TRIM21 depletion negatively regulates osteoclastogenesis via association with JNK2 to reduce nucleation of NFATc1

TRIM21 depletion negatively regulates osteoclastogenesis via association with JNK2 to reduce nucleation of NFATc1 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article TRIM21 depletion negatively regulates osteoclastogenesis via association with JNK2 to reduce nucleation of NFATc1 Xiaohe Wang, Yingming Wang, Gang Yu, Chao Fang, Liang Xu, Di Wu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7540879/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 13 You are reading this latest preprint version Abstract Aberrant activation of osteoclast-mediated bone resorption has been observed in a series of skeletal diseases including osteoporosis and delayed or non-union bone fracture. Ubiquitination-mediated protein degradation has been revealed as an important mechanism for osteoporosis. Trim21 as an important E3 ubiquitin ligase, has been found to be critical for osteoclastogenesis. We have recently demonstrated that Trim21 is a crucial player in fine-tuning bone homeostasis, yet the underlying mechanism that affect bone resorption is largely unknown. Herein, we demonstrate that deletion of Trim21 led to decreased OC formation and activity accompanied by bone mass increase in mice. In addition, unbiased proteomics analysis identified that JNK2, was one of the key substance of Trim21. Mechanistically, we discovered that TRIM21 influences osteoclast differentiation by controlling the degradation of JNK2 via ubiquitin-dependent proteasomal or lysosomal degradation. Protein-protein interaction was further confirmed by immunofluorescence, leading to the modulation of NFATc1 nucleation. Our findings propose TRIM21 as a promising therapeutic target for osteoporosis. Biological sciences/Biochemistry Biological sciences/Cell biology Health sciences/Diseases Biological sciences/Molecular biology TRIM21 osteoclast JNK2 Nfatc1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Osteoclasts (OCs) are large multinucleated cells that play a vital role in bone development and homeostasis. They originate from monocyte-macrophage cells of the hematopoietic lineage, triggered by the macrophage-colony stimulation factor (m-CSF) and receptor activator of nuclear factor-kβ (NF-κβ) ligand (RANKL) 1 , 2 . Excessive bone resorption resulting from increased OC formation and activity is the primary cause of bone loss in conditions like osteoporosis, Paget's disease of bone, bone metastases in cancer, rheumatoid arthritis, and osteoarthritis 3 – 7 . Understanding the mechanisms behind osteoclastogenesis holds significant clinical importance for the treatment of bone loss diseases. The development of drugs targeting osteoclasts is a crucial approach in treating bone loss diseases. Nevertheless, studies have indicated that the effectiveness of currently utilized anti-bone resorption drugs in clinical settings is not entirely satisfactory, and their long-term usage can lead to severe complications. For instance, bisphosphonates carry a risk of inducing osteonecrosis of the jaw (ONJ) and atypical femoral fractures (AFF) 8 . Biological agents like Dinomumab, despite showing promising anti-osteoclast differentiation effects, may trigger antigenicity, potentially causing an immune response in the body 9 . Additionally, long-term use of calcitonin is associated with an increased risk of malignant tumors 10 . Bone-promoting drugs, exemplified by parathyroid hormone analogs (PTHa) such as Teriparatide, offer a viable option. However, the administration of such drugs should not exceed 24 months, and their efficacy diminishes once discontinued, necessitating sequential treatment with anti-bone resorption drugs 11 , 12 . TRIM21 (Triple motif containing 21) is a member of the Class IV TRIM protein family and is known to participate in a wide array of cellular processes, including apoptosis, cell proliferation, immune response, immune cell differentiation, antiviral response, and tumor development 13 – 16 . Macrophages are considered precursor cells for osteoclast differentiation, and recent studies have indicated the significant role of TRIM21 in the maturation and differentiation of macrophages 17 , 18 . However, its role in osteoclast differentiation remains insufficiently explored. In previous study, we investigated the role of TRIM21 in mouse osteoclast differentiation in vitro and in vivo 19 . Firstly, we found the phenotype of increased bone mass of TRIM21 knockout mice by micro-CT analysis, and then found that the number of osteoclasts decreased after TIRM21 knockout by slice TRAP staining. Further in vitro induction of osteoclasts by primary BMM and cell line RAW264.7 confirmed that TRIM21 knockdown inhibited osteoclast differentiation. In this study, we aimed to explore the associated molecular mechanisms. 2. Results 2.1 TMT quantitative protein mass spectrometry detection and bioinformatics analysis We sought to identify the potential substrates or signaling pathway(s) that mediate Trim21-regulated OCs differentiation using tandem mass tagging (TMT)-based quantitative proteomics (Fig. 1 a). A total of 242 proteins were identified in the mouse BMMs derived from Trim21 −/− vs Trim21 +/+ mice. Among the identified proteins, 102 and 140 proteins exhibited increased and decreased, respectively (Fig. 1 b). Next, the volcano plot was sketched to represent the protein abundance changes in BMMs of Trim21 +/+ vs Trim21 −/− mice (Fig. 1 c). To systematically identify differentially expressed proteins (DEPs) involved in OCs differentiation, the heat map was generated to visualize the hierarchical cluster analysis (Fig. 1 d). 2.2 GO function and KEGG pathway enrichment analysis of DEPs As shown in Fig. 2 a, DEPs were mainly distributed in the nucleus (137) and cytoplasm (68). Furthermore, DEPs were found in various cellular components, including the cytoskeleton, lysosomes, mitochondria, plasma membrane, and other regions. This suggests that knockdown of TRIM21 may impact the functionality of the cytoskeleton, lysosomes, and mitochondria. Furthermore, a GO functional analysis was conducted on the aforementioned 242 DEPs, as depicted in Fig. 2 b. In terms of biological processes, the DEPs were primarily associated with metabolism, response to stimuli, development, immunity, cell proliferation, and cell killing. Regarding molecular function, the DEPs were involved in catalytic reactions, functional regulation, transcriptional regulation, and antioxidant activities. In terms of cellular components, the DEPs played a crucial role in cellular structures, organelles, cell membranes, extracellular regions, supramolecular complexes, synapses, and cell junctions. For KEGG pathway enrichment analysis, the top 20 significantly DEPs were selected. Figure 2 c demonstrates that several important pathways, such as protein digestion and absorption, ECM-receptor interaction, and the AGE-RAGE signaling pathway in diabetic complications, displayed significant changes. Additionally, the DEPs were found to be involved in pathways related to NF-kappa B signaling, focal adhesion, and osteoclast differentiation. The protein profiling samples used in this study were obtained from BMMs that were not induced for osteoclast differentiation. Consequently, it should be noted that the top 20 DEPs may not encompass all pathways associated with osteoclast differentiation. To address this limitation, KEGG pathway enrichment analysis was performed on all the differential proteins, enabling the identification of several signaling pathways related to osteoclast differentiation. The DEPs involved in these pathways were subjected to statistical analysis. Figure 2 d illustrates the distribution of these DEPs across various pathways. Specifically, the osteoclast differentiation pathway consisted of 4 differential proteins. The NF-kappa B signaling pathway, Endocytosis, and Ras signaling pathway each comprised 4 differential proteins, while the ECM-receptor-interaction and Lysosome pathway contained 6 differential proteins. Moreover, the PI3K-Akt signaling pathway, Focal adhesion, and Regulation of actin cytoskeleton pathway involved 7 differential proteins. It is important to note that these pathways collectively play a significant role in osteoclast differentiation 20 . 