Treatment Of Pathological Bone Loss Research Articles

7NDprotein exerts inhibitory effects on both osteoclast differentiation invitro and lipopolysaccharide‑induced bone erosion invivo.

Excessive numbers of osteoclasts are responsible for inflammation-induced osteolysis. Identification of osteoclast-targeting agents may facilitate the development of a novel therapeutic approach for the treatment of pathological bone loss. Seven-amino acid truncated (7ND) protein, a mutant form of monocyte chemoattractant protein-1 (MCP-1), functions as a competitive inhibitor of MCP-1. However, the effects of 7ND protein on osteoclast differentiation remain unknown. Therefore, in the present study, the effects of 7ND protein on osteoclast differentiation induced by tumour necrosis factor superfamily member 11 were investigated. In the present study, 7ND protein inhibited the osteoclast differentiation of peripheral blood mononuclear cells without influencing cell proliferation. Furthermore, to evaluate the effects of 7ND protein in vivo, a lipopolysaccharide (LPS)-induced calvarial bone erosion animal model was established. The 7ND protein remarkably attenuated LPS-induced bone resorption, as assessed by micro-computed tomography and histological analysis. Taken together, the present results suggested the feasibility of local delivery of 7ND protein to mitigate osteoclast differentiation and LPS-induced osteolysis, which may represent a potential approach to treat inflammatory bone destruction.

Autophagy mediated by JNK1 resists apoptosis through TRAF3 degradation in osteoclastogenesis

RANKL induces osteoclastogenesis via JNK1 signal that exerts an anti-apoptotic effect during osteoclastogenesis. However, the classic downstream c-Jun/AP-1 pathway is not included in anti-apoptosis of JNK1. Thus, the detail mechanism underlying JNK1-resisted apoptosis remains unknown during RANKL-induced osteoclastogenesis. RANKL-induced autophagy results in the degradation of the osteoclastogenesis-inhibitor TRAF3, and TRAF3 is thought as a regulator of apoptosis. Given the key effect of JNK1 in mediating autophagy, our study aims to investigate the significance of TRAF3 in bridging JNK1-mediated autophagy and apoptotic resistance during osteoclastogenesis. In this study, by using Bone Marrow-derived macrophages (BMMs) as osteoclast precursors (OCPs), we found that RANKL-induced TRAF3 degradation was significantly suppressed by JNK inhibitor (SP600125), which was restored by overexpression of Beclin1 (key autophagic protein). Nevertheless, TRAF3 silencing partially reversed the reduced osteoclastogenesis under SP600125 intervention. Besides, OCP apoptosis was positively regulated by TRAF3 overexpression, regardless of the application of autophagy inhibitor or SP600125. Remarkably, the enhanced apoptosis caused by the pharmacological inhibition of Beclin1 was reversed by TRAF3 silencing. Together, these results suggest that JNK1-mediated autophagy could promote RANKL-induced osteoclastogenesis via enhancing TRAF3 degradation. Importantly, JNK1 could prevent OCP apoptosis through autophagy-TRAF3 signaling, which provides more potential targets for clinical treatment of pathological bone loss.

'Osteoimmunology' Offers New Perspectives for the Treatment of Pathological Bone Loss.

New evidence of the interactions between the immune system and bone has accumulated in bone diseases, including osteoporosis, periodontitis and rheumatoid arthritis. A marked imbalance between bone resorption and formation is central to the onset of pathological bone loss. Osteoimmunology has revealed that the immune system, including T cells, B cells and inflammatory cytokines, is a key regulator of both osteoclasts and osteoblasts. Th1 cells, which differentiate from CD4+T cells, are thought to play a major function during bone loss. Moreover, the correlated expression of Th1 cytokines (interleukin-12 (IL-12), interferon-γ (IFN-γ)) and bone-resorbing cytokines (tumor necrosis factor-α (TNF-α), IL-1) also plays a key role during inflammatory induced bone resorption. Furthermore, a relatively new member of the CD4+T cell family Th17 displays the ability to promote osteoclast activity. The effect of IFN-γ and IL-17 released by Th 17 cells on pre-osteoclast proliferation, differentiation and apoptosis provides the preliminary basis for the immune mechanism of pathological bone loss. The role of B cells in osteoimmunological interactions has long been suspected based on findings of B cells as active regulators of the RANK/RANKL/OPG axis. Pathological bone loss, including osteoporosis and human immunodeficiency virus-associated bone loss, are related to the altered RANKL/OPG through modified production by B cells, supporting this assumption. All of the above evidence may provide new theoretical explanations for the relationship between bone metabolism and the immune system as well as offer perspectives for the prevention and treatment of pathological bone loss.

MiR-33-5p, a novel mechano-sensitive microRNA promotes osteoblast differentiation by targeting Hmga2.

