Clonal hematopoiesis driven by mutated DNMT3A promotes inflammatory bone loss.

Clonal hematopoiesis of indeterminate potential (CHIP) arises from aging-associated acquired mutations in hematopoietic progenitors, which display clonal expansion and produce phenotypically altered leukocytes. We associated CHIP-DNMT3A mutations with a higher prevalence of periodontitis and gingival inflammation among 4,946 community-dwelling adults. To model DNMT3A-driven CHIP, we used mice with the heterozygous loss-of-function mutation R878H, equivalent to the human hotspot mutation R882H. Partial transplantation with Dnmt3aR878H/+ bone marrow (BM) cells resulted in clonal expansion of mutant cells into both myeloid and lymphoid lineages and an elevated abundance of osteoclast precursors in the BM and osteoclastogenic macrophages in the periphery. DNMT3A-driven clonal hematopoiesis in recipient mice promoted naturally occurring periodontitis and aggravated experimentally induced periodontitis and arthritis, associated with enhanced osteoclastogenesis, IL-17-dependent inflammation and neutrophil responses, and impaired regulatory T cell immunosuppressive activity. DNMT3A-driven clonal hematopoiesis and, subsequently, periodontitis were suppressed by rapamycin treatment. DNMT3A-driven CHIP represents a treatable state of maladaptive hematopoiesis promoting inflammatory bone loss.

Identification of an IL-1 receptor mutation driving autoinflammation directs IL-1-targeted drug design.

The interleukin 1 (IL-1) pathway signals through IL-1 receptor type 1 (IL-1R1) and emerges as a central mediator for systemic inflammation. Aberrant IL-1 signaling leads to a range of autoinflammatory diseases. Here, we identified a de novo missense variant in IL-1R1 (p.Lys131Glu) in a patient with chronic recurrent multifocal osteomyelitis (CRMO). Patient PBMCs showed strong inflammatory signatures, particularly in monocytes and neutrophils. The p.Lys131Glu substitution affected a critical positively charged amino acid, which disrupted the binding of the antagonist ligand, IL-1Ra, but not IL-1α or IL-1ß. This resulted in unopposed IL-1 signaling. Mice with a homologous mutation exhibited similar hyperinflammation and greater susceptibility to collagen antibody-induced arthritis, accompanied with pathological osteoclastogenesis. Leveraging the biology of the mutation, we designed an IL-1 therapeutic, which traps IL-1ß and IL-1α, but not IL-1Ra. Collectively, this work provides molecular insights and a potential drug for improved potency and specificity in treating IL-1-driven diseases.

NOTCH2 Hajdu-Cheney Mutations Escape SCFFBW7-Dependent Proteolysis to Promote Osteoporosis.

Hajdu-Cheney syndrome (HCS), a rare autosomal disorder caused by heterozygous mutations in NOTCH2, is clinically characterized by acro-osteolysis, severe osteoporosis, short stature, neurological symptoms, cardiovascular defects, and polycystic kidneys. Recent studies identified that aberrant NOTCH2 signaling and consequent osteoclast hyperactivity are closely associated with the bone-related disorder pathogenesis, but the exact molecular mechanisms remain unclear. Here, we demonstrate that sustained osteoclast activity is largely due to accumulation of NOTCH2 carrying a truncated C terminus that escapes FBW7-mediated ubiquitination and degradation. Mice with osteoclast-specific Fbw7 ablation revealed osteoporotic phenotypes reminiscent of HCS, due to elevated Notch2 signaling. Importantly, administration of Notch inhibitors in Fbw7 conditional knockout mice alleviated progressive bone resorption. These findings highlight the molecular basis of HCS pathogenesis and provide clinical insights into potential targeted therapeutic strategies for skeletal disorders associated with the aberrant FBW7/NOTCH2 pathway as observed in patients with HCS.

AMPKα1 negatively regulates osteoclastogenesis and mitigates pathological bone loss.

