RANK Signaling Pathways Research Articles

Caffeine induces alveolar bone loss in rats submitted to orthodontic movement via activation of receptor activator of nuclear factor ҡB, receptor activator of nuclear factor ҡB ligand, and osteoprotegerin pathway

Caffeine is a widely consumed substance with several effects on bone metabolism. This study aimed to investigate the effect of caffeine on the bone tissue of rats submitted to orthodontic movement. Twenty-five male Wistar rats underwent orthodontic movement (21 days) of the first permanent maxillary molars on the left side. The experimental group (caffeine; n= 13) and control group (n= 12) received caffeine and water, respectively, by gavage. Microcomputed tomography was performed to analyze orthodontic movement. Histologic analysis of the inflammatory infiltrate and osteoclast count by tartrate-resistant acid phosphatase were conducted. Maxilla tissue was evaluated for receptor activator of nuclear factor ҡB (RANK), RANK ligand (RANKL), and osteoprotegerin by immunohistochemistry. Caffeine exhibited a lower bone volume/tissue volume ratio (78.09% ± 5.83%) than the control (86.84% ± 4.89%; P<0.05). Inflammatory infiltrate was increased in the caffeine group compared with the control group (P<0.05). A higher number of tartrate-resistant acid phosphatase-positive cells was observed in the caffeine (9.67 ± 1.73) than in the control group (2.66 ± 0.76; P<0.01). Immunoexpression of RANK and RANKL in the caffeine group was greater than the control (P<0.05). The use of caffeine thermogenic induces alveolar bone loss in rats submitted to orthodontic movement via activation of RANK, RANKL, and osteoprotegerin signaling pathways.

Study on the mechanism of magnesium calcium alloys/mineralized collagen composites mediating macrophage polarization to promote bone repair

Magnesium-based composites are a focal point in biomaterials research. However, the rapid degradation rate of magnesium alloys does not align with the healing time of bone tissue. Additionally, the host reaction caused by magnesium implantation hampers its full osteogenic potential. To maintain an appropriate microenvironment, it is important to enhance both corrosion resistance and osteogenic activity of the magnesium matrix. In this study, a composite scaffold composed of mineralized collagen and magnesium alloy was utilized to investigate the regulatory effect of mineralized collagen on mouse macrophages and evaluate its impact on mouse bone marrow mesenchymal stem cells in terms of osteogenesis, immune response, and macrophage-induced osteogenic differentiation. This experiment examined the biocompatibility of mouse bone marrow mesenchymal stem cells and macrophage-induced osteogenic differentiation in vitro, and examined the expression levels of relevant pathways proteins. Magnesium calcium alloys/mineralized collagen exhibited extensive spreading, facilitated by broad and abundant pseudopodia that firmly adhered them to the material surface and promoted growth and pseudopodia formation. The findings revealed that magnesium calcium alloy/mineralized collagen scaffold materials induced osteogenic differentiation mainly through M2 polarization of macrophages. This effect was mainly mediated by promoting the integrin α2β1-FAK-ERK1/2 signaling pathways and inhibiting the RANK signaling pathways.

Luminal Rank loss decreases cell fitness leading to basal cell bipotency in parous mammary glands

Rank signaling pathway regulates mammary gland homeostasis and epithelial cell differentiation. Although Rank receptor is expressed by basal cells and luminal progenitors, its role in each individual cell lineage remains unclear. By combining temporal/lineage specific Rank genetic deletion with lineage tracing techniques, we found that loss of luminal Rank reduces the luminal progenitor pool and leads to aberrant alveolar-like differentiation with high protein translation capacity in virgin mammary glands. These Rank-deleted luminal cells are unable to expand during the first pregnancy, leading to lactation failure and impairment of protein synthesis potential in the parous stage. The unfit parous Rank-deleted luminal cells in the alveoli are progressively replaced by Rank-proficient cells early during the second pregnancy, thereby restoring lactation. Transcriptomic analysis and functional assays point to the awakening of basal bipotency after pregnancy by the induction of Rank/NF-κB signaling in basal parous cell to restore lactation and tissue homeostasis.

