F-actin Ring Formation Research Articles

Irg1 regulates bone homeostasis via regulating the Grk5 expression

We have previously demonstrated that itaconic acid can regulate osteoclast differentiation in vitro and in vivo, thereby affecting the progression of osteoporosis. The role of Irg1 as itaconic acid catalytic enzyme in bone homeostasis has not been clearly elucidated. Here, we detected enhanced the osteoclast differentiation in Irg1-deficiency BMMs, along with the expression of genes associated with osteoclastogenesis. Irg1 knockout promoted the expression of Nfatc1 and F-actin ring formation, with the inhibited production of itaconate. RNA-seq analysis was carried out and we proved that Grk5 expression was increased the most. Inhibition of Grk5 attenuated the effect of Irg1 in the osteoclastogenesis. However, micro-CT analysis showed no significant difference of bone trabecular structure in Irg1 knockout mice. Moreover, we observed no significant difference of osteoclasts numbers in the femur of Irg1 knockout mice in vivo. And similar bone formation was detected between the Irg1 knockout and WT mice, indicating that irg1 had slight effect on the bone homeostasis under physiological conditions. Surprising, we detected higher level of inflammatory factors in the bone tissues of Irg1 knockout mice. Above all, we for the first time demonstrated that Irg1 knockout promoted the osteoclastogenesis via regulating the Grk5 signaling. Regulation of irg1-Grk5 axis could be effective in treating human diseases under pathological situations in the future.

Exploring the Logic and Conducting a Comprehensive Evaluation of the Adiponectin Receptor Agonists AdipoRon and AdipoAI's Impacts on Bone Metabolism and Repair-A Systematic Review.

Adiponectin replacement therapy shows promising outcomes in various diseases, especially for bone-related disorders. Challenges in using the complete protein have led to alternative approaches, with AdipoRon and AdipoAI emerging as extensively researched drug candidates. Their influence on models of bone-related disorders has progressed considerably but there has been no review of their effectiveness in modulating bone metabolism and repair. This systematic review seeks to address this knowledge gap. Based on preclinical evidence from PubMed, EMBASE, and COCHRANE, ten studies were included following PRISMA guidelines. The JBI Checklist Critical Appraisal Tool assessed the quality of this systematic review. The studies encompassed various animal models, addressing bone defects, osseointegration, diabetes-associated periodontitis, fracture repair, growth retardation, and diabetes-associated peri-implantitis. AdipoRon and AdipoAI demonstrated effectiveness in modulating bone metabolism and repair through diverse pathways, including the activation of AdipoR1/APPL1, inhibition of F-actin ring formation, suppression of IκB-α phosphorylation, p65 nuclear translocation and Wnt5a-Ror2 signaling pathway, reduction of CCL2 secretion and expression, regulation of autophagy via LC3A/B expression, modulation of SDF-1 production, activation of the ERK-1/2 signaling pathway, modulation of bone integration-related markers and osteokines such as RANKL, BMP-2, OPG, OPN, and Runx2, inhibition of RANKL in osteoblasts, and inhibition of podosome formation via the activation of AMPK. While preclinical studies show promise, human trials are crucial to confirm the clinical safety and effectiveness of AdipoRon and AdipoAI. Caution is necessary due to potential off-target effects, especially in bone therapy with multi-target approaches. Structural biology and computational methods can help predict and understand these effects.

NAT10 promotes osteoclastogenesis in inflammatory bone loss by catalyzing Fos mRNA ac4C modification and upregulating MAPK signaling pathway