Osteoclasts are specialized cells responsible for bone resorption, a highly energy-demanding process. Focus on osteoclast metabolism could be a key for the treatment of osteolytic diseases including osteoporosis. In this context, AMP-activated protein kinase α1 (AMPKα1), an energy sensor highly expressed in osteoclasts, participates in the metabolic reconfiguration during osteoclast differentiation and activation. This study aimed to elucidate the role of AMPKα1 during osteoclastogenesis in vitro and its impact in bone loss in vivo. Using LysMcre/0AMPK⍺1f/f animals and LysMcre/0 as control, we evaluated how AMPKα1 interferes with osteoclastogenesis and bone resorption activity in vitro. We found that AMPKα1 is highly expressed in the early stages of osteoclastogenesis. Genetic deletion of AMPKα1 leads to increased gene expression of osteoclast differentiation and fusion markers. In addition, LysMcre/0AMPK⍺1f/f mice had an increased number and size of differentiated osteoclast. Accordingly, AMPKα1 negatively regulates bone resorption in vitro, as evidenced by the area of bone resorption in LysMcre/0AMPK⍺1f/f osteoclasts. Our data further demonstrated that AMPKα1 regulates mitochondrial fusion and fission markers upregulating Mfn2 and downregulating DRP1 (dynamics-related protein 1) and that Ctskcre/0AMPK⍺1f/f osteoclasts lead to an increase in the number of mitochondria in AMPK⍺1-deficient osteoclast. In our in vivo study, femurs from Ctskcre/0AMPK⍺1f/f animals exhibited bone loss associated with the increased number of osteoclasts, and there was no difference between Sham and ovariectomized group. Our data suggest that AMPKα1 acts as a negative regulator of osteoclastogenesis, and the depletion of AMPKα1 in osteoclast leads to a bone loss state similar to that observed after ovariectomy.

Targeting the heparan sulfate-binding site of RAGE with monoclonal antibodies.

Heparan sulfate (HS) plays its biological functions by interacting with hundreds of secreted extracellular and transmembrane proteins. Interaction with HS has been shown to be required for the normal function of many HS-binding proteins. Receptor for advanced glycation end-product (RAGE) is such a protein, whose activation requires HS-induced oligomerization. Using RAGE as an exemplary protein, we show here the workflow of a simple method of developing and characterizing mAbs that targets the HS-binding site. We found that HS-binding site of RAGE is quite immunogenic as 18 out of 94 anti-RAGE mAbs target various epitopes within the HS-binding site. Sequence analysis found that a common feature of anti-HS-binding site mAbs is the presence of abundant acidic residues (range between 6 to 11) in the complementarity determining region, suggesting electrostatic interaction plays an important role in promoting antigen-antibody interaction. Interestingly, mAbs targeting different epitopes within the HS-binding site blocks HS-RAGE interaction to different degrees, and the inhibitory effect is highly consistent among mAbs that target the same epitope. Functional assay revealed that anti-HS-binding site mAbs show different potency in inhibiting osteoclastogenesis, and the inhibitory potency does not have a simple correlation with the affinity and the epitope. Our study demonstrates that developing HS-binding site targeting mAbs should be applicable to most HS-binding proteins. By targeting this unique functional site, these mAbs might find therapeutic applications in treating various human diseases.

Age-related decline in melatonin contributes to enhanced osteoclastogenesis via disruption of redox homeostasis.

BACKGROUND: Increased oxidative stress contributes to enhanced osteoclastogenesis and age-related bone loss. Melatonin (MT) is an endogenous antioxidant and declines with aging. However, it was unclear whether the decline of MT was involved in the enhanced osteoclastogenesis during the aging process. METHODS: The plasma level of MT, oxidative stress status, bone mass, the number of bone marrow-derived monocytes (BMMs) and its osteoclastogenesis were analyzed in young (3-month old) and old (18-month old) mice (n = 6 per group). In vitro, BMMs isolated from aged mice were treated with or without MT, followed by detecting the change of osteoclastogenesis and intracellular reactive oxygen species (ROS) level. Furthermore, old mice were treated with MT for 2 months to investigate the therapeutic effect. RESULTS: The plasma level of MT was markedly lower in aged mice compared with young mice. Age-related decline in MT was accompanied by enhanced oxidative stress, osteoclastogenic potential and bone loss. MT intervention significantly suppressed the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis, decreased intracellular ROS and enhanced antioxidant capacity of BMMs from aged mice. MT supplementation significantly attenuated oxidative stress, osteoclastogenesis, bone loss and deterioration of bone microstructure in aged mice. CONCLUSIONS: These results suggest that age-related decline of MT enhanced osteoclastogenesis via disruption of redox homeostasis. MT may serve as a key regulator in osteoclastogenesis and bone homeostasis, thereby highlighting its potential as a preventive agent for age-related bone loss.

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.

mmu-miR-185 regulates osteoclasts differentiation and migration by targeting Btk.