Panicum miliaceum L. and Bone Healing Properties in Rat Model of Femur Fracture by Activating Phosphate Stimulating Macrophages via BMP2 and RANK Signaling Pathway

Objectives The aim of this study was to investigate the effect of Panicum miliaceum involved in the repair of femur bone fracture in rats. Materials and Methods In this study, 40 Wistar rats in five groups were studied as healthy, rats with lesion induced by surgery in femur (FX), FX rats treated with P. miliaceum extract (FX +100 and 200 P. miliaceum) and FX rats treated with Osteocare syrup (FX + OC). After 40 days, the serum levels of blood calcium (Ca), phosphorus (P), and alkaline phosphatase (ALP) along with activity of glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT) enzymes, and nitric oxide (NO) level were measured. The serum level of cytokines (IL-6, C-reactive protein [CRP], IL-10, IL-1β, and tumor necrosis factor-α [TNF-α]) was also measured by ELISA. The expression of OPG, RANK, RANKL, and BMP-2 genes and proteins were evaluated by real-time PCR and Western blotting techniques, respectively. Results The results showed that P. miliaceum was able to decrease the activity of ALP enzyme in addition to increasing serum Ca and P levels and GPx, SOD, and CAT activity. P. miliaceum reduces serum IL-6, IL-1β, and TNF-α, and NO levels. Also, the extract of this plant could increase the expression of OPG/RANK/BMP-2 pathway genes and proteins. Conclusion P. miliaceum can be used as a prodrug in traditional medicine to repair bone fractures and osteoporosis.

Tereticornate A suppresses RANKL-induced osteoclastogenesis via the downregulation of c-Src and TRAF6 and the inhibition of RANK signaling pathways

Excessive osteoclast differentiation and activation are closely associated with the development and progression of osteoporosis. Natural plant-derived compounds that can inhibit osteoclastogenesis are an efficient strategy for the prevention and treatment of osteoporosis. Tereticornate A (TA) is a natural terpene ester compound extracted from the leaves and branches of Eucalyptus gracilis, with antiviral, antibacterial, and anti-inflammatory activities. However, the effect of TA on osteoclastogenesis and the underlying molecular mechanism remain unclear. Based on the key role of the NF-κB pathway in the regulation of osteoclastogenesis and the observation that TA exhibits an anti-inflammatory effect by inhibiting NF-κB activity, we speculated that TA could exert anti-osteoclastogenesis activity. Herein, TA could inhibit the RANKL-induced osteoclast differentiation and formation of F-actin rings in RAW 264.7 cells. Mechanistically, TA downregulated the expression of c-Src and TRAF6, and also suppressed the RANKL-stimulated canonical RANK signaling pathways, including AKT, MAPK (p38, JNK, and ERK), and NF-κB; ultimately, downregulating the expression of NFATc1 and c-Fos, the key transcriptional factors required for the expression of genes (e.g., TRAP, cathepsin K, β-Integrin, MMP-9, ATP6V0D2, and DC-STAMP) that govern osteoclastogenesis. Our findings demonstrated that TA could effectively inhibit RANKL-induced osteoclastogenesis via the downregulation of c-Src and TRAF6 and the inhibition of RANK signaling pathways. Thus, TA could serve as a novel osteoclastogenesis inhibitor and might have beneficial effects on bone health.

The Roadmap of RANKL/RANK Pathway in Cancer.

The receptor activator of the nuclear factor-κB ligand (RANKL)/RANK signaling pathway was identified in the late 1990s and is the key mediator of bone remodeling. Targeting RANKL with the antibody denosumab is part of the standard of care for bone loss diseases, including bone metastases (BM). Over the last decade, evidence has implicated RANKL/RANK pathway in hormone and HER2-driven breast carcinogenesis and in the acquisition of molecular and phenotypic traits associated with breast cancer (BCa) aggressiveness and poor prognosis. This marked a new era in the research of the therapeutic use of RANKL inhibition in BCa. RANKL/RANK pathway is also an important immune mediator, with anti-RANKL therapy recently linked to improved response to immunotherapy in melanoma, non-small cell lung cancer (NSCLC), and renal cell carcinoma (RCC). This review summarizes and discusses the pre-clinical and clinical evidence of the relevance of the RANKL/RANK pathway in cancer biology and therapeutics, focusing on bone metastatic disease, BCa onset and progression, and immune modulation.

The Role of BMP Signaling in Osteoclast Regulation.

