Osteoclastogenesis Inhibitory Factor Research Articles

Screening for early rheumatoid arthritis using high-frequency ultrasound, serum RANKL, and OPG detection.

To conduct a comparative study of high-frequency ultrasound and magnetic resonance imaging (MRI) combined with serum RANKL and OPG detection, and assess the efficacy of high-frequency ultrasound with RANKL and OPG detection in screening early rheumatoid arthritis (RA). High-frequency ultrasound and MRI were performed on both carpal joints of 60 patients with early RA, and the frequencies of synovitis, joint effusion, tenosynovitis, and bone erosion detected by high-frequency ultrasound and MRI were observed. The serum levels of receptor activator for nuclear factor-κB ligand (RANKL) and osteoclastogenesis inhibitory factor (OPG) were also detected. The serum levels of RANKL and OPG were also detected in 80 normal healthy examinees. The data were recorded and statistically analyzed. The detection rates of carpal synovitis, joint effusion, tenosynovitis, and bone erosion in RA patients by high-frequency ultrasound were 81.66%, 69.16%, 63.33%, and 1.66%, respectively, while the detection rates by MRI were 80.00%, 71.66%, 65.00%, and 15.00%, respectively. There was no significant difference between high-frequency ultrasound and MRI in the detection rates of carpal synovitis, joint effusion, and tenosynovitis in RA patients (P>0.05), while the detection rate of bone erosion by high-frequency ultrasound was significantly lower than that by MRI. The serum levels of RANKL and OPG in RA patients were 231.47 and 68.71, respectively, while the serum levels of RANKL and OPG in normal healthy examinees were 123.51 and 385.05, respectively. The serum RANKL levels of RA patients were significantly higher than those of healthy examinees, while the serum OPG levels of RA patients were significantly lower than those of healthy examinees, which were statistically significant (P < 0.01). The AUC values of the ROC curves obtained by high-frequency ultrasound and MRI combined with serum RANKL and OPG detection in Synovitis modeling were 0.955 and 0.954, respectively. The AUC values of the ROC curves obtained from the joint fusion modeling using high-frequency ultrasound and MRI combined with serum RANKL and OPG detection were 0.949 and 0.950, respectively. The AUC values of the ROC curves obtained from modeling Tenosynovitis using high-frequency ultrasound and MRI combined with serum RANKL and OPG detection were 0.941 and 0.949, respectively. The AUC values of ROC curves obtained by combining high-frequency ultrasound and MRI with serum RANKL and OPG detection in Bone erosion modeling were 0.908 and 0.923, respectively. High-frequency ultrasound combined with serum RANKL and OPG detection has comparable effects to MRI on screening early RA, providing a safe, simple, and cost-effective screening method for the early detection of RA patients. Key Points • High-frequency ultrasound and MRI can effectively detect early lesions of the wrist joints in RA patients. • Ultrasound diagnosis has the advantages of being quick, inexpensive, and repeatable, making it the preferred choice of imaging examination for RA patients at an early stage.

Functionalized calcium phosphate nanoparticles to direct osteoprotegerin to bone lesion sites in a medaka (Oryzias latipes) osteoporosis model.

Calcium phosphate (CaP) is the inorganic part of hard tissues, such as bone, teeth and tendons, and has a high biocompatibility and good biodegradability. Therefore, CaP nanoparticles functionalized with DNA encoding bone anabolic factors are promising carrier-systems for future therapeutic development. Here, we analysed CaP nanoparticles in a genetically modified medaka fish model, where osteoporosis-like lesions can be induced by transgenic expression of receptor activator of nuclear factor kappa-B ligand (Rankl). Rankl-transgenic medaka were used to visualize and understand effects of microinjected functionalized CaP nanoparticles during modulation of osteoclast activity in vivo. For this, we synthetized multi-shell CaP nanoparticles by rapid precipitation of calcium lactate and ammonium hydrogen phosphate followed by the addition of plasmid DNA encoding the osteoclastogenesis inhibitory factor osteoprotegerin-b (Opgb). An additional layer of poly(ethyleneimine) was added to enhance cellular uptake. Integrity of the synthesized nanoparticles was confirmed by dynamic light scattering, scanning electron microscopy and energy dispersive X-ray spectroscopy. Fluorescently labelled CaP nanoparticles were microinjected into the heart, trunk muscle or caudal fins of Rankl-transgenic medaka embryos that expressed fluorescent reporters in various bone cell types. Confocal time-lapse imaging revealed a uniform distribution of CaP nanoparticles in injected tissues and showed that nanoparticles were efficiently taken up by macrophages that subsequently differentiated into bone-resorbing osteoclasts. After Rankl induction, fish injected with Opg-functionalized nanoparticles showed delayed or absent degradation of mineralized matrix, i.e. a lower incidence of osteoporosis-like phenotypes. This is proof of principle that CaP nanoparticles can be used as carriers to efficiently deliver modulatory compounds to osteoclasts and block their activity.

