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Postmenopausal osteoporosis (PMOP) arises from the disruption in bone remodeling caused by estrogen deficiency, leading to a heightened susceptibility to osteoporotic fractures in aging women. Tetrahydroberberine (THB) is a chemical compound extracted from Corydalis yanhusuo , a member of the traditional Chinese medicine series "Zhejiang eight taste", possessing a variety of pharmacological functions such as lowering lipids and preventing muscle atrophy. However, the impact of THB on PMOP has not been systematically explored. In vitro experiments supported that THB suppresses osteoclast formation and resorption of bone concentration-dependently. Further experiments confirmed that these inhibitory effects of THB were related to inhibition on expressions of osteoclast-specific genes, the mitogen-activated protein kinase (MAPK) pathway, and the nuclear factor kappa-B (NF-κB) pathway and an increased apoptosis level in mature osteoclasts. Additionally, THB treatment mitigated the ovariectomy-induced bone loss and improved the skeletal microarchitecture in vivo. In conclusion, THB has such potential to improve the PMOP status.

Osteoclasts play an important role in maintaining the relative stability of bone mass. Abnormal number and function of osteoclasts are closely related to osteoporosis and osteolytic diseases. Thiaplakortone B (TPB), a natural compound derived from the Great Barrier Reef sponge Plakortis lita, has been reported to inhibit the growth of the malaria parasite, Plasmodium falciparum, but its effect on osteoclastogenesis has not been previously investigated. In our study, we found that TPB suppresses the receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclast formation and resorption activity by tartrate-resistant acid phosphatase (TRAcP) staining, immunofluorescence staining of F-actin belts and hydroxyapatite resorption assay. Furthermore, using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting analysis, we discovered that TPB inhibits osteoclast-specific genes and proteins expression. Mechanistically, TPB blocks multiple upstream pathways including calcium oscillation, NF-κB, mitogen-activated protein kinase (MAPK) and nuclear factor of activated T cells 1(NFATc1) signaling pathways. In vivo, TPB could dampen bone loss in an ovariectomy (OVX) mouse model by micro-CT assessment and histological staining. Therefore, TPB may serve as a potential therapeutic candidate for the treatment of osteoporosis and osteolysis., Competing Interests: Conflicts of interest statement The authors declare no conflicts of interest., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The LIM-domain containing protein Ajuba and the scaffold protein SQSTM1/p62 regulate signalling of NF-κB, a transcription factor involved in osteoclast differentiation and survival. The ubiquitin-associated domain of SQSTM1/p62 is frequently mutated in patients with Paget's disease of bone. Here, we report that Ajuba activates NF-κB activity in HEK293 cells, and that co-expression with SQSTM1/p62 inhibits this activation in an UBA domain-dependent manner. SQSTM1/p62 regulates proteins by targeting them to the ubiquitin-proteasome system or the autophagy-lysosome pathway. We show that Ajuba is degraded by autophagy, however co-expression with SQSTM1/p62 (wild type or UBA-deficient) protects Ajuba levels both in cells undergoing autophagy and those exposed to proteasomal stress. Additionally, in unstressed cells co-expression of SQSTM1/p62 reduces the amount of Ajuba present in the nucleus. SQSTM1/p62 with an intact ubiquitin-associated domain forms holding complexes with Ajuba that are not destined for degradation yet inhibit signalling. Thus, in situations with altered levels and localization of SQSTM1/p62 expression, such as osteoclasts in Paget's disease of bone and various cancers, SQSTM1/p62 may compartmentalize Ajuba and thereby impact its cellular functions and disease pathogenesis. In Paget's, ubiquitin-associated domain mutations may lead to increased or prolonged Ajuba-induced NF-κB signalling leading to increased osteoclastogenesis. In cancer, Ajuba expression promotes cell survival. The increased levels of SQSTM1/p62 observed in cancer may enhance Ajuba-mediated cancer cell survival., Competing Interests: No authors have competing interests.

Osteolytic diseases are typified by over-enhanced formation and resorbing function of osteoclasts and have a major impact on human health. Inhibition of osteoclastic differentiation and function is a key strategy for clinical therapy of osteolytic conditions. Maackiain is a natural compound extracted from Sophora flavescens, which has been applied to anti-allergic and anti-tumour treatments. The present results showed that Maackiain could restrain receptor activator of nuclear factor-κB ligand (RANKL)-stimulated osteoclast formation and hydroxyapatite resorption dose-dependently, and interrupt the structures of F-actin belts in the mature osteoclasts. It also repressed the expressions of osteoclast-specific genes and proteins. Furthermore, Maackiain could inhibit RANKL-stimulated NF-κB and calcium signalling pathways, and dampen Nuclear factor of activated T cell cytoplasmic 1 activity, protein expression and translocation into the nucleus. These results revealed that Maackiain may have a potential therapeutic effect on osteoclast-related disorders., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Postmenopausal osteoporosis (PMO) is a progressive disease occurring in elderly postmenopausal women that is characterized by low bone mass and impaired bone quality. Sclareol is a natural product (initially isolated from the leaves and flowers of Salvia Sclarea) that possesses immune-regulation and anti-inflammatory effects, but its role in osteoclastic formation and function as well as the PMO remains unknown. In the current study, we investigated the inhibitory effect of sclareol on osteoclastogenesis and progression of PMO. In vitro, sclareol not only inhibited osteoclast formation but also suppressed osteoclast function. The expression of the receptor activator of NF-κB ligand (RANKL)-induced osteoclast marker gene and protein was also reduced by sclareol treatment. Mechanistically, we found that sclareol inhibits RANKL-induced NF-κB and MAPK/ERK pathway activation. Furthermore, sclareol exerted a protective effect against bone loss in an ovariectomy-induced mouse model. Taken together, our findings suggest that sclareol has potential value as a therapeutic agent for PMO.

