Pycnodysostosis is a rare autosomal recessive skeletal dysplasia resulting from pathogenic variants in the CTSK gene, which encodes cathepsin K, a lysosomal cysteine protease expressed in osteoclasts. Deficiency of this enzyme leads to defective bone resorption and generalized osteosclerosis. We report an 8-year-old girl, the firstborn of a third-degree consanguineous marriage, who presented with poor height and weight gain since birth and delayed developmental milestones, including independent walking at 2 years of age. Physical examination revealed proportionate short stature with microcephaly and brachycephaly. Craniofacial findings included frontal bossing, beaked nose, low-set posteriorly rotated ears, micrognathia and a high-arched fissured palate with dental crowding and supernumerary teeth. Thickened palmar skin, pectus carinatum, and saddle toe deformity were also seen. Fundoscopic examination demonstrated bilateral papilledema. Radiographic evaluation showed diffuse cranial sclerosis, wormian bones, hypoplastic clavicles, acro-osteolysis, and generalized increased bone density. Endocrine assessment revealed isolated low parathyroid hormone levels, an exceptionally rare association. Molecular genetic testing by clinical exome sequencing (CES) identified a homozygous pathogenic variant in exon 6 of the CTSK gene, establishing the diagnosis of pycnodysostosis. The patient was initiated on recombinant growth hormone therapy and referred for multidisciplinary follow-up, including endocrinology, dentistry, orthopedics, and ophthalmology. This case underscores both classical manifestations and unusual findings such as papilledema and low PTH levels. Early diagnosis through careful clinical evaluation and genetic confirmation enables timely initiation of appropriate interventions. Growth hormone therapy demonstrated consistent improvement in linear growth, emphasizing the potential for better long-term outcomes. A coordinated multidisciplinary approach remains essential to enhance prognosis and overall quality of life in children with this rare skeletal dysplasia.

The cancer glycocalyx is characterized by the overexpression of large glycoconjugates, including mucins, proteoglycans, and polysialic acid, which collectively increase glycocalyx thickness and stiffness to promote tumor survival, metastasis, and progression. Previous work demonstrated that enzymatic degradation of cancer cell-surface mucins reduced tumor burden and metastasis in mouse models of breast cancer, thus validating glycocalyx remodeling as a therapeutic strategy. However, this work relied on an engineered bacterial mucin-selective protease, or mucinase, which does not degrade other bulky glycoconjugates and raises immunogenicity concerns because of its bacterial origin. A human enzyme that can degrade cell-surface mucins and other bulky glycoconjugates would address both limitations. We screened all 15 human cathepsins for their ability to degrade purified and cell-surface mucins, since multiple cathepsins have previously been shown to degrade mucins within regions of dense glycosylation. We found that cathepsin K (CTSK) uniquely degrades cell-surface mucins, proteoglycans, and polysialylated glycoproteins, and we demonstrated that CTSK reduces total glycocalyx thickness. These findings establish CTSK as a promising starting point for the development of a glycocalyx-debulking enzyme for cancer therapeutics.

IL-6 blockade at the fracture site accelerates bone healing via inflammatory modulation of sensory nerve CGRP signaling.

