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Abstract Receptor activator of nuclear factor kappa-B ligand (RANKL) initiates a complex signaling cascade that is crucial for inducing osteoclast differentiation and activation. RANKL-induced signaling has been analyzed in detail, and the involvement of TNF receptor-associated factor 6 (TRAF6), calmodulin-dependent protein kinase (CaMK), NF-κB, mitogen-activated protein kinase (MAPK), activator protein-1 (AP-1), and molecules that contain an immunoreceptor tyrosine-based activation motif (ITAM) has been reported. However, the precise molecular steps that regulate RANKL signaling remain largely unknown. Here, we revealed the indispensable role of a class III histone deacetylase (SIRT5) in the processes of RANKL-induced osteoclast differentiation and activation. SIRT5 expression in osteoclasts was increased during osteoclastogenesis upon stimulation with RANKL. The RANKL-induced signaling activation was suppressed in SIRT5-deficient osteoclasts but enhanced by SIRT5 overexpression. Mice with global or conditional monocytic lineage knockout of SIRT5 had increased bone mass and reduced osteoclast numbers. In the cytoplasm, SIRT5 interacted with the scaffold protein JNK-interacting protein 4 (JIP4) to finely regulate MAPK signaling, which was critical for osteoclast differentiation and activation. Pharmacological inhibition of the catalytic activity of SIRT5 effectively reversed bone loss in ovariectomized mice. Taken together, the results of this study reveal that the SIRT5-JIP4 axis is a novel positive regulator that finely regulates RANKL-induced osteoclast differentiation and suggest that targeting this axis is a therapeutic strategy for preventing osteoporotic bone loss. Graphical Abstract

Background: Platelet-rich plasma (PRP) is utilized as an autologous blood product to encourage bone regeneration. The receptor activator of nuclear factor-NB ligand (RANKL) is a key and central regulator of osteoclast homeostasis. A rat model of experimentally generated periodontitis was used to assess the impact of PRP preparation on the expression of the osteoclastogenic and pro-inflammatory markers respectively; RANKL and IL-1β. Material and Methods: To induce periodontitis by silk ligature, thirty-six adult male Sprague Dawleys rats were used and they were allocated into three equal groups (n = 12): group I consisted of intact periodontal tissue, group II; rat-induced periodontitis without treatment by PRP, and group III of periodontitis + 10 µL PRP injection. The rats were sacrificed after both experiment durations (7 and 30 days), and the incisor teeth were fixed and decalcified in HCl for a day and in 10 % EDTA solution for eight weeks at room temperature then samples were processed for H&E stain for bone healing scores and bone cells counting, and the samples were utilized by IHC for detection of both RANKL and IL-1β expression. Results: The PRP enhanced the process of healing on days 7 and 30 showed (Score 10) vs. the control positive group that had a delay in alveolar bone regarded as (Score 4) significantly (P ≤ 0.05). The PRP group attenuated significantly (P ≤ 0.05) the alveolar bone loss by increasing the number of osteoblasts and declining the proliferation of osteoclast vs. the control positive group that revealed bone destruction due to rising osteoclast proliferation and decreasing the osteoblast proliferation significantly (P ≤ 0.05). PRP inhibited the IL-1β expression (score = 0) vs. the control positive group that showed moderate staining of positive cells detected in both inflammatory cells and endothelium (score = 4). Regarding the RANKL expression, the PRP reduced its expression in vs. the control positive group (score = 4 vs. 12 respectively). Conclusion: PRP is an anabolic agent that enhances proliferation of osteoblast and inhibit the osteoclast differentiation by downregulation of IL-1β and RANKL.

Background Osteoporosis (OP) is a chronic disease characterized by decreased bone mass, loss of skeletal structural integrity and increased susceptibility to fracture. Available studies have shown that the pyruvate dehydrogenase kinase (PDK) family is associated with osteoclastogenesis and bone loss, but the specific role of Pdk3 in bone pathology has not been systematically investigated. Methods A cell OP model was established in receptor activator for nuclear factor-κB Ligand (RANKL)-induced bone marrow macrophages (BMMs). Hereafter, the expression levels of Pdk3 and osteoclastogenesis feature genes including nuclear factor of activated T cells 1 (Nfatc1), Cathepsin K (Ctsk), osteoclast associated Ig-like receptor (Oscar) in BMMs-derived osteoclasts were examined based on real-time quantitative PCR and western blotting methods. Further, the phosphorylation of ERK, P65 and JAK/STAT and their correlation was Pdk3 was gauged. In particular, changes in the activity of these signaling pathways were observed by silencing experiments of the Pdk3 gene (using small interfering RNA). Finally, the effects of Pdk3 gene silencing on signaling pathway activity, osteoclastogenesis, and related inflammatory and apoptotic indicators were observed by transfection with PDK3-specific siRNA. Results Following RANKL exposure, the levels of Pdk3 and osteoclastogenesis feature genes were all elevated, and a positive correlation between Pdk3 and osteoclastogenesis feature genes was seen. Meanwhile, ERK, P65 and JAK/STAT phosphorylation was increased by RANKL, and Pdk3 was confirmed to be positively correlated with the phosphorylation of ERK, P65 and JAK/STAT. Additionally, in RANKL-exposed osteoclasts, Pdk3 knockdown diminished the phosphorylation of ERK, P65 and JAK/STAT, reduced the expressions of osteoclastogenesis feature genes. Importantly, knockdown of Pdk3 also reduced the expression of inflammatory cytokines and resulted in elevated levels of Bax and Casp3 expression, as well as downregulation of Bcl2 expression. Conclusion This study reveals for the first time the role of Pdk3 in RANKL-induced osteoclastogenesis and OP. These findings provide a foundation for future studies on the role of Pdk3 in other bone diseases and provide new ideas for the development of OP therapeutics targeting Pdk3.

