldson, Chad; Merry, Taylor; Dalgarno, David; Narula, Surinder; Hatada, Marcos; Lu, Xiaode; van Schravendijk, Marie Rose; Adams, Susan; Violette, Shelia; Smith, Jeremy; Guan, Wei; Bartlett, Catherine; Herson, Jay; Iuliucci, John; Weigele, Manfred; Sawyer, Tomi

2000-01-01

Targeted disruption of the pp60src (Src) gene has implicated this tyrosine kinase in osteoclast-mediated bone resorption and as a therapeutic target for the treatment of osteoporosis and other bone-related diseases. Herein we describe the discovery of a nonpeptide inhibitor (AP22408) of Src that demonstrates in vivo antiresorptive activity. Based on a cocrystal structure of the noncatalytic Src homology 2 (SH2) domain of Src complexed with citrate [in the phosphotyrosine (pTyr) binding pocket], we designed 3′,4′-diphosphonophenylalanine (Dpp) as a pTyr mimic. In addition to its design to bind Src SH2, the Dpp moiety exhibits bone-targeting properties that confer osteoclast selectivity, hence minimizing possible undesired effects on other cells that have Src-dependent activities. The chemical structure AP22408 also illustrates a bicyclic template to replace the post-pTyr sequence of cognate Src SH2 phosphopeptides such as Ac-pTyr-Glu-Glu-Ile (1). An x-ray structure of AP22408 complexed with Lck (S164C) SH2 confirmed molecular interactions of both the Dpp and bicyclic template of AP22408 as predicted from molecular modeling. Relative to the cognate phosphopeptide, AP22408 exhibits significantly increased Src SH2 binding affinity (IC50 = 0.30 μM for AP22408 and 5.5 μM for 1). Furthermore, AP22408 inhibits rabbit osteoclast-mediated resorption of dentine in a cellular assay, exhibits bone-targeting properties based on a hydroxyapatite adsorption assay, and demonstrates in vivo antiresorptive activity in a parathyroid hormone-induced rat model. PMID:10944210

Expression of osteoblast and osteoclast regulatory genes in the bone marrow microenvironment in multiple myeloma

DEFF Research Database (Denmark)

Kristensen, Ida B; Christensen, Jacob Haaber; Lyng, Maria Bibi

2014-01-01

Multiple myeloma (MM) lytic bone disease (LBD) is caused by osteoclast activation and osteoblast inhibition. RANK/RANKL/OPG play central roles in osteoclast activation and Wnt inhibitor DKK1 in osteoblast inhibition. The role of other Wnt inhibitors is less clear. We evaluated gene expression...... of osteoclast regulators (RANK, RANKL, OPG, TRAIL, MIP1A), Wnt inhibitors (DKK1, SFRP2, SFRP3, sclerostin, WIF1) and osteoblast transcription factors (RUNX2, osterix) by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) in the bone marrow (BM) microenvironment using snap-frozen BM biopsies...... radiographs and the bone resorption marker CTX-1. Protein levels were evaluated by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. Among Wnt inhibitors, only SFRP3 and DKK1 were significantly overexpressed in advanced LBD, correlating with protein levels. SFRP3 correlated with CTX-1. Our...

A Dictyostelium secreted factor requires a PTEN-like phosphatase to slow proliferation and induce chemorepulsion.

Science.gov (United States)

Herlihy, Sarah E; Tang, Yitai; Gomer, Richard H

2013-01-01

In Dictyostelium discoideum, AprA and CfaD are secreted proteins that inhibit cell proliferation. We found that the proliferation of cells lacking CnrN, a phosphatase and tensin homolog (PTEN)-like phosphatase, is not inhibited by exogenous AprA and is increased by exogenous CfaD. The expression of CnrN in cnrN cells partially rescues these altered sensitivities, suggesting that CnrN is necessary for the ability of AprA and CfaD to inhibit proliferation. Cells lacking CnrN accumulate normal levels of AprA and CfaD. Like cells lacking AprA and CfaD, cnrN cells proliferate faster and reach a higher maximum cell density than wild type cells, tend to be multinucleate, accumulate normal levels of mass and protein per nucleus, and form less viable spores. When cnrN cells expressing myc-tagged CnrN are stimulated with a mixture of rAprA and rCfaD, levels of membrane-associated myc-CnrN increase. AprA also causes chemorepulsion of Dictyostelium cells, and CnrN is required for this process. Combined, these results suggest that CnrN functions in a signal transduction pathway downstream of AprA and CfaD mediating some, but not all, of the effects of AprA and CfaD.

Exosome-Mediated Genetic Information Transfer, a Missing Piece of Osteoblast-Osteoclast Communication Puzzle.

Science.gov (United States)

Yin, Pengbin; Lv, Houchen; Li, Yi; Deng, Yuan; Zhang, Licheng; Tang, Peifu

2017-01-01

The skeletal system functions and maintains itself based on communication between cells of diverse origins, especially between osteoblasts (OBs) and osteoclasts (OCs), accounting for bone formation and resorption, respectively. Previously, protein-level information exchange has been the research focus, and this has been discussed in detail. The regulative effects of microRNAs (miRNAs) on OB and OC ignite the question as to whether genetic information could be transferred between bone cells. Exosomes, extracellular membrane vesicles 30-100 nm in diameter, have recently been demonstrated to transfer functional proteins, mRNAs, and miRNAs, and serve as mediators of intercellular communication. By reviewing the distinguishing features of exosomes, a hypothesis was formulated and evaluated in this article that exosome-mediated genetic information transfer may represent a novel strategy for OB-OC communication. The exosomes may coordinately regulate these two cells under certain physiological conditions by transferring genetic information. Further research in exosome-shuttered miRNAs in OB-OC communication may add a missing piece to the bone cells communication "puzzle."

Norisoboldine suppresses osteoclast differentiation through preventing the accumulation of TRAF6-TAK1 complexes and activation of MAPKs/NF-κB/c-Fos/NFATc1 Pathways.