2.3 JNK2 is found to be essential in Trim21-mediated osteoclastogenesis The MAPK signaling pathway plays a crucial role in the process of osteoclast differentiation 21 . Among the aforementioned DEPs, we identified a significant protein involved in MAPK signaling, namely MAPK9 (JNK2). This protein governs the JNK signaling pathway, which constitutes one of the three branches of MAPK signaling, alongside JNK1 22 . However, these two proteins play opposing roles in signal transmission 23 – 25 . To validate the findings obtained from the mass spectrometer analysis of the differential protein JNK2, we isolated BMMs from TRIM21 +/+ and Trim21 −/− mice and subjected them to RANKL induction for 0, 1, 3, and 5 days. We employed Western blotting to examine the expression of JNK2 (Fig. 3 a-b). Notably, at the basal level and 3 days after RANKL induction, the Trim21 −/− mice consistently exhibited higher levels of JNK2 expression compared to the TRIM21 +/+ mice. However, the disparity in JNK2 expression between the two groups diminished after 5 days of RANKL induction. In our previous research, we discovered that TRIM21 facilitates the autophagy process in U2OS osteosarcoma cells, wherein autophagy functions similarly to the proteasome in protein degradation 18 . To explore whether TRIM21 regulates JNK2 through autophagy, we employed the autophagy inhibitor 3-MA to observe changes in JNK2 expression (Fig. 3 c,d). Remarkably, in BMMs derived from Trim21 +/+ mice, the presence of 3-MA significantly upregulated JNK2 levels, while its impact on BMMs derived from Trim21 −/− mice was limited. These findings strongly suggest that JNK2 is regulated by the autophagy pathway, and that 3-MA fails to further enhance autophagy inhibition induced by Trim21 −/− mice. 2.4 Inhibition of JNK2 mitigated the effect of TRIM21 knockout on osteoclast differentiation As previously mentioned, we observed an increase in JNK2 expression following TRIM21 knockout, which corresponded to a decrease in osteoclast differentiation. However, further investigation is required to ascertain whether TRIM21 regulates osteoclast differentiation through JNK2. SP600125 is a broad-spectrum inhibitor of JNK, effectively targeting both JNK1 and JNK2 with an IC50 of 40nM 26 . It also exhibits inhibitory effects on FLT3, TRKA, and ERK signals at higher doses. Since our primary objective was to inhibit the function of JNK2, we selected a low dose (50nM) of SP600125 as the final concentration. To evaluate the impact of SP600125 on osteoclast differentiation, we employed TRAP staining. As depicted in Fig. 3 e-f, the knockout of TRIM21 resulted in reduced differentiation of BMMs into osteoclasts. Interestingly, the addition of SP600125 to Trim21 −/− group significantly promoted osteoclast differentiation. Conversely, SP600125 did not enhance osteoclast differentiation in the Trim21 +/+ group and even exhibited a tendency to reduce differentiation. 2.5 Inhibition of JNK2 alleviates the restriction of NFATc1 entry by TRIM21 knockout NFATc1 serves as a pivotal regulator in osteoclast differentiation, acting as a "switch" for this process 27 . During osteoclast differentiation, NFATc1 translocates into the nucleus and orchestrates its own transcription, thereby facilitating a "self-amplifying" effect 28 . The intracellular localization of NFATc1 is primarily influenced by its phosphorylation status. Notably, JNK phosphorylates NFATc1, impeding its nuclear entry 29 . To investigate whether TRIM21 modulates the nuclear localization of NFATc1 through JNK2, we induced BMM cells derived from both Trim21 +/+ mice and Trim21 −/− mice with RANKL. Subsequently, PBS or SP600125 (at a concentration of 50nm/ml) was added simultaneously. After 3 days, immunofluorescence analysis was conducted to assess the nuclear localization of NFATc1. In the Trim21 +/+ group, we observed an increase in NFATc1 expression and its nuclear localization, as indicated by the increased co-localization with DAPI, after 3 days of RANKL induction. However, the addition of SP600125 did not augment this effect and instead displayed a tendency to reduce NFATc1 entry into the nucleus (Fig. 4 a). Conversely, in the Trim21 −/− group, NFATc1 was predominantly distributed in the cytoplasm. Nevertheless, upon inhibiting JNK2, we observed an increase in NFATc1's entry into the nucleus (Fig. 4 b). Quantitative analysis of these observations is presented in Fig. 4 c. In line with this result, we found significantly less protein level of NFATc1 in the Trim21 −/− nuclei, and increased amounts of p-NFATc1 in the Trim21 −/− cytoplasm compared to Trim21 +/+ mice (Fig. 4 d-e). These findings suggest that knockout of TRIM21 can impede osteoclast differentiation by inhibiting the nuclear translocation of NFATc1 through JNK2 . 3. Disccusion In previous studies, we have demonstrated that the depletion of Trim21 leads to reduced formation and activity of osteoclasts in mice, accompanied by an increase in bone mass 19 . However, the underlying mechanisms remain unclear. Our findings suggest that TRIM21 controls JNK2 degradation, thereby regulating NFATc1 nuclear translocation, revealing a potential regulatory role for TRIM21 in bone metabolism. Osteoclast differentiation is governed by various signaling pathways, with the MAPK pathway playing a crucial role 21 . JNK1 and JNK2 are both members of the MAPK family and involved in osteoclastogenesis, they exhibit distinct roles in signal transduction. Through proteomic analysis, we have discovered a significant upregulation of JNK2, another important molecule in the MAPK signaling, after TRIM21 deletion. It is worth noting that our proteomic analysis did not reveal any changes in JNK1. Literature reports indicate that JNK1 and JNK2 play distinct roles in the MAPK signaling, exerting different functions in various cell types 30 , 31 . For instance, in fibroblasts, the knockout of JNK1 and JNK2 exhibit completely opposite phenotypes. The molecular mechanism behind this is that although JNK2 has a higher affinity for c-Jun than JNK1 32,33 , JNK2 promotes the degradation of c-Jun in quiescent cells, while JNK1 phosphorylates c-Jun to activate the MAPK signaling upon stimulation 34 . These findings suggest that an increase in JNK2 does not necessarily facilitate the transmission of the MAPK signal. There is extensive research reporting the promotion of osteoclast differentiation by JNK1. Apart from activating AP-1 proteins, JNK1 can also promote osteoclast differentiation through the Bcl-2-Beclin1-autophagy signaling pathway 35 , 36 . Therefore, it raises the question of whether JNK2 plays a role in inhibiting osteoclast differentiation during the overall process. We have learned that NFATc1, a key transcription factor in osteoclast differentiation, is phosphorylated by numerous kinases, and its phosphorylation status is closely associated with its nuclear localization. According to relevant reports, casein kinase 1 (CK1), glycogen synthase kinase 3 (GSK3), dual-specificity tyrosine-phosphorylation-regulated kinases (DYRKs), cell division control protein 2 homolog (cdc2), and JNK can all phosphorylate NFATc1 37–40 . Interestingly, the three major branches of the MAPK pathway exhibit different regulatory effects on NFAT. For example, JNK1 can enhance NFAT's transcriptional activity, while P38-MAPK can phosphorylate NFAT to inhibit its activity 41 . JNK2 regulates NFAT by phosphorylating specific residues on NFATc1-α, which inhibits its nuclear translocation and transcriptional activity. This phosphorylation disrupts NFAT's interaction with calcineurin, preventing its activation. By retaining