Receptor Activator Of Nuclear factor-κB Ligand Research Articles

MyD88 exacerbates inflammation-induced bone loss by modulating dynamic equilibrium between Th17/Treg cells and subgingival microbiota dysbiosis.

This study aimed to investigate the contribution of myeloid differentiation primary-response gene 88 (MyD88) on the differentiation of T helper type 17 (Th17) and regulatory T (Treg) cells and the emerging subgingival microbiota dysbiosis in Porphyromonas gingivalis-induced experimental periodontitis. Alveolar bone loss, infiltrated inflammatory cells, immunostained cells for tartrate-resistant acid phosphatase (TRAP), the receptor activator of nuclear factor-kB ligand (RANKL), and osteoprotegerin (OPG) were quantified by microcomputerized tomography and histological staining between age- and sex-matched homozygous littermates (wild-type [WT, Myd88+/+] and Myd88-/- on C57BL/6 background). The frequencies of Th17 and Treg cells in cervical lymph nodes (CLNs) and spleen were determined by flow cytometry. Cytokine expression in gingival tissues, CLNs, and spleens were studied by quantitative polymerase chain reaction (qPCR). Analysis of the composition of the subgingival microbiome and functional annotation of prokaryotic taxa (FAPROTAX) analysis were performed. P. gingivalis-infected Myd88-/- mice showed alleviated bone loss, TRAP+ osteoclasts, and RANKL/OPG ratio compared to WT mice. A significantly higher percentage of Foxp3+CD4+ T cells in infected Myd88-/- CLNs and a higher frequency of RORγt+CD4+ T cells in infected WT mice was noted. Increased IL-10 and IL-17a expressions in gingival tissue at D14-D28 then declined in WT mice, whereas an opposite pattern was observed in Myd88-/- mice. The Myd88-/- mice exhibited characteristic increases in gram-positive species and species having probiotic properties, while gram-negative, anaerobic species were noted in WT mice. FAPROTAX analysis revealed increased aerobic chemoheterotrophy in Myd88-/- mice, whereas anaerobic chemoheterotrophy was noted in WT mice after P. gingivalis infection. MyD88 plays an important role in inflammation-induced bone loss by modulating the dynamic equilibrium between Th17/Treg cells and dysbiosis in P. gingivalis-induced experimental periodontitis.

Effect of low-intensity laser therapy on levels of biomarkers during orthodontic tooth movement – A systematic review

This systematic review aimed to explore the association between low-intensity laser therapy (LILT) and various biomarkers, elucidating the potential of this therapeutic modality to expedite orthodontic tooth movement (OTM). A systematic literature search was conducted using electronic databases including PubMed, Central of the Cochrane Library, and Google Scholar databases. Randomized controlled trials (RCTs) and non-randomized controlled clinical trials (nRCTs) using LILT as an adjunct to the standard orthodontic procedures in human and animal subjects as participants were included in the study. The quality of the human and animal studies was assessed using the Revised Cochrane risk of bias tool and Systematic Review Centre for Laboratory Animal Experimentation’s (SYRCLE’s) risk of bias tool respectively. Animal studies revealed increased receptor activator of nuclear factor-kappa B (RANK)/RANK ligand (RANKL), matrix metalloproteinase-9 (MMP-9), and collagen type I expression. Human studies showed elevated interleukin-1-beta (IL-1β), interleukin-16 (IL-16) and RANKL levels, suggesting LILT influences biomarkers associated with bone resorption and connective tissue rearrangement. A high risk of bias was observed in all animal studies and 5 out of 6 human studies. The systematic review concluded that LILT emerges as a promising technique for enhancing orthodontic tooth movement, influencing key biomarkers linked to osteoclastic activity and collagen synthesis. However, the high risk of bias in animal and human studies emphasizes the need for further research to validate findings and optimize laser parameters for clinical benefits.

The histone methyltransferase ASH1L protects against bone loss by inhibiting osteoclastogenesis.

Absent, small, or homeotic1-like (ASH1L) is a histone lysine methyltransferase that generally functions as a transcriptional activator in controlling cell fate. So far, its physiological relevance in bone homeostasis and osteoclast differentiation remains elusive. Here, by conditional deleting Ash1l in osteoclast progenitors of mice, we found ASH1L deficiency resulted in osteoporosis and potentiation of osteoclastogenesis in vivo and in vitro. Mechanistically, ASH1L binds the promoter of the Src homology 3 and cysteine-rich domain 2 (Stac2) and increases the gene's transcription via histone 3 lysine 4 (H3K4) trimethylation modification, thus augmenting the STAC2's protection against receptor activator of nuclear factor kB ligand (RANKL)-initiated inflammation during osteoclast formation. Collectively, we demonstrate the first piece of evidence to prove ASH1L as a critical checkpoint during osteoclastogenesis. The work sheds new light on our understanding about the biological function of ASH1L in bone homeostasis, therefore providing a valuable therapeutic target for the treatment of osteoporosis or inflammatory bone diseases.

