Receptor Activator Of NF-κB Research Articles

25-Hydroxycholesterol induces odontoclastic differentiation through RANK-RANKL upregulation and NF-κB activation in odontoblast-like MDPC-23 cells: An in vitro study.

The physiological effects and cellular mechanism of 25-hydroxycholesterol (25-HC), which is an oxysterol synthesized from cholesterol by cholesterol-25-hydroxylase (CH25H) expressed under inflammatory conditions, are still largely unknown during odontoclastogenesis. This study aimed to evaluate 25-HC-induced odontoclastogenesis and its cellular mechanisms in odontoblast-like MDPC-23 cells. To investigate 25-HC-induced odontoclastogenesis of MDPC-23 cells and its cellular mechanism, haemotoxylin and eosin staining, tartrate-resistant acid phosphatase (TRAP) staining, dentine resorption assay, zymography, reactive oxygen species (ROS) detection, immunocytochemistry, and nuclear translocation were performed. The experimental values are presented as mean ± standard deviation and were compared using analysis of variance, followed by post hoc multiple comparisons (Tukey's test) using SPSS software version 22 (IBM Corp.). A p-value <.05 was considered statistically significant. Lipopolysaccharide or receptor activator of nuclear factor-κB ligand (RANKL) induced the synthesis of 25-HC via the expression of CH25H in MDPC-23 cells (p < .01). Multinucleated giant cells with morphological characteristics and TRAP activity of the odontoclast were increased by 25-HC in MDPC-23 cells (p < .01). Moreover, 25-HC increased dentine resorption through the expression and activity of matrix metalloproteinases in MDPC-23 cells. It not only increased the expression of odontoclastogenic biomarkers but also translocated cytosolic nuclear factor-κB (NF-κB) to the nucleus in MDPC-23 cells. Additionally, 25-HC not only increased the production of ROS (p < .01), expression of inflammatory mediators (p < .01), pro-inflammatory cytokines, receptor activator of NF-κB (RANK), and RANKL but also suppressed the expression of osteoprotegerin (OPG) in MDPC-23 cells. In contrast, CDDO-Me, a chemical NF-κB inhibitor, decreased TRAP activity (p < .01) and downregulated the expression of the odontoclastogenic biomarkers, including RANK and RANKL, in MDPC-23 cells. 25-HC induced odontoclastogenesis by modulating the RANK-RANKL-OPG axis via NF-κB activation in MDPC-23 cells. Therefore, these findings provide that 25-HC derived from cholesterol metabolism may be involved in the pathophysiological etiological factors of internal tooth resorption.

OPG/RANK/RANKL Axis in Egyptian Children With Acute Lymphoblastic Leukemia After Maintenance Therapy: Relationship to Bone Mineral and Vitamin D Status.

Policies to maximize peak bone mass in survivor's children of acute lymphoblastic leukemia (ALL) have been recognized as a priority area for research. The present study aimed to evaluate the relationship between osteoprotegerin (OPG)/receptor activator of nuclear factor kappa-B (RANK)/RANK ligand (RANKL) axis, vitamin D status, and serum magnesium in ALL survivors. Sixty ALL survivors treated with chemotherapy and 60 age and sex-matched controls were included. Vitamin D and parathyroid hormone, RANK, RANKL, and OPG levels were immunoassayed, in addition to serum calcium, phosphorus, magnesium levels, and alkaline phosphatase activity assessment. Furthermore, standard anthropometric measurement, history of fractures since treatment and clinical assessment were recorded. History of bone fractures after the start of therapy was detected in 17 ALL subjects (28.33%). Significantly lower vitamin D, magnesium, calcium, and OPG levels, meanwhile, significantly higher serum parathyroid hormone, RANK, and RANKL levels were detected in survivors compared with the control group. Vitamin D level was significantly positively correlated with magnesium, calcium, and OPG levels. Meanwhile, negatively correlated with RANK and RANKL levels. ALL survivors had a high prevalence of impaired vitamin D status, decreased Mg, and altered OPG/RANK/RANKL axis with impaired bone remodeling. The results herein may open the door for new interventional actions in ALL survivors to protect against bone resorption.

RANKL and RANK in Cancer Therapy.

Receptor activator of nuclear factor-κB (RANK) and its ligand (RANKL) are key regulators of mammalian physiology such as bone metabolism, immune tolerance and antitumor immunity, and mammary gland biology. Here, we explore the multiple functions of RANKL/RANK in physiology and pathophysiology and discuss underlying principles and strategies to modulate the RANKL/RANK pathway as a therapeutic target in immune-mediated cancer treatment.

MicroRNAs-mediated regulation pathways in rheumatic diseases.

