Osteoclasts recycle via osteomorphs during RANKL-stimulated bone resorption.

Osteoclasts are large multinucleated bone-resorbing cells formed by the fusion of monocyte/macrophage-derived precursors that are thought to undergo apoptosis once resorption is complete. Here, by intravital imaging, we reveal that RANKL-stimulated osteoclasts have an alternative cell fate in which they fission into daughter cells called osteomorphs. Inhibiting RANKL blocked this cellular recycling and resulted in osteomorph accumulation. Single-cell RNA sequencing showed that osteomorphs are transcriptionally distinct from osteoclasts and macrophages and express a number of non-canonical osteoclast genes that are associated with structural and functional bone phenotypes when deleted in mice. Furthermore, genetic variation in human orthologs of osteomorph genes causes monogenic skeletal disorders and associates with bone mineral density, a polygenetic skeletal trait. Thus, osteoclasts recycle via osteomorphs, a cell type involved in the regulation of bone resorption that may be targeted for the treatment of skeletal diseases.

14-3-3ζ suppresses RANKL signaling by destabilizing TRAF6.

Macrophages are essential regulators of inflammation and bone loss. Receptor activator of nuclear factor-κß ligand (RANKL), a pro-inflammatory cytokine, is responsible for macrophage differentiation to osteoclasts and bone loss. We recently showed that 14-3-3ζ-knockout (YwhazKO) rats exhibit increased bone loss in the inflammatory arthritis model. 14-3-3ζ is a cytosolic adaptor protein that actively participates in many signaling transductions. However, the role of 14-3-3ζ in RANKL signaling or bone remodeling is unknown. We investigated how 14-3-3ζ affects osteoclast activity by evaluating its role in RANKL signaling. We utilized 14-3-3ζ-deficient primary bone marrow-derived macrophages obtained from wildtype and YwhazKO animals and RAW264.7 cells generated using CRISPR-Cas9. Our results showed that 14-3-3ζ-deficient macrophages, upon RANKL stimulation, have bigger and stronger tartrate-resistant acid phosphatase-positive multinucleated cells and increased bone resorption activity. The presence of 14-3-3ζ suppressed RANKL-induced MAPK and AKT phosphorylation, transcription factors (NFATC1 and p65) nuclear translocation, and subsequently, gene induction (Rank, Acp5, and Ctsk). Mechanistically, 14-3-3ζ interacts with TRAF6, an essential component of the RANKL receptor complex. Upon RANKL stimulation, 14-3-3ζ-TRAF6 interaction was increased, while RANK-TRAF6 interaction was decreased. Importantly, 14-3-3ζ supported TRAF6 ubiquitination and degradation by the proteasomal pathway, thus dampening the downstream RANKL signaling. Together, we show that 14-3-3ζ regulates TRAF6 levels to suppress inflammatory RANKL signaling and osteoclast activity. To the best of our knowledge, this is the first report on 14-3-3ζ regulation of RANKL signaling and osteoclast activation.

Exercise and osteoimmunology in bone remodeling.

Bones can form the scaffolding of the body, support the organism, coordinate somatic movements, and control mineral homeostasis and hematopoiesis. The immune system plays immune supervisory, defensive, and regulatory roles in the organism, which mainly consists of immune organs (spleen, bone marrow, tonsils, lymph nodes, etc.), immune cells (granulocytes, platelets, lymphocytes, etc.), and immune molecules (immune factors, interferons, interleukins, tumor necrosis factors, etc.). Bone and the immune system have long been considered two distinct fields of study, and the bone marrow, as a shared microenvironment between the bone and the immune system, closely links the two. Osteoimmunology organically combines bone and the immune system, elucidates the role of the immune system in bone, and creatively emphasizes its interdisciplinary characteristics and the function of immune cells and factors in maintaining bone homeostasis, providing new perspectives for skeletal-related field research. In recent years, bone immunology has gradually become a hot spot in the study of bone-related diseases. As a new branch of immunology, bone immunology emphasizes that the immune system can directly or indirectly affect bones through the RANKL/RANK/OPG signaling pathway, IL family, TNF-α, TGF-ß, and IFN-γ. These effects are of great significance for understanding inflammatory bone loss caused by various autoimmune or infectious diseases. In addition, as an external environment that plays an important role in immunity and bone, this study pays attention to the role of exercise-mediated bone immunity in bone reconstruction.