Wearing titanium particle-induced osteoclastogenesis, accompanied by peri-implant osteolysis, is the main cause of long-term failure of hip prosthesis. Currently, medications used for the prevention and treatment of peri-implant osteolysis show serious side effects. Therefore, development for more effective new drugs with less side effects is extremely urgent. Vaccarin is a natural flavonoid extracted from Vaccaria segetalis, with various biological functions, including antioxidantory, anti-inflammatory, and promotion of angiogenesis. However, the putative role of vaccarin in the inhibition of titanium particle-induced osteolysis has not been reported. In this study, it was indicated that vaccarin could effectively inhibit RANKL-induced osteoclastogenesis, fusion of F-actin rings, bone resorption, and expression of osteoclast marker genes in a dose-dependent manner in vitro. Moreover, vaccarin could also inhibit RANKL-induced osteoclastogenesis via the inhibition of NF-κB and MAPK (p38, ERK, and JNK) signaling pathways, and inhibit the transcription of downstream transcription factors, such as c-Fos and NFATc1. Consistent with in vitro results, this in vivo study showed that vaccarin exhibited an inhibitory effect on titanium particle-induced osteolysis by antiosteoclastogenesis. In conclusion, vaccarin could be a promising agent for preventing and treating peri-implant osteolysis., (© 2019 Wiley Periodicals, Inc.)

The α,β-unsaturated-enone contained natural products have been reported showing NF-κB inhibition effect. It is well known that NF-κB inhibitors can also be used to inhibit osteoclastogenesis. In a continual discovery new agents for anti-osteoclastogenesis, 8 different type compounds with α,β-unsaturated-enone fragments from our in-house library were evaluated for NF-κB inhibition and anti-osteoclastogenesis. Experimental results indicated five compounds exhibited inhibition of NF-κB signal pathway. Among them, one compound ((E)-2-(4-fluorobenzylidene)-3,4-dihydronaphthalen-1(2H)-one, 6a) simultaneously inhibits both osteoclastogenesis and NF-κB signal pathway. Furthermore, 12 compounds with similar scaffold with 6a were tested for anti-osteoclastogenesis. As a result, 9 compounds inhibited both NF-κB and osteoclastogenesis. Among them, compound 6b is the most potent inhibitor against NF-κB (IC 50  = 2.09 μM) and osteoclast differentiation (IC 50  = 0.86 μM). Further studies show that compound 6b blocks the phosphorylation of both p65 and IκBα, and suppresses NF-κB targeted gene expression without interfering MAPKs and PI3K/Akt signal transduction pathways. This study demonstrates that we can identify promising synthesized compounds with new scaffolds as therapeutic solutions against osteoclastogenesis inspired by the privileged fragment derived from natural leads., (Copyright © 2019. Published by Elsevier Inc.)

Identification of natural compounds that inhibit osteoclastogenesis will facilitate the development of antiresorptive treatment of osteolytic bone diseases. Asiaticoside is a triterpenoid derivative isolated from Centella asiatica, which exhibits varying biological effects like angiogenesis, anti-inflammation, wound healing, and osteogenic differentiation. However, its role in osteoclastogenesis remains unknown. Here, we show that Asiaticoside can suppress RANKL-induced osteoclast formation and bone resorption in a dose-dependent manner. Asiaticoside attenuated the expression of osteoclast marker genes including Ctsk, Atp6v0d2, Nfatc1, Acp5, and Dc-stamp. Furthermore, Asiaticoside inhibited RANKL-mediated NF-κB and NFATc1 activities, and RANKL-induced calcium oscillation. Collectively, this study demonstrates that Asiaticoside inhibited osteoclast formation and function through attenuating RANKL-induced key signaling pathways, which may indicate that Asiaticoside is a potential antiresorptive agent against osteoclast-related osteolytic bone diseases., (© 2018 Wiley Periodicals, Inc.)

Pathological osteolysis is commonly associated with osteoporosis, bone tumors, osteonecrosis, and chronic inflammation. It involves excessive resorption of bone matrix by activated osteoclasts. Suppressing receptor activator of NF-κB ligand (RANKL) signaling pathways has been proposed to be a good target for inhibiting osteoclast differentiation and bone resorption. Bajijiasu-a natural compound derived from Morinda officinalis F. C. How-has previously been shown to have anti-oxidative stress property; however, its effect and molecular mechanism of action on osteoclastogenesis and bone resorption remains unclear. In the present study, we found that Bajijiasu dose-dependently inhibited RANKL-induced osteoclast formation and bone resorption from 0.1 mM, and reached half maximal inhibitory effects (IC 50 ) at 0.4 mM without toxicity. Expression of RANKL-induced osteoclast specific marker genes including cathepsin K (Ctsk), nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), tartrate resistant acid phosphatase (TRAcP), vacuolar-type H⁺-ATPase V0 subunit D2 (V-ATPase d2), and (matrix metalloproteinase-2 (MMP2) was inhibited by Bajijiasu treatment. Luciferase reporter gene studies showed that Bajijiasu could significantly reduce the expression and transcriptional activity of NFAT as well as RANKL-induced NF-κB activation in a dose-dependent manner. Further, Bajijiasu was found to decrease the RANKL-induced phosphorylation of extracellular signal-regulated kinases (ERK), inhibitor of κB-α (IκB-α), NFAT, and V-ATPase d2. Taken together, this study revealed Bajijiasu could attenuate osteoclast formation and bone resorption by mediating RANKL signaling pathways, indicative of a potential effect of Bajijiasu on osteolytic bone diseases., Competing Interests: The authors declare no conflict of interest.