Rheumatoid arthritis (RA) and ankylosing spondylitis (AS) are two common rheumatic disorders marked by persistent inflammatory joint disease. Patients with RA have osteodestructive symptoms, but those with AS have osteoproliferative manifestations. Ligaments, joints, tendons, bones, and muscles are all affected by rheumatic disorders. In recent years, many epigenetic factors contributing to the pathogenesis of rheumatoid disorders have been studied. MicroRNAs (miRNAs) are small, non-coding RNA molecules implicated as potential therapeutic targets or biomarkers in rheumatic diseases. MiRNAs play a critical role in the modulation of bone homeostasis and joint remodeling by controlling fibroblast-like synoviocytes (FLSs), chondrocytes, and osteocytes. Several miRNAs have been shown to be dysregulated in rheumatic diseases, including miR-10a, 16, 17, 18a, 19, 20a, 21, 27a, 29a, 34a, 103a, 125b, 132, 137, 143, 145, 146a, 155, 192, 203, 221, 222, 301a, 346, and 548a.The major molecular pathways governed by miRNAs in these cells are Wnt, bone-morphogenic protein (BMP), nuclear factor (NF)-κB, receptor activator of NF-κB (RANK)-RANK ligand (RANKL), and macrophage colony-stimulating factor (M-CSF) receptor pathway. This review aimed to provide an overview of the most important signaling pathways controlled by miRNAs in rheumatic diseases.

Evaluation of Wnt/β-Catenin Pathway Circulating Proteins in Children and Adolescents with Hemophilia: Correlation with the Degree of Hemophilic Arhtropathy

Introduction: The pathophysiology of hemophilic arthropathy (HAP) establishes a hallmark of heterogeneous mechanisms related with the joint degeneration which has not been fully elucidated especially in reference to the development of inflammatory and catabolic processes occurring within the articular cartilage and synovial membrane. Crucial pathways regulating bone remodeling are the Wnt/β-catenin and the Receptor Activator of Nuclear factor kB (RANK) Ligand (RANKL)/Osteoprotegerin (OPG) systems. The Wnt/β-catenin pathway plays a major role in osteoblast differentiation and calcium homoeostasis. Sclerostin (SOST) and Dickkopf-1 (DKK-1) are soluble antagonists of the Wnt/β-catenin pathway secreted solely by the osteocytes. Osteopontin (OPN) acts as a secreted protein that is referred to many aspects of bone metabolism. OPN participates in bone regulation through neurology, endocrine and immunity. In this context, we aimed to investigate the clinical significance of peripheral biomarkers implicated in HAP severity and correlate with HAP features in children and adolescents with hemophilia A and B. Patients and Methods: Οne hundred sixty two children and adolescents (mean age: 11.7 years, range 1-18.5 years) with severe, moderate and mild (n= 8) hemophilia A or B (n=11), on prophylaxis or on demand treatment (n= 6) were included in the study. Of them, 69 patients with median age of 11 years had no signs of HA (NHAP), 70 patients with median age of 14 years had arthropathy (HAP), while 23 patients with median age of 15.5 years presented with acute bleeding -hemarthrosis or muscle bleed- at the time of the biomarkers’ measurement- (AB). In all patients along with standard care laboratory evaluation, measurements of plasma circulating levels of DKK-1, RANKL, OPG, SOST and OPN were performed using immunoenzymatic techniques. Data are presented as mean ± SEM. Results: We found that a) DKK-1 levels were higher in patients with NHAP and AB compared to patients with HAP, 3.396.0±194.0 pg/mL and 3.221±282.2 pg/mL vs 2.544±172.7 pg/mL (p=0.002); b) SOST levels in patients with NHAP (192.5±27.0 pg/mL), HAP (205.5±22.3 pg/mL) and AB (188.9±30.3 pg/mL) did not differ between the groups of patients (p>0.950); c) OPG levels in patients with NHAP (9.6±0.2 pg/mL), HAP (9.3±0.3 pg/mL) and AB (8.5±0.3 pg/mL) and RANKL levels in patients with NHAP (673.0±53.9 pg/mL), HAP (717.6±53.0 pg/mL) and AB (728.0±75.0 pg/mL) did not differ between the groups of patients (p>0.148 and p>0.515, respectively); d) Similarly, OPN levels in patients with NHAP (57.6±6.9 pg/mL), HAP (65.2±7.3 pg/mL) and AB (59.4±5.9 pg/mL) did not differ between the groups of patients (p>0.822) and e) DKK-1 and OPG levels correlated significantly with FVIII/FIX levels at the time of measurement (r=-0.553, p=0.014 and r=-0.540, p=0.017, respectively). Conclusions: In this study, no significant perturbations of the crucial pathways regulating bone remodeling such as Wnt/β-catenin and RANKL/OPG systems were identified, by measuring circulating levels of key proteins involved in these pathways. Circulating DKK-1 seems to be a more sensitive biomarker indicating a potential decrease in osteoblastic activity. It appears that bone loss is rather rare in well-treated children and adolescents with hemophilic arthropathy comparing to young patients without arthropathy. Further studies are needed in order to determine how biomarkers in serum could play a prognostic role in the evolution of arthropathy from an early age in children and adolescents with hemophilia A or B, as well as the role of FVIII and FIX in bone metabolism, with the aim to ameliorate treatment strategies and prevent joint damage.