Periodontal disease is a chronic inflammatory disorder that irreversibly damages soft tissue. Current treatment options often face challenges such as drug resistance, highlighting the need for alternative therapies with fewer side effects. This study was conducted to explore the therapeutic potential of Hippophae rhamnoides extracted using 70% ethanol (DKB140, with kaempferol as the marker compound) for periodontal disease treatment. The anti-gingivitis and anti-periodontitis effects of DKB140 were demonstrated in RAW 264.7 macrophages and human gingival fibroblasts (hGF-1) using enzyme-linked immunosorbent assay (ELISA), reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and western blot analysis by investigating the NLRP3 signaling pathway and its inhibitory effects on matrix metalloproteinase (MMP) expression and osteoclast differentiation. In RAW 264.7 macrophages, DKB140 attenuated the production of prostaglandin E2 and COX-2 mRNA expression. Furthermore, DKB140 inhibited inflammatory cytokine (IL-6, IL-8, and IL-1β) production and mRNA expression. In hGF-1s, DKB140 downregulated prostaglandin E2, COX-2, and inflammatory cytokine levels. Mechanistic investigations demonstrated that the anti-gingivitis effects of DKB140 were mediated through the NF-κB/MAPK/NLRP3 signaling pathway. These findings were confirmed using an NLRP3 inhibitor. Moreover, DKB140 reduced the expression of collagenases (MMP-1 and MMP-13), gelatinases (MMP-2 and MMP-9), and stromelysins (MMP-3 and MMP-10) in hGF-1s. Furthermore, DKB140 decreased the expression of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), dendritic cell-specific transmembrane protein (DCstamp), and cathepsin K (CTSK), which are responsible for osteoclast differentiation in receptor of nuclear factor kappa-B ligand (RANKL)-stimulated osteoclasts. These findings suggest that DKB140 can function as a dual-functional agent with anti-gingival and anti-periodontal properties.

BackgroundAmiodarone (AMD), a highly effective Class III antiarrhythmic drug, has its clinical utility limited by the risk of inducing a serious adverse effect, amiodarone-induced pulmonary fibrosis (AIPF). The pathogenesis of AIPF remains poorly elucidated, particularly the hub driver genes, which hinders early diagnosis and targeted intervention.MethodsThis study employed an integrative approach combining network toxicology, machine learning (ML), and in vitro validation to identify hub genes in AIPF. Potential AMD targets and pulmonary fibrosis (PF)-related genes were obtained from toxicity databases and transcriptomic data (GEO datasets), respectively, and intersected to identify candidate AIPF targets. Multiple ML models were constructed, and SHAP (Shapley Additive exPlanations) analysis was used to interpret the model and rank feature importance. Molecular docking and dynamics simulations assessed the binding of AMD to the core targets. Key findings were experimentally validated in an AMD-induced human bronchial epithelial (BEAS-2B) cell model using qRT-PCR, Western blot, and functional assays.ResultsBioinformatics analysis identified eight candidate hub genes for AIPF. The glmBoost + GBM model demonstrated superior predictive performance (AUC = 0.845). SHAP interpretability analysis identified Cathepsin K (CTSK), Adenosine A3 Receptor (ADORA3), and Advanced Glycosylation End Product-Specific Receptor (AGER) as the most important predictors. Molecular simulations confirmed stable binding between AMD and these target proteins. In vitro experiments showed that AMD treatment significantly upregulated CTSK and downregulated ADORA3 and AGER at both mRNA and protein levels in BEAS-2B cells, and enhanced cell migration and invasion.ConclusionThis study identifies CTSK, ADORA3, and AGER as key genes in AIPF pathogenesis through a comprehensive bioinformatics and ML approach. Their dysregulation in lung epithelial cells likely promotes fibrosis through modulating extracellular matrix metabolism, inflammation, and cell motility. These findings provide novel insights into AIPF mechanisms and highlight potential biomarkers and therapeutic targets.

Mechanism of Scoparone Against Knee Osteoarthritis: A Study Integrating Network Pharmacology, Animal Experiments, and Molecular Docking.