Osteoporosis, a metabolic bone disease, is characterized by an excessive formation and activation of osteoclasts. Anti-catabolic treatment using natural compounds has been proposed as a potential therapeutic strategy against the osteoclast related osteolytic diseases. In this study, the activity of berberine sulfate (an orally available form of berberine) on osteoclast differentiation and its underlying molecular mechanisms of action were investigated. Using bone marrow macrophages (BMMs) derived osteoclast culture system, we showed that berberine sulfate at the dose of 0.25, 0.5 and 1 μM significantly inhibited the formation of osteoclasts. Notably, berberine sulfate at these doses did not affect the BMM viability. In addition, we observed that berberine sulfate inhibited the expression of osteoclast marker genes, including cathepsin K (Ctsk), nuclear factor of activated T cells cytoplasmic 1 (NFATc1), tartrate resistant acid phosphatase (TRAcP, Acp5) and Vacuolar-type H+-ATPase V0 subunit D2 (V-ATPase d2). Luciferase reporter gene assay and Western blot analysis further revealed that berberine sulfate inhibits receptor for activation of nuclear factor ligand (RANKL)-induced NF-κB and NFAT activity. Taken together, our results suggest that berberine sulfate is a natural compound potentially useful for the treatment of osteoporosis.

Osteolytic bone diseases are commonly presented with enhanced osteoclast formation and bone resorption. Sesquiterpene lactone natural compounds have been found to possess anti-inflammatory and immune-modulation effects. Here, we identified three germacrane sesquiterpenes using computer-based virtual screening for the structural similarity with sesquiterpene lactone, parthenolide. We showed that natural germacrane sesquiterpene compounds A, B, and C inhibit osteoclast formation and bone resorption in a dose-dependent manner, with relative potency compound A > compound C > compound B based on their equimolar concentrations. Mechanistic studies by Luciferase reporter gene assay and Western blot analysis showed that germacrane sesquiterpene compound A inhibits RANKL-induced activation of NF-κB and IκBα degradation. This study reveals that natural germacrane sesquiterpene compounds are inhibitors for osteoclast formation and bone resorption, and provides evidence that naturally-occurring compounds might be beneficial as alternative medicine for the prevention and treatment of osteolysis.

Osteolytic bone diseases are characterized by excessive osteoclast formation and activation. Protein kinase C (PKC)-dependent pathways regulate cell growth, differentiation and apoptosis in many cellular systems, and have been implicated in cancer development and osteoclast formation. A number of PKC inhibitors with anti-cancer properties have been developed, but whether they might also influence osteolysis (a common complication of bone invading cancers) is unclear. We studied the effects of the PKC inhibitor compound, GF109203X on osteoclast formation and activity, processes driven by receptor activator of NFκB ligand (RANKL). We found that GF109203X strongly and dose dependently suppresses osteoclastogenesis and osteoclast activity in RANKL-treated primary mouse bone marrow cells. Consistent with this GF109203X reduced expression of key osteoclastic genes, including cathepsin K, calcitonin receptor, tartrate resistant acid phosphatase (TRAP) and the proton pump subunit V-ATPase-d2 in RANKL-treated primary mouse bone marrow cells. Expression of these proteins is dependent upon RANKL-induced NF-κB and NFAT transcription factor actions; both were reduced in osteoclast progenitor populations by GF109203X treatment, notably NFATc1 levels. Furthermore, we showed that GF109203X inhibits RANKL-induced calcium oscillation. Together, this study shows GF109203X may block osteoclast functions, suggesting that pharmacological blockade of PKC-dependent pathways has therapeutic potential in osteolytic diseases., (© 2014 Wiley Periodicals, Inc.)

The carboxyl terminus of Hsc70-interacting protein (CHIP, also named Stub1), a U-box containing E3 ubiquitin ligase, is involved in degradation of certain oncogenic proteins. Recent studies indicated that CHIP suppresses tumor progression in human cancers by targeting Src-3, hypoxia inducible factor 1α, NF-κB, ErbB2 and c-Myc. Here, we report that CHIP was downregulated, predominantly, in the late stages of human colorectal cancer (CRC), and that the CHIP promoter was hypermethylated in CRC specimens. Overexpression of CHIP in HCT-116 cells resulted in impaired tumor growth in nude mice and decreased abilities of tumor cell migration and invasion. Conversely, depletion of CHIP in HCT-116 cells promoted tumor growth and increased tumor cell migration and invasion. CHIP was further found to negatively regulate NF-κB signaling in HCT-116 cells by promoting ubiquitination and degradation of p65, a subunit of the NF-κB complex. The suppressive effect of CHIP led to decreased expression of NF-κB-targeted oncogenes including Cyclin D1, c-Myc, MMP-2, VEGF and IL-8. We proposed that CHIP inhibits the malignancy of CRC cells, possibly through targeting NF-κB signaling. This study provides functional evidence for CHIP as a potential tumor suppressor in CRC, and CHIP expression may be a marker for stages of CRC.

