RANK mRNA Expression Research Articles

The effect of hyperbaric oxygen therapy on osteoclast formation and bone resorption

Event Abstract Back to Event The effect of hyperbaric oxygen therapy on osteoclast formation and bone resorption HW Al-Hadi1*, SW Fox1 and GR Smerdon2 1 University of Plymouth, School of Biomedical and Biological Science, United Kingdom 2 the Diving Diseases Research Centre, United Kingdom Hyperbaric oxygen therapy (HBO) is the breathing of pure oxygen in a sealed chamber at greater than normal atmospheric pressure. HBO is employed as an adjunctive therapy in a number of skeletal disorders such radio and bisphosphonate induced osteonecrosis of the jaw and chronic osteomyelitis. These disorders are associated with aberrant remodelling and excessive levels of resorption leading to increased fracture risk. The purpose of the present study was to evaluate the effect of HBO, pressure and hyperoxia on RANKL-induced osteoclast formation in RAW 264.7 cells and human peripheral blood monocytes (PBMC). Daily, HBO (2.4 ATA, 97% O2, 90 minutes per day) or hyperoxia (1 ATM, 95% O2, 90 minutes per day) treatments significantly decreased the number of RANKL-induced TRAP positive mononuclear and multinuclear osteoclasts forming in RAW and PBMC cultures (p <0.05). Similarly, HBO and hyperoxia significantly reduced bone resorption as assessed by the bone slice assay. Furthermore, pressure alone (2.4 ATA, 8.8% O2, 90 minutes per day) also significantly suppressed osteoclast differentiation and bone resorption to a similar extent as HBO and hyperoxia. Quantitative PCR analysis of key regulators of osteoclast differentiation (RANK) and multinuclearity (DC-STAMP) indicated that HBO, hyperoxia and pressure all significantly suppressed RANK and DC-STAMP mRNA expression. Interestingly, intermittent HBO, hyperoxia and pressure (daily, 90 minutes per day) also significantly reduced RANKL-induced osteoclast formation in hypoxic conditions (2% O2, 22.5 hours per day). This data suggests that HBO, elevated O2 and pressure suppress osteoclast differentiation and bone resorption in mouse and human monocytes. Furthermore the data indicates that elevated pressure and O2 also directly inhibit osteoclast formation in hypoxic conditions a hallmark of many skeletal disorders. This provides evidence supporting the use of HBO as an adjunctive therapy to prevent bone loss in a range of skeletal disorders associated with low oxygen partial pressure. Keywords: Bones, Bone Research Conference: 2011 joint meeting of the Bone Research Society & the British Orthopaedic Research Society, Cambridge, United Kingdom, 27 Jun - 29 Jun, 2011. Presentation Type: Oral Poster Topic: Abstracts Citation: Al-Hadi H, Fox S and Smerdon G (2011). The effect of hyperbaric oxygen therapy on osteoclast formation and bone resorption. Front. Endocrinol. Conference Abstract: 2011 joint meeting of the Bone Research Society & the British Orthopaedic Research Society. doi: 10.3389/conf.fendo.2011.02.00002 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 30 Sep 2011; Published Online: 30 Sep 2011. * Correspondence: Mrs. HW Al-Hadi, University of Plymouth, School of Biomedical and Biological Science, United Kingdom, hadil.al-hadi@plymouth.ac.uk Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers HW Al-Hadi SW Fox GR Smerdon Google HW Al-Hadi SW Fox GR Smerdon Google Scholar HW Al-Hadi SW Fox GR Smerdon PubMed HW Al-Hadi SW Fox GR Smerdon Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Effect of pioglitazone on receptor activator of nuclear factor-kappa B expression in osteoclast from RAW264.7 cells

To study the effect of pioglitazone, a synthetic peroxisome proliferator-activated receptor γ (PPARγ) agonist, on the RANKL-mediated osteoclastogenesis of osteoclast precursor cells, and to explore the function and mechanism of PPARγ in the osteoclast differentiation. Pioglitazone treatment of RAW264.7 murine macrophages were compared with those of simply cultured control and RANKL-mediated control. Accordingly, the RANKL-mediated cells were cultured with 30 ng/ml RANKL, then induced into significant multinuclear osteoclast formation. And pioglitazone treated cells were exposed to 10 µmol/L pioglitazone during the process of osteoclast differentiation under RANKL. The number of mature osteoclasts was calculated and the mRNA levels of RANK analyzed by real-time quantitative PCR. Pioglitazone significantly inhibited osteoclastogenesis of osteoclast precursor cells, the number of mature osteoclasts of pioglitazone treated group was (176 ± 58)/cm(2) and significantly less than the mature cells of RANKL induced group which number was (322 ± 74)/cm(2) (P < 0.01); and pioglitazone also significantly inhibited the mRNA expression of RANK, a typical differentiated factor of osteoclast, the number of the mRNA expression of RANK of pioglitazone treated group was 2.16 ± 0.74 and significantly less than the number of RANKL induced group (4.94 ± 0.39, P < 0.01). PPARγ agonist inhibited the differentiation of RAW264.7 towards osteoclast. It might be due to the suppression of RANK gene expression in the process of osteoclast differentiation.