IntroductionExcessive osteoclastogenesis is a key driver of inflammatory bone loss. Suppressing osteoclastogenesis has always been considered essential for the treatment of inflammatory bone loss. N-acetyltransferase 10 (NAT10) is the sole enzyme responsible for N4-acetylcytidine (ac4C) modification of mRNA, and is involved in cell development. However, its role in osteoclastogenesis and inflammatory bone loss remained elusive. ObjectivesWe aimed to clarify the regulatory mechanism of NAT10 and ac4C modification in osteoclastogenesis and inflammatory bone loss. MethodsNAT10 expression and ac4C modification during osteoclastogenesis were determined by quantitative real-time PCR (qPCR), western blotting, dot blot and immunofluorescent staining, and the effect of NAT10 inhibition on osteoclast differentiation in vitro was measured by the tartrate-resistant acid phosphatase staining, podosome belts staining assay and bone resorption pit assay. Then, acRIP-qPCR and NAT10RIP-qPCR, ac4C site prediction, mRNA decay assay and luciferase reporter assay were performed to further study the underlying mechanisms. At last, mice models of inflammatory bone loss were applied to verify the therapeutic effect of NAT10 inhibition in vivo. ResultsNAT10 expression was upregulated during osteoclast differentiation and highly expressed in alveolar bone osteoclasts from periodontitis mice. Inhibition of NAT10 notably reduced osteoclast differentiation in vitro, as indicated by great reduction of tartrated resistant acid phosphatse positive multinuclear cells, osteoclast-specific gene expression, F-actin ring formation and bone resorption capacity. Mechanistically, NAT10 catalyzed ac4C modification of Fos (encoding AP-1 component c-Fos) mRNA and maintained its stabilization. Besides, NAT10 promoted MAPK signaling pathway and thereby activated AP-1 (c-Fos/c-Jun) transcription for osteoclastogenesis. Therapeutically, administration of Remodelin, the specific inhibitor of NAT10, remarkably impeded the ligature-induced alveolar bone loss and lipopolysaccharide-induced inflammatory calvarial osteolysis. ConclusionsOur study demonstrated that NAT10-mediated ac4C modification is an important epigenetic regulation of osteoclast differentiation and proposed a promising therapeutic target for inflammatory bone loss.

A p38 MAP kinase inhibitor suppresses osteoclastogenesis and alleviates ovariectomy-induced bone loss through the inhibition of bone turnover

Inhibition of excessive osteoclastic activity is an efficient therapeutic strategy for many bone diseases induced by increased bone resorption, such as osteoporosis. BMS-582949, a clinical p38α inhibitor, is a promising drug in Phase II studies for treating rheumatoid arthritis. However, its function on bone resorption is largely unknown. In this study, we find that BMS-582949 represses RANKL-induced osteoclast differentiation in a dose-dependent manner. Moreover, BMS-582949 inhibits osteoclastic F-actin ring formation and osteoclast-specific gene expression. Mechanically, BMS-582949 treatment attenuates RANKL-mediated osteoclastogenesis through mitogen-activated protein kinases (MAPKs) and protein kinase B (AKT) signaling pathways without disturbing nuclear factor-κB (NF-κB) signaling. Interestingly, BMS-582949 impairs osteoclastic mitochondrial biogenesis and functions, such as oxidative phosphorylation (OXPHOS). Furthermore, BMS-582949 administration prevents bone loss in ovariectomized mouse mode by inhibiting both bone resorption and bone formation in vivo. Taken together, these findings indicate that BMS-582949 may be a potential and effective drug for the therapy of osteolytic diseases.

Cirsilineol inhibits RANKL-induced osteoclast activity and ovariectomy-induced bone loss via NF-κb/ERK/p38 signaling pathways

BackgroundPostmenopausal osteoporosis is a chronic metabolic bone disease caused by excessive osteoclast formation and function. Targeting osteoclast differentiation and activity can modulate bone resorption and alleviate osteoporosis. Cirsilineol, an active constituent of Vestita Wall, has shown numerous biological activities and has been used to treat many metabolic diseases. However, whether cirsilineol inhibits osteoclast activity and prevents postmenopausal osteoporosis still remain unknown.Materials and methodsPrimary bone marrow macrophages (BMMs) and RAW264.7 cells were used. Osteoclast activity was measured by TRAP staining, F-actin staining, and bone resorption assay after BMMs were treated with cirsilineol at concentrations of 0, 1, 2.5 and 5 µM. RT-PCR and western blotting were performed to evaluate the expression of osteoclast-related genes. In addition, female C57BL/6 mice underwent OVX surgery and were treated with cirsilineol (20 mg/kg) to demonstrate the effect of cirsilineol on osteoporosis.ResultsCirsilineol significantly inhibited receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclast differentiation in a concentration- and time-dependent manner, respectively. Additionally, cirsilineol inhibited F-actin ring formation, thus reducing the activation of bone resorption ability. Cirsilineol suppressed the expression of osteoclast-related genes and proteins via blocking nuclear factor (NF)-κb, ERK, and p38 signaling cascades. More importantly, cirsilineol treatment in mice with osteoporosis alleviated osteoclasts hyperactivation and bone mass loss caused by estrogen depletion.ConclusionIn this study, the protective effect of cirsilineol on osteoporosis has been investigated for the first time. In conclusion, our findings prove the inhibitory effect of cirsilineol on osteoclast activity via NF-κb/ERK/p38 signaling pathways and strongapplication of cirsilineol can be proposed as a potential therapeutic strategy.

