Trim21 deficiency alleviates osteoporosis by inhibiting osteoclast differentiation through regulating Txnip

Abstract

Objective

The tripartite motif containing 21 (Trim21), an E3 ubiquitin ligase, plays a crucial role in the progression of various skeletal diseases, particularly in osteoporosis. In our previous study, Trim21 deficiency was shown to exert dual effects by suppressing bone resorption and enhancing osteogenesis. However, the specific mechanism by which Trim21 inhibits osteoclast (OC) differentiation remains unclear. In this study, we utilized a myeloid cell–specific conditional knockout model of Trim21 to investigate the underlying regulatory mechanisms.

Methods

OC-specific Trim21 knockout mice were generated and subjected to ovariectomy (OVX) to establish a model of postmenopausal osteoporosis. Bone mass and OC activity were then evaluated using micro-computed tomography (micro-CT) and tartrate-resistant acid phosphatase (TRAP) staining. Bone marrow-derived macrophages (BMMs) were induced to differentiate into OCs, and gene expression levels were detected by qRT-PCR. Additionally, proteomic analysis was performed to identify downstream regulatory proteins influenced by Trim21.

Results

OC-specific Trim21 deletion alleviated OVX-induced bone loss by inhibiting bone resorption and preserving bone mass. Myeloid-specific Trim21 deletion impaired OC differentiation and suppressed the expression of key OC markers. Thioredoxin-interacting protein (Txnip), was identified as a downstream effector regulated by Trim21.

Conclusion

Trim21 deletion attenuates osteoporosis-induced bone loss, likely by suppressing osteoclast differentiation through modulation of Txnip, thereby presenting a potential novel therapeutic target for osteoporosis treatment.

Supplementary information

The online version contains supplementary material available at 10.1186/s13075-025-03624-6.

Introduction

Osteoporosis is a skeletal disorder characterized by weakened bone strength, leading to an increased risk of fractures. Osteoblasts and osteoclasts (OCs) work in concert to regulate the metabolism. In healthy individuals, bone formation and resorption are tightly regulated to maintain bone homeostasis [1]. Postmenopausal osteoporosis results from excessive OC activity due to estrogen deficiency [2]. OCs, the sole bone-resorbing cells in the body, are multinucleated giant cells derived from the monocytes/macrophages lineage under the stimulation of macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B (NF-κB) ligand (RANKL) [3]. Research has increasingly focused on various signaling pathways in OC differentiation, including NF-κB, mitogen-activated protein kinase (MAPK), and nucleotide-binding oligomerization domain-like receptors (NOD-like receptors, NLRs) signaling pathways [4, 5]. The NOD-like receptor signaling pathway is a key pathway involved in regulating inflammation and immunity [6]. The activation of NOD-like receptors triggers downstream signaling pathways, including NF-κB and MAPK signaling pathways, which are central regulators of OC differentiation [7]. Consequently, inhibiting osteoclastogenesis differentiation presents a promising therapeutic strategy for treating osteoporosis [8].

The tripartite motif containing 21 (Trim21), a member of the tripartite motif protein family, acts as an E3 ubiquitin ligase and has been implicated in various biological processes, including immune response, inflammation, and autophagy [9]. Trim21 has emerged as a key player in bone biology due to its regulatory role in various immune cells’ differentiation and function [10, 11]. Our previous study highlighted the involvement of Trim21 in osteogenesis, particularly through its interaction with the YAP1/β-catenin signaling pathway, which promotes bone formation by enhancing osteoblast activity [12]. In addition, we found that Trim21 influences osteosarcoma cell senescence and proliferation [13], and its knockout enhances Runx2-dependent osteoblast differentiation in osteosarcoma cells [10]. Moreover, a recent study has demonstrated that knockout of Trim21 in macrophages reduces the response to M-CSF via toll-like receptor 2 (TLR2) [14], thereby suppressing osteoclast differentiation. However, the precise role of Trim21 in the process of OC differentiation remains poorly understood.