Inhibition of titanium particle‐induced inflammatory osteolysis through inactivation of cannabinoid receptor 2 by AM630

Wear particle could induce inflammatory osteolysis and is the primary pathological factor for aseptic loosening. Although it is known that cannabinoid receptor 2 (CB2) inhibits osteoclast differentiation, the effect on inflammatory osteolysis induced by wear particles remains unclear. This study examined the effect of CB2 in the regulation of osteoclast differentiation in a murine macrophage cell line (RAW264.7), which has been shown to be stimulated by titanium (Ti) particles and receptor activator of the NF-kappaB ligand (RANKL). Results showed that CB2 expression in RAW cells cultured with Ti particles and RANKL. CB2 inactivation by AM630, a CB2 selective antagonist, effectively inhibited osteoclastogenesis in the differentiation medium system. AM630 treatment (> or =100 nM) significantly reduced the number of tartrate-resistant acid phosphatase-positive cells when compared with the control. Real-time reverse transcription polymerase chain reaction analysis revealed that AM630 (100 nM) inhibited mRNA expression of RANK and cathepsin K in RAW cells stimulated by Ti particles and RANKL. Moreover, enzyme-linked immunosorbent assay showed that AM630 (100 nM) reduced protein expression of interleukin-1beta and tumor necrosis factor-alpha in RAW cells cultured with Ti particles. In addition, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide revealed that AM630 had no toxic effect on RAW cells. These results suggested that CB2 inactivation by AM630 could provide a promising therapeutic target for treating or preventing aseptic loosening.

Effects and mechanism of aromatic aminoketone SY0916 on osteoclastic bone destruction.

To study the effects and mechanism of aromatic aminoketone (SY0916) on bone destruction in vitro. MC3T3-E1 cells and bone marrow cells were co-cultured to obtain purified osteoclasts. The proliferation of osteoclast-like cells (OCLs) was determined by MTT assay. The number of osteoclasts was measured by tartrate-resistant acid phosphatase (TRAP) staining. The functioning of osteoclasts was determined by measuring the area of bone resorption pits on bone slices. MMP-9 secretion by osteoclasts was measured by an ELISA kit. Osteoclast apoptosis was detected by flow cytometry using an AnnexinV-FITC kit. Gene expression of RANK and MMP-9 in osteoclasts as well as RANKL and OPG in MC3T3-E1 cells was determined by real-time PCR. SY0916 significantly inhibited the proliferation of OCLs, decreased both the total and average area of bone resorption pits, and dramatically inhibited the number of osteoclasts between concentrations of 0.01 and 10 micromol/L. Furthermore, SY0916 reversed IL-1 beta-mediated inhibition of osteoclast apoptosis and shortened osteoclast lifespan. In addition, SY0916 significantly inhibited the mRNA expression of RANK, RANKL, OPG, and MMP-9. However, the inhibition of OPG was weaker than that of RANKL. Accordingly, the ratio of RANKL to OPG mRNA expression in MC3T3-E1 cells was significantly decreased by SY0916. Meanwhile, the expression of MMP-9 protein in osteoclasts was inhibited by SY0916 between 0.01 and 10 micromol/L. SY0916 prevents osteoclastic bone destruction by inhibiting the proliferation and function of osteoclasts. The underlying mechanism for this effect involves the regulation of the RANKL-OPG-RANK axis, which determines the direction of bone metabolism.