The novel small molecule E0924G dually regulates bone formation and bone resorption through activating the PPARδ signaling pathway to prevent bone loss in ovariectomized rats and senile mice

Osteoporosis is particularly prevalent among postmenopausal women and the elderly. In the present study, we investigated the effect of the novel small molecule E0924G (N-(4-methoxy-pyridine-2-yl)-5-methylfuran-2-formamide) on osteoporosis. E0924G significantly increased the protein expression levels of osteoprotegerin (OPG) and runt-related transcription factor 2 (RUNX2), and thus significantly promoted osteogenesis in MC3T3-E1 cells. E0924G also significantly decreased osteoclast differentiation and inhibited bone resorption and F-actin ring formation in receptor activator of NF-κB ligand (RANKL)-induced osteoclasts from RAW264.7 macrophages. Importantly, oral administration of E0924G in both ovariectomized (OVX) rats and SAMP6 senile mice significantly increased bone mineral density and decreased bone loss compared to OVX controls or SAMR1 mice. Further mechanistic studies showed that E0924G could bind to and then activate peroxisome proliferator-activated receptor delta (PPARδ), and the pro-osteoblast effect and the inhibition of osteoclast differentiation induced by E0924G were significantly abolished when PPARδ was knocked down or inhibited. In conclusion, these data strongly suggest that E0924G has the potential to prevent OVX-induced and age-related osteoporosis by dual regulation of bone formation and bone resorption through activation of the PPARδ signaling pathway.

Germacrone alleviates breast cancer-associated osteolysis by inhibiting osteoclastogenesis via inhibition of MAPK/NF-κB signaling pathways.

Bone is one of the most frequent sites for metastasis in breast cancer patients. Bone metastasis significantly reduces the survival time and the life quality of breast cancer patients. Germacrone (GM) can serve humans as an anti-cancer and anti-inflammation agent, but its effect on breast cancer-induced osteolysis remains unclear. This study aims to investigate the functions and mechanisms of GM in alleviating breast cancer-induced osteolysis. The effects of GM on osteoclast differentiation, bone resorption, F-actin ring formation, and gene expression were examined in vitro. RNA-sequencing and Western Blot were conducted to explore the regulatory mechanisms of GM on osteoclastogenesis. The effects of GM on breast cancer-induced osteoclastogenesis, and breast cancer cell malignant behaviors were also evaluated. The in vivo efficacy of GM in the ovariectomy model and breast cancer bone metastasis model with micro-CT and histomorphometry. GM inhibited osteoclastogenesis, bone resorption and F-actin ring formation in vitro. Meanwhile, GM inhibited the expression of osteoclast-related genes. RNA-seq analysis and Western Blot confirmed that GM inhibited osteoclastogenesis via inhibition of MAPK/NF-κB signaling pathways. The in vivo mouse osteoporosis model further confirmed that GM inhibited osteolysis. In addition, GM suppressed the capability of proliferation, migration, and invasion and promoted the apoptosis of MDA-MB-231 cells. Furthermore, GM could inhibit MDA-MB-231 cell-induced osteoclastogenesis in vitro and alleviate breast cancer-associated osteolysis in vivo human MDA-MB-231 breast cancer bone metastasis-bearing mouse models. Our findings identify that GM can be a promising therapeutic agent for patients with breast cancer osteolytic bone metastasis.

The effect of Schizophyllan on the differentiation of osteoclasts and osteoblasts

Schizophyllan (SPG), a β-glucan from Schizophyllum commune, is recognized for its antioxidant, immunoregulatory, and anticancer activities. In this study, its effects on bone cells, particularly osteoclasts and osteoblasts, were examined. We demonstrated that SPG dose-dependently inhibited osteoclastogenesis and reduced gene expression associated with osteoclast differentiation. SPG also decreased bone resorption and F-actin ring formation. This inhibition could have been due to the downregulation of transcription factors c-Fos and nuclear factor of activated T cells 1 (NFATc1) via the MAPKs (JNK and p38), IκBα, and PGC1β/PPARγ pathways. In coculture, SPG lowered osteoclastogenic activity in calvaria-derived osteoblasts by reducing macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor–κB ligand (RANKL) expression. In addition, SPG slightly enhanced osteoblast differentiation, as evidenced by increased differentiation marker gene expression and alizarin red staining. It also exhibited antiresorptive effects in a lipopolysaccharide-induced calvarial bone loss model. These results indicated a dual role of SPG in bone cell regulation by suppressing osteoclastogenesis and promoting osteoblast differentiation. Thus, SPG could be a therapeutic agent for bone resorption-related diseases such as osteoporosis, rheumatoid arthritis, and periodontitis.

