RANKL Activity Research Articles

Differential but complementary roles of HIF-1α and HIF-2α in the regulation of bone homeostasis

Bone is a highly dynamic tissue undergoing continuous formation and resorption. Here, we investigated differential but complementary roles of hypoxia-inducible factor (HIF)-1α and HIF-2α in regulating bone remodeling. Using RNA-seq analysis, we identified that specific genes involved in regulating osteoblast differentiation were similarly but slightly differently governed by HIF-1α and HIF-2α. We found that increased HIF-1α expression inhibited osteoblast differentiation via inhibiting RUNX2 function by upregulation of Twist2, confirmed using Hif1a conditional knockout (KO) mouse. Ectopic expression of HIF-1α via adenovirus transduction resulted in the increased expression and activity of RANKL, while knockdown of Hif1a expression via siRNA or osteoblast-specific depletion of Hif1a in conditional KO mice had no discernible effect on osteoblast-mediated osteoclast activation. The unexpected outcome was elucidated by the upregulation of HIF-2α upon Hif1a overexpression, providing evidence that Hif2a is a transcriptional target of HIF-1α in regulating RANKL expression, verified through an experiment of HIF-2α knockdown after HIF-1α overexpression. The above results were validated in an ovariectomized- and aging-induced osteoporosis model using Hif1a conditional KO mice. Our findings conclude that HIF-1α plays an important role in regulating bone homeostasis by controlling osteoblast differentiation, and in influencing osteoclast formation through the regulation of RANKL secretion via HIF-2α modulation.

Melatonin Decreases Alveolar Bone Loss in Rats with Experimental Periodontitis and Osteoporosis: A Morphometric and Histopathologic Study.

Periodontitis and post-menopausal osteoporosis include common chronic bone disorders worldwide, with similar etiopathogenetic events. This study evaluated the effect of systemic melatonin administration on the alveolar bone destruction of periodontitis progression in an experimental periodontitis model in osteoporotic rats. Forty-four Wistar rats were randomly divided into six experimental groups: control (C; n = 6); osteoporosis (O; n = 6); ligated periodontitis (LP; n = 8); osteoporosis- and periodontitis-induced (O+LP; n = 8); osteoporosis- and periodontitis-induced through 30 mg/kg/day melatonin administration (ML30; n = 8); and osteoporosis- and periodontitis-induced through 50 mg/kg/day melatonin administration (ML50; n = 8). The rats underwent bilateraloophorectomy and were maintained for 4 months to induce osteoporosis. After 4 months, 4-0 silk ligatures were placed submarginally around the mandibular first molar of each rat to induce experimental periodontitis, and melatonin was administered in the ML30 and ML50 groups for 30 days. Changes in alveolar bone levels were clinically measured, and tissues were histopathologically examined. Osteoclastic activity in the LP and O+LP groups was significantly higher than in the other groups (p < 0.05), but was similar in the C, O, and ML30 groups (p > 0.05). RANKL activity was the highest in the O+LP group, while melatonin decreased RANKL activity in the melatonin-administered groups (p < 0.05). Systemically administered melatonin significantly decreased alveolar bone loss in the ML30 and ML50 groups compared with that in the periodontitis groups (p < 0.05). Melatonin inhibited alveolar bone destruction by decreasing the RANKL expression and inflammatory cell infiltration and increased osteoblastic activity in a rat model with osteoporosis and periodontitis.

Hypoxia caused by unilateral nasal obstruction decreases mandibular density in rats through inhibition of Cyp1a1 expression.