Icariin regulates RANKL-induced osteoclast differentiation via the ERα/c-Src/RANK signaling

Osteoporosis (OP) is a common metabolic bone disease. Excessive osteoclastic activity significantly contributes to the development of OP. Icariin (ICA) is a flavonol glycoside derived from herbal plants and possesses curative effects on postmenopausal OP and bone fracture. This study aimed to investigate the effects of ICA on osteoclast differentiation induced by receptor activator of nuclear factor kappa B (RANK) ligand (RANKL) and the involvement of estrogen receptor α (ERα) and RANK signaling cascade in this process. RANKL was used to induce the differentiation of RAW264.7 cells to into osteoclasts. Small interfering RNA technique was used to knockdown ERα in cells. Cell counting kit-8 assay was performed to determine the cytotoxicity of ICA. The number of tartrate-resistant acid phosphatase (TRAP)-positive cells was quantified by TRAP staining. RANKL induced the differentiation of RAW264.7 cells into osteoclasts, while ICA abolished the pro-osteoporotic effect of RANKL. Moreover, ERα knockdown abolished the effects of ICA on RANKL-induced osteoclastogenesis. Further exploration revealed that ICA inhibited the phosphorylation of c-Src in osteoclasts via regulating ERα, while inactivation of c-Src reversed ERα knockdown-promoted osteoclastogenesis. Lastly, ICA inhibited the activation of the mitogen-activated protein kinase signaling pathway and downregulated the expressions of target osteoclastogenic proteins in RANKL-treated RAW 264.7 cells, while ERα knockdown almost completely diminished the effects of ICA. ICA inhibited RANKL-induced osteoclast differentiation via regulating the ERα/c-Src/RANK signaling. These findings elucidated a novel mechanism by which ICA exerts an anti-osteoporotic effect.

Melittin Treats Periprosthetic Osteolysis in a Rat Model by Inhibiting the NF-kB Pathway and Regulating the Ratio of Receptor Activator of Nuclear Factor Kappa B Ligand/Osteoprotegerin

BackgroundAseptic loosening around the prosthesis is a common cause of failure in total joint arthroplasty. Polyethylene wear particles trigger the release of inflammatory factors by macrophages. Key mediators involved in osteoclastogenesis include interleukin-6, tumor necrosis factor-α, receptor activator of nuclear factor kappa B (RANK), RANK ligand (RANKL), and bone protection hormone (Osteoprotegerin [OPG]). The purpose of our experiment was to see whether melittin can slow down the release of inflammatory mediators through the NF-kB pathway, regulate the RANKL/OPG ratio, reduce osteoclast formation, and delay the onset of arthritis in rats. MethodsA total of 20 male Sprague-Dawley rats (10 months, Specific Pathogen Free, 350 g ± 20 g) were randomly divided into 5 groups: sham group, model group, melittin concentration 1 group (0.2 mg/kg), concentration 2 group (0.4 mg/kg), and concentration 3 group (0.6 mg/kg). All rats were implanted with TA2 high-purity titanium rods. A drill was used to create a bone canal along the long axis of the femur in the intercondylar notch. The model group and experimental groups were exposed to polyethylene particles, while the sham group did not receive any particles. ResultsThe melittin group exhibited significantly increased serum levels of serum P, calcium-phosphorus product, OPG, PINP, PINP/CTX-I, and OPG/RANKKL (P < .05). In the experimental group, micro computed tomography scanning results revealed a decrease in the amount of bone defect around the prosthesis. Immunofluorescence analysis demonstrated a decrease in the expression of IKKα and P65, while the expression of OPG showed an upward trend. Both Hematoxylin-Eosin and Tartrate-Resistant Acid Phosphatase staining revealed less osteoclast and inflammatory cell infiltration in bone resorption pits. ConclusionsOur study demonstrates that melittin has the ability to inhibit the NF-kB pathway in a rat model, and reduce the impact of RANKL/OPG, thereby delaying osteoclast activity and alleviating periprosthetic osteolysis.

A Comparative Study for Localization of Odontoclast in Crown and Root of Physiological Resorbed Primary Teeth

Root and crown resorption is a physiologic event for the primary teeth. It is still unclear whether odontoclasts, the cells which resorb the dental har d tissue, are different from the osteoclasts, the cells that resorb bone. Dental tissue resorption seems to be initiated and regulated by the stellate reticulum and the dental follicle of the underlying permanent tooth via the secretion of stimulatory molecules, i.e. cytokines and transcription factors. The primary teeth resorption process is regulated in a manner similar to bone remodeling, involving the same receptor ligand system known as RANK/RANKL (receptor activator of nuclear factor-kappa B/ RANK Ligand), which represent two cytokine-like proteins of the tumor necrosis factor superfamily, are localized on bone cells and dental cells .They are crucial for the regulation of osteoclastic/odontoclast cell differentiation and also for the upregulation of mature osteoclasts/odontoclasts mediated by cell-to-cell contact and a subsequent cascade of diverse intracellular signaling processes .The aim of the present study was to localize and compare the IHC rea ctions for RANKL along root surface and the crown of human phs iological resorbed primary teeth. Fifteen human upper deciduous (second molar) teeth ,undergoing root and crown resorption wer e used for immunohistochemical study to identify RANKL expre ssion. The results demonstrated a high mean of expression of R ANKL in root as compared with crown in human primary shedding teeth. The present study concludes that RANKL play a role in res orption process of the primary teeth.