The RANKL-induced NF-κB signaling pathway is essential for osteoclastogenesis. This study aims to identify specific inhibitors targeting NF-κB signaling pathway, which might serve as useful small molecule inhibitors for the treatment and alleviation of osteoclast-mediated bone lytic diseases. By screening for compounds that selectively inhibit RANKL-induced NF-κB activation in RAW264.7 cells as monitored by luciferase reporter gene assay, we identified SC-514, a specific inhibitor of IKKβ, as a candidate compound targeting osteoclastogenesis. SC-514 dose-dependently inhibits RANKL-induced osteoclastogenesis with an IC50 of <5μM. At high concentrations, SC-514 (≥12.5μM) induced apoptosis and caspase 3 activation in RAW264.7 cells. Moreover, SC-514 specifically suppressed NF-κB activity owing to delayed RANKL-induced degradation of IκBα and inhibition of p65 nuclear translocation. Taken together, our results indicate that SC-514 impairs RANKL-induced osteoclastogenesis and NF-κB activation. Thus, targeting IKKβ by SC-514 presents as a potential treatment for osteoclast-related disorders such as osteoporosis and cancer-induced bone loss., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Cancer chemotherapy with methotrexate (MTX) is known to cause bone loss. However, the underlying mechanisms remain unclear. This study investigated the potential role of MTX-induced pro-inflammatory cytokines and activation of NF-κB in the associated osteoclastogenesis in rats. MTX (0.75 mg/kg per day) was administered for 5 days, and bone and bone marrow specimens were collected on days 6, 9, and 14. Compared with a normal control, MTX increased the density of osteoclasts within the metaphyseal bone and the osteoclast formation potential of marrow cells on day 9. RT-PCR analysis of mRNA expression for pro-osteoclastogenic cytokines in the metaphysis indicated that, although the receptor activator of NF-κB ligand/osteoprotegerin axis was unaffected, expression of tumor necrosis factor (TNF)-α, IL-1, and IL-6 increased on day 9. Enzyme-linked immunosorbent assay analysis of plasma showed increased levels of TNF-α on day 6 and of IL-6 on day 14. Plasma from treated rats induced osteoclast formation from normal bone marrow cells, which was attenuated by a TNF-α-neutralizing antibody. Indicative of a role for NF-κB signaling, plasma on day 6 increased NF-κB activation in RAW(264.7) cells, and plasma-induced osteoclastogenesis was abolished in the presence of the NF-κB inhibitor, parthenolide. Our results demonstrate mechanisms for MTX-induced osteoclastogenesis and show that MTX induces osteoclast differentiation by generating a pro-osteoclastogenic environment in both bone and the circulation, specifically with increased TNF-α levels and activation of NF-κB., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Osteolytic bone diseases including osteoporosis are commonly accompanied with enhanced osteoclast formation and bone resorption. Naringin, a natural occurring flavonoid has been found to protect against retinoic acid-induced osteoporosis and improve bone quality in rats. Here, we showed that naringin perturbs osteoclast formation and bone resorption by inhibiting RANK-mediated NF-κB and ERK signaling. Naringin suppressed gene expression of key osteoclast marker genes. Naringin was found to inhibit RANKL-induced activation of NF-κB by suppressing RANKL-mediated IκB-α degradation. In addition, naringin inhibited RANKL-induced phosphorylation of ERK. This study identifies naringin as an inhibitor for osteoclast formation and bone resorption, and provides evidence that natural compounds such as naringin might be beneficial as an alternative medicine for the prevention and treatment of osteolysis., (Copyright © 2011. Published by Elsevier B.V.)

Osteolytic bone diseases such as osteoporosis have a common pathological feature in which osteoclastic bone resorption outstrips bone synthesis. Osteoclast formation and activation are regulated by receptor activator of nuclear factor κB ligand (RANKL). The induction of RANKL-signaling pathways occurs following the interaction of RANKL to its cognate receptor, RANK. This specific binding drives the activation of downstream signaling pathways; which ultimately induce the formation and activation of osteoclasts. In this study, we showed that a natural immunomodulator, mangiferin, inhibits osteoclast formation and bone resorption by attenuating RANKL-induced signaling. Mangiferin diminished the expression of osteoclast marker genes, including cathepsin K, calcitonin receptor, DC-STAMP, and V-ATPase d2. Mechanistic studies revealed that mangiferin inhibits RANKL-induced activation of NF-κB, concomitant with the inhibition of IκB-α degradation, and p65 nuclear translocation. In addition, mangiferin also exhibited an inhibitory effect on RANKL-induced ERK phosphorylation. Collectively, our data demonstrates that mangiferin exhibits anti-resorptive properties, suggesting the potential application of mangiferin for the treatment and prevention of bone diseases involving excessive osteoclastic bone resorption.

Receptor activator NF-kappaB ligand (RANKL)-activated signaling is essential for osteoclast differentiation, activation and survival. Caffeic acid phenethyl ester (CAPE), a natural NF-kappaB inhibitor from honeybee propolis has been shown to have anti-tumor and anti-inflammatory properties. In this study, we investigated the effect of CAPE on the regulation of RANKL-induced osteoclastogenesis, bone resorption and signaling pathways. Low concentrations of CAPE (<1 microM) dose dependently inhibited RANKL-induced osteoclastogenesis in RAW264.7 cell and bone marrow macrophage (BMM) cultures, as well as decreasing the capacity of human osteoclasts to resorb bone. CAPE inhibited both constitutive and RANKL-induced NF-kappaB and NFAT activation, concomitant with delayed IkappaBalpha degradation and inhibition of p65 nuclear translocation. At higher concentrations, CAPE induced apoptosis and caspase 3 activities of RAW264.7 and disrupts the microtubule network in osteoclast like (OCL) cells. Taken together, our findings demonstrate that inhibition of NF-kappaB and NFAT activation by CAPE results in the attenuation of osteoclastogenesis and bone resorption, implying that CAPE is a potential treatment for osteolytic bone diseases.

