Receptor Activator Of Nuclear Factor Kappa Beta Ligand Research Articles

New mechanistic understanding of osteoclast differentiation and bone resorption mediated by P2X7 receptors and PI3K-Akt-GSK3β signaling.

Osteoporosis is a global health issue characterized by decreased bone mass and microstructural degradation, leading to an increased risk of fractures. This study aims to explore the molecular mechanism by which P2X7 receptors influence osteoclast formation and bone resorption through the PI3K-Akt-GSK3β signaling pathway. An osteoporosis mouse model was generated through ovariectomy (OVX) in normal C57BL/6 and P2X7f/f; LysM-cre mice. Osteoclasts were isolated for transcriptomic analysis, and differentially expressed genes were selected for functional enrichment analysis. Metabolite analysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and multivariate statistical analysis and pattern recognition were used to identify differential lipid metabolism markers and their distribution. Bioinformatics analyses were conducted using the Encyclopedia of Genes and Genomes database and the MetaboAnalyst database to assess potential biomarkers and create a metabolic pathway map. Osteoclast precursor cells were used for in vitro cell experiments, evaluating cell viability and proliferation using the Cell Counting Kit 8 (CCK-8) assay. Osteoclast precursor cells were induced to differentiate into osteoclasts using macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-beta ligand (RANKL), and tartrate-resistant acid phosphatase (TRAP) staining was performed to compare differentiation morphology, size, and quantity between different groups. Western blot analysis was used to assess the expression of differentiation markers, fusion gene markers, and bone resorption ability markers in osteoclasts. Immunofluorescence staining was employed to examine the spatial distribution and quantity of osteoclast cell skeletons, P2X7 protein, and cell nuclei, while pit assay was used to evaluate osteoclast bone resorption ability. Finally, in vivo animal experiments, including micro computed tomography (micro-CT), hematoxylin and eosin (HE) staining, TRAP staining, and immunohistochemistry, were conducted to observe bone tissue morphology, osteoclast differentiation, and the phosphorylation level of the PI3K-Akt-GSK3β signaling pathway. Transcriptomic and metabolomic data collectively reveal that the P2X7 receptor can impact the pathogenesis of osteoporosis through the PI3K-Akt-GSK3β signaling pathway. Subsequent in vitro experiments showed that cells in the Sh-P2X7 + Recilisib group exhibited increased proliferative activity (1.15 versus 0.59), higher absorbance levels (0.68 versus 0.34), and a significant increase in resorption pit area (13.94 versus 3.50). Expression levels of osteoclast differentiation-related proteins MMP-9, CK, and NFATc1 were markedly elevated (MMP-9: 1.72 versus 0.96; CK: 2.54 versus 0.95; NFATc1: 3.05 versus 0.95), along with increased fluorescent intensity of F-actin rings. In contrast, the OE-P2X7 + LY294002 group showed decreased proliferative activity (0.64 versus 1.29), reduced absorbance (0.34 versus 0.82), and a significant decrease in resorption pit area (5.01 versus 14.96), accompanied by weakened expression of MMP-9, CK, and NFATc1 (MMP-9: 1.14 versus 1.79; CK: 1.26 versus 2.75; NFATc1: 1.17 versus 2.90) and decreased F-actin fluorescent intensity. Furthermore, in vivo animal experiments demonstrated that compared with the wild type (WT) + Sham group, mice in the WT + OVX group exhibited significantly increased levels of CTX and NTX in serum (CTX: 587.17 versus 129.33; NTX: 386.00 versus 98.83), a notable decrease in calcium deposition (19.67 versus 53.83), significant reduction in bone density, increased trabecular separation, and lowered bone mineral density (BMD). When compared with the KO + OVX group, mice in the KO + OVX + recilisib group showed a substantial increase in CTX and NTX levels in serum (CTX: 503.50 versus 209.83; NTX: 339.83 versus 127.00), further reduction in calcium deposition (29.67 versus 45.33), as well as decreased bone density, increased trabecular separation, and reduced BMD. P2X7 receptors positively regulate osteoclast formation and bone resorption by activating the PI3K-Akt-GSK3β signaling pathway.

Abstract 178: Adipose tissue upregulates expression of RANKL in B-cell acute lymphoblastic leukemia through prostaglandin E2 signaling

