Soluble RANKL Research Articles

Osteoclast regulation of osteoblasts via RANK‑RANKL reverse signal transduction invitro.

The treatment of osteoporosis typically inhibits the activity of osteoclasts, which subsequently results in the suppression of bone formation and maintenance, however the underlying mechanism remains to be elucidated. The receptor activator of nuclear factor κ-B ligand (RANKL)-receptor activator of nuclear factor κ-B (RANK) signaling axis is important in the osteoblast regulation of osteoclasts. RANKL surface-bound molecules expressed on T cells stimulate a reverse signaling transduction in order to regulate the T cells, therefore the present study hypothesized that RANKL expressed on osteoblasts may transfer reverse signals to regulate osteoblasts. A series of experiments were designed to test the hypothesis, using MTT, stealth RNA interference, reverse transcription-quantitative polymerase chain reaction, western blot analysis, alkaline phosphatase activity assay and alizarin red staining. The present study observed the role of RANK-RANKL reverse signaling on osteoblasts, regulated by osteoclasts. Osteoblasts were treated with recombinant RANK proteins. The soluble RANK enhanced the mineralization of osteoblasts. When the RANKL was knocked down in the osteoblast, RANK demonstrated a weak osteogenic effect on the RANKL-deficient osteoblast compared with the wild-type osteoblast which served as a control. Addition of soluble RANK activated the p38 mitogen activated protein kinase (MAPK) signaling pathway in the osteoblast and blocking this same pathway in E1 cells reduced the effect of RANK. In the co-culture system of osteoblasts and osteoclasts, p38 MAPK in E1 cells was phosphorylated a short time following co-culture and the phosphorylation then blocked by abundant soluble RANKL. The findings suggested that RANKL expressed on osteoblasts transferred reverse signals from the exterior of the cell to the interior, which regulated the osteoblasts.

Plasma levels of sRANKL and OPG are associated with atherogenic cytokines in patients with intermediate cardiovascular risk.

Osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) are regulators of bone remodeling, but are also considered to play important roles in coronary artery disease (CAD). This study evaluated potential associations of soluble (s) RANKL and OPG with atherosclerosis-relevant cytokines. Blood was collected from 414 individuals who presented to our hospital with intermediate likelihood for CAD for further examination. Plasma concentrations of total sRANKL, OPG, and 20 cytokines were measured using sandwich-type enzyme-linked immunoassays (ELISAs; OPG and sRANKL) and Luminex laser-based fluorescence analysis and correlated with each other. The plasma levels of interferon-γ (IFN-γ) and the T-helper cell 2 cytokines interleukin-4 (IL-4) and IL-13 showed a positive correlation with sRANKL. The association with sRANKL levels was negative for IFN-γ-induced protein-10 (IP-10) and monocyte chemotactic protein-1 (MCP-1). The strongest independent association with sRANKL in multivariable analyses was found for IFN-γ (positive) and IP-10 (negative), while IL-13 showed a positive and independent association with OPG plasma levels. OPG and sRANKL plasma levels correlate strongly and independently with specific circulating atherosclerosis-related cytokines in patients with intermediate cardiovascular risk.

Enhancing hRANKL production in Escherichia coli by optimizing culture conditions and lysing program

RANKL/RANK/OPG axis is important in bone metabolism regulation, and becomes a popular research area in bone diseases. RANKL is a critical part of RANKL/RANK/OPG axis, and widely required in bone metabolism research. However, the yield of recombinant soluble human RANKL (hRANKL) in Escherichia coli is much lower than mouse RANKL (mRANKL). In this study, by adjusting and stabilizing the pH value of LB medium at 7.5, lowering the inducing temperature to 16 ℃ and optimizing the lysis program, the yield of soluble hRANKL increased by approximately 5 to 12-fold over the non-adjusted group. Our experiment effectively enhanced soluble hRANKL expression in E. coli and might constitute a meaningful attempt to obtain soluble expression of recombinant protein in E. coli.

