[miRNA Is Involved in the Pathogenesis of Multiple Diseases by Targeting Osteoprotegerin]. / miRNAé€šè¿‡é¶å‘éª¨ä¿æŠ¤ç´ å‚ä¸Žå¤šç§ç–¾ç— çš„å‘ç— æœºåˆ¶.

As a member of the tumor necrosis factor receptor family, osteoprotegerin (OPG) is highly expressed in adults in the lung, heart, kidney, liver, spleen, thymus, prostate, ovary, small intestines, thyroid gland, lymph nodes, trachea, adrenal gland, the testis, and bone marrow. Together with the receptor activator of nuclear factor-κB (RANK) and the receptor activator of nuclear factor-κB ligand (RANKL), it forms the RANK/RANKL/OPG pathway, which plays an important role in the molecular mechanism of the development of various diseases. MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs performing regulatory functions in eukaryotes, with a size of about 20-25 nucleotides. miRNA genes are transcribed into primary transcripts by RNA polymerase, bind to RNA-induced silencing complexes, identify target mRNAs through complementary base pairing, with a single miRNA being capable of targeting hundreds of mRNAs, and influence the expression of many genes through pathways involved in functional interactions. In recent years, a large number of studies have been done to explore the mechanism of action of miRNA in diseases through miRNA isolation, miRNA quantification, miRNA spectrum analysis, miRNA target detection, in vitro and in vivo regulation of miRNA levels, and other technologies. It was found that miRNA can play a key role in the pathogenesis of osteoporosis, rheumatoid arthritis, and other diseases by targeting OPG. The purpose of this review is to explore the interaction between miRNA and OPG in various diseases, and to propose new ideas for studying the mechanism of action of OPG in diseases.

Bone Remodeling and Bone Structural Genes in Legg-Calvé-Perthes Disease: The OPG rs2073618 and IL-6 rs1800795 Are Associated with High Risk in Mexican Patients.

Legg-Calve-Perthes disease (LCPD) is an idiopathic avascular necrosis of the pediatric femoral head. Bone remodeling and bone structural genes have the potential to contribute to the progression of LCPD when there is disequilibrium between bone resorption and bone formation. A case-control study was performed to search for associations of several common polymorphisms in the genes Receptor Activator for Nuclear Factor κappa B (RANK), Receptor Activator for Nuclear Factor κappa B Ligand (RANKL), osteoprotegerin (OPG), interleukin (IL)-6, and type 1 collagen (COL1A1) with LCPD susceptibility in Mexican children. A total of 23 children with LCPD and 46 healthy controls were genotyped for seven polymorphisms (rs3018362, rs12585014, rs2073618, rs1800795, rs1800796, rs1800012, and rs2586498) in the RANK, RANKL, OPG, IL-6, and COL1A1 genes by real-time polymerase chain reaction with TaqMan probes. The variant allele (C) of IL-6 rs1800795 was associated with increased risk of LCPD (odds ratio [OR]: 3.8, 95% confidence interval [CI]: [1.08-13.54], p = 0.033), adjusting data by body mass index (BMI) and coagulation factor V (FV), the association with increased risk remained (OR: 4.9, 95% CI: [1.14-21.04], p = 0.025). The OPG polymorphism rs2073618, specifically GC-GG carriers, was associated with a more than fourfold increased risk of developing LCPD (OR: 4.34, 95% CI: [1.04-18.12], p = 0.033) when data were adjusted by BMI-FV. There was no significant association between RANK rs3018362, RANKL rs12585014, IL-6 rs1800796, COL1A1 rs1800012, and rs2586498 polymorphisms and LCPD in a sample of Mexican children. The rs1800975 and rs2037618 polymorphisms in the IL-6 and OPG genes, respectively, are informative markers of increased risk of LCPD in Mexican children.

Investigating the Effects and Mechanisms of Cyclomorusin on Osteoclasts in a High Glucose Environment.