Abstract Obesity increases the risk of developing cancer and worsens treatment outcome. Our lab has shown that adipose tissue contributes to B-cell acute lymphoblastic leukemia (B-ALL) resistance to chemotherapy both in vitro and in vivo. However, the mechanisms by which adipose tissue causes ALL cell resistance are complex and not completely understood. We therefore implanted obese mice (n=3) with syngeneic BCR/ABL B-ALL cells and performed single-cell RNA sequencing (scRNAseq) of ALL collected from epididymal adipose tissue vs. bone marrow. Our data uncovered a strong adipose microenvironment effect on the ALL transcriptome, identifying 577 differentially expressed genes using a log2 fold threshold of 0.25 and Bonferroni corrected p-values <0.05. Notably, we found that ALL cells in adipose tissue upregulate expression of Tnfsf11, the gene that encodes receptor activator of nuclear factor kappa beta ligand (RANKL). 93% of ALL cells in adipose tissue express RANKL compared to 33% in marrow, along with a log2 fold expression increase of 2.12 (p<1 × 10−100, Wilcoxon test). Because ALL expression of RANKL has been linked to bone resorption and CNS invasion, both of which have increased incidence in obese patients, we elected to explore this further. Ex vivo culture with human visceral adipose tissue validated ALL cell RANKL upregulation, demonstrating a 1.63 and 17.5-fold increase in gene expression after 48 hours in BV173 and RS4;11 B-ALL cell lines, respectively (n=4-6, both p<0.01, paired t test). Soluble RANKL was not detected in media from ALL cells cultured alone, detectable in media conditioned by human adipose tissue (0.018 pM), but increased in media from adipose-ALL cocultures (RS4;11: 0.065 pM, BV173: 0.317 pM, n=1). One potential candidate signal to stimulate ALL cell RANKL expression is the inflammatory mediator prostaglandin E2 (PGE2), which is known to be released by adipose tissue. Physiological concentrations of PGE2 (200 ng/mL) caused a similar increase in ALL cell RANKL gene expression as adipose tissue in both cell lines (n=6). PGE2 treatment also caused a mild resistance of ALL cells to daunorubicin chemotherapy in vitro, increasing the EC50 from 27±4 to 53±12 nM in RS4;11 (n=6, p<0.05), and from 39±11 to 62±19 nM in BV173 (n=6, p=0.06). Adipose tissue induction of RANKL expression was partially blocked by the addition of PGE2 receptor EP2 and EP4 blockers. Taken together, our results suggest that PGE2 produced by the adipose microenvironment activates ALL cell RANKL expression, which could potentially contribute to CNS invasion, bone breakdown, and chemoresistance. Citation Format: Michael Cohen, Jia Tan, Tyler Kuk, Blesscamal Nadeak, In Sook Ahn, Xia Yang, Etan Orgel, Steven D. Mittelman. Adipose tissue upregulates expression of RANKL in B-cell acute lymphoblastic leukemia through prostaglandin E2 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 178.

Osteogenic Differentiation and Proliferation of Apical Papilla Stem Cells Using Chitosan-Coated Nanohydroxyapatite and Bioactive Glass Nanoparticles.

The aim of this study was to evaluate the osteogenic differentiation ability and proliferation of apical papilla stem cells (SCAPs) using chitosan-coated nanohydroxyapatite and bioactive glass nanoparticles. Hydroxyapatite, chitosan-coated nanohydroxyapatite, and bioactive glass 45S5 nanoparticles were prepared and characterized using a transmission electron microscope and X-ray diffraction. SCAPs were harvested from freshly extracted impacted wisdom teeth, cultured, and characterized using flow cytometric analysis. Tested nanomaterials were mixed and samples were classified into five equal groups as follows: negative control group: SCAP with Dulbecco's modified eagle's medium, positive control group: SCAP with inductive media, first experimental group: nanohydroxyapatite with SCAP, second experimental group: chitosan-coated nanohydroxyapatite with SCAP, third experimental group: bioactive glass nanoparticles with SCAP. Osteoblastic differentiation was assessed using an alkaline phosphatase (ALP) assay. Receptor activator of nuclear factor kappa beta ligand (RANKL) expression was evaluated using specific polyclonal antibodies by fluorescence microscope. The proliferation of SCAP was assessed using cell count and viability of trypan blue in addition to an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Isolated SCAP showed a nonhematopoietic origin. Chitosan-coated nanohydroxyapatite showed the highest ALP concentration followed by nanobioactive glass, nanohydroxyapatite, and negative control. Chitosan-coated nanohydroxyapatite showed the highest H score followed by nanobioactive glass, nanohydroxyapatite, and negative control in RANKL expression. Chitosan-coated nanohydroxyapatite showed the highest viable cell count. SCAP isolation is achievable from extracted fully impacted immature third molars. All tested biomaterials have the ability to induce osteogenic differentiation and proliferation of SCAP. Composite nanoparticle materials show better osteogenic differentiation and proliferation of SCAP than single nanoparticles.

A chlorogenic acid-conjugated nanomicelle attenuates disease severity in experimental arthritis.

Rheumatoid arthritis (RA) is a systemic immune disorder marked by synovitis, bone damage, and cartilage erosion, leading to increased socio-economic burdens and reduced quality of life. Despite its unknown cause, advancements in understanding its pathophysiology have facilitated novel therapeutic approaches. Current treatments, including disease-modifying anti-rheumatic drugs (DMARDs) and biologics, often result in low efficacy and unnecessary side effects. To address the limitations of these drugs, carrier-based drug delivery systems, such as nanomicelles, have emerged as a promising solution. In this study, nanomicelles were synthesised utilizing PLGA (poly(lactic-co-glycolic acid)) as a backbone; this backbone is conjugated with chlorogenic acid (CGA), which is known for suppressing inflammation, and incorporates methotrexate (MTX), a model drug that is established for RA treatment. The nanomicelles were extensively characterized in terms of size, charge, drug loading, and drug-release behaviour. The in vivo assessment of MTX-PLGA-b-CGA nanomicelles in a collagen-induced arthritis model demonstrated a remarkable reduction in joint swelling, cartilage erosion, and disease severity. Furthermore, histological findings confirmed cartilage integrity and reduced expression of key pro-inflammatory markers, including receptor activator of nuclear factor kappa beta ligand (RANKL) and tumor necrosis factor (TNF-α). The approach based on the MTX-PLGA-b-CGA nanomicelles presents a biocompatible and potentially effective therapeutic strategy for management of the severity and progression of RA, providing a hopeful alternative for RA treatment.