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Zhi-Feng Wei

Full Text Available Norisoboldine (NOR is the main alkaloid constituent in the dry root of Lindera aggregata (Sims Kosterm. (L. strychnifolia Vill.. As reported previously, orally administered NOR displayed a robust inhibition of joint bone destruction present in both mouse collagen-induced arthritis and rat adjuvant-induced arthritis with lower efficacious doses than that required for ameliorating systemic inflammation. This attracted us to assess the effects of NOR on differentiation and function of osteoclasts, primary effector cells for inflammatory bone destruction, to get insight into its anti-rheumatoid arthritis mechanisms. Both RAW264.7 cells and mouse bone marrow-derived macrophages (BMMs were stimulated with RANKL (100 ng/mL to establish osteoclast differentiation models. ELISA, RT-PCR, gelatin zymography, western blotting, immunoprecipitation and EMSA were used to reveal related signalling pathways. NOR (10 and 30 µM, without significant cytotoxicity, showed significant reduction of the number of osteoclasts and the resorption pit areas, and it targeted osteoclast differentiation at the early stage. In conjunction with the anti-resorption effect of NOR, mRNA levels of cathepsin K and MMP-9 were decreased, and the activity of MMP-9 was attenuated. Furthermore, our mechanistic studies indicated that NOR obviously suppressed the ubiquitination of TRAF6, the accumulation of TRAF6-TAK1 complexes and the activation of ERK and p38 MAPK, and reduced the nuclear translocation of NF-κB-p65 and DNA-binding activity of NF-κB. However, NOR had little effect on expressions of TRAF6 or the phosphorylation and degradation of IκBα. Moreover, NOR markedly inhibited expressions of transcription factor NFATc1, but not c-Fos. Intriguingly, the subsequent nuclear translocations of c-Fos and NFATc1 were substantially down-regulated. Hence, we demonstrated for the first time that preventing the differentiation and function of osteoclasts at the early stage was an

Osteoclastic bone degradation and the role of different cysteine proteinases and matrix metalloproteinases: differences between calvaria and long bone

NARCIS (Netherlands)

Everts, Vincent; Korper, Wolf; Hoeben, Kees A.; Jansen, Ineke D. C.; Bromme, Dieter; Cleutjens, Kitty B. J. M.; Heeneman, Sylvia; Peters, Christoph; Reinheckel, Thomas; Saftig, Paul; Beertsen, Wouter

2006-01-01

Osteoclastic bone degradation involves the activity of cathepsin K. We found that in addition to this enzyme other, yet unknown, cysteine proteinases participate in digestion. The results support the notion that osteoclasts from different bone sites use different enzymes to degrade the collagenous

Impact of Bisphosphonate on Orthodontic tooth movement and osteoclastic count: An Animal Study.

Science.gov (United States)

Venkataramana, V; Chidambaram, S; Reddy, B Vishnuvardhan; Goud, E V Soma Shekara; Arafath, Mohammed; Krishnan6, Santhana

2014-04-01

Background : The aim of the current study is to examine the effect of systemically administered BP-Pamidronate, on Orthodontic Tooth Movement (OTM) along with osteoclastic quantification in New Zealand white rabbits. Materials & Methods : Twenty rabbits used in the study, were equally divided into 2 groups ; Group-1 as Control & Group-2 as Experimental. A sentalloy NITI closed coil spring (GAC International, USA) of 100 gram force, ligated between the lower first molar and the anterior most incisors of the rabbit has served as orthodontic force element. The BP- Pamidronate was administered at the dosage of 1.5 mg/kg body intra-peritonially, on the 1st, 7th and 14th day of the experiment. On the 21st day both group of animals were sacrificed, mandibles were dissected. The formed diastema between the 1st and 2nd molar was measured on the dissected mandibles using standard metric scale, which is considered as the OTM in the mesial direction. Next, the alveolar bone regions along with intact mesial surfaces were processed for histological investigation (osteoclastic count). Results : The student 't' test has been done to compare the mean values of molar tooth movement and osteoclastic count. Parameter :1 molar tooth movement has shown a significant difference between the control (3.750 ± 0.548 mm) and the experimental group (3.050 ± 0.556 mm) with calculated 'p' value (p-value <0.05) is significant at 0.0110 level. Parameter : 2 osteoclastic count has shown a significant difference between the control (13.335000 ± 0.735856 per square mm.) and the experimental group (11.426900 ± 1.49369 per square mm) calculated 'p' value (p-value <0.05) is significant at 0.003 level. Conclusion : The molar tooth movement and the osteoclastic count were significantly reduced in BP - Pamidronate administered animals than non-drug recipients. How to cite the article: Venkataramana V, Chidambaram S, Reddy BV, Goud EV, Arafath M, Krishnan S. Impact of Bisphosphonate on Orthodontic tooth

Osteomacs interact with megakaryocytes and osteoblasts to regulate murine hematopoietic stem cell function.

Science.gov (United States)

Mohamad, Safa F; Xu, Linlin; Ghosh, Joydeep; Childress, Paul J; Abeysekera, Irushi; Himes, Evan R; Wu, Hao; Alvarez, Marta B; Davis, Korbin M; Aguilar-Perez, Alexandra; Hong, Jung Min; Bruzzaniti, Angela; Kacena, Melissa A; Srour, Edward F

2017-12-12

Networking between hematopoietic stem cells (HSCs) and cells of the hematopoietic niche is critical for stem cell function and maintenance of the stem cell pool. We characterized calvariae-resident osteomacs (OMs) and their interaction with megakaryocytes to sustain HSC function and identified distinguishing properties between OMs and bone marrow (BM)-derived macrophages. OMs, identified as CD45 + F4/80 + cells, were easily detectable (3%-5%) in neonatal calvarial cells. Coculture of neonatal calvarial cells with megakaryocytes for 7 days increased OM three- to sixfold, demonstrating that megakaryocytes regulate OM proliferation. OMs were required for the hematopoiesis-enhancing activity of osteoblasts, and this activity was augmented by megakaryocytes. Serial transplantation demonstrated that HSC repopulating potential was best maintained by in vitro cultures containing osteoblasts, OMs, and megakaryocytes. With or without megakaryocytes, BM-derived macrophages were unable to functionally substitute for neonatal calvarial cell-associated OMs. In addition, OMs differentiated into multinucleated, tartrate resistant acid phosphatase-positive osteoclasts capable of bone resorption. Nine-color flow cytometric analysis revealed that although BM-derived macrophages and OMs share many cell surface phenotypic similarities (CD45, F4/80, CD68, CD11b, Mac2, and Gr-1), only a subgroup of OMs coexpressed M-CSFR and CD166, thus providing a unique profile for OMs. CD169 was expressed by both OMs and BM-derived macrophages and therefore was not a distinguishing marker between these 2 cell types. These results demonstrate that OMs support HSC function and illustrate that megakaryocytes significantly augment the synergistic activity of osteoblasts and OMs. Furthermore, this report establishes for the first time that the crosstalk between OMs, osteoblasts, and megakaryocytes is a novel network supporting HSC function.