Oral breathing has an important impact on morphology and bone mineral density (BMD) in a mandible. This study aimed to investigate the hub genes and mechanism regulating the mandibular BMD decrease induced by nasal obstruction. A unilateral nasal obstruction model was established in 1-week-old Wistar rats by electrocautery obstruction. BMD of the mandible was determined by micro-computed tomography. Transcriptome analysis was performed to identify differentially expressed genes (DEGs). Hub genes were identified by building protein-protein interaction network and verified by western blot. A hypoxic cell model was established in bone marrow mesenchymal stem cells (BMSCs) by using CoCl2. The expression of hypoxia-inducible factor-1α (HIF-1α), NF-kB ligand-receptor activator (RANKL), osteoprotegerin (OPG), and Cyp1a1 was detected by western blot. The mandibular BMD of rats in the unilateral nasal obstruction group was significantly decreased. A total of 38 DEGs were identified in nasal obstruction rats compared with normal rats. A ratio of RANKL/OPG in the mandible was elevated by nasal obstruction, while the Cyp1a1 was decreased. In vitro, the HIF-1α expression and RANKL/OPG ratio were upregulated by hypoxia while the Cyp1a1 expression was decreased. Pretreatment with Cyp1a1 activator, FICZ, could increase the expression of Cyp1a1 while attenuating the activation of HIF-1α and RANKL. Respiratory changes caused by nasal obstruction contribute to the decrease in Cyp1a1 expression in the mandible of juvenile rats, which is associated with disturbances in bone homeostasis controlled by the RANKL/OPG ratio.

Anti-Inflammatory Effect of Specialized Proresolving Lipid Mediators on Mesenchymal Stem Cells: An In Vitro Study

An interconnection between tissue inflammation and regeneration has been established through the regulation of defense and repair mechanisms within diseased dental tissue triggered by the release of immune-resolvent mediators. To better our understanding of the role of specific pro-resolving mediators (SPMs) in inflamed human bone marrow-derived mesenchymal stem cells (hBMMSCs), we studied the effects of Resolvin E1 (RvE1) and Maresin 1 (MaR1) in lipopoly-saccharide (LPS) stimulated hBMMSCs. The hBMMSCs were divided into five different groups, each of which was treated with or without SPMs. Group-1: negative control (no LPS stimulation), Group-2: positive control (LPS-stimulated), Group-3: RvE1 100 nM + 1 μg/mL LPS, Group-4: MaR1 100 nM + 1 µg/mL LPS, and Group-5: RvE1 100 nM + MaR1100 nM + 1 μg/mL LPS. Cell proliferation, apoptosis, migration, colony formation, Western blotting, cytokine array, and LC/MS analysis were all performed on each group to determine the impact of SPMs on inflammatory stem cells. According to our data, RvE1 plus MaR1 effectively reduced inflammation in hBMMSCs. In particular, IL-4, 1L-10, and TGF-β1 activation and downregulation of RANKL, TNF-α, and IFN-γ compared to groups receiving single SPM were shown to be significantly different (Group 3 and 4). In addition, the LC/MS analysis revealed the differentially regulated peptide's role in immunological pathways that define the cellular state against inflammation. Inflamed hBMMSCs treated with a combination of Resolvin E1 (RvE1) and Maresin 1 (MaR1) promoted the highest inflammatory resolution compared to the other groups; this finding suggests a potential new approach of treating bacterially induced dental infections.

PSAT197 Treatment of Paget's Disease of the Skull With Denosumab: A Case Report

Abstract Background Paget's disease of bone (PDB) is a common skeletal disorder characterized by increased osteoclastic activity and bone resorption followed by increased osteoblastic bone formation leading to an abnormality in modeling and remodeling of bone. Bisphosphonates are the most common treatments for PDB, with zoledronic acid being the most effective medication, but treatment would be difficult if bisphosphonates are contraindicated. Clinical case: We report a case of PDB of the skull in a 75-year-old woman presenting with a persistent headache that was successfully treated with denosumab. The patient had been complaining of severe headaches since December 2020. Her skull x-ray and laboratory results [calcium 8.2 mg/dL (range: 8.5-10.5 mg/dL), albumin 4 g/dL (range: 3.4- 4.8 g/dL), alkaline phosphatase 132 U/L (range: 35-104 U/L)] were consistent with PDB. Brain MRI and bone scintigraphy also showed skull changes consistent with PDB. Importantly, a DXA scan from July 2020 showed osteopenia, and her primary care provider treated it with denosumab in the first week of December 2020. At the time of PDB diagnosis in January 2021, zoledronic acid could not be administered because of mild hypocalcemia that was corrected with calcium carbonate. However, the headaches resolved, alkaline phosphatase normalized, and zoledronic acid was put on hold. Up until January 2022, she remains asymptomatic and alkaline phosphatase remains normal. Conclusion This case illustrates the successful management of PDB of the skull with denosumab, a human monoclonal antibody against RANKL. To the best of our knowledge, there have not been reported cases of full recovery of PBD with denosumab. Although PBD pathophysiology remains poorly understood, this case suggests that increased RANKL activity might mediate PBD directly or indirectly. Thereby, denosumab might be an effective treatment for some cases of PDB. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.