Efficacy and Mechanisms of Oleuropein in Postmenopausal Osteoporosis.

Postmenopausal osteoporosis (PMOP) has a supernal morbidity rate in elderly females. To appraise the effects of oleuropein on bone densitometry, bone metabolic index, oxidative stress, and inflammatory index in PMOP. In addition, the mechanism of olive bittersweet preventing bone loss was explored. We grouped 80 salubrious female Sprague-Dawley rats into four teams: (1) sham operation team (sham, N = 20), (2) ovariectomy (OVX, N = 20), (3) castrated mice fed with oleuropein (OVX+ole, N = 20), and (4) castrated mice fed with estrogen (OVX+E2, N = 20). The ovariectomized SD rats were continuously raised with 200 μg/kg/dose of oleuropein. Bone mineral density and bone metabolism indexes were recorded. In order to assess the effectiveness of oleuropein on osteopenia, an enzyme-linked immunosorbent assay (ELISA) was devoted to examining the bone marrow indexes. The bone metabolism standards of PMOP rats were appraised by assessing serum levels of calcium, alkaline phosphatase (ALP), phosphorus, malondialdehyde (MDA), and nitrate content by experimental detection methods and levels of osteoclastogenesis inhibitory factor (OPG) and receptor activator for nuclear factor-κB ligand (RANKL) by ELISA. The OPG-RANK-RANKL signal passage was examined by Western blot (WB). We measured bone mineral density using dual-energy X-rays. Our animal experimental results indicated that oleuropein could significantly improve the bone mineral density of ovariectomized SD rats. In the meantime, it could reduce ending interleukin-6 (IL-6), malondialdehyde (MDA), nitrate, alkaline phosphatase (ALP), and phosphorus (P) serum concentration and would not affect Ca2+ concentration. In cell experiments, oleuropein also can promote the proliferation of osteoblasts. Furthermore, it can promote the expression of OPG protein and mRNA. In reverse, it inhibits the expression of RANKL protein and mRNA. Oleuropein can not only improve the inflammatory and oxidative indexes of castrated rats but also prevent osteoporosis. Oleuropein avoids bone resorption by regulating OPG/RANKL expression.

Effects of Long-Term Treatment with Different Types of Anti-Epileptic Drugs on Vitamin D2 and Osteoprotegrin Serum Levels in Iraqi Patients

Epilepsy is a chronic neurological illness had been described as repeated seizures associated with abnormal neurological activities in the brain. Epilepsy affects nearly fifty million people worldwide, with 85 percent of them living in developing countries. Vitamin D2 (VD2) is ergocalciferol (made from ergosterol). Genetic disorders related to vitamin D metabolism are dysfunction of generally loss osteoid mineralization the leading cause to bone disorders including: rickets or osteomalacia. Osteoprotegerin (OPG) is an osteoclastogenesis inhibitory factor (OCIF), OPG has essential role in bone metabolism and bone mass level. The aim of the present study is to investigate the effects of long-term treatment with anti-epileptic drugs (old Vs new antiepileptics) on vitamin D2 and osteoprotegrin levels among Iraqi epileptic patients. The study included fifty-one epileptic outpatients, whom attending the Consultant Clinic of Baghdad Teaching Hospital at the Medical City –Complex for the period from October/2021 to December/2021. The selected patients were on antiepileptic drugs for more than 2years, hence were grouped according to their antiepileptic therapy into: Group-1: 24 epileptic patients on old antiepileptic drugs (Carbamazepine or Valproate). Group-2: 27 epileptic patients on new antiepileptic drugs (Levetiracetam), to be compared with Group-3: 28 apparently healthy control subjects, with age and sex matching to that of patients. Serum was obtained from their blood specimens to measure: serum VD2 and OPG concentrations among the study groups participants, using specific ELISA Kits. Data analysis revealed that the mean vitamin D2 levels were not significantly different between group -1 (old antiepileptic drugs) and group-2 (new antiepileptic drugs), whereas, both of the patients groups expressed significantly lower values than the control group. However, serum osteoprotegrin levels were significantly elevated in group-1 compared to group-2. Furthermore, both of the epileptic patients groups had significantly higher values compared to the healthy subjects group. Keywords: Osteomalacia, antiepileptic drugs, Vitamin D2, Osteoprotegrin.