Proteasome inhibitors represent a promising therapy for the treatment of relapsed and/or refractory multiple myeloma, a disease that is concomitant with osteolysis and enhanced osteoclast formation. While blockade of the proteosome pathway has been recently shown to influence osteoclast formation and function, the precise molecular cascade underlying these effects is presently unclear. Here, we provide evidence that proteasome inhibitors directly impair osteoclast formation and function via the disruption of key RANK-mediated signaling cascades. Disruption of the proteosome pathway using selective inhibitors (MG-132, MG-115, and epoxomicin) resulted in the accumulation of p62 and CYLD, and altered the subcellular targeting and distribution of p62 and TRAF6 in osteoclast-like cells. Proteosome inhibition also blocked RANKL-induced NF-kappaB activation, IkappaBalpha degradation and nuclear translocation of p65. The disruption in RANK-signaling correlated dose-dependently with an impairment in osteoclastogenesis, with relative potency epoxomicin > MG-132 > MG-115 based on equimolar concentrations. In addition, these inhibitors were found to impact osteoclastic microtubule organization and attenuate bone resorption. Based on these data we propose that deregulation of key RANK-mediated signaling cascades (p62, TRAF6, CYLD, and IkappaBalpha) underscores proteasome-mediated inhibition of osteolytic bone conditions.

Previously reported Sequestosome 1(SQSTM1)/p62 gene mutations associated with Paget's disease of bone (PDB) cluster in, or cause deletion of, the ubiquitin-associated (UBA) domain. The aims of this study were to examine the prevalence of SQSTM1 mutations in Australian patients, genotype/phenotype correlations and the functional consequences of a novel point mutation (P364S) located upstream of the UBA. Mutation screening of the SQSTM1 gene was conducted on 49 kindreds with PDB. In addition, 194 subjects with apparently sporadic PDB were screened for the common P392L mutation by restriction enzyme digestion. HEK293 cells stably expressing RANK were co-transfected with expression plasmids for SQSTM1 (wildtype or mutant) or empty vector and a NF-kappaB luciferase reporter gene. GST-SQSTM1 (wildtype and mutant) proteins were used in pull-down assays to compare monoubiquitin-binding ability. We identified SQSTM1 mutations in 12 of 49 families screened (24.5%), comprising 9 families with the P392L mutation and 1 family each with the following mutations: K378X, 390X, and a novel P364S mutation in exon 7, upstream of the UBA. The P392L mutation was found in 9 of 194 (4.6%) patients with sporadic disease. Subjects with SQSTM1 mutations had more extensive disease, but not earlier onset, compared with subjects without mutations. In functional studies, the P364S mutation increased NF-kappaB activation compared with wildtype SQSTM1 but did not reduce ubiquitin binding. This suggests that increased NF-kappaB signaling, but not the impairment of ubiquitin binding, may be essential in the pathogenesis of PDB associated with SQSTM1 mutations.

Osteoclasts are responsible for bone resorption and play a pivotal role in the pathogenesis of osteolytic disorders. NF-kappaB is a set of nuclear factors that bind to consensus DNA sequences called kappaB sites, and is essential for osteoclast formation and survival. NF-kappaB signalling pathways are strictly regulated to maintain bone homeostasis by cytokines such as RANKL, TNF-alpha and IL-1, which differentially regulate classical and/or alternative NF-kappaB pathways in osteoclastic cells. These pathways are also modulated by NF-kappaB mediators, including TRAF6, aPKC, p62/SQSTM1 and deubiquitinating enzyme CYLD that are involved in the ubiquitin-proteasome system during RANK-mediated osteoclastogenesis. Abnormal activation of NF-kappaB signalling in osteoclasts has been associated with excessive osteoclastic activity, and frequently observed in osteolytic conditions, including periprosthetic osteolysis, arthritis, Paget's disease of bone, and periodontitis. NF-kappaB modulators such as parthenolide and NEMO-binding domain peptide demonstrate therapeutic effects on inflammation-induced bone destruction in mouse models. Unravelling the structure and function of NF-kappaB pathways in osteoclasts and other cell types will be important in developing new strategies for treatments of bone diseases.

Unlabelled: Sequestosome 1/p62 (p62) mutations are associated with PDB; however, there are limited data regarding functional consequences. We report a novel mutation in exon 7 (K378X) in a patient with polyostotic Paget's disease of bone. p62 mutants increased NF-kappaB activation and significantly potentiated osteoclast formation and bone resorption in human primary cell cultures., Introduction: Sequestosome 1/p62 (p62) mutations are associated with Paget's disease of bone (PDB); however, there are limited data regarding functional consequences. One report has linked the common P392L mutation in the p62 ubiquitin binding associated (UBA) domain with increases in NF-kappaB activity, a transcription factor essential for osteoclastogenesis. To further clarify the functional impact of p62 mutations associated with PDB, we assessed the effect of p62 mutation (a novel mutation: K378X, and previously reported mutations: P392L and E396X) on RANK-induced NF-kappaB activation and compared this with the effect of wildtype p62. In addition, we studied the effect of p62 mutation on osteoclast formation and bone resorption., Materials and Methods: We performed co-transfection experiments with expression plasmids for p62 (wildtype or mutated) and RANK and an NF-kappaB luciferase reporter gene. Luciferase activities were recorded after addition of luciferin to cellular lysates. RAW(264.7) cells stably expressing enhanced green fluorescent protein (EGFP)-tagged p62 (wildtype, K378X, or P392L) or EGFP alone were assessed for changes in cell proliferation. Additionally, these cells were stimulated with RANKL to produce osteoclast-like cells (OLCs). Primary human monocytes collected from the K378X-affected patient and a control subject were stimulated to form OLCs and bone resorption data were obtained., Results: The novel mutation introduces a premature stop codon in place of Lys-378 and thereby eliminates the entire p62 UBA domain; this and two additional natural mutations (P392L, E396X) increased NF-kappaB activation compared with wildtype p62. Wildtype p62 consistently inhibited NF-kappaB activation compared with empty vector. UBA mutations (K378X and P392L) significantly increased the number of OLCs formed in response to RANKL and also the number of nuclei of the OLCs. K378X-affected human monocytes formed more OLCs with more nuclei and increased bone resorption compared with control monocytes., Conclusions: Our data show that mutation of the p62 UBA domain results in increased activation of NF-kappaB and osteoclast formation and function compared with wildtype p62. These results may partially explain the mechanism by which p62 mutation contributes to the pathogenesis of PDB.