Serum levels of soluble receptor activator of NF-κβ ligand are under dual, immune and neural effects in postmenopausal women

Background: The receptor activator of NF-κβ (RANK)/RANK ligand/ osteoprotegerin system is essential for osteoclast maturation and activation to induce bone loss, which plays a role in postmenopausal osteoporosis. Estrogen deficiency in postmenopausal women is associated with T-cell mediated inflammation with increased proinflammatory cytokines, such as IL-17A, IL-1, IL-6 and tumor necrosis factor alpha. Bone repair and remodeling processes highlight the role of nerve growth factor β (NGFβ), whose non-neural production is associated with T-cell mediated inflammation. Aims: To investigate the involvement of IL-17A and NGFβ cytokines in the elevation of serum soluble RANK ligand levels in postmenopausal and control women. Methods: Fifty-two postmenopausal and 37 control women were studied for age, body mass index, homeostasis model assessment (HOMA) index, serum levels of IL-17A, NGFβ, soluble RANK ligand (measured by indirect enzyme-linked immunosorbent assay) and estrogen (measured by chemiluminescence assay). Two- and three-way ANOVA (expressed as median with Q1 to Q3) and linear regression analysis were used to evaluate the strength of relationship between soluble RANK ligand levels as dependent and age, serum IL-17A and NGFβ levels as independent variables. Results: A significant difference in age, serum NGFβ and estrogen levels could be demonstrated between postmenopausal and control women (with borderline significance in HOMA indices). The differences in serum IL-17A and NGFβ levels were relevant between categories for body mass index in postmenopausal and control women, but in soluble RANK ligand levels only in postmenopausal women. The increase in soluble RANK ligand levels was greater in elevated IL-17A [101(291.75 to 39.92) vs. 45.24(95.56 to 25.34) ng/ml, p&lt;0.015] and elevated NGFβ [222.2(142.75 to 1086.87) vs. 52.44(25.87 to 76.88) ng/ml, p&lt;0.0001] categories compared to low categories in postmenopause, but not in controls. Both elevated IL-17A and NGFβ levels resulted in the greatest increase in serum levels of soluble RANK ligand [252.27(159.9 to 1099.64) vs. 41.4(35.91 to 59.94) ng/ml, p&lt;0.0001] compared to elevated IL-17A levels alone. Conclusion: Both IL-17A and NGFβ cytokines were involved in the increased serum levels of soluble RANK ligand in postmenopausal women. Their joint involvement in postmenopausal osteoporosis highlighted that neurogenic inflammation may play a critical role in bone loss via greater elevated soluble RANK ligand levels induced by concomitant elevated IL-17A and NGFβ levels.

Role of Actinomyces spp. and related organisms in the development of medication-related osteonecrosis of the jaw (MRONJ): Clinical evidence based on a case series.

Medication-related osteonecrosis of the jaw (MRONJ) is an increasingly common consequence of antiresorptive treatment, which often leads to the development of necrotic exposed bone surfaces with inflammatory processes affecting the jawbone. Although the development of MRONJ is often associated with the inflammatory response or infections caused by the colonizing members of the oral microbiota, the exact pathogenesis of MRONJ is still not fully understood. In the present paper, we aimed to provide additional, microbiological culture-supported evidence, supporting the "infection hypothesis" that Actinomyces spp. and related organisms may play an important pathogenic role in the development of MRONJ and the resulting bone necrosis. In our case series, all patients presented with similar underlying conditions and anamnestic data, and have received antiresorptive medications (bisphosphonates or a RANK ligand (RANKL) inhibitor) to prevent the occurrence or progression of bone metastases, secondary to prostate cancer. Nevertheless, a few years into antiresorptive drug therapy, varying stages of MRONJ was identified in the mentioned patients. In all three cases, quantitative microbiological culture of the necrotic bone samples yielded a complex microbiota, dominated by Actinomyces and Schaalia spp. with high colony counts. Additionally, our followed-up case series document the treatment of these patients with a combination of surgical intervention and long-term antibiotic therapy, where favourable clinical responses were seen is all cases. If the "infection hypothesis" is valid, it may have significant consequences in the preventative and therapeutic strategies associated with this disease.

TIFA contributes to periodontitis in diabetic mice via activating the NF‑κB signaling pathway.