MicroRNAs (miRNAs) interfere with the translation of specific target mRNAs and are thought to thereby regulate many cellular processes. However, the role of miRNAs in osteoblast mechanotransduction remains to be defined. In this study, we investigated the ability of a miRNA to respond to different mechanical environments and regulate mechano-induced osteoblast differentiation. First, we demonstrated that miR-33-5p expressed by osteoblasts is sensitive to multiple mechanical environments, microgravity and fluid shear stress. We then confirmed the ability of miR-33-5p to promote osteoblast differentiation. Microgravity or fluid shear stress influences osteoblast differentiation partially via miR-33-5p. Through bioinformatics analysis and a luciferase assay, we subsequently confirmed that Hmga2 is a target gene of miR-33-5p that negatively regulates osteoblast differentiation. Moreover, miR-33-5p regulates osteoblast differentiation partially via Hmga2. In summary, our findings demonstrate that miR-33-5p is a novel mechano-sensitive miRNA that can promote osteoblast differentiation and participate in the regulation of differentiation induced by changes in the mechanical environment, suggesting this miRNA as a potential target for the treatment of pathological bone loss.

Endocytotic Uptake of Zoledronic Acid by Tubular Cells May Explain Its Renal Effects in Cancer Patients Receiving High Doses of the Compound

Zoledronic acid, a highly potent nitrogen-containing bisphosphonate used for the treatment of pathological bone loss, is excreted unmetabolized via the kidney if not bound to the bone. In cancer patients receiving high doses of the compound renal excretion may be associated with acute tubular necrosis. The question of how zoledronic acid is internalized by renal tubular cells has not been answered until now. In the current work, using a primary human tubular cell culture system, the pathway of cellular uptake of zoledronic acid (fluorescently/radiolabeled) and its cytotoxicity were investigated. Previous studies in our laboratory have shown that this primary cell culture model consistently mimics the physiological characteristics of molecular uptake/transport of the epithelium in vivo. Zoledronic acid was found to be taken up by tubular cells via fluid-phase-endocytosis (from apical and basolateral side) as evidenced by its co-localization with dextran. Cellular uptake and the resulting intracellular level was twice as high from the apical side compared to the basolateral side. Furthermore, the intracellular zoledronic acid level was found to be dependent on the administered concentration and not saturable. Cytotoxic effects however, were only seen at higher administration doses and/or after longer incubation times. Although zoledronic acid is taken up by tubular cells, no net tubular transport could be measured. It is concluded that fluid-phase-endocytosis of zoledronic acid and cellular accumulation at high doses may be responsible for the acute tubular necrosis observed in some cancer patients receiving high doses of the compound.

Ampelopsis brevipedunculata extract prevents bone loss by inhibiting osteoclastogenesis in vitro and in vivo.

Osteoclasts play a critical role in bone resorbing disorders such as osteoporosis, periodontitis, and rheumatoid arthritis. Therefore, discovery of agents capable of suppressing osteoclast differentiation may aid the development of a therapeutic access for the treatment of pathological bone loss. Ampelopsis brevipedunculata has been used as herbal folk medicine to treat liver diseases and inflammation in Asia. However, its effects on osteoclast differentiation are unknown. We were aimed to investigate the anti-osteoclastogenic activity in vitro and in vivo and to elucidate the underlying mechanism of Ampelopsis brevipedunculata extract (ABE). In this study, ABE inhibited receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation, the formation of filamentous actin rings and the bone resorbing activity of mature osteoclasts. ABE inhibited RANKL-induced p38 and IκB phosphorylation and IκB degradation. Also, ABE suppressed the mRNA and protein expression of nuclear factor of activated T cells c1 (NFATc1) and c-Fos, and the mRNA expression of genes required for cell fusion and bone resorption, such as osteoclast-associated receptor (OSCAR), tartrate resistant acid phosphatase (TRAP), cathepsin K, dendritic cell-specific transmembrane protein (DC-STAMP), β3-integrin and osteoclast stimulatory transmembrane protein (OC-STAMP). Furthermore, results of micro-CT and histologic analysis indicated that ABE remarkably prevented lipopolysaccharide (LPS)-induced bone erosion. These results demonstrate that ABE prevents LPS-induced bone erosion through inhibition of osteoclast differentiation and function, suggesting the promise of ABE as a potential cure for various osteoclast-associated bone diseases.