BACKGROUND: Bones undergo a constant remodeling, a process involving osteoclast-mediated bone resorption and osteoblast-mediated bone formation, crucial for maintaining healthy bone mass. We previously observed that miR-185 depletion may promote bone formation by regulating Bgn expression and the BMP/Smad signaling pathway. However, the effects of miR-185-5p on the osteoclasts and bone remodeling have not been elucidated, warranting further exploration. METHODS: Tartrate-resistant acid phosphatase staining was utilized to assess the differentiation ability of bone marrow mononuclear macrophages (BMMs) from mmu-miR-185 gene knockout (KO) mice and wild-type (WT) mice. A reverse transcriptase-quantitative PCR was conducted to compare differences in miR-185-5p and osteoclast marker molecules, including Trap, Dcstamp, Ctsk and Nfatc1, between the KO group and WT group BMMs. Western blot analysis was employed to observe the expression of osteoclast marker molecules. A cell-counting kit-8 was used to analyze cell proliferation ability. Transwell experiments were conducted to detect cell migration. Dual-luciferase reporter assays were employed to confirm whether Btk is a downstream target gene of miR-185-5p. RESULTS: miR-185 depletion promoted osteoclast differentiation in bone marrow-derived monocytes/macrophages. Overexpression of miR-185-5p in RAW264.7 cells inhibited differentiation and migration of osteoclasts. Furthermore, Btk was identified as a downstream target gene of miR-185-5p, suggesting that miR-185-5p may inhibit osteoclast differentiation and migration by targeting Btk. CONCLUSIONS: miR-185 regulates osteoclasts differentiation, with overexpression of miR-185-5p inhibiting osteoclast differentiation and migration in vitro. Additionally, miR-185-5p may modulate osteoclastic differentiation and migration by regulating Btk expression.

The stiffness and collagen control differentiation of osteoclasts with an altered expression of c-Src in podosome.

Osteoclasts are hematopoietic cells attached to the bones containing type I collagen-deposited hydroxyapatite during bone resorption. Two major elements determine the stiffness of bones: regular calcified bone (bone that is resorbable by osteoclasts) and un-calcified osteoid bone (bone that is un-resorbable by osteoclasts). The osteolytic cytokine RANKL promotes osteoclast differentiation; however, the roles of the physical interactions of osteoclasts with calcified and un-calcified bone at the sealing zones and the subsequent cellular signaling remain unclear. In this study, we investigated podosomes, actin-rich adhesion structures (actin-ring) in the sealing zone that participates in sensing hard stiffness with collagen in the physical environment during osteoclast differentiation. RANKL-induced osteoclast differentiation induction was promoted when Raw264.7 cells were cultured on collagen-coated plastic dishes but not on non-coated plastic dishes, which was associated with the increased expression of podosome-related genes and Src. In contrast, when cells were cultured on collagen gel, expression of podosome-related genes and Src were not upregulated. The induction of podosome-related genes and Src requires hard stiffness with RGD-containing substratum and integrin-mediated F-actin polymerization. These results indicate that osteoclasts sense both the RGD sequence and stiffness of calcified collagen through their podosome components regulating osteoclast differentiation via the c-Src pathway.

Neutrophil extracellular traps inhibit osteoclastogenesis.

Neutrophil extracellular traps (NETs) released by neutrophils upon inflammation or infection, act as an innate immune defense against pathogens. NETs also influence inflammatory responses and cell differentiation in host cells. Osteoclasts, which are derived from myeloid stem cells, are critical for the bone remodeling by destroying bone. In the present study, we explores the impact of NETs, induced by the inflammatory agent calcium ionophore A23187, on the differentiation and activation of osteoclasts, potentially through suppressing RANK expression. Our results collectively suggested that the inhibition of RANKL-mediated osteoclastogenesis by NETs might lead to the suppression of excessive bone resorption during inflammation.

A novel method to efficiently differentiate human osteoclasts from blood-derived monocytes.