Effects of Pulsed Electromagnetic Fields on the mRNA Expression of RANK and CAII in Ovariectomized Rat Osteoclast-Like Cell

This study was designed to determine the effects of pulsed electromagnetic fields (PEMF) on the mRNA expression of the receptor activator of NF-κ-B (RANK) and carbonic anhydrase II (CA II) in ovariectomized rat osteoclast-like cells. Marrow cells were harvested from femora and tibiae of rats, from which the ovaries had been totally excised, and cultured in 6-well chamber slides. After 1 day of incubation, the marrow cells were exposed to PEMF for 3 days with 3.8 mT, 8 Hz, and 40 min per day. Osteoclast-like cells were confirmed by both tartrate resistant acid phosphatase (TRAP) stain and bone resorption assay. The expression of RANK and CA II mRNA was determined with real-time fluorescent-nested quantitative polymerase chain reaction. Compared with the sham group, the level of serum estradiol in the ovariectomized group was significantly decreased ( p < 0.05). The numbers of multinucleated, TRAP-positive osteoclast-like cells and resorption pits formed were observed. In invitro study, the expression of RANK and CA II were measured in sham, ovariectomized without PEMF, and ovariectomized with PEMF treatment. Compared with the ovariectomized (PEMF) experimental group and sham group, CA II mRNA expression was significantly increased in the ovariectomized control group ( p < 0.05, 0.01, respectively). Compared with the sham group, RANK mRNA expression was significantly increased in the ovariectomized control group ( p < 0.05). These data suggest that PEMF could regulate the expression of RANK and CA II mRNA in the marrow culture system.

Receptor activator of nuclear factor‐kappa B ligand induces osteoclast formation in RAW 264.7 macrophage cells via augmented production of macrophage–colony‐stimulating factor

RAW 264.7 macrophage cells differentiate into osteoclast-like cells in the presence of RANKL. Participation of M-CSF in RANKL-induced osteoclast formation of RAW 264.7 cells was examined. TRAP-positive osteoclast-like cells appeared in RAW 264.7 cells cultured in the presence of RANKL. RANKL-induced osteoclast formation was markedly inhibited by anti-M-CSF antibody. RANKL augmented M-CSF mRNA expression and M-CSF production in RAW 264.7 cells. Further, anti-M-CSF antibody inhibited the expression of RANK, c-fms, c-fos and TRAP mRNA in RANKL-stimulated RAW 264.7 cells. However, anti-M-CSF antibody did not affect the expression of DC-STAMP in the stimulated cells. Therefore, RANKL was suggested to induce osteoclast formation in RAW 264.7 cells via augmented production of M-CSF. The putative role of M-CSF in RANKL-induced osteoclast formation of RAW 264.7 cells is discussed.

Regulation of RANK expression mediated by L‐ascorbic acid in RAW264.7 cells:involvement of cNOS, NF‐kB and Ikaros

L‐ascorbic acid (AsA) has been known to induce osteoclastic differentiation through osteoclast differentiation factor in some osteogenic cells. However, the precise mechanism of AsA in osteoclastogenesis is not completely understood. This study investigated the effect of AsA on the expression of receptor activator of nuclear factor‐kappa B (RANK) which is indispensable for osteoclast differentiation. Our data showed that AsA treatment up‐regulated the expression of RANK mRNA and protein in RAW 264.7 cells, which was inhibited by DPI, cNOS inhibitor. AsA also induced the expression of cNOS protein, indicating that cNOS is involved in AsA‐induced RANK expression. The level of RANK expression was decreased in Ikaros dominant negative isoform‐overexpressed RAW 264.7 cells compared with that of control cells. In addition, AsA dose‐dependently increased the DNA binding affinity of the Ikaros transcription factor, suggesting that Ikaros acts as a positive regulator on RANK expression. DPI also inhibited the nuclear expression of NF‐kB p65 induced by AsA. Furthermore, DPI significantly decreased the DNA binding activity of NF‐kB induced by AsA, implicating that cNOS is clearly related to NF‐kB activity. Overall, the present data demonstrate that AsA might stimulate osteoclastic differentiation by enhancing RANK expression and this process might be related to cNOS, NF‐kB and ikaros activation.

Biomarkers of Vorinostat Resistance in Cutaneous T-Cell Lymphoma.