Unlabelled: Effective treatment for bacteria-induced bone lytic diseases is not yet available. In this study, we showed that PAR, an NF-kappaB inhibitor found in medicinal herbs, can block LPS-induced osteolysis. PAR does this by inhibiting osteoclastogenesis and bone resorption and promoting apoptosis of osteoclasts through the suppression of NF-kappaB activity., Introduction: Osteolysis induced by chronic gram-negative bacterial infection underlies many bone diseases such as osteomyelitis, septic arthritis, and periodontitis. Drugs that inhibit lipopolysaccharide (LPS)-induced osteolysis are critically needed for the prevention of bone destruction in infective bone diseases. In this study, we investigated the effect of parthenolide (PAR) on LPS-induced osteolysis in vivo and studied its role in osteoclastogenesis, bone resorption, apoptosis, and NF-kappaB activity., Materials and Methods: The LPS-induced osteolysis in the mouse calvarium model was used to examine the effect of PAR in vivo. RANKL-induced osteoclast differentiation from RAW264.7 cells and bone resorption assays were used to assess the effect of PAR in vitro. Assays for NF-kappaB activation, p65 translocation, and IkappaB-alpha degradation were used to determine the mechanism of action of PAR in osteoclasts and their precursors. Flow cytometry and confocal microscopic analysis were used to examine cell apoptosis. Semiquantitative RT-PCR was performed to examine the effect of PAR on gene expression of RANK and TRAF6., Results: We found that PAR (0.5 and 1 mg/kg), injected simultaneously with LPS (25 mg/kg) or 3 days later, blocked the LPS-induced osteolysis in the mouse calvarium model. In vitro studies showed that low concentrations of PAR (<1 microM) inhibited in vitro osteoclastogenesis and osteoclastic bone resorption, whereas higher concentrations (>5 microM) triggered apoptotic cell death of osteoclasts and their precursor cells in a dose-dependent manner. Furthermore, PAR inhibited LPS-induced NF-kappaB activation, p65 translocation, and IkappaB-alpha degradation both in mature osteoclasts and their precursors in a time- and dose-dependent manner. In addition, PAR inhibited NF-kappaB activation induced by osteoclastogenic factors RANKL, interleukin (IL)-1beta, or TNF-alpha to varying degrees and reduced the gene expression of RANK and TRAF6., Conclusion: The NF-kappaB pathway is known to mediate both osteoclast differentiation and survival. These findings indicate that PAR blocks LPS-induced osteolysis through the suppression of NF-kappaB activity and suggest that it might have therapeutic value in bacteria-induced bone destruction.

Unlabelled: The mechanism by which TPA-induced PKC activity modulates osteoclastogenesis is not clear. Using a RAW(264.7) cell culture system and assays for NF-kappaB nuclear translocation, NF-kappaB reporter gene activity, and MAPK assays, we demonstrated that TPA inhibits osteoclastogenesis through the suppression of RANKL-induced NF-kappaB activation., Introduction: The protein kinase C (PKC) pathway has been suggested to be an important regulator of osteoclastic bone resorption. The role of PKC in RANKL-induced osteoclastogenesis, however, is not clear. In this study, we examined the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC activator, on osteoclastogenesis and studied its role in RANKL-induced signaling., Materials and Methods: RANKL-induced RAW(264.7) cell differentiation into osteoclast-like cells was used to assess the effect of TPA on osteoclastogenesis. Assays for NF-kappaB nuclear translocation, NF-kappaB reporter gene activity, protein kinase activity, and Western blotting were used to examine the effects of TPA on RANKL-induced NF-kappaB, c-Jun N-terminal kinase (JNK), and MEK/ERK and p38 signal transduction pathways., Results: We found that TPA inhibited RANKL-induced RAW(264.7) cell differentiation into osteoclasts in a dose-dependent manner. Time course analysis showed that the inhibitory effect of TPA on RANKL-induced osteoclastogenesis occurs predominantly at an early stage of osteoclast differentiation. TPA alone had little effect on NF-kappaB activation in RAW(264.7) cells, but it suppresses the RANKL-induced NF-kappaB activation in a dose-dependent fashion. Interestingly, the suppressive effect of TPA on RANKL-induced NF-kappaB activation was prevented by a conventional PKC inhibitor, Go6976. Supershift studies revealed that the RANKL-induced DNA binding of NF-kappaB complexes consisted of C-Rel, NF-kappaB1 (p50), and RelA (p65). In addition, TPA induced the activation of JNK in RAW(264.7) cells but had little effect on RANKL-induced activation of JNK. TPA also inhibited RANKL-induced activation of ERK but had little effect on p38 activation., Conclusion: Given that NF-kappaB activation is obligatory for osteoclast differentiation, our studies imply that inhibition of osteoclastogenesis by TPA is, at least in part, caused by the suppression of RANKL-induced activation of NF-kappaB during an early stage of osteoclastogenesis. Selective modulation of RANKL signaling pathways by PKC activators may have important therapeutic implications for the treatment of bone diseases associated with enhanced bone resorption.