Angiotensin II/Angiotensin II Receptor Blockade Affects Osteoporosis via the AT1/AT2-Mediated cAMP-Dependent PKA Pathway

Animal studies have reported on the benefits of ARB on bone mass. However, the underlying mechanism for angiotensin II (AngII)/AngII receptor blockade (ARB) in regulating bone mass remains elusive. Since high levels of plasma and urine cAMP are observed in osteoporotic and hypertensive patients, we hypothesized that cAMP may be an important molecule for the downstream events of the activation of AT receptors, members of the G-protein-coupled receptor family, in regulating bone turnover. In this study, micro-CT and X-ray analyses indicated that AngII decreased bone mass via biasing bone resorption over bone formation in osteoporotic mice. However, these adverse effects were blocked by olmesartan and PD123319. In vitro, AngII was shown to downregulate osteogenic differentiation and matrix mineralization, but to upregulate osteoclastic activity by mainly affecting osteoblasts producing osteoclastogenesis-associated key soluble factors, including M-CSF and RANKL. Similarly, ARB treatment exhibited antagonistic effects on AngII. In conclusion, osteoblasts are the directly targeted cells. ARB1 exhibits a greater capacity to increase bone mass than ARB2. The cAMP-dependent PKA pathway plays an important role in AngII/ARB on changing bone mass.

Murine Rankl-/- Mesenchymal Stromal Cells Display an Osteogenic Differentiation Defect Improved by a RANKL-Expressing Lentiviral Vector.

Autosomal recessive osteopetrosis (ARO) is a severe bone disease characterized by increased bone density due to impairment in osteoclast resorptive function or differentiation. Hematopoietic stem cell transplantation is the only available treatment; however, this therapy is not effective in RANKL-dependent ARO, since in bone this gene is mainly expressed by cells of mesenchymal origin. Of note, whether lack of RANKL production might cause a defect also in the bone marrow (BM) stromal compartment, possibly contributing to the pathology, is unknown. To verify this possibility, we generated and characterized BM mesenchymal stromal cell (BM-MSC) lines from wild type and Rankl-/- mice, and found that Rankl-/- BM-MSCs displayed reduced clonogenicity and osteogenic capacity. The differentiation defect was significantly improved by lentiviral transduction of Rankl-/- BM-MSCs with a vector stably expressing human soluble RANKL (hsRANKL). Expression of Rankl receptor, Rank, on the cytoplasmic membrane of BM-MSCs pointed to the existence of an autocrine loop possibly activated by the secreted cytokine. Based on the close resemblance of RANKL-defective osteopetrosis in humans and mice, we expect that our results are also relevant for RANKL-dependent ARO patients. Data obtained in vitro after transduction with a lentiviral vector expressing hsRANKL would suggest that restoration of RANKL production might not only rescue the defective osteoclastogenesis of this ARO form, but also improve a less obvious defect in the osteoblast lineage, thus possibly achieving higher benefit for the patients, when the approach is translated to clinics. Stem Cells 2017;35:1365-1377.

Relationship between insulin resistance, hs-CRP, and body fat and serum osteoprotegerin/RANKL in prediabetic patients

Receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPN) are soluble members of the tumor necrosis factor superfamily. Growing evidence suggest that there is link between inflammation, insulin resistance and OPG, soluble RANKL (sRANKL). We aimed to ascertain whether OPG and sRANKL levels are altered in prediabetic subjects and there is association between OPG/sRANKL and metabolic parameters. Forty prediabetic subjects and 40 age- and BMI-matched controls were recruited for this cross-sectional study. Circulating OPG, sRANKL were measured using ELISA. Anthropometric and metabolic parameters were also determined. Circulating sRANKL (97.74±17.67 vs. 55.00±11.19 pg/mL, P=0.010) and OPG (261.54±74.55 vs. 159.23±52.91 pg/mL, P=0.020) levels were found to be significantly higher in diabetic subjects compared with control subjects. There was a positive correlation between sRANKL and OPG. sRANKL also positively correlated with BMI, insulin resistance marker HOMA-IR, inflammatory marker hs-CRP. Logistic regression analyses revealed that the odds ratio was increased for prediabetes in subjects with having elevated sRANKL levels. Increased sRANKL and OPG levels were associated with prediabetic subjects. sRANKL and OPG may play a role in the pathogenesis of diabetes as well as metabolic disturbance.

Lutein Enhances Bone Mass by Stimulating Bone Formation and Suppressing Bone Resorption in Growing Mice.