Risk association of RANKL and OPG gene polymorphism with breast cancer to bone metastasis in Pashtun population of Khyber Pakhtunkhwa, Pakistan.

The receptor activator NF-κB ligand (RANKL) and Osteoprotegrin (OPG) single nucleotide polymorphisms (SNPs) have been associated with the risk of breast cancer to bone metastasis. This study was designed to investigate the association of RANKL and OPG gene polymorphisms with breast to bone metastasis in Pashtun population of Khyber Pakhtunkhwa, Pakistan. A total of 215 participants were enrolled containing 106 breast cancer patients, 58 breast to bone metastasis and 51 age and gender matched healthy controls. RANKL (rs9533156) and OPG (rs2073618, rs3102735) polymorphisms were genotyped in genomic DNA, using Tetra-ARMS PCR protocol. The results were analyzed among the three groups and P-value less then 0.05 were considered statistically significant. Our results displayed significant association of OPG (rs3102735) risk allele and corresponding genotypes in breast cancer vs healthy controls, bone metastasis vs healthy controls and breast cancer vs breast to bone metastasis as a disease risk. However, there was no association observed for OPG (rs2073618) risk allele and corresponding genotypes with the diseases risk. Similarly, RANKL (rs9533156) risk allele and corresponding genotypes in breast cancer vs healthy controls, bone metastasis vs healthy controls and breast cancer vs breast to bone metastasis exhibited significant association except for the risk allele carrying genotypes in breast to bone metastasis. OPG (rs3102735) and RANKL (rs9533156) exhibited significant association with breast to bone metastasis while OPG (rs2073618) didn't show significant association with breast to bone metastasis in Pashtun population of Pakistan. However, this study unlocks more questions to investigate the exact scenario of genetic predisposition of breast to bone metastasis.

Roles of osteoprotegerin in endocrine and metabolic disorders through receptor activator of nuclear factor kappa-B ligand/receptor activator of nuclear factor kappa-B signaling.

Endocrine and metabolic diseases show increasing incidence and high treatment costs worldwide. Due to the complexity of their etiology and mechanism, therapeutic strategies are still lacking. Osteoprotegerin (OPG), a member of the tumor necrosis factor receptor superfamily, appears to be a potential candidate for the treatment of these diseases. Studies based on clinical analysis and rodent animal models reveal the roles of OPG in various endocrine and metabolic processes or disorders, such as bone remodeling, vascular calcification, and β-cell proliferation, through the receptor activator of nuclear factor kappa-B ligand (RANKL) and the receptor activator of NF-κB (RANK). Thus, in this review, we mainly focus on relevant diseases, including osteoporosis, cardiovascular disease (CVD), diabetes, and gestational diabetes mellitus (GDM), to summarize the effects of the RANKL/RANK/OPG system in endocrine and metabolic tissues and diseases, thereby providing a comprehensive insight into OPG as a potential drug for endocrine and metabolic diseases.

Salivary Gene Expression of RANK, RANKL, and OPG in Type 1 Diabetes Mellitus and Periodontal Disease Patients.

The relationship between type 1 diabetes mellitus (T1DM) and periodontal disease may exhibit by the alteration of bone metabolism. However, evidence for this relationship is scarce and inconclusive. Thus, the aims of the present study were to investigate salivary receptor activator of nuclear factor kappa-β (RANK), receptor activator of nuclear factor kappa-β ligand (RANKL), osteoprotegerin (OPG) gene expression and the RANKL:OPG ratio in T1DM and non-T1DM. Secondary objective was to determine the relationships of RANK, RANKL and OPG gene expression to clinical parameters of T1DM and periodontal disease. Twenty patients with T1DM and twenty age-matched non-T1DM were recruited. Clinical periodontal parameters were measured. Total RNA was isolated from non-stimulated saliva, and the relative gene expressions of RANK, RANKL, OPG and RANKL:OPG ratio were determined by quantitative real-time polymerase chain reaction. The T1DM group had significantly higher mean periodontal parameters than the non-T1DM group, while the mean plaque scores of both groups were not significantly different. There was a trend of higher relative gene expression of RANK, RANKL, and the RANKL:OPG ratio and lower expression of OPG in T1DM group but no statistic significant different when compared to non-T1DM. In the T1DM group, RANKL:OPG correlated with the percentage of bleeding sites, whereas RANK, RANKL, and HbA1c levels correlated with pocket depth. Bone metabolisms demonstrating by decreased OPG gene expression and upregulated of RANK, RANKL, RANKL:OPG with higher pocket depth and bleeding in T1DM may play an important role in periodontal destruction in T1DM.