Effect of methoxy-polyethylene glycol-polylactic acid-bone protective protein nano micelle complex on osteoprotegerin and bone mineral density in osteoporotic rats

This study assessed the effect of nanomicelle complex Methoxy-polyethylene glycol Polylactic acid bone protective protein on OPG and bone mineral density in osteoporotic rats. 35 rats were assigned into group A, B, C, D and E. Except for group A, Osteoarthritis (OA) rat models were established in all other groups. After modeling, group C, D and E were injected with PEG+OPG plasmid, PLL+OPG plasmid and PEG-PLL+ plasmid at 40 mg/kg dose of nanoparticles into mice via tail vein. The other groups were injected with an equal volume of NaCl solution to observe the bone density and OPG level. The bone trabeculae in the femoral tissues of group A were mostly plate-shaped and dense and uniform. Group B bones were sparse without new bone, while the trabecular bone in group C increased, the gap among trabecular bones decreased. The number of trabecular bones increased in group D, and new bone trabecula from group E increased, and arrangement was neat. Bone mineral density (BMD) and BMc levels in the femoral tissue from rats in group B decreased relative to group A (P &lt;0.05), and the levels of BMD and BMc in the femoral tissue from rats in group C increased (P &lt;0.05). BMD and BMc from mouse femur tissue were lower than group E (P &lt;0.05). Moreover, Group B had a lower maximum load than group A (P &lt;0.05) and group E was higher than group D (P &lt;0.05). The femoral tissue structure of rats in group A was complete and bone matrix was arranged well without cavities. The number of bone cells in group B was reduced, while the bone matrix was disorderly distributed, and bones were fractured and more cavities were formed. Compared with group B, groups C, and D, and E had increased bone cells number and reduced bone matrix disorder improvement voids. Immunohistochemistry showed that, OPG increased in group B, and RANKL decreased (P &lt;0.05), which was different from group A (P &lt;0.05). OPG protein and mRNA from C, D and E groups decreased, and RANKL levels increased (P &lt;0.05). PEG-PLL-OPG (PPO) significantly improved OP rat bone mineral density via regulation of BMD, BMc and biomechanical indicators, and the mechanism is related to increased OPG expression and reduced RANKL activity.

Metabolic Bone Disease of Prematurity.

Metabolic Bone Disease of Prematurity.

The Effect of Two Different Doses of Astaxanthin on Alveolar Bone Loss in an Experimental Model of Periodontitis in Diabetic Rats.

This study evaluated the effects of astaxanthin (ASX) on alveolar bone loss, receptor activator of nuclear factor-κB ligand (RANKL), and osteoprotegerin (OPG) activity in ligature-induced periodontitis in diabetic rats. Diabetes mellitus (DM) was induced with 50 mg/kg intraperitoneal streptozotocin in 40 male Wistar rats. The Wistar rats were divided into six experimental groups: non-ligated (NL; n = 6); ligature only (L; n = 6); DM only (D; n = 6); DM + ligature (DP; n = 6); DM + ligature + 1 mg/kg/day ASX (ASX 1 group; n = 8); and DM + ligature + astaxanthin 5 mg/kg/day ASX (ASX 5 group; n = 8). Silk ligatures were placed along the gingival margin of the left mandibular first molar tooth. The study duration was 11 days, after which the animals were euthanised. Changes in alveolar bone levels were clinically measured, and RANKL and OPG activities were immunohistochemically examined. Alveolar bone loss was the most significant in the DP group (p < 0.05). Decreased alveolar bone loss was observed in the ASX 5 group (p < 0.05). Although RANKL activity was highest in the DP group, it was observed at lower levels in the groups to which ASX was administered. OPG activity did not differ between groups (p > 0.05). The results of this study suggested that 1 and 5 mg/kg ASX administration reduced RANKL activity and alveolar bone loss in rats with experimentally induced periodontitis.