Ugonin L inhibits osteoclast formation and promotes osteoclast apoptosis by inhibiting the MAPK and NF-κB pathways

Bone loss is a major issue for patients with osteoporosis, arthritis, periodontitis, and bone metastasis; however, anti-resorption drugs used to treat bone loss have been linked to a variety of adverse effects. Helminthostachys zeylanica (L.) Hook, belonging to the family Ophioglossaceae, is commonly used in traditional Chinese medicine to treat inflammation and liver problems. In the current study, ugonin L extracted from H. zeylanica was shown to reduce the receptor activator of nuclear factor kappa beta ligand (RANKL)-induced osteoclastogenesis in RAW264.7 cells in a concentration-dependent manner. Ugonin L treatment also inhibited the mRNA expression of osteoclast markers. Ugonin L was also shown to promote cell apoptosis in mature osteoclasts and suppress RANKL-induced ERK, p38, JNK, and NF-κB activation. Taken together, ugonin L appears to be a promising candidate for the development of novel anti-resorption therapies.

Vibration therapy as an effective approach to improve bone healing in diabetic rats.

ObjectiveTo investigate the effects of vibration therapy on fracture healing in diabetic and non-diabetic rats.Methods148 rats underwent fracture surgery and were assigned to four groups: (1) SHAM: weight-matched non-diabetic rats, (2) SHAM+VT: non-diabetic rats treated with vibration therapy (VT), (3) DM: diabetic rats, and (4) DM+VT: diabetic rats treated with VT. Thirty days after diabetes induction with streptozotocin, animals underwent bone fracture, followed by surgical stabilization. Three days after bone fracture, rats began VT. Bone healing was assessed on days 14 and 28 post-fracture by serum bone marker analysis, and femurs collected for dual-energy X-ray absorptiometry, micro-computed tomography, histology, and gene expression.ResultsOur results are based on 88 animals. Diabetes led to a dramatic impairment of bone healing as demonstrated by a 17% reduction in bone mineral density and decreases in formation-related microstructural parameters compared to non-diabetic control rats (81% reduction in bone callus volume, 69% reduction in woven bone fraction, 39% reduction in trabecular thickness, and 45% in trabecular number). These changes were accompanied by a significant decrease in the expression of osteoblast-related genes (Runx2, Col1a1, Osx), as well as a 92% reduction in serum insulin-like growth factor I (IGF-1) levels. On the other hand, resorption-related parameters were increased in diabetic rats, including a 20% increase in the callus porosity, a 33% increase in trabecular separation, and a 318% increase in serum C terminal telopeptide of type 1 collagen levels. VT augmented osteogenic and chondrogenic cell proliferation at the fracture callus in diabetic rats; increased circulating IGF-1 by 668%, callus volume by 52%, callus bone mineral content by 90%, and callus area by 72%; and was associated with a 19% reduction in circulating receptor activator of nuclear factor kappa beta ligand (RANK-L).ConclusionsDiabetes had detrimental effects on bone healing. Vibration therapy was effective at counteracting the significant disruption in bone repair induced by diabetes, but did not improve fracture healing in non-diabetic control rats. The mechanical stimulus not only improved bone callus quality and quantity, but also partially restored the serum levels of IGF-1 and RANK-L, inducing bone formation and mineralization, thus creating conditions for adequate fracture repair in diabetic rats.

Bone Marrow Mesenchymal Stem Cell-Derived Small Extracellular Vesicles Promote Periodontal Regeneration.

Bone marrow mesenchymal stem cell-derived small extracellular vesicles (BMSC-sEVs) can be used as a potential cell-free strategy for periodontal tissue regeneration, and we aim to investigate the effect and possible mechanism of BMSC-sEV in periodontal tissue regeneration in this study. The BMSC-sEV was isolated by the Exosome Isolation™ reagent and identified by transmission electron microscopy, nanoparticle tracking analysis, and Western blotting. The human periodontal ligament cells (hPDLCs) were cocultured with BMSC-sEV in vitro to detect the effects of BMSC-sEV on hPDLC migration, proliferation, and differentiation. The BMSC-sEV loaded by hydrogel was injected into experimental periodontitis rats to verify the therapeutic effect and possible mechanism. The results showed that BMSC-sEVs were 30-150 nm vesicles and expressed the exosome protein CD63 and tumor susceptibility 101 (TSG101), which could promote the migration, proliferation, osteogenic differentiation of hPDLCs. The BMSC-sEV-hydrogel had a therapeutic effect on periodontitis rats. After administration for 4-8 weeks, microcomputed tomography and histological analysis showed that alveolar bone loss, inflammatory infiltration, and collagen destruction in the BMSC-sEV-hydrogel group were less than that in the phosphate-buffered saline (PBS)-hydrogel group and periodontitis group. Further immunohistochemical and immunofluorescent staining revealed that the number of tartrate-resistant acid phosphatase-positive cells and the expression ratio of osteoprotegerin (OPG) and receptor-activator of nuclear factor kappa beta ligand (RANKL) in the BMSC-sEV-hydrogel group were lower than that in the PBS-hydrogel group and periodontitis group, while the expression of transforming growth factor-beta 1 (TGF-β1) and the ratio of macrophage type 2 and macrophage type 1 (M2/M1) were opposite. Therefore, BMSC-sEV can promote the regeneration of periodontal tissues, and that may be partly due to BMSC-sEV involvement in the OPG-RANKL-RANK signaling pathway to regulate the function of osteoclasts and affect the macrophage polarization and TGF-β1 expression to modulate the inflammatory immune response, thereby inhibiting the development of periodontitis and immune damage of periodontal tissue. Impact statement Bone marrow mesenchymal stem cell-derived small extracellular vesicles (BMSCs-sEVs) have been proven to have similar functions to BMSCs, such as promoting the regeneration of heart, liver, kidney, and bone tissue and regulating immune responses. BMSCs are candidate seed cells of periodontal regeneration, but it is unclear about the role of BMSC-sEV on periodontal regeneration. In this study, we explored the effects and possible mechanism of BMSC-sEV on periodontal regeneration. The results of this study provide the evidence of BMSC-sEV as a cell-free strategy for periodontal regeneration.