Excessive osteoclast formation and function are considered as the main causes of bone lytic disorders such as osteoporosis and osteolysis. Therefore, the osteoclast is a potential therapeutic target for the treatment of osteoporosis or other osteoclast‐related diseases. Helvolic acid (HA), a mycotoxin originally isolated from Aspergillus fumigatus , has been discovered as an effective broad‐spectrum antibacterial agent and has a wide range of pharmacological properties. Herein, for the first time, HA was demonstrated to be capable of significantly inhibiting receptor activator of nuclear factor‐κB ligand (RANKL)‐induced osteoclastogenesis and bone resorption in vitro by suppressing nuclear factor of activated T cells 1 (NFATc1) activation. This inhibition was followed by the dramatically decreased expression of NFATc1‐targeted genes including Ctr (encoding calcitonin receptor), Acp5 (encoding tartrate‐resistant acid phosphatase [TRAcP]), Ctsk (encoding cathepsin K), Atp6v0d2 (encoding the vacuolar H+ ATPase V0 subunit d2 [V‐ATPase‐d2]) and Mmp9 (encoding matrix metallopeptidase 9) which are osteoclastic‐specific genes required for osteoclast formation and function. Mechanistically, HA was shown to greatly attenuate multiple upstream pathways including extracellular signal‐regulated kinase (ERK) phosphorylation, c‐Fos signaling, and intracellular Ca 2+ oscillation, but had little effect on nuclear factor‐κB (NF‐κB) activation. In addition, HA also diminished the RANKL‐induced generation of intracellular reactive oxygen species. Taken together, our study indicated HA effectively suppressed RANKL‐induced osteoclast formation and function. Thus, we propose that HA can be potentially used in the development of a novel drug for osteoclast‐related bone diseases. Helvolic acid (HA), a mycotoxin originally isolated from Aspergillus fumigatus, is demonstrated to be capable of significantly inhibiting receptor activator of nuclear factor‐κB ligand (RANKL)‐induced osteoclastogenesis and bone resorption in vitro by suppressing nuclear factor of activated T cells 1 (NFATc1) activation without affecting nuclear factor‐κB (NF‐κB) activation. Mechanistically, HA greatly attenuated multiple upstream pathways of NFATc1, including extracellular‐signal‐regulated kinase (ERK) phosphorylation, c‐Fos signaling, and intracellular Ca2+oscillation. Taken together, HA can be potentially used in the development of a novel drug for osteoclast‐related bone diseases. [ABSTRACT FROM AUTHOR]

In mice, CD49fhi mammary stem cells (MaSCs) asymmetrically divide to generate CD49f+ committed progenitor cells that differentiate into CD49f− phenotypes of the milk-secreting tissue at the onset of pregnancy. We show CD49f+ primary mammary epithelial cells (PMECs) isolated from lactating tissue uniquely respond to pregnancy-associated hormones (PAH) compared with CD49f+ cells from nonlactating tissue. Differentiation of CD49f+ PMEC in extracellular matrix produces CD49f− luminal cells to form differentiated alveoli. The PAH prolactin and placental lactogen specifically stimulate division of CD49f− luminal cells, while receptor activator of nuclear factor (NF)-κB ligand (RANKL) specifically stimulates division of basal CD49f+ cells. In nondifferentiating conditions, we observed a greater proportion of multipotent self-renewing cells, and RANKL treatment activated the RANK pathway in these cultures. Furthermore, we observed the deposition of calcium nodules in a proportion of these cells. These data imply that a MaSC unique to the lactating breast exists in humans, which generates progeny with discrete lineages and distinct response to PAH. S TEM C ELLS 2012;30:1255-1264 [ABSTRACT FROM AUTHOR]