Orcinol gentiobioside inhibits RANKL-induced osteoclastogenesis by promoting apoptosis and suppressing autophagy via the JNK1 signaling

Ethnopharmacological relevanceOsteoporosis (OP) is a metabolic disorder characterized by disrupted osteoclastic bone resorption and osteoblastic bone formation. Curculigo orchioides Gaertn has a long history of application in traditional Chinese and Indian medicine for treating OP. Orcinol gentiobioside (OGB) is a principal active constituent derived from Curculigo orchioides Gaertn and has been shown to have anti-OP activity. However, the therapeutic efficacy and mechanism of OGB in modulating osteoclastic bone resorption remain undefined. Aim of the studyTo evaluate the effect of OGB on the formation, differentiation and function of osteoclasts derived from bone marrow macrophages (BMMs), and further elucidate the underlying action mechanism of OGB in OP. Materials and methodsOsteoclasts derived from BMMs were utilized to evaluate the effect of OGB on osteoclast formation, differentiation and bone resorption. Tartrate-resistant acid phosphatase (TRAP) staining and activity assays were conducted to denote the activity of osteoclasts. Osteoclast-related genes and proteins were determined by RT-PCR and Western blotting assays. The formation of the F-actin ring was observed by confocal laser microscopy, and bone resorption pits were observed by inverted microscopy. The target of OGB in osteoclasts was predicted by using molecular docking and further verified by Cellular Thermal Shift Assay (CETSA) and reversal effects of the target activator. The apoptosis of osteoclasts was analyzed by flow cytometry, and autophagic flux in osteoclasts was determined by confocal laser microscopy. ResultsOGB inhibited osteoclast formation and differentiation, osteoclast-related genes and proteins expression, F-actin ring formation, and bone resorption activity. Molecular docking and CETSA analysis demonstrated that OGB exhibited good affinity for c-Jun N-terminal Kinase 1 (JNK1). In addition, OGB induced apoptosis and inhibited autophagy in osteoclasts, and the JNK agonist anisomycin reversed the increase in apoptosis and inhibition of autophagy induced by OGB in osteoclasts. ConclusionOGB inhibited osteoclastogenesis by promoting apoptosis and diminishing autophagy via JNK1 signaling.

Adipose Tissue-Derived Mesenchymal Stem Cell Inhibits Osteoclast Differentiation through Tumor Necrosis Factor Stimulated Gene-6.

Mesenchymal stem cells (MSCs) have been highlighted as a potent therapeutic option for conditions with excessive osteoclast activity such as systemic and local bone loss in rheumatic disease. In addition to their immunomodulatory functions, MSCs also directly suppress osteoclast differentiation and activation by secreting osteoprotegerin (OPG) and IL-10 but the underlying mechanisms are still to be clarified. Tumor necrosis factor-stimulated gene-6 (TSG-6) is a potent anti-inflammatory molecule that inhibits osteoclast activation and has been shown to mediate MSC's immunomodulatory functions. In this study, we aimed to determine whether adipose tissue-derived MSC (ADMSC) inhibits the differentiation from osteoclast precursors to mature osteoclasts through TSG-6. Human ADMSCs were co-cultured with bone marrow-derived monocyte/macrophage (BMMs) from DBA/1J or B6 mouse in the presence of osteoclastogenic condition (M-CSF 10 ng/mL and RANKL 10 ng/mL). In some co-culture groups, ADMSCs were transfected with siRNA targeting TSG-6 or OPG to determine their role in osteoclastogenesis. Tartrate-resistant acid phosphatase (TRAP) activity in culture supernatant and mRNA expression of osteoclast markers were investigated. TRAP+ multinucleated cells and F-actin ring formation were counted. ADMSCs significantly inhibited osteoclast differentiation under osteoclastogenic conditions. Suppression of TSG-6 significantly reversed the inhibition of osteoclast differentiation in a degree similar to that of OPG based on TRAP activity, mRNA expression of osteoclast markers, and numbers of TRAP+ multinucleated cell and F-actin ring formation. This study demonstrated that ADMSCs inhibit osteoclast differentiation through TSG-6 under osteoclastogenic conditions.

Anti-osteoporosis activity of casticin in ovariectomized rats.