Diabetes mellitus is an endocrine disease characterized by prolonged hyperglycemia. Prolonged high blood sugar levels interfere with the differentiation and maturation process of OBs and OCs, leading to the onset of osteoporosis. However, OCs differentiation and maturation is a complex regulatory process. In this study, we used a co-culture system of RAW264.7 and MC3T3-E1 cells under HG concentration to explore the effect of CYM on OCs in a HG environment. The effects of CYM on the formation and function of OCs were observed using TRAP-positive cell counts and bone resorption pits. Then, mRNA and protein expression levels of OCs-related genes were detected by real-time qPCR and western blotting. The results showed that CYM had an inhibitory effect on OCs differentiation and bone resorption, reduced mRNAs expression of OCs-associated genes, and downregulated RANKL/RANK/TRAF6 pathway that mediates OCs differentiation. CYM could be a promising natural compound against diabetic osteoporosis.

RANK/RANKL axis promotes migration, invasion, and metastasis of osteosarcoma via activating NF-κB pathway.

Osteosarcoma (OS) is one of the most prevalent primary bone tumors with a high degree of metastasis and poor prognosis. Epithelial-to-mesenchymal transition (EMT) is a cellular mechanism that contributes to the invasion and metastasis of cancer cells, and OS cells have been reported to exhibit EMT-like characteristics. Our previous studies have shown that the interaction between tumor necrosis factor superfamily member 11 (TNFRSF11A; also known as RANK) and its ligand TNFSF11 (also known as RANKL) promotes the EMT process in breast cancer cells. However, whether the interaction between RANK and RANKL enhances aggressive behavior by inducing EMT in OS cells has not yet been elucidated. In this study, we showed that the interaction between RANK and RANKL increased the migration, invasion, and metastasis of OS cells by promoting EMT. Importantly, we clarified that the RANK/RANKL axis induces EMT by activating the nuclear factor-kappa B (NF-κB) pathway. Furthermore, the NF-κB inhibitor dimethyl fumarate (DMF) suppressed migration, invasion, and EMT in OS cells. Our results suggest that the RANK/RANKL axis may serve as a potential tumor marker and promising therapeutic target for OS metastasis. Furthermore, DMF may have clinical applications in the treatment of lung metastasis in patients with OS.

Downregulation of osteoprotegerin in colorectal cancer cells promotes liver metastasis via activating tumor-associated macrophage.

Osteoprotegerin (OPG) is a secreted cytokine that functions as a decoy receptor for receptor activator of nuclear factor kappa-B (RANK) ligand (RANKL). Anti-RANKL treatment for bone metastasis has been widely accepted for solid tumors. However, the mechanism of OPG-RANKL-RANK signaling in systemic colorectal cancer (CRC) metastasis remains unclear. In this study, we investigated the relevance and function of OPG expression in CRC liver metastasis. First, we performed in silico analysis using The Cancer Genome Atlas public database and found that lower OPG expression in CRC was associated with poor overall survival. Immunohistochemistry analyses using resected specimen from patients with CRC in our institute confirmed the result. Patient-matched primary CRC and liver metastases showed a significant downregulation of OPG expression in metastatic lesions. In CRC cell lines, OPG expression did not suppress cell proliferation and migration. However, OPG expression inhibited macrophage migration by suppressing the RANKL-RANK pathway. Moreover, in vivo mouse liver metastasis models showed that OPG expression in CRC cells suppressed liver metastases. In addition, treatment with an anti-RANKL neutralizing antibody also suppressed liver metastases. These results showed that downregulation of OPG expression in CRC cells promotes liver metastasis by activating tumor-associated macrophage, which can become a candidate for targeted therapy with anti-RANKL neutralizing antibody for CRC liver metastasis.

In vivo and in vitro action mechanism of treatment of glucocorticoid-induced osteoporosis by regulation of osteoprotegerin/receptor activator of nuclear factor-κB pathways by denshensu.

The research aimed to discuss the action mechanism of the treatment of glucocorticoid-induced osteoporosis (GIOP) by denshensu. In the research, 60 rats were purchased and divided into a control group, model group, estradiol group, and denshensu treatment group. Except for the control group, GIOP models were established for all other groups, and then the structural changes of osseous tissues as well as osteoprotegerin (OPG), expression of receptor activator of nuclear factor-κB ligands (RANKL) were detected. Besides, the changes in osteoclasts were observed by bone marrow-derived mononuclear phagocytes in vitro. The results showed that the micro-structure of bone trabeculae, bone mineral density (BMD), and bone metabolic markers of rats in the denshensu treatment group were enhanced significantly, while trabecular separation and structural model index were reduced (P<0.05). OPG messenger ribonucleic acid (mRNA) and protein levels in the hypothalamus and femur tissues were increased, while RANKL content was remarkably decreased (P<0.05). In addition, in vitro experiments revealed that denshensu inhibited the differentiation of positive osteoclasts, and osteoclast-related genes were reduced (P<0.05). To conclude, denshensu might inhibit the expressions of OPG and RANKL and further play a role in treating GIOP.

