RANKL Treatment Research Articles

Isofraxidin Inhibits Receptor Activator of Nuclear Factor-κB Ligand–Induced Osteoclastogenesis in Bone Marrow–Derived Macrophages Isolated from Sprague–Dawley Rats by Regulating NF-κB/NFATc1 and Akt/NFATc1 Signaling Pathways

Osteoporosis is a common bone disease that is characterized by decreased bone mass and fragility fractures. Isofraxidin is a hydroxy coumarin with several biological and pharmacological activities including an anti-osteoarthritis effect. However, the role of isofraxidin in osteoporosis has not yet been investigated. In the present study, we used receptor activator of nuclear factor-κB ligand (RANKL) to induce osteoclast formation in primary bone marrow macrophages (BMMs). Our results showed that RANKL treatment significantly increased tartrate-resistant acid phosphatase (TRAP) activity, as well as the expression of osteoclastogenesis-related markers including MMP-9, c-Src, and cathepsin K at both mRNA and protein levels; however, these effects were inhibited by isofraxidin in BMMs. In addition, luciferase reporter assay demonstrated that isofraxidin treatment suppressed the RANKL-induced an increase in nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) transcriptional activity. Besides, the decreased expression level of IκBα and increased levels of p-p65, p-IκBα, and p-Akt in RANKL-induced BMMs were attenuated by isofraxidin. Moreover, NFATc1 overexpression rescued the anti-osteoclastogenic effect of isofraxidin with increased expression levels of MMP-9, c-Src, and cathepsin K. Taken together, these findings indicated that isofraxidin inhibited RANKL-induced osteoclast formation in BMMs via inhibiting the activation of NF-κB/NFATc1 and Akt/NFATc1 signaling pathways. Thus, isofraxidin might be a therapeutic agent for the treatment of osteoporosis.

Characterization of Extracellular Vesicles in Osteoporotic Patients Compared to Osteopenic and Healthy Controls.

Extracellular vesicles (EVs) are mediators of a range of pathological conditions. However, their role in bone loss disease has not been well understood. In this study we characterized plasma EVs of 54 osteoporotic (OP) postmenopausal women compared to 48 osteopenic (OPN) and 44 healthy controls (CN), and we investigated their effects on osteoclasts and osteoblasts. We found no differences between the three groups in terms of anthropometric measurements and biochemical evaluation of serum calcium, phosphate, creatinine, PTH, 25-hydroxy vitamin D and bone biomarkers, except for an increase of CTX level in OP group. FACS analysis revealed that OP patients presented a significantly increased number of EVsand RANKL+ EVs compared with both CN and OPN subjects. Total EVs are negatively associated with the lumbar spine T-score and femoral neck T-score. Only in the OPN patients we observed a positive association between the total number of EVs and RANKL+ EVs with the serum RANKL. In vitro studies revealed that OP EVs supported osteoclastogenesis of healthy donor peripheral blood mononuclear cells at the same level observed following RANKL and M-CSF treatment, reduced the ability of mesenchymal stem cells to differentiate into osteoblasts, while inducing an increase of OSTERIX and RANKL expression in mature osteoblasts. The analysis of miRNome revealed that miR-1246 and miR-1224-5p were the most upregulated and downregulated in OP EVs; the modulated EV-miRNAs in OP and OPN compared to CN are related to osteoclast differentiation, interleukin-13 production and regulation of canonical WNT pathway. A proteomic comparison between OPN and CN EVs evidenced a decrease in fibrinogen, vitronectin, and clusterin and an increase in coagulation factors and apolipoprotein, which was also upregulated in OP EVs. Interestingly, an increase in RANKL+ EVs and exosomal miR-1246 was also observed in samples from patients affected by Gorham-Stout disease, suggesting that EVs could be good candidate as bone loss disease biomarkers. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Circular RNA atlas in osteoclast differentiation with and without alendronate treatment