Abstract Background Estrogen deficiency-mediated hyperactive osteoclast represents the leading role during the onset of postmenopausal osteoporosis. The activation of a series of signaling cascades triggered by RANKL-RANK interaction is crucial mechanism underlying osteoclastogenesis. Vorinostat (SAHA) is a broad-spectrum pan-histone deacetylase inhibitor (HDACi) and its effect on osteoporosis remains elusive. Methods The effects of SAHA on osteoclast maturation and bone resorptive activity were evaluated using in vitro osteoclastogenesis assay. To investigate the effect of SAHA on the osteoclast gene networks during osteoclast differentiation, we performed high-throughput transcriptome sequencing. Molecular docking and the assessment of RANKL-induced signaling cascades were conducted to confirm the underlying regulatory mechanism of SAHA on the action of RANKL-activated osteoclasts. Finally, we took advantage of a mouse model of estrogen-deficient osteoporosis to explore the clinical potential of SAHA. Results We showed here that SAHA suppressed RANKL-induced osteoclast differentiation concentration-dependently and disrupted osteoclastic bone resorption in vitro. Mechanistically, SAHA specifically bound to the predicted binding site of RANKL and blunt the interaction between RANKL and RANK. Then, by interfering with downstream NF-κB and MAPK signaling pathway activation, SAHA negatively regulated the activity of NFATc1, thus resulting in a significant reduction of osteoclast-specific gene transcripts and functional osteoclast-related protein expression. Moreover, we found a significant anti-osteoporotic role of SAHA in ovariectomized mice, which was probably realized through the inhibition of osteoclast formation and hyperactivation. Conclusion These data reveal a high affinity between SAHA and RANKL, which results in blockade of RANKL-RANK interaction and thereby interferes with RANKL-induced signaling cascades and osteoclastic bone resorption, supporting a novel strategy for SAHA application as a promising therapeutic agent for osteoporosis.

Introduction: Osteopenia and osteoporosis are well-known hemophilia A comorbidities. The pathogenesis of bone turnover alteration resulting in reduced bone mass includes impaired osteoblastic differentiation and disinhibition of RANKL-induced osteoclastogenesis as a result of a low FVIII level.Aim: To evaluate the bone mineral density (BMD) in adult patients with severe hemophilia A and assess a possible correlation with the bone remodeling biomarkers OPG/RANKL, CTX-1, osteocalcin, and Vit D.Materials and methods: 28 male subjects with severe hemophilia A and 33 age-matched controls were recruited. The biomarkers were tested with the ELISA assay and BMD with DEXA of the lumbar spine (LS) and total hip (TH).Results: The patients had lower LS-BMD (−0.955±0.145 vs. 1.118±0.079, p=0.05) and TH-BMD (−0.840±0.147 vs. 0.951±0.075, p=0.05) than those of the controls. The TH T-scores were −1.41±0.91 vs. 0.4±0.49 (p=0.05) and the LS T-scores −1.16±1.046 vs. 0.14±0.72 (p=0.05). 66.6% of patients under 50 years had osteopenia and 8.3% had osteoporosis. Fifty percent of those over 50 years old had osteopenia and 20% had osteoporosis. We found significantly higher OPG levels (123.69±107.05 vs. 41.98±18.95, p=0.05) than that in controls and lower sRANKL levels (23.49±29.39 vs. 131.32±201.27, p=0.05) and sRANKL/OPG ratio (0.27±0.35 vs. 5.28±10.01, p=0.05) than those in controls. A positive correlation was found between sRANKL and the BMD T-score of lumbar spine (p=0.001) in the patient group.Conclusions: sRANKL level and ratio can be used as predictors of low BMD.

Сахарный диабет справедливо называют неинфекционной эпидемией 21 века. Вместе с тем сердечно-сосудистые заболевания по-прежнему лидируют среди причин смертности в мире. При сочетании сахарного диабета и облитерирующего атеросклероза периферических артерий риск потери нижней конечности и смерти увеличивается многократно. Существует множество концепций механизма развития этих заболеваний. В последние годы появляется все больше данных о схожести процессов развития атеросклероза и костного остеопороза. Связующим звеном может быть остеопротегерин (OPG). Получены сведения о остеопротегерине как антирезорбтивном факторе, участвующем в системе RANKL-RANK-OPG. В патофизиологических условиях OPG экспрессируется в сосудистой стенке, в том числе обнаруживается на разных этапах формирования атеросклеротической бляшки. Сигнальный путь RANKL-RANK-OPG участвует в процессе ремоделирования костной ткани. Механизмы развития атеросклероза и остеопороза схожи, однако требуют дальнейшего изучения.