Xanthoxyletin blocks the RANK/RANKL signaling pathway to suppress the growth of human pancreatic cancer cells.

Xanthoxyletin is a vital plant-derived bioactive coumarin. It has been shown to exhibit anticancer effects against different human cancers. Nonetheless, the anticancer effects of xanthoxyletin against human pancreatic cancer cells have not been evaluated. Against this backdrop, the present study was designed to evaluate the anticancer effects of xanthoxyletin in human pancreatic cancer cells and to decipher the underlying molecular mechanisms. The results revealed a significant (p < 0.05) upregulation of receptor activator of NF-kappaB (RANK), receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG) in human pancreatic tissues and cell lines at both transcriptional and translational levels. The administration of pancreatic cancer cells with xanthoxyletin diminished the viability of Capan-2 cells in a concentration-dependent manner and led to a significant decline in RANK, RANKL, and OPG expression. Silencing of RANK and xanthoxyletin treatment declined the viability of Capan-2 pancreatic cancer cells via induction of apoptosis. However, pancreatic cancer cells overexpressing RANK could rescue the growth inhibitory effects. Collectively, xanthoxyletin targets the RANK/RANKL signaling pathway in pancreatic cancer cells to induce cell apoptosis and may prove to be an important lead molecule.

RANK Expression as an Independent Predictor for Response to Neoadjuvant Chemotherapy in Luminal-Like Breast Cancer: A Translational Insight from the GeparX Trial.

PURPOSE: The GeparX study investigated whether denosumab as add-on treatment to nab-paclitaxel-based neoadjuvant chemotherapy (NACT) with two different schedules (125 mg/m² weekly vs. day 1, 8 every 22 days) may increase pathologic complete response (pCR) rate. The addition of denosumab to NACT did not improve pCR rates as recently published. In this study, we investigated whether receptor activator of nuclear factor-kappa B (RANK) expression, as part of the denosumab target pathway: (i) may retrospectively identify a subgroup of patients with additional clinical benefit of denosumab or (ii) may predict response to nab-paclitaxel NACT. EXPERIMENTAL DESIGN: RANK protein was IHC-stained on pre-therapeutic core biopsies from patients of the GeparX study (n = 667) with the antibody RANK/Envision System HRP (DAB) and was analyzed for the percentage of membranous RANK tumor cell staining (>5% RANKhigh vs. ≤5% RANKlow). RESULTS: We could not identify any patient subgroup with differential response under denosumab add-on treatment in patients with RANKhigh expression [139/667, 20.8%; OR, 0.86; 95% confidence interval (CI), 0.44-1.68; P = 0.667] or RANKlow expression (528/667 (79.2%) OR, 1.10; 95% CI, 0.78-1.56; P = 0.589; Pinteraction = 0.528). However, the pCR rate was higher in the RANKhigh subgroup compared with RANKlow (50% vs. 39%; OR, 1.52; 95% CI, 1.04-2.21; P = 0.037). RANK expression constituted an independent predictor of response to NACT frequently in patients with luminal-like subtype (HR+/HER2-; OR, 2.98; 95% CI, 1.30-6.79; P = 0.010). No predictive value of RANK expression among the different nab-paclitaxel regimens was observed. CONCLUSION: We report RANK expression to be an independent predictive biomarker for response to NACT in patients with luminal-like breast cancer.

Bone destruction in chronic otitis media is not mediated by the RANKL pathway or estrogen receptor-alpha.

BACKGROUND: Chronic otitis media with or without cholesteatoma progresses with various degrees of bone resorption and remodeling. Estrogen mediates osteoprotective effects through the receptor activator of NF-κB ligand (RANKL) pathway, which is mainly mediated by estrogen receptor-alpha (ER-α). OBJECTIVES: The present study investigated the expression patterns of receptor activator of NF-κB (RANK), osteoprotegerin (OPG), RANKL, and ER-α in pathological tissue from patients with chronic otitis media to determine the roles of those factors in osteolytic mechanisms underlying the pathogenesis of chronic otitis media. METHODS: Normal and pathological specimens from 18 patients with chronic otitis media were examined. RESULTS: There were no significant differences in RANK, OPG, RANKL, or ER-α mRNA expression between normal and pathological specimens of epithelial tissue. CONCLUSIONS: Our findings suggested that RANK, OPG, RANKL, and ER-α are not associated with the bone destruction in chronic otitis media; other cytokines may directly activate the osteoclasts in chronic otitis media.