A supra-cellular model for coupling of bone resorption to formation during remodeling

DEFF Research Database (Denmark)

Jensen, Pia Rosgaard; Andersen, Thomas Levin; Pennypacker, Brenda L

2014-01-01

The bone matrix is maintained functional through the combined action of bone resorbing osteoclasts and bone forming osteoblasts, in so-called bone remodeling units. The coupling of these two activities is critical for securing bone replenishment and involves osteogenic factors released by the ost......The bone matrix is maintained functional through the combined action of bone resorbing osteoclasts and bone forming osteoblasts, in so-called bone remodeling units. The coupling of these two activities is critical for securing bone replenishment and involves osteogenic factors released...... by the osteoclasts. However, the osteoclasts are separated from the mature bone forming osteoblasts in time and space. Therefore the target cell of these osteoclastic factors has remained unknown. Recent explorations of the physical microenvironment of osteoclasts revealed a cell layer lining the bone marrow...... and forming a canopy over the whole remodeling surface, spanning from the osteoclasts to the bone forming osteoblasts. Several observations show that these canopy cells are a source of osteoblast progenitors, and we hypothesized therefore that they are the likely cells targeted by the osteogenic factors...

The Anti-Periodontitis Effects of Ethanol Extract Prepared Using Lactobacillus paracasei subsp. paracasei NTU 101

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Te-Hua Liu

2018-04-01

Full Text Available Poor oral health and related diseases, including caries, periodontal disease, and oral cancer, are highly prevalent across the world, particularly in the elderly. This study aimed to investigate the anti-periodontitis activity of fermented skim milk produced using the promising probiotic Lactobacillus paracasei subsp. paracasei NTU 101 (NTU101FM. An initial analysis found that an ethanol extract of NTU101FM displayed anti-oxidative activities. Further investigation of pathogen growth inhibition zones, minimum inhibitory concentrations (MICs, and minimum bactericidal concentrations (MBCs revealed that the NTU101FM ethanol extract also had anti-periodontal pathogen activities. In addition, the NTU101FM ethanol extract significantly decreased the release of pro-inflammatory cytokines induced by lipopolysaccharide (LPS in RAW 264.7 macrophage cells. Finally, the NTU101FM ethanol extract was found to inhibit receptor activator of nuclear factor-κB ligand (RANKL-induced osteoclast differentiation by reducing tartrate-resistant acid phosphatase (TRAP activity and the number of TRAP-positive multinucleated osteoclasts. In summary, our study demonstrated that ethanol extract prepared from NTU101FM has potential use as an anti-periodontitis agent.

A secreted factor represses cell proliferation in Dictyostelium.

Science.gov (United States)

Brock, Debra A; Gomer, Richard H

2005-10-01

Many cells appear to secrete factors called chalones that limit their proliferation, but in most cases the factors have not been identified. We found that growing Dictyostelium cells secrete a 60 kDa protein called AprA for autocrine proliferation repressor. AprA has similarity to putative bacterial proteins of unknown function. Compared with wild-type cells, aprA-null cells proliferate faster, while AprA overexpressing cells proliferate slower. Growing wild-type cells secrete a factor that inhibits the proliferation of wild-type and aprA- cells; this activity is not secreted by aprA- cells. AprA purified by immunoprecipitation also slows the proliferation of wild-type and aprA- cells. Compared with wild type, there is a higher percentage of multinucleate cells in the aprA- population, and when starved, aprA- cells form abnormal structures that contain fewer spores. AprA may thus decrease the number of multinucleate cells and increase spore production. Together, the data suggest that AprA functions as part of a Dictyostelium chalone.

PU.1 target genes undergo Tet2-coupled demethylation and DNMT3b-mediated methylation in monocyte-to-osteoclast differentiation

DEFF Research Database (Denmark)

de la Rica, Lorenzo; Rodríguez-Ubreva, Javier; García, Mireia

2013-01-01

to osteoclasts is a unique terminal differentiation process within the hematopoietic system. This differentiation model is relevant to autoimmune disease and cancer, and there is abundant knowledge on the sets of transcription factors involved.......DNA methylation is a key epigenetic mechanism for driving and stabilizing cell-fate decisions. Local deposition and removal of DNA methylation are tightly coupled with transcription factor binding, although the relationship varies with the specific differentiation process. Conversion of monocytes...

A Dictyostelium secreted factor requires a PTEN-like phosphatase to slow proliferation and induce chemorepulsion.

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Sarah E Herlihy

Full Text Available In Dictyostelium discoideum, AprA and CfaD are secreted proteins that inhibit cell proliferation. We found that the proliferation of cells lacking CnrN, a phosphatase and tensin homolog (PTEN-like phosphatase, is not inhibited by exogenous AprA and is increased by exogenous CfaD. The expression of CnrN in cnrN cells partially rescues these altered sensitivities, suggesting that CnrN is necessary for the ability of AprA and CfaD to inhibit proliferation. Cells lacking CnrN accumulate normal levels of AprA and CfaD. Like cells lacking AprA and CfaD, cnrN cells proliferate faster and reach a higher maximum cell density than wild type cells, tend to be multinucleate, accumulate normal levels of mass and protein per nucleus, and form less viable spores. When cnrN cells expressing myc-tagged CnrN are stimulated with a mixture of rAprA and rCfaD, levels of membrane-associated myc-CnrN increase. AprA also causes chemorepulsion of Dictyostelium cells, and CnrN is required for this process. Combined, these results suggest that CnrN functions in a signal transduction pathway downstream of AprA and CfaD mediating some, but not all, of the effects of AprA and CfaD.

The vitamin D analogue ED71 but Not 1,25(OH2D3 targets HIF1α protein in osteoclasts.

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Yuiko Sato

Full Text Available Although both an active form of the vitamin D metabolite, 1,25(OH2D3, and the vitamin D analogue, ED71 have been used to treat osteoporosis, anti-bone resorbing activity is reportedly seen only in ED71- but not in 1,25(OH2D3 -treated patients. In addition, how ED71 inhibits osteoclast activity in patients has not been fully characterized. Recently, HIF1α expression in osteoclasts was demonstrated to be required for development of post-menopausal osteoporosis. Here we show that ED71 but not 1,25(OH2D3, suppress HIF1α protein expression in osteoclasts in vitro. We found that 1,25(OH2D3 or ED71 function in osteoclasts requires the vitamin D receptor (VDR. ED71 was significantly less effective in inhibiting M-CSF and RANKL-stimulated osteoclastogenesis than was 1,25(OH2D3 in vitro. Downregulation of c-Fos protein and induction of Ifnβ mRNA in osteoclasts, both of which reportedly block osteoclastogenesis induced by 1,25(OH2D3 in vitro, were both significantly higher following treatment with 1,25(OH2D3 than with ED71. Thus, suppression of HIF1α protein activity in osteoclasts in vitro, which is more efficiently achieved by ED71 rather than by 1,25(OH2D3, could be a reliable read-out in either developing or screening reagents targeting osteoporosis.

