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.

Keywords: Macrophage; Osteoclast; Osteoporosis; Trim21; Txnip.

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

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

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

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