Effects of blue-green LED lights with two perceived illuminance (human and poultry) on immune performance and skeletal development of layer chickens

Light is one of the essential environmental factors in the production process of laying hens, which can directly affect their behavior, growth and development, and production performance. The spectral sensitivity of humans is different from that of poultry, and the perceived illuminance units of human and poultry are lux and clux, respectively. If the light management of laying hen production is carried out according to human perceived illuminance, the growth and development of laying hens during pullet rearing may be adversely affected due to the discomfort of the perceived illuminance. Preliminary research has found that blue-green LED light can improve the immune function of laying hens during the brooding and rearing periods. However, the differences of the effects caused by blue-green light on the immune performance and bone development of laying hens during pullet rearing are still unclear for the 2 spectral sensitivities. A total of 120 Jinghong layer chickens were raised from 1 d to 13 wk of age in one of three groups with a white LED light (light intensity unit lux, WL) group, a blue-green LED light (light intensity unit lux, HBGL) group, and blue-green LED light (light intensity unit clux, PBGL) group, and unlimited feed and water were provided during the whole experiment. At 7 and 13 wk of age, the immune performance, bone parameters, and related gene expression were investigated. The results showed that compared with the WL groups, HBGL and PBGL increased the immunoglobulin A (IgA) content at 13 wk of age and the IgM content at 7 wk of age (P < 0.05). The bone mineral density (BMD) at 7 and 13 wk of age and tibial strength (TS) at 13 wk of age of the pullets in the WL group were significantly higher than those in the HBGL and PBGL group (P < 0.05). Osteoclastogenesis inhibitory factor gene (OPG mRNA) expression was increased in the layer chickens at the age of 7 and 13 wk for the WL group (P < 0.05). Compared with the WL group and PBGL group, the melanopsin gene (OPN4 mRNA) transcription level of hypothalamus and pineal gland of the chickens under HBGL significantly increased at 7 and 13 wk of age (P < 0.05). In conclusion, blue-green LED light with two perceived illuminance (human and poultry) can increase the Ig content and the immune performance of layer chickens, and blue-green LED light (light intensity unit lux) can promote the expression of OPN4 gene in the hypothalamus and pineal gland. In addition, white LED light can enhance bone quality by increasing tibia OPG gene expression.

Paradoxical effects of osteoprotegerin on vascular function: inhibiting inflammation while promoting oxidative stress?

Osteoprotegerin (OPG), also known as osteoclastogenesis inhibitory factor or tumor necrosis factor receptor superfamily member 11B, is well known as a modulator of bone remodeling. The contribution of OPG to cardiovascular disease (CVD) has been suggested, but its molecular mechanism is complex and remains unclear. In the present study, Alves-Lopes et al. (Clin. Sci. (Lond.) (2021) 135(20): https://doi.org/10.1042/CS20210643) reported the critical role of syndecan-1 (SDC-1, also known as CD138), a surface protein part of the endothelial glycocalyx, in OPG-induced vascular dysfunction. The authors found that in endothelial cells (ECs), through SDC-1, OPG increased eNOS Thr495 phosphorylation, thereby inhibiting eNOS activity. Furthermore, the OPG–SDC-1 interaction increased reactive oxygen species (ROS) production through NOX1/4 activation. Both the reduced eNOS activity and induced ROS production inhibited NO production and impaired EC function. In vascular smooth muscle cells (VSMCs), the OPG–SDC-1 interaction increased ROS production through NOX1/4 activation, subsequently increased MLC phosphorylation-mediated Rho kinase-MYPT1 regulation, leading to increased vascular contraction. Ultilizing wire myography and mechanistic studies, the authors nicely provide the evidence that SDC-1 plays a crucial role in OPG-induced vascular dysfunction. As we mentioned above, the molecular mechanism and roles of OPG in cardiovascular system are complex and somewhat confusing. In this commentary, we briefly summarize the OPG-mediated signaling pathways in cardiovascular system.

H19 Promotes HCC Bone Metastasis Through Reducing Osteoprotegerin Expression in a Protein Phosphatase 1 Catalytic Subunit Alpha/p38 Mitogen‐Activated Protein Kinase–Dependent Manner and Sponging microRNA 200b‐3p