BACKGROUND: Osteoclasts are the tissue-specific macrophage population of the bone and unique in their bone-resorbing activity. Hence, they are fundamental for bone physiology in health and disease. However, efficient protocols for the isolation and study of primary human osteoclasts are scarce. In this study, we aimed to establish a protocol, which enables the efficient differentiation of functional human osteoclasts from monocytes. RESULTS: Human monocytes were isolated through a double-density gradient from donor blood. Compared to standard differentiation schemes in polystyrene cell culture dishes, the yield of multinuclear osteoclasts was significantly increased upon initial differentiation of monocytes to macrophages in fluorinated ethylene propylene (FEP) Teflon bags. This initial differentiation phase was then followed by the development of terminal osteoclasts by addition of Receptor Activator of NF-κB Ligand (RANKL). High concentrations of RANKL and Macrophage colony-stimulating factor (M-CSF) as well as an intermediate cell density further supported efficient cell differentiation. The generated cells were highly positive for CD45, CD14 as well as the osteoclast markers CD51/ITGAV and Cathepsin K/CTSK, thus identifying them as osteoclasts. The bone resorption of the osteoclasts was significantly increased when the cells were differentiated from macrophages derived from Teflon bags compared to macrophages derived from conventional cell culture plates. CONCLUSION: Our study has established a novel protocol for the isolation of primary human osteoclasts that improves osteoclastogenesis in comparison to the conventionally used cultivation approach.

Bioactive Self-Regulated Liquified Microcompartments to Bioengineer Bone-Like Microtissues.

Designing a microenvironment that drives autonomous stromal cell differentiation toward osteogenesis while recapitulating the complexity of bone tissue remains challenging. In the current study, bone-like microtissues are created using electrohydrodynamic atomization to form two distinct liquefied microcapsules (mCAPs): i) hydroxypyridinone (HOPO)-modified gelatin (GH mCAPs, 7.5% w/v), and ii) HOPO-modified gelatin and dopamine-modified gelatin (GH+GD mCAPs, 7.5%+1.5% w/v). The ability of HOPO to coordinate with iron ions at physiological pH allows the formation of a semipermeable micro-hydrogel shell. In turn, the dopamine affinity for calcium ions sets a bioactive milieu for bone-like microtissues. After 21 days post encapsulation, GH and GH+GD mCAPs potentiate autonomous osteogenic differentiation of mesenchymal stem cells accompanied by collagen type-I gene upregulation, increased alkaline phosphatase (ALP) expression, and formation of mineralized extracellular matrix. However, the GH+GD mCAPs show higher levels of osteogenic markers starting on day 14, translating into a more advanced and organized mineralized matrix. The GH+GD system also shows upregulation of the receptor activator of nuclear factor kappa-B ligand (RANK-L) gene, enabling the autonomous osteoclastic differentiation of monocytes. These catechol-based mCAPs offer a promising approach to designing multifunctional and autonomous bone-like microtissues to study in vitro bone-related processes at the cell-tissue interface, angiogenesis, and osteoclastogenesis.

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.

Role of Piezo1 in modulating the RANKL/OPG ratio in mouse osteoblast cells exposed to Porphyromonas gingivalis lipopolysaccharide and mechanical stress.

AIMS: Excessive occlusal force with periodontitis leads to rapid alveolar bone resorption. However, the molecular mechanism by which inflammation and mechanical stress cause bone resorption remains unclear. We examined the role of Piezo1, a mechanosensitive ion channel expressed on osteoblasts, in the changes in the receptor activator of nuclear factor-kappa B ligand (RANKL)/osteoprotegerin (OPG) ratio in mouse MC3T3-E1 osteoblast-like cells under Porphyromonas gingivalis lipopolysaccharide (P.g.-LPS) and mechanical stress. METHODS: To investigate the effect of P.g.-LPS and mechanical stress on the RANKL/OPG ratio and Piezo1 expression, we stimulated MC3T3-E1 cells with P.g.-LPS. After 3 days in culture, shear stress, a form of mechanical stress, was applied to the cells using an orbital shaker. Subsequently, to investigate the role of Piezo1 in the change of RANKL/OPG ratio, we inhibited Piezo1 function by knockdown via Piezo1 siRNA transfection or by adding GsMTx4, a Piezo1 antagonist. RESULTS: The RANKL/OPG ratio significantly increased in MC3T3-E1 cells cultured in a medium containing P.g.-LPS and undergoing mechanical stress compared to cells treated with P.g.-LPS or mechanical stress alone. However, the expression of Piezo1 was not increased by P.g.-LPS and mechanical stress. In addition, phosphorylation of MEK/ERK was induced in the cells under P.g.-LPS and mechanical stress. MC3T3-E1 cells treated with P.g.-LPS and mechanical stress when cocultured with RAW264.7 cells induced their differentiation into osteoclast-like cells. The increased RANKL/OPG ratio was suppressed by either Piezo1 knockdown or the addition of GsMTx4. Furthermore, GsMTx4 inhibited the phosphorylation of MEK/ERK. CONCLUSION: These findings suggest that P.g.-LPS and Piezo1-mediated mechanical stress induce MEK/ERK phosphorylation and increase RANKL expression in osteoblasts. Consequently, this leads to the differentiation of osteoclast precursor cells into osteoclasts.