Lutein is a member of the xanthophyll family of carotenoids, which are known to prevent hypoxia-induced cell damage in the eye by removing free radicals. However, its role in other tissues is controversial, and the effects of lutein on bone tissues are unknown. To identify a possible role of lutein in bone tissues, we examined the effects of lutein on bone formation and bone resorption and on femoral bone mass in mice. Lutein enhanced the formation of mineralized bone nodules in cultures of osteoblasts. On the other hand, lutein clearly suppressed 1α, 25-dihydroxyvitamin D3-induced bone resorption as measured by pit formation in organ culture of mouse calvaria. In co-cultures of bone marrow cells and osteoblasts, lutein suppressed 1α, 25-dihydroxyvitamin D3-induced osteoclast formation. In cultures of bone marrow macrophages, lutein suppressed soluble RANKL, the receptor activator of nuclear factor-kappaB (NF-κB) ligand, induced osteoclast formation. When five-week-old male mice were orally administered lutein for 4 weeks, the femoral bone mass was clearly enhanced in cortical bone, as measured by bone mineral density in dual X-ray absorptiometry and micro computed tomography (µCT) analyses. The present study indicates that lutein enhances bone mass in growing mice by suppressing bone resorption and stimulating bone formation. Lutein may be a natural agent that promotes bone turnover and may be beneficial for bone health in humans.

Lenalidomide, Adriamycin, and Dexamethasone (RAD) As Induction Therapy for Patients with Newly Diagnosed Multiple Myeloma Who Are Eligible for Autologous Transplantation (ASCT): A Phase 2 Study from the Greek Myeloma Study Group

RAD combination is highly effective for patients with relapsed/refractory multiple myeloma (MM), but there is limited data for its efficacy on newly-diagnosed patients (NDMM). Although lenalidomide exerts its function, at least partially, through the inhibition of interactions between myeloma and stromal cells, there is no information for the effect of RAD on bone metabolism and angiogenesis in NDMM. The primary endpoint of this phase 2 study was the assessment of overall response rate (ORR) after 4 cycles of RAD induction in NDMM patients who are eligible for ASCT. Secondary endpoints included toxicity, PFS, TTP, and TtNT. Exploratory endpoints included: i) the yield of stem cell collection after RAD; ii) the effects of RAD on biochemical markers of bone metabolism: CTX, TRACP-5b, bone-alkaline phosphatase (bALP), P1NP, osteocalcin, soluble RANKL, osteoprotegerin (OPG) and dickkopf-1 (Dkk-1) and iii) the effects of RAD on angiogenic cytokines: angiopoietin- (Angp) 1 & -2, angiogenin (Ang), VEGF and bFGF. Main inclusion criteria were: i) NDMM patients eligible for ΑSCT; ii) Κarnofsky performance status ≥ 60; iii) platelet count ≥100x10 9 /L, neutrophil count ≥1.5x10 9 /L. Main exclusion criteria included: i) serum ALT or AST >3-fold or bilirubin >2-fold the upper normal limit; ii) eGFR 2 , on days 1-4 of each cycle. RAD was repeated on day 28 if the neutrophil count was >1.5x10 9 /L and the platelet count was >50x10 9 /L. All patients received anti-coagulation with LMWH and monthly zoledronic acid. Between November 2014 and February 2016, 45 patients were enrolled. Median age was 56 years (range: 37-69 years); 28 (62%) patients had IgG, 12 (27%) IgA, 4 (9%) light chain only and one IgM MM; 18 (40%) patients had ISS-1, 22 (49%) ISS-2 and 5 (11%) ISS-3 disease. Osteolytic lesions were present in 33 (73%) patients. Seven (15.5%) patients had elevated serum LDH (>250 U/l) and 3 (6.6%) had hypercalcemia (>11 mg/dl). All but one patient completed 4 cycles of RAD. Best response included one (2.2%) CR, 8 (17.8%) VGPRs, 21 (46.7%) PRs, for an ORR of 66.7%, while 14 (31%) patients had stable disease and one progressed during the 4 th cycle of treatment. Adverse events of grade 3 or 4 included: anemia (4 patients, 9%), neutropenia (3 patients, 6.6%), febrile neutropenia (one patient), hypocalcemia (one patient), acute renal failure (one patient), pulmonary embolism (one patient), fatigue (one patient) and pathological fracture (one patient). Forty (89%) patients had adequate stem cell collection post-RAD induction (mean±SD: 8.94±6.50 x10 6 /kg CD34+ cells). Three patients failed to collect adequate number of stem cells and two patients refused to proceed to stem cell collection. Patients at baseline had elevated levels of CTX, TRACP-5b, sRANKL/OPG, Dkk-1, Ang, VEGF, VEGF-A, bFGF and reduced levels of Angp-1/Angp-2, bALP and P1NP compared to 30 healthy subjects of similar age and gender (p We conclude that RAD resulted in successful induction for NDMM patients and produced an ORR of approximately 67%. RAD reduced bone resorption and increased bone formation; the latter has not been previously described with lenalidomide-based regimens. Furthermore, RAD reduced angiogenic cytokines and this supports the action of the regimen also through the disruption of the interactions between myeloma and stromal cells. Disclosures Terpos: Celgene: Honoraria; Amgen: Consultancy, Honoraria, Other: Travel expenses, Research Funding; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: Travel expenses, Research Funding; BMS: Consultancy, Honoraria; Novartis: Honoraria; Genesis: Consultancy, Honoraria, Other: Travel expenses, Research Funding. Katodritou: Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Genesis: Honoraria, Research Funding; Takeda: Consultancy, Honoraria. Symeonidis: Amgen: Honoraria; Roche: Honoraria; Takeda: Consultancy, Honoraria; Genesis: Honoraria. Kourakli: Genesis: Consultancy, Honoraria. Kastritis: Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Genesis: Consultancy, Honoraria. Dimopoulos: Genesis: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees.