Abstract Background and Aims Limitations of the conventional diagnostic techniques urged researchers to seek novel methods to predict, diagnose, and monitor periodontal disease. Use of the biomarkers available in oral fluids could be a revolutionary surrogate for the manual probing/diagnostic radiograph. Several salivary biomarkers have the potential to accurately discriminate periodontal health and disease. This study aimed to determine the diagnostic sensitivity and specificity of salivary interleukin (IL)‐17, receptor activator of nuclear factor‐κB ligand (RANKL), osteoprotegerin (OPG), RANKL/OPG for differentiating (1) periodontal health from disease and (2) stable and unstable periodontitis. Methods Participants with periodontitis (n = 50) and gingivitis (n = 25), both diseases represented the cases, and subjects with healthy periodontium (n = 15) as a control were recruited for this study. Periodontitis cases were further equally subdivided into stable and unstable. Whole unstimulated salivary sample were collected from all participants. Periodontal parameters including bleeding on probing, probing pocket depth, clinical attachment loss, and number of missing teeth were recorded. The protein levels of salivary IL‐17, RANKL, and OPG were determined by using enzyme‐linked immunosorbent assays technique. Results Salivary IL‐17, OPG, RANKL, and RANKL/OPG showed high sensitivity and specificity to differentiate periodontal health from gingivitis and periodontitis. Similar pattern was observed in discriminating stable and unstable periodontitis. Salivary IL‐17 and RANKL showed a good accuracy to differentiate gingivitis from periodontitis. However, OPG and RANKL/OPG did not exhibit enough sensitivity and specificity to differentiate the latter conditions. Conclusion Salivary IL‐17, RANKL, OPG, and RANKL/OPG system are potential candidates for differentiating periodontal health and disease and discriminate stable and unstable periodontitis.

With over 2.3 million new cases and 685,000 deaths, breast cancer is the most commonly diagnosed cancer worldwide, and its burden has been rising over the past decades. In India, the incidence increased significantly, almost 50%, between 1965 and 1985. As per the 2020 Globocan information, in India, BC accounted for 13.5% of all cancer cases and 10.6% of all deaths. Compared to Western countries, the poor survival of patients with breast cancer in India is due to several contributing factors such as lack of awareness, earlier onset of disease, delayed initiation and late stage of disease at presentation, and lack of consistent treatment regime. Under these circumstances, it is even more imperative to identify novel targets for breast cancer and put them to clinical use. In a recent report by Ciscar et al., the authors reported that RANK and receptor activator of NFκB ligands (RANKL) status might be important predictors in estrogen receptor (ER)-negative breast cancer. In analyses of more than 2,000 breast cancer samples, the authors confirmed that RANK expression was associated with ER− tumors, while RANKL was rarely found in tumor cells. This also let to conclude that RANK expression is an independent poor prognostic factor in breast cancer, particularly in ER− BC postmenopausal women, and is associated with poor survival. This seminal work has enormous outcomes in the clinical management of breast cancer. This also implies that RANK+ ER- breast cancer might benefit from the recently FDA-approved compound “Denosumab.” Denosumab is used to treat bone loss in men treated for prostate cancer and those who have osteoporosis. It is an inhibitor of RANKL, which is an essential factor initiating bone turnover. RANKL inhibition blocks osteoclast maturation, function, and survival, thus reducing bone resorption. The same mechanism might be beneficial in these RANK+ ER- breast cancer. Clinical trials are underway to test if denosumab can be re-purposed for this subset of breast cancer.

Context: Osteoporosis occurs not only in the lumbar bone, femur, and radius but also occurs in the mandibular bone. Therefore, osteoporosis is a disease that required attention from the dentists. Aims: To determine the ratio of Receptor Activator of Nuclear Factor kB-Ligand:Osteoprotegerin (RANKL:OPG) expression and the ratio of osteoclasts:osteoblasts number in the mandibular bone of the osteoporosis rat models. Methods: The research subjects used were 20 Rattus novergicus, which were randomly divided into two groups, namely ovariectomy (OVX) and sham surgery (SHS). Twelve weeks after surgery, the animals were terminated and mandibular bone specimens were taken for histological examination of osteoclasts, osteoblasts, RANKL, and OPG expression. An examination of estrogen levels in the blood of the research subjects was also carried out. All data obtained were tested statistically using a t-test. Results: There was a decrease in the amount of estrogen in the OVX group rather than SHS (p = 0.005). In the SHS group, the RANKL:OPG expression value ratio was 0.51 ± 0.15, while in the OVX group was 0.86 ± 0.22 (p = 0.001). In the SHS group, the ratio of osteoclasts:osteoblasts number was 0.012 ± 0.004, while in the OVX group was 0.061 ± 0.023 (p = 0.001). Conclusions: A decrease in estrogen significantly induces the ratio of RANKL:OPG and osteoclast:osteoblasts in the mandibular bone.

The goal of this research was to investigate at the expression profiles of heat shock protein 70 (HSP70) and receptor activator of nuclear factor-kappa B ligand (RANKL) in term and preterm infants. Preterm birth (n=13) and term birth (n=13) cord blood samples were studied. In the cord blood serum protein expression levels of the HSP70 and RANKL were measured by ELISA. When compared to the term birth group, preterm birth had greater RANKL concentrations. When comparing the preterm birth study group to the term birth study group, protein expression of HSP70 and RANKL was not shown to be associated. These results indicate that HSP70 and RANKL levels in cord blood may affect both prenatal and postnatal treatment approaches. [Med-Science 2022; 11(2.000): 462-4]

Abstract Background: Multiple myeloma is a clonal plasma cell neoplasm which affects 1% of all malignancies. In this article we reviewed epidemiology, symptoms with particular attention to renal failure and its manifestation in multiple myeloma (MM). Material and methods: This paper was based on medical articles collected in PubMed from 2001 to 2022, medical websites and books. The research has been done by looking through key words such as: „multiple myeloma”, „myeloma cast nephropathy”, „bortezomib”, „RANKL” Results: Multiple myeloma has indolent course and many patients present renal disorders at the moment of diagnosis. The use of novel therapies can reverse renal failure. Conclusions: Treatment of the patients with MM and kidney disease is challenging for health care professionals. Doctors have to adjust treatment to provide the best therapeutic effects.