Profiling gene expression with DNA microarray technology has elucidated novel therapeutic targets and led the approval of a number of targeted therapeutic agents for the treatment of cancer. Vorinostat (suberoylanilide hydroxamic acid, SAHA) is a pan-histone deacetylase (HDAC) inhibitor that has demonstrated an overall response rate of approximately 24–30% in two phase II studies of cutaneous T cell lymphoma (CTCL) patients. There are currently no known specific biomarkers to indicate resistance to vorinostat. To identify genes resistant to vorinostat we compared profiles using the Aligent whole human genome oligo microarrays containing ∼41,000 genes/transcripts in vitro in vorinostat-resistant MJ and -sensitive HH CTCL cell lines treated with 1 μM of vorinostat for 24 hours and compared them to patients' peripheral blood mononuclear cells (PBMCs) before and during oral therapy. There were 3151 (7.7%) genes/transcripts differentially expressed in vitro in treated resistant MJ cells compared to untreated vehicle control (p < 0.001). We also studied differential gene expression in two clinically resistant Sézary patients' PBMCs taken at baseline and four weeks after oral vorinostat (400 mg daily or 300 mg bid 3 days/wk). In patients' PBMCs, 585 (1.4%) and 2744 (6.7%) differentially expressed genes/transcripts (p < 0.001) were identified, respectively. Genes that were up-regulated both in vitro and in vivo included a tumor necrosis factor receptor super-family member 11a (TNFRSF11a or RANK), matrix metallopeptidase 9 (MMP9), suppressor of cytokine signaling 3 (SOCS3), vinculin (VCL) and KIAA1840. Genes that were down-regulated in both included adenylate kinase 3-like 1 (AK3L1), leucine rich repeat and fibronectin type III domain containing 4 (LRFN4), and AL359650. Increased RANK, MMP9 and SOCS3 mRNA expression in MJ compared to HH cells and in three resistant versus three vorinostat responding Sézary patients' PBMCs was confirmed using quantitative real-time PCR. In conclusion, our results suggest that oligonucleotide microarray analysis may identify biomarkers of resistance to vorinostat which would be helpful to select patients who may not benefit from treatment. These findings provide the rationale for future functional studies and development of more effective use of HDAC inhibitors for CTCL patients.

RANKL acts directly on RANK‐expressing prostate tumor cells and mediates migration and expression of tumor metastasis genes

Metastases to bone are a frequent complication of human prostate cancer and result in the development of osteoblastic lesions that include an underlying osteoclastic component. Previous studies in rodent models of breast and prostate cancer have established that receptor activator of NF-kappaB ligand (RANKL) inhibition decreases bone lesion development and tumor growth in bone. RANK is essential for osteoclast differentiation, activation, and survival via its expression on osteoclasts and their precursors. RANK expression has also been observed in some tumor cell types such as breast and colon, suggesting that RANKL may play a direct role on tumor cells. Male CB17 severe combined immunodeficient (SCID) mice were injected with PC3 cells intratibially and treated with either PBS or human osteprotegerin (OPG)-Fc, a RANKL antagonist. The formation of osteolytic lesions was analyzed by X-ray, and local and systemic levels of RANKL and OPG were analyzed. RANK mRNA and protein expression were assessed on multiple prostate cancer cell lines, and events downstream of RANK activation were studied in PC3 cells in vitro. OPG-Fc treatment of PC3 tumor-bearing mice decreased lesion formation and tumor burden. Systemic and local levels of RANKL expression were increased in PC3 tumor bearing mice. PC3 cells responded to RANKL by activating multiple signaling pathways which resulted in significant changes in expression of genes involved in osteolysis and migration. RANK activation via RANKL resulted in increased invasion of PC3 cells through a collagen matrix. These data demonstrate that host stromal RANKL is induced systemically and locally as a result of PC3 prostate tumor growth within the skeleton. RANK is expressed on prostate cancer cells and promotes invasion in a RANKL-dependent manner.

Hydrostatic Pressure Induces Cytokine Production in Human Periodontal Ligament Cells

Periodontal tissue has a unique structure in that the human periodontal ligament (hPDL) lies between the hard tissues of cementum and alveolar bone. Although the role of cytokines in hPDL function is not clearly understood, we investigated the effect of mechanical stress as hydrostatic pressure (HP) on cytokine expression in hPDL cells.The hPDL cells were obtained from a healthy maxillary third molar. After the 3rd to 4th passage, the cells were exposed to HP ranging from 1 MPa to 6 MPa as previously described. The expression of cytokine mRNA was determined by RT-PCR and cytokines in the culture supernatants were assessed by enzyme-linked immunosorbent assay (ELISA).The exposure to 6 MPa of HP caused no morphological changes of hPDL cells, and did not affect cellular viability. No expression of IL-1β, IL-6, IL-8, TNF-α, RANK, RANKL or OPG mRNA was observed in the control cells under atmospheric pressure, whereas in hPDL cells treated with HP, enhancement of IL-6, IL-8, RANKL and OPG mRNA expression was observed between 10 and 60 minutes after the exposure to HP. After the exposure to HP, the production of IL-6 and TNF-α were induced significantly in hPDL cells, but IL-1β and IL-8 were not produced.These results suggest that hPDL cells may play a role in the production of cytokines in response to mechanical stress in vivo.