Diabetic periodontitis (DP) refers to destruction of periodontal tissue and absorption of bone tissue in diabetic patients. Tumor necrosis factor receptor‑associated factor (TRAF)‑interacting protein with forkhead‑associated domain (TIFA) as a crucial regulator of inflammation activates the NF‑κB signaling pathway to regulate cell biological behavior. However, the function and mechanism of TIFA on DP suffer from a lack of research. In the present study, TIFA was upregulated in the periodontal tissue of a DP mouse model. In addition, the expression of TIFA in RAW264.7 cells was induced by high glucose (HG) culture and increased by lipopolysaccharide (LPS) from Porphyromonasgingivalis treatment in a time‑dependent manner. Knockdown of TIFA significantly reduced the levels of inflammatory cytokines, including TNF‑α, IL‑6, IL‑1β and monocyte chemoattractant protein‑1, in HG and LPS‑induced RAW264.7 cells. The nuclear translocation of NF‑κB p65 was induced by HG and LPS and was clearly suppressed by absence of TIFA. The expression of downstream factors Nod‑like receptor family pyrin domain‑containing 3 and apoptosis‑associated speck‑like protein was inhibited by silencing TIFA. Moreover, TIFA was increased by receptor activator of NF‑κB (RANK) ligand (RANKL) in a concentration dependent manner. The expression of cathepsin K, MMP9 and nuclear factor of activated T cells cytoplasmic 1 was downregulated by depletion of TIFA. RANKL‑induced osteoclast differentiation was inhibited by silencing of TIFA. Meanwhile, the decrease of TIFA blocked activation of the NF‑κB pathway in RANKL‑treated RAW264.7 cells. In conclusion, TIFA as a promoter regulates the inflammation and osteoclast differentiation via activating the NF‑κB signaling pathway.

An LGR4 agonist activates the GSK‑3β pathway to inhibit RANK‑RANKL signaling during osteoclastogenesis in bone marrow‑derived macrophages.

The binding between receptor‑activated nuclear factor‑κB (RANK) and the RANK ligand (RANKL) during osteoclast development is an important target for drugs that treat osteoporosis. The leucine‑rich repeat‑containing G‑protein‑coupled receptor 4 (LGR4) acts as a negative regulator of RANK‑RANKL that suppresses canonical RANK signaling during osteoclast differentiation. Therefore, LGR4 agonists may be useful in inhibiting osteoclastogenesis and effectively treating osteoporosis. In the present study, bone marrow‑derived macrophages and a mouse model of RANKL‑induced bone loss were used to investigate the effect of mutant RANKL (MT RANKL), which was previously developed based on the crystal structure of the RANKL complex. In the present study, the binding affinity of wild‑type (WT) RANKL and MT RANKL to RANK and LGR4 was determined using microscale thermophoresis analysis, and the effect of the ligands on the AKT‑glycogen synthase kinase‑3β (GSK‑3β)‑nuclear factor of activated T cells, cytoplasmic, calcineurin‑dependent 1 (NFATc1) signaling cascade was investigated using western blotting and confocal microscopy. In addition, the expression of LGR4 and the colocalization of LGR4 with MT RANKL were analyzed in a mouse model of RANKL‑induced bone loss. The results showed that in osteoclast precursor cells, MT RANKL bound with high affinity to LGR4 and increased GSK‑3β phosphorylation independently of AKT, resulting in the inhibition of NFATc1 nuclear translocation. In the mouse model, MT RANKL colocalized with LGR4 and inhibited bone resorption. These results indicated that MT RANKL may inhibit RANKL‑induced osteoclastogenesis through an LGR4‑dependent pathway and this could be exploited to develop new therapies for osteoporosis.

Effect of internal heat-type acupuncture on bone remodeling in a glucocorticoid-induced osteoporosis model rabbit by regulating the triplet of OPG-RANKL-RANK

Introduction: Internal heat-type acupuncture therapy is a new therapeutic method that integrates acupuncture and heat therapy and effectively reduces local muscle inflammation. It has a good effect on the clinical application of osteoporosis. The purpose of this study was to assess the improvement of glucocorticoid-induced osteoporosis (GIOP) after internal heat-type acupuncture treatment as well as its role in promoting the balance of bone remodeling in the GIOP rabbit model by regulating the triplet of osteoprotegerin(OPG)-receptor activator of nuclear factor-KB ligand (RANKL)-receptor activator of nuclear factor-KB (RANK). Methods: The rabbits were divided into the control, model (GIOP), GIOP+Alendronate, and GIOP+Internal heat-type acupuncture groups, with 8 rabbits in each group. At the end of the treatment, all rabbits were sacrificed. The pathologic changes of lumbar vertebrae were observed by x-ray, the morphology of lumbar trabecular bone was observed by HE staining, and the apoptosis of lumbar vertebrae was detected by Tunel. The protein expressions of OPG, RANKL, and RANK in lumbar vertebrae were detected by immunohistochemistry staining, immunofluorescence staining, and Western blotting. Results: Internal heat-type acupuncture partly prevented osteopenia among GIOP-induced rabbits, improved the morphology of lumbar vertebrae, and inhibited the apoptosis of lumbar vertebrae osteocytes. Moreover, by increasing the protein expression level of OPG and reducing the protein expression of RANKL and RANK, internal heat-type acupuncture effectively promoted the balance of bone remodeling and eventually achieved the treatment of GIOP. Conclusion: Internal heat-type acupuncture therapy may promote the balance of bone remodeling by regulating the triplet of osteoprotegerin-receptor activator of nuclear factor-KB ligand-receptor activator of nuclear factor-KB, thereby treating osteoporosis which was induced by glucocorticoid.