Evaluation of gingival crevicular fluid and peri-implant crevicular fluid levels of sclerostin, TWEAK, RANKL and OPG

BackgroundThe combination of local and systemic factors play role in the pathogenesis of periodontal and peri-implant diseases. Host-derived enzymes, cytokines and other proinflammatory mediators play an integral role in this destruction. The aim of this study is to evaluate gingival crevicular fluid (GCF) and peri-implant crevicular (PICF) fluid levels of sclerostin, TNF-related weak inducer of apoptosis (TWEAK), receptor activator of nuclear factor kappa-beta ligand (RANKL) and osteoprotegerin OPG in periodontal and peri-implant tissues in disease and health conditions and also to assess the potential for use as biomarkers. Materials and methodsThe study population was consisted of 50 women and 41 men, in the total of 91 individuals, with a mean age of 51.84 ± 14.05. Periodontitis (n = 22), periodontal health (n = 17), peri-implantitis (n = 27) and peri-implant health (n = 25) groups were established according to clinical and radiographic examination results of 39 teeth and 52 implants restored with fixed prosthetic restorations. In all groups, periodontal and peri-implant parameters (probing depth, gingival recession, gingival bleeding time index, gingival index, and plaque index) were recorded and GCF and PICF samples were also collected. Sclerostin, TWEAK, RANKL and OPG levels in GCF and PICF were measured with ELISA tests. ResultsPeri-implantitis group presented significantly higher levels of Sclerostin (p = 0.002), TWEAK(p < 0.0001), RANKL(p < 0.0001), and OPG (p = 0.037) compared to peri-implant health group. Similarly, significantly higher levels of TWEAK (p = 0.001), RANKL(p < 0.0001), and OPG(p = 0.025) were detected in periodontitis group when compared to periodontal health group. Statistically significant correlations were also noted between biochemical parameters and clinical parameters. ConclusionFindings of this study evaluating four different bone metabolism related proteins at the same time, suggests levels of sclerostin may be a biomarker for peri-implant disease presenting significantly higher levels in the peri-implantitis group than in the peri-implant health group. Moreover, levels of TWEAK can be a good indicator for both periodontal and peri-implant disease, due to the correlations with periodontal clinical parameters and the higher levels of TWEAK in diseased sites compared to the healthy sites for both dental implants and teeth.

Russian federal clinical guidelines on the diagnostics, treatment, and prevention of osteoporosis

Screening using the Fracture Risk Assessment Tool (FRAX) is recommended in all postmenopausal woman and mеn over 50 (A1) in order to identify individuals with high probability of fractures. It is recommended to diagnose osteoporosis and start treatment in patients with fragility fracture of large bones of the skeleton and/or high individual probability of major fragility fractures (FRAX) and/or detected decrease in bone mineral density (BMD) up to –2.5 T-score as assessed by DXA in the femoral neck and/or lumbar vertebrae (A1). Patients with back pain, lifetime height loss of 4 cm or height loss of 2 cm since a previous medical examination, those who receive glucocorticoids, patients with long lasting decompensated type 2 diabetes mellitus, or those receiving insulin therapy, as well as patients who were previously diagnosed with fragility fractures at the other sites are advised to underwent standard lateral X-ray imaging of the spine (Th4—L5) in order to verify the presence of compression vertebral fractures (B1).&#x0D; Dual-energy X-ray absorptiometry (DXA) is recommended for individuals whose 10-year probability of major osteoporotic fracture (FRAX) falls within the medium risk group (B1). It is recommended to include the trabecular bone score (TBS) the FRAX algorithm in order to improve the sensitivity of this method (B1).&#x0D; Laboratory testing is recommended for the differential diagnosis with other causes of increased skeletal fragility in all patients with newly diagnosed osteoporosis and when previously prescribed antiosteoporostic treatment was ineffective (B1).&#x0D; Bisphosphonates (BPs), antibodies to receptor activator of nuclear factor kappa-beta ligand (RANKL) (denosumab), or parathyroid hormone analogue (teriparatide) are equally recommended to prevent fragility fractures and increase BMD in patients with osteoporosis (A1). Denosumab is also recommended to prevent BMD loss and fractures in females receiving aromatase inhibitors therapy for breast cancer and males with prostate cancer receiving hormone-deprivation therapy and having no bone metastases (A1). Since teriparatide has the anabolic effect, it is recommended as the first line treatment in patients with severe osteoporosis having history of vertebral fractures, in the individuals with very high risk of fragility fractures, or in the cases when antiresorptive treatment was ineffective (B1). All medications for treatment of osteoporosis are recommended in combination with calcium and vitamin D supplements (A1).