Hepatocyte growth factor is protective in early stage but bone-destructive in late stage of experimental periodontitis.

BACKGROUND AND OBJECTIVE: Clinical studies found high levels of hepatocyte growth factor (HGF) expression in patients with periodontitis. Studies suggest that HGF plays an important role in periodontitis, is involved in inflammation, and modulates alveolar bone integrity in periodontitis. This study aims to investigate the effects and mechanisms of HGF in the progression of experimental periodontitis. METHODS: We used silk thread ligation to induce periodontitis in HGF-overexpressing transgenic (HGF-Tg) and wild-type C57BL/6J mice. The effects of HGF overexpression on alveolar bone destruction were assessed by microcomputed tomography imaging at baseline and on days 7, 14, 21, and 28. We analyzed the cytokines (IL-6 and TNF-α) and lymphocytes in periodontitis tissues by enzyme-linked immunosorbent assay and flow cytometry. The effects of HGF on alveolar bone destruction were further tested by quantifying the systemic bone metabolism markers CTXI and PINP and by RNA sequencing for the signaling pathways involved in bone destruction. Western blotting and immunohistochemistry were performed to further elucidate the involved signaling pathways. RESULTS: We found that experimental periodontitis increased HGF production in periodontitis tissues; however, the effects of HGF overexpression were inconsistent with disease progression. In the early stage of periodontitis, periodontal inflammation and alveolar bone destruction were significantly lower in HGF-Tg mice than in wild-type mice. In the late stage, HGF-Tg mice showed higher inflammatory responses and progressively aggravated bone destruction with continued stimulation of inflammation. We identified the IL-17/RANKL/TRAF6 pathway as a signaling pathway involved in the HGF effects on the progression of periodontitis. CONCLUSION: HGF plays divergent effects in the progression of experimental periodontitis and accelerates osteoclastic activity and bone destruction in the late stage of inflammation.

The impact of arthritogenic viruses in oral tissues.

Arthritis and periodontitis are inflammatory diseases that share several immunopathogenic features. The expansion in the study of virus-induced arthritis has shed light on how this condition could impact other parts of the human body, including the mouth. Viral arthritis is an inflammatory joint disease caused by several viruses, most notably the alphaviruses Chikungunya virus (CHIKV), Sindbis virus (SINV), Ross River virus (RRV), Mayaro virus (MAYV), and O'nyong'nyong virus (ONNV). These viruses can induce an upsurge of matrix metalloproteinases and immune-inflammatory mediators such as Interleukin-6 (IL6), IL-1ß, tumor necrosis factor, chemokine ligand 2, and receptor activator of nuclear factor kappa-B ligand in the joint and serum of infected individuals. This can lead to the influx of inflammatory cells to the joints and associated muscles as well as osteoclast activation and differentiation, culminating in clinical signs of swelling, pain, and bone resorption. Moreover, several data indicate that these viral infections can affect other sites of the body, including the mouth. The human oral cavity is a rich and diverse microbial ecosystem, and viral infection can disrupt the balance of microbial species, causing local dysbiosis. Such events can result in oral mucosal damage and gingival bleeding, which are indicative of periodontitis. Additionally, infection by RRV, CHIKV, SINV, MAYV, or ONNV can trigger the formation of osteoclasts and upregulate pro-osteoclastogenic inflammatory mediators, interfering with osteoclast activation. As a result, these viruses may be linked to systemic conditions, including oral manifestations. Therefore, this review focuses on the involvement of alphavirus infections in joint and oral health, acting as potential agents associated with oral mucosal inflammation and alveolar bone loss. The findings of this review demonstrate how alphavirus infections could be linked to the comorbidity between arthritis and periodontitis and may provide a better understanding of potential therapeutic management for both conditions.

BASP1 down-regulates RANKL-induced osteoclastogenesis.