BackgroundAlendronate (AL) is the most widely used bisphosphonate in the treatment of osteoporosis (OP). However, the role of circular RNAs (circRNAs) in the treatment of OP with AL remains unclear.MethodsIn this study, we showed that osteoclast (OC) precursors (OPCSs) could be induced into OCs with macrophage colony-stimulating factor (MCSF) and receptor activator of nuclear factor-κB ligand (RANKL) treatment. Subsequently, the OCs were treated with AL. OC differentiation-related biomarkers including RANK, tartrate-resistant acid phosphatase (TRAP), and cathepsin K (CTSK) were analyzed with TRAP staining, quantitative real-time (qPCR), and western blotting. Differentially expressed circRNAs (DECs) were identified among the OPCS, OC, and OC + AL groups. In addition, the expression levels of 10 DECs related to OC differentiation were verified by qPCR.ResultsTRAP staining showed that MCSF and RANKL treatment effectively induced OPCSs to differentiate into OCs. In addition, qPCR and western blot analysis revealed that the three biomarkers of OC (RANK, TRAP, and CTSK) were expressed significantly more in the OC group than those in the OPCS group. In contrast, the mRNA and protein expression levels of these three biomarkers decreased significantly in OCs treated with AL compared with those non-treated OCs. GO analysis of the DECs in the OPCS group vs. the OC group revealed that their functions were mainly related to cell, cell part, binding, and single-organism terms. KEGG analysis of the top 20 DECs in a comparison between the OPCS and OC groups showed that genes involved in mitogen-activated protein kinase signaling were the most common. Results of functional analyses of DECs in an OC vs. OC + AL comparison were similar to those in the OPCS vs. OC comparison. Finally, qPCR showed that, in the OC + AL vs. OC group comparison, the expression levels of seven and three DECs significantly decreased and increased, respectively.ConclusionsHaving successfully induced OPCSs to differentiate into OCs, we showed that AL suppresses the differentiation of OPCS into OC and that 10 DECs were involved in the regulation of this process. This indicates that these DECs might be important to the treatment of OP.

Gingipains promote RANKL-induced osteoclastogenesis through the enhancement of integrin β3 in RAW264.7 cells.

As a crucial virulence factor of Porphyromonas gingivalis, gingipains play an important role in periodontal destruction. This study aimed to investigate the effect of gingipains on osteoclastogenesis. We used RAW264.7 cells as osteoclast precursors in our study. In experimental groups, cells were treated with gingipains and/or receptor activator of nuclear factor-κB ligand (RANKL). Tartrate-resistant acid phosphatase (TRAP) activity staining assay showed osteoclast precursors and RANKL-induced mature osteoclasts were increased in a gingipains dose-dependent manner. Real-time reverse transcription polymerase chain reaction analysis demonstrated that gingipains upregulated osteoclastic genes including the protease cathepsin K (Ctsk), matrix metalloprotein 9 (Mmp9), nuclear factor of activated T cells 1 (Nfatc1) and acid phosphatase 5, tartrate resistant (Acp5) in a time-dependent manner. Western blotting assays presented upregulated expressions of TNF receptor-activating factor 6 (TRAF6) and integrin β3 induced by gingipains and RANKL compared to RANKL alone. Enhanced integrin-related signaling was also demonstrated by elevated phosphorylations of FAK and paxillin compared to control. Moreover, the pit resorption assays showed that gingipains augmented bone resorptive function of osteoclasts induced by RANKL. When we used Cilengitide to block integrin αvβ3, gingipains reversed the reduction of formation and resorptive function in RANKL-induced osteoclasts, as they enhanced integrin αvβ3 levels more than RANKL treatment alone. In conclusion, our data suggest that gingipains augmented the differentiation and function of mature osteoclasts induced by RANKL through the increase in integrin αvβ3.

Combining RANK/RANKL and ERBB-2 targeting as a novel strategy in ERBB-2-positive breast carcinomas

BackgroundERBB-2 is overexpressed in about 20% of breast cancers (BCs), indicating poor prognosis. The receptor activator of nuclear factor-κB (RANK) pathway is implicated in ERBB-2 (+) BC. The purpose of this study was to elucidate the underlying molecular mechanism of this interaction and the beneficial impact of dual targeting of RANK and ERBB-2 pathways.MethodsWe used SKBR3, MCF7, MDA-MB-453, and BT-474 human BC cell lines. We examined RANK and RANKL expression using RT-PCR, Western blot, and immunofluorescence. The evaluation of RANK expression in a cohort of BC patients was performed using immunohistochemistry. The interaction between RANK and ERBB family members was detected using proximity ligation assay (PLA), which enables the visualization of interacting proteins. We used inhibitors of both pathways [trastuzumab (T), pertuzumab (P), denosumab (D)]. NF-κB pathway activation was studied using Western blot. Cell growth and viability was evaluated using XTT, flow cytometry, and clonogenic assay. For cell migration evaluation, scratch assay was performed. Data were analyzed by one-way ANOVA.ResultsCell lines express RANK and RANKL. RANK immunostaining was also detected in human BC tissue samples. RANK receptor dimerizes with ERBB family members. RANK/ERBB-2 dimer number seems to be associated with ERBB-2 expression (SKBR3, 5.4; BT-474, 8.2; MCF7, 0.7; MDA-MB-453, 0.3). RANK/ERBB-2 dimers were decreased in the presence of the inhibitors D, T, and P, while they were increased after RANKL (R) treatment in SKBR3 (m, 5.4; D, 1.2; T, 1.9; DT, 0.6; TP, 1; DTP, 0.4; R, 11.8) and BT-474 (m, 8.2; D, 3.1; T, 4.3; DT, 0.7; TP, 3.4; DTP, 3.2; R, 11.6). Combination targeting of SKBR3 further decreased NF-κB pathway activation compared to single targeting. In SKBR3, RANKL and ERBB-2 blockage resulted in reduced cell proliferation, increased apoptosis, and lower metastatic potential compared to mock cells (m) and reversed values in RANKL presence. The combination treatment of SKBR3 with D, T, and P had an advantage in functional traits compared to single targeting. Denosumab suppressed NF-κB signaling and diminished proliferation rate in MDA-MB-453 cells. MCF7 did not correspond to inhibitors.ConclusionsThe results indicate a novel physical and molecular association between ERBB-2 and RANK pathways that affects ERBB-2 (+) BC growth. We also present data suggesting that the combination of anti-ERBB-2 agents and RANKL inhibitors have a potential direct anti-tumor effect and should be further tested in certain BC patients.