ADR3, a next generation i-body to human RANKL, inhibits osteoclast formation and bone resorption.

Osteoporosis is a chronic skeletal condition characterized by low bone mass and deteriorated microarchitecture of bone tissue and puts tens of millions of people at high risk of fractures. New therapeutic agents like i-bodies, a class of next-generation single-domain antibodies, are needed to overcome some limitations of conventional treatments. An i-body is a human immunoglobulin scaffold with two long binding loops that mimic the shape and position of those found in shark antibodies, the variable new antigen receptors of sharks. Its small size (∼12 kDa) and long binding loops provide access to drug targets, which are considered undruggable by traditional monoclonal antibodies. Here, we have successfully identified a human receptor activator of nuclear factor-κB ligand (RANKL) i-body, ADR3, which demonstrates a high binding affinity to human RANKL (hRANKL) with no adverse effect on the survival or proliferation of bone marrow-derived macrophages. Differential scanning fluorimetry suggested that ADR3 is stable and able to tolerate a wide range of physical environments (including both temperature and pH). In addition, in vitro studies showed a dose-dependent inhibitory effect of ADR3 on osteoclast differentiation, podosome belt formation, and bone resorption activity. Further investigation on the mechanism of action of ADR3 revealed that it can inhibit hRANKL-mediated signaling pathways, supporting the in vitro functional observations. These clues collectively indicate that hRANKL antagonist ADR3 attenuates osteoclast differentiation and bone resorption, with the potential to serve as a novel therapeutic to protect against bone loss.

Macrophage expressed tartrate-resistant acid phosphatase 5 promotes pulmonary fibrosis progression.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disorder involving scarring of pulmonary tissue and a subsequent decrease in respiratory capacity, ultimately resulting in death. Tartrate resistant acid phosphatase 5 (ACP5) plays a role in IPF but the exact mechanisms are yet to be elucidated. In this study, we have utilized various perturbations of the bleomycin mouse model of IPF including genetic knockout, RANKL inhibition, and macrophage adoptive transfer to further understand ACP5's role in pulmonary fibrosis. Genetic ablation of Acp5 decreased immune cell recruitment to the lungs and reduced the levels of hydroxyproline (reflecting extracellular matrix-production) as well as histological damage. Additionally, gene expression profiling of murine lung tissue revealed downregulation of genes including Ccl13, Mmp13, and Il-1α that encodes proteins specifically related to immune cell recruitment and macrophage/fibroblast interactions. Furthermore, antibody-based neutralization of RANKL, an important inducer of Acp5 expression, reduced immune cell recruitment but did not decrease fibrotic lung development. Adoptive transfer of Acp5-/- bone marrow-derived monocyte (BMDM) macrophages 7 or 14 days after bleomycin administration resulted in reductions of cytokine production and decreased levels of lung damage, compared to adoptive transfer of WT control macrophages. Taken together, the data presented in this study suggest that macrophage derived ACP5 plays an important role in development of pulmonary fibrosis and could present a tractable target for therapeutic intervention in IPF.

Recent advances of NFATc1 in rheumatoid arthritis-related bone destruction: mechanisms and potential therapeutic targets.