Ovariectomy induces bone loss via microbial-dependent trafficking of intestinal TNF+ T cells and Th17 cells.

Estrogen deficiency causes a gut microbiome-dependent expansion of BM Th17 cells and TNF-α-producing T cells. The resulting increased BM levels of IL-17a (IL-17) and TNF stimulate RANKL expression and activity, causing bone loss. However, the origin of BM Th17 cells and TNF+ T cells is unknown. Here, we show that ovariectomy (ovx) expanded intestinal Th17 cells and TNF+ T cells, increased their S1P receptor 1-mediated (S1PR1-mediated) egress from the intestine, and enhanced their subsequent influx into the BM through CXCR3- and CCL20-mediated mechanisms. Demonstrating the functional relevance of T cell trafficking, blockade of Th17 cell and TNF+ T cell egress from the gut or their influx into the BM prevented ovx-induced bone loss. Therefore, intestinal T cells are a proximal target of sex steroid deficiency relevant for bone loss. Blockade of intestinal T cell migration may represent a therapeutic strategy for the treatment of postmenopausal bone loss.

Inverse behavior of IL-23R and IL-17RA in chronic and aggressive periodontitis

PurposePeriodontitis is associated with a dysbiosis of periodontopathic bacteria, which stimulate the interleukin (IL)-23/IL-17 axis that plays an essential role in the immunopathogenesis of this disease, leading to alveolar bone destruction through receptor activator of nuclear factor κB ligand (RANKL). IL-23 receptor mRNA (IL-23R) has been identified in periodontitis, and IL-17 receptor A mRNA (IL-17RA) and its protein have not yet been evaluated in patients with periodontitis. In this study was measure IL-23R and IL-17RA in gingival tissue (GT) from patients with generalized chronic periodontitis (GCP) and generalized aggressive periodontitis (GAP) and to explore correlations with clinical parameters.MethodsWe included 16 healthy subjects (HS), 18 patients with GCP, and 14 with GAP. GT samples were collected during periodontal surgery. Both IL-23R and IL-17RA were detected by enzyme-linked immunosorbent assay.ResultsThe results were analyzed with Mann-Whitney U test and Spearman' rank correlation coefficients using SPSS version 25.0. We found lower IL-23R levels in patients with GCP and GAP than in HS. Contrarily, we observed higher IL-17RA levels in GCP and GAP patients than in HS. Moreover, we found negative correlations between IL-23R in GT and probing depth and clinical attachment loss (CAL). Likewise, a positive correlation of IL-17RA in GT with CAL was found.ConclusionsThe results of these findings suggest that the reverse behavior between IL-23R and IL-17RA in periodontitis patients may also be involved with the activation of RANKL, which promotes alveolar bone loss.

Assessment of the prevalence of osteoporosis among children with hemophilia and its relation to serum 25(oh) vitamin D and serum rankl