NFATc1-α in the cytoplasm, JNK2 reduces its transcriptional activity. Inhibition of JNK2 leads to increased nuclear accumulation and enhanced NFATc1-α activity 41 – 43 . In most cases, the phosphorylation of NFATc1 prevents its nuclear translocation and gene transcription regulation, making increased phosphorylation of NFATc1 a crucial step in inhibiting osteoclast differentiation 27 , 44 . Based on this, we hypothesize that the increase in JNK2 may inhibit osteoclast differentiation by suppressing the nuclear translocation of NFATc1. To test this hypothesis, we induced osteoclast differentiation and simultaneously treated the cells with the JNK inhibitor sp600125. Through immunofluorescence and TRAP staining, we confirmed that a low dose of sp600125 (a broad-spectrum JNK inhibitor that can also inhibit the ERK and p38-MAPK pathways at high doses) promotes the nuclear translocation of NFATc1 and osteoclast differentiation in TRIM21-depleted cells. It is worth noting that the effect of sp600125 on the Trim21 +/+ group was not significant, possibly because sp600125 inhibits the activity of both JNK1 and JNK2. This suggests that JNK1 and JNK2 have different roles in osteoclast differentiation, and in the absence of TRIM21, the inhibitory effect of JNK2 on osteoclasts outweighs the promoting effect of JNK1. In addition to the alterations in the osteoclast signaling pathway involving JNK2, our study also identified several pathway changes associated with osteoclast differentiation through protein profiling and bioinformatics analysis. These include the antioxidant signaling pathway, cell adhesion, mitochondrial oxidative phosphorylation, PI3K-Akt signaling pathway, and Toll-like receptor signaling pathway 18 . 4. Conclusion At the molecular level, our research further confirms that TRIM21 knockout leads to the upregulation of JNK2, which phosphorylates NFATc1, thereby limiting its nuclear translocation and transcriptional activity (Fig. 5 ). These findings hold promise in providing novel theoretical support and research groundwork for the prevention and treatment of osteoporosis. However, this study also has limitations. For instance, it remains unclear whether JNK2 upregulation is the sole factor responsible for the inhibition of osteoclastogenesis following TRIM21 depletion. Further investigations are needed to explore whether other molecular pathways interact with TRIM21 to jointly regulate osteoclast differentiation. Future studies using advanced gene editing techniques and animal models could help address these questions, providing more precise molecular targets for the treatment of bone diseases. 5. Methods and materials 5.1 Ethics declarations All animal procedures were approved by the Institutional Animal Care and Use Committee of Jinan University (Approval number: IACUC-20211216-15) and conformed to the “Guide for the Care and Use of Laboratory Animals” of the National Institute of Health in China. C57BL/6 mice with an average body weight of approximately 20–25 g were first anesthetized by controlled CO₂ inhalation until loss of consciousness and then humanely euthanized by continued CO₂ exposure followed by cervical dislocation to ensure death, in accordance with institutional ethical guidelines and the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals. All authors complied with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. 5.2 Osteoclastogenesis in BMMs Establishment of global Trim21 knockout mice was performed as described in our previous study 19 . BMMs were derived from 4 to 8-week-old Trim21 +/+ and Trim21 −/− mice, and then were incubated in α-MEM containing 30 ng/mL M-CSF (MCE, Cat#HY-P70553, USA) for 4 days to generate BMMs. To generate mature OCs, BMMs were cultured with 30 ng/ml M-CSF and 100 ng/ml RANKL for at least 5 days, followed by a series of experiments. The same volume of PBS containing 30 ng/ml M-CSF was applied as control. 5.3 Tandem mass tag-based quantitative proteomics Tandem mass tag (TMT)-based quantitative proteomic analysis was performed by Shanghai Applied Protein Technology Company (Shanghai, China). In brief, proteins were extracted using SDT-lysis buffer (4% sodium dodecyl sulfate (SDS), 100mM Tris/HCl pH7.6, 0.1M Dithiothreitol (DTT)), and protein content was determined using the BCA method. Next, protein samples were digested using the filter-aided proteome preparation (FASP) method as previously described 45 , and were desalted on C18 Cartridges (Empore™ SPE Cartridges C18, standard density), dried under vacuum, and then resuspended in 0.1% (v/v) formic acid. Each set of eluted peptides was labeled with a unique TMT isobaric tag (TMT126-128 for Trim21 +/+ , TMT129-131 for Trim21 −/− ) and analyzed by a Q Exactive mass spectrometer (MS, Thermo Fisher Scientific) coupled with an Easy-nLC 1000 system (Thermo Fisher Scientific). MS raw data were analyzed by Proteome Discoverer (Thermo Fisher Scientific, version 1.4) and then subjected to a database search using the MASCOT search engine (Matrix Science, Boston, MA, USA, version 2.2) for peptide identification. 5.4 Bioinformatics analysis Protein clustering analysis was conducted using the Complexheatmap R package (R Version 3.4) to classify and generate a hierarchical clustering heatmap based on both sample and protein expression dimensions. Subcellular localization analysis was performed using the CELLO method ( http://cello.life.nctu.edu.tw/ ) to predict the subcellular localization of differentially expressed proteins. For Gene Ontology (GO) functional annotation, significant differentially expressed proteins were subjected to sequence alignment (Blast), GO term extraction (Mapping), GO annotation (Annotation), and supplementary InterProScan annotation (Annotation Augmentation) using Blast2GO. KEGG pathway annotation for the significantly differentially expressed proteins was carried out using the KAAS (KEGG Automatic Annotation Server) software 46 – 48 . 5.5 Tartrate-resistant acid phosphatase (TRAP) staining Cells and mice knee sections were fixed and stained with TRAP solution using a kit according to the manufacturer’s instructions (Sigma-Aldrich, Cat#387A, Germany). Cells with more than 3 nuclei were considered as OCs. Five high-power fields (200 ×) were randomly selected for OCs counting. TRAP-positive cells were visualized, and the number of OCs/per field and nucleus number of per TRAP + cell was quantified by ImageJ software (National Institutes of Health, Bethesda, MD, USA). 5.6 Immunoblotting assay Protein concentration was determined by the BCA method and 20 ~ 50 µg of protein was firstly separated by 8 ~ 15% SDS-PAGE gels (Beyotime, Cat# P0012A, China), followed by the visualization with primary and secondary antibodies using a Tanon 5200 Luminescent Imaging Workstation (Tanon, China) as described previously 49 . Antibodies used were listed as follows: GAPDH (CST, Cat# 2118, USA, 1:1000), Trim21 (Novus, Cat# NBP1-33548, China, 1:1000), CTSK (Abcam, Cat# ab19027, UK, 1:1000), NFATC1 (SANTA, Cat# sc-7294, USA, 1:1000). 5.7 Immunofluorescence (IF) staining for cells OCs induced as described above were then fixed with 4% PFA for 20 min, permeabilized with 0.1% (v/v) Triton X-100 for 10 min, and blocked with 5% skim milk for 1h. Cells were then incubated with primary antibodies (NFATc1, CST, Cat# 14074, USA, 1:50) at 4°C overnight, washed with phosphate-buffered saline with Tween 20 (PBST) 5 min for 3 times, and then incubated with a fluorescent secondary anti-mouse antibody (Alexa Fluor 488, green, CST, Cat# 8878, USA, 1:100). F-actin (only for OCs) was stained with Rhodamine Phalloidin (Invitrogen, Cat# R415, USA) in the dark for 1 h followed by 4′, 6-diamidino-2-phenylindole (DAPI) staining for 10 min. The images were captured using a Laser Scan Confocal Microscope (Zeiss LSM 880, Germany). F-actin rings were visualized, and the number of F-actin ring per field (4 ×) was quantified by ImageJ software. 