Glucocorticoid Regulation of Osteoclast Differentiation and Expression of Receptor Activator of Nuclear Factor-κB (NF-κB) Ligand, Osteoprotegerin, and Receptor Activator of NF-κB in Mouse Calvarial Bones

In the present study, dexamethasone treatment of neonatal mouse calvarial bones increased mRNA expression of tartrate-resistant acid phosphatase, calcitonin receptor (CTR), cathepsin K, carbonic anhydrase II, osteoprotegerin (OPG), and receptor activator of nuclear factor-kappaB (RANK) as well as mRNA and protein expression of RANK ligand (RANKL). The increase in OPG mRNA noted with dexamethasone was in contrast to 1,25(OH)(2)-vitamin D3 (D3) treatment, which decreased OPG expression. Stimulation of (45)Ca release by dexamethasone and hydrocortisone in calvariae was blocked by OPG. Stimulation of RANKL, RANK, OPG, and CTR mRNA expression by dexamethasone in calvariae was blocked by the glucocorticoid receptor antagonist RU 38,486. Greater than additive potentiations of CTR mRNA and RANKL mRNA and protein were observed when D3 and dexamethasone were combined. Vitamin D receptor mRNA was increased by dexamethasone and D3, whereas glucocorticoid receptor (GR) mRNA was decreased by dexamethasone and unaffected by D3. No synergistic interaction between dexamethasone and D3 on either vitamin D receptor or GR mRNA expression was noted. The data demonstrate that dexamethasone-induced bone resorption in calvarial bones is associated with increased differentiation of osteoclasts and regulation of the RANKL-RANK-OPG system. The increase in OPG expression and the decrease of GR expression noted with dexamethasone offer an explanation for why bone breakdown in mouse calvariae treated with glucocorticoids is less than that caused by resorptive agents like D3. The synergistic stimulation of RANKL by dexamethasone and D3 offers an explanation of how glucocorticoids and D3 interact to potentiate bone resorption.

Inhibition of Hormone and Cytokine-stimulated Osteoclastogenesis and Bone Resorption by Interleukin-4 and Interleukin-13 Is Associated with Increased Osteoprotegerin and Decreased RANKL and RANK in a STAT6-dependent Pathway

Interleukin (IL)-4 and IL-13 are cytokines that inhibit bone resorption. Data showing an inhibitory effect of IL-4 and IL-13 on RANK mRNA in mouse calvariae were first reported at the 22nd American Society for Bone and Mineral Research Meeting (Lerner, U.H., and Conaway, H. H. 2000) J. Bone Min. Res. 15, Suppl. 1, Abstr. SU 230). In the present study, release of 45Ca from cultured mouse calvarial bones stimulated by different cytokines, peptides, and steroid hormones was inhibited by IL-4 and IL-13. IL-4 and IL-13 decreased receptor activator of nuclear factor-kappaB ligand (RANKL) and RANK mRNA and increased osteoprotegerin (OPG) mRNA in calvariae. Additionally, the cytokines decreased RANKL protein and increased OPG protein in calvarial bones. In osteoblasts isolated from calvariae, both an increase in RANKL mRNA and a decrease in OPG mRNA and protein elicited by vitamin D3 were reversed by IL-4 and IL-13. IL-4 and IL-13 decreased the number of tartrate-resistant acid phosphatase positive multinucleated cells and the mRNA expression of calcitonin receptor, tartrate-resistant acid phosphatase, and cathepsin K in mouse spleen cells and bone marrow macrophages (BMM) treated with macrophage colony-stimulating factor and RANKL. Inhibition of mRNA for RANK and the transcription factor NFAT2 was also noted in spleen cell and BMM cultures treated with IL-4 and IL-13. In addition, RANK mRNA and RANK protein were decreased by IL-4 and IL-13 in RAW 264.7 cells. Osteoblasts, spleen cells, and BMM expressed mRNA for the four proteins making up the IL-4 and IL-13 receptors. No effects by IL-4 on bone resorption and osteoclast formation or on RANKL and RANK mRNA expression were seen in Stat6-/- mice. The data indicate that IL-4 and IL-13, via a STAT6-dependent pathway, inhibit osteoclast differentiation and bone resorption by activating receptors on osteoblasts and osteoclasts that affect the RANKL/RANK/OPG system.