Cord blood-derived macrophage-lineage cells rapidly stimulate osteoblastic maturation in mesenchymal stem cells in a glycoprotein-130 dependent manner.

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Tania J Fernandes

Full Text Available In bone, depletion of osteoclasts reduces bone formation in vivo, as does osteal macrophage depletion. How osteoclasts and macrophages promote the action of bone forming osteoblasts is, however, unclear. Since recruitment and differentiation of multi-potential stromal cells/mesenchymal stem cells (MSC generates new active osteoblasts, we investigated whether human osteoclasts and macrophages (generated from cord blood-derived hematopoietic progenitors induce osteoblastic maturation in adipose tissue-derived MSC. When treated with an osteogenic stimulus (ascorbate, dexamethasone and β-glycerophosphate these MSC form matrix-mineralising, alkaline phosphatase-expressing osteoblastic cells. Cord blood-derived progenitors were treated with macrophage colony stimulating factor (M-CSF to form immature proliferating macrophages, or with M-CSF plus receptor activator of NFκB ligand (RANKL to form osteoclasts; culture medium was conditioned for 3 days by these cells to study their production of osteoblastic factors. Both osteoclast- and macrophage-conditioned medium (CM greatly enhanced MSC osteoblastic differentiation in both the presence and absence of osteogenic medium, evident by increased alkaline phosphatase levels within 4 days and increased mineralisation within 14 days. These CM effects were completely ablated by antibodies blocking gp130 or oncostatin M (OSM, and OSM was detectable in both CM. Recombinant OSM very potently stimulated osteoblastic maturation of these MSC and enhanced bone morphogenetic protein-2 (BMP-2 actions on MSC. To determine the influence of macrophage activation on this OSM-dependent activity, CM was collected from macrophage populations treated with M-CSF plus IL-4 (to induce alternative activation or with GM-CSF, IFNγ and LPS to cause classical activation. CM from IL-4 treated macrophages stimulated osteoblastic maturation in MSC, while CM from classically-activated macrophages did not. Thus, macrophage-lineage cells

Involvement of plant stem cells or stem cell-like cells in dedifferentiation

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Fangwei eJiang

2015-11-01

Full Text Available Dedifferentiation is the transformation of cells from a given differentiated state to a less differentiated or stem cell-like state. Stem cell-related genes play important roles in dedifferentiation, which exhibits similar histone modification and DNA methylation features to stem cell maintenance. Hence, stem cell-related factors possibly synergistically function to provide a specific niche beneficial to dedifferentiation. During callus formation in Arabidopsis petioles, cells adjacent to procambium cells (stem cell-like cells are dedifferentiated and survive more easily than other cell types. This finding indicates that stem cells or stem cell-like cells may influence the dedifferentiating niche. In this paper, we provide a brief overview of stem cell maintenance and dedifferentiation regulation. We also summarize current knowledge of genetic and epigenetic mechanisms underlying the balance between differentiation and dedifferentiation. Furthermore, we discuss the correlation of stem cells or stem cell-like cells with dedifferentiation.

Induction of Functional Hair-Cell-Like Cells from Mouse Cochlear Multipotent Cells

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Quanwen Liu

2016-01-01

Full Text Available In this paper, we developed a two-step-induction method of generating functional hair cells from inner ear multipotent cells. Multipotent cells from the inner ear were established and induced initially into progenitor cells committed to the inner ear cell lineage on the poly-L-lysine substratum. Subsequently, the committed progenitor cells were cultured on the mitotically inactivated chicken utricle stromal cells and induced into hair-cell-like cells containing characteristic stereocilia bundles. The hair-cell-like cells exhibited rapid permeation of FM1-43FX. The whole-cell patch-clamp technique was used to measure the membrane currents of cells differentiated for 7 days on chicken utricle stromal cells and analyze the biophysical properties of the hair-cell-like cells by recording membrane properties of cells. The results suggested that the hair-cell-like cells derived from inner ear multipotent cells were functional following differentiation in an enabling environment.

Concise Review: Stem Cells in Osteoimmunology.

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Fierro, Fernando A; Nolta, Jan A; Adamopoulos, Iannis E

2017-06-01

Bone remodeling is a lifelong process in which mature bone tissue is removed from the skeleton by bone resorption and is replenished by new during ossification or bone formation. The remodeling cycle requires both the differentiation and activation of two cell types with opposing functions; the osteoclast, which orchestrates bone resorption, and the osteoblast, which orchestrates bone formation. The differentiation of these cells from their respective precursors is a process which has been overshadowed by enigma, particularly because the precise osteoclast precursor has not been identified and because the identification of skeletal stem cells, which give rise to osteoblasts, is very recent. Latest advances in the area of stem cell biology have enabled us to gain a better understanding of how these differentiation processes occur in physiological and pathological conditions. In this review we postulate that modulation of stem cells during inflammatory conditions is a necessary prerequisite of bone remodeling and therefore an essential new component to the field of osteoimmunology. In this context, we highlight the role of transcription factor nuclear factor of activated T cells cytoplasmic 1 (NFATc1), because it directly links inflammation with differentiation of osteoclasts and osteoblasts. Stem Cells 2017;35:1461-1467. © 2017 The Authors Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Toll-Like Receptor 2 Stimulation of Osteoblasts Mediates Staphylococcus Aureus Induced Bone Resorption and Osteoclastogenesis through Enhanced RANKL

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Kassem, Ali; Lindholm, Catharina; Lerner, Ulf H

2016-01-01

Severe Staphylococcus aureus (S. aureus) infections pose an immense threat to population health and constitute a great burden for the health care worldwide. Inter alia, S. aureus septic arthritis is a disease with high mortality and morbidity caused by destruction of the infected joints and systemic bone loss, osteoporosis. Toll-Like receptors (TLRs) are innate immune cell receptors recognizing a variety of microbial molecules and structures. S. aureus recognition via TLR2 initiates a signaling cascade resulting in production of various cytokines, but the mechanisms by which S. aureus causes rapid and excessive bone loss are still unclear. We, therefore, investigated how S. aureus regulates periosteal/endosteal osteoclast formation and bone resorption. S. aureus stimulation of neonatal mouse parietal bone induced ex vivo bone resorption and osteoclastic gene expression. This effect was associated with increased mRNA and protein expression of receptor activator of NF-kB ligand (RANKL) without significant change in osteoprotegerin (OPG) expression. Bone resorption induced by S. aureus was abolished by OPG. S. aureus increased the expression of osteoclastogenic cytokines and prostaglandins in the parietal bones but the stimulatory effect of S. aureus on bone resorption and Tnfsf11 mRNA expression was independent of these cytokines and prostaglandins. Stimulation of isolated periosteal osteoblasts with S. aureus also resulted in increased expression of Tnfsf11 mRNA, an effect lost in osteoblasts from Tlr2 knockout mice. S. aureus stimulated osteoclastogenesis in isolated periosteal cells without affecting RANKL-stimulated resorption. In contrast, S. aureus inhibited RANKL-induced osteoclast formation in bone marrow macrophages. These data show that S. aureus enhances bone resorption and periosteal osteoclast formation by increasing osteoblast RANKL production through TLR2. Our study indicates the importance of using different in vitro approaches for studies of how S

Biology of Bone Tissue: Structure, Function, and Factors That Influence Bone Cells.