In this study, we present novel insights into the role of Trim21 in regulating OC differentiation and bone loss. Using an ovariectomy (OVX)-induced osteoporosis model in conditional knockout mice, we demonstrated that Trim21 deletion in OCs suppresses bone resorption and mitigates estrogen deficiency–induced osteoporosis. In addition, Trim21 deficiency in myeloid cells impaired osteoclastogenesis. Proteomics profiling revealed that the NOD-like pathway is involved in Trim21-regulated osteoclastogenesis, likely through its binding to thioredoxin-interacting protein (Txnip). Our results offer a novel mechanism by which Trim21 serves as a therapeutic target for treating OC-driven bone loss disorders.

Material and methods

Generation of OC- and myeloid-specific conditional Trim21 knockout mice

To generate Trim21 conditional knockout mice in myeloid cells and OCs (Lyz2-Cre-tdTomato; Trim21^f/f, Trim21^ΔLyz2, and Trim21^ΔCtsk), Trim21^f/f mice were crossed with either Lyz2-Cre or Ctsk-Cre mice, followed by genotyping was performed using a Quick Mouse Genotyping Kit (Beyotime, China). The primers used for polymerase chain reaction (PCR) genotyping are listed in Table S1. After breeding, all mice were housed and experiments were performed in the specific pathogen-free conditions animal facility at Jinan University’s Animal Experiment Center. All animal procedures were approved by the Institutional Animal Care and Use Committee of Jinan University (Approval No: IACUC-20230912–08) and conformed to the NIH"Guide for the Care and Use of Laboratory Animals."All mice were on a C57BL/6 background.

Establishment of the OVX-induced osteoporosis model

Female Trim21^f/f and Trim21^ΔCtsk mice (12 weeks old) were randomly divided into two groups: the sham group (n = 5) and the OVX group (n = 5). Mice in the OVX group underwent bilateral ovariectomy under sodium pentobarbital anesthesia to induce osteoporosis, while the sham group had their ovaries temporarily exteriorized and then repositioned, with subsequent suturing of muscle and skin incisions. All mice sacrificed eight weeks after surgery, and the hind limbs were harvested for micro-computed tomography (micro-CT) analysis and histological examination.

Micro-CT analysis

After sacrifice, hind limbs were harvested and fixed in 4% PFA for 48 h, then stored in 70% ethanol. The limbs were scanned using high-resolution ex vivo micro-CT (SkyScan 1176, Belgium) and sagittal images of the limbs were reconstructed following our previously established protocol [12]. Trabecular bone in the region 0.1 mm to 1.0 mm above the growth plate at the distal femur and cortical bone in the femoral shaft were reconstructed in three dimensions (3D). Morphometric parameters were quantitatively analyzed using manufacturer-recommended software, and the following indices were assessed: bone volume per tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), and bone mineral density (BMD).

Histological and immunological staining

After sacrifice, hind limbs were fixed in 4% PFA for 48 h, decalcified in 0.5 M EDTA (pH 7.4) for 3 weeks, and processed into either paraffin or frozen sections. For frozen sections, tissues were dehydrated with 20% sucrose and 2% polyvinylpyrrolidone (PVP), followed by embedding in a medium containing 20% sucrose, 2% PVP, and 8% gelatin. Frozen Sects. (75 μm thick) or paraffin Sects. (6 μm thick) were subjected to further experiments. Every two out of six consecutive paraffin sections were stained with tartrate-resistant acid phosphatase (TRAP) (Sigma-Aldrich, Germany) and hematoxylin and eosin (H&E) (ServiceBio, China) following the manufacturers’ protocols. Subsequent image analysis was performed using ImageJ software (NIH, USA).

For immunofluorescence (IF) staining, frozen sections were incubated overnight at 4 °C with anti-Trim21 (NOVUS, USA), followed by incubation with Alexa Fluor 647-conjugated secondary antibody (CST, USA). Nucleus were counterstained with DAPI (CST, USA). Fluorescence images were acquired using a laser confocal microscope (Zeiss LSM 880, Germany; Leica STELLARIS 5, Germany).