Regulation of osteoclastogenesis by gap junction communication

Receptor activator of NF-kappaB ligand (RANKL) is crucial in osteoclastogenesis but signaling events involved in osteoclast differentiation are far from complete and other signals may play a role in osteoclastogenesis. A more direct pathway for cellular crosstalk is provided by gap junction intercellular channel, which allows adjacent cells to exchange second messengers, ions, and cellular metabolites. Here we have investigated the role of gap junction communication in osteoclastogenesis in mouse bone marrow cultures. Immunoreactive sites for the gap junction protein connexin 43 (Cx43) were detected in the marrow stromal cells and in mature osteoclasts. Carbenoxolone (CBX) functionally blocked gap junction communication as demonstrated by a scrape loading Lucifer Yellow dye transfer technique. CBX caused a dose-dependent inhibition (significant > or = 90 microM) of the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells formed in 7- to 8-day marrow cultures stimulated by parathyroid hormone (PTH; 10 nM) or forskolin (FSK; 1 microM). Furthermore, CBX (100 microM) significantly inhibited prostaglandin E2 (PGE2; 10 microM) and 1,25(OH)2-vitamin D3 stimulated osteoclast differentiation in the mouse bone marrow cultures. Consequently, quantitative real-time polymerase chain reaction (PCR) analysis demonstrated that CBX downregulated the expression of osteoclast phenotypic markers, but without having any significant effects on RANK, RANKL, and osteoprotegerin (OPG) mRNA expression. However, the results demonstrated that CBX significantly inhibits RANKL-stimulated (100 ng/ml) osteoclastogenesis in the mouse bone marrow cultures. Taken together, our results suggests that gap junctional diffusion of messenger molecules interacts with signaling pathways downstream RANKL in osteoclast differentiation. Further studies are required to define the precise mechanisms and molecular targets involved.

Down-regulation of osteoprotegerin production in bone marrow macrophages by macrophage colony-stimulating factor

Macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) induce the differentiation of bone marrow macrophages (BMMs) into osteoclasts. To delineate mechanisms involved, the effect of M-CSF on the production of osteoprotegerin (OPG), decoy receptor of RANKL, in BMMs was investigated. Mouse bone marrow cells were cultured with M-CSF for 4 days and adherent cells formed were used as BMMs. BMMs were cultured with or without M-CSF, and analyzed for expression of OPG and receptor activator of NF-κB (RANK; receptor for RANKL) mRNAs by real-time polymerase chain reaction and secretion of OPG by enzyme-linked immunosorbent assay. BMMs expressed macrophage markers, CD115 (c-fms), Mac-1 and F4/80, and showed phagocytotic activity. In addition, BMMs expressed OPG mRNA and secreted OPG into medium. M-CSF inhibited both the OPG mRNA expression and the OPG secretion dose-dependently and reversibly. The expression of RANK mRNA was not significantly affected by M-CSF. The results showed that M-CSF suppresses the OPG production in BMMs, which may increase the sensitivity of BMMs to RANKL.

Menaquinone-7 regulates the expressions of osteocalcin, OPG, RANKL and RANK in osteoblastic MC3T3E1 cells

Epidemiological studies show that dietary intake of natto, which contains significant amount of vitamin K(2), reduces the risk of bone formation loss. However, many confounding factors, such as calcium and isoflavone, are found in natto, because it is made from soybeans. In this study, the direct effects of MK-7, a vitamin K(2) analogue, were assessed in osteoblasts. Osteoblastic MC3T3E1 cells were cultured with or without MK-7 for 10 days and the number of cells was calculated. The cell count was not different between MK-7 treated cells and control cells for 1, 2, and 4 days. However, it was significantly suppressed in MK-7 treated cells at 10 days, suggesting that MK-7 suppressed cell proliferation. Real-time PCR analysis showed that mRNAs of osteocalcin (OC), osteoprotegerin (OPG), and the receptor activator of the NFkappaB ligand (RANKL) were induced after MK-7 administration to the culture medium. RANK mRNA expression was also enhanced by MK-7 administration. Immunocytochemical analysis showed that MK-7 increased the protein levels of OC and RANKL. RANK protein was also enhanced, but this induction was suppressed by anti-RANK antibody administration. This suppression was recovered when anti-RANK antibody and MK-7 were administered. These observations suggest that MK-7 may directly affect MC3T3E1 cells and stimulate osteoblastic differentiation, not proliferation.

Ascorbic acid inhibits the formation and function of osteoclasts from RAW264.7 cells induced by receptor activated nuclear factor kappaB ligand in vitro