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease characterized by inflammation of the synovial tissue and joint bone destruction, often leading to significant disability. The main pathological manifestation of joint deformity in RA patients is bone destruction, which occurs due to the differentiation and proliferation of osteoclasts. The transcription factor nuclear factor-activated T cell 1 (NFATc1) plays a crucial role in this process. The regulation of NFATc1 in osteoclast differentiation is influenced by three main factors. Firstly, NFATc1 is activated through the upstream nuclear factor kappa-B ligand (RANKL)/RANK signaling pathway. Secondly, the Ca2+-related co-stimulatory signaling pathway amplifies NFATc1 activity. Finally, negative regulation of NFATc1 occurs through the action of cytokines such as B-cell Lymphoma 6 (Bcl-6), interferon regulatory factor 8 (IRF8), MAF basic leucine zipper transcription factor B (MafB), and LIM homeobox 2 (Lhx2). These three phases collectively govern NFATc1 transcription and subsequently affect the expression of downstream target genes including TRAF6 and NF-κB. Ultimately, this intricate regulatory network mediates osteoclast differentiation, fusion, and the degradation of both organic and inorganic components of the bone matrix. This review provides a comprehensive summary of recent advances in understanding the mechanism of NFATc1 in the context of RA-related bone destruction and discusses potential therapeutic agents that target NFATc1, with the aim of offering valuable insights for future research in the field of RA. To assess their potential as therapeutic agents for RA, we conducted a drug-like analysis of potential drugs with precise structures.

Exploring the therapeutic potential of isoorientin in the treatment of osteoporosis: a study using network pharmacology and experimental validation.

BACKGROUND: Isoorientin (ISO) is a glycosylated flavonoid with antitumor, anti-inflammatory, and antioxidant properties. However, its effects on bone metabolism remain largely unknown. METHODS: In this study, we aimed to investigate the effects of ISO on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation in vitro and bone loss in post-ovariectomy (OVX) rats, as well as to elucidate the underlying mechanism. First, network pharmacology analysis indicated that MAPK1 and AKT1 may be potential therapeutic targets of ISO and that ISO has potential regulatory effects on the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathways, as well as oxidative stress. ISO was added to RAW264.7 cells stimulated by RANKL, and its effects on osteoclast differentiation were evaluated using tartrate-resistant acid phosphatase (TRAP) staining, TRAP activity measurement, and F-actin ring analysis. Reactive oxygen species (ROS) production in osteoclasts was detected using a ROS assay kit. The effects of ISO on RANKL-triggered molecular cascade response were further investigated by Western blotting, quantitative real-time polymerase chain reaction, and immunofluorescence staining. In addition, the therapeutic effects of ISO were evaluated in vivo. RESULTS: ISO inhibited osteoclastogenesis in a time- and concentration-dependent manner. Mechanistically, ISO downregulated the expression of the main transcription factor for osteoclast differentiation by inhibiting MAPK and PI3K/AKT1 signaling pathways. Moreover, ISO exhibited protective effects in OVX-induced bone loss rats. This was consistent with the results derived from network pharmacology. CONCLUSION: Our findings suggest a potential therapeutic utility of ISO in the management of osteoclast-associated bone diseases, including osteoporosis.

Dickkopf-1 (DKK1) blockade mitigates osteogenesis imperfecta (OI) related bone disease.