OC differentiation from bone marrow-derived macrophages (BMMs)

Trim21^f/f and Trim21^ΔLyz2 mice, aged 4–8 weeks, were sacrificed, and their hind limbs were collected. Bone marrow stromal cells were flushed from the femurs and tibiae and cultured in α-MEM containing 30 ng/mL M-CSF (MCE, USA) for 4 days to generate BMMs. To generate mature OCs, BMMs were cultured with 30 ng/mL M-CSF and 50 ng/mL receptor activator of RANKL for at least 5 days, followed by further experimentation. PBS containing 30 ng/mL M-CSF was used as negative control.

Real-Time quantitative Polymerase Chain Reaction (RT-qPCR)

Total RNA was extracted using TRIzol (TaKaRa, Japan) and then reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher, USA) following our previously established protocols [10]. Briefly, 1 mL of TRIzol was added to each well of a 6-well plate to lyse the cells and isolate RNA, which was then used for cDNA synthesis and subsequent RT-qPCR analysis. RT-qPCR was performed on a QuantStudio 5 system using Fast SYBR Green PCR Master Mix (Thermo Fisher, USA) with 500 nmol/L primers. The primers used are listed in Table S2. The relative expression levels of target genes were calculated using the 2^−ΔΔCt method, normalized to Gapdh expression.

Western blotting (WB) analysis

Protein concentrations were determined using the BCA method, and 20–50 μg of protein was separated on 10% SDS-PAGE (Beyotime, China) gels and transferred to PVDF membranes, followed by visualization with primary and secondary antibodies using the Tanon 5200 Chemiluminescent Imaging System (Tanon, China). The following antibodies were used: anti-Trim21 (Novus, USA), anti-Txnip (Abcam, UK), anti-Gapdh (CST, USA), and HRP-conjugated anti-rabbit IgG (CST, USA).

Proteomic analysis

We extracted BMMs from Trim21^ΔLyz2 (n = 3) and Trim21^f/f (n = 3) mice for proteomic analysis. Quantitative proteomics was conducted by Shanghai Applied Protein Technology Company (Shanghai, China). Briefly, proteins were extracted using SDT lysis buffer (4% SDS, 100 mmol/L Tris/HCl pH 7.6, 0.1 mol/L DTT), and protein concentration was measured using the BCA method. Proteins were digested using the filter-aided sample preparation method and analyzed by mass spectrometry with data-independent acquisition (400 ng peptides per sample) [15]. To identify significantly differentially expressed proteins, we defined up-regulated proteins as those with a fold change (FC) > 1.5 and down-regulated proteins as those with FC < 0.67, with statistical significance set at p < 0.05 (T-test). These differentially expressed proteins were visualized in a volcano plot, showing the relationship between protein expression changes (log₂FC) and statistical significance (-log₁₀P-value). In the volcano plot visualization, significantly up-regulated proteins (FC > 1.5, p < 0.05) are shown in orange, while significantly down-regulated proteins (FC < 0.67, p < 0.05) appear in purple. Proteins that did not show significant differential expression are depicted in gray. Pathway analysis was performed using the Kyoto Encyclopedia of Genes and Genomes database (http://www.genome.jp/kegg/pathway.html).

Statistical analysis

All data are presented as mean ± standard deviation (SD), with sample sizes indicated in the figures or legends. Statistical significance between two groups was performed using Student’s t-test, while two-way ANOVA was used for comparisons between multiple groups. A value of p < 0.05 was considered statistically significant. Significance levels are indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns not significant.

Results

OC-specific deletion of Trim21 alleviates OVX-induced osteoporosis

Trim21 knockout has been shown to promote osteogenic differentiation of bone marrow mesenchymal stem cells via regulating the YAP1/β-catenin signaling pathway, enhancing bone formation activity. Additionally, the absence of Trim21 is found to inhibit bone loss induced by inflammation [12]. However, the precise role of Trim21 in regulating osteoclastogenesis remains unclear. We first extracted BMMs from Trim21 global knockout mice for proteomic analysis (Fig. 1A) and found that differentially expressed proteins were enriched in pathways related to OC differentiation (Fig. 1B). The results were confirmed by TRAP and immunofluorescence staining. Compared with the Trim21^+/+ group, the Trim21^−/− group showed an absence of multinucleated cell formation (Fig. S1A) and reduced nuclear translocation of Nfatc1 (Fig. S1B), indicating that global Trim21 knockout strongly inhibits osteoclast differentiation.

Fig. 1.