RANKL treatment of vascular endothelial cells leading to paracrine pro-calcific signaling involves ROS production.

Numerous studies have highlighted the causal link between over-production of reactive oxygen species (ROS) and cardiovascular complications such as vascular calcification (VC). Receptor-activator of nuclear factor-κB ligand (RANKL) has previously been shown to act on endothelial cells, eliciting the production/release of paracrine pro-calcific signals that act, in-turn, upon underlying vascular smooth muscle cells (VSMCs) to induce osteoblastic differentiation and VC. A role for endothelial ROS signaling in this process has not been established however. In the current paper, we investigate the possibility that RANKL leads to ROS signaling within the endothelial layer as part of the RANKL-driven VC signaling cascade. Human aortic endothelial cells (HAECs) were treated with RANKL (25ng/ml, 72h) and monitored for ROS production, in parallel with various pro-calcific signaling indices. Antioxidant co-treatments included TRAIL (5ng/ml), apocynin (10mM) and N-acetylcysteine (5mM). Treatment of HAECs with RANKL-induced robust ROS production. This surge could be partially attenuated by TRAIL and strongly attenuated by apocynin and N-acetylcysteine. RANKL also elicited a range of signaling events in HAECs that we have previously demonstrated are coupled to osteoblastic differentiation in underlying VSMCs. These include non-canonical NF-κB/p52 activation, elevated BMP-2 release and increased alkaline phosphatase (ALP) enzyme activity (cellular and extracellular). Importantly, these RANKL-induced signaling events could be completely prevented by co-treatment of HAECs with antioxidants. In summary, RANKL elicits ROS generation in HAECs with direct consequences for generation of paracrine pro-calcific signals known to effect calcification in underlying VSMCs.

Effects of TGF‑β1 on the migration and morphology of RAW264.7 cells in vitro.

Osteoclasts (OCs) differentiate from monocyte/macrophage‑lineage hematopoietic precursor cells, which are known as OC precursors (OCPs). Several studies have investigated cell chemotaxis in the bone microenvironment; however, OCP migration ability in the bone microenvironment during OC differentiation is yet to be elucidated. As an initial investigation of this characteristic, the present study aimed to determine the effects of transforming growth factor (TGF)‑β1 on OCP migration in vitro. Pre‑osteoclastic RAW264.7 cells were cultured with and without TGF‑β1 (2, 5 or 20 ng/ml), receptor activator of NF‑κB ligand (RANKL; 50 ng/ml), and/or SB431542 (10 µM), a potent and specific inhibitor of TGF‑β1 receptor kinase activity. Cell proliferation was significantly inhibited in the presence of TGF‑β1 for 3 days, and the effect was reversed by SB431542. Tartrate‑resistant acid phosphatase (TRAP) activity in RAW264.7 cells was significantly increased by RANKL treatment, compared with TRAP activity in control cells on day 3. The highest TRAP activity in RAW264.7 cells was induced by the combined treatment with TGF‑β1 (2 ng/ml) and RANKL. When TGF‑β1 signaling was inhibited by addition of SB431542 to the medium during culture, OC differentiation was notably suppressed. These findings suggest that TGF‑β1 accelerates RANKL‑induced OC differentiation, but does not act in a dose‑dependent manner. The migration of RAW264.7 cells was promoted at 24 h, but was suppressed at 72 h, during RANKL‑induced osteoclast differentiation in the presence of TGF‑β1. These results were accompanied with the increased expression of small G‑proteins, RhoA and Rac, at 24 h, but their expression decreased at 72 h. RAW264.7 cells treated with TGF‑β1 for 24 h underwent morphological changes, from round to polygonal morphology. Furthermore, protrusions were completely lost and the cell morphology reverted from polygonal to round after TGF‑β1 treatment for 72 h. Therefore, our findings indicated that OCP migration may be modified by differentiation in vitro.