Soluble RANKL Cleaved from Activated Lymphocytes by TNF-α-Converting Enzyme Contributes to Osteoclastogenesis in Periodontitis.

Host immune responses play a key role in promoting bone resorption in periodontitis via receptor activator of NF-κB ligand (RANKL)-dependent osteoclastogenesis. Both membrane-bound RANKL (mRANKL) expressed on lymphocytes and soluble RANKL (sRANKL) are found in periodontal lesions. However, the underlying mechanism and cellular source of sRANKL release and its biological role in periodontitis are unclear. TNF-α-converting enzyme (TACE) is reported to cleave the following: 1) precursor TNF-α with release of mature, soluble TNF-α and 2) mRANKL with release of sRANKL. Both soluble TNF-α and sRANKL are found in the periodontitis lesion, leading to the hypothesis that TACE expressed on lymphocytes is engaged in RANKL shedding and that the resulting sRANKL induces osteoclastogenesis. In the current study, upon stimulating PBLs with mitogens in vitro, RANKL expression, sRANKL secretion, and TACE expression were all upregulated. Among the four putative mRANKL sheddases examined in neutralization assays, TACE was the only functional sheddase able to cleave mRANKL expressed on PBL. Moreover, PBL culture supernatant stimulated with mitogens in the presence of anti-TACE Ab or anti-RANKL Ab showed a marked reduction of osteoclastogenesis from osteoclast precursors, indicating that TACE-mediated sRANKL may possess sufficient osteoclastogenic activity. According to double-color confocal microscopy, B cells expressed a more pronounced level of RANKL and TACE expression than T cells or monocytes in periodontally diseased gingiva. Conditioned medium of patients' gingival lymphocyte culture increased in vitro osteoclastogenic activity, which was suppressed by the addition of anti-TACE Ab and anti-RANKL Ab. Therefore, TACE-mediated cleavage of sRANKL from activated lymphocytes, especially B cells, can promote osteoclastogenesis in periodontitis.