Background and Aims Bone is the second most frequent site of metastasis for HCC, which leads to an extremely poor prognosis. HCC bone metastasis is typically osteolytic, involving the activation of osteoclasts. Long noncoding RNA H19 plays an important role in the pathogenesis of human cancers. Nonetheless, the mechanism underlying the participation of H19 in HCC bone metastasis remains unclear. Approach and Results The current study established a mouse HCC bone metastasis model by using serial intracardiac injection and cell isolation to obtain cells with distinct bone metastasis ability. H19 was highly expressed in these cells and in clinical HCC bone metastasis specimens. Both osteoclastogenesis in vitro and HCC bone metastasis in vivo were promoted by H19 overexpression, whereas these processes were suppressed by H19 knockdown. H19 overexpression attenuated p38 phosphorylation and further down‐regulated the expression of osteoprotegerin (OPG), also known as osteoclastogenesis inhibitory factor. However, up‐regulated OPG expression as well as suppressed osteoclastogenesis caused by H19 knockdown were recovered by p38 interference, indicating that p38 mitogen‐activated protein kinase (MAPK)–OPG contributed to H19‐promoted HCC bone metastasis. Furthermore, we demonstrated that H19 inhibited the expression of OPG by binding with protein phosphatase 1 catalytic subunit alpha (PPP1CA), which dephosphorylates p38. SB‐203580‐mediated inactivation of p38MAPK reversed the down‐regulation of HCC bone metastasis caused by H19 knockdown in vivo. Additionally, H19 enhanced cell migration and invasion by up‐regulating zinc finger E‐box binding homeobox 1 through the sequestration of microRNA (miR) 200b‐3p. Conclusions H19 plays a critical role in HCC bone metastasis by reducing OPG expression, which is mediated by the PPP1CA‐induced inactivation of the p38MAPK pathway; and H19 also functions as a sponge for miR‐200b‐3p.

Abstract 333: Development of a biomimetic 3D scaffold to study breast cancer bone metastasis

Abstract Cancer metastasis is the leading cause of morbidity and cancer-related death. Bone is one of the most favored sites of metastatic spread, particularly from breast cancer. However, our understanding on how the host tissue can impact tumor growth is weak. This is hampered by the lack of a reliable in vitro model ables to replicate the complex microenvironment. Indeed, bone microenvironment provides physical support to cells but it can also affect cell behaviour and phenotype. We developed a hybrid 3D collagen-based scaffold functionalized with hydroxyapatite material (HA) in order to mimic natural bone features. Scaffolds were synthesized in our laboratory through collagen solution mixed and cross-linked with hydroxyapatite. Then, we cultured MDA-MB-231, a triple negative breast cancer cell line and MCF-7, an ER+ breast cancer cell line in collagen or hybrid scaffold, in order to evaluate how extracellular matrix affects cell behavior. Cell growth was evaluated by MTT cell-proliferation assay in a time course analysis. Moreover, we evaluated cell morphology and cell disposition within the scaffold by confocal microscopy. Finally, gene profiling was evaluated by qRT-PCR. MCF-7 cells acquire an organized and linear structure, whereas MDA-MB-231 an epithelial-like morphology, when cultured in hybrid scaffold. MDA-MB-231 cells proliferate faster than MCF-7 in both models. However, this is not affected by hydroxyapatite since the proliferation rate is similar when cells are cultured in collagen or hybrid scaffold. MDA-MB-231 grown in hybrid scaffold show a significant increase in RANKL/OPG ratio, pathway strictly involved in bone homeostasis. Moreover, they show a significant increased expression in JAG1, MMP2 and SNAIL1. For MCF-7, we observe a decrease in CDH1/VIM expression ratio and a significant decrease in OPG, osteoclastogenesis inhibitory factor. Overall, these results suggest a shift induced by hydroxyapatite scaffold towards an osteolytic phenotype. Whereas collagen-scaffold could better simulate in vivo primary tumor (Liverani C. et al, Sci Rep 9, 12263, 2019), we highlight that hybrid hydroxyapatite/collagen scaffold is more suitable to study breast cancer cells behaviour in bone metastasis. Then, we will implement it with a direct co-culture of osteoclast and osteoblast cells, in order to better investigate the contribution of the host microenvironment in bone metastasis. This model could provide a reliable 3D in vitro model for the study of bone metastasis mechanisms and for drug screening assays. Citation Format: Chiara Spadazzi, Laura Mercatali, Alessandro De Vita, Chiara Liverani, Claudia Cocchi, Giacomo Miserocchi, Alberto Bongiovanni, Federica Recine, Sebastiano Calpona, Nada Riva, Giandomenico Di Menna, Lorena Gurrieri, Valentina Fausti, Silvia Angela De Bonis, Giovanni Martinelli, Nicola Baldini, Toni Ibrahim. Development of a biomimetic 3D scaffold to study breast cancer bone metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 333.