ODP085 Denosumab Induced Severe Hypophosphatemia

Abstract Introduction Denosumab is a receptor activator of nuclear factor kappa-B (RANK) ligand inhibitor used for treatment of osteoporosis and bone metastases in patients with solid tumors. Denosumab induced hypophosphatemia has been reported in less than 1% of patients, and in most cases described as mild to moderate in severity. We present a case of severe hypophosphatemia after denosumab treatment for diffuse bone metastases due to prostate cancer. Case report A 78-year-old male patient with history of diffuse bone metastases due to prostate cancer, schizophrenia on treatment and intermittent alcohol use, presented to the hospital with suicidal ideation and worsening fatigue. Patient was found to have a phosphorus levels of &amp;lt;1. 0 mg/dL (2.5-4.6 mg/dL), corrected calcium of 8.2 mg/dL (8.4-10.5 mg/dl), vitamin D 25 OH of 36.34 ng/mL (30-100 ng/mL), magnesium of 2.3 mg/dL (1.7-2.5 mg/dL), parathyroid hormone (iPTH) level of 379 pg/mL (10-65pg/mL) and serum creatinine of 1.1 mg/dL (0.5-1.2 mg/dL). Urinary studies showed 24 hour urinary phosphate 1282 mg/24h (360-1600 mg/24h), urine creatinine 1570 mg/24h (500-2150 mg/24h). There was no evidence of acidosis, chronic kidney disease, preexisting osteomalacia or vitamin D deficiency. The patient had no recent alcohol consumption and his alcohol level was undetectable. After reviewing the outside medical records it was noted that the patient had received denosumab 120 mg, 11 days prior to the hospital admission, as part of the management of bone metastases due to prostate cancer. The patient's baseline phosphorus prior to denosumab was in the low normal range. The severe hypophosphatemia and secondary hyperparathyroidism were attributed to denosumab administration. Patient was initially treated with intravenous phosphate and then transitioned to oral phosphate along with elemental calcium and calcitriol during the course of his hospitalization. Patient's phosphorus improved to 2.3 mg/dL by the time of discharge. Discussion In patients treated with denosumab, the primary concern is potential post-treatment hypocalcemia, however, our case brings attention to severe hypophosphatemia as a serious side effect to treatment with denosumab. Multiple specialties are involved in the treatment of osteoporosis and metastatic bone lesions, and each should be aware that denosumab can lead to lower serum phosphate levels due to its effects on bone resorption and reduction of proximal tubular reabsorption of the phosphate resulting in clinical symptoms of muscle weakness, bone pain, fractures and even stupor, coma and death. We therefore conclude that monitoring phosphate levels should be part of the pre- and post-treatment evaluation in patients who are candidates for treatment with denosumab, particularly high-dose denosumab. Presentation: No date and time listed

Hypoxia-inducible factor 1α enhances RANKL-induced osteoclast differentiation by upregulating the MAPK pathway

Hypoxia (low-oxygen tension) and excessive osteoclast activation are common conditions in many bone loss diseases, such as osteoporosis, rheumatoid arthritis (RA), and pathologic fractures. Hypoxia-inducible factor 1 alpha (HIF1α) regulates cellular responses to hypoxic conditions. However, it is not yet known how HIF1α directly affects osteoclast differentiation and activation. This study sought to. explore the effects of HIF1α on osteoclast differentiation and it's molecular mechanisms. L-mimosine, a prolyl hydroxylase (PHDs) domain inhibitor, was used to stabilize HIF1α in normoxia. In the presence of receptor activator of nuclear factor-kB (NF-kB) ligand (RANKL), RAW264.7 cells were cultured and stimulated by treatment with L-mimosine at several doses to maintain various levels of intracellular HIF1α. The multi-nucleated cells were assessed by a tartrate-resistant acid phosphatase (TRAP) and F-actin ring staining assays. The osteoclast-specific genes, such as Cathepsin K, β3-Integrin, TRAP, c-Fos, nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), and matrix metallo-proteinase 9 (MMP9), were analyzed by real time-polymerase chain reaction (RT-PCR). The expression of relevant proteins was analyzed by Western blot. L-mimosine increased the content of intracellular HIF1α in a dose-dependent manner, which in turn promoted RANKL-induced osteoclast formation and relevant protein expression by upregulating the mitogen-activated protein kinase (MAPK) pathways. Our findings suggest that HIF1α directly increases the osteoclast differentiation of RANKL-mediated RAW264.7 cells in vitro by upregulating the MAPK pathways.