Loss of Trim21 alleviates OVX-induced osteoporosis via targeting OCs. A Flowchart for proteomic analysis of BMMs derived from Trim21 knockout mice; B Statistics of differentially expressed proteins involved in OC differentiation-related pathways; C Representative micro-CT images of different genotypes of mice subjected to OVX and Sham operations, including sagittal sections of the femur and 3D reconstructions of cancellous and cortical bone; D, E Parameters analysis of different genotypes of mice subjected to OVX and Sham operations, including BV/TV, Tb.Th, Tb.N, and BMD; G, F Representative TRAP staining images (G) and quantitative analyses of histomorphometric bone parameters (F) of different genotypes of mice with OVX and Sham operations, including OC surface/bone surface (OC.S/BS) and the number of OCs for each bone parameter (Oc.N/BPm). Scale as shown in the images. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns not significant

Next, we generated OC-specific Trim21 knockout mice (Trim21^ΔCtsk)[12] and subjected them to OVX to induce estrogen-deficient osteoporosis. We collected uterine tissue from mice eight weeks post-surgery and observed varying degrees of atrophy in the uteri of the OVX group (Fig. S2). Micro-CT scanning and 3D reconstruction of the hind limbs showed significant trabecular bone loss in the OVX group compared to the Sham group (Fig. 1C). Interestingly, in mice with the Trim21^ΔCtsk genotypes, trabecular bone loss was mitigated, and increased trabecular volume compared to the Trim21^f/f group. Subsequently, quantitative analysis of the trabecular bone in different groups was assessed based on the parameters, including BV/TV, Tb.Th, Tb.N, Tb.Sp, and BMD (Fig. 1D, Fig. S3A). In agreement with the reconstructed images, Trim21^ΔCtsk mice exhibited higher bone mass, a greater number of trabeculae, and a more uniform trabecular structure. However, no significant differences were observed in cortical bone thickness between the groups. Furthermore, H&E staining further confirmed that Trim21^ΔCtsk mice had more robust trabecular bone structures (Fig. S3B). TRAP staining of tibiae revealed a significant reduction of TRAP-positive OCs on the trabecular bone surface in Trim21^ΔCtsk mice compared to Trim21^f/f mice (Fig. 1E). Besides, OC. S/BS and OC. N/B.Pm were also lower in the Trim21^ΔCtsk mice (Fig. 1F). These results indicated that Trim21 depletion can alleviate osteoporosis induced by estrogen deficiency.

Myeloid-specific deletion of Trim21 impairs OC differentiation

Our previous study demonstrated elevated TRIM21 expression in the bones of osteoporotic patients [12]. Next, we aimed to investigate whether knocking out Trim21 in OC precursors, specifically BMMs, affects osteoclast differentiation. Therefore, we generated myeloid-specific Trim21 conditional knockout mice (Trim21^ΔLyz2 and Lyz2-Cre-tdTomato; Trim21^f/f) (Fig. S4), where myeloid cells were labeled with red fluorescence and other cell types with green fluorescence (Fig. 2A). In non-knockout mice, Trim21 expression colocalized with tdTomato, whereas this colocalization was largely absent in Lyz2-Cre-tdTomato; Trim21^f/f mice (Fig. 2B), confirming efficient Trim21 deletion in myeloid cells.

Fig. 2.

Conditional knockout Trim21 in myeloid cells inhibits OC differentiation. A Construction of myeloid-specific Trim21 knockout mice. Four distinct genotypes were successfully obtained: Trim21^f/f, Trim21^ΔLyz2, Lyz2-tdTomato, and Lyz2-tdTomato; Trim21^f/f; B Representative fluorescence images and quantification analysis of Trim21 expression in fluorescent reporter mice. GP: growth plate, TB: trabecular bone; C,D Immunoblotting images and quantification analysis of Trim21 expression in myeloid-specific Trim21 knockout mice; E Trim21 expression in myeloid-specific Trim21 knockout mice by RT-qPCR; F Representative fluorescence images and quantification analysis of Nfatc1 expression in myeloid-specific Trim21 knockout mice; G TRAP staining of BMMs derived from Trim21^f/f or Trim21^ΔLyz2 with or without RANKL and quantification of TRAP-positive OCs and the number of nuclei per TRAP⁺ cell; H Representative images of F-actin ring formation of BMMs derived from Trim21^f/f or Trim21^ΔLyz2 with or without RANKL; I Expression of key genes inducing OCs differentiation from BMMs, including Ctsk, Nfatc1, Acp5, Atp6v0d2, and Mmp9. Scale as shown in the images. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns not significant