Combining denosumab (D) and trastuzumab (T) as a novel treatment strategy in HER2-positive (+) breast carcinomas (BC).

e12506 Background: HER-2 is overexpressed in 20–30% of BC, indicating poor prognosis. RANKL/RANK pathway is implicated in HER-2(+) BC. The molecular mechanism and the potential of dual targeting have been evaluated and showed promising results. Methods: We used SKBR3, BT474, MCF7, MDA-MB-453 human BC cell lines. We examined the expression of RANK and RANKL using RT-PCR, Western-Blot and immunofluorescence. The interaction between RANK and HER-2 was detected using Proximity Ligation Assay (PLA) which enables the visualization of interacting proteins. NF-κB pathway activation was studied using Western-Blot. We used inhibitors of both pathways. Cell viability and cell migration was evaluated using XTT and scratch assay, respectively. Results: Cell lines differentially express RANK and RANKL. RANK receptor dimerizes with HER-2. RANK/ HER-2 dimer number depends on HER-2 expression (SKBR3: 5.4, BT474: 8.2, MCF7: 0.7, MDA-MB-453: 0.3). RANK/HER-2 dimers are decreased in the presence of the inhibitors D and T, while they are increased after RANKL (R) treatment in SKBR3 (m:5.4, D:1.2, T:1.9, DT:0.6, R:11.8,) and BT474 (m:8.2, D:3.1, T:4.3, DT:0.7, R:11.6). MCF7 and MDA-MB-453 do not correspond to inhibitors. The combination treatment of SKBR3 with the inhibitors further decreased NF-κB pathway activation compared to single targeting. SKBR3, after inhibitors treatment, also exhibit reduced cell proliferation (m:100%, D:84.7%, T:51.57%, DT:48.61%, R:111%) and lower metastatic potential (m:26.02%, D:33.56%, T:16.54%, DT:13.75%, R:32.36%) compared to mock cells (m) and increased values in RANKL presence. The combination treatment of SKBR3 with D and T has an advantage in functional proliferation assays compared to single targeting. Conclusions: The results indicate a direct association between HER-2 and RANKL/RANK pathway in HER-2(+) BC growth. We also present data suggesting that the combination of trastuzumab and denosumab may have therapeutic benefit in certain BC patients.

Receptor activator of NF-κB (RANK)-mediated induction of metastatic spread and association with poor prognosis in renal cell carcinoma

BackgroundInhibition of the receptor activator of NF-κB ligand (RANKL) has become a standard of care supportive treatment to prevent skeletal related events in cancer patients. Moreover, RANKL inhibition has been implicated with better survival outcome in lung cancer, while RANKL expression induces tumor progression and metastatic spread in vivo in breast cancer. Whether RANK/RANKL may have an impact on the pathogenesis of clear cell renal cell carcinoma (ccRCC) is currently unknown. Patients and MethodsA retrospective tissue micro array (TMA)-study was carried out determining the expression of RANK/RANKL in primary tumors of 306 ccRCC patients. Additionally, 24 ccRCC cell lines were employed for in vitro analyses of the RANK/RANKL axis including cell proliferation, migration and anchorage independent growth. ResultsRANK (+) vs. RANK (−) tumors had both worse cancer specific survival (CSS) (6.3 vs. 1.3 years; p < 0.001) and recurrence free survival (RFS) (9.9 vs. 5.8 years; p < 0.001). RANK (+) (HR 2.21; p < 0.001) was an independent prognostic factor for CSS and RFS (HR 4.98; p < 0.001). RANKL treatment resulted in increased proliferation, soft agar growth, and colony formation of RANK (+) RCC cell lines, which could be reversed by treatment with an NF-κB inhibitor and with a combination of osteoprotegrin and RANKL in vitro. ConclusionsRANK is expressed in ccRCC tissue, correlates with clinicopathological features, survival outcome, and when stimulated with RANKL can induce ccRCC progression in vitro. Consequently, RANKL inhibition combined with standard of care treatment may be a promising approach to improve ccRCC patient's survival.