Postmenopausal osteoporosis (PMPO) is the most familiar type of osteoporosis, a silent bone disease. Casticin, a natural flavonoid constituent, improves osteoporosis in animal model. Nevertheless, the potential mechanism remains to be further explored. A model of PMPO was established in rats treated with ovariectomy (OVX) and RAW 264.7 cells induced with receptor activator of nuclear factor kappa-B ligand (RANKL). The effect and potential mechanism of casticin on PMPO were addressed by pathological staining, measurement of bone mineral density (BMD), three-point bending test, serum biochemical detection, filamentous-actin (F-actin) ring staining, TRAcP staining, reverse transcription quantitative polymerase chain reaction, western blot and examination of oxidative stress indicators. The casticin treatment increased the femoral trabecular area, bone maturity, BMD, elastic modulus, maximum load, the level of calcium and estrogen with the reduced concentrations of alkaline phosphatase (ALP) and tumor necrosis factor (TNF)-α in OVX rats. An enhancement in the F-actin ring formation, TRAcP staining and the relative mRNA expression of NFATc1 and TRAP was observed in RANKL-induced RAW 264.7 cells, which was declined by the treatment of casticin. Moreover, the casticin treatment reversed the reduced the relative protein expression of Nrf2 and HO-1 and the concentrations of superoxide dismutase and glutathione peroxidase, and the increased content of malondialdehyde both in vivo and in vitro. Casticin improved bone density, bone biomechanics, the level of calcium and estrogen, the release of pro-inflammatory factor and oxidative stress to alleviate osteoporosis, which was associated with the upregulation of Nrf2/HO-1 pathway.

Trachelogenin alleviates osteoarthritis by inhibiting osteoclastogenesis and enhancing chondrocyte survival

BackgroundOsteoarthritis (OA) is a prevalent global health concern associated with the loss of articular cartilage and subchondral bone. The lack of disease-modifying drugs for OA necessitates the exploration of novel therapeutic options. Our previous study has demonstrated that traditional Chinese medical herb Trachelospermum jasminoides (Lindl.) Lem. extract suppressed osteoclastogenesis and identified trachelogenin (TCG) as a representative compound. Here, we delved into TCG’s potential to alleviate OA.MethodsWe initially validated the in vivo efficacy of TCG in alleviating OA using a rat OA model. Subsequently, we isolated primary bone marrow-derived macrophages in vitro to investigate TCG's impact on osteoclastogenesis. We further employed a small molecule pull-down assay to verify TCG's binding target within osteoclasts. Finally, we isolated primary mouse chondrocytes in vitro to study TCG's regulatory effects and mechanisms on chondrocyte survival.ResultsTCG preserved subchondral bone integrity and protected articular cartilage in a rat OA model. Subsequently, in vitro experiments unveiled TCG's capability to inhibit osteoclastogenesis and function through binding to Ras association proximate 1 (Rap1) and inhibiting its activation. Further study demonstrated that TCG inhibited Rap1/integrin αvβ3/c-Src/Pyk2 signaling cascade, and consequently led to failed F-actin ring formation. Besides, TCG promoted the proliferation of mouse primary chondrocytes while suppressing apoptosis in vitro. This is attributed to TCG's ability to upregulate HIF1α, thereby promoting glycolysis.ConclusionTCG exerted inhibitory effects on osteoclastogenesis through binding to Rap1 and inhibiting Rap1 activation, consequently preventing subchondral bone loss. Moreover, TCG enhanced chondrocyte survival by upregulating HIF1α and promoting glycolysis. These dual mechanisms collectively provide a novel approach to prevented against cartilage degradation.

The Role of Rosavin in the Pathophysiology of Bone Metabolism.

Rosavin, a phenylpropanoid in Rhodiola rosea's rhizome, and an adaptogen, is known for enhancing the body's response to environmental stress. It significantly affects cellular metabolism in health and many diseases, particularly influencing bone tissue metabolism. In vitro, rosavin inhibits osteoclastogenesis, disrupts F-actin ring formation, and reduces the expression of osteoclastogenesis-related genes such as cathepsin K, calcitonin receptor (CTR), tumor necrosis factor receptor-associated factor 6 (TRAF6), tartrate-resistant acid phosphatase (TRAP), and matrix metallopeptidase 9 (MMP-9). It also impedes the nuclear factor of activated T-cell cytoplasmic 1 (NFATc1), c-Fos, the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and mitogen-activated protein kinase (MAPK) signaling pathways and blocks phosphorylation processes crucial for bone resorption. Moreover, rosavin promotes osteogenesis and osteoblast differentiation and increases mouse runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) expression. In vivo studies show its effectiveness in enhancing bone mineral density (BMD) in postmenopausal osteoporosis (PMOP) mice, restraining osteoclast maturation, and increasing the active osteoblast percentage in bone tissue. It modulates mRNA expressions by increasing eukaryotic translation elongation factor 2 (EEF2) and decreasing histone deacetylase 1 (HDAC1), thereby activating osteoprotective epigenetic mechanisms, and alters many serum markers, including decreasing cross-linked C-telopeptide of type I collagen (CTX-1), tartrate-resistant acid phosphatase 5b (TRACP5b), receptor activator for nuclear factor κ B ligand (RANKL), macrophage-colony-stimulating factor (M-CSF), and TRAP, while increasing alkaline phosphatase (ALP) and OCN. Additionally, when combined with zinc and probiotics, it reduces pro-osteoporotic matrix metallopeptidase 3 (MMP-3), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α), and enhances anti-osteoporotic interleukin 10 (IL-10) and tissue inhibitor of metalloproteinase 3 (TIMP3) expressions. This paper aims to systematically review rosavin's impact on bone tissue metabolism, exploring its potential in osteoporosis prevention and treatment, and suggesting future research directions.

