Selenoprotein S deficiency induces matrix degradation via TGF-β pathway leading to cartilage damage

Selenoprotein S deficiency induces matrix degradation via TGF-β pathway leading to cartilage damage | 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 Research Article Selenoprotein S deficiency induces matrix degradation via TGF-β pathway leading to cartilage damage Hui Wang, Yinan Liu, Meng Zhang, Mengying Wang, Yawen Shi, Jian Sun, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9267022/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Background: The deficiency of trace element selenium is a global nutritional issue. Selenium exerts its biological functions in the human body through selenoproteins, which play crucial roles in bone and cartilage development. Selenoprotein S (SelS), a key selenoprotein involved in the regulation of oxidative stress and inflammation, has an unclear role in cartilage development. The purpose of this study was to investigate the effects of SelS deficiency on cartilage matrix degradation. Methods: Chondrocytes with SelS gene knockdown and mice with SelS gene knockout were constructed. The mRNA and protein levels of COL II, MMP3, MMP10, and MMP13 were measured by RT-qPCR and western blotting, respectively. Changes in cartilage morphology and matrix composition were evaluated using histological staining techniques including toluidine blue (TB), saffron O-fast green, and sirius red staining. The expression levels of MMP3, MMP13, MMP19 and COL II in the knee joints of mice were detected by immunohistochemical (IHC) staining. A TGF-β pathway inhibitor (GW788388) was applied to SelS knockdown chondrocytes to verify whether the TGF-βsignaling pathway is involved in the matrix degradation induced by SelS deficiency. Results: Compared with the sh-NC group, the mRNA and protein levels of COL Ⅱ were decreased in the sh-Sels group. The expression levels of Mmp3 , Mmp10 and Mmp13 were also increased significantly, while the metallopeptidase inhibitor Timp2 was downregulated in the sh-Sels group. SelS gene knockout in mice did not affect body length or weight but resulted in reduced proteoglycan and collagen contents in articular cartilage. Compared with wild-type mice, the expression of COL Ⅱ was decreased, while the expressions of MMP3, MMP13, and MMP19 were increased in the articular cartilage of SelS knockout mice. Furthermore, treatment with the TGF-β pathway inhibitor (GW788388) partially rescued the reduction in Col2a1 expression induced by SelS deficiency. Conclusion: SelS plays a crucial role in maintaining cartilage homeostasis, and its deficiency results in cartilage matrix degradation. The TGF-β signaling pathway is involved in the degradation of cartilage matrix caused by SelS silencing. Selenoprotein S cartilage type Ⅱ collagen matrix metalloproteinases TGF-β Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1 Introduction Selenoprotein S (SelS), also known as SEPS1 or VIMP, is a member of the selenoprotein family. The SelS gene is located on chromosome 15 in humans. SelS contains a selenocysteine (Sec) residue at position 188 of its amino acid sequence, which is encoded by the UGA codon, typically a stop signal in translation. However, selenoproteins, including SelS, possess specialized translational machinery that recognizes the UGA codon as a Sec incorporation signal. The presence of Sec is crucial for SelS function, enabling it to participate in redox reactions and contributing to unique biological activities [ 1 ] . Initially, SelS was identified as a glucose-regulating protein because of its inverse correlation with blood glucose and insulin levels [ 2 ] . Subsequent studies demonstrated that SelS, as a component of the ER-associated protein degradation (ERAD) complex, facilitates the transfer of misfolded proteins from the ER to the cytosol for degradation [ 3 ] . Moreover, SelS has been shown to regulate inflammation by inhibiting NF-κB activity and reducing the production of proinflammatory cytokines [ 4 , 5 ] . Given its roles in glucose regulation, oxidative stress management, ERAD, and inflammation modulation, SelS has garnered significant attention in research related to diabetes, Alzheimer's disease, cardiovascular disease, and cancer [ 2 , 6 – 9 ] . The expression of SelS is regulated by various factors, including cellular stress, inflammation, and selenium intake, and exhibits tissue-specific patterns, with higher levels observed in secretory tissues such as the liver, pancreas, colon, and small intestine [ 1 ] . In recent years, the role of selenium in bone and cartilage diseases has received increasing attention. Selenium deficiency has been associated with the onset and progression of diseases such as Kashin-Beck disease (KBD), osteoarthritis (OA), rheumatoid arthritis (RA), and osteoporosis (OP) [ 10 – 13 ] . Feeding mice a low-selenium diet induced the formation of fibrocartilage on the joint surface, ultimately resulting in degenerative changes in articular cartilage [ 14 ] . Selenium deficiency diet decreased femoral trabecular bone volume/total volume and trabecular number while increasing trabecular separation in mice, suggesting that selenium deficiency enhances bone resorption and damages bone microstructure [ 15 ] . In a rat model, a two-generation selenium-deficient diet caused growth retardation, epiphyseal plate lesions, and reduced expression of GPx1 and COL II in chondrocytes, indicating diminished anabolic and antioxidant capacity in chondrocytes [ 16 ] . Selenium exerts its biological functions in the body primarily through selenoproteins. After absorption and transformation, selenium