Giant Cells Tumour

Background: The first line of treatment of Giant Cells Tumor (GCT) consists in the curettage and coverage with bone graft. New treatments as Denosumab have shown great results as adjuvants in its treatment, and is gaining popularity in the treatment, especially because the high rates of local recurrence after surgery and the limitations of the technique in very aggressive tumors, or in those located in complex regions. This monoclonal antibody inhibits Receptor Activator of Nuclear Factor Kappa Beta Ligand (RANKL) and can only be administered in skeletally mature patients, so it is contraindicated during pregnancy. There are other adjuvant therapies to surgery, such as cryotherapy with liquid nitrogen or the phenolization, which can be equally effective. Methods: We present the case of a pregnant woman of 28 years old diagnosed with an aggressive GCT of proximal tibia during the first trimester of pregnancy. On this occasion, surgical treatment was performed by curettage and high speed milling. Adjuvant treatment was added with liquid nitrogen and the cavity was filled in with demineralized bone matrix and hydroxyapatite. Based on our experience and on a revision of the literature, there are few cases described of the treatment of a GCT in a woman during the pregnancy. Results: The patient's symptoms disappeared after the intervention. There have not been shown signs of recurrence after 10 months of clinical follow-up and simple radiology controls. The treatment did not produce adverse effects in the fetus during pregnancy neither breastfeeding. Conclusions: Indications of Denosumab in the treatment of GCT include unresectable tumors or those in which its resection leads to high morbidity, as well as tumors in the spine, sacrum, pelvis and challenging lesions located in the upper and lower extremities. If we cannot opt for this drug for medical or other reasons, cryotherapy with liquid nitrogen may be effective in its treatment

Influence of obesity on experimental periodontitis in rats: histopathological, histometric and immunohistochemical study.

This study assessed the influence of obesity on the progression of ligature-induced periodontitis in rats. Forty-eight adult Wistar rats were randomly divided into two groups: the HL group (n=24) was fed high-fat animal food to induce obesity, and the NL group (n=24) was fed normolipidic animal food. Obesity was induced within a period of 120days, and the induction of experimental periodontitis (EP) was subsequently performed for 30days. The animals were euthanized after 7, 15, and 30days, and the jaws were removed for histopathological, histometric, and immunohistochemical analyses. Tartrate-resistant acid phosphatase (TRAP), receptor activator of nuclear factor kappa beta ligand (RANKL), and osteoprotegerin (OPG) were analyzed via immunolabeling. Histological findings indicated that the inflammation was more extensive and lasted longer in the HL⁄EP; however, advanced destruction also occurred in the NL/EP. Greater bone loss was verified in the HL/EP group (2.28±0.35) in the period of 7days than in the NL/EP group (1.2±0.29). High immunolabeling was identified in the HL/EP group in the initial periods for RANKL and TRAP, whereas the NL⁄EP group presented with moderate immunolabeling for both factors. The HL/EP and NL/EP groups showed low immunolabeling for OPG. Obesity induced by a high-fat diet influenced alveolar bone metabolism when associated with experimental periodontitis and caused a more severe local inflammatory response and alveolar bone loss. Obesity is related to greater alveolar bone loss and an accentuated local inflammatory response, which may be reflected in the clinical severity of periodontitis and dental loss.

Abstract 1041: PTHrP(12-48) localization is intracellular and it inhibits cell proliferation and osteoclastogenesis