Osteoporosis is one of the comorbidities that complicating hemophilia. Recurrent hemarthrosis, chronic arthropathy and immobilization all are factors that make patients with hemophilia prone to this complication. Till recently the major emphasis of the osteoporosis diagnosis is the dual-energy X-ray absorptiometry (DEXA) scan, however, the definition of osteoporosis in pediatrics not only low bone mineral density measured by DEXA scan but also required the presence of clinically significant fracture history. Investigation target is evaluating the osteoporosis prevalence between cases with hemophilia, assessing the risk factors increasing its incidence including diet habits, the severity of hemophilia, duration between the first diagnosis of the disease and enrollment in the study and chronic hepatitis C infection. Also, considering the role of vitamin D deficiency, and another potential indirect mechanism mediated through Receptor activator of NF-Kappa-B, the Receptor activator of NF-Kappa-B ligand (RANK-RANKL) pathway has been suggested in the pathogenesis of bone disease and osteoclastic activity but remains controversial.Thirty-nine hemophilia pediatric patients were recruited from hematology clinic, Cairo university hospital, history taking, and examination were done focusing on the musculoskeletal system and dietetic data. Twenty normal age-matching children enrolled as a control group. DEXA scan results showed normal bone mineral density in 28 patients (71.8%) and osteoporosis in 11 patients (28.2%). The median Z score of patients was -1.40 (-2.2 – -0.6). There was a statistically significant decrease of bone mineral density in patients with hemophilia comparing with a control group with P-value &lt;0.001, also we observed significantly higher serum RANKL in the group of patients with low bone mineral density ensuring the relation between RANKL and osteoclastic activity.

Local Production of Osteoprotegerin by Osteoblasts Suppresses Bone Resorption

SUMMARYOsteoprotegerin (OPG) inhibits the ability of receptor activator of nuclear factor κB (NF-κB) ligand (RANKL) to stimulate the differentiation, activity, and survival of bone-resorbing osteoclasts. Genetic studies in mice show that osteocytes are an important source of RANKL, but the cellular sources of OPG are unclear. We use conditional deletion of Tnfrsf11b, which encodes OPG, from different cell populations to identify functionally relevant sources of OPG in mice. Deletion from B lymphocytes and osteocytes, two cell types commonly thought to supply OPG, has little or no impact on bone mass. By contrast, deletion of Tnfrsf11b from osteoblasts increases bone resorption and reduces bone mass to an extent similar to germline deletion, demonstrating that osteoblasts are an essential source of OPG. These results suggest that, in addition to producing new bone matrix, osteoblasts also play an active role in terminating the resorption phase of the bone remodeling cycle by suppressing RANKL activity.

Pluripotent anti-inflammatory immunomodulatory effects of papaverine against cerebral ischemic-reperfusion injury

The mechanism of the papaverine (PV) for the treatment of cerebral ischemia remains unclear. A total of 42 mice induced with focal cerebral ischemia were randomly divided into three groups: PV,baicalin (BA)and vehicle group. Both PV and BA could significantly reduce the ischemic infarct volume (P < 0.05). Pathway enrichment analysis was performed on MetaCore™ to search the molecular pathways associated with the gene expression profile of PV, compared with vehicle and BA. Compared with vehicle, we found that 60% of the top 10 pathways in PV group were related to immune response. The top 10 biological processes of the targeted pathways were mainly related to the multiple immunomodulatory process of neuro-vascular inflammation, including immune_Th17-deried cytokins, regulation of angiogenesis, cell adhesion_Leucocyte chemotaxis, antigen presentation, cell adhesion_synaptic contact, and inflammation related to Amphoterin signaling. Moreover, compared with BA, 17 pathways of PV were identified, and 58.82% (10/17) were also related to immune response, especially, half of the top 10 pathways with the lower p-value. In these top 10 pathways, 4 were the cytokine-mediated signaling pathways, which play key role in inflammation, like IL-17 signaling pathways with the activation of G-CSF,IL-23,RANKL, p38MAPK and NF-κB.These findings indicate that PV may be an efficacious pluripotent anti-inflammatory agent against cerebral ischemic-reperfusion injury by targeting on multiple immunomodulatory process of neuro-vascular inflammation.

Kurkumin’in Osteosarkomada Doğal RANKL İnhibitörü Olarak Biyolojik Değerlendirilmesi

Osteosarcoma is an important health problem among children and teenagers. Metastases is observed in about twenty percent of osteosarcoma patients. RANK/RANKL/OPG signaling pathway is involved in formation of osteosarcoma and its metastases. Especially, the overexpression of RANKL is related to osteoclast and osteosarcoma formation. Therefore, inhibition of RANKL activity has been significant treatment method of osteosarcoma. In this study, curcumin was evaluated as RANKL inhibitor in osteosarcoma cells. In this context, anti-proliferative and anti-invasive properties of the curcumin were determined with in-vitro and computational assays in Saos-2 cell line. Obtained results showed that curcumin may has a potential for treatment of osteosarcoma and its metastases.