Early identification of a 12-bp tandem duplication in TNFRSF11A encoding receptor activator of nuclear factor-kappa B (RANK): Clinical characterization and response to bisphosphonate therapy.

INTRODUCTION: Ultra-rare mendelian osteolytic disorders caused by different length in-frame activating duplications within exon 1 of TNFRSF11A encoding receptor activator of nuclear factor-kappa B (RANK) comprise familial expansile osteolysis (FEO), expansile skeletal hyperphosphatasia (ESH), early-onset familial Paget's disease of bone (PDB2), juvenile Paget's disease 2 (JPD2), and panostotic expansile bone disease (PEBD). FEO typically presents with childhood-onset deafness followed by resorption of permanent dentition, and then appendicular bone pain, fractures, and deformities from progressive focal expansile osteolytic lesions emerging from a background of generalized high bone turnover. An 18-bp duplication in TNFRSF11A has been reported in all kindreds with FEO, whereas a 12-bp duplication was found in the young man with PEBD complicated by a massive jaw tumor. We report the clinical course and successful treatment with bisphosphonates of a girl with the 12-bp duplication yet a skeletal phenotype seemingly milder than PEBD. CASE PRESENTATION AND DISCUSSION: This 10-year-old girl presented for dental and orthodontic treatment and was found to have progressive external tooth root resorption. Speech delay was identified at age 18 months, and audiological evaluation showed both conductive and sensorineural hearing loss subsequently treated with a cochlear implant at age 3 years. Biochemical studies indicated increased bone turnover with elevated urinary N-telopeptide levels and serum alkaline phosphatase in the upper normal range. Low lumbar spine bone mineral density (BMD) was revealed by dual-energy X-ray absorptiometry, but whole-body Technetium-99 m bone scintigraphy was normal. Genetic testing identified the identical de novo 12-bp duplication within exon 1 of TNFRSF11A harbored by the young man with PEBD and massive jaw tumor. Bisphosphonate treatment, initiated with one dose of intravenous zoledronic acid that caused prolonged hypocalcemia, then comprised weekly oral alendronate that decreased bone turnover markers and normalized her BMD. CONCLUSION: Constitutive activation of RANK signaling should be considered a possible cause in any young person with rapid bone turnover, particularly in the context of early-onset deafness and/or root resorption of permanent teeth. Early diagnosis and anti-resorptive treatment, given judiciously to avoid sudden and prolonged hypocalcemia, may prevent further skeletal disease.

Familial Paget's disease of bone with ocular manifestations and a novel TNFRSF11A duplication variant (72dup27).

INTRODUCTION: Paget's disease of bone (PDB) is a skeletal disorder characterized by disorganized bone remodeling due to abnormal osteoclasts. Tumor necrosis factor receptor superfamily member 11A (TNFRSF11A) gene encodes the receptor activator of nuclear factor kappa B (RANK), which has a critical role in osteoclast function. There are five types of rare PDB and related osteolytic disorders due to TNFRSF11A tandem duplication variants so far, including familial expansile osteolysis (84dup18), expansile skeletal hyperphosphatasia (84dup15), early-onset familial PDB (77dup27), juvenile PDB (87dup15), and panostotic expansile bone disease (90dup12). MATERIALS AND METHODS: We reviewed a Japanese family with PDB, and performed whole-genome sequencing to identify a causative variant. RESULTS: This family had bone symptoms, hyperphosphatasia, hearing loss, tooth loss, and ocular manifestations such as angioid streaks or early-onset glaucoma. We identified a novel duplication variant of TNFRSF11A (72dup27). Angioid streaks were recognized in Juvenile Paget's disease due to loss-of-function variants in the gene TNFRSF11B, and thought to be specific for this disease. However, the novel recognition of angioid streaks in our family raised the possibility of occurrence even in bone disorders due to TNFRSF11A duplication variants and the association of RANKL-RANK signal pathway as the pathogenesis. Glaucoma has conversely not been reported in any case of Paget's disease. It is not certain whether glaucoma is coincidental or specific for PDB with 72dup27. CONCLUSION: Our new findings might suggest a broad spectrum of phenotypes in bone disorders with TNFRSF11A duplication variants.