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Florencio-Silva, Rinaldo; Sasso, Gisela Rodrigues da Silva; Sasso-Cerri, Estela; Simões, Manuel Jesus; Cerri, Paulo Sérgio

2015-01-01

Bone tissue is continuously remodeled through the concerted actions of bone cells, which include bone resorption by osteoclasts and bone formation by osteoblasts, whereas osteocytes act as mechanosensors and orchestrators of the bone remodeling process. This process is under the control of local (e.g., growth factors and cytokines) and systemic (e.g., calcitonin and estrogens) factors that all together contribute for bone homeostasis. An imbalance between bone resorption and formation can result in bone diseases including osteoporosis. Recently, it has been recognized that, during bone remodeling, there are an intricate communication among bone cells. For instance, the coupling from bone resorption to bone formation is achieved by interaction between osteoclasts and osteoblasts. Moreover, osteocytes produce factors that influence osteoblast and osteoclast activities, whereas osteocyte apoptosis is followed by osteoclastic bone resorption. The increasing knowledge about the structure and functions of bone cells contributed to a better understanding of bone biology. It has been suggested that there is a complex communication between bone cells and other organs, indicating the dynamic nature of bone tissue. In this review, we discuss the current data about the structure and functions of bone cells and the factors that influence bone remodeling.

Molecular Signaling Pathways Mediating Osteoclastogenesis Induced by Prostate Cancer Cells

International Nuclear Information System (INIS)

Rafiei, Shahrzad; Komarova, Svetlana V

2013-01-01

Advanced prostate cancer commonly metastasizes to bone leading to osteoblastic and osteolytic lesions. Although an osteolytic component governed by activation of bone resorbing osteoclasts is prominent in prostate cancer metastasis, the molecular mechanisms of prostate cancer-induced osteoclastogenesis are not well-understood. We studied the effect of soluble mediators released from human prostate carcinoma cells on osteoclast formation from mouse bone marrow and RAW 264.7 monocytes. Soluble factors released from human prostate carcinoma cells significantly increased viability of naïve bone marrow monocytes, as well as osteoclastogenesis from precursors primed with receptor activator of nuclear factor κ-B ligand (RANKL). The prostate cancer-induced osteoclastogenesis was not mediated by RANKL as it was not inhibited by osteoprotegerin (OPG). However inhibition of TGFβ receptor I (TβRI), or macrophage-colony stimulating factor (MCSF) resulted in attenuation of prostate cancer-induced osteoclastogenesis. We characterized the signaling pathways induced in osteoclast precursors by soluble mediators released from human prostate carcinoma cells. Prostate cancer factors increased basal calcium levels and calcium fluctuations, induced nuclear localization of nuclear factor of activated t-cells (NFAT)c1, and activated prolonged phosphorylation of ERK1/2 in RANKL-primed osteoclast precursors. Inhibition of calcium signaling, NFATc1 activation, and ERK1/2 phosphorylation significantly reduced the ability of prostate cancer mediators to stimulate osteoclastogenesis. This study reveals the molecular mechanisms underlying the direct osteoclastogenic effect of prostate cancer derived factors, which may be beneficial in developing novel osteoclast-targeting therapeutic approaches

The Modulatory Effects of Mesenchymal Stem Cells on Osteoclastogenesis

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Wessam E. Sharaf-Eldin

2016-01-01

Full Text Available The effect of mesenchymal stem cells (MSCs on bone formation has been extensively demonstrated through several in vitro and in vivo studies. However, few studies addressed the effect of MSCs on osteoclastogenesis and bone resorption. Under physiological conditions, MSCs support osteoclastogenesis through producing the main osteoclastogenic cytokines, RANKL and M-CSF. However, during inflammation, MSCs suppress osteoclast formation and activity, partly via secretion of the key anti-osteoclastogenic factor, osteoprotegerin (OPG. In vitro, co-culture of MSCs with osteoclasts in the presence of high concentrations of osteoclast-inducing factors might reflect the in vivo inflammatory pathology and prompt MSCs to exert an osteoclastogenic suppressive effect. MSCs thus seem to have a dual effect, by stimulating or inhibiting osteoclastogenesis, depending on the inflammatory milieu. This effect of MSCs on osteoclast formation seems to mirror the effect of MSCs on other immune cells, and may be exploited for the therapeutic potential of MSCs in bone loss associated inflammatory diseases.

Neovascular niche for human myeloma cells in immunodeficient mouse bone.

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Hirono Iriuchishima

Full Text Available The interaction with bone marrow (BM plays a crucial role in pathophysiological features of multiple myeloma (MM, including cell proliferation, chemoresistance, and bone lesion progression. To characterize the MM-BM interactions, we utilized an in vivo experimental model for human MM in which a GFP-expressing human MM cell line is transplanted into NOG mice (the NOG-hMM model. Transplanted MM cells preferentially engrafted at the metaphyseal region of the BM endosteum and formed a complex with osteoblasts and osteoclasts. A subpopulation of MM cells expressed VE-cadherin after transplantation and formed endothelial-like structures in the BM. CD138(+ myeloma cells in the BM were reduced by p53-dependent apoptosis following administration of the nitrogen mustard derivative bendamustine to mice in the NOG-hMM model. Bendamustine maintained the osteoblast lining on the bone surface and protected extracellular matrix structures. Furthermore, bendamustine suppressed the growth of osteoclasts and mesenchymal cells in the NOG-hMM model. Since VE-cadherin(+ MM cells were chemoresistant, hypoxic, and HIF-2α-positive compared to the VE-cadherin(- population, VE-cadherin induction might depend on the oxygenation status. The NOG-hMM model described here is a useful system to analyze the dynamics of MM pathophysiology, interactions of MM cells with other cellular compartments, and the utility of novel anti-MM therapies.

Giant cell angiofibroma or localized periorbital lymphedema?