Abstract Periodontitis, a bacterium-induced inflammatory disease that is characterized by alveolar bone loss, is highly prevalent worldwide. Elucidating the underlying mechanisms of alveolar bone loss in periodontitis is crucial for understanding its pathogenesis. Classically, bone cells, such as osteoclasts, osteoblasts and bone marrow stromal cells, are thought to dominate the development of bone destruction in periodontitis. Recently, osteocytes, the cells embedded in the mineral matrix, have gained attention. This review demonstrates the key contributing role of osteocytes in periodontitis, especially in alveolar bone loss. Osteocytes not only initiate physiological bone remodeling but also assist in inflammation-related changes in bone remodeling. The latest evidence suggests that osteocytes are involved in regulating bone anabolism and catabolism in the progression of periodontitis. The altered secretion of receptor activator of NF-κB ligand (RANKL), sclerostin and Dickkopf-related protein 1 (DKK1) by osteocytes affects the balance of bone resorption and formation and promotes bone loss. In addition, the accumulation of prematurely senescent and apoptotic osteocytes observed in alveolar bone may exacerbate local destruction. Based on their communication with the bloodstream, it is noteworthy that osteocytes may participate in the interaction between local periodontitis lesions and systemic diseases. Overall, further investigations of osteocytes may provide vital insights that improve our understanding of the pathophysiology of periodontitis.

Osteocytes, which develop from osteoblasts, are recognized as the main cells embedded in mature bone tissue. The traditional notion is that osteocytes exclusively play a structural role, however, with the development of related research in recent years, the role of osteocytes in bone metabolism has been explored. Periodontitis is a chronic inflammatory disease initiated by plaque biofilm, and is the main cause of adult tooth loss. Clinically, periodontitis primarily manifests as attachment loss, bleeding on probing and other symptoms. Alveolar bone resorption is the most characteristic pathological change. Current research demonstrated that osteocytes sense mechanical stress, participate in bone remodeling, regulate mineral balance, and participate in endocrine function. Thus, these cells play an important role in bone homeostasis and systemic metabolic balance. Osteocytes are actively involved in the development of periodontitis through the high expression of receptor activator of nuclear factor kappa B ligand (RANKL), secretion of sclerostin, and effect on apoptosis, senescence and autophagy. In the future, the detection of bone cell metabolism-related products will have certain application prospects for the clinical evaluation of periodontitis prevention and treatment. Therefore, this paper reviewed the role of osteocytes in bone homeostasis and the relationship between osteocytes and periodontitis, to provide new ideas for the prevention and treatment of periodontitis.

Parathyroid hormone (PTH) is a key regulator of bone turnover. Depending on the duration of action, the hormone causes catabolic and anabolic effects by binding with specific receptors (PTHR1) in the bone. Various cells expressing PTHR1 on their surface are involved in the process – osteoblasts, osteocytes, bone marrow stromal cells, T-lymphocytes and macrophages. In physiological conditions PTH balances the bone metabolism. Intermittent pharmacological doses of PTH lead to the prevalence of bone formation and are used in the treatment of osteoporosis. Persistently elevated levels of PTH stimulate bone resorption by impacting mainly the cortical bone. New imaging and analysis techniques show that high PTH levels can also have an adverse effect on trabecular microarchitecture. Primary hyperparathyroidism (PHPT) is a disease characterized by increased bone metabolism, decreased bone mineral density (BMD), inadequate osteoid mineralization and an increased risk of fractures. Prolonged overproduction of PTH leads to stimulation of bone resorption and defects in bone formation, mainly causing loss of cortical bone mass, while in the trabecular bone predominate demineralization processes. One explanation of these findings is the enhanced stimulation of RANKL expression by osteoblasts with decreased OPG expression and bone formation at the same time.