Current indications for denosumab in benign bone tumours.

Denosumab is a fully humanised monoclonal antibody to RANK ligand, inhibiting the RANK-RANKL pathway. It promotes the apoptosis of osteoclast-like giant cells, a secondary ossification and connective tissue formation. Given its high efficacy, denosumab is the standard treatment of unresectable or metastatic giant cell tumour of bone (GCTB) requiring morbid surgery. Neoadjuvant administration of denosumab may be justified to enable the resection of the tumour in certain cases; it should be considered, however, with caution for joint-saving surgery due to high local recurrence rates. In cases of unresectable or metastatic GCTB, however, denosumab treatment should be administered for years or even as a lifelong therapy. This poses many yet unanswered questions concerning the frequency of denosumab treatment as well as the ratio of the adverse events in the following years. Denosumab suppresses, not directly targets, the neoplastic stromal cells of GCTB. Ongoing in vitro studies suggest that other drugs alone or in combination (e.g. sunitinib) with denosumab may target both the neoplastic and the giant cells. Promising results have been reported regarding the off-label use of denosumab in other giant cell-rich tumours/tumour-like lesions, i.e. aneurysmal bone cysts and central giant cell granulomas. Data are derived, however, mostly from case reports and case series. Large prospective clinical trials are needed to evaluate the role and also the side effects of denosumab in the treatment of these rare diseases.

In Vitro Analysis of Bone Remodeling Alterations in Hemophilia: Influence of Coagulation Factors on Bone Cells

Background. Today, one of the main comorbidities affecting the quality of life of the haemophilia patient is bone pathology. Hemophilia A (HA) is associated with reduced bone mass and mineral density (BMD). Due to the rarity of the disease and the heterogeneity among the studies, pathogenesis of bone loss is still under investigation and both processes of bone resorption and bone formation could be hypothesized to be altered. FVIII deficiency could alter BMD directly affecting bone cells or indirectly by decreasing thrombin generation. The reduction of BMD seems due to perturbations of the Receptor Activator of Nuclear factor-B RANK Ligand (RANKL) and osteoprotegerin (OPG) pathways. In haemophilia alterations of bone remodeling, osteoclasts seem to play a major role. Osteoclasts are multinucleated cells, which derive from the CD14+ monocyte/macrophage lineage. Until recently, the identity of osteoclast progenitors has not been well defined, but evidences report that CD16−CD14+ rather than CD16+CD14+ monocytes were prone to differentiate into osteoclasts (Komano et al. Arthritis Res Ther. 2006). Objectives. To dissect the mechanism of bone loss in hemophilia, we studied the effects of coagulation factors, such as factor VIII, von Willebrand factor (VWF), activated factor X (FXa) and thrombin, on both osteoclasts and osteoblasts biology and we characterized the osteoclastogenic potential of HA patients' osteoclast precursors. Patients/Methods: Peripheral Blood Mononuclear Cells (PBMC) isolated from healthy donors were induced to differentiate into osteoclasts and treated with plasma derived VWF/FVIII complex, human rVWF, human full length rFVIII, activated FX and thrombin. Osteoclastogenesis and expression analysis of RANK, TRAF6 (TNF receptor-associated factor 6), TCIRG1 (T Cell Immune Regulator 1) and osteoclasts protease CTSK (cathepsin K) expression were assessed. Moreover, in vitro assays assessed the osteoclastogenic potential of PBMC isolated from different hemophilic A patients. FACS analyses of osteoclast precursors isolated from patients were performed. Osteoblasts differentiation, mineralization and genes expression (Alkaline Phosphatase and COL1A2) were performed in the presence of aforementioned coagulation factors. Results. We showed a significant reduction of mature osteoclasts after treatment of HD-PBMC with FVIII, VWF, FVIII/VWF, FXa and thrombin. VWF appears to play a major role to regulate osteoclast differentation from healthy donor-derived PBMC. Indeed, it inhibits by itself ~45% the osteoclastogenesis comparable to OPG, and even more if is complexed with FVIII (53% inhibition). About 50% and 70% reduced levels of osteoclast differentiation were also revealed following treatment with FXa and thrombin, respectively. Interestingly, PBMC from HA patients showed increased ability to form mature osteoclasts compared to those obtained from healthy controls. Osteoclast precursors (CD16−CD14+CD11b+) are significantly higher in HA patients than age and sex matched controls. Moreover, transcriptional analysis revealed increased RANK, TRAF6, CTSK and TCIRG1 genes expression in adult hemophilia patient's osteoclasts compared to matched controls. FVIII and VWF treatments led also to a statistically significant reduction of ALP positivity in control osteoblasts; opposite effect was shown following thrombin treatment. Conclusions. All these data support that bone loss observed in haemophilia patients could be related to increased osteoclast formation and activity and that coagulation factors directly impact on bone cells. These results may have important implications in the clinical management of hemophilic patients to prevent bleeding as well as to preserve bone health.