BACKGROUND: The current treatment of osteogenesis imperfecta (OI) is imperfect. Our study thus delves into the potential of using Dickkopf-1 antisense (DKK1-AS) to treat OI. METHODS: We analysed serum DKK1 levels and their correlation with lumbar spine and hip T-scores in OI patients. Comparative analyses were conducted involving bone marrow stromal cells (BMSCs) and bone tissues from wild-type mice, untreated OI mice, and OI mice treated with DKK1-ASor DKK1-sense (DKK1-S). RESULTS: Significant inverse correlations were noted between serum DKK1 levels and lumbar spine (correlation coefficient = - 0.679, p = 0.043) as well as hip T-scores (correlation coefficient = - 0.689, p = 0.042) in OI patients. DKK1-AS improved bone mineral density (p = 0.002), trabecular bone volume/total volume fraction (p < 0.001), trabecular separation (p = 0.010), trabecular thickness (p = 0.001), trabecular number (p < 0.001), and cortical thickness (p < 0.001) in OI mice. DKK1-AS enhanced the transcription of collagen 1α1, osteocalcin, runx2, and osterix in BMSC from OI mice (all p < 0.001), resulting in a higher von Kossa-stained matrix area (p < 0.001) in ex vivo osteogenesis assays. DKK1-AS also reduced osteoclast numbers (p < 0.001), increased ß-catenin and T-cell factor 4 immunostaining reactivity (both p < 0.001), enhanced mineral apposition rate and bone formation rate per bone surface (both p < 0.001), and decreased osteoclast area (p < 0.001) in OI mice. DKK1-AS upregulated osteoprotegerin and downregulated nuclear factor-kappa B ligand transcription (both p < 0.001). Bone tissues from OI mice treated with DKK1-AS exhibited significantly higher breaking force compared to untreated OI mice (p < 0.001). CONCLUSIONS: Our study elucidates that DKK1-AS has the capability to enhance bone mechanical properties, restore the transcription of osteogenic genes, promote osteogenesis, and inhibit osteoclastogenesis in OI mice.

Ctdnep1 phosphatase is required for negative regulation of RANKL-induced osteoclast differentiation in RAW264.7 cells.

Osteoclasts are multinucleated cells with bone resorption activity. Excessive osteoclast activity has been implicated in osteoporosis, rheumatoid arthritis, and bone destruction due to bone metastases from cancer, making osteoclasts essential target cells in bone and joint diseases. C-terminal domain nuclear envelope phosphatase 1 (Ctdnep1, formerly Dullard) is a negative regulator of transforming growth factor (TGF)-ß superfamily signaling and regulates endochondral ossification in mesenchymal cells during skeletal development. In this study, we investigated the role of Ctdnep1 in the Receptor activator of nuclear factor-kappa B ligand (RANKL)-induced RAW264.7 osteoclast differentiation. Expression of Ctdnep1 did not change during osteoclast differentiation; Ctdnep1 protein localized to the cytoplasm before and after osteoclast differentiation. Small interfering RNA-mediated knockdown of Ctdnep1 increased tartrate-resistant acid phosphatase-positive multinucleated osteoclasts and the expression of osteoclast marker genes, including Acp5, Ctsk, and Nfatc1. Interestingly, the knockdown of Ctdnep1 increased the protein level of Nfatc1 in cells unstimulated with RANKL. Knockdown of Ctdnep1 also enhanced calcium-resorbing activity. Mechanistically, the knockdown of Ctdnep1 increased the phosphorylation of RANKL signaling components. These results suggest that Ctdnep1 negatively regulates osteoclast differentiation by suppressing the RANKL signaling pathway.

Unique cartilage matrix-associated protein inhibits osteoclast differentiation by alleviating RANKL-induced reactive oxygen species.

Unique cartilage matrix-associated protein (UCMA) is a γ-carboxyglutamic acid-rich secretory protein primarily expressed in adult cartilage. UCMA promotes osteoblast differentiation and reduces high glucose-induced reactive oxygen species (ROS) production in osteoblasts; however, its role in osteoclasts remains unclear. Since Ucma is not expressed in osteoclasts, treatment with recombinant UCMA protein (rUCMA) was employed to investigate the effect of UCMA on osteoclasts. The rUCMA-treated osteoclasts exhibited significantly reduced osteoclast differentiation, resorption activity, and osteoclast-specific gene expression. Moreover, rUCMA treatment reduced RANKL-induced ROS production and increased the expression of antioxidant genes in osteoclasts. This study demonstrates that UCMA effectively inhibits RANKL-stimulated osteoclast differentiation and oxidative stress.

Bone-Targeted Biomimetic Nanogels Re-Establish Osteoblast/Osteoclast Balance to Treat Postmenopausal Osteoporosis.