Abstract Background The present study was to determine the effect of local anti-RANKL antibody administration in the presence or absence of microRNA-146a on ligature-induced peri-implant bone resorption, and the potential role of TLR2/4 signaling in such effect. Results Titanium implants were placed in the left maxilla alveolar bone 6 weeks after extraction of first and second molars in C57/BL6 wild-type (WT) and TLR2−/− TLR4−/− (TLR2/4 KO) mice. Silk ligatures were tied around the implants 4 weeks after implantation. Anti-RANKL antibody (500 μg/mL) with or without microRNA 146a (miR-146a) (100 nM) was injected into palatal gingiva around implant on days 3, 6, and 9 during 2 weeks of ligation period. Bone resorption around the implants was assessed by 2D imaging using area measurement and 3D imaging using micro-computed tomography (μCT). Real-time quantitative PCR (RT-qPCR) was used to determine the peri-implant gingival mRNA expression levels of pro-inflammatory cytokines (TNF-α) and osteoclastogenesis-related cytokines (RANKL). In both WT and TLR2/4 KO mice, the bone resorption around implants was significantly increased in the ligation only group when compared to the non-ligation group, but TLR2/4 KO mice showed significantly less bone loss compared to WT mice after ligation. As expected, gingival injection of anti-RANKL antibody significantly reduced bone loss compared with the ligation only group in both WT and TLR2/4 KO mice. Moreover, injection of miR-146a in addition to anti-RANKL antibody significantly enhanced the inhibition of bone loss in WT mice but not in TLR2/4 KO mice. Gingival mRNA expressions of RANKL were significantly reduced by anti-RANKL antibody treatment in both WT and TLR2/4 KO mice but were not affected by the additional miR-146a treatment. Gingival mRNA expression of TNF-α was significantly reduced by miR-146a treatment in WT mice but not in TLR2/4 KO mice. The number of gingival inflammatory cell infiltration and peri-implant TRAP-positive cell formation was significantly reduced by the additional miR-146a treatment in WT mice but not in TLR2/4 KO mice. Conclusions This study suggests that anti-inflammatory miR-146a enhance anti-RANKL-induced inhibition of peri-implant bone resorption through the regulation of TLR2/4 signaling and inhibition of TNF-α expression.

چکیده مقدمه: بتا تالاسمی‎ ماژور نوعی اختلال اتوزومال مغلوب است که با اریتروپوئز غیر موثر شناخته می‎شود. تزریق مداوم و ناتوانی بدن در حذف آهن در بدن منجر به اضافه بار آهن شده که باعث ایجاد صدمه به بافت قلب می‎شود. یکی از مسیرهای تنظیمی‎بیماریهای قلبی با واسطه تجمع آهن، مسیر Osteoprotegrin/RANK(Receptor Activator of Nuclear Factor k_B / RANKL Receptor Activator of Nuclear Factor Ligandمی‎باشد.گیرنده فاکتور هسته‎ایی کاپا_B(RANKL)/ لیگاند فعال کننده فاکتور هسته ایی کاپا_B(RANK) و استئوپروتگرین((OPG هستند. هدف از این مطالعه بررسی سطح سرمی‎RANKL و ارتباط آن با هیپرتروفی بطن چپ (LVH) و اختلال عملکرد دیاستولیک وکسر جهشی وفشار خون ریوی و شاخص MRIT*2در بیماران تالاسمی‎ماژور می‎باشد. روش کار: این پژوهش در سال 1396 به روش توصیفی روی 82 بیمار مبتلا به تالاسمی‎ماژور کیلینک سرور مشهد انجام شد.تمام بیماران، مورد بررسی کامل و غیر تهاجمی(اکوکاردیوگرافی کامل 2 بعدی و داپلرM-Mode رنگی وMRIT*2)قرار گرفتند. برای اندازه گیری سطح سرمی‎RANKL از تکنیک الایزا استفاده شد.جهت تجزیه و تحلیل آماری از spss20 استفاده شد. نتایج: از مجموع 82 بیمار 36 زن و 46 مرد بودند.میانگین سنی بیماران83/6±69/23 سال بود. هایپرتروفی بطن چپ و اختلال عملکرد دیاستولیک به ترتیب در 24 بیمار(30/29%) و 23 نفر(28%)وجود داشت. ارتباط معناداری بین سرم RANKL با هایپرتروفی بطن چپ ، اختلال عملکرد دیاستولیک ،کسر جهشی ،فشار خون ریوی ، شاخص MRIT*2وجود نداشت(05/0 نتیجه‌گیری: یافته‎های پژوهشی نشان داد که بین سطح RANKLسرم با شدت درگیری قلب هایپرتروفی بطن چپ، نقص عملکرد دیاستولیک،کسر جهشی ،فشار خون ریوی، شاخص MRIT*2ارتباط معنا داری وجود ندارد که این یافته پژوهش‎های بیشتری را در این مورد فرا میخواند.

Abstract Background Osteopenia have been well documented in adolescent idiopathic scoliosis (AIS). Adiponectin has been shown to be inversely proportional to body mass index and to affect bone metabolism. However, the circulating levels of adiponectin and the relationship between adiponectin and low bone mass in AIS remain unclear. Methods A total of 563 AIS and 281 age-matched controls were recruited for this study. Anthropometry and bone mass were measured in all participants. Plasma adiponectin levels were determined by enzyme-linked immunosorbent assay (ELISA) in the AIS and control groups. An improved multiplex ligation detection reaction was performed to study on single nucleotide polymorphism. Facet joints were collected and used to measure the microstructure, the expression of RANKL, OPG, osteoblast-related genes, inflammatory factors, adiponectin and its receptors by qPCR, western blotting and immunohistochemistry. Furthermore, primary cells were extracted from facet joints to observe the reaction after adiponectin stimulation. Results Compared with the controls, lower body mass index and a marked increase in circulating adiponectin were observed in AIS osteopenia (17.09 ± 1.09 kg/m2 and 21.63 ± 10.30 mg/L). A significant difference in the presence of rs7639352 was detected in the AIS osteopenia, AIS normal bone mass and control groups. The T allele showed a significant higher proportion in AIS osteopenia than AIS normal bone mass and control groups (41.75% vs 31.3% vs 25.7%, p