To observe the influence of ascorbic acid (AA) on osteoclastogenesis induced by receptor activated nuclear factor kappaB ligand (RANKL) in RAW264.7 cells and to study the mechanism of AA in osteoclast differentiation. Mouse mononuclear cells of the line RAW264.7 were cultured. AA and/or RANKL were added. Cellular respiration of RAW264.7 cells was assayed by MTT cell proliferation method. RAW264.7 cells were inoculated and RANKL and AA of different concentrations were added. TRAP staining was used to observe the formation of osteoclasts. RAW264.7 cells were inoculated on OAAS, RANKL and AA were added. The number of bone absorption pits was counted. RT-PCR was used to measure the expression of RANK, matrix metalloproteinase 9, TRAF6, TRAP, integrin alpha v, integrin beta 3, cathepsin K, and carbonic anhydrase II (CA II). AA of the concentration of 500 microg/ml significantly inhibited the proliferation of RAW264.7 cells (0.13 +/- 0.05 vs 1.11 +/- 0.22, P < 0.05). RANKL significantly inhibited the proliferation of RAW264.7 cells as well (1.58 +/- 0.22 vs 1.26 +/- 0.17). AA showed no synergistic action to RANKL. AA alone could not induce the formation of TRAP-positive osteoclasts from RAW264.7 cells and 0 approximately 100 microg/ml AA inhibited the RANKL-induced formation of osteoclast-like multinuclear cells from RAW264.7 cells. AA alone up-regulated the mRNA expression of CA II, cathepsin K, and TRAP by 10, 1.5, and 1.5 times respectively. RANKL plus AA significantly down-regulated the mRNA expression of CAII and RANK by 60% and 20% respectively. No bone absorption pit was seen after addition of AA. The number of pits on OAAS after addition of AA + RANKL was significantly lower than that after addition of RANKL alone (P < 0.05). Western blotting showed that no difference was seen in the protein expression of CA I between the RANKL group and the control group, however, the protein expression of CA II in the low concentration AA + RANKL group was significantly lower than that in the RANKL alone group (P < 0.05). AA directly inhibits RANKL induced osteoclast formation and its function in vitro.

Increased expression of IL-6 and RANK mRNA in human trabecular bone from fragility fracture of the femoral neck

Previous studies have implicated pro-inflammatory cytokines in the bone loss of estrogen deficiency. The aim of this study was to investigate the expression of key regulatory molecules of bone remodeling in the trabecular bone microenvironment in osteoporosis. Bone samples were taken from the intertrochanteric region of the proximal femur of patients undergoing total hip arthroplasty for a subcapital fragility fracture of the femoral neck (#NOF). For comparison, samples were taken from age-matched control individuals at routine autopsy. Expression of RANKL, RANK, osteoprotegerin (OPG), IL-6, IL-11, osteocalcin (OCN), and calcitonin receptor (CTR) messenger RNA (mRNA) species were analyzed and the data were nonparametrically distributed. The median expression of the proresorptive genes, RANK and IL-6, were significantly elevated in the fracture group compared to an age-matched control group (2.2 [1.9–2.9; 25th–75th percentiles] > 1.0 [0.4–2.1], P < 0.03; 3.9 [1.8–6.2] > 0.8 [0.7–1.5], P < 0.002, respectively). In contrast, there were no significant differences in expression of RANKL, OPG, CTR, or OCN mRNA between the #NOF and control groups. The median RANKL/OPG mRNA ratio was significantly greater in hip fracture bone than in bone from controls (4.8 [3.8–7.6] > 3.2 [2.1–4.0], P < 0.05). IL-6 mRNA levels associated strongly with RANKL mRNA levels in the #NOF group ( r = 0.77, P < 0.001), but not in the control group. A strong positive association was found between IL-11 mRNA levels and RANKL mRNA levels in the #NOF group ( r = 0.81, P < 0.001), consistent with the apparent coordinated regulation of IL-6 and IL-11 in bone samples from the #NOF group ( r = 0.93, P < 0.0001). These data suggest a relative increase in the expression of the molecular promoters of osteoclast formation and activity in #NOF bone, which may lead to the imbalance between bone formation and resorption associated with fragility fracture.

Protective effects of IL-1Ra or vIL-10 gene transfer on a murine model of wear debris-induced osteolysis.

The current study evaluated the protective effects of anti-inflammatory cytokine gene transfer on osteolysis provoked by orthopedic biomaterial particles using a murine model of inflammatory bone loss. A section of bone was surgically implanted into an air pouch established on a syngeneic recipient mouse. Inflammation was provoked by introduction of ultra-high-molecular-weight polyethylene (UHMWPE) particles into the pouch, and retroviruses encoding for interleukin-1 receptor antagonist (hIL-1Ra), viral interleukin-10 (vIL-10), or LacZ genes were injected. Pouch fluid and tissue were harvested 7 days later for histological and molecular analyses. The results indicated that IL-1Ra or vIL-10 gene transfer significantly inhibited IL-1beta and tumor necrosis factor (TNF) expression at both mRNA and protein levels. There were significantly lower mRNA expressions of calcitonin receptor and cathepsin K in RNA isolated from hIL-1Ra- or vIL-10-transduced pouches than LacZ-transduced and virus-free controls. Both anti-inflammatory cytokine gene transfers significantly reduced the mRNA expression of M-CSF (70-90%) and RANK (>65%) in comparison with LacZ- and virus-free controls. Histological examination showed that hIL-1Ra or vIL-10 gene transfer dramatically abolished UHMWPE-induced inflammatory cellular infiltration and bone pit erosion compared to LacZ-transduced and virus-free controls. Histochemical staining revealed significantly fewer osteoclast-like cells in samples treated with IL-1Ra or vIL-10 gene transfer. In addition, bone collagen content was markedly preserved in the groups with anti-inflammatory cytokine gene transfers compared with the other two groups. Overall, retrovirus-mediated hIL-1Ra or vIL-10 gene transfer effectively protected against UHMWPE-particle-induced bone resorption, probably due to the inhibition of IL-1/TNF-induced M-CSF production and the consequent osteoclast recruitment and maturation.