The osteogenic effects of Bone Morphogenetic Proteins (BMPs) were delineated in 1965 when Urist et al. showed that BMPs could induce ectopic bone formation. In subsequent decades, the effects of BMPs on bone formation and maintenance were established. BMPs induce proliferation in osteoprogenitor cells and increase mineralization activity in osteoblasts. The role of BMPs in bone homeostasis and repair led to the approval of BMP2 by the Federal Drug Administration (FDA) for anterior lumbar interbody fusion (ALIF) to increase the bone formation in the treated area. However, the use of BMP2 for treatment of degenerative bone diseases such as osteoporosis is still uncertain as patients treated with BMP2 results in the stimulation of not only osteoblast mineralization, but also osteoclast absorption, leading to early bone graft subsidence. The increase in absorption activity is the result of direct stimulation of osteoclasts by BMP2 working synergistically with the RANK signaling pathway. The dual effect of BMPs on bone resorption and mineralization highlights the essential role of BMP-signaling in bone homeostasis, making it a putative therapeutic target for diseases like osteoporosis. Before the BMP pathway can be utilized in the treatment of osteoporosis a better understanding of how BMP-signaling regulates osteoclasts must be established.

RANK signaling increases after anti-HER2 therapy contributing to the emergence of resistance in HER2-positive breast cancer

BackgroundAround 15–20% of primary breast cancers are characterized by HER2 protein overexpression and/or HER2 gene amplification. Despite the successful development of anti-HER2 drugs, intrinsic and acquired resistance represents a major hurdle. This study was performed to analyze the RANK pathway contribution in HER2-positive breast cancer and anti-HER2 therapy resistance.MethodsRANK and RANKL protein expression was assessed in samples from HER2-positive breast cancer patients resistant to anti-HER2 therapy and treatment-naive patients. RANK and RANKL gene expression was analyzed in paired samples from patients treated with neoadjuvant dual HER2-blockade (lapatinib and trastuzumab) from the SOLTI-1114 PAMELA trial. Additionally, HER2-positive breast cancer cell lines were used to modulate RANK expression and analyze in vitro the contribution of RANK signaling to anti-HER2 resistance and downstream signaling.ResultsRANK and RANKL proteins are more frequently detected in HER2-positive tumors that have acquired resistance to anti-HER2 therapies than in treatment-naive ones. RANK (but not RANKL) gene expression increased after dual anti-HER2 neoadjuvant therapy in the cohort from the SOLTI-1114 PAMELA trial. Results in HER2-positive breast cancer cell lines recapitulate the clinical observations, with increased RANK expression observed after short-term treatment with the HER2 inhibitor lapatinib or dual anti-HER2 therapy and in lapatinib-resistant cells. After RANKL stimulation, lapatinib-resistant cells show increased NF-κB activation compared to their sensitive counterparts, confirming the enhanced functionality of the RANK pathway in anti-HER2-resistant breast cancer. Overactivation of the RANK signaling pathway enhances ERK and NF-κB signaling and increases lapatinib resistance in different HER2-positive breast cancer cell lines, whereas RANK loss sensitizes lapatinib-resistant cells to the drug. Our results indicate that ErbB signaling is required for RANK/RANKL-driven activation of ERK in several HER2-positive cell lines. In contrast, lapatinib is not able to counteract the NF-κB activation elicited after RANKL treatment in RANK-overexpressing cells. Finally, we show that RANK binds to HER2 in breast cancer cells and that enhanced RANK pathway activation alters HER2 phosphorylation status.ConclusionsOur data support a physical and functional link between RANK and HER2 signaling in breast cancer and demonstrate that increased RANK signaling may contribute to the development of lapatinib resistance through NF-κB activation. Whether HER2-positive breast cancer patients with tumoral RANK expression might benefit from dual HER2 and RANK inhibition therapy remains to be elucidated.

Inhibitory Effect of a Rosmarinic Acid-Enriched Fraction Prepared from Nga-Mon (Perilla frutescens) Seed Meal on Osteoclastogenesis through the RANK Signaling Pathway.