Next, bone marrow stromal cells were isolated from the mice and induced into macrophages under M-CSF stimulation. The protein and mRNA levels of Trim21 were significantly decreased in the Trim21^ΔLyz2 mice (Fig. 2C-E), confirming the successful deletion of Trim21 in myeloid cells. In addition, we observed impaired osteoclastogenesis in Trim21 myeloid knockout mice at the animal level. The immunofluorescence analysis showed that Nfatc1 expression was significantly reduced in Trim21 myeloid knockout mice, indicating that OC differentiation was impaired (Fig. 2F). Next, M-CSF-induced macrophages were incubated with or without RANKL to examine the effect of Trim21 on osteoclastogenesis. BMMs isolated from Trim21^ΔLyz2 mice exhibited a marked suppression of RANKL-induced osteoclastogenesis, as evidenced by a decrease in TRAP-positive cells and defective F-actin ring formation (Fig. 2G,H). RT-qPCR results demonstrated that the transcription levels of several key OC markers, including Ctsk, Nfatc1, Acp5, Atp6v0d2, and Mmp9, were significantly elevated in Trim21^f/f mice compared to Trim21^ΔLyz2 mice (Fig. 2I). Collectively, these results demonstrate that knockout of Trim21 in myeloid cells inhibits OC differentiation.

Trim21 regulates Txnip to affect OC differentiation

To investigate the mechanisms by which Trim21 regulates osteoclastogenesis, we isolated and purified BMMs derived from Trim21^ΔLyz2 and Trim21^f/f mice, followed by proteomic analysis (Fig. 3A). The results showed that, compared with Trim21^f/f mice, Trim21^ΔLyz2 mice exhibited significant upregulation of 171 proteins and downregulation of 348 proteins, as shown in the volcano plot (Fig. 3B). KEGG pathway enrichment analysis revealed that the majority of differentially expressed proteins were associated with the NOD-like receptor signaling pathway (Fig. 3C). Since NOD-like receptor pathways are crucial for macrophage function and OC differentiation [16, 17], we generated a differential protein heat map based on fold changes (Fig. 3D). This analysis identified a marked increase in thioredoxin-interacting protein (Txnip), a key downstream effector within the NOD-like receptor pathway [18] (Fig. 3D).

Fig. 3.

Trim21 affects OC differentiation by regulating the content of Txnip. A Flowchart for proteomic analysis of BMMs derived from myeloid-specific Trim21 knockout mice; B Volcano plot of significantly differentially expressed proteins, with a fold change cutoff set at greater than 1.5 or less than 0.67, and a P-value threshold of < 0.05; C KEGG enrichment analysis of differentially expressed proteins in BMMs, only showing the TOP10 signaling pathways; D Differentially expressed proteins in the NOD-like receptor signaling pathway; E Immunoblotting images and quantification analysis of Txnip expression in BMMs derived from myeloid-specific Trim21 knockout mice; F Representative fluorescence images and quantitative analysis of Txnip expression in myeloid-specific Trim21 knockout mice. GP: growth plate. Scale as shown in the images. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns not significant

To further validate our findings, we investigated the regulatory relationship between Trim21 and Txnip both in vitro and in vivo. Immunoblotting analysis revealed that Trim21 deficiency resulted in elevated TXNIP levels in BMMs isolated from Trim21^ΔLyz2 mice (Fig. 3E). Consistently, immunofluorescence images confirmed that Txnip content in myeloid-specific Trim21 knockout mice was significantly higher compared to non-Trim21 knockout mice (Fig. 3F). These results suggest that Trim21 influences OC differentiation by regulating Txnip level.