631. Rankl Knock-Out Mesenchymal Stromal Cells Have an Unexpected Osteogenic Differentiation Defect Which Is Improved by a RANKL-Expressing Lentiviral Vector

Osteoclast-poor RANKL-dependent Autosomal Recessive Osteopetrosis (ARO) is a rare bone disease characterized by an increase in bone density due to the failure of bone resorption by impaired osteoclast formation. Hematopoietic stem cell transplantation is not an effective therapy for this ARO form, since in bone RANKL is produced mainly by cells of mesenchymal origin. Therefore Mesenchymal Stromal Cells (MSC) transplantation together with a gene-therapy strategy to correct RANKL defect in MSC could represent a possible effective therapy. Of note, whether also MSC, besides the osteoclasts, are affected by RANKL deficiency is unknown. To verify this, we established and characterized bone marrow derived MSC (BM-MSC) lines from the Rankl−/− (KO) mouse model, which recapitulates the human disease, and from wild type (WT) mice. No differences were found between KO and WT MSC in terms of morphology, immunophenotype and proliferation capacity. However, KO MSC displayed a reduced clonogenic potential with a decrease in stemness genes expression. KO MSC were able to normally differentiate towards the adipogenic and chondrogenic lineages, while showed a significantly impaired osteogenic differentiation capacity compared to WT MSC, as demonstrated by reduced Alizarin Red staining (ARS) and expression of osteogenic genes. To confirm that this alteration was due to the lack of functional RANKL, we developed a third generation lentiviral vector expressing human soluble RANKL (hsRL) for the genetic correction of KO MSC. We first investigated lentiviral transduction in 293T cells to optimize transduction efficiency at different multiplicity of infection (MOI) ranging from 1 to 100. hsRL production increased proportionally to the MOI and was stable over time. However, the higher the MOI the higher the cytotoxicity observed. Based on these data, we performed a lentiviral hsRL transduction in KO MSC at 20 and 50 MOI, to define the optimal transduction conditions. After transduction 99.5% of MSC were GFP+. While in Rankl−/− control cells the cytokine was not detected, in corrected cells hsRL production and secretion was measurable and comparable to sRL levels in WT mouse. KO MSC stably expressing hsRL showed an improved osteogenic differentiation capacity compared to untransduced KO MSC, as demonstrated by increased ARS and expression of osteogenic genes. Moreover, the expression of RANK receptor in both MSC suggested an autocrine role of sRL as possible mechanism. Our data suggest that restoration of RANKL production in lentiviral-transduced KO MSC might not only allow osteoclast differentiation in Rankl−/− mice upon transplantation, but also improve the osteogenic differentiation defect of KO MSC.

Data in support of the bone analysis of NOD-SCID mice treated with zoledronic acid and prednisolone.

This paper reports data on the bone, specifically the tibia and mandible, of nonobese diabetic mice with severe combined immunodeficiency disease (NOD–SCID mice) treated with zoledronic acid (ZA) and prednisolone (PSL). The data described here are related to the research article titled “Zoledronic acid basically increases circulating soluble RANKL level in mice, and in glucocorticoid-administrated mice, more increases lymphocytes derived sRANKL by bacterial endotoxic stimuli” [1]. The present data and the NOD–SCID mice experiments described contain insights into the role of bone-remodeling factors induced by ZA treatment.

Zoledronic acid increases the circulating soluble RANKL level in mice, with a further increase in lymphocyte-derived soluble RANKL in zoledronic acid- and glucocorticoid-treated mice stimulated with bacterial lipopolysaccharide

The nitrogen-containing bisphosphonate (BP) zoledronic acid (ZA) is a potent antiresorptive drug used in conjunction with standard cancer therapy to treat osteolysis or hypercalcemia due to malignancy. However, it is unclear how ZA influences the circulating levels of bone remodeling factors. The aim of this study was to evaluate the effects of ZA on the serum levels of soluble receptor activator of NF-kB ligand (sRANKL) and osteoprotegerin (OPG). The following four groups of C57BL/6 mice were used (five mice per group): (1) the placebo+phosphate-buffered saline (PBS) group, in which placebo-treated mice were injected once weekly with PBS for 4weeks; (2) the placebo+ZA group, in which placebo-treated mice were injected once weekly with ZA for 4weeks; (3) the prednisolone (PSL)+PBS group, in which PSL-treated mice were injected once weekly with PBS for 4weeks; and (4) the PSL+ZA group, in which PSL-treated mice were injected once weekly with ZA for 4weeks. At the 3-week time point, all mice were subjected to oral inflammatory stimulation with bacterial lipopolysaccharide (LPS). The sera of these mice were obtained every week and the levels of sRANKL and OPG were measured using enzyme-linked immunosorbent assay. At the time of sacrifice, femurs were prepared for micro-computed tomography (micro-CT), histological, and histomorphometric analyses. Our data indicated that ZA administration remarkably reduced bone turnover and significantly increased the basal level of sRANKL. Interestingly, the PSL+ZA group showed a dramatically elevated sRANKL level after LPS stimulation. In contrast, the PSL+ZA group in nonobese diabetic mice with severe combined immunodeficiency disease (NOD-SCID mice), which are characterized by the absence of functional T- and B-lymphocytes, showed no increase in the sRANKL level. Our data suggest that, particularly with combination treatment of ZA and glucocorticoids, surviving lymphocytes might be the source of inflammation-induced sRANKL. Thus, circulating sRANKL levels might be modulated by ZA.