5.8 Statistical analysis All data are expressed as mean ± standard deviation (SD) with sample sizes indicated in either the figures and/or legends. For comparisons between the two groups, statistical analyses were performed by Student’s t-test. One-way ANOVA was used to compare the effects of more than two groups. A p-value of less than 0.05 was considered statistically significant. Declarations Declaration of competing interest The authors declare no competing interests. Funding Declaration This work was financially supported by the National Natural Science Foundation of China (82072470), Guangdong Basic and Applied Basic Research Foundation (2023B1515020007). Author Contribution Qi-Chun Zhao, Ying-Ming Wang made substantial contributions to conception, design, drafting, and revision. Xiao-he Wang made substantial contributions to the drafting, revision, acquisition of data, or analysis and interpretation of data. Gang Yu, Chao Fang, Liang Xu, Di Wu, Yan Yan assisted in acquisition of data and contributed to revision. Data Availability The data supporting the findings of this study are available from the corresponding author upon reasonable request. References Ash, P., Loutit, J. F. & Townsend, K. M. 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09:53:15","extension":"html","order_by":32,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":128356,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7540879/v1/e99e3e04fee8def015974571.html"},{"id":94747318,"identity":"fba71134-3259-4790-afe2-a466417fd6f1","added_by":"auto","created_at":"2025-10-30 09:53:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3196970,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTMT quantitative protein mass spectrometry detection and bioinformatics analysis.\u003c/strong\u003e a Schematic diagram showing TMT-based quantitative proteomics for the identification of differentially expressed proteins (DEPs) in BMMs derived from Trim21+/+ and Trim21-/- mice. b Schematic diagram illustrating the downregulated (Trim21-/-: Trim21+/+ \u0026lt; 0.8, p \u0026lt; 0.05) and upregulated (Trim21-/-: Trim21+/+ \u0026gt; 1.2, p \u0026lt; 0.05) DEPs identified in the BMMs of Trim21+/+ and Trim21-/- mice. c The volcano plots of DEPs in BMMs of Trim21+/+ and Trim21-/- mice. d Heat map analysis of DEPs in the BMMs. Three replicates of each group were included.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7540879/v1/3d390c85e5ce1b50e812feb4.png"},{"id":94747313,"identity":"86381a32-4b2c-4647-b0cc-b67606ab59d6","added_by":"auto","created_at":"2025-10-30 09:53:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3004967,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKEGG pathway and GO enrichment analysis of DEP.\u003c/strong\u003e a Subcellular localization chart of differentially expressed proteins. b KEGG enrichment analysis of the DEPs in the BMMs after osteoclastogenesis. c After KEGG annotation of all significantly differentially expressed proteins, the pathways related to osteoclast differentiation were selected and differential protein statistics were performed. The X-axis represents the number of differential proteins with up-or downregulation, and the Y-axis represents the pathway annotation entry. d GO functional analysis for DEPs after osteoclastogenesis in the BMMs. BP：Biological Process; CC：Cellular Component; MF：Molecular Function.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7540879/v1/69d62c826e907e1b29f6d86f.png"},{"id":94747333,"identity":"32d0f966-601b-4802-899f-6bd15ac6673b","added_by":"auto","created_at":"2025-10-30 09:53:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4675422,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eJNK is found to be essential in Trim21-mediated osteoclastogenesis.\u003c/strong\u003e a, b Immunoblotting analysis and quantification of JNK2 expression in the BMMs isolated from Trim21+/+ and Trim21-/- mice and stimulated with RANKL for 0, 1, 3, and 5 days. b Immunoblotting analysis and quantification of JNK2 expression in the BMMs isolated from Trim21+/+ and Trim21-/- mice treated with 3-MA or not (n = 3). Original blots are presented in Supplementary material. c, d TRAP staining and quantification analysis of BMMs isolated from Trim21+/+ and Trim21-/- mice after 5 days of RANKL-induction with or without SP600125 (n = 3). e Representative images of TRAP-stained cells in BMM-derived OCs from WT and KO mice treated with SP600125 or not. F Quantitative analysis of the number and size of TRAP-positive multinuclear cells (n = 3). Scale bar, 200 μm. n = 3. All bar graphs are presented as mean ± SD. * p\u0026lt; 0.05 ; ** p\u0026lt; 0.01; *** p\u0026lt; 0.001; **** p\u0026lt; 0.0001; n.s., not significant by Student’s t-test.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7540879/v1/7d7e4ab97be782b8e5abbc13.png"},{"id":94747327,"identity":"b9fed371-8999-4c91-89a9-42cf142f480a","added_by":"auto","created_at":"2025-10-30 09:53:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1517769,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of JNK2 alleviates the restriction of NFATc1 entry by TRIM21 knockout.\u003c/strong\u003e a The NFATc1 nuclear translocation of BMMs isolated from Trim21+/+ mice was examined by immunofluorescence. Scale bar, 50 μm. b The NFATc1 nuclear translocation of BMMs isolated from Trim21-/- mice was examined by immunofluorescence. Scale bar, 50 μm. c The nuclear/total integrated optical density (IOD) ratio was obtained to compare NFATc1 nuclear translocation BMMs (n = 3). d NFATc1 and p-NFATc1 protein level in cytoplasm and nucleus of osteoclasts measured by Western blot. e Quantitative analysis of d (n = 3). Original blots are presented in Supplementary material. All bar graphs are presented as mean ± SD. * p\u0026lt; 0.05 ; ** p\u0026lt; 0.01; *** p\u0026lt; 0.001; **** p\u0026lt; 0.0001; n.s., not significant by Student’s t-test.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-7540879/v1/e724e61b979681718506256f.png"},{"id":94747326,"identity":"fa2b01b9-072e-4e9b-a626-2b2363851e33","added_by":"auto","created_at":"2025-10-30 09:53:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":48418079,"visible":true,"origin":"","legend":"\u003cp\u003eTRIM21 knockout leads to the upregulation of JNK2, which phosphorylates NFATc1, thereby limiting its nuclear translocation and transcriptional activity.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-7540879/v1/de7078749970926ecb416482.png"},{"id":94984664,"identity":"e047161e-bbb3-4f7f-a44e-14522dfaa4c7","added_by":"auto","created_at":"2025-11-03 06:54:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":40726791,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7540879/v1/4ab9d2e2-9dcf-4ade-8a7b-c182c81dc66e.pdf"},{"id":94823194,"identity":"d810f802-242b-40be-8d04-c88a0c696adb","added_by":"auto","created_at":"2025-10-31 06:46:41","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10457,"visible":true,"origin":"","legend":"","description":"","filename":"ConflictofInterest.docx","url":"https://assets-eu.researchsquare.com/files/rs-7540879/v1/64b6d22425f971e623548ae8.docx"},{"id":94747316,"identity":"dd4f44c3-5259-4081-942f-152425ec52e9","added_by":"auto","created_at":"2025-10-30 09:53:11","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":91831,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterialoriginalblots.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7540879/v1/8731ec35c1a494f56749543a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"TRIM21 depletion negatively regulates osteoclastogenesis via association with JNK2 to reduce nucleation of NFATc1","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eOsteoclasts (OCs) are large multinucleated cells that play a vital role in bone development and homeostasis. They originate from monocyte-macrophage cells of the hematopoietic lineage, triggered by the macrophage-colony stimulation factor (m-CSF) and receptor activator of nuclear factor-kβ (NF-κβ) ligand (RANKL)\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Excessive bone resorption resulting from increased OC formation and activity is the primary cause of bone loss in conditions like osteoporosis, Paget's disease of bone, bone metastases in cancer, rheumatoid arthritis, and osteoarthritis\u003csup\u003e\u003cspan additionalcitationids=\"CR4 CR5 CR6\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Understanding the mechanisms behind osteoclastogenesis holds significant clinical importance for the treatment of bone loss diseases.