Osteoblast derived-neurotrophin‑3 induces cartilage removal proteases and osteoclast-mediated function at injured growth plate in rats.

Faulty bony repair causes dysrepair of injured growth plate cartilage and bone growth defects in children; however, the underlying mechanisms are unclear. Recently, we observed the prominent induction of neurotrophin‑3 (NT-3) and its important roles as an osteogenic and angiogenic factor promoting the bony repair. The current study investigated its roles in regulating injury site remodelling. In a rat tibial growth plate drill-hole injury repair model, NT-3 was expressed prominently in osteoblasts at the injury site. Recombinant NT-3 (rhNT-3) systemic treatment enhanced, but NT-3 immunoneutralization attenuated, expression of cartilage-removal proteases (MMP-9 and MMP-13), presence of bone-resorbing osteoclasts and expression of osteoclast protease cathepsin K, and remodelling at the injury site. NT-3 was also highly induced in cultured mineralizing rat bone marrow stromal cells, and the conditioned medium augmented osteoclast formation and resorptive activity, an ability that was blocked by presence of anti-NT-3 antibody. Moreover, NT-3 and receptor TrkC were induced during osteoclastogenesis, and rhNT-3 treatment activated TrkC downstream kinase Erk1/2 in differentiating osteoclasts although rhNT-3 alone did not affect activation of osteoclastogenic transcription factors NF-κB or NFAT in RAW264.7 osteoclast precursor cells. Furthermore, rhNT-3 treatment increased, but NT-3 neutralization reduced, expression of osteoclastogenic cytokines (RANKL, TNF-α, and IL-1) in mineralizing osteoblasts and in growth plate injury site, and rhNT-3 augmented the induction of these cytokines caused by RANKL treatment in RAW264.7 cells. Thus, injury site osteoblast-derived NT-3 is important in promoting growth plate injury site remodelling, as it induces cartilage proteases for cartilage removal and augments osteoclastogenesis and resorption both directly (involving activing Erk1/2 and substantiating RANKL-induced increased expression of osteoclastogenic signals in differentiating osteoclasts) and indirectly (inducing osteoclastogenic signals in osteoblasts).

Serum calcium concentration is maintained when bone resorption is suppressed by osteoprotegerin in young growing male rats.

Serum calcium (Ca) is maintained in a narrow range through regulation of Ca metabolism in the intestine, kidney, and bone. Calcium is incorporated and resorbed from bone during bone remodeling via cellular processes as well as by exchange. Both routes contribute to calcium homeostasis. To assess the magnitude of bone turnover contribution to calcium homeostasis we labeled bone with a Ca tracer and measured Ca release following stimulation or suppression of bone resorption. Young growing male rats (n = 162) were dosed with 45Ca to label skeletal Ca. After a one-month period to allow the label to incorporate into the skeleton, rats were treated with a bone resorption antagonist (OPG), a bone resorption agonist (RANKL), or vehicle control (PBS). Serum and urine 45Ca and total Ca, and serum TRACP5b (a bone resorption biomarker), were monitored for 45 days following treatment. Tracer data were analyzed by a compartmental model using WinSAAM to quantify dynamic changes in Ca metabolism and identify sites of change following treatment. In RANKL treated rats, both serum 45Ca and serum TRACP5b were increased by >70% due to a 25-fold increase in bone resorption. In OPG treated rats, both serum 45Ca and serum TRACP5b were suppressed by >70% due to a 75% decrease in bone resorption, a 3-fold increase in bone formation, and a 50% increase in absorption. Because TRACP5b and 45Ca responded similarly, we conclude that Ca release from bone into serum occurs mostly via osteoclast-mediated bone resorption. However, because serum Ca concentration did not change with altered resorption in response to either RANKL or OPG treatment, we also conclude that serum Ca concentration under normal dietary conditions in young growing male rats is maintained by processes in addition to cellular bone resorption.

PRMT1 mediates RANKL-induced osteoclastogenesis and contributes to bone loss in ovariectomized mice