MicroRNA-9 ameliorates destructive arthritis through down-regulation of NF-κB1-RANKL pathway in fibroblast-like synoviocytes

We investigated the effect of miR-9 on fibroblast-like synoviocytes (FLS) from RA patients and animal arthritis model. The binding of miR-9 to NF-κB1 3’UTR was analyzed by luciferase reporter assay and immunoprecipitation. ChIP assay and luciferase promoter assay were performed to identify the binding of NF-κB1 to RANKL promoter and its activity. FLS were treated with miR-9/anti-miR-9 to evaluate cell proliferation and the expression of RANKL. Therapeutic effect of intra-articular miR-9 was evaluated in type-II collagen-induced arthritis in rats. miR-9 bound to the 3’-UTR of NF-κB1 and downregulated NF-κB1. NF-κB1 bound to RANKL promoter and increased the promoter activity of RANKL. RANKL was downregulated by miR-9. Proliferation of FLS was increased by miR-9 inhibitor. miR-9 dampened experimental arthritis by lowering inflammatory state, reducing RANKL and osteoclasts formation. Our findings revealed miR-9-NF-κB1-RANKL pathway in RA-FLS, further, miR-9 ameliorated inflammatory arthritis in vivo which propose therapeutic implications of miR- 9 in RA.

EZH2 Supports Osteoclast Differentiation and Bone Resorption Via Epigenetic and Cytoplasmic Targets.

Key osteoclast (OCL) regulatory gene promoters in bone marrow-derived monocytes harbor bivalent histone modifications that combine activating Histone 3 lysine 4 tri-methyl (H3K4me3) and repressive H3K27me3 marks, which upon RANKL stimulation resolve into repressive or activating architecture. Enhancer of zeste homologue 2 (EZH2) is the histone methyltransferase component of the polycomb repressive complex 2, which catalyzes H3K27me3 modifications. Immunofluorescence microscopy reveals that EZH2 localization during murine osteoclastogenesis is dynamically regulated. Using EZH2 knockdown and small molecule EZH2 inhibitor GSK126, we show that EZH2 plays a critical epigenetic role in OCL precursors (OCLp) during the first 24 hours of RANKL activation. RANKL triggers EZH2 translocation into the nucleus where it represses OCL-negative regulators MafB, Irf8, and Arg1. Consistent with its cytoplasmic localization in OCLp, EZH2 methyltransferase activity is required during early RANKL signaling for phosphorylation of AKT, resulting in downstream activation of the mTOR complex, which is essential for induction of OCL differentiation. Inhibition of RANKL-induced pmTOR-pS6RP signaling by GSK126 altered the translation ratio of the C/EBPβ-LAP and C/EBPβ-LIP isoforms and reduced nuclear translocation of the inhibitory C/EBPβ-LIP, which is necessary for transcriptional repression of the OCL negative-regulatory transcription factor MafB. EZH2 in multinucleated OCL is primarily cytoplasmic and mature OCL cultured on bone segments in the presence of GSK126 exhibit defective cytoskeletal architecture and reduced resorptive activity. Here we present new evidence that EZH2 plays epigenetic and cytoplasmic roles during OCL differentiation by suppressing MafB transcription and regulating early phases of PI3K-AKT-mTOR-mediated RANKL signaling, respectively. Consistent with its cytoplasmic localization, EZH2 is required for cytoskeletal dynamics during resorption by mature OCL. Thus, EZH2 exhibits complex roles in supporting osteoclast differentiation and function. © 2019 American Society for Bone and Mineral Research.