\u003c/p\u003e\u003cp\u003eThe development of drugs targeting osteoclasts is a crucial approach in treating bone loss diseases. Nevertheless, studies have indicated that the effectiveness of currently utilized anti-bone resorption drugs in clinical settings is not entirely satisfactory, and their long-term usage can lead to severe complications. For instance, bisphosphonates carry a risk of inducing osteonecrosis of the jaw (ONJ) and atypical femoral fractures (AFF)\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Biological agents like Dinomumab, despite showing promising anti-osteoclast differentiation effects, may trigger antigenicity, potentially causing an immune response in the body\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Additionally, long-term use of calcitonin is associated with an increased risk of malignant tumors\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Bone-promoting drugs, exemplified by parathyroid hormone analogs (PTHa) such as Teriparatide, offer a viable option. However, the administration of such drugs should not exceed 24 months, and their efficacy diminishes once discontinued, necessitating sequential treatment with anti-bone resorption drugs\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eTRIM21 (Triple motif containing 21) is a member of the Class IV TRIM protein family and is known to participate in a wide array of cellular processes, including apoptosis, cell proliferation, immune response, immune cell differentiation, antiviral response, and tumor development\u003csup\u003e\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Macrophages are considered precursor cells for osteoclast differentiation, and recent studies have indicated the significant role of TRIM21 in the maturation and differentiation of macrophages\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. However, its role in osteoclast differentiation remains insufficiently explored.\u003c/p\u003e\u003cp\u003eIn previous study, we investigated the role of TRIM21 in mouse osteoclast differentiation in vitro and in vivo\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Firstly, we found the phenotype of increased bone mass of TRIM21 knockout mice by micro-CT analysis, and then found that the number of osteoclasts decreased after TIRM21 knockout by slice TRAP staining. Further in vitro induction of osteoclasts by primary BMM and cell line RAW264.7 confirmed that TRIM21 knockdown inhibited osteoclast differentiation. In this study, we aimed to explore the associated molecular mechanisms.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 TMT quantitative protein mass spectrometry detection and bioinformatics analysis\u003c/h2\u003e\u003cp\u003eWe sought to identify the potential substrates or signaling pathway(s) that mediate Trim21-regulated OCs differentiation using tandem mass tagging (TMT)-based quantitative proteomics (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). A total of 242 proteins were identified in the mouse BMMs derived from \u003cem\u003eTrim21\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e vs \u003cem\u003eTrim21\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e mice. Among the identified proteins, 102 and 140 proteins exhibited increased and decreased, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Next, the volcano plot was sketched to represent the protein abundance changes in BMMs of \u003cem\u003eTrim21\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e vs \u003cem\u003eTrim21\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). To systematically identify differentially expressed proteins (DEPs) involved in OCs differentiation, the heat map was generated to visualize the hierarchical cluster analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 GO function and KEGG pathway enrichment analysis of DEPs\u003c/h2\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, DEPs were mainly distributed in the nucleus (137) and cytoplasm (68). Furthermore, DEPs were found in various cellular components, including the cytoskeleton, lysosomes, mitochondria, plasma membrane, and other regions. This suggests that knockdown of TRIM21 may impact the functionality of the cytoskeleton, lysosomes, and mitochondria.\u003c/p\u003e\u003cp\u003eFurthermore, a GO functional analysis was conducted on the aforementioned 242 DEPs, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb. In terms of biological processes, the DEPs were primarily associated with metabolism, response to stimuli, development, immunity, cell proliferation, and cell killing. Regarding molecular function, the DEPs were involved in catalytic reactions, functional regulation, transcriptional regulation, and antioxidant activities. In terms of cellular components, the DEPs played a crucial role in cellular structures, organelles, cell membranes, extracellular regions, supramolecular complexes, synapses, and cell junctions.\u003c/p\u003e\u003cp\u003eFor KEGG pathway enrichment analysis, the top 20 significantly DEPs were selected. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec demonstrates that several important pathways, such as protein digestion and absorption, ECM-receptor interaction, and the AGE-RAGE signaling pathway in diabetic complications, displayed significant changes. Additionally, the DEPs were found to be involved in pathways related to NF-kappa B signaling, focal adhesion, and osteoclast differentiation.\u003c/p\u003e\u003cp\u003eThe protein profiling samples used in this study were obtained from BMMs that were not induced for osteoclast differentiation. Consequently, it should be noted that the top 20 DEPs may not encompass all pathways associated with osteoclast differentiation. To address this limitation, KEGG pathway enrichment analysis was performed on all the differential proteins, enabling the identification of several signaling pathways related to osteoclast differentiation. The DEPs involved in these pathways were subjected to statistical analysis. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed illustrates the distribution of these DEPs across various pathways. Specifically, the osteoclast differentiation pathway consisted of 4 differential proteins. The NF-kappa B signaling pathway, Endocytosis, and Ras signaling pathway each comprised 4 differential proteins, while the ECM-receptor-interaction and Lysosome pathway contained 6 differential proteins. Moreover, the PI3K-Akt signaling pathway, Focal adhesion, and Regulation of actin cytoskeleton pathway involved 7 differential proteins. It is important to note that these pathways collectively play a significant role in osteoclast differentiation\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 JNK2 is found to be essential in Trim21-mediated osteoclastogenesis\u003c/h2\u003e\u003cp\u003eThe MAPK signaling pathway plays a crucial role in the process of osteoclast differentiation\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Among the aforementioned DEPs, we identified a significant protein involved in MAPK signaling, namely MAPK9 (JNK2). This protein governs the JNK signaling pathway, which constitutes one of the three branches of MAPK signaling, alongside JNK1\u003csup\u003e22\u003c/sup\u003e. However, these two proteins play opposing roles in signal transmission\u003csup\u003e\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. To validate the findings obtained from the mass spectrometer analysis of the differential protein JNK2, we isolated BMMs from \u003cem\u003eTRIM21\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eTrim21\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice and subjected them to RANKL induction for 0, 1, 3, and 5 days. We employed Western blotting to examine the expression of JNK2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-b). Notably, at the basal level and 3 days after RANKL induction, the \u003cem\u003eTrim21\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice consistently exhibited higher levels of JNK2 expression compared to the \u003cem\u003eTRIM21\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e mice. However, the disparity in JNK2 expression between the two groups diminished after 5 days of RANKL induction.