Insufficient bone formation and excessive bone resorption caused by estrogen deficiency are the major factors resulting in the incidence of postmenopausal osteoporosis (PMOP). The existing drugs usually fail to re-establish the osteoblast/osteoclast balance from both sides and generate side-effects owing to the lack of bone-targeting ability. Here, engineered cell-membrane-coated nanogels PNG@mR&C capable of scavenging receptor activator of nuclear factor-κB ligand (RANKL) and responsively releasing therapeutic PTH 1-34 in the bone microenvironment are prepared from RANK and CXCR4 overexpressed bone mesenchymal stem cell (BMSC) membrane-coated chitosan biopolymers. The CXCR4 on the coated-membranes confer bone-targeting ability, and abundant RANK effectively absorb RANKL to inhibit osteoclastogenesis. Meanwhile, the release of PTH 1-34 triggered by osteoclast-mediated acid microenvironment promote osteogenesis. In addition, the dose and frequency are greatly reduced due to the smart release property, prolonged circulation time, and bone-specific accumulation. Thus, PNG@mR&C exhibits satisfactory therapeutic effects in the ovariectomized (OVX) mouse model. This study provides a new paradigm re-establishing the bone metabolic homeostasis from multitargets and shows great promise for the treatment of PMOP.

The abortive SARS-CoV-2 infection of osteoclast precursors promotes their differentiation into osteoclasts.

The Coronavirus Disease 2019 (COVID-19) pandemic has resulted in the loss of millions of lives, although a majority of those infected have managed to survive. Consequently, a set of outcomes, identified as long COVID, is now emerging. While the primary target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the respiratory system, the impact of COVID-19 extends to various body parts, including the bone. This study aims to investigate the effects of acute SARS-CoV-2 infection on osteoclastogenesis, utilizing both ancestral and Omicron viral strains. Monocyte-derived macrophages, which serve as precursors to osteoclasts, were exposed to both viral variants. However, the infection proved abortive, even though ACE2 receptor expression increased postinfection, with no significant impact on cellular viability and redox balance. Both SARS-CoV-2 strains heightened osteoclast formation in a dose-dependent manner, as well as CD51/61 expression and bone resorptive ability. Notably, SARS-CoV-2 induced early pro-inflammatory M1 macrophage polarization, shifting toward an M2-like profile. Osteoclastogenesis-related genes (RANK, NFATc1, DC-STAMP, MMP9) were upregulated, and surprisingly, SARS-CoV-2 variants promoted RANKL-independent osteoclast formation. This thorough investigation illuminates the intricate interplay between SARS-CoV-2 and osteoclast precursors, suggesting potential implications for bone homeostasis and opening new avenues for therapeutic exploration in COVID-19.

Is RANKL a potential molecular target in osteoarthritis?

OBJECTIVE: Osteoarthritis (OA) is a disease of joints, in which the bone under the articular cartilage undergoes increased remodelling activity. The question is whether a better understanding of the causes and mechanisms of bone remodelling can predict disease-modifying treatments. DESIGN: This review summarises the current understanding of the aetiology of OA, with an emphasis on events in the subchondral bone (SCB), and the cells and cytokines involved, to seek an answer to this question. RESULTS: SCB remodelling across OA changes the microstructure of the SCB, which alters the load-bearing properties of the joint and seems to have an important role in the initiation and progression of OA. Bone remodelling is tightly controlled by numerous cytokines, of which Receptor Activator of NFκB ligand (RANKL) and osteoprotegerin are central factors in almost all known bone conditions. In terms of finding therapeutic options for OA, an important question is whether controlling the rate of SCB remodelling would be beneficial. The role of RANKL in the pathogenesis and progression of OA and the effect of its neutralisation remain to be clarified. CONCLUSIONS: This review further makes the case for SCB remodelling as important in OA and for additional study of RANKL in OA, both its pathophysiological role and its potential as an OA disease target.

The novel cytotoxic polybisphosphonate osteodex decreases bone resorption by enhancing cell death of mature osteoclasts without affecting osteoclastogenesis of RANKL-stimulated mouse bone marrow macrophages.