The aim of this study is to determine antioxidant and anti-inflammatory activities relating to the antiosteoporosis effects of various perilla seed meal (PSM) fractions. The remaining waste of perilla seed obtained from cold oil compression was extracted with 70% ethanol and sequentially fractionated according to solvent polarity with hexane, dichloromethane, ethyl acetate, and water. The results indicated that the seed-meal ethyl acetate fraction (SMEF) exhibited the highest antioxidant and anti-inflammatory activities, and rosmarinic acid (RA) content. The signaling pathways induced by the receptor activator of the nuclear factor kappa B (NF-κB) ligand (RANKL) that trigger reactive oxygen species (ROS) and several transcription factors, leading to the induction of osteoclastogenesis, were also investigated. The SMEF clearly showed attenuated RANKL-induced tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts and TRAP activity. A Western blot analysis showed that the SMEF significantly downregulated RANKL-induced NF-κB, AP-1 activation, and the nuclear factor of activated T-cell 1 (NFATc1) expression. SMEF also suppressed RANKL-induced osteoclast-specific marker gene-like MMP-9 using zymography. Furthermore, the SMEF showed inhibition of RANKL-induced ROS production in RAW 264.7 cells. The results suggest that the SMEF, which contained high quantities of RA, could be developed as a natural active pharmaceutical ingredient for osteoclastogenic protection and health promotion.

Progress on signal pathways related to bone metabolism in animals

Bone is a hard organ that makes up vertebrate endoskeleton, which plays a role in movement, support and protection for the body. The normal growth and development of bone is in the dynamic balance of bone metabolism, which is composed of bone formation and bone absorption. This balance is very important for maintaining bone mass and mineral homeostasis. In the process of bone growth and metabolism, there are many signaling pathways regulating bone formation and absorption, such as BMP (bone morphogenetic protein)/SMADs, TGF-β (transforming growth factor β), Wnt/β-catenin, OPG (osteoprotegerin)/RANKL (receptor activator of NF-κB ligand)/RANK (receptor activator of NF-κB), FGF (fibroblast growth factor) and Notch signaling pathway. These signaling pathways have complex regulatory mechanisms and are involved in the regulation of bone metabolism. In this review, we summarize the mechanism and research progress of signal pathways that play key regulatory roles in the process of animal bone metabolism, thereby laying a foundation for research in animal bone metabolism.

Ellagic acid blocks RANKL–RANK interaction and suppresses RANKL-induced osteoclastogenesis by inhibiting RANK signaling pathways

Ellagic acid (EA) is a naturally occurring polyphenolic compound that has been shown to exhibit diverse beneficial pharmacological activities including anti-osteoclastogenesis effect. However, the molecular mechanism by which EA inhibits osteoclastogenesis remains to be elucidated. The protein-protein interaction between receptor activator of nuclear factor (NF)-κB ligand (RANKL) and its receptor RANK contributes to osteoclast differentiation and activation in bone remodeling, and is regarded as an important therapeutic target for the treatment of osteoporosis. The current study is focused on investigating whether EA can directly bind to RANKL and/or RANK and block the interaction between RANKL and RANK, thereby inhibiting downstream signaling pathways. Interestingly, we found that EA had strong affinities to RANK and RANKL, with the estimated equilibrium dissociation constants (KD) of 2.485 × 10−11 and 1.688 × 10−9 M, respectively, and could disrupt the interaction between RANKL and RANK, thereby inhibiting RANKL-induced canonical RANK signaling pathways (p65, JNK, ERK, and p38) and expression of downstream master transcriptional factors (NFATc1 and c-Fos) and osteoclast-specific genes and proteins (TRAP, c-Src, and cathepsin K), which could ultimately suppress RANKL-induced osteoclast differentiation and F-actin ring formation. Taken together, our results revealed that EA could block RANKL–RANK interaction and suppress RANKL-induced osteoclastogenesis by inhibiting RANK signaling pathways in RAW 264.7 murine macrophages.

MiR-143 Inhibits Osteoclastogenesis by Targeting RANK and NF-κB and MAPK Signaling Pathways.