Objective(s): To investigate the effect of cocoa on orthodontic tooth movement (OTM) rate, osteoprotegerin (OPG), and receptor activator of nuclear factor κ β ligand (RANKL) levels after OTM.Materials and Methods: A total of 24 Sprague-Dawley rats were included in the study. They were equally divided into two groups: cocoa and control. The upper incisors of all rats were subjected to 35 cN orthodontic force and moved distally with a stainless steel 3-spin coil spring. During OTM, the cocoa group was given 4.8 g of unsweetened cocoa once a day. At 4 subsequent time points (0, 1, 7, and 14 days), the OTM rate was determined by measuring the distance between the mesial tips using a digital caliper, while OPG and RANKL levels were examined based on their gingival crevicular fluid through specific enzyme-linked immunosorbent assay (ELISA). Data gathered were analyzed through independent t-test (P

Inflammatory osteolysis is governed by exacerbated osteoclastogenesis. Ample evidence points to central role of NF-κB in such pathologic responses, yet the precise mechanisms underpinning specificity of these responses remain unclear. We propose that motifs of the scaffold protein IKKγ/NEMO partly facilitate such functions. As proof-of-principle, we used site-specific mutagenesis to examine the role of NEMO in mediating RANKL-induced signaling in mouse bone marrow macrophages, known as osteoclast precursors. We identified lysine (K)270 as a target regulating RANKL signaling as K270A substitution results in exuberant osteoclastogenesis in vitro and murine inflammatory osteolysis in vivo. Mechanistically, we discovered that K270A mutation disrupts autophagy, stabilizes NEMO, and elevates inflammatory burden. Specifically, K270A directly or indirectly hinders binding of NEMO to ISG15, a ubiquitin-like protein, which we show targets the modified proteins to autophagy-mediated lysosomal degradation. Taken together, our findings suggest that NEMO serves as a toolkit to fine-tune specific signals in physiologic and pathologic conditions.

Abstract Background Cherubism is a rare autosomal dominant disorder of the jaws caused by mutation of the SH3BP2 gene. The bone is replaced by a fibrous granuloma containing multinucleated giant cells. Cells of the cherubism granuloma have never been systematically analyzed. Hence, the aim of this study was to characterize the cells in human cherubism granulomas, to determine the osteoclastic characteristics of the multinucleated giant cells and to investigate the potential role of TNF-α in human cherubism. Results Seven granulomas were analyzed in pathology, molecular biology and immunohistochemistry. Granulomas were composed mainly of macrophages or osteoclasts within a fibroblastic tissue, with few lymphoid cells. Myeloid differentiation and nuclear NFATc1 localization were both associated with disease aggressiveness. OPG and RANKL immunohistochemical expression was unexpected in our specimens. Five granuloma cells were cultured in standard and osteoclastogenic media. In culture, cherubism cells were able to differentiate into active osteoclasts, in both osteoclastogenic and standard media. IL-6 was the major cytokine present in the culture supernatants. Conclusion Multinucleated giant cells from cherubism granulomas are CD68 positive cells, which differentiate into macrophages in non-aggressive cherubism and into osteoclasts in aggressive cherubism, stimulated by the NFATc1 pathway. This latter differentiation appears to involve a disturbed RANK-L/RANK/OPG pathway and be less TNF-α dependent than the cherubism mouse model.

Objective(s): The study aimed to uncover the underlying mechanism linking wear particles to osteoclast differentiation, and we explored the effect of titanium particles of different sizes on CD147 expression and autophagy in macrophages. Materials and Methods: Effects of titanium particles on CD147 and RANKL mRNA were detected by QPCR; protein level of CD147 and Beclin-1 were detected by Western blot; soluble RANKL were detected by ELISA. To determine the effect of CD147 and autophagy, KG-1a cells were transfected with siRNA-CD147 or treated with autophagy inhibitor CQ (chloroquine), and then co-cultured with different sizes of titanium particles.Results: Our results showed that 0.2-1.2 µm and 1.2-10 µm titanium particles up-regulate CD147 to activate autophagy, which increase the level of soluble RANKL to promote osteoclastogenesis. Suppression of CD147 with siRNA could diminish particle-induced autophagy and soluble RANKL expression. In addition, CQ could dramatically reduce particle-induced soluble RANKL expression. Conclusion: Our findings suggested a possible mechanism underlying wear debris-induced osteolysis and identified CD147 as a potential therapeutic target in aseptic loosening.

Objective To investigate the effect and potential molecular mechanisms of isorhamnetin (ISO) extracted from Ginkgo biloba on the differentiation of osteoclasts. Methods Osteoclast precursor RAW264.7 cells were induced with RANKL to differentiate into mature osteoclasts. Different concentrations of ISO were added to RAW264.7 cells to determine its effect on osteoclast differentiation. CCK8 was used to evaluate the effect of ISO on cytotoxicity. The im⁃ pact of ISO on the osteoclast differentiation process was investigated by analyzing tartrate resistance and bone resorption lacuna. Real⁃time PCR was performed to analyze the levels of differentiation marker genes, including tartrate resistant acid phosphatase (Trap), cathepsin K (Ctsk), and matrix metalloproteinase 9 (MMP⁃9); differentiation⁃related transcription factors, including the proto⁃oncogene protein c⁃Fos, nuclear factor of activated T⁃cells cytoplasmic 1(NFATc1); and the levels of downstream NF⁃κB p65 signaling pathway phosphorylation. Using the above⁃described method, we verified that ISO exerted an inhibitory effect on osteoclast differentiation and explored related molecular mechanisms. Results Dif⁃ ferent concentrations of ISO (1⁃10 μM) had no cytotoxic effects on RAW264.7 cells, inhibited TRAP activity and decreased the number of bone resorption lacuna during osteoclast differentiation. When applied at a concentration of 10 μM, its inhibitory effect was significant. In addition, ISO significantly reduced the expression levels of Trap, Ctsk, MMP⁃9, c⁃Fos, NFATc1 and NF⁃ κB p65 mRNA. Conclusion ISO extracted from Ginkgo biloba extract exerted an inhibitory effect on osteoclast differentiation, and the mechanism underlying its activity may involve the inhibition of the classical NF⁃κB pathway.