Discussion

In osteoporosis, particularly the postmenopausal form, OCs are overactivated, leading to an imbalance between OC-mediated bone resorption and osteoblast-mediated bone formation. Therefore, therapeutic strategies that simultaneously inhibit bone resorption while promoting bone formation are crucial for delaying the progression of osteoporosis [19]. Our previous studies have identified Trim21 as a key regulator of both bone formation and resorption. Trim21 knockout promotes the osteogenic differentiation of BMSCs via the Yap1/β-catenin signaling and inhibits OC function. However, the specific mechanism by which Trim21 affects OC differentiation remains unclear. In the present study, the loss of Trim21 is demonstrated to regulate OC differentiation by the use of global Trim21, OC-specific Trim21, and myeloid-specific Trim21 knockout mice (Figs. 1, 2 and 3).

The mechanism by which Trim21 affects bone metabolism appears to be multifactorial, involving the degradation of cytosolic proteins essential for osteoclastogenesis [20, 21]. In OCs, Trim21 may influence critical signaling pathways related to cell differentiation and function. As an E3 ubiquitin ligase, Trim21 mediates the degradation of target proteins via the ubiquitin–proteasome system [22]. Moreover, osteoclast bone resorption critically depends on proteolytic mechanisms such as autophagy and lysosomal degradation [23, 24]. Trim21 may also impact the degradation of specific cytosolic proteins essential for osteoclastogenesis. Our proteomic analysis indicates that the deletion of Trim21 significantly affects the NOD-like receptor pathway, leading to the upregulation of Txnip, an important regulator in this pathway (Fig. 3C,D). Research indicates that Txnip plays a key role in bone metabolism, in various bone-related diseases, including osteoporosis, osteoarthritis, and rheumatoid arthritis [25]. Our previous study demonstrated that Trim21 regulates the Txnip/P21 axis and influences the senescence of osteosarcoma cells [13]. In addition, we have demonstrated that Trim21 can directly regulate Yap1 [12], and we also identified Yap1 as an upstream regulator of Txnip [26]. Herein, we further confirmed a regulatory effect of Trim21 on Txnip expression through immunoblotting and immunofluorescence (Fig. 3E-G). Taken together, these lines of evidence suggest that Trim21 regulates Txnip through an indirect mechanism rather than direct interaction.

In addition to our findings, several recent studies also explored the involvement of Txnip in osteoclastogenesis. For instance, Txnip has been considered a critical regulator of OC proliferation and differentiation, with its expression progressively decreasing during OC differentiation [26, 27]. In another study, the activity of OC differentiation markers significantly increased when OC precursors were co-cultured with Txnip-silenced osteoblasts [28]. Moreover, osteoclast differentiation was found to be suppressed in Txnip-overexpressing macrophages, an effect attributed to the inhibition of AP-1 activation by RANKL, thereby preventing the initiation of osteoclastogenesis [29]. However, the specific mechanism by which Txnip influences OC differentiation requires further exploration. One possibility is that elevated Txnip levels disrupt the intracellular redox balance [30, 31] thereby impairing BMMs function. Alternatively, altered Txnip expression may interfere with glucose metabolism in BMMs [32, 33], leading to insufficient energy supply required for osteoclast differentiation (Fig. 4).

Fig. 4.

Schematic representation of Trim21 regulating OC differentiation via Txnip. Normal bone remodeling is maintained by a balance between osteoblast-mediated bone formation and OC-mediated bone resorption. Trim21 is essential for sustaining the basal expression of key OC biomarkers, including Nfatc1, Ctsk, and Acp5. The loss of Trim21 results in reduced degradation of Txnip, ultimately alleviating bone loss by suppressing the expression of OC marker genes

Currently, no Trim21-specific inhibitors or modulators have been reported in clinical trials. Furthermore, major drug databases, including DrugBank, Therapeutic Target Database, and PharmGKB, contain no entries related to compounds targeting TRIM21. However, toxicogenomics resources (Comparative Toxicogenomics Database) indicate that certain substances may influence the expression of Trim21 [34]. This highlights the challenges in the development and clinical application of Trim21-targeting drugs, indicating that much work remains to be done.