NCX1 disturbs calcium homeostasis and promotes RANKL-induced osteoclast differentiation by regulating JNK/c-Fos/NFATc1 signaling pathway in multiple myeloma

Although several types of calcium channels abnormalities have been shown to promote myeloma bone disease (MBD), the relationship between Na+/Ca2+ exchanger 1 (NCX1) and MBD remains unexplored. Here, we examined the role of NCX1 in the development of multiple myeloma (MM), with a special focus on the underlying effects involved osteoclast differentiation. Firstly, we detected NCX1 protein highly expressed in BM tissues of MM patients, and its expression was positively correlated with serum calcium and the percentage of BM CD138+ cells. In vitro, NCX1 suppression with the inhibitor KB-R7943 reduced cell viability of MM cells and caused apoptosis. Extracellular high Ca2+ environment increased the level of intracellular Ca2+ in MM cells through gating the calcium influx, with subsequently promoting the expression of NCX1 and osteoclastogenesis-related genes (receptor activator of nuclear factor-κB (RANKL), nuclear factor of activated T cell cytoplasmic 1 (NFATc1), and proto-oncogene Fos (c-Fos). This phenomenon could be reversed by KB-R7943 or calcium chelation. Furthermore, NCX1 overexpression in MM cells accelerated osteoclastogenesis, while NCX1 knockdown or suppression resulted in the opposite effect. Mechanistically, we further investigated the related mechanisms of NCX1 regulating osteoclast differentiation using RNA sequencing, western blotting and Enzyme linked immunosorbent assay, and found that NCX1 modulated osteoclast differentiation in MM though JNK/c-Fos/NFATc1 signaling pathway. In conclusion, the Ca2+/NCX1-mediated signaling participates in the osteoclasts-myeloma cell interactions, which represents a promising target for future therapeutic intervention in MBD.

Complement receptor C5aR1 on osteoblasts regulates osteoclastogenesis in experimental postmenopausal osteoporosis.

In recent years, evidence has accumulated that the complement system, an integral part of innate immunity, may be involved in the regulation of bone homeostasis as well as inflammatory bone loss, for example, in rheumatoid arthritis and periodontitis. Complement may also contribute to osteoporosis development, but investigation of the mechanism is limited. Using mice with a conditional deletion of the complement anaphylatoxin receptor C5aR1, we here demonstrated that C5aR1 in osteoblasts (C5aR1 Runx2-Cre mice) or osteoclasts (C5aR1 LysM-Cre mice) did not affect physiological bone turnover or age-related bone loss in either sex, as confirmed by micro-computed tomography, histomorphometry, and biomechanical analyses of the bone and by the measurement of bone turnover markers in the blood serum. When female mice were subjected to ovariectomy (OVX), a common model for postmenopausal osteoporosis, significant bone loss was induced in C5aR1 fl/fl and C5aR1 LysM-Cre mice, as demonstrated by a significantly reduced bone volume fraction, trabecular number and thickness as well as an increased trabecular separation in the trabecular bone compartment. Confirming this, the osteoclast number and the receptor activator of nuclear factor k-B (RANK) ligand (RANKL) serum level were significantly elevated in these mouse lines. By contrast, C5aR1 Runx2-Cre mice were protected from bone loss after OVX and the serum RANKL concentration was not increased after OVX. These data suggested that bone cell-specific C5aR1 may be redundant in bone homeostasis regulation under physiological conditions. However, C5aR1 on osteoblasts was crucial for the induction of bone resorption under osteoporotic conditions by stimulating RANKL release, whereas C5aR1 on osteoclasts did not regulate OVX-induced bone loss. Therefore, our results implicate C5aR1 on osteoblasts as a potential target for treating postmenopausal osteoporosis.

Monotropein Protects against Inflammatory Bone Loss and Suppresses Osteoclast Formation and Bone Resorption by Inhibiting NFATc1 via NF-κB and Akt/GSK-3β Pathway.