The cytokine RANKL (Receptor Activator of NFκB Ligand) is the key driver of differentiation of monocytes/macrophages to form multi-nucleated, bone-resorbing osteoclasts, a process that is accompanied by significant changes in gene expression. We show that exposure to RANKL rapidly down-regulates expression of Brain Acid Soluble Protein 1 (BASP1) in cultured primary mouse bone marrow macrophages (BMMs), and that this reduced expression is causally linked to the osteoclastogenic process in vitro. Knocking down BASP1 expression in BMMs or eliminating its expression in these cells or in RAW 264.7 cells enhanced RANKL-induced osteoclastogenesis, promoted cell-cell fusion, and generated cultures containing larger osteoclasts with increased mineral degrading abilities relative to controls. Expression of exogenous BASP1 in BMMs undergoing osteoclastogenic differentiation produced the opposite effects. Upon exposure to RANKL, primary mouse BMMs in which BASP1 had been knocked down exhibited increased expression of the key osteoclastogenic transcription factor Nfatc1and of its downstream target genes Dc-stamp, Ctsk, Itgb3, and Mmp9 relative to controls. The knock-down cells also exhibited increased sensitivity to the pro-osteoclastogenic effects of RANKL. We conclude that BASP1 is a negative regulator of RANKL-induced osteoclastogenesis, which down-regulates the pro-osteoclastogenic gene expression pattern induced by this cytokine. Decreased expression of BASP1 upon exposure of BMMs to RANKL removes a negative regulator of osteoclastogenesis and promotes this process.

Sucrosephenylpropanoid esters and isoflavonoids isolated from Belamcanda chinensis roots and their potential anti-osteoclastogenic activity.

Repeated chromatography of the CH2Cl2 and EtOAc soluble fractions from the methanol extract of Belamcanda chinensis root yielded six new sucrosephenylpropanoid esters (1-6) and twenty-one known compounds (7-27). The structures of 1-6 were elucidated using diverse nuclear magnetic resonance (NMR) techniques and high-resolution mass spectrometry (HRMS) data analysis, together with chemical methods. All the twenty-seven isolated compounds were evaluated for their anti-osteoclastogenic activities. Preliminary screening results revealed that compounds 1 and 19 exhibited strong effects against RANKL-induced osteoclast formation in RAW264.7 cells. In addition, the treatment of mouse bone marrow macrophages (BMMs) with compounds 1 and 19 significantly decreased RANKL-induced TRAP-positive multinucleated osteoclast formation in a concentration-dependent manner without affecting cell viability. Further bioassay investigation showed that compounds 1 and 19 inhibited the expression of some osteoclast-specific marker genes and the transcription factor nuclear factor of activated T cells cytoplasmic 1 (NFATc1) in response to RANKL. To the best of our knowledge, this is the first investigation of anti-osteoclastogenic activity for compounds isolated from B. chinensis.

Macrolide sesquiterpene pyridine alkaloids from Celastrus monospermus and evaluation of their immunosuppressive and anti-osteoclastogenesis activities.

Phytochemical investigation of the stems of Celastrus monospermus Roxb enabled isolation and identification of fifteen new macrolide sesquiterpene pyridine alkaloids (1-15) along with five known analogues. Their structures were elucidated by comprehensive spectroscopic analysis (NMR, HRESIMS, IR, UV), chemical hydrolysis, and single crystal X-ray diffraction analysis. Bioassay of the abundant isolates revealed that seven compounds inhibited the proliferation of B lymphocytes with IC50 values ranging between 1.4 and 19.9 µM. Among them, celasmondine C (3) could significantly promote the apoptosis of activated B lymphocyte, especially late-stage apoptosis. Besides, compounds 3, 16, and 20 exhibited potent suppression of osteoclast formation at a concentration of 1.0 µM. This investigation enriched the chemical diversity of macrolide sesquiterpene pyridine alkaloids, and supported evidence for the development of new immunosuppressive and anti-osteoclastogenesis agents.

Discovery of TCP-(MP)-caffeic acid analogs as a new class of agents for treatment of osteoclastic bone loss.

Inhibition of LSD1 was proposed as promising and attractive therapies for treating osteoporosis. Here, we synthesized a series of novel TCP-(MP)-Caffeic acid analogs as potential LSD1 inhibitors to assess their inhibitory effects on osteoclastogenesis by using TRAP-staining assay and try to explore the preliminary SAR. Among them, TCP-MP-CA (11a) demonstrated osteoclastic bone loss both in vitro and in vivo, showing a significant improvement in the in vivo effects compared to the LSD1 inhibitor GSK-LSD1. Additionally, we elucidated a mechanism that 11a and its precursor that 11e directly bind to LSD1/CoREST complex through FAD to inhibit LSD1 demethylation activity and influence its downstream IκB/NF-κB signaling pathway, and thus regulate osteoclastic bone loss. These findings suggested 11a or 11e as potential novel candidates for treating osteoclastic bone loss, and a concept for further development of TCP-(MP)-Caffeic acid analogs for therapeutic use in osteoporosis clinics.