Chlorogenic Acid Inhibits Osteoclast Differentiation and Bone Resorption by Down-Regulation of Receptor Activator of Nuclear Factor Kappa-B Ligand-Induced Nuclear Factor of Activated T Cells c1 Expression

Excessive osteoclastic bone resorption plays a critical role in inflammation-induced bone loss such as rheumatoid arthritis and periodontal bone erosion. Therefore, identification of osteoclast targeted-agents may be a therapeutic approach to the treatment of pathological bone loss. In this study, we isolated chlorogenic acid (CGA) from fructus of Gardenia jasminoides to discover anti-bone resorptive agents. CGA is a polyphenol with anti-inflammatory and anti-oxidant activities, however, its effects on osteoclast differentiation is unknown. Thus, we investigated the effect of CGA in receptor activator of nuclear factor-kappa B (NF-κB) ligand (RANKL)-induced osteoclast differentiation and RANKL signaling. CGA dose-dependently inhibited RANKL-mediated osteoclast differentiation in bone marrow macrophages (BMMs) without any evidence of cytotoxicity. CGA inhibited the phosphorylation of p38, Akt, extracellular signal-regulated kinase (ERK), and inhibitor of nuclear factor-kappa B (IκB), and IκB degradation by RANKL treatment. CGA suppressed the mRNA expression of nuclear factor of activated T cells c1 (NFATc1), TRAP and OSCAR in RANKL-treated bone marrow macrophages (BMMs). Also, overexpression of NFATc1 in BMMs blocked the inhibitory effect of CGA on RANKL-mediated osteoclast differentiation. Furthermore, to evaluate the effects of CGA in vivo, lipopolysaccharide (LPS)-induced bone erosion study was carried out. CGA remarkably attenuated LPS-induced bone loss based on micro-computed tomography and histologic analysis of femurs. Taken together, our findings suggest that CGA may be a potential treatment option for osteoclast-related diseases with inflammatory bone destruction.

P36 Hydrogen sulfide inhibits RANKL-induced ROS production and osteoclast differentiation

Osteoclasts (OC), the bone resorbing cells, are multinucleated cells arising from the fusion of circulating monocytic precursors. Once differentiated, they settle into bone and break down bone mineral matrix, thereby playing a vital role in physiological bone turnover.In post-menopausal osteoporosis or in inflammatory bone disease, OC are activated leading to an excess bone erosion which in not balanced by an equal bone formation by osteoblasts, a process that leads to a loss of bone mass, increased bone fragility and fracture risk. The rate of monocyte differentiation into OC has been shown to be a critical step in pathological bone erosion.Here we sought to investigate whether hydrogen sulfide affect OC differentiation and function. Human osteoclast precursors were isolated from peripheral blood obtained from healthy donors by immunomagnetic positive selection of CD11b+ cells. Mature OC were obtained in vitro by culturing CD11b+ cells for 8–10 days in the presence of M-CSF (10 ng/ml) and RANKL (75 ng/ml). The effect of hydrogen sulfide was evaluated by treating the cells with NaHS in the range of concentrations between 50 and 300 μg/ml.We first assessed whether NaHS affect cell survival and proliferation. LDH activity assay revealed no cytotoxic effect of NaHS at 50, 10, 200 or 300 μg/ml at 24 h after stimulation, while 72 h after stimulation the concentration of 300 μg/ml induced a 25% increase in cell death. Cell proliferation was evaluated by 3 H-timidine uptake: consistent with citotoxicity data, NaHS did not affect the proliferation of CD11b+ cells in response to M-CSF stimulation up to a concentration of 200 μg/ml. Evaluation of apoptosis by Annexin V/PI further confirmed that NaHS did not induced cell death at 50, 100 or 200 μg/ml while induced a 30% increase of apoptosis at the concentration of 300 μg/ml. To assess the effect of NaHS on OC differentiation, TRAP + mature OC were counted at the end of the culture. NaHS dose-dependently inhibited OC formation, leading to a significant decrease in OC number starting a 100 μg/ml. Functional Pit Assay confirmed that NaHS significantly inhibits the ability of OC to break down a bone-like mineral matrix. Attesting to specificity, similar data were obtained in the presence of the H 2 S-donor GYY4137. Therefore, hydrogen sulfide inhibits OC differentiation and function in a range of concentration which does not affect cell survival. As RANKL signaling is critically dependent on ROS generation in OC progenitor cells, we evaluated the effect of NaHS on RANKL-dependent ROS generation by Dichlorofluorescein staining. As expected, RANKL (100 μg/ml, 30 min) induced a significant production of intracellular ROS in OC precursors cells; however, NaHS (200 μg/ml), significantly inhibited RANKL-induced ROS generation.In conclusion, Hydrogen sulfide dose-dependently prevents OC differentiation and function and this effect is associated to a significant inhibition of RANKL-induced ROS production. These findings suggest a possible therapeutical role for hydrogen sulfide donors in the treatment of pathological bone loss.