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Lynch, Michael C; Chung, Catherine G; Specht, Charles S; Wilkinson, Michael; Clarke, Loren E

2013-12-01

Giant cell angiofibroma represents a rare soft tissue neoplasm with a predilection for the orbit. We recently encountered a mass removed from the lower eyelid of a 56-year-old female that histopathologically resembled giant cell angiofibroma. The process consisted of haphazardly arranged CD34-positive spindled and multinucleated cells within an edematous, densely vascular stroma. However, the patient had recently undergone laryngectomy and radiotherapy for a laryngeal squamous cell carcinoma. A similar mass had arisen on the contralateral eyelid, and both had developed several months post-therapy. Lymphedema of the orbit can present as tumor-like nodules and in some cases may share histopathologic features purported to be characteristic of giant cell angiofibroma. A relationship between giant cell angiofibroma and lymphedema has not been established, but our case suggests there may be one. The potential overlap of these two conditions should be recognized, as should other entities that may enter the differential diagnosis. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Inhibited osteoclastic bone resorption through alendronate treatment in rats reduces severe osteoarthritis progression.

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Siebelt, M; Waarsing, J H; Groen, H C; Müller, C; Koelewijn, S J; de Blois, E; Verhaar, J A N; de Jong, M; Weinans, H

2014-09-01

Osteoarthritis (OA) is a non-rheumatoid joint disease characterized by progressive degeneration of extra-cellular cartilage matrix (ECM), enhanced subchondral bone remodeling, osteophyte formation and synovial thickening. Alendronate (ALN) is a potent inhibitor of osteoclastic bone resorption and results in reduced bone remodeling. This study investigated the effects of pre-emptive use of ALN on OA related osteoclastic subchondral bone resorption in an in vivo rat model for severe OA. Using multi-modality imaging we measured effects of ALN treatment within cartilage and synovium. Severe osteoarthritis was induced in left rat knees using papain injections in combination with a moderate running protocol. Twenty rats were treated with subcutaneous ALN injections and compared to twenty untreated controls. Animals were longitudinally monitored for 12weeks with in vivo μCT to measure subchondral bone changes and SPECT/CT to determine synovial macrophage activation using a folate-based radiotracer. Articular cartilage was analyzed at 6 and 12weeks with ex vivo contrast enhanced μCT and histology to measure sulfated-glycosaminoglycan (sGAG) content and cartilage thickness. ALN treatment successfully inhibited subchondral bone remodeling. As a result we found less subchondral plate porosity and reduced osteophytosis. ALN treatment did not reduce subchondral sclerosis. However, after the OA induction phase, ALN treatment protected cartilage ECM from degradation and reduced synovial macrophage activation. Surprisingly, ALN treatment also improved sGAG content of tibia cartilage in healthy joints. Our data was consistent with the hypothesis that osteoclastic bone resorption might play an important role in OA and may be a driving force for progression of the disease. However, our study suggest that this effect might not solely be effects on osteoclastic activity, since ALN treatment also influenced macrophage functioning. Additionally, ALN treatment and physical activity

Inhibition of prostate cancer osteoblastic progression with VEGF121/rGel, a single agent targeting osteoblasts, osteoclasts, and tumor neovasculature.

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Mohamedali, Khalid A; Li, Zhi Gang; Starbuck, Michael W; Wan, Xinhai; Yang, Jun; Kim, Sehoon; Zhang, Wendy; Rosenblum, Michael G; Navone, Nora M

2011-04-15

A hallmark of prostate cancer (PCa) progression is the development of osteoblastic bone metastases, which respond poorly to available therapies. We previously reported that VEGF(121)/rGel targets osteoclast precursors and tumor neovasculature. Here we tested the hypothesis that targeting nontumor cells expressing these receptors can inhibit tumor progression in a clinically relevant model of osteoblastic PCa. Cells from MDA PCa 118b, a PCa xenograft obtained from a bone metastasis in a patient with castrate-resistant PCa, were injected into the femurs of mice. Osteoblastic progression was monitored following systemic administration of VEGF(121)/rGel. VEGF(121)/rGel was cytotoxic in vitro to osteoblast precursor cells. This cytotoxicity was specific as VEGF(121)/rGel internalization into osteoblasts was VEGF(121) receptor driven. Furthermore, VEGF(121)/rGel significantly inhibited PCa-induced bone formation in a mouse calvaria culture assay. In vivo, VEGF(121)/rGel significantly inhibited the osteoblastic progression of PCa cells in the femurs of nude mice. Microcomputed tomographic analysis revealed that VEGF(121)/rGel restored the bone volume fraction of tumor-bearing femurs to values similar to those of the contralateral (non-tumor-bearing) femurs. VEGF(121)/rGel significantly reduced the number of tumor-associated osteoclasts but did not change the numbers of peritumoral osteoblasts. Importantly, VEGF(121)/rGel-treated mice had significantly less tumor burden than control mice. Our results thus indicate that VEGF(121)/rGel inhibits osteoblastic tumor progression by targeting angiogenesis, osteoclastogenesis, and bone formation. Targeting VEGF receptor (VEGFR)-1- or VEGFR-2-expressing cells is effective in controlling the osteoblastic progression of PCa in bone. These findings provide the basis for an effective multitargeted approach for metastatic PCa. ©2011 AACR.

Inhibition of prostate cancer osteoblastic progression with VEGF121/rGel, a single agent targeting osteoblasts, osteoclasts, and tumor neovasculature

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Mohamedali, Khalid A.; Li, Zhi Gang; Starbuck, Michael W.; Wan, Xinhai; Yang, Jun; Kim, Sehoon; Zhang, Wendy; Rosenblum, Michael G.; Navone, Nora M.

2011-01-01

Purpose A hallmark of prostate cancer (PCa) progression is the development of osteoblastic bone metastases, which respond poorly to available therapies. We previously reported that VEGF121/rGel targets osteoclast precursors and tumor neovasculature. Here we tested the hypothesis that targeting non-tumor cells expressing these receptors can inhibit tumor progression in a clinically relevant model of osteoblastic PCa. Experimental Design Cells from MDA PCa 118b, a PCa xenograft obtained from a bone metastasis in a patient with castrate-resistant PCa, were injected into the femurs of mice. Osteoblastic progression was monitored following systemic administration of VEGF121/rGel. Results VEGF121/rGel was cytotoxic in vitro to osteoblast precursor cells. This cytotoxicity was specific as VEGF121/rGel internalization into osteoblasts was VEGF121 receptor driven. Furthermore, VEGF121/rGel significantly inhibited PCa-induced bone formation in a mouse calvaria culture assay. In vivo, VEGF121/rGel significantly inhibited the osteoblastic progression of PCa cells in the femurs of nude mice. Microcomputed tomography analysis revealed that VEGF121/rGel restored the bone volume fraction of tumor-bearing femurs to values similar to those of the contralateral (non–tumor bearing) femurs. VEGF121/rGel significantly reduced the number of tumor-associated osteoclasts but did not change the numbers of peritumoral osteoblasts. Importantly, VEGF121/rGel-treated mice had significantly less tumor burden than control mice. Our results thus indicate that VEGF121/rGel inhibits osteoblastic tumor progression by targeting angiogenesis, osteoclastogenesis, and bone formation. Conclusions Targeting VEGFR-1 – or VEGFR-2–expressing cells is effective in controlling the osteoblastic progression of PCa in bone. These findings provide the basis for an effective multitargeted approach for metastatic PCa. PMID:21343372