Protein arginine methylation is a novel form of posttranslational modification mediated by protein arginine methyltransferase (PRMTs). PRMT1, a major isoform of the PRMT family, is responsible for various biological functions, including cellular differentiation. Although the important function that PRMT1 plays in various tissues is being increasingly recognized, its role in receptor activation of NF-κB ligand (RANKL)-induced osteoclastogenesis or osteoporosis has not yet been described. Here, we show that PRMT1 is essential for RANKL-induced osteoclastogenesis in vitro and for bone loss in vivo. RANKL treatment increased the expression of PRMT1 and its nuclear localization in bone marrow-derived macrophages (BMDMs) in a c-Jun N-terminal kinase (JNK)-dependent manner. Silencing PRMT1 attenuated RANKL-induced osteoclastogenesis by decreasing tartrate-resistant acid phosphatase (TRAP)-positive cells and inhibiting F-actin ring formation and bone resorption, which was confirmed in a separate experiment using haploinsufficient cells from PRMT1+/- mice. Our results also revealed that PRMT1 regulates the transcription activity of NF-κB by directly interacting with it in RANKL-treated BMDMs. An in vivo study showed that the haploinsufficiency of PRMT1 reduced the enzyme activity of TRAP and increased the bone mineral density in the metaphysis of ovariectomized (OVX) mice. Finally, treatment with estrogen (E2) downregulated the RANKL-induced expression of PRMT1, suggesting that estrogen may exert an inhibitory effect on osteoclastogenesis by suppressing PRMT1 expression. Our results suggest that PRMT1 plays an important role in the progression of osteoporosis and that it might be a good therapeutic target for postmenopausal osteoporosis.

Actin-binding LIM protein 1 regulates receptor activator of NF-κB ligand-mediated osteoclast differentiation and motility.

Actin-binding LIM protein 1 (ABLIM1), a member of the LIM-domain protein family, mediates interactions between actin filaments and cytoplasmic targets. However, the role of ABLIM1 in osteoclast and bone metabolism has not been reported. In the present study, we investigated the role of ABLIM1 in the receptor activator of NF-κB ligand (RANKL)-mediated osteoclastogenesis. ABLIM1 expression was induced by RANKL treatment and knockdown of ABLIM1 by retrovirus infection containing Ablim1-specific short hairpin RNA (shAblim1) decreased mature osteoclast formation and bone resorption activity in a RANKL-dose dependent manner. Coincident with the downregulated expression of osteoclast differentiation marker genes, the expression levels of c-Fos and the nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), critical transcription factors of osteoclastogenesis, were also decreased in shAblim1-infected osteoclasts during RANKL-mediated osteoclast differentiation. In addition, the motility of preosteoclast was reduced by ABLIM1 knockdown via modulation of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt/Rac1 signaling pathway, suggesting another regulatory mechanism of ABLIM1 in osteoclast formation. These data demonstrated that ABLIM1 is a positive regulator of RANKL-mediated osteoclast formation via the modulation of the differentiation and PI3K/Akt/Rac1-dependent motility.

LRP1 Suppresses Bone Resorption in Mice by Inhibiting the RANKL-Stimulated NF-κB and p38 Pathways During Osteoclastogenesis.

Single-nucleotide polymorphisms in the LRP1 gene coding sequence are associated with low bone mass, and cell culture studies suggest that LRP1 plays a role in osteoblast proliferation and osteoblast-mediated osteoclastogenesis. However, the in vivo function of LRP1 in bone homeostasis has not been explored. In this work, we studied the osteoclast-specific role of LRP1 in bone homeostasis using a Ctsk-Cre;Lrp1f/f mouse model on the C57BL/6J background. These mice had a dramatically decreased trabecular bone mass with markedly more osteoclasts, while the osteoblast activity was unaffected or slightly increased. The cortical bone parameters were largely unaltered. Upon RANKL treatment, Lrp1-deficient bone marrow monocytes more efficiently differentiated into osteoclasts and showed elevated p65 NFκB and p38 signaling. Consistently, Lrp1-overexpressing Raw264.7 cells were desensitized to RANKL-induced p38 and p65 activation and osteoclastogenesis. Moreover, RANKL treatment led to a sharp decrease of LRP1 protein and RNA in BMMs. Overall, our data suggest that osteoclast-expressed LRP1 is a crucial regulator of bone mass. It inhibits the NFκB and p38 pathways and lessens the efficiency of RANKL-induced osteoclastogenesis. © 2018 American Society for Bone and Mineral Research.

CCL4 enhances preosteoclast migration and its receptor CCR5 downregulation by RANKL promotes osteoclastogenesis

Chemokine CCL4 (MIP-1β) is released from osteoblast cells to restore the homeostasis of hematopoietic stem cells during the activation of bone marrow. In this study, we investigated the function of CCL4 and its receptor CCR5 during osteoclastogenesis. CCL4 promoted the migration and viability of preosteoclast cells. However, CCL4 had no direct effect on the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation in mouse preosteoclast cells. In addition, CCR5 expression was rapidly reduced by RANKL treatment, which was recovered by IFN-γ during osteoclastogenesis. CCR5 downregulation by RANKL was mediated by MEK and JNK in preosteoclast cells and promoted osteoclastogenesis. These results suggest that CCL4 can enhance the recruitment of preosteoclasts to bone in the early stage, and the reduction of CCR5 promotes osteoclastogenesis when RANKL is prevalent.