Osteoclastogenesis is decreased by cysteine proteinase inhibitors

The effects of cystatin C and other cysteine proteinase inhibitors on osteoclast formation and differentiation have been investigated. Cystatin C decreased osteoclast formation stimulated by parathyroid hormone (PTH), 1,25(OH) 2-vitamin D3 or interleukin-6 (IL-6) (in the presence of its soluble receptor) as assessed by the number of tartrate-resistant acid phosphatase (TRAP +) multinucleated cells in mouse bone marrow cultures. The inhibitory effect was associated with decreased mRNA expression for the calcitonin receptor as well as decreased number of specific binding sites for 125I-calcitonin, and without any effect on the mRNA expression of receptor activator of nuclear factor κB (NF-κB) ligand (RANKL). Similarly, the cysteine proteinase inhibitors leupeptin, E-64 and benzyloxycarbonyl-Phe-Ala-diazomethane (Z-FA-CHN 2) decreased PTH-stimulated formation of TRAP + multinucleated cells and binding of 125I-calcitonin. A peptidyl derivative synthesized to mimic part of the proteinase-binding site of cystatin C (benzyloxycarbonyl-Arg-Leu-Val-Gly-diazomethane, or Z-RLVG-CHN 2) also decreased PTH-stimulated osteoclast formation. In a 9-day culture, addition of cystatin C during the last 5 days was sufficient to cause substantial inhibition of osteoclast formation. Cystatin C-induced decrease of osteoclast formation was associated with enhanced number of F4/80-positive macrophages and increased mRNA expression of the macrophage receptor c-fms in the bone marrow culture. Osteoclast formation in mouse bone marrow cultures as well as in mouse spleen cell cultures, stimulated by macrophage colony-stimulating factor (M-CSF) and RANKL was also decreased by different cysteine proteinase inhibitors. In addition, cystatin C inhibited M-CSF/RANKL induction of calcitonin receptor mRNA in spleen cell cultures. The inhibitory effect by cystatin C in spleen cells was associated with decreased mRNA expression of RANK and the transcription factor NFAT2. It is concluded that cysteine proteinase inhibitors decrease formation of osteoclasts by interfering at a late stage of pre-osteoclast differentiation.

Vascular Endothelial Growth Factor Up-regulates Expression of Receptor Activator of NF-κB (RANK) in Endothelial Cells: CONCOMITANT INCREASE OF ANGIOGENIC RESPONSES TO RANK LIGAND

Vascular endothelial growth factor (VEGF) is known as a key regulator of angiogenesis during endochondral bone formation. Recently, we demonstrated that TNF-related activation-induced cytokine (TRANCE or RANKL), which is essential for bone remodeling, also had an angiogenic activity. Here we report that VEGF up-regulates expression of receptor activator of NF-kappa B (RANK) and increases angiogenic responses of endothelial cells to TRANCE. Treatment of human umbilical vein endothelial cells (HUVECs) with VEGF increased both RANK mRNA and surface protein expression. Although placenta growth factor specific to VEGF receptor-1 had no significant effect on RANK expression, inhibition of downstream signaling molecules of the VEGF receptor-2 (Flk-1/KDR) such as Src, phospholipase C, protein kinase C, and phosphatidylinositol 3'-kinase suppressed VEGF-stimulated RANK expression in HUVECs. Moreover, the MEK inhibitor PD98059 or expression of dominant negative MEK1 inhibited induction of RANK by VEGF but not the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM). VEGF potentiated TRANCE-induced ERK activation and tube formation via RANK up-regulation in HUVECs. Together, these results show that VEGF enhances RANK expression in endothelial cells through Flk-1/KDR-protein kinase C-ERK signaling pathway, suggesting that VEGF plays an important role in modulating the angiogenic action of TRANCE under physiological or pathological conditions.