It has previously been demonstrated that the polybisphosphonate osteodex (ODX) inhibits bone resorption in organ-cultured mouse calvarial bone. In this study, we further investigate the effects by ODX on osteoclast differentiation, formation, and function in several different bone organ and cell cultures. Zoledronic acid (ZOL) was used for comparison. In retinoid-stimulated mouse calvarial organ cultures, ODX and ZOL significantly reduced the numbers of periosteal osteoclasts without affecting Tnfsf11 or Tnfrsf11b mRNA expression. ODX and ZOL also drastically reduced the numbers of osteoclasts in cell cultures isolated from the calvarial bone and in vitamin D3-stimulated mouse crude bone marrow cell cultures. These data suggest that ODX can inhibit osteoclast formation by inhibiting the differentiation of osteoclast progenitor cells or by directly targeting mature osteoclasts. We therefore assessed if osteoclast formation in purified bone marrow macrophage cultures stimulated by RANKL was inhibited by ODX and ZOL and found that the initial formation of mature osteoclasts was not affected, but that the bisphosphonates enhanced cell death of mature osteoclasts. In agreement with these findings, ODX and ZOL did not affect the mRNA expression of the osteoclastic genes Acp5 and Ctsk and the osteoclastogenic transcription factor Nfatc1. When bone marrow macrophages were incubated on bone slices, ODX and ZOL inhibited RANKL-stimulated bone resorption. In conclusion, ODX does not inhibit osteoclast formation but inhibits osteoclastic bone resorption by decreasing osteoclast numbers through enhanced cell death of mature osteoclasts.

Effect of RANKL on Lower Depressive Symptoms In Hemodialysis Patients.

Depression and osteoporosis are common diseases in dialysis patients. In addition, patients with osteoporosis are more susceptible to depression. Contrary to previous anti-osteoporosis agents, denosumab and romosozumab could be used in dialysis patients and have similar action mechanisms for blocking RANKL. RANKL causes bone resorption after binding RANKL, but binding with OPG leads to suppress of bone resorption. In recent mice study, inhibition of RANKL with denosumab improved depressive-like phenotype. Besides, it was found that OPG was associated with depression. Therefore, this study aimed to investigate the association of depressive symptoms with RANKL and OPG in hemodialysis patients. We conducted a cross-sectional study with a total of 172 hemodialysis patients. The participants were measured for plasma RANKL, OPG, MMP-2, and MMP-9 levels. Logistic regression analysis was performed to evaluate the effect of RANKL and OPG on the presence of depressive symptoms. The depressive symptoms were observed in 90 (52.3%) subjects. RANKL tertile 3 had negative association with BDI score (ß - 4.527, 95% CI - 8.310 to - 0.743) in univariate analysis, and this association persisted even after multivariate adjustments (ß - 5.603, 95% CI - 9.715 to -1.491) in linear regression. In logistic regression between RANKL tertiles and depressive symptoms, RANKL tertile 3 had significantly lower unadjusted OR (0.40, 95% CI 0.19-0.86), and multivariate-adjusted OR (0.31, 95% CI 0.12-0.82) for depressive symptoms. OPG was not significantly associated with depressive symptoms. Higher plasma RANKL concentrations were significantly associated with lower depressive symptoms in HD patients.Trial registration WHO registry, No. KCT0003281, date: January 12, 2017.

Preventive Effects of Dental Pulp Stem Cell-conditioned Media on Anti-RANKL Antibody-Related Osteonecrosis of the Jaw.