Giant cell tumor (GCT) is a rare, usually benign but locally aggressive neoplasm. Recent studies suggest new approaches in light of the elucidation of molecular pathways in bone. The osteolytic nature of GCT is caused by the receptor for activating nuclear factor-kB ligand (RANKL) associated osteoclasts. Denosumab is a monoclonal antibody that affects GCT through RANKL and it prevents normal and neoplastic osteolysis. The aim of this study is to evaluate the histopathologic alterations due to denosumab treatment and the efficiency of this drug in GCT therapy. Ten patients had been treated with denosumab and were included in the study. Pretreatment biopsies were interpreted as conventional GCTs and posttreatment biopsies of the ten patients’ GCTs were classified in accordance with the grading system. Only one patient had tumor remaining after treatment. There is limited data on histopathologic alterations that follow denosumab treatment. The bone pathologist should keep these changes in mind because they mimic different types of bone tumors. Furthermore, there is no widely accepted grading system to evaluate the effect of denosumab in GCT. Our study suggested a scheme that would be helpful to evaluate the efficiency of denosumab treatment in GCT.

Blood calcium homeostasis is critical for biological function. Caldecrin, or chymotrypsin-like elastase, was originally identified in the pancreas as a serum calcium-decreasing factor. The serum calcium-decreasing activity of caldecrin requires the trypsin-mediated activation of the protein. Protease activity-deficient mature caldecrin can also reduce serum calcium concentration, indicating that structural processing is necessary for serum calcium-decreasing activity. Caldecrin suppresses the differentiation of bone-resorbing osteoclasts from bone marrow macrophages (BMMs) by inhibiting receptor activator of NF-κB ligand (RANKL)-induced nuclear factor of activated T-cell cytoplasmic 1 expression via the Syk–PLCγ–Ca2+ oscillation-calcineurin signaling pathway. It also suppresses mature osteoclastic bone resorption by RANKL-stimulated TRAF6–c-Src–Syk–calcium entry and actin ring formation. Caldecrin inhibits lipopolysaccharide (LPS)-induced osteoclast formation in RANKL-primed BMMs by inducing the NF-κB negative regulator A20. In addition, caldecrin suppresses LPS-mediated M1 macrophage polarization through the immunoreceptor triggering receptor expressed on myeloid cells (TREM) 2, suggesting that caldecrin may function as an anti-osteoclastogenic and anti-inflammatory factor via TREM2. The ectopic intramuscular expression of caldecrin cDNA prevents bone resorption in ovariectomized mice, and the administration of caldecrin protein also prevents skeletal muscle destruction in dystrophic mice. In vivo and in vitro studies have indicated that caldecrin is a unique multifunctional protease and a possible therapeutic target for skeletal and inflammatory diseases.

Introducción: En la enfermedad renal crónica (ERC) se producen alteraciones del metabolismo óseo y mineral que favorecen la calcificación de tejidos blandos. Alteraciones del sistema RANK/RANKL/OPG podrían estar favoreciendo la calcificación vascular, importante causa de morbimortalidad en la ERC.Objetivo: Valorar en un modelo experimental in vivo de insuficiencia renal crónica el efecto de la progresión de la misma sobre la calcificación vascular y sobre la pérdida de hueso correlacionando estos cambios con alteraciones en el sistema RANK/RANKL/OPG, utilizando un sistema in vitro para confirmar los hallazgos encontrados. Material y métodos: Se utilizaron dos modelos de calcificación vascular: un modelo in vivo en ratas con insuficiencia renal crónica alimentadas con dieta con diferente contenido en fósforo, y un modelo in vitro en células de músculo liso vascular (CMLV) sometidas a diferentes estímulos calcificantes. Resultados: A las 20 semanas, un 50% de los animales con dieta alta en fósforo presentó calcificaciones aórticas que se acompañó de aumento en la expresión aórtica de RANKL. Por el contrario, la OPG disminuyó como consecuencia probablemente del componente inflamatorio. A las 20 semanas en la tibia aumentó la expresión de RANKL y OPG, mientras que el aumento de OPG ocurrió en fases más tempranas. En CMLV la adición de suero urémico y medio calcificante indujo un incremento del contenido de calcio y de la expresión de RANKL y OPG. La adición de OPG y el silenciamiento de RANK inhibieron este aumento. Conclusiones: Nuestros resultados confirman la participación del eje RANK/RANKL/OPG en el proceso de calcificación vascular.