This study aims to investigate whether Scutellariae Radix-Gardeniae Fructus(S-G) drug pairs alleviate joint inflammation and bone erosion in collagen-induced arthritis(CIA) mice by regulating sphingosine kinase 1(SphK1)-mediated glycolysis and inhibiting the differentiation of osteoclasts(OCs) induced by receptor activator of nuclear factor-κB ligand(RANKL). Potential therapeutic targets of S-G drug pairs were screened via network pharmacology, followed by Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment and molecular docking analyses. The effects of S-G drug pairs on bone erosion, inflammatory cytokines, glycolysis-related factors, and OCs differentiation were evaluated using both the CIA mouse model and the RAW264.7 cell in vitro induction system. In vivo results showed that S-G drug pairs alleviated joint swelling and synovial inflammation, reduced OCs number, and modulated the expression of key cytokines, including RANKL, osteoprotegerin(OPG), interleukin(IL)-6, and IL-10. In vitro findings further demonstrated that serum containing the drug significantly inhibited RANKL-induced OCs differentiation, reduced F-actin ring formation, and lowered levels of lactate acid(LA) and lactate dehydrogenase A(LDH-A). Additionally, it suppressed the expression of glycolysis-and bone resorption-related proteins and genes such as SphK1, pyruvate kinase M2(PKM2), oncoprotein c-Myc(c-Myc), and cathepsin K(CTSK), while also weakening the interaction between SphK1 and PKM2. In conclusion, S-G drug pairs could inhibit OCs differentiation by regulating SphK1-mediated glycolytic metabolism, thereby mitigating rheumatoid arthritis-induced bone erosion.

PurposeCompromise in trabecular meshwork (TM) function due to extracellular matrix (ECM) accumulation contributes to increased intraocular pressure (IOP) in primary open-angle glaucoma (POAG). Our previous study demonstrated the role of cathepsin K (CTSK), a potent collagenase, on ECM homeostasis, actin bundling in TM, and IOP regulation. This study was designed to understand the response of TM cells to the loss of CTSK function.MethodsNormal primary human TM (HTM) cells transfected with either small interfering RNA (siRNA) against CTSK (siCTSK) or scrambled siRNA (siScr) as a control were screened using mass spectrometry–based quantitative proteomics for changes in protein levels. Immunofluorescence imaging was used to identify changes in the distribution of differentially expressed proteins. Flow cytometry analysis provided evidence for the cell death mechanism resulting from the loss of CTSK function. Biochemical analysis was performed to quantify filamentous actin, assess BMP1 activity, and measure calcium levels.ResultsCTSK loss significantly disrupted collagen biogenesis and ECM remodeling and increased intracellular calcium levels and the expression of calcium-regulatory proteins. Actin polymerization was increased due to protein kinase D1 (PRKD1) activation through the slingshot phosphatase 1 (SSH1)/cofilin pathway, promoting focal adhesion maturation. Despite increased apoptotic markers (CASP3, CASP7, TRADD, PPM1F), caspase-3/-7 activation was not induced, suggesting apoptosis-independent cellular remodeling. Notably, RhoQ and myosin motor proteins were significantly downregulated, indicating altered mechanotransduction.ConclusionsOur findings underscore the multifaceted role of CTSK in maintaining critical cellular processes within the TM. Specifically, we have shown that CTSK is closely involved in regulating ECM homeostasis, influencing calcium signaling, and governing cytoskeletal dynamics and TM cellularity.

Background and AimsLiver fibrosis is characterized by the excessive deposition of extracellular matrix, a process primarily driven by activated hepatic stellate cells (HSCs), and currently lacks effective therapy. Cathepsin K (CTSK) exhibits context-dependent roles across organ systems in fibrosis, but its function in liver fibrosis is unclear. This study aimed to investigate the role and underlying mechanisms of CTSK during liver fibrosis.MethodsCTSK expression was analyzed in human fibrotic liver samples via transcriptomic analysis and confirmed in murine fibrosis models. The function of CTSK was investigated in both primary HSCs and LX-2 cells by assessing its effects on cell activation, proliferation, apoptosis, and the underlying signaling pathways following CTSK overexpression. The therapeutic potential was evaluated using an adeno-associated virus serotype 8 to overexpress CTSK in two etiologically distinct murine fibrosis models.ResultsCTSK was upregulated in activated HSCs and fibrotic livers. Furthermore, we discovered that it mediates a negative feedback loop to inhibit the TGF-β/Smad pathway via Smad7/Smurf2-dependent TGF-β receptor-I degradation, thereby suppressing HSC activation and proliferation. CTSK also induced mitochondrial apoptosis through Bax/Bcl-2 imbalance and caspase-3 activation. Together, these actions contribute to the anti-fibrotic effect of CTSK. Notably, adeno-associated virus serotype 8-mediated CTSK overexpression attenuated liver fibrosis across multiple murine models.ConclusionsOur study demonstrates that elevated CTSK functions as an endogenous protective factor that attenuates liver fibrosis. CTSK mediates negative feedback inhibition of the TGF-β pathway while concurrently promoting the mitochondrial apoptosis pathway. The dual anti-fibrotic mechanisms identify CTSK as a promising therapeutic target for liver fibrosis.