Abstract Background: PTHrP is produced by various osteotropic malignancies and promotes progression of bone metastasis. PTHrP(12-48) is a unique, proteolytically-derived fragment of full length PTHrP and is the first PTHrP fragment, identified in the serum of breast cancer patients. Serum PTHrP(12-48) levels in breast cancer patients, correlate with a high sensitivity and specificity with the presence of bone metastasis. The purpose of this study was to define the role of PTHrP(12-48) in the metastatic breast tumor microenvironment. PTHrP(12-48) activity at the cellular level, was examined by PTHrP(12-48) agonist/antagonist activity at the PTH1 receptor (PTH1R), examination of subcellular localization and the effects of PTHrP(12-48) on cell proliferation, survival and osteoclast differentiation. Methods: To test the agonist/antagonist activity of PTHrP(12-48), c-AMP and IP3 assays were performed in cells expressing the PTH1R, in the presence of potent PTH1R agonists (PTH 1-34) and antagonists (PTH7-34). Immunolocalization of PTHrP(12-48) was examined by immunohistochemistry (IHC) using a specific anti-PTHrP(12-48) antibody and by immunofluorescence in PTH1R expressing human cells and human peripheral blood mononuclear cell (PBMC)-derived osteoclasts, which lack PTH1R. Cellular proliferation was measured in PTH1R expressing and PTH1R lacking cells. In addition, the effect of PTHrP(12-48) on human osteoclast precursor survival and osteoclast differentiation was assessed in receptor activator of nuclear factor kappa beta ligand (RANKL) treated PBMC cultures. Results: The data demonstrate that PTHrP(12-48) is not an agonist or an antagonist of the PTH1R receptor. IHC demonstrated positive immunostaining in primary breast tumors and bone metastases, whereas normal breast tissue was negative. Immunofluorescence successfully identified the cytoplasmic localization of PTHrP(12-48), 90 minutes after addition of ligand in PTH1R expressing cells. In contrast, no cytoplasmic staining was observed in PTH1R lacking cells. In addition PTHrP(12-48) significantly inhibited serum-induced proliferation of PTH1R expressing cells, but not PTH1R lacking cells. Interestingly, although human osteoclast precursors lack PTH1R expression, PTHrP(12-48) treatment strongly inhibited the survival and proliferation of PBMCs as well as their differentiation into osteoclasts. Conclusions: PTHrP(12-48) is a circulating bone metastasis biomarker that is expressed by primary breast tumors as well as bone metastasis. PTHrP(12-48) does not interact productively with the PTH1R in terms of second messenger activation. However, treatment of cells with PTHrP(12-48) results in cellular uptake by PTH1R expressing cells, with a resultant inhibition of serum-induced cell proliferation and osteoclastogenesis. Citation Format: Varinder Kaur, Archana Kamalakar, Nisreen Akel, Kim Leitzel, Allan Lipton, Larry J. Suva. PTHrP(12-48) localization is intracellular and it inhibits cell proliferation and osteoclastogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1041. doi:10.1158/1538-7445.AM2015-1041

Effects of plyometric exercise session on markers of bone turnover in boys and young men.

The acute exercise effects on bone markers in adults are unclear, while in children, there are no such data. To investigate the acute response of biochemical markers of bone turnover to a high-impact exercise session consisting of high-mechanical loading in boys and young men. Twelve boys (10.2 ± 0.4 years) and 14 men (22.0 ± 0.8 years) underwent a protocol of plyometric jumping exercises (total 144 jumps). Venous blood samples were collected pre-, 5 min, 1 and 24 h post-exercise, and analyzed for markers of bone formation and resorption: bone-specific alkaline phosphatase (bone ALP), osteoprotegerin (OPG), amino-terminal cross-linking telopeptide (NTx), and receptor activator of nuclear factor kappa beta ligand (RANKL). Boys had higher resting bone ALP (111.9 ± 29.2 vs. 30.6 ± 11.2 µg/L, p < 0.05) and NTx levels (49.8 ± 13.2 vs. 21.7 ± 5.9 nM BCE, p < 0.05) than men but no group differences were observed in resting OPG or RANKL. Following exercise (24 h), bone ALP and NTx increased in both boys and men (bone ALP: 24.1 vs. 9.9%, respectively; NTx: 23.5 vs. -5%, respectively), although the group-by-time interaction was not statistically significant. OPG increased significantly (p < 0.05) in both groups (5.7 and 16.1 %, respectively). Even one session of plyometric exercises appear to stimulate bone formation in boys and men, as reflected by the increase in bone ALP and OPG. The boys' response appears more pronounced than the men's, suggesting that during growth, cellular bone activities respond with greater magnitude to mechanical stimuli.

Tamibarotene modulates the local immune response in experimental periodontitis.

Tamibarotene (Am80), a synthetic retinoic acid receptor (RAR), is an agonist with high specificity for RARα and RARβ. Retinoid agonists have been shown to inhibit Th17 cell polarization and to enhance forkhead box P3 (Foxp3) expression during the course of inflammatory diseases. The aim of this study was to evaluate the previously unrecognized role of Am80 in regulating the immune responses of periodontitis within the oral microenvironment. The experimental model of periodontitis in mice was induced by oral infection with Porphyromonas gingivalis (P. gingivalis) W83. Our results indicated that Am80 effectively suppressed alveolar bone resorption induced by P. gingivalis W83 and decreased the number of osteoclasts. We clarified that these effects were closely associated with the reduced percentage of CD4(+) retinoid-related orphan receptor (ROR)γt(+) cells and increased the percentage of CD4(+) Foxp3(+) cells in the gingival tissues, cervical lymph nodes (CLNs), and spleen. Furthermore, in P. gingivalis-infected mice, Am80 down-regulated mRNA expression levels of interleukin-17A (IL-17A), receptor activator of nuclear factor-kappa beta ligand (RANKL), monocyte chemotactic protein-1 (MCP-1), IL-6, and IL-1β. Simultaneously, Am80 up-regulated expression levels of IL-10 and transforming growth factor-β1 (TGF-β1) in gingival tissues and the CLNs. Our results suggest that Am80 could protect against periodontal bone resorption, primarily through the modulation of immune responses in the oral microenvironment, and demonstrate the potential of Am80 as a novel clinical strategy for preventing periodontitis.