Monotropein (Mon) is a kind of iridoid glycoside plant secondary metabolite primarily present in some edible and medicinal plants. The aim of this study was to investigate the effect of Mon on lipopolysaccharide (LPS)-induced inflammatory bone loss in mice and osteoclasts (OCs) derived from bone marrow-derived macrophages (BMMs), and explore the mechanisms underlying the effect of Mon on LPS-induced osteoclastogenesis. It was found that Mon markedly attenuated deterioration of the bone micro-architecture, enhanced tissue mineral content (TMC) and bone volume/total volume (BV/TV), reduced structure model index (SMI) and trabecular separation/spacing (Tb.Sp) in the bone tissue and decreased the activities of tartrate resistant acid phosphatase-5b (TRACP-5b), receptor activator NF-κB (RANK), and receptor activator NF-κB ligand (RANKL) as well as the serum levels of interleukin 6 (IL-6) and interleukin 1β (IL-1β) in LPS-treated mice. In addition, Mon treatment reduced the number of TRAP positive OCs in the bone tissue of LPS-treated mice and also exerted a stronger inhibitory effect on formation, differentiation, and F-actin ring construction of OCs derived from BMMs. Mon significantly inhibited the expression of the nuclear factor of activated T-cells c1 (NFATc1) and the immediate early gene (C-Fos) and nuclear translocation of NFATc1 in LPS-treated OCs, thereby inhibiting the expression of matrix metalloproteinase-9 (MMP-9), cathepsin K (CtsK), and TRAP. Mon significantly inhibited the expression of TRAF6, phosphorylation of P65, and degradation of IKBα, thus inhibiting the activation of NF-κB pathway in LPS-induced inflammatory mice and OCs derived from BMMs, and also inhibited LPS-induced phosphorylation of protein kinase B (Akt) and Glycogen synthase kinase 3β (GSK-3β) in OCs derived from BMMs. In conclusion, these results suggested that Mon could effectively inhibit osteoclastogenesis both in vitro and in vivo and therefore may prove to be potential option for prevention and treatment of osteoclastic bone resorption-related diseases.

An ankylosing spondylitis risk variant alters osteoclast differentiation.

To explore whether the variants in non MHC proteasome gene are associated with AS and explain the role of the variant in the disease. Case-control analysis to identify AS predisposition genes; dual-luciferase reporter assay, immunoblot analysis and osteoclastogenesis assays to detect the function of the positive variant. Affected individuals were diagnosed according to the modified New York Criteria by at least two experienced rheumatologists, and rechecked by another rheumatologist. The study included 1037 AS patients and 1014 no rheumatic and arthritis disease controls. The main age of AS onset is between 16 and 35 years old. HLA-B27-positive subjects comprised 90.0% of patients. A nonsynonymous SNP rs12717 in proteasome gene PSMB1 significantly associated with AS. Individuals with CC genotype had a higher onset risk compared with those with GG/GC genotypes (OR = 1.89, P = 0.0047). We also discovered that PSMB1 regulates the receptor activator of nuclear factor-κB (RANK)/RANK ligand (RANKL) signalling pathway and the disease-associated variant PSMB1-Pro11 significantly inhibits RANKL-induced NF-κB pathway in osteoclast differentiation via the degradation of IKK-β compared with PSMB1-Ala11. RANKL induced osteoclast differentiation was significantly lower in primary monocyte osteoclast precursor from individuals with genotype PSMB131C/31C compared with individuals with genotype PSMB131G/31G. These results reveal a novel understanding of the bone formation and reabsorbing imbalance in AS. The new bone formation phenotype can be attributed to the inhibition of osteoclast differentiation by a more functional PSMB1 gene.

HSP90 drives the Rab11a-mediated vesicular transport of the cell surface receptors in osteoclasts.

Rab11a, which ubiquitously localizes to early and recycling endosomes, is required for regulating the vesicular transport of cellular cargos. Interestingly, our previous study revealed that Rab11a served as a negative regulator of osteoclastogenesis by facilitating the lysosomal proteolysis of (1) colony-stimulating factor-1 (c-fms) receptor and (2) receptor activator of nuclear factor-κB (RANK) receptor, thereby resulting in inhibition of osteoclast (OC) differentiation, maturation, and bone-resorbing activity. However, the molecular mechanisms of how Rab11a negatively affected osteoclastogenesis were largely unknown. Heat shock protein (HSP90), including two isoforms HSP90αand HSP90β, necessitates the stability, maturation, and activity of a broad range of its clients, and is essentially required for a vast array of signal transduction pathways in nonstressful conditions. Furthermore, cumulative evidence suggests that HSP90 is a vital element of the vesicular transport network. Indeed, our recent study revealed thatHSP90, a novel effector protein of Rab11b, modulatedRab11b-mediated osteoclastogenesis. In this study, we also found that Rab11a interacted with both HSP90α and HSP90β in OCs. Upon blockade of HSP90 ATPase activity by a specific inhibitor(17-allylamino-demethoxygeldanamycin), we showed that (1) the ATPase domain of HSP90 was a prerequisite for the interaction between HSP90 and Rab11a, and (2) the interaction of HSP90 to Rab11a sufficiently maintained the inhibitory effects of Rab11a on osteoclastogenesis. Altogether, our findings undoubtedly indicate a novel role of HSP90 in regulating Rab11a-mediated osteoclastogenesis.