RANKL-Independent Osteoclastogenesis: A Long-Standing Controversy.

RANKL-Independent Osteoclastogenesis: A Long-Standing Controversy.

Changes in RANKL and osteoprotegerin expression after chronic exposure to indoor air pollution as a result of cooking with biomass fuel.

The impact of indoor air pollution as a result of cooking with unprocessed biomass on membrane-bound and serum receptor activator of nuclear factor-kappa ligand 1 (RANKL), its soluble decoy receptor osteoprotegerin (OPG) and osteoclast precursor CD14(+) CD16(+) monocytes was investigated. Seventy-four pre-menopausal women from eastern India using biomass and 65 control women who cooked with cleaner liquefied petroleum gas were enrolled. PM10 and PM2.5 levels in their indoor air were measured with real-time aerosol monitors. The levels of membrane-bound RANKL on leukocytes and percentage CD14(+) CD16(+) monocytes in the subjects' blood were assayed by flow cytometry. Soluble RANKL and OPG in serum were measured by ELISA. The results showed that PM10 and PM2.5 levels were significantly higher in the indoor air of biomass-using households. Compared with the control women, the levels of CD4(+) and CD19(+) lymphocytes and circulating granulocytes with elevated levels of membrane-bound RANKL were higher in biomass users. The serum levels of RANKL were increased by 41% whereas serum OPG was reduced by 22% among biomass users. The absolute number of CD14(+) CD16(+) monocytes was significantly increased in biomass users than the control women. After controlling for potential confounders, PM10 and PM2.5 levels were found to be positively associated with leukocyte and serum RANKL and CD14(+) CD16(+) monocyte levels, but negatively with serum OPG. From these results, we can conclude that chronic exposure to biomass smoke increased membrane-bound and soluble RANKL and circulating osteoclast precursors but decreased OPG, suggesting an increased risk of bone resorption and consequent osteoporosis in biomass-exposed women of a child-bearing age. Copyright © 2015 John Wiley & Sons, Ltd.

Unique Distal Enhancers Linked to the Mouse Tnfsf11 Gene Direct Tissue-Specific and Inflammation-Induced Expression of RANKL.

Receptor activator of nuclear factor κB ligand (RANKL) is expressed by a number of cell types to participate in diverse physiological functions. We have previously identified 10 distal RANKL enhancers. Earlier studies have shown that RL-D5 is a multifunctional RANKL enhancer. Deletion of RL-D5 from the mouse genome leads to lower skeletal and lymphoid tissue RANKL, causing a high bone mass phenotype. Herein, we determine the physiological role and lineage specificity of 2 additional RANKL enhancers, RL-D6 and RL-T1, which are located 83 and 123 kb upstream of the gene's transcriptional start site, respectively. Lack of RL-D6 or RL-T1 did not alter skeletal RANKL or bone mineral density up to 48 weeks of age. Although both RL-D5 and RL-T1 contributed to activation induction of T-cell RANKL, RL-T1 knockout mice had drastically low lymphocyte and lymphoid tissue RANKL levels, indicating that RL-T1 is the major regulator of lymphocyte RANKL. Moreover, RL-T1 knockout mice had lower circulating soluble RANKL, suggesting that lymphocytes are important sources of circulating soluble RANKL. Under physiological conditions, lack of RL-D6 did not alter RANKL expression. However, lack of RL-D5 or RL-D6, but not of RL-T1, blunted the oncostatin M and lipopolysaccharide induction of RANKL ex vivo and in vivo, suggesting that RL-D5 and RL-D6 coregulate the inflammation-mediated induction of RANKL in osteocytes and osteoblasts while lack of RL-D6 did not alter secondary hyperparathyroidism or lactation induction of RANKL or bone loss. These results suggest that although RL-D5 mediates RANKL expression in multiple lineages, other cell type- or factor-specific enhancers are required for its appropriate control, demonstrating the cell type-specific and complex regulation of RANKL expression.