Pycnodysostosis is a rare, autosomal recessive illness that is generally pathognomonic. It is characterized by the postnatal onset of short limbs, small stature, and global hyperostosis, as well as acro-osteolysis with sclerosis of the terminal phalanges. It has been shown that approximately 30% of patients have parental consanguinity. This condition is brought on by a mutation in the cathepsin K (CTSK) gene. To date, 34 distinct CTSK mutations have been found in patients. This lysosomal enzyme helps break down bone matrix proteins, including some forms of collagen, and is mostly present in osteoclasts. Between 90 and 95 percent of all organic bone matrix is made up of type 1 collagen, which is still uncleaved. Unusual bone and dental development results from the accumulation of undigested collagen fibrils by these patients’ fibroblasts. Pycnodysostosis is a clinical characteristic that only occurs when cathepsin K is completely lost. About 10% of patients are found to have mental impairment. We present an infant with pycnodysostosis, undiagnosed before presentation at birth.

The xenogeneic collagen matrix (XCM) is widely used for keratinized mucosa augmentation around natural teeth and dental implants due to its consistent efficacy and the advantage of avoiding a second surgical site. However, the shrinkage rate of XCM after surgery exceeds 50%, which is not conducive to accurate preoperative design. This study aimed to investigate factors influencing XCM shrinkage.Fifteen participants with buccal keratinized tissue width (KTW) <2 mm around mandibular single implants underwent XCM-based KTW augmentation. Clinical parameters were recorded, and peri-implant crevicular fluid (PICF) was collected preoperatively and at 3-month follow-ups. Twenty cytokines in PICF were assessed using a commercial kit, with Spearman tests evaluating correlations between cytokines, clinical parameters, and shrinkage predictors.XCM significantly increased KTW from 1.39 ± 0.26 mm to 4.13 ± 1.19 mm at 3 months (p < 0.001), with a shrinkage rate of 60.12 ± 12%. The PICF showed significant decreases in C-reactive protein (CRP), interleukin-6 (IL-6), calprotectin (CLP), and keratin type II cytoskeletal 1 (K2C1) (p < 0.05) and a significant increase in osteoprotegerin (OPG) at 3 months (p < 0.05). Preoperative keratinized tissue thickness (KTT) and cathepsin K (CTSK), along with K2C1 at 3 months, correlated with XCM shrinkage.KTW augmentation alters cytokine expression. Thin preoperative KTT and high CTSK in PICF may predict high postoperative XCM shrinkage.Gingival phenotype significantly impacts XCM shrinkage after augmentation. PICF cytokine expression could serve as a predictive biomarker.The Chinese Clinical Trial Registry, no. ChiCTR2500100933 (last updated on 17/04/2025).