Medication-related osteonecrosis of the jaw is a serious disease occurring in patients with cancer and osteoporosis, who are undergoing treatment with antiresorptive agents (ARAs) such as bisphosphonate (BP) or denosumab, an antibody targeting receptor activator of NF-κB ligand. Recently, stem cell-based therapy has been shown to be effective in preventing the development of bisphosphonate-related osteonecrosis of the jaw. However, studies on denosumab-related osteonecrosis of the jaw (DRONJ) remain limited. Here, the efficacy of treatment with dental pulp stem cell conditioned media (DPSC-CM) in preventing DRONJ in a murine model was evaluated. Local administration of DPSC-CM into the extraction socket of a mouse with DRONJ decreased the number of empty osteocyte lacunae and the prevalence of ONJ. In tissues surrounding the extraction sockets in the DPSC-CM-treated group, the expression of inflammatory cytokines was attenuated and that of osteogenesis-related molecules was enhanced compared to that in the control group. Further, the expression of Wnt signaling molecules, which had been suppressed, was improved. These findings collectively suggest that DPSC-CM prevents ONJ development in a murine DRONJ model.

Pan-histone deacetylase inhibitor vorinostat suppresses osteoclastic bone resorption through modulation of RANKL-evoked signaling and ameliorates ovariectomy-induced bone loss.

BACKGROUND: Estrogen deficiency-mediated hyperactive osteoclast represents the leading role during the onset of postmenopausal osteoporosis. The activation of a series of signaling cascades triggered by RANKL-RANK interaction is crucial mechanism underlying osteoclastogenesis. Vorinostat (SAHA) is a broad-spectrum pan-histone deacetylase inhibitor (HDACi) and its effect on osteoporosis remains elusive. METHODS: The effects of SAHA on osteoclast maturation and bone resorptive activity were evaluated using in vitro osteoclastogenesis assay. To investigate the effect of SAHA on the osteoclast gene networks during osteoclast differentiation, we performed high-throughput transcriptome sequencing. Molecular docking and the assessment of RANKL-induced signaling cascades were conducted to confirm the underlying regulatory mechanism of SAHA on the action of RANKL-activated osteoclasts. Finally, we took advantage of a mouse model of estrogen-deficient osteoporosis to explore the clinical potential of SAHA. RESULTS: We showed here that SAHA suppressed RANKL-induced osteoclast differentiation concentration-dependently and disrupted osteoclastic bone resorption in vitro. Mechanistically, SAHA specifically bound to the predicted binding site of RANKL and blunt the interaction between RANKL and RANK. Then, by interfering with downstream NF-κB and MAPK signaling pathway activation, SAHA negatively regulated the activity of NFATc1, thus resulting in a significant reduction of osteoclast-specific gene transcripts and functional osteoclast-related protein expression. Moreover, we found a significant anti-osteoporotic role of SAHA in ovariectomized mice, which was probably realized through the inhibition of osteoclast formation and hyperactivation. CONCLUSION: These data reveal a high affinity between SAHA and RANKL, which results in blockade of RANKL-RANK interaction and thereby interferes with RANKL-induced signaling cascades and osteoclastic bone resorption, supporting a novel strategy for SAHA application as a promising therapeutic agent for osteoporosis.

Novel inhibitor N-cyclopropyl-4-((4-((4-(trifluoromethyl)phenyl)sulfonyl)piperazin-1-yl)methyl)benzamide attenuates RANKL-mediated osteoclast differentiation in vitro.

Both cyclopropyl amide and piperazine sulfonamide functional groups are known for their various biological properties used for drug development. Herein, we synthesized nine new derivatives with different substituent groups incorporating these moieties and screened them for their anti-osteoclast differentiation activity. After analyzing the structure-activity relationship (SAR), the inhibitory effect against osteoclastogenesis was determined to be dependent on the lipophilicity of the compound. Derivative 5b emerged as the most effective dose-dependent inhibitor after TRAP staining with an IC50 of 0.64 µM against RANKL-induced osteoclast cells. 5b was also able to suppress F-acting ring formation and bone resorption activity of osteoclasts in vitro. Finally, well-acknowledged gene and protein osteoclast-specific marker expression levels were decreased after 5b administration on primary murine osteoclast cells.

The effect of interleukin-20 on periodontal tissue destruction in individuals with periodontitis.