Trolox Prevents Osteoclastogenesis by Suppressing RANKL Expression and Signaling

Excessive receptor activator of NF-kappaB ligand (RANKL) signaling causes enhanced osteoclast formation and bone resorption. Thus, down-regulation of RANKL expression or its downstream signals may be a therapeutic approach to the treatment of pathological bone loss. In this study, we investigated the effects of Trolox, a water-soluble vitamin E analogue, on osteoclastogenesis and RANKL signaling. Trolox potently inhibited interleukin-1-induced osteoclast formation in bone marrow cell-osteoblast coculture by abrogating RANKL induction in osteoblasts. This RANKL reduction was attributed to the reduced production of prostaglandin E(2) via a down-regulation of cyclooxygenase-2 activity. We also found that Trolox inhibited osteoclast formation from bone marrow macrophages induced by macrophage colony-stimulating factor plus RANKL in a reversible manner. Trolox was effective only when present during the early stage of culture, which implies that it targets early osteoclast precursors. Pretreatment with Trolox did not affect RANKL-induced early signaling pathways, including MAPKs, NF-kappaB, and Akt. We found that Trolox down-regulated the induction by RANKL of c-Fos protein by suppressing its translation. Ectopic overexpression of c-Fos rescued the inhibition of osteoclastogenesis by Trolox in bone marrow macrophages. Trolox also suppressed interleukin-1-induced osteoclast formation and bone loss in mouse calvarial bone. Taken together, our findings indicate that Trolox prevents osteoclast formation and bone loss by inhibiting both RANKL induction in osteoblasts and c-Fos expression in osteoclast precursors.

Novel vitamin D 3 analogs, 1α, 25(OH) 2D 3-26, 23-lactam (DLAMs), antagonize bone resorption via suppressing RANKL expression in osteoblasts

Novel vitamin D analogs, 1α, 25-dihydroxyvitamin D 3-26, 23-lactam (DLAMs) with a lactam moiety in the side chain, were synthesized and examined for their function in bone. In computer docking simulation, DLAM-1P binds to vitamin D receptor (VDR), and its lactam moiety may interfere with VDR helix-12 folding. In co-cultures of mouse bone marrow cells and osteoblasts, (23S, 25S)-DLAM-1P dose-dependently suppressed osteoclast differentiation induced by 1α, 25-dihydroxyvitamin D 3 [1α, 25(OH) 2D 3]. Its stereoisomer (23R, 25R)-DLAM-1P did not affect the osteoclast differentiation. In osteoblasts, (23S, 25S)-DLAM-1P suppressed 1α, 25(OH) 2D 3-induced mRNA expression of the receptor activator of NF-κB ligand (RANKL). In an organ culture using mouse calvaria, bone-resorbing activity induced by 1α, 25(OH) 2D 3 was clearly suppressed by (23S, 25S)-DLAM-1P. The other analog, (23S, 25S)-DLAM-2P, showed a similar activity to (23S, 25S)-DLAM-1P. Therefore, DLAMs act on osteoblasts as an antagonist of 1α, 25(OH) 2D 3 to suppress RANKL-dependent osteoclast formation, suggesting them as a novel candidate for the treatment of pathological bone loss.

Effects of receptor activator of NFκB (RANK) signaling blockade on fracture healing

As dominant regulators of osteoclastogenesis and bone resorption, receptor activator of NFκB (RANK), receptor activator of NFκB ligand, and OPG have been identified as ideal drug targets for the treatment of metabolic bone disease. One concern regarding the therapeutic use of RANK signaling inhibitors is their effect on fracture healing. Therefore we tested if uncoupling and osteoclast depletion via RANK blockade affects callus formation, maturation and matrix remodeling, as well as union rates in a mouse tibia fracture model. Low dose (1 mg/kg i.p.) RANK:Fc therapy had no effect on callus formation, matrix maturation and remodeling, and resulted in 100% bony union by day 28. High dose RANK:Fc treatment (10 mg/kg i.p.) effectively eliminated osteoclasts at the fracture site on day 14, with no significant effects on fracture healing. When therapy was discontinued, normal numbers of osteoclasts were observed at the fracture site by day 28. However, continuous therapy resulted in a large osteopetrotic callus consisting of both mineralized and unmineralized matrix that was void of osteoclasts, but bony union was unaffected at day 28. We also evaluated this process in the complete absence of RANK signaling using RANK −/− mice. These animals exhibited significant radiographic and histologic evidence of callus formation, indicating that RANK signaling is not required for fracture callus formation. However, only 33% of RANK −/− animals formed bony unions compared to 100% of the osteopetrotic control mice. This defect was most likely a result of decreased blood flow, as evidenced by fewer blood vessels in the RANK −/− animals. Together, these data imply that osteoclast depletion via inhibition of RANK signaling is a viable option for the treatment of pathological bone loss since no adverse effects on fracture healing are observed when therapy is discontinued.