Activation of the non-canonical NF-κB/p52 pathway in vascular endothelial cells by RANKL elicits pro-calcific signalling in co-cultured smooth muscle cells

BackgroundThe intimal endothelium is known to condition the underlying medial smooth muscle cell (SMC) layer of the vessel wall, and is highly responsive to receptor-activator of nuclear factor-κB ligand (RANKL) and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), pro-calcific and anti-calcific agents, respectively. In this paper, we tested the hypothesis that RANKL-induced activation of endothelial NF-κB signalling is essential for pro-calcific activation of the underlying SMCs. MethodsFor these studies, human aortic endothelial and smooth muscle cell mono-cultures (HAECs, HASMCs) were treated with RANKL (0–25 ng/ml ± 5 ng/ml TRAIL) for 72 h. Non-contact transwell HAEC:HASMC co-cultures were also employed in which the luminal HAECs were treated with RANKL (± 5 ng/ml TRAIL), followed by analysis of pro-calcific markers in the underlying subluminal HASMCs. ResultsTreatment of either HAECs or HASMCs with RANKL activated the non-canonical NF-κB/p52 and canonical NF-κB/p65 pathways in both cell types. In RANKL ± TRAIL-treated HAECs, recombinant TRAIL, previously demonstrated by our group to strongly attenuate the pro-calcific signalling effects of RANKL, was shown to specifically block the RANKL-mediated activation of non-canonical NF-κB/p52, clearly pointing to the mechanistic relevance of this specific pathway to RANKL function within endothelial cells. In a final series of HAEC:HASMC transwell co-culture experiments, RANKL treatment of HAECs that had been genetically silenced (via siRNA) for the NF-κB2 gene (the molecular forerunner to NF-κB/p52 generation) exhibited strongly attenuated pro-calcific activation of underlying HASMCs relative to scrambled siRNA controls. SummaryThese in vitro observations provide valuable mechanistic insights into how RANKL may potentially act upon endothelial cells through activation of the alternative NF-κB pathway to alter endothelial paracrine signalling and elicit pro-calcific responses within underlying vascular smooth muscle cells.

MTORC1 impedes osteoclast differentiation via calcineurin and NFATc1

Rapamycins are immunosuppressant and anti-cancer drugs that inhibit the kinase mTOR. Clinically, they often cause bone pain, bone necrosis, and high bone turnover, yet the mechanisms are unclear. Here we show that mTORC1 activity is high in osteoclast precursors but downregulated upon RANKL treatment. Loss-of-function genetic models reveal that while early Raptor deletion in hematopoietic stem cells blunts osteoclastogenesis due to compromised proliferation/survival, late Raptor deletion in osteoclast precursors instead augments osteoclastogenesis. Gain-of-function genetic models by TSC1 deletion in HSCs or osteoclast precursors cause constitutive mTORC1 activation, impairing osteoclastogenesis. Pharmacologically, rapamycin treatment at low but clinically relevant doses exacerbates osteoclast differentiation and bone resorption, leading to bone loss. Mechanistically, RANKL inactivates mTORC1 via calcineurin-mediated mTORC1 dephosphorylation, consequently activating NFATc1 by reducing mTORC1-mediated NFATc1 phosphorylation. These findings uncover biphasic roles of mTORC1 in osteoclastogenesis, dosage-dependent effects of rapamycin on bone, and a previously unrecognized calcineurin–mTORC1–NFATc1 phosphorylation-regulatory signaling cascade.

Effect of l-caldesmon on osteoclastogenesis in RANKL-induced RAW264.7 cells.