Effects of VEGF on Bone Cells’ Metabolism and Activity

The treatments of bone loss have been extended to the molecular level and previous research has found that vascular endothelia growth factor (VEGF) can enhance the vascular network building around bone injury to aid healing progress. In this manuscript, we discussed the effects of VEGF on bone cells' metabolism and activity. VEGF could enhance osteogenesis by inducing neovascularization and direct effects on bone cells via regulation on genes osteoprotegerin (OPG)/the receptor activator of nuclear factor kappa beta ligand (RANKL), alkaline phosphatase (ALP), osteopontin (OPN) and osteocalcin (OCN), without causing abnormal cell metabolism, which may shed lights on new clinical treatments of bone loss.

SERUM CONCENTRATIONS OF PROTEIN REGULATORS OSTEOBLASTOGENESIS AND OSTEOCLASTOGENESIS IN PATIENTS WITH ENDOGENOUS HYPERCORTICISM

Purpose. Endogenous Cushing’s syndrome (CS), usually affecting young and otherwise healthy patients, is a good model to validate the effects of supraphysiological levels of glucocorticoids in humans. This study evaluates circulating levels of extracellular antagonists of Wnt/ß-catenin signaling pathway (sclerostin, Dickkopf1 (Dkkl), secretedfrizzled-related protein 1 (SFRP1)) along with osteoprotegerin (OPG) and soluble receptor activator of nuclear factor kappa-beta ligand (RANKL) in patients with CS as compared to healthy individuals. Materials and methods. Forty patients with clinically and biochemically evident CS and 40 sex, age and body-mass index matched healthy individuals provided fasting serum samples (8:00-10:00AM) for measurement of sclerostin, SFRP1 and Dkkl, RANKL., OPG along with bone turnover markers. Serum samples on RANKL., OPG., Dkkl, SFRP1, sclerostin were frozen and then concurrently measured by an enzyme immunoassay (ELISA) using commercially available reagents. Serum samples on osteocalcin (OC), carboxyterminal cross-linked telopeptide of type I collagen (CTx), cortisol in serum and saliva were assayed by electrochemiluminescence (ECLIA) Cobas e601 Roche. Urinary free cortisol (24hUFC) was measured by an immunochemiluminescence assay (extraction with diethyl ether) on a Vitros ECi. All participants were questioned regarding any recent low traumatic fractures. Patients with CS underwent standard spinal radiographs in anterior-posterior and lateral positions of the vertebrae Th4-L4 (Axiom Icons R200 "Siemens"). Results. Patents with CS (30 (26-40) years old with 24hUFC 2575 (1184-4228) nmol/l (Me (Q25-Q75)) had suppressed OC and normal CTx levels as compared to healthy subjects. A significant correlation, which we observed between OC and CTx (po=0.724 (p&lt;0.001)) among the healthy volunteers, weakened to a non-significant level (po - 0.285 (p=0.083)) when analyzing patients with CS only. 24hUFC correlated with OC po = - 0.464 p=0.003, but not with CTx po= 0.245 (p=0.132) in patients with CS. Patients with CS had higher sclerostin levels versus healthy control subjects (p=0.032). Differences in sclerostin were due to the lack of lower sclerostin values rather than an increase in protein levels above the upper-limits of the healthy control individuals. Sclerostin levels higher than 662 pg/ml were four times more frequent in patients with CS as compared to healthy subjects (OR=4,19, 95% CI 1,44-12,22), p=0,006. Dkk1, SFRP1 did not differ from the control group. Patients with CS had a significantly lower level of RANKL (0.083 (0.075 0.093) pmol/L) as compared to healthy subjects (0.106 (0.089 0.131) pmol/L) p&lt;0.001. Conversely, no difference was found between the OPG level in patients with CS (6.65 (4.92-7.66) pmol/L) and healthy individuals (5.77 (5.00-6.40) pmol/L), p=0.14. RANKL was lower (p=0.02) and OPG was higher (p=0.04) in patients with CS and low traumatic fractures (n=19) versus patients without fractures (n=21). Conclusions. Patients with CS have higher sclerostin level as compared to healthy subjects. Hypercotisolism prevents the normal physiological suppression of sclerostin rather than raising its absolute level. Of all the tested proteins (sclerostin, Dkk1, SFRP1, RANKL., OPG) only sclerostin seems to be a promising therapeutic approach to treating osteoporosis in patients with endogenous CS.

Iron Chelation Therapy in Upper Egyptian Transfusion-dependent Pediatric Homozygous β-Thalassemia Major

Bone disease in beta-thalassemia major (betaTM) remains poorly understood. Receptor activator of nuclear factor-kappabeta ligand (RANKL) regulates osteoclast formation and function. RANKL activity is balanced by interaction with its receptor (RANK) and binding to osteoprotegerin (OPG). L-Carnitine (LC) enhances osteoblastic activity by furnishing fuel. This study hypothesized that abnormal bone metabolism in betaTM involves imbalanced RANKL/OPG and LC/free fatty acids (FFAs) metabolism. Sixty-nine transfusion-dependent betaTM patients and 15 healthy controls were enrolled. One group of patients (n=34) received desferrioxamine (DFO) and the other (n=35) did not. Serum OPG, soluble RANKL (sRANKL), FFAs, LC [total LC (TC), free LC (FC), and esterified LC (EC)], calcium, and inorganic phosphate were measured by specific immuno and colorimetric assays; bone mineral density was examined by dual x-ray absorptiometry. Patients showed lower levels of OPG, TC, FC, EC and higher levels of sRANKL, sRANKL/OPG ratio, and FFAs than controls. Patients on DFO showed lower levels of OPG, TC, FC and higher levels of sRANKL, sRANKL/OPG ratio, and FFAs than those without chelation. In patients, sRANKL correlated negatively with TC and OPG and FC correlated positively with OPG and negatively with sRANKL, sRANKL/OPG ratio, and FFAs. In conclusion, altered bone metabolism owing to imbalanced osteoclastic bone resorption versus constructive osteoblastic activities in betaTM pediatric patients could be due to abnormal sRANKL-OPG and LC-FFAs systems that were worsened by DFO.