Response of Osteoblasts and Osteoclasts to Low-Intensity Pulsed Ultrasound: In Vitro and In Vivo Analyses Using Goldfish Scales

Objectives: Low-intensity pulsed ultrasound (LIPUS), which is known as a noninvasive therapy, promotes the repair of bone fractures. Until now, studies of LIPUS have focused on the proliferation and the differentiation of osteoblasts. However, the effects of LIPUS on osteoclasts are still not fully understood. Therefore, we examined the effects of LIPUS on osteoclastogenesis using goldfish scales with both osteoclasts and osteoblasts as a model of bone. Materials and Methods: Ultrasound was generated by the Sonic Accelerated Fracture Healing System (SAFHS 4000J; Teijin Pharma, Ltd). Scales were collected from goldfish under anesthesia; they were then treated with LIPUS for 20 minutes, incubated at 15°C for 3, 6, 12, and 24 hours in L-15 medium, and their mRNA expression was measured. Next, the influence of LIPUS on scale regeneration was investigated to confirm the results obtained from an in vitro experiment. Results and Discussion: After 6 hours of incubation following LIPUS treatment, RANKL mRNA increased significantly. Resulting from the increased RANKL mRNA level, the expression of transcription-regulating factors significantly increased after 6 hours of incubation, and the mRNA expression of functional genes was significantly upregulated after 12 hours of incubation. However, the mRNA expression of osteoprotegerin, which is known as an osteoclastogenesis inhibitory factor, also significantly increased after 6 hours of incubation. Thus, we determined that LIPUS moderately activates osteoclasts via RANK/RANKL signaling and may induce bone formation. Next, we performed an in vivo experiment with goldfish. After 2 weeks of daily LIPUS exposure, the regeneration rate of the LIPUS-treated scales was significantly higher than that of control scales. These data support the hypothesis that LIPUS moderately activates osteoclasts and induces bone formation.

Therapeutic and Preventive Effects of Osteoclastogenesis Inhibitory Factor on Osteolysis, Proliferation of Mammary Tumor Cell and Induction of Cancer Stem Cells in the Bone Microenvironment.

Background: We examined the effects of recombinant human osteoclastogenesis inhibitory factor (hOCIF) on osteolysis, proliferation of mammary tumor cells, and induction of cancer stem cells (CSCs) in the tumor-bone and tumor-subcutaneous microenvironments (TB- and TS-microE). Methods: Mouse mammary tumor cells were transplanted onto the calvaria or into a subcutaneous lesion of female mice, creating a TB-microE and a TS-microE, and the mice were then treated with hOCIF. To investigate the preventive effects of hOCIF, mice were treated with hOCIF before tumor cell implantation onto the calvaria (Pre), after (Post), and both before and after (Whole). The number of CSCs and cytokine levels were evaluated by IHC and ELISA assay, respectively. Results: hOCIF suppressed osteolysis, and growth of mammary tumors in the TB-microE, but not in the TS-microE. In the Pre, Post, and Whole groups, hOCIF suppressed osteolysis, and cell proliferation. hOCIF increased mouse osteoprotegrin (mOPG) levels in vivo, which suppressed mammary tumor cell proliferation in vitro. These preventive effects were observed in the dose-dependent. hOCIF did not affect the induction of CSCs in either microenvironment. Conclusion: While receptor activator of NF-κB ligand (RANKL) targeting therapy may not affect the induction of CSCs, RANKL is a potential target for prevention as well as treatment of breast cancer bone metastasis.

Milk basic protein supplementation exerts an anti-inflammatory effect in a food-allergic enteropathy model mouse

To examine novel functions of milk basic protein (MBP) in T-cell-related inflammatory diseases, such as autoimmune diseases and allergies, we evaluated the effects of MBP on the causative responses of ovalbumin (OVA)-specific T cells in a food-allergic enteropathy model, OVA23-3 mice, which express an OVA-specific T-cell receptor gene. The OVA-specific CD4+ T cells of the mesenteric lymph nodes (MLN) from OVA23-3 mice were cultured with CD11c+ dendritic cells of MLN from BALB/cA mice in the absence or presence of MBP following stimulation with OVA; then the levels of CD69 expression and the levels of cytokine production by CD4+ T cells were measured to evaluate activation. The effects of MBP supplementation of OVA 23-3 mice were assessed by feeding a diet containing OVA (OVA diet) with or without MBP for 28 d. Intestinal inflammation, together with activation and cytokine production of CD4+ T cells by MLN, as well as femoral bone mineral density, were measured. In in vitro culture, MBP inhibited excess activation and IL-4 production by CD4+ T cells. The supplementation of MBP to the OVA diet attenuated OVA-specific IgE production in OVA-diet-fed OVA23-3 mice and slightly resolved developing enteropathy caused by excess IL-4 production by CD4+ T cells. Feeding OVA diet to OVA23-3 mice exhibited bone loss accompanied with enteropathy, whereas MBP supplementation prevented bone loss and increased osteoprotegerin, an osteoclastogenesis inhibitory factor, in the mice. The inhibition of T-cell-activation in both MLN and bone marrow by MBP supplementation may help prevent increased IgE levels caused by excessive IL-4 production and bone loss accompanied by enteropathy. Our findings show that MBP may help attenuate both T-cell-related inflammation and bone loss.