Histopathologic and transcriptomic phenotypes of a conditional RANKL transgenic mouse thymus.

Although conventional knockout and transgenic mouse models have significantly advanced our understanding of Receptor Activator of NF-κB Ligand (RANKL) signaling in intra-thymic crosstalk that establishes self-tolerance and later stages of lymphopoiesis, the unique advantages of conditional mouse transgenesis have yet to be explored. A main advantage of conditional transgenesis is the ability to express a transgene in a spatiotemporal restricted manner, enabling the induction (or de-induction) of transgene expression during predetermined stages of embryogenesis or during defined postnatal developmental or physiological states, such as puberty, adulthood, and pregnancy. Here, we describe the K5: RANKL bigenic mouse, in which transgene derived RANKL expression is induced by doxycycline and targeted to cytokeratin 5 positive medullary thymic epithelial cells (mTECs). Short-term doxycycline induction reveals that RANKL transgene expression is significantly induced in the thymic medulla and only in response to doxycycline. Prolonged doxycycline induction in the K5: RANKL bigenic results in a significantly enlarged thymus in which mTECs are hyperproliferative. Flow cytometry showed that there is a marked enrichment of CD4+ and CD8+ single positive thymocytes with a concomitant depletion of CD4+ CD8+ double positives. Furthermore, there is an increase in the number of FOXP3+ T regulatory (Treg) cells and Ulex Europaeus Agglutinin 1+ (UEA1+) mTECs. Transcriptomics revealed that a remarkable array of signals-cytokines, chemokines, growth factors, transcription factors, and morphogens-are governed by RANKL and drive in part the K5: RANKL thymic phenotype. Extended doxycycline administration to 6-weeks results in a K5: RANKL thymus that begins to display distinct histopathological features, such as medullary epithelial hyperplasia, extensive immune cell infiltration, and central tissue necrosis. As there are intense efforts to develop clinical approaches to restore thymic medullary function in the adult to treat immunopathological conditions in which immune cell function is compromised following cancer therapy or toxin exposure, an improved molecular understanding of RANKL's involvement in thymic medulla enlargement will be required. We believe the versatility of the conditional K5: RANKL mouse represents a tractable model system to assist in addressing this requirement as well as many other questions related to RANKL's role in thymic normal physiology and disease processes.

Analysis of RANK-c interaction partners identifies TRAF3 as a critical regulator of breast cancer aggressiveness.

Breast cancer is a highly heterogeneous disease both at the histological and molecular levels. We have previously shown that RANK-c is a regulator of NF-κB signaling and exerts a suppressive effect on aggressive properties of ER negative breast cancer cells, while there is an opposite effect on ER positive cell lines. In order to identify molecular determinants that govern the opposing function of RANK-c in breast cancer cells we employed the two cell lines with the highest degree of phenotypic divergence upon RANK-c-expression (SKBR3 and BT474) and identified proteins that interact with RANK-c by affinity-enrichment mass spectrometry (AE-MS) analysis. Annotating enriched proteins with NF-κB signaling pathway revealed TRAF3 as an interacting partner of RANK-c in SKBR3 cell protein lysates, but not in BT474 breast cancer cells in which RANK-c induces cell aggressiveness. To determine the role of TRAF3 in the phenotype of BT474-RANK-c cells, we reconstructed the TRAF3/RANK-c interaction both in parental BT474 and RANK-c expressing cells and tested for aggressive properties through colony formation, migration and invasion assays. TRAF3 forced expression was able to reverse BT474 phenotypic changes imposed by RANK-c, rendering cells less aggressive. Finally, TRAF3 gene expression data and TRAF3 immunohistochemical (IHC) analysis on breast cancer samples indicated that TRAF3 expression correlates with Overall Survival (OS), Recurrence Free Survival (RFS) and several clinicopathological parameters (histological grade, proliferation index) of breast cancer disease.

Osteoclast-poor osteopetrosis.