Cannabidiol (CBD) effects on bone metabolism in postmenopausal osteoporosis remain unclear. While endocannabinoids and phytocannabinoids bind to receptors in bone cells, direct evidence of CBD’s bone-protective effects is lacking. We evaluated the effects of CBD on bone metabolism in ovariectomized (OVX) rat model of estrogen deficiency. Twelve-week study with treatment initiated 2 wk after the surgery was conducted. Five experimental groups were established: sham-operated with vehicle (SHM/VEH), sham with CBD (SHM/CBD5), OVX with vehicle (OVX/VEH), OVX with 17β-estradiol (OVX/E2), and OVX with CBD (OVX/CBD5). Cannabidiol was administered at 5 mg/kg/d via osmotic pumps. Micro-CT of the distal femur revealed that trabecular bone mass in OVX/CBD5 decreased similarly to OVX/VEH, indicating no protective effect. Serum bone turnover markers showed increased bone resorption in OVX/CBD5 compared to OVX/VEH. Gene expression analysis revealed that estrogen significantly reduced Ctsk gene expression compared to OVX/VEH, while CBD showed no significant differences. No significant changes were observed in cannabinoid receptor expression or bone metabolism in sham-operated rats receiving CBD. While CBD (5 mg/kg/d) was well-tolerated, it did not mitigate OVX-induced bone loss in skeletally mature rats. Consequently, CBD should not be considered a monotherapy for postmenopausal osteoporosis, though it appears safe for other potential medical applications.

Cystatins, endogenous inhibitors of cysteine proteases, regulate extracellular matrix degradation. Their plant-derived homologs (phytocystatins) include MaquiCPI-3, a recombinant protein obtained from Aristotelia chilensis (maqui berry). This study aimed to investigate the inhibitory effect of MaquiCPI-3 on human cathepsin K (CTSK) activity and its cytotoxicity and impact on the proliferation, migration, and osteogenic differentiation of human dental pulp cells (hDPCs). The inhibitory activity of MaquiCPI-3 against CTSK was measured using a spectrofluorometer with the fluorogenic substrate Z-Phe-Arg-AMC. The hDPCs from third molars were characterized by flow cytometry for mesenchymal (CD90, CD73, CD105) and hematopoietic (CD34, CD45) markers. The hDPCs, either exposed to MaquiCPI-3 or left untreated (control), were assessed for viability (MTT assay), proliferation (bromodeoxyuridine incorporation), chemotaxis (Transwell assay), mineralized nodule formation (Alizarin Red S staining), alkaline phosphatase activity (thymolphthalein release), and expression of mineralization-related genes (qPCR). Data were analyzed using one- or two-way ANOVA with appropriate post hoc tests or nonparametric alternatives (α=0.05). MaquiCPI-3 potently inhibited CTSK (Ki=1.72 nM, Ki,app=2.08 nM), showed no cytotoxicity, and significantly enhanced ALP activity, mineralized nodule formation, and expression of BMP-2 and osteocalcin, stimulating no hDPC proliferation or migration when compared with the control. MaquiCPI-3 increased no cell proliferation or migration, its ability to inhibit CTSK activity and induce an osteogenic phenotype shows promising potential therapeutic strategies aimed at repairing and regenerating pulp and periapical tissues and controlling bone resorption.