\u003c/p\u003e\u003cp\u003eIn our previous research, we discovered that TRIM21 facilitates the autophagy process in U2OS osteosarcoma cells, wherein autophagy functions similarly to the proteasome in protein degradation\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. To explore whether TRIM21 regulates JNK2 through autophagy, we employed the autophagy inhibitor 3-MA to observe changes in JNK2 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec,d). Remarkably, in BMMs derived from \u003cem\u003eTrim21\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e mice, the presence of 3-MA significantly upregulated JNK2 levels, while its impact on BMMs derived from \u003cem\u003eTrim21\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice was limited. These findings strongly suggest that JNK2 is regulated by the autophagy pathway, and that 3-MA fails to further enhance autophagy inhibition induced by \u003cem\u003eTrim21\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Inhibition of JNK2 mitigated the effect of TRIM21 knockout on osteoclast differentiation\u003c/h2\u003e\u003cp\u003eAs previously mentioned, we observed an increase in JNK2 expression following TRIM21 knockout, which corresponded to a decrease in osteoclast differentiation. However, further investigation is required to ascertain whether TRIM21 regulates osteoclast differentiation through JNK2. SP600125 is a broad-spectrum inhibitor of JNK, effectively targeting both JNK1 and JNK2 with an IC50 of 40nM\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. It also exhibits inhibitory effects on FLT3, TRKA, and ERK signals at higher doses. Since our primary objective was to inhibit the function of JNK2, we selected a low dose (50nM) of SP600125 as the final concentration. To evaluate the impact of SP600125 on osteoclast differentiation, we employed TRAP staining. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee-f, the knockout of TRIM21 resulted in reduced differentiation of BMMs into osteoclasts. Interestingly, the addition of SP600125 to \u003cem\u003eTrim21\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e group significantly promoted osteoclast differentiation. Conversely, SP600125 did not enhance osteoclast differentiation in the \u003cem\u003eTrim21\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e group and even exhibited a tendency to reduce differentiation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Inhibition of JNK2 alleviates the restriction of NFATc1 entry by TRIM21 knockout\u003c/h2\u003e\u003cp\u003eNFATc1 serves as a pivotal regulator in osteoclast differentiation, acting as a \"switch\" for this process\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. During osteoclast differentiation, NFATc1 translocates into the nucleus and orchestrates its own transcription, thereby facilitating a \"self-amplifying\" effect\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. The intracellular localization of NFATc1 is primarily influenced by its phosphorylation status. Notably, JNK phosphorylates NFATc1, impeding its nuclear entry\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. To investigate whether TRIM21 modulates the nuclear localization of NFATc1 through JNK2, we induced BMM cells derived from both \u003cem\u003eTrim21\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e mice and \u003cem\u003eTrim21\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice with RANKL. Subsequently, PBS or SP600125 (at a concentration of 50nm/ml) was added simultaneously. After 3 days, immunofluorescence analysis was conducted to assess the nuclear localization of NFATc1.\u003c/p\u003e\u003cp\u003eIn the \u003cem\u003eTrim21\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e group, we observed an increase in NFATc1 expression and its nuclear localization, as indicated by the increased co-localization with DAPI, after 3 days of RANKL induction. However, the addition of SP600125 did not augment this effect and instead displayed a tendency to reduce NFATc1 entry into the nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Conversely, in the \u003cem\u003eTrim21\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e group, NFATc1 was predominantly distributed in the cytoplasm. Nevertheless, upon inhibiting JNK2, we observed an increase in NFATc1's entry into the nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Quantitative analysis of these observations is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec. In line with this result, we found significantly less protein level of NFATc1 in the \u003cem\u003eTrim21\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e nuclei, and increased amounts of p-NFATc1 in the \u003cem\u003eTrim21\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e cytoplasm compared to \u003cem\u003eTrim21\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed-e). These findings suggest that knockout of TRIM21 can impede osteoclast differentiation by inhibiting the nuclear translocation of NFATc1 through JNK2 .\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Disccusion","content":"\u003cp\u003eIn previous studies, we have demonstrated that the depletion of Trim21 leads to reduced formation and activity of osteoclasts in mice, accompanied by an increase in bone mass\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. However, the underlying mechanisms remain unclear. Our findings suggest that TRIM21 controls JNK2 degradation, thereby regulating NFATc1 nuclear translocation, revealing a potential regulatory role for TRIM21 in bone metabolism.\u003c/p\u003e\u003cp\u003eOsteoclast differentiation is governed by various signaling pathways, with the MAPK pathway playing a crucial role\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. JNK1 and JNK2 are both members of the MAPK family and involved in osteoclastogenesis, they exhibit distinct roles in signal transduction. Through proteomic analysis, we have discovered a significant upregulation of JNK2, another important molecule in the MAPK signaling, after TRIM21 deletion. It is worth noting that our proteomic analysis did not reveal any changes in JNK1.\u003c/p\u003e\u003cp\u003eLiterature reports indicate that JNK1 and JNK2 play distinct roles in the MAPK signaling, exerting different functions in various cell types\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. For instance, in fibroblasts, the knockout of JNK1 and JNK2 exhibit completely opposite phenotypes. The molecular mechanism behind this is that although JNK2 has a higher affinity for c-Jun than JNK1\u003csup\u003e32,33\u003c/sup\u003e, JNK2 promotes the degradation of c-Jun in quiescent cells, while JNK1 phosphorylates c-Jun to activate the MAPK signaling upon stimulation\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. These findings suggest that an increase in JNK2 does not necessarily facilitate the transmission of the MAPK signal. There is extensive research reporting the promotion of osteoclast differentiation by JNK1. Apart from activating AP-1 proteins, JNK1 can also promote osteoclast differentiation through the Bcl-2-Beclin1-autophagy signaling pathway\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Therefore, it raises the question of whether JNK2 plays a role in inhibiting osteoclast differentiation during the overall process.