Paracrine osteoprotegerin and β-catenin stabilization support synovial sarcomagenesis in periosteal cells.

Synovial sarcoma (SS) is an aggressive soft-tissue sarcoma that is often discovered during adolescence and young adulthood. Despite the name, synovial sarcoma does not typically arise from a synoviocyte but instead arises in close proximity to bones. Previous work demonstrated that mice expressing the characteristic SS18-SSX fusion oncogene in myogenic factor 5-expressing (Myf5-expressing) cells develop fully penetrant sarcomagenesis, suggesting skeletal muscle progenitor cell origin. However, Myf5 is not restricted to committed myoblasts in embryos but is also expressed in multipotent mesenchymal progenitors. Here, we demonstrated that human SS and mouse tumors arising from SS18-SSX expression in the embryonic, but not postnatal, Myf5 lineage share an anatomic location that is frequently adjacent to bone. Additionally, we showed that SS can originate from periosteal cells expressing SS18-SSX alone and from preosteoblasts expressing the fusion oncogene accompanied by the added stabilization of β-catenin, which is a common secondary change in SS. Expression and secretion of the osteoclastogenesis inhibitory factor osteoprotegerin enabled early growth of SS18-SSX2-transformed cells, indicating a paracrine link between the bone and synovial sarcomagenesis. These findings explain the skeletal contact frequently observed in human SS and may provide alternate means of enabling SS18-SSX-driven oncogenesis in cells as differentiated as preosteoblasts.

Effects of low-intensity pulsed ultrasound on osteoclasts: Analysis with goldfish scales as a model of bone.

The effects of low-intensity pulsed ultrasound (LIPUS) on osteoclastogenesis were examined using fish scales that had both osteoclasts and osteoblasts. The binding of the receptor activator of NF-κB ligand (RANKL) in osteoblasts to the receptor activator of NF-κB (RANK) in osteoclasts induced osteoclastogenesis. Therefore, we focused on RANK/RANKL signaling. After 6 h of incubation following LIPUS treatment, mRNA expression of RANKL increased significantly. Resulting from the increased RANKL mRNA level, the expression of transcription-regulating factors significantly increased after 6 h of incubation, and then the mRNA expression of functional genes was significantly up-regulated after 12 h of incubation. However, the mRNA expression of osteoprotegerin (OPG), which is known as an osteoclastogenesis inhibitory factor, also significantly increased after 6 h of incubation and tended to further increase after 12 h of incubation. At 24 h of incubation, osteoclastic functional genes' mRNA expression decreased to the level of the control. Furthermore, we performed an in vivo experiment with goldfish. Two weeks after daily LIPUS exposure, osteoclastic marker enzymes tended to decrease while osteoblastic marker enzymes were activated. The regeneration rate of the LIPUS-treated scales was significantly higher than that of the control scales. Thus, LIPUS moderately activates osteoclasts and induces bone formation.

Sodium fluoride induces hypercalcemia resulting from the upregulation of both osteoblastic and osteoclastic activities in goldfish, Carassius auratus

The influence of sodium fluoride (NaF) on calcium metabolism was examined in goldfish (fresh water teleost). At 2days after administration of NaF (500ng/g body weight; 5μg/g body weight) (around 10−5 to 10−4M in goldfish), we indicated that plasma calcium levels upregulated in both doses of NaF-treated goldfish. To examine the mechanism of hypercalcemia by NaF treatments, therefore, direct effects of NaF on osteoblasts and osteoclasts in goldfish were investigated by an original assay system using teleost scale which has osteoblasts, osteoclasts and bone matrix. Alkaline phosphatase activity in the scales increased with the treatment of NaF (10−6 and 10−5M) during 6h of incubation. Also, tartrate-resistant acid phosphatase activity increased after exposure to NaF (10−5M) at the 6h of incubation. To investigate the osteoclastic activation, the mRNA expression of osteoclastogenesis related factors were examined. The receptor activator of the nuclear factor-κB ligand (RANKL) which is known as a factor for osteoclastogenesis, increased in the NaF-treated scales after 6h of incubation. The ratio of RANKL/osteoprotegerin (osteoclastogenesis inhibitory factor) significantly increased after 6h of incubation. Resulting from the increase of RANKL mRNA level, the expression of transcription-regulating factors was significantly increased. Furthermore, the expression of functional genes, cathepsin K and matrix metalloproteinase-9 mRNA, was significantly increased. In our knowledge, this is the first report concerning the effects of NaF on osteoblasts and osteoclasts in teleosts. We concluded that NaF influences calcium metabolism via osteoclastic activation in goldfish.