SAT-499 Denosumab for Treatment of Hypercalcemia in Myelofibrosis

Introduction Hypercalcemia is a rare complication of myeloproliferative neoplasms. As a result, the underlying mechanisms and optimal treatment options remain under investigation. We present a case of hypercalcemia in post-essential thrombocythemia (ET) related myelofibrosis that was responsive to treatment with denosumab 60mg. Clinical Case A 65-year-old woman with ET for 10 years and recent progression to myelofibrosis (confirmed by bone marrow biopsy) was admitted for failure to thrive. Significant labs include: Ca 11.5 mg/dL (n: 8.5-10.5 mg/dL), corrected Ca 13.2 mg/dL, phosphorous 3.7 mg/dL (n: 2.4-4.7 mg/dL), PTH 5 pg/mL (n: 16-87 pg/mL), 25-OH Vitamin D 20.2 ng/mL (n: 30-100 ng/mL), 1,25 Vitamin D: 6.5 pg/mL (n: 19.9-79.3 pg/mL), PTHrP <2 pmol/L (n: <2 pmol/L), Vitamin A 9.4 ug/dL (n: 36.4-108.0 ug/dL), bone specific alkaline phosphatase 58 ug/L (n: 8.1-31.6 ug/L), TSH 0.16 uIU/mL (n: 0.34-5.60 uIU/mL), pancytopenia on CBC with 3% blasts on differential, LDH 3157 U/L (n: 100-200 U/L), and uric acid 7.4 mg/dL (n: 2.7-7.3 mg/dL). There were no lytic lesions on CT head/chest/abdomen/pelvis. Corrected Ca levels improved to 11 mg/dL after i.v. fluids and calcitonin. Denosumab 60 mg was administered on day 9 of hospitalization when corrected Ca reached 14.5 mg/dL. Cholecalciferol 1000 IU daily was started. Ca levels normalized three days after denosumab. However, clinical course then rapidly deteriorated with transformation into acute leukemia with hemophagocytic lymphohistiocytosis (HLH), tumor lysis syndrome, septic shock, and acute renal failure. Venous ionized Ca levels dropped to 0.80 mmol/L (n: 1.14-1.29 mmol/L) six days after denosumab dose. She required i.v. calcium gluconate, oral Ca, and adjusted Ca bath in dialysis to correct hypocalcemia. The patient died on day 19 of hospitalization. Conclusion While hypercalcemia is uncommon in myelofibrosis, it needs to be recognized as a potential complication. Although the underlying pathogenesis remains unknown, suggested mechanisms include: ectopic 1,25 Vitamin D production, osteolytic metastases, immobilization, and RANKL activation. Our patient did not have known bone metastases, prolonged periods of immobility or elevated 1,25 Vitamin D levels. However, our patient’s response to denosumab supports a RANKL mediated process. It is likely that there is an extensive release of cytokines in myelofibrosis even in the absence of osteolytic lesions. This would shift the RANKL/OPG balance towards osteoclast differentiation and subsequent hypercalcemia. Our case illustrates effective treatment of hypercalcemia in myelofibrosis with denosumab 60 mg. Low dose denosumab can be considered for treatment in myelofibrosis patients with a high risk of acute clinical decompensation. However, more research to elucidate the underlying mechanism and optimal treatment of hypercalcemia in myeloproliferative disorders is necessary.

Anti-receptor activator of nuclear factor κB ligand antibody treatment increases osteoclastogenesis-promoting IL-8 in patients with rheumatoid arthritis.

The receptor activator of nuclear factor κB ligand (RANKL) is an important factor for osteoclastogenesis and contributes to the pathology of rheumatoid arthritis (RA); thus, the anti-RANKL antibody (Ab) has been expected to protect joint destruction in RA patients. IL-8 also has osteoclastogenic activity; however, the role of IL-8 in the bone pathology of RA as well as the relation between IL-8 and RANKL remain unclear. In the present study, clinical observation revealed serum IL-8 levels of 611 pg ml−1 in RA patients with anti-RANKL Ab and 266 pg ml−1 in the same patients without anti-RANKL Ab. In vitro assay showed that anti-RANKL Ab induced production of IL-8 from pre-osteoclast-like cells (OCLs), and IL-8 promoted the formation of OCLs from peripheral monocytes even without RANKL activity. We further showed that treatment with FK506 (tacrolimus) possibly inhibits the increase in IL-8 levels in RA patients with anti-RANKL Ab, and in vitro assay confirmed that FK506 suppressed IL-8 production in pre-OCLs. These results suggest that inhibition of RANKL induces the change in osteoclastogenesis-promoting factor from RANKL to IL-8, and FK506 may be a valuable combination drug to support the use of anti-RANKL Ab in treatment of RA.