Optimization of the Static Human Osteoblast/Osteoclast Co-culture System

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James Jam Jolly

2018-03-01

Full Text Available Osteoblasts (OBs and osteoclasts (OCs are 2 major groups of bone cells. Their cell-to-cell interactions are important to ensure the continuity of the bone-remodeling process. Therefore, the present study was carried out to optimize an OB/OC co-culture system utilizing the human OB cell line hFOB 1.19 and OCs extracted from peripheral blood mononuclear cells (PBMNCs. It was a 2-step procedure, involving the optimization of the OB culture and the co-culture of the OBs with PBMNCs at an optimum ratio. Firstly, pre-OBs were cultured to 90% confluency and the time required for differentiation was determined. OB differentiation was determined using the van Gieson staining to detect the presence of collagen and Alizarin Red for calcium. Secondly, OBs and OCs were co-cultured at the ratios of 1 OC: 1 OB, 1 OC: 4 OBs, 2 OCs: 1 OB, and 1 OC: 2 OBs. Tartrate-resistant acid phosphatase (TRAP staining was used to detect the differentiation of the OCs. The results showed that collagen was present on day 1, whereas calcium was detected as early as day 3. Based on the result of TRAP staining, 1 OC: 2 OBs was taken as the most appropriate ratio. No macrophage colony-stimulating factor and receptor activator of the nuclear factor-κB ligand were added because they were provided by the OBs. In conclusion, these optimization processes are vital as they ensure the exact time point and ratio of the OB/OC co-culture in order to produce a reliable and reproducible co-culture system.

Zoledronate induces apoptosis in cells from fibro-cellular membrane of unicameral bone cyst (UBC).

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Yu, John; Chang, Seong-Sil; Suratwala, Sanjeev; Chung, Woo-Sik; Abdelmessieh, Peter; Lee, Hahn-Jun; Yang, Jay; Lee, Francis Young-In

2005-09-01

Unicameral bone cyst (UBC) is a benign cystic lesion in children which is prone to fracture. Various treatments are available, but recurrence after different types of percutaneous injection therapy can cause bone destruction and pathologic fracture. The potential therapeutic effects of anti-resorptive agents, such as bisphosphonates, have not been investigated for UBC. The objective of this study was to characterize the cells from the fibro-cellular membrane of unicameral bone cyst (UBC cells) and to determine whether zoledronate, a nitrogen-containing bisphosphonate, could induce apoptosis in UBC cells. Flow cytometry and immunoblotting were performed in order to determine whether zoledronate induced apoptosis. Cells derived from normal human trabecular bones were used as controls against UBC cells to compare the effect of zoledronate in inducing apoptosis. Immunohisto/cytochemistry (IHC/ICC) and mini-array analyses were performed on tissues and cultured cells. Isolated peripheral blood mononuclear cells were incubated with conditioned media from the UBC cells to determine whether they are capable of inducing osteoclastogenesis. UBC membrane is composed of cells staining positively with CD68, SDF-1, STRO-1 and RANKL, but in vitro cells showed no staining with antibodies to CD68 and STRO-1, suggesting that there was a clonal selection of stromal cells during cell culture. UBC cells also express RUNX2 (runt-related transcription factor-2, core binding factor-1), a key transcription factor for osteoblastic differentiation. In addition, media collected from UBC cells induced a generation of multi-nucleated osteoclast-like cells of peripheral blood mononuclear cells. Zoledronate induced apoptosis of UBC cells in a dose-dependent manner. Apoptosis was evidenced by induction of the active cleaved form of caspase-3. The baseline apoptotic fractions were similar in UBC cells and trabecular bone cells. However, in the overall apoptotic fractions in this study, trabecular

A global downregulation of microRNAs occurs in human quiescent satellite cells during myogenesis

NARCIS (Netherlands)

Koning, Merel; Werker, Paul M N; van Luyn, Marja J A; Krenning, Guido; Harmsen, Martin C

2012-01-01

During myogenesis, human satellite cells differentiate and form multinucleated myotubes, while a fraction of the human satellite cells enter quiescence. These quiescent satellite cells are able to activate, proliferate and contribute to muscle regeneration. Post-transcriptional regulation of

Ibandronate promotes osteogenic differentiation of periodontal ligament stem cells by regulating the expression of microRNAs

Energy Technology Data Exchange (ETDEWEB)

Zhou, Qiang [Department of General Dentistry and Emergency, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Zhao, Zhi-Ning [Clinical Laboratory, 451 Hospital of Chinese PLA, Xi' an 710054 (China); Cheng, Jing-Tao [Department of Special Dentistry, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Zhang, Bin [Department of Orthodontics, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Xu, Jie [Department of Periodontology, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Huang, Fei; Zhao, Rui-Ni [Department of General Dentistry and Emergency, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Chen, Yong-Jin, E-mail: cyj1229@fmmu.edu.cn [Department of General Dentistry and Emergency, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China)

2011-01-07

Research highlights: {yields} Ibandronate significantly promote the proliferation of PDLSC cells. {yields} Ibandronate enhanced the expression of ALP, COL-1, OPG, OCN, Runx2. {yields} The expression of a class of miRNAs, e.g., miR-18a, miR-133a, miR-141 and miR-19a, was significantly modified in PDLSC cells cultured with ibandronate. {yields} Ibandronate regulates the expression of diverse bone formation-related genes via miRNAs in PDLSCs. {yields} Ibandronate can suppress the activity of osteoclast while promoting the proliferation of osteoblast by regulating the expression of microRNAs. -- Abstract: Bisphosphonates (BPs) have a profound effect on bone resorption and are widely used to treat osteoclast-mediated bone diseases. They suppress bone resorption by inhibiting the activity of mature osteoclasts and/or the formation of new osteoclasts. Osteoblasts may be an alternative target for BPs. Periodontal ligament stem cells (PDLSCs) exhibit osteoblast-like features and are capable of differentiating into osteoblasts or cementoblasts. This study aimed to determine the effects of ibandronate, a nitrogen-containing BP, on the proliferation and the differentiation of PDLSCs and to identify the microRNAs (miRNAs) that mediate these effects. The PDLSCs were treated with ibandronate, and cell proliferation was measured using the MTT (3-dimethylthiazol-2,5-diphenyltetrazolium bromide) assay. The expression of genes and miRNAs involved in osteoblastic differentiation was assayed using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). Ibandronate promoted the proliferation of PDLSCs and enhanced the expression of alkaline phosphatase (ALP), type I collagen (COL-1), osteoprotegerin (OPG), osteocalcin (OCN), and Runx2. The expression of miRNAs, including miR-18a, miR-133a, miR-141 and miR-19a, was significantly altered in the PDLSCs cultured with ibandronate. In PDLSCs, ibandronate regulates the expression of diverse bone formation