Despite the promising therapeutic potential Trim21 holds in the treatment of skeletal diseases, its clinical application remains challenging. As an E3 ubiquitin ligase, Trim21 may ubiquitinate off-target proteins, potentially leading to a range of unintended cellular effects [35]. Furthermore, Trim21 is involved in multiple physiological processes beyond protein degradation, including innate immune responses, inflammatory regulation, and apoptosis. Consequently, systemic inhibition of Trim21 could elicit complex immunological effects [36, 37], possibly aggravating skeletal inflammation or impairing immune homeostasis. These concerns underscore the need for further in-depth investigation to clarify the safety and feasibility of Trim21-targeted therapies.

Taken together, our study provides novel insights into the role of Trim21 in osteoclastogenesis and bone resorption, likely through the modulation of Txnip expression. These findings suggest that Trim21 may represent a promising therapeutic target for disorders characterized by excessive bone resorption. Future research should aim to further elucidate the molecular mechanisms by which Trim21 regulates osteoclast function and to assess the therapeutic potential of Trim21 inhibitors or modulators in clinical applications.

Conclusion

In conclusion, the deletion of Trim21 in OCs and myeloid cells confers protection against bone loss by inhibiting OC differentiation and function through the regulation of Txnip levels. These findings not only deepen our understanding of the molecular mechanisms governing OC differentiation but also suggest new avenues for therapeutic intervention in bone diseases. These findings suggest that Trim21 could serve as a potential therapeutic target for diseases characterized by excessive bone resorption.

Abbreviations

Trim21

Tripartite motif containing 21

OC

Osteoclast

OVX

Ovariectomy

Micro-CT

Micro-computed tomography

TRAP

Tartrate-resistant acid phosphatase

BMMs

Bone marrow-derived macrophages

Txnip

Thioredoxin-interacting protein

M-CSF

Macrophage colony-stimulating factor

NF-κB

Nuclear factor kappa-B

RANKL

Receptor activator of nuclear factor kappa-B ligand

MAPK

Mitogen-activated protein kinase

NOD-like receptors

Nucleotide-binding oligomerization domain-like receptors

TLR2

Toll-like receptor 2

PCR

Polymerase chain reaction

BV/TV

Bone volume per tissue volume

Tb.Th

Trabecular thickness

Tb.N

Trabecular number

Tb.Sp

Trabecular separation

BMD

Bone mineral density

PVP

Polyvinylpyrrolidone

H&E

Hematoxylin and eosin

IF

Immunofluorescence

RT-qPCR

Real-time quantitative polymerase chain reaction

WB

Western blotting

SD

Standard deviation

OC.S/BS

OC surface/bone surface

Oc.N/BPm

OCs for each bone parameter

Authors’ contributions

Ya-Chen Peng, Yong-Sheng Ye, and Qin-Xiao Hu contributed equally to this work and share first authorship. All authors have made substantial contributions to data analysis and took part in drafting the article or revising it critically for important intellectual content and agreed to submit it to the current journal. Huan-Tian Zhang, Zhen-Gang Zha, and Ri-Xu Liu made substantial contributions to conception, design, drafting, and revision. Ya-Chen Peng, Yong-Sheng Ye, and Qin-Xiao Hu made substantial contributions to the drafting, revision, acquisition of data, or analysis and interpretation of data. Zhi-Quan Hao, Zhen-Yan Li, Luo-Yong Jiang, and Hao-Ran Peng assisted in acquisition of data and contributed to revision.

Funding

This work was financially supported by the National Natural Science Foundation of China (82072470 and 82401021), Guangdong Basic and Applied Basic Research Foundation (2023B1515020007, 2021A1515012154, 2024A1515012751, and 2025A1515012745), Macau Foundation for Development of Science and Technology (0008/2024/RIA1), Science and Technology Projects in Guangzhou (2024A04J6408, 2023A03J1014, 2024A04J3317 and 2024A03J1213)， Health Science and Technology Project in Guangzhou (20241A011074), Plan on enhancing scientific research in GMU.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

All animal procedures were approved by the Institutional Animal Care and Use Committee of Jinan University (Approval No: IACUC-20230912–08) and conformed to the NIH"Guide for the Care and Use of Laboratory Animals."

Consent for publication

All the authors have agreed that the study should be submitted to Journal.

Competing interests

The authors declare no competing interests.