Osteopetrosis (OPT) is a rare inherited bone disease characterized by a bone resorption defect, due to osteoclast malfunction (in osteoclast-rich, oc-rich, OPT forms) or absence (in oc-poor OPT forms). This causes severe clinical abnormalities, including increased bone density, lack of bone marrow cavity, stunted growth, macrocephaly, progressive deafness, blindness, hepatosplenomegaly, and severe anemia. The oc-poor subtype of OPT is ultra-rare in humans. It is caused by mutations in either the tumor necrosis factor ligand superfamily member 11 (TNFSF11) gene, encoding RANKL (Receptor Activator of Nuclear factor-kappa B [NF-κB] Ligand) which is expressed on cells of mesenchymal origin and lymphocytes, or the TNFRSF member 11A (TNFRSF11A) gene, encoding the RANKL functional receptor RANK which is expressed on cells of myeloid lineage including osteoclasts. Clinical presentation is usually severe with onset in early infancy or in fetal life, although as more patients are reported, expressivity is variable. Phenotypic variability of RANK-deficient OPT sometimes includes hypogammaglobulinemia or radiological features of dysosteosclerosis. Disease progression is somewhat slower in RANKL-deficient OPT than in other 'malignant' subtypes of OPT. While both RANKL and RANK are essential for normal bone turnover, hematopoietic stem cell transplantation (HSCT) is the treatment of choice only for patients with the RANK-deficient form of oc-poor OPT. So far, there is no cure for RANKL-deficient OPT.

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.

RANKL Impairs the TLR4 Pathway by Increasing TRAF6 and RANK Interaction in Macrophages.

High serum levels of osteoprotegerin (OPG) are found in patients with obesity, type 2 diabetes, sepsis, or septic shock and are associated with a high mortality rate in stroke. The primary known function of OPG is to bind to the receptor activator of NF-κB ligand (RANKL), and by doing so, it inhibits the binding between RANKL and its receptor (RANK). TLR4 signaling in macrophages involves TRAF6 recruitment and contributes to low-grade chronic inflammation in adipose tissue. LPS is a classical activator of the TLR4 pathway and induces the expression of inflammatory cytokines in macrophages. We have previously observed that in the presence of RANKL, there is no LPS-induced activation of TLR4 in macrophages. In this study, we investigated the crosstalk between RANK and TLR4 pathways in macrophages stimulated with both RANKL and LPS to unveil the role of OPG in inflammatory processes. We found that RANKL inhibits TLR4 activation by binding to RANK, promoting the binding between TRAF6 and RANK, lowering TLR4 activation and the expression of proinflammatory mediators. Furthermore, high OPG levels aggravate inflammation by inhibiting RANKL. Our findings elect RANKL as a candidate for drug development as a way to mitigate the impact of obesity-induced inflammation in patients.

Hormone and receptor activator of NF-κB (RANK) pathway gene expression in plasma and mammographic breast density in postmenopausal women.

BACKGROUND: Hormones impact breast tissue proliferation. Studies investigating the associations of circulating hormone levels with mammographic breast density have reported conflicting results. Due to the limited number of studies, we investigated the associations of hormone gene expression as well as their downstream mediators within the plasma with mammographic breast density in postmenopausal women. METHODS: We recruited postmenopausal women at their annual screening mammogram at Washington University School of Medicine, St. Louis. We used the NanoString nCounter platform to quantify gene expression of hormones (prolactin, progesterone receptor (PGR), estrogen receptor 1 (ESR1), signal transducer and activator of transcription (STAT1 and STAT5), and receptor activator of nuclear factor-kB (RANK) pathway markers (RANK, RANKL, osteoprotegerin, TNFRSF18, and TNFRSF13B) in plasma. We used Volpara to measure volumetric percent density, dense volume, and non-dense volume. Linear regression models, adjusted for confounders, were used to evaluate associations between gene expression (linear fold change) and mammographic breast density. RESULTS: One unit increase in ESR1, RANK, and TNFRSF18 gene expression was associated with 8% (95% CI 0-15%, p value = 0.05), 10% (95% CI 0-20%, p value = 0.04) and % (95% CI 0-9%, p value = 0.04) higher volumetric percent density, respectively. There were no associations between gene expression of other markers and volumetric percent density. One unit increase in osteoprotegerin and PGR gene expression was associated with 12% (95% CI 4-19%, p value = 0.003) and 7% (95% CI 0-13%, p value = 0.04) lower non-dense volume, respectively. CONCLUSION: These findings provide new insight on the associations of plasma hormonal and RANK pathway gene expression with mammographic breast density in postmenopausal women and require confirmation in other studies.