Erythropoietin Treatment in Mice Enriches Bone Marrow Osteoclast Precursors, As Reflected By Increased Macrophage Colony-Stimulating Factor (MCSF) and Elevated Expression of Osteoclast Markers in Monocytes

The impact of erythropoietin (EPO) on erythropoiesis has long been established. However, recent studies have indicated that EPO may also exert regulatory effects on other hematopoietic lineages, including the monocyte lineage. In that respect, we have previously shown that EPO treatment in mice induces bone loss. Here, we studied the effect of EPO on bone marrow (BM) monocytes and its potential implications for osteoclastogenesis. To address the dynamic changes in monocyte populations following EPO administration, we injected EPO (180U) once, or 3 times on alternating days for one week. Mice were sacrificed 16 h after EPO injection. Our results revealed a significant 20 percent increase in the monocyte population (TER119 -, LY6G -, CD11b +) 16 hours after EPO injection, and a twofold increase in the levels of these cells after one week of 3 injections of EPO. Soluble macrophage colony-stimulating factor (MCSF) protein levels in the bone marrow were increased 1.5-fold 16 h after EPO injection, while MCSF mRNA expression levels remained unchanged. After 1-week of EPO administration (3 injections), although the global BM expression of MCSF was reduced, we observed a two-fold increase in MCSF expression levels in T cells (CD3 +). To further elucidate the functional consequences of EPO-induced monocyte expansion, we isolated monocytes from mice treated with EPO for one week. We characterized expression levels of key osteoclastic markers in these cells. Indeed, we found that in BM monocytes, EPO treatment led to the upregulation of Lymphocyte function-associated antigen 1 (LFA-1) and Receptor Activator of Nuclear Factor κ B (RANK). The former is implicated in osteoclastogenesis via its interaction with intercellular adhesion molecule-1 (ICAM−1), while the latter is the surface receptor for RANK ligand which regulates osteoclast differentiation and activation. These data suggest that EPO not only influences BM monocyte expansion, but also contributes to the regulation of osteoclast differentiation, thus linking erythropoiesis to bone homeostasis. Furthermore, we investigated the early effects of EPO treatment on the expression of transcription factors implicated in monocyte differentiation. Remarkably, we found that EPO administration for 16 hours resulted in the downregulation of KLF2 and KLF4, transcription factors involved in monocytic lineage commitment. The rapid EPO-induced expansion of the monocytic population, coupled with the EPO-regulated expression of MSCF, osteoclastic markers, and transcription factors, suggests a role for EPO in modulating monocyte-to-osteoclast differentiation. The current study opens avenues for further investigating the crosstalk between erythropoiesis, myelopoiesis, and bone homeostasis.

Impact of Mechanical Strain and Nicotinamide on RUNX2-Deficient Osteoblast Mimicking Cleidocranial Dysplasia

Cleidocranial dysplasia (CCD) is a rare genetic defect caused by a heterozygous mutation of runt-related transcription factor 2 (RUNX2), which is important for osteoblast and skeletal development. RUNX2-deficiency causes extra- and intra-oral malformations that often require orthodontic treatment. Nicotinamide (NAM) affects bone remodelling processes. As these are crucial for orthodontic therapy, NAM could improve orthodontic treatment in CCD patients. This study investigates the effect of NAM in control and RUNX2-deficient osteoblasts under mechanical strain mimicking orthodontic treatment. First, the optimal NAM concentration and the differences in the expression profile of control and RUNX2-deficient osteoblasts were determined. Subsequently, osteoblasts were exposed to tensile and compressive strain with and without NAM, and the expression of genes critically involved in bone remodelling was investigated. NAM increased the expression of bone remodelling genes. RUNX2-deficient osteoblasts expressed more receptor activator of NFkB ligand (RANKL) and interleukin-6 (IL6), but less colony-stimulating factor-1 (CSF1). Most of the positive effects of NAM on bone remodelling genes were impaired by mechanical loading. In conclusion, NAM stimulated osteoblast differentiation by increasing the expression of RUNX2 and regulated the expression of osteoclastogenic factors. However, the positive effects of NAM on bone metabolism were impaired by mechanical loading and RUNX2 deficiency.