\u003c/p\u003e\u003cp\u003eWe have learned that NFATc1, a key transcription factor in osteoclast differentiation, is phosphorylated by numerous kinases, and its phosphorylation status is closely associated with its nuclear localization. According to relevant reports, casein kinase 1 (CK1), glycogen synthase kinase 3 (GSK3), dual-specificity tyrosine-phosphorylation-regulated kinases (DYRKs), cell division control protein 2 homolog (cdc2), and JNK can all phosphorylate NFATc1\u003csup\u003e37\u0026ndash;40\u003c/sup\u003e. Interestingly, the three major branches of the MAPK pathway exhibit different regulatory effects on NFAT. For example, JNK1 can enhance NFAT's transcriptional activity, while P38-MAPK can phosphorylate NFAT to inhibit its activity\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. JNK2 regulates NFAT by phosphorylating specific residues on NFATc1-α, which inhibits its nuclear translocation and transcriptional activity. This phosphorylation disrupts NFAT's interaction with calcineurin, preventing its activation. By retaining NFATc1-α in the cytoplasm, JNK2 reduces its transcriptional activity. Inhibition of JNK2 leads to increased nuclear accumulation and enhanced NFATc1-α activity\u003csup\u003e\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. In most cases, the phosphorylation of NFATc1 prevents its nuclear translocation and gene transcription regulation, making increased phosphorylation of NFATc1 a crucial step in inhibiting osteoclast differentiation\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Based on this, we hypothesize that the increase in JNK2 may inhibit osteoclast differentiation by suppressing the nuclear translocation of NFATc1. To test this hypothesis, we induced osteoclast differentiation and simultaneously treated the cells with the JNK inhibitor sp600125. Through immunofluorescence and TRAP staining, we confirmed that a low dose of sp600125 (a broad-spectrum JNK inhibitor that can also inhibit the ERK and p38-MAPK pathways at high doses) promotes the nuclear translocation of NFATc1 and osteoclast differentiation in TRIM21-depleted cells. It is worth noting that the effect of sp600125 on the \u003cem\u003eTrim21\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e group was not significant, possibly because sp600125 inhibits the activity of both JNK1 and JNK2. This suggests that JNK1 and JNK2 have different roles in osteoclast differentiation, and in the absence of TRIM21, the inhibitory effect of JNK2 on osteoclasts outweighs the promoting effect of JNK1.\u003c/p\u003e\u003cp\u003eIn addition to the alterations in the osteoclast signaling pathway involving JNK2, our study also identified several pathway changes associated with osteoclast differentiation through protein profiling and bioinformatics analysis. These include the antioxidant signaling pathway, cell adhesion, mitochondrial oxidative phosphorylation, PI3K-Akt signaling pathway, and Toll-like receptor signaling pathway\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eAt the molecular level, our research further confirms that TRIM21 knockout leads to the upregulation of JNK2, which phosphorylates NFATc1, thereby limiting its nuclear translocation and transcriptional activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These findings hold promise in providing novel theoretical support and research groundwork for the prevention and treatment of osteoporosis.\u003c/p\u003e\u003cp\u003eHowever, this study also has limitations. For instance, it remains unclear whether JNK2 upregulation is the sole factor responsible for the inhibition of osteoclastogenesis following TRIM21 depletion. Further investigations are needed to explore whether other molecular pathways interact with TRIM21 to jointly regulate osteoclast differentiation. Future studies using advanced gene editing techniques and animal models could help address these questions, providing more precise molecular targets for the treatment of bone diseases.\u003c/p\u003e"},{"header":"5. Methods and materials","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e5.1 Ethics declarations\u003c/h2\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e All animal procedures were approved by the Institutional Animal Care and Use Committee of Jinan University (Approval number: IACUC-20211216-15) and conformed to the \u0026ldquo;Guide for the Care and Use of Laboratory Animals\u0026rdquo; of the National Institute of Health in China. C57BL/6 mice with an average body weight of approximately 20\u0026ndash;25 g were first anesthetized by controlled CO₂ inhalation until loss of consciousness and then humanely euthanized by continued CO₂ exposure followed by cervical dislocation to ensure death, in accordance with institutional ethical guidelines and the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals. All authors complied with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e5.2 Osteoclastogenesis in BMMs\u003c/h2\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eEstablishment of global Trim21 knockout mice was performed as described in our previous study\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. BMMs were derived from 4 to 8-week-old \u003cem\u003eTrim21\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e and Trim21\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, and then were incubated in α-MEM containing 30 ng/mL M-CSF (MCE, Cat#HY-P70553, USA) for 4 days to generate BMMs. To generate mature OCs, BMMs were cultured with 30 ng/ml M-CSF and 100 ng/ml RANKL for at least 5 days, followed by a series of experiments. The same volume of PBS containing 30 ng/ml M-CSF was applied as control.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e5.3 Tandem mass tag-based quantitative proteomics\u003c/h2\u003e\u003cp\u003eTandem mass tag (TMT)-based quantitative proteomic analysis was performed by Shanghai Applied Protein Technology Company (Shanghai, China). In brief, proteins were extracted using SDT-lysis buffer (4% sodium dodecyl sulfate (SDS), 100mM Tris/HCl pH7.6, 0.1M Dithiothreitol (DTT)), and protein content was determined using the BCA method. Next, protein samples were digested using the filter-aided proteome preparation (FASP) method as previously described\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, and were desalted on C18 Cartridges (Empore\u0026trade; SPE Cartridges C18, standard density), dried under vacuum, and then resuspended in 0.1% (v/v) formic acid. Each set of eluted peptides was labeled with a unique TMT isobaric tag (TMT126-128 for \u003cem\u003eTrim21\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e, TMT129-131 for \u003cem\u003eTrim21\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) and analyzed by a Q Exactive mass spectrometer (MS, Thermo Fisher Scientific) coupled with an Easy-nLC 1000 system (Thermo Fisher Scientific). MS raw data were analyzed by Proteome Discoverer (Thermo Fisher Scientific, version 1.4) and then subjected to a database search using the MASCOT search engine (Matrix Science, Boston, MA, USA, version 2.2) for peptide identification.