Elevated serum osteoprotegerin levels in women: friend or foe?

osteoprotegerin has emerged as a new candidate for the treatment of osteoporosis. However, high levels of osteoprotegerin have been linked to vascular calcification, an independent and well-defined risk factor for cardiovascular disease (CVD) and mortality. Thus, the action of osteoprotegerin in these situations has been questioned. to evaluate the effect of osteoprotegerin (OPG) on the human body, especially in bone tissue and in vascular diseases. the scientific databases consulted were PubMed-Medline and Cochrane, using keywords (MeSH terms) grouped into the following syntaxes: (Osteoprotegerin OR Osteoclastogenesis Inhibitory Factor OR Receptors, Tumor Necrosis Factor, Member 11b OR Tumor Necrosis Factor Receptor Superfamily, Member 11b OR FDCR-1 Protein OR FDCR 1 Protein OR OCIF Protein OR Follicular Dendritic Cell-Derived Receptor-1) AND (Bones AND Bone OR Bones AND Bone Tissue OR Bones OR Bone Tissue OR Cardiovascular Diseases). Osteoprotegerin is present in various organs and binds to two ligands: nuclear factor kB (RANKL) related to the differentiation of osteoclasts, and tumor necrosis factor related to the apoptosis-inducing ligand (TRAIL). OPG inhibits the regulation effects of nuclear factor kB on inflammation and on the skeletal and vascular systems, preventing the apoptosis induced by TRAIL, being related to the preservation of bone tissue. a deeper knowledge of the mechanisms involved in the association between OPG serum levels, bone integrity and cardiovascular disease can provide important data for future therapeutic interventions.

RANK, RANKL, and OPG in recurrent solid/multicystic ameloblastoma: their distribution patterns and biologic significance.

To determine the distribution patterns of bone resorption regulators, receptor activator of nuclear factor κ-B (RANK), RANK ligand (RANKL), and osteoprotegerin (OPG) in recurrent ameloblastoma (RAs) and to clarify their impact on the biologic behavior of these neoplasms. Fifteen paraffin-embedded RA cases were subjected to immunohistochemistry for expression of RANK, RANKL, and OPG. The RANK-RANKL-OPG triad was heterogeneously detected in RA samples. RANK, essential for osteoclast differentiation, was strongly expressed in tumoral epithelium. Conversely, RANKL, an osteoclast activator, was markedly underexpressed, and protein localization was predominantly stromal. OPG, an osteoclastogenesis inhibitory factor, was detected in neoplastic epithelium more than in stroma, suggesting functional inactivation of RANKL. Most RA (n= 12/15; 80%) exhibited a bimolecular spatial expression pattern, the most common being RANK-positive/OPG-positive (n= 8/15; 53.3%). All three proteins showed no significant correlation with the clinical/histopathologic parameters in RA patients (P>.05). The RANK(+)/RANKL(low/-)/OPG(+) phenotype observed in RA suggests an altered local bone metabolism characterized by low bone resorptive activity in these recurrent tumors.

Porphyromonas gingivalis-derived Lysine Gingipain Enhances Osteoclast Differentiation Induced by Tumor Necrosis Factor-α and Interleukin-1β but Suppresses That by Interleukin-17A: IMPORTANCE OF PROTEOLYTIC DEGRADATION OF OSTEOPROTEGERIN BY LYSINE GINGIPAIN

Periodontitis is a chronic inflammatory disease accompanied by alveolar bone resorption by osteoclasts. Porphyromonas gingivalis, an etiological agent for periodontitis, produces cysteine proteases called gingipains, which are classified based on their cleavage site specificity (i.e. arginine (Rgps) and lysine (Kgps) gingipains). We previously reported that Kgp degraded osteoprotegerin (OPG), an osteoclastogenesis inhibitory factor secreted by osteoblasts, and enhanced osteoclastogenesis induced by various Toll-like receptor (TLR) ligands (Yasuhara, R., Miyamoto, Y., Takami, M., Imamura, T., Potempa, J., Yoshimura, K., and Kamijo, R. (2009) Lysine-specific gingipain promotes lipopolysaccharide- and active-vitamin D3-induced osteoclast differentiation by degrading osteoprotegerin. Biochem. J. 419, 159-166). Osteoclastogenesis is induced not only by TLR ligands but also by proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-17A, in inflammatory conditions, such as periodontitis. Although Kgp augmented osteoclastogenesis induced by TNF-α and IL-1β in co-cultures of mouse osteoblasts and bone marrow cells, it suppressed that induced by IL-17A. In a comparison of proteolytic degradation of these cytokines by Kgp in a cell-free system with that of OPG, TNF-α and IL-1β were less susceptible, whereas IL-17A and OPG were equally susceptible to degradation by Kgp. These results indicate that the enhancing effect of Kgp on cytokine-induced osteoclastogenesis is dependent on the difference in degradation efficiency between each cytokine and OPG. In addition, elucidation of the N-terminal amino acid sequences of OPG fragments revealed that Kgp primarily cleaved OPG in its death domain homologous region, which might prevent dimer formation of OPG required for inhibition of receptor activator of nuclear factor κB ligand. Collectively, our results suggest that degradation of OPG by Kgp is a crucial event in the development of osteoclastogenesis and bone loss in periodontitis.