Hydroxychavicol Inhibits In Vitro Osteoclastogenesis via the Suppression of NF-κB Signaling Pathway.

Hydroxychavicol, a primary active phenolic compound of betel leaves, previously inhibited bone loss in vivo by stimulating osteogenesis. However, the effect of hydroxychavicol on bone remodeling induced by osteoclasts is unknown. In this study, the anti-osteoclastogenic effects of hydroxychavicol and its mechanism were investigated in receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclasts. Hydroxychavicol reduced the number of tartrate resistance acid phosphatase (TRAP)-positive multinucleated, F-actin ring formation and bone-resorbing activity of osteoclasts differentiated from RAW264.7 cells in a concentration-dependent manner. Furthermore, hydroxychavicol decreased the expression of osteoclast-specific genes, including cathepsin K, MMP-9, and dendritic cell-specific transmembrane protein (DC-STAMP). For mechanistic studies, hydroxychavicol suppressed RANKL-induced expression of major transcription factors, including the nuclear factor of activated T-cells 1 (NFATc1), c-Fos, and c-Jun. At the early stage of osteoclast differentiation, hydroxychavicol blocked the phosphorylation of NF-κB subunits (p65 and Iκβα). This blockade led to the decrease of nuclear translocation of p65 induced by RANKL. In addition, the anti-osteoclastogenic effect of hydroxychavicol was confirmed by the inhibition of TRAP-positive multinucleated differentiation from human peripheral mononuclear cells (PBMCs). In conclusion, hydroxychavicol inhibits osteoclastogenesis by abrogating RANKL-induced NFATc1 expression by suppressing the NF-κB signaling pathway in vitro.

Reversible SAHH inhibitor ameliorates MIA-induced osteoarthritis of rats through suppressing MEK/ERK pathway

Osteoarthritis (OA) is characterized by gradual articular cartilage degradation, accompanied by persistent low-grade joint inflammation, correlating with radiographic and pain-related progression. The latent therapeutic potential of DZ2002, a reversible inhibitor of S-adenosyl-L-homocysteine hydrolase (SAHH), holds promise for OA intervention. This study endeavored to examine the therapeutic efficacy of DZ2002 within the milieu of OA. The cytotoxicity of DZ2002 was evaluated using the MTT assay on bone marrow-derived macrophages. The inhibitory impact of DZ2002 during the process of osteoclastogenesis was assessed using TRAP staining, analysis of bone resorption pits, and F-actin ring formation. Mechanistic insights were derived from qPCR and Western blot analyses. Through the intra-articular injection of monosodium iodoacetate (MIA), an experimental rat model of OA was successfully instituted. This was subsequently accompanied by a series of assessments including Von Frey filament testing, analysis of weight-bearing behaviors, and micro-CT imaging, all aimed at assessing the effectiveness of DZ2002. The findings emphasized the effectiveness of DZ2002 in mitigating osteoclastogenesis induced by M-CSF/RANKL, evident through a reduction in TRAP-positive OCs and bone resorption. Moreover, DZ2002 modulated bone resorption-associated gene and protein expression (CTSK, CTR, Integrin β3) via the MEK/ERK pathway. Encouragingly, DZ2002 also alleviates MIA-induced pain, cartilage degradation, and bone loss. In conclusion, DZ2002 emerges as a potential therapeutic contender for OA, as evidenced by its capacity to hinder in vitro M-CSF/RANKL-induced osteoclastogenesis and mitigate in vivo osteoarthritis progression. This newfound perspective provides substantial support for considering DZ2002 as a compelling agent for osteoarthritis intervention.