Sex Differences in Bone, Muscle, and Inflammatory Markers and Their Associations with Muscle Performance Variables.

The importance of various markers such as Sclerostin, Dickkopf-1 (DKK-1), Irisin, receptor activator of NF-kB ligand (RANKL), and Vitamin D have been well studied in bone metabolism. Additionally, inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and Interleukin 6 (IL-6) have been shown to hinder muscle protein synthesis, leading to the loss of muscle and strength. However, a research gap exists in understanding their role in muscle function and physical activity. Therefore, this study aims to explore the serum levels of Sclerostin, DKK-1, Irisin, IL-6, RANKL, Vitamin D, and TNF-α and assess their relationships with upper- and lower-body strength in young adults. In this study, 38 college-aged students (18-23 years), males and females, participated and completed the protocols. The participants' lower and upper body strength were assessed by the vertical jump test (Just Jump, Probotic, AL) with a Tendo FitroDyne (Tendo Sports Machines, Trencin, Slovak Republic) and handgrip (HG) dynamometry (Takei Scientific Instruments, Yashiroda, Japan), respectively. Fasting morning blood samples were analyzed for serum levels of biomarkers by ELISA. The results indicate significant sex differences in Sclerostin, DKK-1, Irisin, and Vitamin D levels (p < 0.05). Furthermore, a positive association was observed between Sclerostin, DKK-1, and Vitamin D, with lower body muscle performance variables (p < 0.05). Conversely, a significant negative correlation was observed between TNF-α and lower-body muscle performance variables (p < 0.05). The results suggest that these markers may have a distinct effect on muscle performance, underscoring the need for further investigation to elucidate the concept of muscle-bone crosstalk.

SAT258 Successful Treatment Of Adult T-cell Leukemia-lymphoma Induced Hypercalcemia With Denosumab

Abstract Disclosure: S. Yuksel: None. R. Lopez Fanas: None. D. Schwartz: None. V. Tabatabaie: None. S. Sachdev: None. Introduction: Adult T-cell leukemia/lymphoma (ATLL) is a rare, aggressive lymphoproliferative disorder associated with human T-cell leukemia virus type 1 (HTLV-1), often presenting with severe refractory hypercalcemia. Here, we present a rare case of bisphosphonate-resistant ATLL-associated hypercalcemia successfully treated with denosumab (Dmab). Clinical Case: A 47-year-old woman from Jamaica presented with pruritic rash of left forearm and bilateral lower extremity (LE) pain for 6 weeks. Laboratory evaluation noted serum Ca of 16.2 mg/dL (n 9-11), PTH 12.5 pg/mL (n 16-65), PTHrP 24 pg/mL (n 11-20) and 25(OH)D 16 ng/mL (n 30-60). 1,25(OH)2D level was normal. Cr was 1.5 mg/dL (n 0.7-1.2). X-rays demonstrated punctate lytic lesions throughout LEs. The patient received IV fluids, calcitonin, pamidronate 60 mg IV, and ergocalciferol 50,000 IU. Corrected Ca (cCa) improved to 9.3 mg/dL. However, hypercalcemia recurred after 2 days. HTLV-1 serology was positive. Flow cytometry and bone marrow biopsy were consistent with ATLL. When Ca rose to 14.3 mg/dL, Dmab 60 mg SC was administered. Within 1 week, she developed asymptomatic hypocalcemia with cCa of 7.1 mg/dL requiring oral calcium citrate and calcitriol. Stable normocalcemia was achieved after 10 days. Hospital course was complicated by pathologic right wrist fracture. Palliative radiation to LE was initiated. She was discharged on calcium citrate 2850 mg TID, calcitriol 0.25 mcg BID and ergocalciferol 50,000 IU weekly. The patient was treated with 6 cycles of etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab, as well as intrathecal methotrexate for CNS prophylaxis. She received 120 mg of Dmab 6 months after the first dose. Ca remained stable. Conclusion: ATLL-associated hypercalcemia is mediated by increased secretion of PTHrP, and RANK ligand and proinflammatory cytokines that promote RANK expression. Dmab is an effective treatment for ATLL-associated hypercalcemia as it specifically targets RANK ligand. A dose of 120 mg Dmab is recommended for patients with bisphosphonate-resistant hypercalcemia of malignancy and GFR greater than 30 mL/min. In another published case report on the use of Dmab in ATLL-associated hypercalcemia, treatment with Dmab 120 mg SC resulted in protracted hypocalcemia lasting 5 months despite calcium carbonate and Vitamin D supplements (1). Our patient had a significantly shorter hypocalcemia course likely due to reduced Dmab dose and more aggressive proactive treatment with ergocalciferol, calcium citrate and calcitriol. This case supports the successful use of Dmab in bisphosphonate-resistant ATLL-associated hypercalcemia. Protracted Dmab-induced hypocalcemia may be mitigated by Dmab dose reduction even in the presence of normal GFR and early aggressive treatment with calcium citrate and calcitriol. Reference: (1) Japp et al. AACE Clin Case Rep.2019;5:e210-213 Presentation: Saturday, June 17, 2023

The effect of osteocyte-derived RANKL on bone graft remodeling: An in vivo experimental study.