Identification of a novel TNFRSF11A gene variant in a rare case of familial osteopetrosis autosomal recessive type 7

Osteopetrosis is a group of rare genetic disorders characterized by defective osteoclast differentiation that leads to abnormally dense bones resulting in frequent fractures in patients. Among the various types of osteopetrosis, based on inheritance patterns, the autosomal recessive (ARO) type is the least frequently encountered. Among the AROs, a particular sub-type, known as autosomal recessive osteopetrosis type 7 (ARO7), is characterized by osteoclast-poor osteopetrosis with hypo-gammaglobulinemia. This disease is caused by abnormalities in the receptor activator of nuclear factor kappa-Β (RANK) protein, which is encoded by the TNFSFR11A gene. We describe a clinically diagnosed case of ARO7, the product of a consanguineous marriage, who presented with clinical features typical of ARO7 including elevated bone density, optic nerve atrophy and macrocephaly. Whole exome sequencing revealed the presence of a previously unreported homozygous pathogenic frameshift variation c.1527_1528insG (p.Ser511ValfsX20) in exon 9 of the TNFRSF11A gene in the proband, which was confirmed by Sanger sequencing and was found in heterozygous state in both the parents.. Neurosurgical intervention performed to relieve optic canal decompression included bifrontal craniotomy with orbitofrontal Bandau advancement and cranial remodelling procedures. This was followed by hematopoietic stem cell transplantation, which is standard of care for ARO7 patients, from an unrelated donor. However, the patient eventually developed aspiration pneumonia with sepsis coupled with pulmonary haemorrhage and respiratory failure that proved to be fatal. Despite the unfortunate outcome, this study highlights the need for timely genetic diagnosis and improved treatment options for patients with ARO7.

MiR-124-3p Reduces Bone Mineral Density in Postmenopausal Osteoporosis Rats Through Regulating Signal Transducer and Activator of Transcription 3 Pathway

We aimed to study the effect of miR-124-3p on postmenopausal osteoporosis (POP) rats through regulating the signal transducer and activator of transcription 3 (STAT3) pathway. Rats were randomly divided into normal group, model group and miR-124-3p antagomir group. Enzyme-linked immunosorbent assay (ELISA) was performed to determine the levels of receptor activator of nuclear factor-κB (RANK) and osteoprotegerin (OPG). BMD of femur was significantly lower in model group and miR-124-3p antagomir group than that in normal group at 12 weeks after modeling, while it was significantly higher in miR-124-3p antagomir group than that in model group. Positive expression of BMP2 was obviously higher in miR-124-3p antagomir group than that in model group. Protein expression of p-STAT3 was evidently lower in miR-124-3p antagomir group than that in model group. Besides, POP rats have significantly increased level of miR-124-3p compared with that in normal group. In model group and miR-124-3p antagomir group, the content of OPG was remarkably lower, and the content of RANK was remarkably higher than those in normal group. In miR-124-3p antagomir group, the content of OPG was remarkably higher, and RANK was remarkably lower than those in model group. MiR-124-3p reduces BMD in POP rats through up-regulating the STAT3 pathway.

Evaluation of an association between RANKL and OPG with bone disease in people with cystic fibrosis

BackgroundAs people with Cystic Fibrosis (CF) live longer, extra-pulmonary complications such as CF-related bone disease (CFBD) are becoming increasingly important. The etiology of CFBD is poorly understood but is likely multifactorial. Bones undergo continuous remodeling via pathways including RANK (receptor activator of NF-κB)/sRANKL (soluble ligand)/OPG (osteoprotegerin). We sought to examine the association between sRANKL (stimulant of osteoclastogenesis) and OPG levels (inhibitor of osteoclast formation) and CFBD to investigate their potential utility as biomarkers of bone turnover in people with CF. MethodsWe evaluated sRANKL and OPG in plasma from people with CF and healthy controls (HC) and compared levels in those with CF to bone mineral density results. We used univariable and multivariable analysis to account for factors that may impact sRANKL and OPG. ResultsWe found a higher median [IQR] sRANKL 10,896pg/mL [5,781–24,243] CF; 2,406pg.mL [659.50–5,042] HC; p= 0.0009), lower OPG 56.68pg/mL [36.28–124.70] CF; 583.20pg/mL [421.30–675.10] HC; p < 0.0001), and higher RANKL/OPG in people with CF no BD than in HC (p < 0.0001). Furthermore, we found a higher RANKL/OPG ratio 407.50pg/mL [214.40–602.60] CFBD; 177.70pg/mL [131.50–239.70] CF no BD; p = 0.007) in people with CFBD versus CF without bone disease. This difference persisted after adjusting for variables thought to impact bone health. ConclusionsThe current screening recommendations of imaging for CFBD may miss important markers of bone turnover such as the RANKL/OPG ratio. These findings support the investigation of therapies that modulate the RANK/RANKL/OPG pathway as potential therapeutic targets for bone disease in CF.