Water Extract of Desalted Salicornia europaea Inhibits RANKL-Induced Osteoclast Differentiation and Prevents Bone Loss in Ovariectomized Mice

Osteoporosis, which is often associated with increased osteoclast activity due to menopause or aging, was the main focus of this study. We investigated the inhibitory effects of water extract of desalted Salicornia europaea L. (WSE) on osteoclast differentiation and bone loss in ovariectomized mice. Our findings revealed that WSE effectively inhibited RANKL-induced osteoclast differentiation, as demonstrated by TRAP staining, and also suppressed bone resorption and F-actin ring formation in a dose-dependent manner. The expression levels of genes related to osteoclast differentiation, including NFATc1, ACP5, Ctsk, and DCSTAMP, were downregulated by WSE. Oral administration of WSE improved bone density and structural parameters in ovariectomized mice. Dicaffeoylquinic acids (DCQAs) and saponins were detected in WSE, with 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA being isolated and identified. All tested DCQAs, including the aforementioned types, inhibited osteoclast differentiation, bone resorption, and the expression of osteoclast-related genes. Furthermore, WSE and DCQAs reduced ROS production mediated by RANKL. These results indicate the potential of WSE and its components, DCQAs, as preventive or therapeutic agents against osteoporosis and related conditions.

Extracellular Vesicles from Human Adipose Tissue-Derived Mesenchymal Stem Cells Suppress RANKL-Induced Osteoclast Differentiation via miR122-5p.

Researchers are increasingly interested in cell therapy using mesenchymal stem cells (MSCs) as an alternative remedy for osteoporosis, with fewer side effects. Thus, we isolated and characterized extracellular vesicles (EVs) from human adipose tissue-derived MSCs (hMSCs) and investigated their inhibitory effects on RANKL-induced osteoclast differentiation. Purified EVs were collected from the supernatant of hMSCs by tangential flow filtration. Characterization of EVs included typical evaluation of the size and concentration of EVs by nanoparticle tracking analysis and morphology analysis using transmission electron microscopy. hMSC-EVs inhibited RANKL-induced differentiation of bone marrow-derived macrophages (BMDMs) into osteoclasts in a dose-dependent manner. F-actin ring formation and bone resorption were also reduced by EV treatment of osteoclasts. In addition, EVs decreased RANKL-induced phosphorylation of p38 and JNK and expression of osteoclastogenesis-related genes in BMDMs treated with RANKL. To elucidate which part of the hMSC-EVs plays a role in the inhibition of osteoclast differentiation, we analyzed miRNA profiles in hMSC-EVs. The results showed that has-miR122-5p was present at significantly high read counts. Overexpression of miR122-5p in BMDMs significantly inhibited RANKL-induced osteoclast differentiation and induced defects in F-actin ring formation and bone resorption. Our results also revealed that RANKL-induced phosphorylation of p38 and JNK and osteoclast-specific gene expression was decreased by miR122-5p transfection, which was consistent with the results of hMSC-EVs. These findings suggest that hMSC-EVs containing miR122-5p inhibit RANKL-induced osteoclast differentiation via the downregulation of molecular mechanisms and could be a preventive candidate for destructive bone diseases.

Exploring the Anti-Osteoporotic Potential of Daucosterol: Impact on Osteoclast and Osteoblast Activities.

Osteoporosis is a debilitating condition characterized by reduced bone mass and density, leading to compromised structural integrity of the bones. While conventional treatments, such as bisphosphonates and selective estrogen receptor modulators (SERMs), have been employed to mitigate bone loss, their effectiveness is often compromised by a spectrum of adverse side effects, ranging from gastrointestinal discomfort and musculoskeletal pain to more severe concerns like atypical fractures and hormonal imbalances. Daucosterol (DC), a natural compound derived from various plant sources, has recently garnered considerable attention in the field of pharmacology. In this study, we investigated the anti-osteoporosis potential of DC by characterizing its role in osteoclasts, osteoblasts, and lipopolysaccharide (LPS)-induced osteoporosis. The inhibitory effect of DC on osteoclast differentiation was determined by tartrate-resistant acid phosphatase (TRAP) staining, F-actin ring formation by fluorescent staining, and bone resorption by pit formation assay. In addition, the calcification nodule deposition effect of osteoblasts was determined by Alizarin red S staining. The effective mechanisms of both cells were verified by Western blot and reverse transcription polymerase chain reaction (RT-PCR). To confirm the effect of DC in vivo, DC was administered to a model of osteoporosis by intraperitoneal administration of LPS. The anti-osteoporosis effect was then characterized by micro-CT and serum analysis. The results showed that DC effectively inhibited osteoclast differentiation at an early stage, promoted osteoblast activity, and inhibited LPS-induced bone density loss. The results of this study suggest that DC can treat osteoporosis through osteoclast and osteoblast regulation, and therefore may be considered as a new therapeutic alternative for osteoporosis patients in the future.

Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells.

Cell spreading and migration play central roles in many physiological and pathophysiological processes. We have previously shown that MFN2 regulates the migration of human neutrophil-like cells via suppressing Rac activation. Here, we show that in mouse embryonic fibroblasts, MFN2 suppresses RhoA activation and supports cell polarization. After initial spreading, the wild-type cells polarize and migrate, whereas the Mfn2-/- cells maintain a circular shape. Increased cytosolic Ca2+ resulting from the loss of Mfn2 is directly responsible for this phenotype, which can be rescued by expressing an artificial tether to bring mitochondria and endoplasmic reticulum to close vicinity. Elevated cytosolic Ca2+ activates Ca2+/calmodulin-dependent protein kinase II, RhoA, and myosin light-chain kinase, causing an overactivation of nonmuscle myosin II, leading to a formation of a prominent F-actin ring at the cell periphery and increased cell contractility. The peripheral actin band alters cell physics and is dependent on substrate rigidity. Our results provide a novel molecular basis to understand how MFN2 regulates distinct signaling pathways in different cells and tissue environments, which is instrumental in understanding and treating MFN2-related diseases.

Pharmacological inhibition of protein S-palmitoylation suppresses osteoclastogenesis and ameliorates ovariectomy-induced bone loss

BackgroundExcessive osteoclast formation disrupts bone homeostasis, thereby significantly contributing to pathological bone loss associated with a variety of diseases. Protein S-palmitoylation is a reversible post-translational lipid modification catalyzed by ZDHHC family of palmitoyl acyltransferases, which plays an important role in various physiological and pathological processes. However, the role of palmitoylation in osteoclastogenesis has never been explored. Consequently, it is unclear whether this process can be targeted to treat osteolytic bone diseases that are mainly caused by excessive osteoclast formation. Materials and methodsIn this study, we employed acyl-biotin exchange (ABE) assay to reveal protein S-palmitoylation in differentiating osteoclasts (OCs). We utilized 2-bromopalmitic acid (2-BP), a pharmacological inhibitor of protein S-palmitoylation, to inhibit protein palmitoylation in mouse bone marrow-derived macrophages (BMMs), and tested its effect on receptor activator of nuclear factor κβ ligand (RANKL)-induced osteoclast differentiation and activity by TRAP staining, phalloidin staining, qPCR analyses, and pit formation assays. We also evaluated the protective effect of 2-BP against estrogen deficiency-induced bone loss and bone resorption in ovariectomized (OVX) mice using μCT, H&E staining, TRAP staining, and ELISA assay. Furthermore, we performed western blot analyses to explore the molecular mechanism underlying the inhibitory effect of 2-BP on osteoclastogenesis. ResultsWe found that many proteins were palmitoylated in differentiating OCs and that pharmacological inhibition of palmitoylation impeded RANKL-induced osteoclastogenesis, osteoclast-specific gene expression, F-actin ring formation and osteoclastic bone resorption in vitro, and to a lesser extent, osteoblast formation from MC3T3-E1 cells. Furthermore, we demonstrated that administration of 2-BP protected mice from ovariectomy-induced osteoporosis and bone resorption in vivo. Mechanistically, we showed that 2-BP treatment inhibited osteoclastogenesis partly by downregulating the expression of c-Fos and NFATc1 without overtly affecting RANKL-induced activation of osteoclastogenic AKT, MAPK, and NF-κB pathways. ConclusionPharmacological inhibition of palmitoylation potently suppresses RANKL-mediated osteoclast differentiation in vitro and protects mice against OVX-induced osteoporosis in vivo. Mechanistically, palmitoylation regulates osteoclast differentiation partly by promoting the expression of c-Fos and NFATc1. Thus, palmitoylation plays a key role in promoting osteoclast differentiation and activity, and could serve as a potential therapeutic target for the treatment of osteoporosis and other osteoclast-related diseases. The translational potential of this articleThe translation potential of this article is that we first revealed palmitoylation as a key mechanism regulating osteoclast differentiation, and therefore provided a potential therapeutic target for treating osteolytic bone diseases.