To explore the effect of miRNA-143 on osteoclast formation and provide new ideas for the treatment of osteoporosis. Mice macrophage lines RAW264.7 cells after transfection were divided into four groups: control group, RANKL group, RANKL combined with miR-143 mimics group and RANKL combined with miR-NC group. TARCP staining was used to observe the effect of miR-143 on osteoclast formation. The expression of RANK, TRAF6 and NFATc-1 in the upstream of RANKL pathway was detected by real-time quantitative PCR (RT qPCR) and Western blotting (WB). The binding of miR-143 to TNFRSF11A was detected by double Luciferase Reporter Analysis. The effect of miR-143 on the expression of NF-κB (p65, I-κB-α) signal pathway in osteoclasts was detected. The effects of I-BET151 on the expression of osteoclast-specific genes TRACP, MMP 9, CtsK and c-Src were detected. The positive level of osteoclasts in RANKL group and RANKL combined with miR-NC group was significantly higher than that of RANKL combined with miR-143 mimics group and control group (P < 0.05). The expression levels of RANK, TRAF6, NFATc-1, TRACP, MMP-9, CtsK and c-Src in RANKL group and RANKL combined with miR-NC group were significantly higher than those of RANKL combined with miR-143 mimics group and control group (P < 0.05). The expression levels of I-κB-α were significantly lower than that of RANKL combined with miR-143 mimics group and control group (P<0.05). MiR-143 can inhibit the expression of RANK, TRAF6 and downstream NFATc-1 in the RANKL pathway, thereby inhibiting the RANK/RANKL pathway. MiR-143 can inhibit the signal pathway of NF-κB (p65, I-κB-α). MiR-143 inhibits the expression of osteoclast-specific genes TRACP, MMP 9, CtsK and c-Src. That is to say, miR-143 inhibits osteoclast formation by targeting RANK, NF- κB and MAPK signaling pathways.

Water Extract of Lysimachia christinae Inhibits Trabecular Bone Loss and Fat Accumulation in Ovariectomized Mice.

In Asia, extracts of Lysimachia christinae have been used for liver or urinogenital system-related diseases in traditional medicine. In this study, we investigated the effects of the water extract of L. christinae (WELC) on receptor activator of nuclear factor-kappa Β ligand (RANKL)-induced osteoclastic differentiation of bone marrow macrophages, and on osteoporosis and obesity in ovariectomy mice. RANK signaling pathways related to osteoclast differentiation were examined by real-time polymerase chain reaction (PCR) and western blot analysis. Additionally, we performed micro-computed tomography to assess trabecular bone loss, histological analysis for fat accumulation in adipose, liver, and bone tissues, and phytochemical profiling for WELC characterization. WELC significantly inhibited osteoclast differentiation by downregulating RANKL-induced mitogen-activated protein kinase (MAPK)/c-Fos/nuclear factor of activated T-cells (NFAT) signaling in osteoclast precursors and ovariectomy-induced trabecular loss by suppressing osteolcastic bone resorption. WELC markedly decreased ovariectomy-induced body weight gain and fat accumulation in adipose, liver, and bone tissues. Furthermore, ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS) identified 16 phytochemicals in WELC when compared with the mass fragmentation of standard chemicals. Collectively, these results suggest that WELC might possess beneficial effects on postmenopausal osteoporosis by inhibiting osteoclast differentiation and obesity by suppressing fat accumulation.

Acanthopanax senticosus aqueous extract ameliorates ovariectomy-induced bone loss in middle-aged mice by inhibiting the receptor activator of nuclear factor-κB ligand-induced osteoclastogenesis.

Acanthopanax senticosus (Ciwujia) has broad-spectrum pharmacological activities, including osteoprotective effects. However, the mechanisms underlying these effects remain unclear. We investigated whether Acanthopanax senticosus aqueous extract (ASAE) ameliorates ovariectomy-induced bone loss in middle-aged mice through inhibition of osteoclastogenesis. In vitro, ASAE significantly suppressed the receptor activator of nuclear factor-κB ligand (RANKL)-stimulated osteoclast differentiation and formation of F-actin rings by downregulating the expression of the nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), c-Fos, and osteoclastogenesis-related marker genes and proteins, including c-Src, tartrate-resistant acid phosphatase (TRAP), cathepsin K, β3-integrin, and matrix metallopeptidase-9 (MMP-9). This was achieved by inhibiting RANK signaling pathways, including p65, c-Jun N-terminal kinase, extracellular signal-regulated kinase, and p38 in osteoclast precursors. In vivo, ASAE markedly ameliorated bone loss in ovariectomized (OVX) middle-aged mice. ASAE significantly inhibited the serum levels of tartrate-resistant acid phosphatase 5b (TRACP-5b) and RANKL, whereas it increased those of osteocalcin, procollagen 1 N-terminal peptide (P1NP), and osteoprotegerin in OVX mice. ASAE significantly inhibited the OVX-induced expression of osteoclast-specific proteins and genes in the femur. In conclusion, ASAE prevents ovariectomy-induced bone loss in middle-aged mice by inhibiting RANKL-induced osteoclastogenesis through suppression of RANK signaling pathways and could be potentially used in mediated treatment of osteoclast-related diseases (e.g., osteoporosis).