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e5.4 Bioinformatics analysis\u003c/h2\u003e\u003cp\u003eProtein clustering analysis was conducted using the Complexheatmap R package (R Version 3.4) to classify and generate a hierarchical clustering heatmap based on both sample and protein expression dimensions. Subcellular localization analysis was performed using the CELLO method (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://cello.life.nctu.edu.tw/\u003c/span\u003e\u003cspan address=\"http://cello.life.nctu.edu.tw/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to predict the subcellular localization of differentially expressed proteins. For Gene Ontology (GO) functional annotation, significant differentially expressed proteins were subjected to sequence alignment (Blast), GO term extraction (Mapping), GO annotation (Annotation), and supplementary InterProScan annotation (Annotation Augmentation) using Blast2GO. KEGG pathway annotation for the significantly differentially expressed proteins was carried out using the KAAS (KEGG Automatic Annotation Server) software\u003csup\u003e\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e5.5 Tartrate-resistant acid phosphatase (TRAP) staining\u003c/h2\u003e\u003cp\u003e Cells and mice knee sections were fixed and stained with TRAP solution using a kit according to the manufacturer\u0026rsquo;s instructions (Sigma-Aldrich, Cat#387A, Germany). Cells with more than 3 nuclei were considered as OCs. Five high-power fields (200 \u0026times;) were randomly selected for OCs counting. TRAP-positive cells were visualized, and the number of OCs/per field and nucleus number of per TRAP\u0026thinsp;+\u0026thinsp;cell was quantified by ImageJ software (National Institutes of Health, Bethesda, MD, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e5.6 Immunoblotting assay\u003c/h2\u003e\u003cp\u003eProtein concentration was determined by the BCA method and 20\u0026thinsp;~\u0026thinsp;50 \u0026micro;g of protein was firstly separated by 8\u0026thinsp;~\u0026thinsp;15% SDS-PAGE gels (Beyotime, Cat# P0012A, China), followed by the visualization with primary and secondary antibodies using a Tanon 5200 Luminescent Imaging Workstation (Tanon, China) as described previously\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Antibodies used were listed as follows: GAPDH (CST, Cat# 2118, USA, 1:1000), Trim21 (Novus, Cat# NBP1-33548, China, 1:1000), CTSK (Abcam, Cat# ab19027, UK, 1:1000), NFATC1 (SANTA, Cat# sc-7294, USA, 1:1000).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e5.7 Immunofluorescence (IF) staining for cells\u003c/h2\u003e\u003cp\u003eOCs induced as described above were then fixed with 4% PFA for 20 min, permeabilized with 0.1% (v/v) Triton X-100 for 10 min, and blocked with 5% skim milk for 1h. Cells were then incubated with primary antibodies (NFATc1, CST, Cat# 14074, USA, 1:50) at 4\u0026deg;C overnight, washed with phosphate-buffered saline with Tween 20 (PBST) 5 min for 3 times, and then incubated with a fluorescent secondary anti-mouse antibody (Alexa Fluor 488, green, CST, Cat# 8878, USA, 1:100). F-actin (only for OCs) was stained with Rhodamine Phalloidin (Invitrogen, Cat# R415, USA) in the dark for 1 h followed by 4\u0026prime;, 6-diamidino-2-phenylindole (DAPI) staining for 10 min. The images were captured using a Laser Scan Confocal Microscope (Zeiss LSM 880, Germany). F-actin rings were visualized, and the number of F-actin ring per field (4 \u0026times;) was quantified by ImageJ software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e5.8 Statistical analysis\u003c/h2\u003e\u003cp\u003eAll data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) with sample sizes indicated in either the figures and/or legends. For comparisons between the two groups, statistical analyses were performed by Student\u0026rsquo;s t-test. One-way ANOVA was used to compare the effects of more than two groups. A p-value of less than 0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003ch2\u003eFunding Declaration\u003c/h2\u003e\u003cp\u003eThis work was financially supported by the National Natural Science Foundation of China (82072470), Guangdong Basic and Applied Basic Research Foundation (2023B1515020007).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eQi-Chun Zhao, Ying-Ming Wang made substantial contributions to conception, design, drafting, and revision. Xiao-he Wang made substantial contributions to the drafting, revision, acquisition of data, or analysis and interpretation of data. Gang Yu, Chao Fang, Liang Xu, Di Wu, Yan Yan assisted in acquisition of data and contributed to revision.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data supporting the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAsh, P., Loutit, J. F. \u0026amp; Townsend, K. M. Osteoclasts derived from haematopoietic stem cells. \u003cem\u003eNature\u003c/em\u003e \u003cb\u003e283\u003c/b\u003e, 669\u0026ndash;670. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/283669a0\u003c/span\u003e\u003cspan address=\"10.1038/283669a0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1980).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBoyle, W. J., Simonet, W. S. \u0026amp; Lacey, D. L. 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Gang Yu, Chao Fang, Yan Yan assisted in acquisition of data and contributed to revision.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"TRIM21, osteoclast, JNK2, Nfatc1","lastPublishedDoi":"10.21203/rs.3.rs-7540879/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7540879/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAberrant activation of osteoclast-mediated bone resorption has been observed in a series of skeletal diseases including osteoporosis and delayed or non-union bone fracture. Ubiquitination-mediated protein degradation has been revealed as an important mechanism for osteoporosis. Trim21 as an important E3 ubiquitin ligase, has been found to be critical for osteoclastogenesis. We have recently demonstrated that Trim21 is a crucial player in fine-tuning bone homeostasis, yet the underlying mechanism that affect bone resorption is largely unknown. Herein, we demonstrate that deletion of Trim21 led to decreased OC formation and activity accompanied by bone mass increase in mice. In addition, unbiased proteomics analysis identified that JNK2, was one of the key substance of Trim21. Mechanistically, we discovered that TRIM21 influences osteoclast differentiation by controlling the degradation of JNK2 via ubiquitin-dependent proteasomal or lysosomal degradation. Protein-protein interaction was further confirmed by immunofluorescence, leading to the modulation of NFATc1 nucleation. Our findings propose TRIM21 as a promising therapeutic target for osteoporosis.\u003c/p\u003e","manuscriptTitle":"TRIM21 depletion negatively regulates osteoclastogenesis via association with JNK2 to reduce nucleation of NFATc1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-30 09:53:06","doi":"10.21203/rs.3.rs-7540879/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-07T19:46:20+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-06T07:08:23+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-30T12:24:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-21T10:56:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"91972552502319364441414723139354099058","date":"2025-10-17T03:33:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"102020819336037782678974659289369393895","date":"2025-10-17T01:53:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"288229946847996226410399190548789171551","date":"2025-10-16T08:12:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"59387504360067487955859787400213628119","date":"2025-10-16T05:51:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-16T02:59:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-14T09:16:33+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-13T19:08:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-10T15:17:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-10-10T14:47:03+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d69b809d-a107-4bc5-8b94-71a2cc41322a","owner":[],"postedDate":"October 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":56879911,"name":"Biological sciences/Biochemistry"},{"id":56879912,"name":"Biological sciences/Cell biology"},{"id":56879913,"name":"Health sciences/Diseases"},{"id":56879914,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2025-11-07T19:53:22+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-30 09:53:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7540879","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7540879","identity":"rs-7540879","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}