Static and Dynamic Hypergravity Responses of Osteoblasts and Osteoclasts in Medaka Scales

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 osteoblastic and osteoclastic activities were measured. Osteoblastic activity significantly increased under 2- to 4-G loading by both centrifugation and vibration. In contrast, we found that osteoclastic activity significantly decreased under 2- and 3-G loading in response to both centrifugation and vibration. Under 4-G loading, osteoclastic activity also decreased on centrifugation, but significantly increased under 4-G loading by vibration, concomitant with markedly increased osteoblastic activity. Expression of the receptor activator of the NF-κB ligand (RANKL), an activation factor of osteoclasts expressed in osteoblasts, increased significantly under 4-G loading by vibration but was unchanged by centrifugal loading. A protein sequence similar to osteoprotegerin (OPG), which is known as an osteoclastogenesis inhibitory factor, was found in medaka using our sequence analysis. The ratio of RANKL/OPG-like mRNAs in the vibration-loaded scales was significantly higher than that in the control scales, although there was no difference between centrifugal loaded scales and the control scales. Accordingly, medaka scales provide a useful model by which to analyze bone metabolism in response to physical strain.

Trans-10,cis-12 Conjugated linoleic acid promotes bone formation by inhibiting adipogenesis by peroxisome proliferator activated receptor-γ-dependent mechanisms and by directly enhancing osteoblastogenesis from bone marrow mesenchymal stem cells

The bone undergoes continuous remodeling of osteoblastic bone formation and osteoclastic bone resorption to maintain proper bone mass. It is also reported that bone marrow adiposity has a reciprocal role in osteoblasts due to their same origin from mesenchymal stem cells. In addition, one of the key mediators of adipogenesis, peroxisome-proliferator activated receptor-γ (PPARγ), plays a significant role in osteoblastogenesis in bone marrow mesenchymal stem cells. One dietary component that is known to have significant impact on adiposity and bone mass is conjugated linoleic acid (CLA). However, the link between controlling adiposity to improving bone mass by CLA has not been studied intensively. Thus, the purpose of this study is to determine the role of CLA on bone marrow adiposity and bone formation using murine mesenchymal stem cells. The results confirmed that the trans-10,cis-12 CLA, but not the cis-9,trans-11 CLA isomer, significantly inhibited adipogenesis and promoted osteoblastogenesis from mesenchymal stem cells. The inhibition of adipogenesis by the trans-10,cis-12 CLA was mediated by PPARγ; however, the trans-10,cis-12 CLA had a direct effect on osteoblastogenesis which was independent to PPARγ in this model. The trans-10,cis-12 CLA also had significant effects on osteoclastogenesis inhibitory factor, which suggests potential influence of CLA on osteoclastogenesis. Overall, the results suggest that the trans-10,cis-12, but not the cis-9,trans-11 CLA isomer, has a positive impact on bone health by both PPARγ mediated and independent mechanisms in mesenchymal stem cells.

Bisphosphonate- and statin-induced enhancement of OPG expression and inhibition of CD9, M-CSF, and RANKL expressions via inhibition of the Ras/MEK/ERK pathway and activation of p38MAPK in mouse bone marrow stromal cell line ST2

Osteoclast differentiation is influenced by receptor activator of the NF-κB ligand (RANKL), macrophage colony-stimulating factor (M-CSF), and CD9, which are expressed on bone marrow stromal cells and osteoblasts. In addition, osteoprotegerin (OPG) is known as an osteoclastogenesis inhibitory factor. In this study, we investigated whether bisphosphonates and statins increase OPG expression and inhibit the expression of CD9, M-CSF, and RANKL in the bone marrow-derived stromal cell line ST2. We found that bisphosphonates and statins enhanced OPG mRNA expression and inhibited the expression of CD9, M-CSF, and RANKL mRNA. Futhermore, bisphosphonates and statins decreased the membrane localization of Ras and phosphorylated ERK1/2, and activated the p38MAPK. This indicates that bisphosphonates and statins enhanced OPG expression, and inhibited the expression of CD9, M-CSF, and RANKL through blocking the Ras/ERK pathway and activating p38MAPK. Accordingly, we believe that its clinical applications will be investigated in the future for the development of osteoporosis therapy.