Oxidation derivative of (-)-epigallocatechin-3-gallate (EGCG) inhibits RANKL-induced osteoclastogenesis by suppressing RANK signaling pathways in RAW 264.7 cells

Tea consumption has positive effects on the skeletal system and prevents postmenopausal osteoporosis, mainly by inhibiting osteoclastogenesis. In green tea, (-)-epigallocatechin-3-gallate (EGCG) is the most abundant and active compound and has been shown to inhibit RANKL-induced osteoclast formation. Taking into account the highly oxidizable and unstable nature of EGCG, we hypothesized that EGCG oxidation product exhibits greater anti-osteoclastogenesis potential than EGCG. In this study, we successfully isolated and identified an EGCG oxidation derivative, (-)-gallocatechin gallate (compound 2), using a chemical oxidation strategy. We then compared the ability of compound 2 and EGCG to inhibit RANKL-induced osteoclastogenesis in RAW 264.7 cells. The results of TRAP staining and F-actin ring immunofluorescent staining showed that compound 2 exhibits stronger inhibition of RANKL-induced osteoclast differentiation and F-actin ring formation, respectively, than EGCG. Additionally, quantitative real-time PCR (qRT-PCR) and western blotting analyses showed that compound 2 significantly and more strongly inhibited the expression of osteoclastogenesis-related marker genes and proteins, including c-Src, TRAP, cathepsin K, β3-Integrin, and MMP-9, compared with EGCG. Furthermore, compound 2 significantly suppressed RANKL-induced expression of NFATc1 and c-Fos, the master transcriptional regulators of osteoclastogenesis, more strongly than EGCG. Mechanistically, molecular interaction assays showed that compound 2 binds to RANK with high affinity (KD = 189 nM) and blocks RANKL–RANK interactions, thereby suppressing RANKL-induced early RANK signaling pathways including p65, JNK, ERK, and p38 in osteoclast precursors. Taken together, this study demonstrates for the first time that an oxidation derivative of EGCG (compound 2) inhibits RANKL-induced osteoclastogenesis by suppressing RANK signaling pathways in RAW 264.7 cells.

Oleanolic acid inhibits RANKL-induced osteoclastogenesis via ER alpha/miR-503/RANK signaling pathway in RAW264.7 cells

Oleanolic acid (OA) has recently become a research hotspot in the treatment of many human diseases, especially osteoporosis and arthritis. However, the mechanisms are not elucidated completely. We aimed to elucidate the target and the mechanism via which OA inhibited osteoclast differentiation. We used TRAP staining and toluidine blue dye to test OA effect on osteoclastogenesis and bone resorption respectively. We detected the expression level of osteoclast differentiation related genes, estrogen receptor alpha (ERα) and miR-503. We blocked ERα with its specific blocker, methylpiperidino pyrazole (MPP). We antagonized the function of miR-503 with antagomir-503-5p. RT-PCR and ELISA kits were used to investigate the effects of OA on miR-503 formation and maturation-relevant enzymes Dicer and Drosha at gene and protein levels. The data suggested that OA inhibited osteoclastogenesis and bone resorption. OA upregulated ERα and miR-503 expression levels, inhibited RANK expression. MPP significantly attenuated the OA effect including inhibiting osteoclastogenesis, inhibiting bone resorption and up-regulating miR-503 expression. It showed that ERα was the target of OA and OA up-regulated miR-503 expression through ERα. Antagomir-503-5p inhibited the function of miR-503 and attenuated the inhibition of OA on osteoclastogenesis, suggesting that OA inhibited osteoclast by up-regulating miR-503 expression. In addition, OA up-regulated miR-503 by up-regulating Dicer expression. In conclusion, OA inhibits RANKL-induced osteoclastogenesis via ERα/miR-503/RANK signaling pathway in RAW264.7 cells.

Inhibitory effects of methyl-3,5-di-O-caffeoyl-epi-quinate on RANKL-induced osteoclast differentiation

In this study, we have shown that methyl-3,5-di-O-caffeoyl-epi-quinate, a naturally occurring compound isolated from Ainsliaea acerifolia, inhibits receptor activator of nuclear factor-κB ligand (RANKL)-induced formation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts and the expression of osteoclast marker genes. Methyl-3,5-di-O-caffeoyl-epi-quinate also inhibited RANKL-induced activation of p38, Akt and extracellular signal-regulated kinase (ERK) as well as the expression of nuclear factor of activated T-cell (NFATc1), the key regulator of osteoclast differentiation. Negative regulators for osteoclast differentiation was upregulated by methyl-3,5-di-O-caffeoyl-epi-quinate. Collectively, our results suggested that methyl-3,5-di-O-caffeoyl-epi-quinate suppresses osteoclast differentiation via downregulation of RANK signaling pathways and NFATc1.