Cancer cachexia is a complex syndrome marked by weight loss and muscle wasting, significantly impacting patient quality of life and survival. Mechanistically, it is characterized by suppressed protein synthesis and enhanced muscle catabolism, with the role of endoplasmic reticulum (ER) stress and unfolded protein response (UPR) becoming increasingly evident. This study aimed to explore ER stress-tolerant factors in muscle wasting and evaluate their potential to prevent muscle loss in cancer cachexia. A genome-wide CRISPR screening was conducted in the context of ER stress-mediated growth inhibition of C2C12 myoblasts. The candidate genes resistant to ER stress were further evaluated in C2C12 myotubes treated with conditioned medium of Lewis lung adenocarcinoma (LLC) cells. Twelve-week-old male mice were administered LLC cells and shRNA against Naa35 via adeno-associated virus. Four weeks later, tibialis anterior (TA) muscles were analysed for muscle mass, grip strength and molecular changes with quantitative polymerase chain reaction, western blotting and histological analysis. CRISPR screening identified Naa35, Naa38 and Naa30, all three components of N-terminal acetyltransferase C, as key molecules for resistance to ER stress. The atrophic muscles of mice bearing LLC demonstrated an elevation of UPR, as well as 1.64-fold upregulation of Naa35 protein (p = 0.0072). Among the three branches of the UPR, an ATF6 inhibitor, AEBSF, abolished upregulation of Naa35, Naa38 and Naa30, and an ATF6 activator, AA147, induced Naa35 expression in a dose-dependent manner (p < 0.001). In cells treated with LLC conditioned medium, Naa35 knockdown reduced the amount of cathepsin K (CTSK) protein, which subsequently resulted in the CTSK-mediated proteolysis of insulin receptor substrate 1. In LLC-bearing mice, Naa35 knockdown led to a 65.4% reduction in CTSK protein expression (p < 0.001) and preservation of the phosphorylation levels of protein kinase B (p < 0.0324) and anabolic-related S6 kinase (p < 0.0375). Concurrently, the expression of catabolism-related genes was repressed (MuRF1, p < 0.0015; MAFbx1, p < 0.0265). These alterations were associated with the restoration of TA muscle mass (2.52 ± 0.19 vs. 3.72 ± 0.45 mg/g, p = 0.0004), fibre area (1741 ± 992 vs. 2099 ± 1264 mm2, p < 0.0001), grip strength in all four limbs (0.0328 ± 0.0076 vs. 0.0506 ± 0.0130 N/g, p = 0.0295) and wire mesh hanging time (496 ± 331 vs. 1038 ± 370 s, p = 0.0406). Inhibition of N-terminal acetyltransferase C prevents ER stress-induced muscle wasting via the downregulation of CTSK and subsequent activation of the anabolic pathway. This suggests that N-terminal acetyltransferase C is a potential therapeutic target for combating muscle wasting in cancer cachexia.

Osteoporosis is a metabolic bone disease characterized by dysregulated osteoclast activity, resulting in increased bone degradation and compromised bone microarchitecture. While the interconnection between osteoclast differentiation and cellular energy metabolism has become increasingly recognized, the role of pyrimidine metabolism in this process remains largely undefined. Integrative multi-omics analyses were performed to characterize transcriptional and metabolic alterations during receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation. The effects of uridine (UD) on osteoclast development and resorptive function were assessed in vitro using RAW264.7 cells and bone marrow-derived macrophages (BMMs). In vivo effects of UD on bone loss were evaluated in an ovariectomized (OVX) mouse model. Integrative analyses revealed distinct metabolic remodeling during osteoclast differentiation and identified UD as a pivotal metabolite that showed a significant decline upon RANKL stimulation. Experimental evidence indicated that exogenous UD supplementation significantly suppressed osteoclast development and resorptive function, along with a reduction in the expression of nuclear factor of activated T cells c1 (NFATc1) and cathepsin K (CTSK). In OVX mice, UD administration improved trabecular microarchitecture, reduced osteoclast burden, and mitigated bone loss. Mechanistically, UD inhibited phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) phosphorylation, facilitated Forkhead box O (FoxO) nuclear translocation, and suppressed reactive oxygen species (ROS) accumulation, thereby preventing NFATc1 activation and nuclear import. Collectively, this research identifies a novel metabolic-signaling interplay linking pyrimidine metabolism with osteoclast differentiation and highlights UD as a promising metabolic regulator for the treatment and prevention of osteoporosis.

Cathepsin K-lineage cells and mechanical loading independently modulate bone mass in the murine tibia.

Osteoporosis therapies remain limited by non-specific osteoclast inhibition, which disrupts physiological bone remodeling and increases fracture risk. Here we show that selectively targeting mature osteoclasts via enzyme-responsive nanoparticles could eliminate pathological bone resorption while generating apoptotic bodies (ABs) to promote regeneration. We design OsteoSAVE, a peptide-based nanoparticle integrating a cathepsin K (CTSK)-cleavable linker, a bone-targeting motif, and a self-assembling biphenyl core. Upon CTSK-mediated hydrolysis, OsteoSAVE transforms into nanofibers within osteoclast lysosomes, inducing apoptosis and releasing ABs enriched with PDGF-BB. Mechanistic studies confirm CTSK-selective cleavage and charge-driven cellular specificity. In ovariectomized mice, OsteoSAVE restores bone density by coupling osteoclast elimination with ABs-mediated osteogenesis via PI3K/AKT activation in mesenchymal stem cells. This work establishes enzyme-triggered supramolecular self-assembly as a paradigm for dual-action therapies that recalibrate bone homeostasis without disrupting physiological processes.