Receptor activator NF-κB ligand (RANKL)-activated signaling is essential for osteoclast differentiation, activation and survival. Caffeic acid phenethyl ester (CAPE), a natural NF-κB inhibitor from honeybee propolis has been shown to have anti-tumor and anti-inflammatory properties. In this study, we investigated the effect of CAPE on the regulation of RANKL-induced osteoclastogenesis, bone resorption and signaling pathways. Low concentrations of CAPE (<1 µM) dose dependently inhibited RANKL-induced osteoclastogenesis in RAW264.7 cell and bone marrow macrophage (BMM) cultures, as well as decreasing the capacity of human osteoclasts to resorb bone. CAPE inhibited both constitutive and RANKL-induced NF-κB and NFAT activation, concomitant with delayed IκBα degradation and inhibition of p65 nuclear translocation. At higher concentrations, CAPE induced apoptosis and caspase 3 activities of RAW264.7 and disrupts the microtubule network in osteoclast like (OCL) cells. Taken together, our findings demonstrate that inhibition of NF-κB and NFAT activation by CAPE results in the attenuation of osteoclastogenesis and bone resorption, implying that CAPE is a potential treatment for osteolytic bone diseases. J. Cell. Physiol. 221: 642–649, 2009. © 2009 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Obacunone (OB) targets macrophage migration inhibitory factor (MIF) to impede osteoclastogenesis and alleviate osteoporosis. [Display omitted] • Obacunone (OB) effectively inhibits osteoclast formation and function in vitro. • OB attenuates RANKL-induced osteoclast differentiation signaling pathways. • Macrophage migration inhibitory factor (MIF) is identified as the molecular target of OB. • OB alleviates estrogen deficiency-induced bone loss by impeding excessive osteoclast activity. • OB may serve as an alternative therapeutic candidate for osteoporosis. Osteoporosis is the most common bone disorder where the hyperactive osteoclasts represent the leading role during the pathogenesis. Targeting hyperactive osteoclasts is currently the primary therapeutic strategy. However, concerns about the long-term efficacy and side effects of current frontline treatments persist. Alternative therapeutic agents are still needed. Obacunone (OB) is a small molecule with a broad spectrum of biological activities, particularly antioxidant and anti-inflammatory effects. This study aims to examine OB's therapeutic potential on osteoporosis and explore the rudimentary mechanisms. Osteoclast formation and osteoclastic resorption assays were carried out to examine OB's inhibitory effects in vitro , followed by the in-vivo studies of OB's therapeutic effects on ovariectomy-induced osteoporotic preclinical model. To further study the underlying mechanisms, mRNA sequencing and analysis were used to investigate the changes of downstream pathways. The molecular targets of OB were predicted, and in-silico docking analysis was performed. Ligand-target binding was verified by surface plasmon resonance (SPR) assay and Western Blotting assay. The results indicated that OB suppressed the formation of osteoclast and its resorptive function in vitro. Mechanistically, OB interacts with macrophage migration inhibitory factor (MIF) which attenuates receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL)-induced signaling pathways, including reactive oxygen species (ROS), NF-κB pathway, and mitogen-activated protein kinases (MAPKs). These effects eventually caused the diminished expression level of the master transcriptional factor of osteoclastogenesis, nuclear factor of activated T cells 1 (NFATc1), and its downstream osteoclast-specific proteins. Furthermore, our data revealed that OB alleviated estrogen deficiency-induced osteoporosis by targeting MIF and thus inhibiting hyperactive osteoclasts in vivo. These results together implicated that OB may represent as a therapeutic candidate for bone disorders caused by osteoclasts, such as osteoporosis. [ABSTRACT FROM AUTHOR]

Receptor activator of NF-κB ligand (RANKL) is essential for osteoclastogenesis. Targeting RANKL signaling pathways has been an encouraging strategy for treating lytic bone diseases such as osteoporosis and rheumatoid arthritis (RA). Sinomenine (SIN), derived from Chinese medicinal plant Sinomenioum acutum, is an active compound to treat RA, but its effect on osteoclasts has been hitherto unknown. In the present study, SIN was found to ameliorate M. tuberculosis H37Ra (Mt)-induced bone loss in rats with a decreased serum level of TRACP5b and RANKL, and an increased level of osteoprotegerin (OPG). In vitro study also showed that SIN could inhibit RANKL-induced osteoclast formation and bone resorption. The osteoclastic specific marker genes induced by RANKL including c-Src, MMP-9, TRACP were inhibited by SIN in a dose dependent manner. Signal transduction studies showed that SIN could obviously reduce the expression of RANK adaptor molecule TRAF6 and down-regulate RANKL-induced NF-κB activation. It decreased the RANKL-induced p38, JNK posphorylation but not ERK1/2 posphorylation. SIN could also reduce RANKL-mediated calcium influx which is associated with TRAF6/c-Src complex. Finally, SIN suppressed RANKL induced AP-1 and NFAT transcription, as well as the gene expression of NFATc1 and AP-1 components (Fra-1, Fra-2, c-Fos). The protein expression of c-Fos and TRAF6 were also inhibited by SIN after RANKL stimulation. Taken together, SIN could attenuate osteoclast formation and Mt-induced bone loss by mediating RANKL signaling pathways. [ABSTRACT FROM AUTHOR]

Abstract: Osteoclast formation is central to bone metabolism, occurring when myelomonocytic progenitors are stimulated by membrane-bound receptor activator of NFκB ligand (RANKL) on osteoblasts. Osteolytic hormones induce osteoblast RANKL expression, and reduce production of RANKL decoy receptor osteoprotegerin (OPG). However, rather than RANKL and OPG mRNA or protein levels, to measure hormonally-induced osteoclastogenic stimuli the net RANKL activity at the osteoblast surface needs to be determined. To estimate this we developed a cell reporter approach employing pre-osteoclast RAW264.7 cells transfected with luciferase reporter constructs controlled by NFκB (NFκB-RAW) or NFATc1 (NFAT-RAW)-binding promoter elements. Strong signals were induced in these cells by recombinant RANKL over 24h. When NFκB-RAW cells were co-cultured on osteoblastic cells (primary osteoblasts or Kusa O cells) stimulated by osteolytic factors 1,25(OH)2 vitamin D3 (1,25(OH)2D3) and prostaglandin E2 (PGE2), a strong dose dependent signal in NFκB-RAW cells was induced. These signals were completely blocked by soluble recombinant RANKL receptor, RANK.Fc. This osteoblastic RANKL activity was sustained for 3days in Kusa O cells; with 1,25(OH)2D3 withdrawal, RANKL-induced signal was still detectable 24h later. However, conditioned medium from stimulated osteoblasts induced no signal. TGFβ treatment inhibited osteoclast formation supported by 1,25(OH)2D3-treated Kusa O cells, and likewise blocked RANKL-dependent signals in NFAT-RAW co-cultured with these cells. These data indicate net RANKL stimulus at the osteoblast surface is increased by 1,25(OH)2D3 and PGE2, and suppressed by TGFβ, in line with their effects on RANKL mRNA levels. These results demonstrate the utility of this simple co-culture-based reporter assay for osteoblast RANKL activity. [Copyright &y& Elsevier]