Histological Grade and Tumor Stage Are Correlated with Expression of Receptor Activator of Nuclear Factor Kappa b (Rank) in Epithelial Ovarian Cancers.

The receptor activator of nuclear factor kappa B (RANK) is becoming recognized as a master regulator of tumorigenesis, yet its role in gynecological cancers remains mostly unexplored. We investigated whether there is a gradation of RANK protein and mRNA expression in epithelial ovarian cancer (EOC) according to malignancy and tumor staging. Immunohistochemical expression of RANK was examined in a cohort of 135 (benign n = 29, borderline n= 23 and malignant n = 83) EOCs. Wild type and truncated RANK mRNA isoform quantification was performed in a cohort of 168 (benign n = 26, borderline n = 13 and malignant n = 129) EOCs. RANK protein and mRNA values were increased in malignant vs. benign or borderline conditions across serous, mucinous and endometrioid cancer subtypes. Additionally, a trend of increased RANK values with staging was observed for the mucinous and serous histotype. Thus, increased expression of RANK appears associated with the evolution of disease to the onset of malignancy in EOC. Moreover, in some EOC histotypes, RANK expression is additionally associated with clinicopathological markers of tumor aggressiveness, suggesting a role in further progression of tumor activity.

Bone health in pediatric transfusion-dependent beta-thalassemia: Circulating osteoprotegerin and RANKL system.

INTRODUCTION: While the mechanism of bone disease in thalassemia is multifactorial and still under investigation, the receptor activator of nuclear factor kappa B (RANK), receptor activator of nuclear factor kappa B ligand (RANKL), and osteoprotegerin (OPG) have pivotal roles in regulating bone metabolism. This study aimed to measure RANKL and OPG serum levels, and to detect the incidence of RANKL rs9533156, OPG rs2073618, and OPG rs2073617 genotypes in pediatric ß-thalassemia patients and to assess their relation to bone mineral density. METHODS: Sixty patients with transfusion-dependent ß-thalassemia (TBT) patients ages 5 to 14 years were included, and 60 healthy, age- and sex-matched volunteers contributed as a control group. The patients were scanned for bone mineral density. RESULTS: The mean of spine dual-energy X-ray absorptiometry (DXA) Z-score in patients was -1.66 ± 1.02 standard deviation (SD). Twenty-four of them had low spine DXA Z-scores. The patients showed significantly lower OPG levels and OPG/RANKLs ratios than the control group (3.28 ± 9.11 ng/ml and 11.38 ± 14.93 ng/ml, and 0.01 ± 0.03 and 0.07 ± 0.09, respectively). The RANKL SNP rs9533156 TC heterozygous genotype was detected more with statistical significance in patients than controls. The incidence of OPG rs2073618 and OPG rs2073617 genotypes were 2.3 times and 1.9 times more frequent in patients than controls, respectively. CONCLUSION: The RANK/RANKL/OPG system may have an important role in regulating bone metabolism in TBT patients, although further studies are needed to clarify its role.

The RANK/RANKL/OPG system and tumor bone metastasis: Potential mechanisms and therapeutic strategies.

With the markedly increased diagnosis and incidence of cancer in the population, tumor bone metastasis has become a frequent event in tumor patients. Healthy bone integrity is maintained by a delicate balance between bone formation and bone resorption. Unfortunately, many tumors, such as prostate and breast, often metastasize to the bone, and the alterations to the bone homeostasis can particularly favor tumor homing and consequent osteolytic or osteoblastic lesions. Receptor activator of NF-κB ligand (RANKL), its receptor RANK, and osteoprotegerin (OPG) are involved in the regulation of the activation, differentiation, and survival of osteoclasts, which play critical roles in bone metastasis formation. High rates of osteoclastic bone resorption significantly increase fracture risk, cause severe bone pain, and contribute to homing tumor cells in bone and bone marrow. Consequently, suppression of the RANK/RANKL/OPG system and osteoclastic activity can not only ameliorate bone resorption but may also prevent tumor bone metastases. This review summarizes the important role of the RANK/RANKL/OPG system and osteoclasts in bone homeostasis and its effect on tumor bone metastasis and discusses therapeutic strategies based on RANKL inhibition.