Eudebeiolide B is a eudesmane-type sesquiterpenoid compound isolated from Salvia plebeia R. Br., and little is known about its biological activity. In this study, we investigated the effects of eudebeiolide B on osteoblast differentiation, receptor activator nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis in vitro and ovariectomy-induced bone loss in vivo. Eudebeiolide B induced the expression of alkaline phosphatase (ALP) and calcium accumulation during MC3T3-E1 osteoblast differentiation. In mouse bone marrow macrophages (BMMs), eudebeiolide B suppressed RANKL-induced osteoclast differentiation of BMMs and bone resorption. Eudebeiolide B downregulated the expression of nuclear factor of activated T-cells 1 (NFATc1) and c-fos, transcription factors induced by RANKL. Moreover, eudebeiolide B attenuated the RANKL-induced expression of osteoclastogenesis-related genes, including cathepsin K (Ctsk), matrix metalloproteinase 9 (MMP9) and dendrocyte expressed seven transmembrane protein (DC-STAMP). Regarding the molecular mechanism, eudebeiolide B inhibited the phosphorylation of Akt and NF-κB p65. In addition, it downregulated the expression of cAMP response element-binding protein (CREB), Bruton’s tyrosine kinase (Btk) and phospholipase Cγ2 (PLCγ2) in RANKL-induced calcium signaling. In an ovariectomized (OVX) mouse model, intragastric injection of eudebeiolide B prevented OVX-induced bone loss, as shown by bone mineral density and contents, microarchitecture parameters and serum levels of bone turnover markers. Eudebeiolide B not only promoted osteoblast differentiation but inhibited RANKL-induced osteoclastogenesis through calcium signaling and prevented OVX-induced bone loss. Therefore, eudebeiolide B may be a new therapeutic agent for osteoclast-related diseases, including osteoporosis, rheumatoid arthritis and periodontitis.

Breast cancer development in BRCA1/2 mutation carriers is a net consequence of cell-autonomous and cell nonautonomous factors which may serve as excellent targets for cancer prevention. In light of our previous data we sought to investigate the consequences of the BRCA-mutation carrier state on RANKL/osteoprotegerin (OPG) signalling. We analysed serum levels of RANKL, OPG, RANKL/OPG complex, oestradiol (E2), and progesterone (P) during menstrual cycle progression in 391 BRCA1/2-mutation carriers and 782 noncarriers. These studies were complemented by analyses of RANKL and OPG in the serum and mammary tissues of female cynomolgus macaques (n = 88) and serum RANKL and OPG in postmenopausal women (n = 150). BRCA-mutation carriers had lower mean values of free serum OPG in particular in BRCA1-mutation carriers (p = 0.018) compared with controls. Among BRCA1/2 mutation carriers, lower OPG levels were associated with germline mutation locations known to confer an increased breast cancer risk (p = 0.003). P is associated with low OPG levels in serum and tissue, particularly in BRCA-mutation carriers (rho = −0.216; p = 0.002). Serum OPG levels were inversely correlated (rho = −0.545, p

Objective(s):miR-125b has been identified as a tumor suppressor in many tumors, but its role in giant cell tumor (GCT) of bone remains poorly understood. The current study aimed to investigate the potential role and mechanism of miR-125b in GCT. Materials and Methods:Expression levels of miR-125b in GCT tissues were determined using RT-PCR. The cell proliferation was surveyed by direct cell counting, MTS and CCK-8, and the apoptotic cells were evaluated by Annexin V-FITC and propidium iodine staining assay. The target gene expression was determined using RT-PCR and western blot. Parathyroid hormone 1 receptor (PTH1R) 3’-UTR was cloned into luciferase reporter plasmid to confirm direct targeting. Results: We found that miR-125b was significantly down-regulated in GCT tissues. Using both gain- and loss-of-function analyses, we further revealed that miR-125b suppressed GCT stromal cell proliferation and induced cell apoptosis. Furthermore, we revealed that PTH/PTHrP type 1 receptor is a direct and functional target of miR-125b. Conclusion:Our results suggest that miR-125b acts as a tumor suppressor through suppression of the PTH1R/RANKL signaling pathway. These findings contribute to our understanding of the functions of miR-125b in GCT.

Objective: to evaluate the osteoclastogenesis regulatory system – osteoprotegerin/receptor activator of nuclear factor-κβ ligand (OPG/RANKL) system – in men with different clinical types of ankylosing spondylitis (AS).Subjects and methods. The osteoclastogenesis regulatory system was studied in 60 men, including 40 patients diagnosed with AS complying with the 1984 New York criteria and 20 men without AS. RANKL, a major stimulant of osteoclastogenesis, and OPG, a decoy receptor that binds RANKL and, accordingly, blocks osteoclastogenesis, were investigated.Results. It was shown that in the patients with AS, RANKL concentrations were normal and the content of OPG and OPG/RANKL ratio proved to be significantly higher than those in the men without AS. The highest OPG concentrations were recorded in patients with the axial form of this disease, its moderate activity and early X-ray stage. No relationship was found between the level of RANKL and the clinical characteristics of AS; however, there was a tendency to a slight increase in its concentration in patients with extraskeletal manifestations of AS, its high activity, high functional class, and late X-ray stage.Conclusion. The considerable increase in OPG levels and OPG/RANKL ratio was ascertained to be associated to the fact that 94% of the patients with late-stage AS characterized by the presence of numerous syndesmophytes. These changes must be compensatory in response to modestly increased RANKL level and enhanced bone resorption.