BACKGROUND AND OBJECTIVE: Periodontitis is an inflammatory disease that destroys periodontal tissues. Interleukin-20 (IL-20), on the other hand, is known as a potent angiogenic, chemotactic, and pro-inflammatory cytokine associated with various chronic inflammatory disorders. IL-20 has a significant role in the regulation of osteoclastogenesis and osteoblastogenesis. This study aimed to evaluate the effect of IL-20 on periodontal destruction. METHODS: In this study, a total of 60 participants were included, 30 of whom were systemically and periodontally healthy (control group), and 30 were systemically healthy but had periodontitis (periodontitis group). Gingival crevicular fluid (GCF) and serum samples were collected from the participants for biochemical analysis. Enzyme-linked immunosorbent assay was used to determine the levels of IL-20, tumor necrosis factor (TNF)-α, IL1ß/IL-10, RANKL/osteoprotegerin (OPG), and matrix metalloproteinase-8 (MMP8). For statistical analysis, the independent t-test, Pearson correlation coefficients, and the Chi-square test were used. RESULTS: GCF IL-20, RANKL, RANKL/OPG, serum IL-20, RANKL, RANKL/OPG, MMP-8, TNF-α, IL-1B, and IL-1ß/IL-10 values were found to be statistically significantly higher in the periodontitis group than in the control group. GCF OPG and serum IL-10 values were found to be statistically significantly higher in the control group than in the periodontitis group. No statistically significant difference was observed between the groups in serum OPG values. A statistically significantly positive correlation was observed between serum IL-20 value and serum RANKL, RANKL/OPG, MMP-8, TNF-α, IL-1ß values, and periodontal clinical parameters. The ROC curves showed: AUC = 0.788 for GCF IL-20, and AUC = 1.000 for serum IL-20. CONCLUSION: According to the results of the study, IL-20 was found to be associated with periodontitis. The role of IL-20 in periodontal pathogenesis is related to osteoclastogenesis and collagen degradation. It is conceivable that IL-20 may increase bone destruction by both affecting the RANKL/OPG ratio and proinflammatory cytokines.

Dementia exacerbates periodontal bone loss in females.

BACKGROUND: Periodontitis is a chronic inflammatory disease defined by the pathologic loss of the periodontal ligament and alveolar bone in relation to aging. Although clinical cohort studies reported that periodontitis is significantly elevated in males compared to females, emerging evidence indicates that females with dementia are at a greater risk for periodontitis and decreased alveolar bone. OBJECTIVE: This study aimed to evaluate whether dementia is a potential sex-dependent risk factor for periodontal bone loss using an experimental model of periodontitis induced in the triple transgenic (3x-Tg) dementia-like mice and clinical samples collected from senior 65 plus age patients with diagnosed dementia. MATERIALS AND METHODS: We induced periodontitis in dementia-like triple-transgenic (3x-Tg) male and female mice and age-matched wild-type (WT) control mice by ligature placement. Then, alveolar bone loss and osteoclast activity were evaluated using micro-CT and in situ imaging assays. In addition, we performed dental examinations on patients with diagnosed dementia. Finally, dementia-associated Aß42 and p-Tau (T181) and osteoclastogenic receptor activator of nuclear factor kappa-Β ligand (RANKL) in gingival crevicular fluid (GCF) collected from mice and clinical samples were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Alveolar bone loss and in situ osteoclast activity were significantly elevated in periodontal lesions of 3x-Tg females but not males, compared to wild-type control mice. In addition, we also observed that the probing pocket depth (PPD) was also significantly elevated in female patients with dementia. Using ELISA assay, we observed that females had elevated levels of osteoclastogenic RANKL and dementia-associated Aß42 and p-Tau (T181) in the GCF collected from experimental periodontitis lesions and clinical samples. CONCLUSION: Altogether, we demonstrate that females with dementia have an increased risk for periodontal bone loss compared to males.