Phospholipase C signaling activated by parathyroid hormone mediates the rapid osteoclastogenesis in the fracture healing of orchiectomized mice

BackgroundThe age-related osteoporosis is an increasing risk severely threatening the live quality of aged people. Human parathyroid hormone (hPTH) is applied to the therapy of osteoporosis successfully, however, the mechanism, especially the signaling pathway activated in the healing fracture by PTH is still unknown.MethodsThe once daily injections of hPTH(1–34) and GR (1–34) (the PLC deficient analog) into the orchiectomized male mice with bone fracture, were started at the second day after fracture and lasted for 4 weeks. To explore the role of phospholipase C signaling in the androgen-deficient fracture healing, the fracture healing were evaluated via radiography, micro-CT, biomechanics testing, serum biochemistry, bone marrow cell culture and gene expression quantification.ResultsAfter two weeks of fracture, both peptides significantly increased bone mineral density (BMD), bone mass content (BMC) and bone volume (BV/TV) in the healing area. However, compared to hPTH(1–34), GR(1–34) induced more woven bones, the higher BMC and BMD, as well as the less serum TRAP and osteoclasts. After four weeks of treatment, the effects of hPTH(1–34) on fracture healing showed no difference to those of GR(1–34). Consistently, GR(1–34) induced the similar osteogenesis but less osteoclastogenesis under the ex vivo condition immediately after administration compared to hPTH(1–34), which was verified by the weaker activation of RANKL, NFATC1, TRAP and Cathepsin K in GR(1–34) treatment.ConclusionThese results indicated that the PLC signaling activated by the intermittent injection of hPTH(1–34) leads to the bone resorption by rapidly activating the osteoclastogenesis in the fracture healing zone.

Cell viability assessed in a reproducible model of human osteoblasts derived from human adipose-derived stem cells.

Human adipose tissue-derived stem cells (hASCs) have been subjected to extensive investigation because of their self-renewal properties and potential to restore damaged tissues. In the literature, there are several protocols for differentiating hASCs into osteoblasts, but there is no report on the control of cell viability during this process. In this study, we used osteoblasts derived from hASCs of patients undergoing abdominoplasty. The cells were observed at the beginning and end of bone matrix formation, and the expression of proteins involved in this process, including alkaline phosphatase and osteocalcin, was assessed. RANKL, Osterix, Runx2, Collagen3A1, Osteopontin and BSP expression levels were analyzed using real-time PCR, in addition to a quantitative assessment of protein levels of the markers CD45, CD105, STRO-1, and Nanog, using immunofluorescence. Rhodamine (Rho123), cytochrome-c, caspase-3, P-27, cyclin D1, and autophagy cell markers were analyzed by flow cytometry to demonstrate potential cellular activity and the absence of apoptotic and tumor cell processes before and after cell differentiation. The formation of bone matrix, along with calcium nodules, was observed after 16 days of osteoinduction. The gene expression levels of RANKL, Osterix, Runx2, Collagen3A1, Osteopontin, BSP and alkaline phosphatase activity were also elevated after 16 days of osteoinduction, whereas the level of osteocalcin was higher after 21 days of osteoinduction. Our data also showed that the cells had a high mitochondrial membrane potential and a low expression of apoptotic and tumor markers, both before and after differentiation. Cells were viable after the different phases of differentiation. This proposed methodology, using markers to evaluate cell viability, is therefore successful in assessing different phases of stem cell isolation and differentiation.