Non-muscle caldesmon (l-CaD) is involved in the regulation of actin cytoskeletal remodeling in the podosome formation, but its function in osteoclastogenesis remains to be determined. In this study, RANKL-induced differentiation of RAW264.7 murine macrophages to osteoclast-like cells (OCs) was used as a model to determine the physiological role of l-CaD and its phosphorylation in osteoclastogenesis. Upon RANKL treatment, RAW264.7 cells undergo cell-cell fusion into multinucleate, and TRAP-positive large OCs with a concomitant increase of l-CaD expression. Using gain- and loss-of-function in OC precursor cells followed by RANKL induction, we showed that the expression of l-CaD in response to RANKL activation is an important event for osteoclastogenesis, and bone resorption. To determine the effect of l-CaD phosphorylation in osteoclastogenesis, three decoy peptides of l-CaD were used with, respectively, Ser-to-Ala mutations at the Erk- and Pak1-mediated phosphorylation sites, and Ser-to-Asp mutation at the Erk-mediated phosphorylation sites. Both the former two peptides competed with the C-terminal segment of l-CaD for F-actin binding and accelerated formation of podosome-like structures in RANKL-induced OCs, while the third peptide did not significantly affect the F-actin binding of l-CaD, and decreased the formation of podosome-like structures in OCs. With the experiments using dephosphorylated and phosphorylated l-CaD mutants, we further showed that dephosphorylated l-CaD mutant facilitated RANKL-induced TRAP activity with an increased cell fusion index, whereas phosphorylated l-CaD decreased the TRAP activity and cell fusion. Our findings suggested that both the level of l-CaD expression and the extent of l-CaD phosphorylation play a role in RANKL-induced osteoclast differentiation.

Cordycepin Accelerates Osteoblast Mineralization and Attenuates Osteoclast Differentiation In Vitro.

Bone homeostasis destruction is triggered by the uncontrolled activity of osteoblasts and osteoclasts. Targeting both the regulation of bone formation and resorption is a promising strategy for treating bone disorders. Cordycepin is a major component of Chinese caterpillar fungus Cordyceps militaris. It exerts a variety of biological actions in various cells and animal models. However, its function on bone metabolism remains unclear. In the present study, we discovered a dual-action function of cordycepin in murine MC3T3-E1 and RAW264.7 cells. MC3T3-E1 cells were cultured in an osteogenic medium in the presence of 1 μM cordycepin for up two weeks. Cordycepin was used for effects of osteoblast and osteoclast differentiation. Cell viability was measured using the MTT assay. Osteoblast differentiation was confirmed by alizarin red staining, ALP activity, western blot, and real-time PCR. Osteoclast differentiation and autophagic activity were confirmed via TRAP staining, pit formation assay, confocal microscopy, western blot, and real-time PCR. Cordycepin promoted osteoblast differentiation, matrix mineralization, and induction of osteoblast markers via BMP2/Runx2/Osterix pathway. On the other hand, RAW264.7 cells were differentiated into osteoclast by RANKL treatment for 72 h. 1 μM cordycepin significantly inhibited RANKL-induced osteoclast formation and resorption activity through disturbing the actin ring-formatted sealing zone and activating cathepsin K and MMP9. These findings indicate that cordycepin might be an innovative dual-action therapeutic agent for bone disease caused by an imbalance of osteoblasts and osteoclasts.

TRAIL attenuates RANKL-mediated osteoblastic signalling in vascular cell mono-culture and co-culture models

Background and objectivesVascular calcification (VC) is a major risk factor for elevated cardiovascular morbidity/mortality. Underlying this process is osteoblastic signalling within the vessel wall involving complex and interlinked roles for receptor-activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG), and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). RANKL promotes vascular cell osteoblastic differentiation, whilst OPG acts as a neutralizing decoy receptor for RANKL (and TRAIL). With respect to TRAIL, much recent evidence points to a vasoprotective role for this ligand, albeit via unknown mechanisms. In order to shed more light on TRAILs vasoprotective role therefore, we employed in vitro cell models to test the hypothesis that TRAIL can counteract the RANKL-mediated signalling that occurs between the vascular cells that comprise the vessel wall.Methods and resultsHuman aortic endothelial and smooth muscle cell mono-cultures (HAECs, HASMCs) were treated with RANKL (0–25 ng/mL ± 5 ng/mL TRAIL) for 72 hr. Furthermore, to better recapitulate the paracrine signalling that exists between endothelial and smooth muscle cells within the vessel wall, non-contact transwell HAEC:HASMC co-cultures were also employed and involved RANKL treatment of HAECs (±TRAIL), subsequently followed by analysis of pro-calcific markers in the underlying subluminal HASMCs. RANKL elicited robust osteoblastic signalling across both mono- and co-culture models (e.g. increased BMP-2, alkaline phosphatase/ALP, Runx2, and Sox9, in conjunction with decreased OPG). Importantly, several RANKL actions (e.g. increased BMP-2 release from mono-cultured HAECs or increased ALP/Sox9 levels in co-cultured HASMCs) could be strongly blocked by co-incubation with TRAIL. In summary, this paper clearly demonstrates that RANKL can elicit pro-osteoblastic signalling in HAECs and HASMCs both directly and across paracrine signalling axes. Moreover, within these contexts we present clear evidence that TRAIL can block several key signalling actions of RANKL in vascular cells, providing further evidence of its vasoprotective potential.