Autologous bone is considered the gold standard for grafting, yet it suffers from a tendency to undergo resorption over time. While the exact mechanisms of this resorption remain elusive, osteocytes have been shown to play an important role in stimulating osteoclastic activity through their expression of receptor activator of NF-κB (RANK) ligand (RANKL). The aim of this study was to assess the function of osteocyte-derived RANKL in bone graft remodeling. In Tnfsf11fl/fl ;Dmp1-Cre mice without osteocyte-specific RANKL as well as in Dmp1-Cre control mice, 2.6 mm calvarial bone disks were harvested and transplanted into mice with matching genetic backgrounds either subcutaneously or subperiosteally, creating 4 groups in total. Histology and micro-computed tomography of the grafts and the donor regions were performed 28 days after grafting. Histology revealed marked resorption of subcutaneous control Dmp1-Cre grafts and new bone formation around subperiosteal Dmp1-Cre grafts. In contrast, Tnfsf11fl/fl ;Dmp1-Cre grafts showed effectively neither signs of bone resorption nor formation. Quantitative micro-computed tomography revealed a significant difference in residual graft area between subcutaneous and subperiosteal Dmp1-Cre grafts (p < .01). This difference was not observed between subcutaneous and subperiosteal Tnfsf11fl/fl ;Dmp1-Cre grafts (p = .17). Residual graft volume (p = .08) and thickness (p = .13) did not differ significantly among the groups. Donor area regeneration was comparable between Tnfsf11fl/fl ;Dmp1-Cre and Dmp1-Cre mice and restricted to the defect margins. The results suggest an active function of osteocyte-derived RANKL in bone graft remodeling.

RANK ligand converts the NCoR/HDAC3 co-repressor to a PGC1β- and RNA-dependent co-activator of osteoclast gene expression

The nuclear receptor co-repressor (NCoR) complex mediates transcriptional repression dependent on histone deacetylation by histone deacetylase 3 (HDAC3) as a component of the complex. Unexpectedly, we found that signaling by the receptor activator of nuclear factor κB (RANK) converts the NCoR/HDAC3 co-repressor complex to a co-activator of AP-1 and NF-κB target genes that are required for mouse osteoclast differentiation. Accordingly, the dominant function of NCoR/HDAC3 complexes in response to RANK signaling is to activate, rather than repress, gene expression. Mechanistically, RANK signaling promotes RNA-dependent interaction of the transcriptional co-activator PGC1β with the NCoR/HDAC3 complex, resulting in the activation of PGC1β and inhibition of HDAC3 activity for acetylated histone H3. Non-coding RNAs Dancr and Rnu12, which are associated with altered human bone homeostasis, promote NCoR/HDAC3 complex assembly and are necessary for RANKL-induced osteoclast differentiation invitro. These findings may be prototypic for signal-dependent functions of NCoR in other biological contexts.

Garlic extract enhances bioceramic bone scaffolds through upregulating ALP & BGLAP expression in hMSC-monocyte co-culture

Bone homeostasis is predicated by osteoblast and osteoclast cell cycles where gene expressions are responsible for their differentiation from human mesenchymal stem cells (hMSC) and monocytes, respectively. The pro-osteogenic potential of an hMSC-monocyte co-culture can be measured through complementary DNA (mRNA synthesis) within the nucleus, known as quantitative polymerase chain reaction (qPCR). Through this technique, the effects of garlic extract (allicin) release from calcium phosphate bone scaffolds on gene expression of bone forming and bone remodeling cells was explored. Results show this complex biomaterial system enhances hMSC differentiation through the upregulation of bone-forming proteins. Osteoblastic gene markers alkaline phosphatase (ALP) and osteocalcin (BGLAP), are respectively upregulated by 3-fold and 1.6-fold by day 14. These mature osteoblasts then upregulate the receptor activator of nuclear factor-kB ligand (RANKL) which recruits osteoclast cells, as captured by a nearly 2-fold higher osteoclast expression of tartrate-resistance acid-phosphatase (ACP5). This also activates antagonist osteoprotegerin (OPG) expression in osteoblasts, decreasing osteoclast resorption potential and ACP5 expression by day 21. The pro-osteogenic environment with garlic extract release is further quantified by a 4× increase in phosphatase activity and visibly captured in immunofluorescent tagged confocal images. Also corroborated by enhanced collagen formation in a preliminary in vivo rat distal femur model, this work collectively reveals how garlic extract can enhance bioceramic scaffolds for bone tissue regenerative applications.