Precision-targeting and dual silencing osteoclastogenesis and inflammatory pathways for the treatment of radiation-induced bone deterioration.

NFATc1-targeted siRNA microdroplets prevent radiation-induced osteoporosis by coordinating bone-immune crosstalk.

Objective: To explore the effect of hairy and enhancer of split related protein 2 (Hey2) on osteoclast differentiation through the activation of nuclear factor of activated T cells cytoplasmic 1 (NFATc1). Methods: RAW264.7 cells were induced with receptor activator of NF-κB ligand (RANKL) to differentiate into osteoclasts. Experimental groups were divided by different concentrations of RANKL (0, 10, 20, 50 μg/L) and different processing time (0, 3, 5, 7 days). Hey2 overexpression experiment was grouped as follows: blank control group, RANKL group, empty plasmid vector control group (Hey2-NC+RANKL), Hey2 overexpression group (Hey2-OE+RANKL); similarly, groups in Hey2 knockdown experiment were as follows: blank control group, RANKL group, negative control group (si-NC+RANKL), Hey2 knockdown group (si-Hey2+RANKL). Chromatin immunoprecipitation experiment groups were divided as non-specific IgG control group (IgG control group), non-specific IgG group (IgG RANKL group), Hey2-specific antibody control group (anti-Hey2 control group), Hey2-specific antibody group (anti-Hey2-RANKL group). For the different RANKL concentration groups and different induction time groups, real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the mRNA expressions of nuclear factor of NFATc1, cathepsin K (CTSK), and cellular feline osteosarcoma oncogene (c-Fos) and tartrate-resistant acid phosphatase (TRAP) staining was used to assess the formation of multinucleated osteoclasts. After Hey2 overexpression or knockdown, RT-qPCR and Western blotting were used to detect the gene and protein expressions of NFATc1, c-Fos, and CTSK. TRAP staining was used to evaluate the formation of multinucleated osteoclasts. Bioinformatics prediction (NCBI, JASPAR) and chromatin immunoprecipitation (ChIP) assay were used to validate the binding of Hey2 to the NFATc1 promoter region. Results: During the osteoclastic differentiation of RAW 264.7 cells induced by RANKL, the expression of Hey2 could be detected, and the expression level of Hey2 decreased with the increase of RANKL concentration and induction time. In the 50 μg/L RANKL group, the expression levels of Hey2 gene (0.18±0.00) and protein (0.22±0.02) were significantly lower than those in the control group (1.00±0.00, 0.52±0.01) (t=41.67, 12.88; both P<0.001). In the 50 μg/L RANKL group inducted for 5 days, the expression levels of Hey2 gene (0.27±0.02) and protein (0.79±0.01) were significantly lower than those in the control group (1.00±0.00, 1.15±0.02) (t=11.47, 108.60; both P<0.001). Hey2 overexpression significantly reduced the gene and protein expressions of NFATc1, c-Fos, and CTSK, as well as the production of TRAP-positive cells (all P<0.05). Hey2 knockdown significantly increased the gene and protein expressions of NFATc1, c-Fos, and CTSK, as well as the production of TRAP-positive cells (all P<0.05). After inducing RAW264.7 cells with 50 μg/L RANKL for 1 day, ChIP results showed that among the two sample groups treated with Hey2 antibody, the detection level of the NFATc1 promoter region (-400 to -200 bp) in the anti-Hey2-RANKL group (18.06±0.06) was significantly higher than that in the anti-Hey2 control group (13.37±0.36) (t=12.56, P<0.001). Conclusions: Hey2 can bind to the downstream target gene NFATc1 at -400 to -200 bp region of the promoter. As a transcriptional repressor, Hey2 inhibits osteoclast differentiation.