Afatinib ameliorates osteoclast differentiation and function through downregulation of RANK signaling pathways.

Non-small-cell lung cancer (NSCLC) is the third most common cancer that spreads to the bone, resulting in osteolytic lesions caused by hyperactivation of osteoclasts. Activating mutations in epidermal growth factor receptor-tyrosine kinase (EGF-TK) are frequently associated with NSCLC, and afatinib is a first-line therapeutic drug, irreversibly targeting EGF-TK. However, the effects of afatinib on osteoclast differentiation and activation as well as the underlying mechanism remain unclear. In this study, afatinib significantly suppressed receptor activator of nuclear factor κB (RANK) ligand (RANKL)-induced osteoclast formation in bone marrow macrophages (BMMs). Consistently, afatinib inhibited the expression of osteoclast marker genes, whereas, it upregulated the expression of negative modulator genes. The bone resorbing activity of osteoclasts was also abrogated by afatinib. In addition, afatinib significantly inhibited RANKL-mediated Akt/protein kinase B and c-Jun N-terminal kinase phosphorylation. These results suggest that afatinib substantially suppresses osteoclastogenesis by downregulating RANK signaling pathways, and thus may reduce osteolysis after bone metastasis.

RANK as a therapeutic target in cancer.

The RANK signaling pathway has emerged as a new target in breast cancer as receptor activator of nuclear factor κB ligand (RANKL) and its receptor RANK mediate the pro-tumorigenic role of progesterone in the mammary gland. Thousands of cancer patients worldwide are already taking RANKL inhibitors for the management of bone metastasis, given the relevance of this pathway in osteoclastogenesis and bone resorption. RANK signaling also has multiple divergent effects in immunity and inflammation, both in the generation of active immune responses and in the induction of tolerance: it is required for lymph node organogenesis, thymic medullary epithelial development and self-tolerance, and regulates activation of several immune cells and inflammatory processes. The RANK pathway interferes with mammary epithelial differentiation and mediates the major proliferative response of mammary epithelium to progesterone and progesterone-driven expansion of mammary stem cells; it also controls hair follicle and epidermal stem cell homeostasis, pointing to RANK as a key regulator of epithelial stemness. Here we revisit the main functions of RANK signaling in bone remodeling, immune cells and epithelial differentiation. We also discuss the mechanistic evidence that supports its pleiotropic effects on cancer: from bone metastasis to immune and cancer-cell-dependent effects.

Regulation of Osteoblast Migration Involving Receptor Activator of Nuclear Factor-kappa B (RANK) Signaling.

Bone remodeling requires osteoclast activation, resorption, and reversal, prior to osteoblast migration into the bone pit. The Receptor Activator of NF-κB (RANK) signaling pathway plays an important role in bone remodeling. Two components of the RANK signaling pathway, RANK Ligand (RANKL) and the decoy receptor Osteoprotegerin (OPG), are expressed predominantly on the surface of osteoblasts, while RANK is principally expressed on the surface of osteoclasts. However, RANK has also been reported to be expressed on the surface of osteoblasts and osteosarcoma tumor cells. Treatment with soluble RANKL (sRANKL) of both normal osteoblasts and osteosarcoma tumor cells activated phosphorylation of ERK, p38(MAPK) , Akt, and p65(NF-κB). However, modified Boyden chamber assays and wound repair assays showed differential response to sRANKL-induced chemotactic migration in normal osteoblasts and osteosarcoma tumor cells. In contrast to previously published results, both normal osteoblasts and osteosarcoma tumor cells responded to sRANKL-induced chemotactic migration but the normal osteoblasts did so only in the presence of an ERK pathway inhibitor. For both normal and tumor cells, the chemotactic response could be blocked by inhibiting the PI3K/Akt or p65(NF-κB) pathway. Response to sRANKL in normal and tumor cells suggests a role for RANK/ERK-mediated signaling in normal osteoblasts chemotactic migration during bone remodeling that is altered or lost during osteosarcoma tumorigenesis.