Nanoemulsified Genistein and Vitamin D Mediated Epigenetic Regulation to Inhibit Osteoporosis.

The imbalance in the bone remodeling process with more bone resorption by osteoclasts compared to bone formation by osteoblasts results in a metabolic bone disorder known as osteoporosis. This condition reduces the bone mineral density and increases the risk of fractures due to low bone mass and disrupted bone microarchitecture. Osteoclastogenesis increases when the receptor activator NFκB ligand (RANKL) on the osteoblast surface binds to the receptor activator NFκB (RANK) on the osteoclast surface and the function of the decoy receptor of RANKL, osteoprotegrin, is compromised due to external stimuli such as heparin and lipopolysaccharides. The RANK/RANKL axis promotes the nuclear factor kappa B (NFκB) expression, which in turn increases the histone methyltransferase activity of EzH2 and EzH1 for the epigenetic regulation of osteoclastogenesis-related genes. Genistein counteracts NFκB-induced osteoclastogenesis and downstream signaling through the direct regulation of histone methyltransferase, EzH2 and EzH1, transcription. However, genistein possesses limitations like low bioavailability, low water solubility, high estrogen activity, and thyroid side effects, which obstruct its therapeutic usage. Here, the nanoemulsified formulation of genistein with vitamin D was utilized to circumvent the limitations of genistein so that it can be utilized for therapeutic purposes in osteoporosis management. The nanoemulsification of genistein and vitamin D was performed through the spontaneous emulsification using Tween 80 and medium chain triglyceride oil as an organic phase. The physiologically stable and biocompatible combination of the genistein and vitamin D nanoemulsion (GVNE) exhibited the controlled release pattern of genistein with Korsmeyer-Peppas and Higuchi models under different pH conditions (7.4, 6.5, and 1.2). The GVNE potentially enhanced the therapeutic efficacy under in vitro osteoporosis models and helped restore disease parameters like alkaline phosphatase activity, tartrate-resistant acid phosphatase activity, and the formation of multinuclear giant cells. Molecularly, the GVNE overturned the LPS-induced osteoclastogenesis by downregulation of NFκB expression along with its binding on EzH2 and EzH1 promoters. GVNE effects on the osteoporosis model established it as an efficient antiosteoporotic therapy. This nanonutraceutical-based formulation provides an epigenetic regulation of osteoporosis management and opens new avenues for alternate epigenetic therapies for osteoporosis.

The Effect of Osteoprotectin (OPG)/Receptor Activator of Nuclear Factor-κB Ligand (RANKL)/Receptor Activator of Nuclear Factor-κB (RANK) Gene Methylation on Aortic Valve Calcified.

To evaluate the effect of the methylation of osteoprotectin (OPG)/receptor activator of nuclear factor-κB ligand (RANKL)/receptor activator of nuclear factor-κB (RANK) pathway on aortic valve calcification, the aortic valve tissue was collected from 38 aortic stenosis (AS) patients who underwent valve replacement. OPG and RANKL gene methylation, RT-PCR, and ELISA were performed. Hematoxylin-eosin staining (HE), alizarin red-S staining, and immunohistochemically staining of OPG, RANKL, and CD68 were simultaneously performed. The patients were divided into noncalcified group (n = 21) and calcified group (n = 17). The methylation rate of OPG gene in noncalcified group was higher than that in calcified group (P = 0.027). The methylation degree of RANKL gene was generally lower, but the noncalcified group was still higher than that in the calcified group (P = 0.025). RT-PCR analysis showed that the mRNA expression of OPG and RANKL was higher in calcified group than in noncalcified group (P = 0.007 and P = 0.036, respectively), and the mRNA expression was negatively correlated with the gene methylation rate. The protein expression of OPG and RANKL was detected by immunohistochemistry and ELISA, showing significantly increased in calcified group (P = 0.004 and P = 0.042, respectively). Soluble RANKL (sRANKL) in CD68-positive group was significantly different from that in negative group (0.1243 ± 0.0321 vs 0.0984 ± 0.0218 pg/mL, P = 0.007). There was no significant difference in OPG value between positive group (1.9411 ± 0.4554 ng/mL) and negative group (1.8422 ± 0.5218 ng/mL, P = 0.587). In conclusion, the degree of methylation of OPG and RANKL genes may play an important role in regulating valve calcification in AS patients.