Topography Influences Adherent Cell Regulation of Osteoclastogenesis

The importance of osteoclast-mediated bone resorption in the process of osseointegration has not been widely considered. In this study, cell culture was used to investigate the hypothesis that the function of implant-adherent bone marrow stromal cells (BMSCs) in osteoclastogenesis is influenced by surface topography. BMSCs isolated from femur and tibia of Sprague-Dawley rats were seeded onto 3 types of titanium surfaces (smooth, micro, and nano) and a control surface (tissue culture plastic) with or without osteogenic supplements. After 3 to 14 d, conditioned medium (CM) was collected. Subsequently, rat bone marrow–derived macrophages (BMMs) were cultured in media supplemented with soluble receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) as well as BMSC CM from each of the 4 surfaces. Gene expression levels of soluble RANKL, osteoprotegerin, tumor necrosis factor α, and M-CSF in cultured BMSCs at different time points were measured by real-time polymerase chain reaction. The number of differentiated osteoclastic cells was determined after tartrate-resistant acid phosphatase staining. Analysis of variance and t test were used for statistical analysis. The expression of prominent osteoclast-promoting factors tumor necrosis factor α and M-CSF was increased by BMSCs cultured on both micro- and nanoscale titanium topographies (P < 0.01). BMSC CM contained a heat-labile factor that increased BMMs osteoclastogenesis. CM from both micro- and nanoscale surface-adherent BMSCs increased the osteoclast number (P < 0.01). Difference in surface topography altered BMSC phenotype and influenced BMM osteoclastogenesis. Local signaling by implant-adherent cells at the implant-bone interface may indirectly control osteoclastogenesis and bone accrual around endosseous implants.

Myristoleic acid inhibits osteoclast formation and bone resorption by suppressing the RANKL activation of Src and Pyk2

Cytoskeletal changes in osteoclasts such as formation of actin ring is required for bone-resorbing activity. The tyrosine kinase Src is a key player in massive cytoskeletal change of osteoclasts, thereby in bone destruction. In order for Src to be activated, trafficking to the inner plasma membrane via myristoylation is of importance. A previous study reported that myristoleic acid derived from myristic acid, inhibited N-myristoyl-transferase, an essential enzyme for myristoylation process. This prompted us to investigate whether myristoleic acid could affect osteoclastogenesis. Indeed, we observed that myristoleic acid inhibited RANKL-induced osteoclast formation in vitro, especially, at later stages of differentiation. Myristoleic acid attenuated the tyrosine phosphorylation of c-Src and Pyk2, which associates with Src, by RANKL. When myristoleic acid was co-administered with soluble RANKL into mice, RANKL-induced bone loss was substantially prevented. Bone dissection clearly revealed that the number of multinucleated osteoclasts was significantly diminished by myristoleic acid. On the other hand, myristoleic acid treatment had little or no influence on early osteoclast differentiation markers, such as c-Fos and NFATc1, and proteins related to cytoskeletal rearrangement, including DC-STAMP, integrin αv and integrin β3 in vitro. Taken together, our data suggest that myristoleic acid is capable of blocking the formation of large multinucleated osteoclasts and bone resorption likely through suppressing activation of Src and Pyk2.

RANKL/RANK interaction promotes the growth of cervical cancer cells by strengthening the dialogue between cervical cancer cells and regulation of IL-8 secretion.