RANKL inhibition in the treatment of bone metastases

Bone metastases are common in patients with advanced malignancies and are associated with skeletal-related events, cancer progression, and death. Receptor activator of nuclear factor kappa beta ligand (RANKL) and its receptor, RANK, key mediators of osteoclast differentiation and function, play a pivotal role in bone destruction induced by metastatic bone tumors. The present review summarizes the contribution of RANKL to pathologic bone disease and presents early clinical data on RANKL inhibition in human metastatic cancer. RANKL/RANK interactions are essential for normal bone homeostasis. Binding of RANKL to RANK induces osteolytic bone resorption, a process that occurs in excess when tumor cells are present in bone. In cancer patients, increased tumor-induced osteolysis that is measurable using bone resorption markers can be used to monitor response to treatment or predict tumor progression. Denosumab, a novel, fully human monoclonal antibody specific to RANKL, suppresses bone resorption markers in patients with a variety of metastatic tumors and is being investigated in multiple clinical trials for the prevention and treatment of bone metastases. RANKL is an appropriate target to reduce the osteolytic bone damage caused by bone metastases. Clinical trials are ongoing to assess the safety and efficacy of denosumab for the treatment of bone resorption in patients with metastatic cancers.

The Evolving Systemic and Local Biomarker Milieu at Different Stages of Disease Progression in Rat Adjuvant-Induced Arthritis

Rats with adjuvant-induced arthritis (AIA) were necropsied on 14 occasions during preclinical, acute clinical and chronic clinical stages of AIA progression to characterize local (joint protein extracts) and systemic (serum) levels of mediators regulating inflammation and bone erosion in conjunction with lymphoid tissue-specific leukocyte kinetics. Systemic increases in alpha1 acid glycoprotein, tumor necrosis factor-alpha (TNFalpha), interleukin (IL)-17, transforming growth factor beta (TGFbeta), and chemokine (C-C motif) ligand 2 (CCL2) together with local IL-1alpha/beta and TGFbeta enrichment and local lymphoid hyperplasia preceded the onset of clinical disease and joint damage. Systemic upregulation of TNFalpha, IL-6, IL-17, TGFbeta, IL-18, CCL2, receptor activator of nuclear factor-kappabeta ligand (RANKL), and prostaglandin E(2) during acute and/or chronic AIA coincided with systemic leukocytosis and CD4+ T cell increase in blood and spleen. In contrast, progression of joint erosions during clinical AIA was associated with intra-articular increases in IL-1alpha/beta, IL-6, RANKL, IL-17, TGFbeta, CCL2, and KC/GRO and also a dramatic decline in osteoprotegerin. These data indicate that systemic and local events in inflammatory arthritis are discrete processes, driven by multiple cellular and humoral mediators with distinct kinetic profiles.

Ovariectomy increases vascular calcification via the OPG/RANKL cytokine signalling pathway

Observational studies suggest a strong relationship between menopause and vascular calcification. Receptor activator of nuclear factor-kappaBeta ligand (RANKL) and osteoprotegerin (OPG) are critical regulators of bone remodelling and modulate vascular calcification. We assessed the hypothesis that ovariectomy increases vascular calcification via the OPG/RANKL axis. Age-matched sexually mature rabbits were randomized to ovariectomy (OVX, n = 12) or sham procedure (SHAM, n = 12). One month post-procedure, atherosclerosis was induced by 15 months 0.2%-cholesterol diet and endothelial balloon denudations (at months 1 and 3). Aortic atherosclerosis was assessed in vivo by magnetic resonance imaging (MRI) at months 9 and 15. At sacrifice, aortas were harvested for ex vivo microcomputed tomography (microCT) and molecular analysis of the vascular tissue. Vascular calcification density and calcific particle number were significantly greater in OVX than SHAM (8.4 +/- 2.8 vs. 1.9 +/- 0.6 mg cm(-3), P = 0.042, and 94 +/- 26 vs. 33 +/- 7 particles cm(-3), P = 0.046, respectively). Calcification morphology, as assessed by the arc angle subtended by the largest calcific particle, showed no difference between groups (OVX 33 +/- 7 degrees vs. SHAM 33 +/- 5 degrees , P = 0.99). By Western blot analysis, OVX increased the vascular OPG:RANKL ratio by 66%, P = 0.029, primarily by decreasing RANKL (P = 0.019). At month 9, MRI demonstrated no difference in atheroma volume between OVX and SHAM, and no significant change was seen by the end of the study. In contrast to bone, vascular OPG:RANKL ratio increased in response to ovariectomy with a corresponding fourfold increase in arterial calcification. This diametrical organ-specific response may explain the comorbid association of osteoporosis with calcifying atherosclerosis in post-menopausal women.