Ibandronate promotes osteogenic differentiation of periodontal ligament stem cells by regulating the expression of microRNAs

International Nuclear Information System (INIS)

Zhou, Qiang; Zhao, Zhi-Ning; Cheng, Jing-Tao; Zhang, Bin; Xu, Jie; Huang, Fei; Zhao, Rui-Ni; Chen, Yong-Jin

2011-01-01

Research highlights: → Ibandronate significantly promote the proliferation of PDLSC cells. → Ibandronate enhanced the expression of ALP, COL-1, OPG, OCN, Runx2. → The expression of a class of miRNAs, e.g., miR-18a, miR-133a, miR-141 and miR-19a, was significantly modified in PDLSC cells cultured with ibandronate. → Ibandronate regulates the expression of diverse bone formation-related genes via miRNAs in PDLSCs. → Ibandronate can suppress the activity of osteoclast while promoting the proliferation of osteoblast by regulating the expression of microRNAs. -- Abstract: Bisphosphonates (BPs) have a profound effect on bone resorption and are widely used to treat osteoclast-mediated bone diseases. They suppress bone resorption by inhibiting the activity of mature osteoclasts and/or the formation of new osteoclasts. Osteoblasts may be an alternative target for BPs. Periodontal ligament stem cells (PDLSCs) exhibit osteoblast-like features and are capable of differentiating into osteoblasts or cementoblasts. This study aimed to determine the effects of ibandronate, a nitrogen-containing BP, on the proliferation and the differentiation of PDLSCs and to identify the microRNAs (miRNAs) that mediate these effects. The PDLSCs were treated with ibandronate, and cell proliferation was measured using the MTT (3-dimethylthiazol-2,5-diphenyltetrazolium bromide) assay. The expression of genes and miRNAs involved in osteoblastic differentiation was assayed using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). Ibandronate promoted the proliferation of PDLSCs and enhanced the expression of alkaline phosphatase (ALP), type I collagen (COL-1), osteoprotegerin (OPG), osteocalcin (OCN), and Runx2. The expression of miRNAs, including miR-18a, miR-133a, miR-141 and miR-19a, was significantly altered in the PDLSCs cultured with ibandronate. In PDLSCs, ibandronate regulates the expression of diverse bone formation-related genes via miRNAs. The exact

Biology of Bone Tissue: Structure, Function, and Factors That Influence Bone Cells

Directory of Open Access Journals (Sweden)

Rinaldo Florencio-Silva

2015-01-01

Full Text Available Bone tissue is continuously remodeled through the concerted actions of bone cells, which include bone resorption by osteoclasts and bone formation by osteoblasts, whereas osteocytes act as mechanosensors and orchestrators of the bone remodeling process. This process is under the control of local (e.g., growth factors and cytokines and systemic (e.g., calcitonin and estrogens factors that all together contribute for bone homeostasis. An imbalance between bone resorption and formation can result in bone diseases including osteoporosis. Recently, it has been recognized that, during bone remodeling, there are an intricate communication among bone cells. For instance, the coupling from bone resorption to bone formation is achieved by interaction between osteoclasts and osteoblasts. Moreover, osteocytes produce factors that influence osteoblast and osteoclast activities, whereas osteocyte apoptosis is followed by osteoclastic bone resorption. The increasing knowledge about the structure and functions of bone cells contributed to a better understanding of bone biology. It has been suggested that there is a complex communication between bone cells and other organs, indicating the dynamic nature of bone tissue. In this review, we discuss the current data about the structure and functions of bone cells and the factors that influence bone remodeling.

Breast Cancer Stimulation of Osteolysis

National Research Council Canada - National Science Library

Oursler, Merry

1998-01-01

.... We have determined the tumors make IGF-I, IGF-II, M-CSF, PTHrP, TGF-Beta, and TNF-alpha. We are examining the effects of these growth factors on bone resorption and survival of avian osteoclasts and mouse osteoclast-like cells in vitro...

Cytokinetic effects of razoxane and relevance to radiopotentiation in a human adenocarcinoma cell line

International Nuclear Information System (INIS)

Norimah Yusof.

1982-01-01

Razoxane (+-1,2-bis (3,5-dioxopiperazin-1-yl)propane) or ICRF 159 has been shown to enhance the X-radiation sensitivity of a human colorectal adenocarcinoma cell line (HT29R) in culture if the cells were incubated with the drug for more than ten hours prior irradiation. Razoxane was shown not to interfere with repair of sublethal of potentially lethal damage of radiation. The radiosensitizing effect is therefore attributed to a reduced ability to accumulate such damage. As the effect was shown greater in exponential phase, which consists of greater proportion of dividing cells than plateau phase cells, it is believed that the drug (0.1 - 100 μg/ml) potentiates the effect of X-radiation in HT29R cells by pertubation in the cell cycle kinetics. Razoxane at low and high concentrations (5 and 100 μg/ml) was shown to delay the cell progression on cells entering mitosis. Chromosomes of drug-treated cells seemed to be unable to condense at the premitosis stage and cells with chromosomes resembling those in prophase were allowed to proceed through mitosis but progressed very slowly so that accumulation of mitotic cells were seen. Cells were than allowed to complete mitosis but frequently failed to undergo cytokinesis and subsequently produced many giant multinucleate cells. It is likely that the cells in mitosis and multinucleate cells are more radiosensitive. However, the mitotic delay was found to be reversible following progressive drug inactivation in the cells. Cells appeared to return to normal cell progression rate and division while the radiopotentiating effect was disappearing. The mechanism of drug action in inhibiting chromosome condensation was shown to involve the drug interference with histone H 1 phosphorylation. The inhibition of H 1 phosphorylation might reduce the cross-linking of the histone on DNA strands and thus preventing them from participating in supercoiling of the chromosome. (author)