Receptor activator for nuclear factor κB ligand (RANKL) is a member of the tumor necrosis factor (TNF) family. The interaction between RANKL and its receptor RANK plays an important role in the development and function of diverse tissues. However, the expression and role of RANKL in cervical cancer are still unknown. In the present study, we found that RANKL and RANK were highly co-expressed in cervical cancer. HeLa and SiHa cells secreted soluble RANKL (sRANKL), expressed member RANKL (mRANKL) and RANK. Recombinant human RANKL protein had no effect on the viability of HeLa and SiHa cells. Yet, blocking RANKL with an anti-human RANKL neutralizing antibody (α-RANKL) or recombinant human osteoprotegrin (OPG) protein resulted in the downregulation of Ki-67 and B-cell lymphoma 2 (Bcl-2) expression and an increase in Fas and Fas ligand (FasL) expression, as well as a high level of viability and a low level of apoptosis in the HeLa and SiHa cells. In addition, α-RANKL led to a decrease in IL-8 secretion. Recombinant human IL-8 protein reversed the effect of α-RANKL on the expression of proliferation- and apoptosis‑related molecules, and proliferation and apoptosis in the HeLa and SiHa cells. The present study suggests that a high level of mRANKL/RANK expression in cervical cancer lesions plays an important role in the rapid growth of cervical cancer cells possibly through strengthening the dialogue between cervical cancer cells and regulation of IL-8 secretion, which may be a possible target for cervical cancer therapy.

Overexpression of Arl6ip5 in osteoblast regulates RANKL subcellualr localization

The osteoblastic expression of RANKL, which is essential for the communication between osteoblastic cells and osteoclastogenic cells, is stimulated by locally acting or circulating osteotropic cytokines and hormones such as PTH and 1,25-(OH)2-D3 during the bone remodeling process. However, mechanisms those control subcellular trafficking events, membrane expression and extracellular secretion of the newly synthesized RANKL are still not well understood. In our previous study, we have found that the deficiency of osteoblastic Arl6ip5 (ADP-ribosylation-like factor 6 interacting protein 5), an endoplasmic reticulum (ER)-localized protein belonging to the prenylated rab-acceptor-family, enhanced osteoclastogenesis by increasing RANKL transcription in an ER stress dependent signaling. Here we found that over-expression of hemagglutinin (HA)-tagged Arl6ip5 in UAMS32 stromal/osteoblastic cells inhibited osteoclastogenesis, decreased the amount of soluble RANKL in culture supernatant and increased RANKL retention in ER. Moreover, Arl6ip5 bound with RANKL and disturbed the RANKL-OPG complex in UAMS-32 cells. Finally, 1 to 36 amino acid deletion on the NH2 lumen terminus of Arl6ip5 impaired the interaction between Arl6ip5 and RANKL, restored the level of soluble RANKL and the osteoclastogenic ability. These findings indicated that Arl6ip5 was an anti-catabolic factor by binding with RANKL and disturbing its subcellular trafficking in osteoblast.

Denosumab versus zoledronic acid in patients previously treated with zoledronic acid.

Denosumab and zoledronic acid are potent antiresorptives. In this study in patients pre-treated with zoledronic acid, denosumab achieved similar increases with zoledronic acid in lumbar spine BMD despite the more prominent reduction of bone turnover markers. Denosumab reversibly reduced endogenous RANKL. We aimed to compare yearly changes in lumbar spine (LS) bone mineral density (BMD), bone turnover markers, free soluble receptor activator of nuclear factor kappaB ligand (sRANKL) and sclerostin levels between denosumab and zoledronic acid. Postmenopausal women with low bone mass previously treated with zoledronic acid for 1 year were assigned to denosumab injection (n = 32) or zoledronic acid infusion (n = 26). Procollagen type 1 N-terminal propeptide (P1NP), C-terminal cross-linking telopeptide of type 1 collagen (CTx), sRANKL, and sclerostin levels were measured in serum samples obtained at baseline and 3, 6, and 12 months after denosumab injection or zoledronic acid infusion. LS BMD was measured at baseline and 12 months. The mean LS increase was 4.5 and 4.4% with denosumab and zoledronic acid, respectively (p = 0.560). Denosumab caused a larger decrease in CTx at 3 months (p < 0.001) and P1NP at 3 (p < 0.001), 6 (p = 0.021), and 12 months (p = 0.042). Denosumab significantly decreased sRANKL by 87.4% at 3 months (p < 0.001). Sclerostin levels were not changed with either intervention (p = 0.162 and p = 0.214, respectively). In patients previously treated with zoledronic acid, denosumab reduces bone turnover more than zoledronic acid, but the increases in LS BMD are comparable. Furthermore, denosumab administration results in reversible inhibition of the metabolically significant endogenous free soluble RANKL levels. Serum sclerostin is not affected by either agent.