is transported via the liver to various organs and tissues for the synthesis of selenoproteins. The most direct consequence of low selenium diet is insufficient expression of selenoproteins. Extensive researches have demonstrated the critical role of selenoproteins in bone and cartilage development. Selenoprotein synthesis depends on the Trsp gene, which encodes selenocysteine tRNA [Ser]Sec , which is essential for incorporating Sec residues into selenoproteins. Mice with osteo-chondroprogenitor-specific deletion of Trsp exhibited growth retardation, abnormal epiphyseal growth plates, delayed skeletal ossification, and significant chondronecrosis in articular, auricular, and tracheal cartilages [ 17 ] . The relationship between selenoproteins and bone development was further supported by observations of growth retardation and delayed bone maturation in patients with SBP2 deficiency, a key trans-acting factor for Sec insertion into selenoproteins [ 18 ] . Knockout of SelP, which is responsible for selenium transport, leads to bone loss [ 19 ] . Selenium deficiency induces chondrocyte apoptosis associated with SelM [ 20 ] . Transcriptomic analysis of selenoproteins in chondrocytes from patients with KBD indicates that abnormal expression of GPX and DIO family selenoproteins may contribute to the pathogenesis of KBD [ 21 ] . Knockdown of GPx1 or SelO has been shown to reduce the expression of Sox9, Col II, and aggrecan, inhibit glycosaminoglycans (GAGs) accumulation and chondrocyte proliferation, and induce cell apoptosis [ 22 , 23 ] .The effects of abnormal SelS expression on bone and cartilage formation and development remain poorly understood. KBD, an endemic osteoarthritis, is prevalent in low-selenium areas. KBD patients have lower levels of blood selenium, hair selenium, and urine selenium compared to people from non-KBD areas. Therefore, selenium deficiency is widely recognized as a major contributing factor to KBD. Decreased expression of SelS has been observed in the articular cartilage of KBD patients. Initially, it was hypothesized that reduced SelS expression would induce ER stress and subsequent chondrocyte death. However, experimental evidence suggests that SelS knockdown does not trigger ER stress in chondrocytes [ 24 ] . To further elucidate the impact of SelS knockdown on hypertrophic chondrocytes, which are the primary damaged cells in KBD, RNA sequencing was performed, and differentially expressed genes were identified. The results demonstrated that SelS knockdown significantly upregulated the expression of matrix metalloproteinases ( Mmp3 , Mmp10 , Mmp13 , and Mmp19 ) while downregulating the expression of type II collagen ( Col2a1 ) in hypertrophic chondrocytes. Degradation of extracellular matrix (ECM) in cartilage, characterized by elevated MMPs expression and reduced type II collagen (COL II) and proteoglycan levels, represents a critical pathological feature of KBD [ 25 ] . Therefore, we hypothesized that decreased SelS expression might contribute to cartilage damage via ECM degradation. Thus, the objective of this study was to verify the relationship between SelS and cartilage matrix degradation and investigate its underlying mechanism. 2 Methods 2.1 Cell culture and treatment The origin of ATDC5 cells and construction process of SelS knockdown cells were described in our previous paper [ 24 ] . The negative control group and the SelS gene knockdown group were expressed as sh-NC and sh-Sels, respectively. The cells were cultured in DMEM/F-12 medium (HyClone, Logan, UT, USA) containing 5% fetal bovine serum (FBS) (Gibco, Gaithersburg, PA, USA) at 37℃ and 5% CO 2 . The lentivirus used for SelS knockdown was purchased from Shanghai Obio Technology Company (Shanghai, China) and the overexpression plasmid was purchased from Hanbio Biotechnology (Shanghai, China). 2.2 Origin and breeding of mice C57BL/6 Sels +/− mice were purchased from GemPharmatech LLC(Nanjing, China). All the mice were housed in the pathogen-free Animal Center of Xi’an jiaotong University under controlled temperature conditions (23°C) with ad libitum access to food and water. Wild type and homozygous mice were generated by crossing heterozygous mice. Neonatal mice were toe-clipped and genotyped on the 10th day after birth. The mice were sacrificed at 7 weeks of age, and the knee joint and costal cartilage were collected for subsequent experiments. All animal experiments were approved by the Animal Experimental Ethics Committee of Xi’an Jiaotong University. 2.3 DNA extraction and genotype identification Genomic DNA was extracted using the TIANamp Genomic DNA kit (DP304, TIANGEN BIOTECH, Beijing, China). After the DNA was extracted, PCR amplification was performed. The genotyping strategy and primer information are shown in Figure S1 . The amplified DNA was separated by 1% agarose gel electrophoresis. In addition to DNA identification, RIPA lysate was used to extract costal cartilage proteins, and the protein level of SelS was detected by western blotting. The genotype identification and western blotting results are shown in Fig. 1 . 2.4 Hematoxylin & eosin (H&E) staining and toluidine blue (TB) staining The knee joints of the mice were embedded in paraffin wax and cut into 5µm sections. After being dewaxed in xylene and hydrated in gradient ethanol, the samples were stained with hematoxylin for 5 minutes to dye the nuclei. Next, the sections were treated with hydrochloric ethanol for a few seconds to remove excess dye and further treated with ammonia liquor. For eosin staining, the sections were stained with eosin for 1 minute. For TB staining, the sections were stained with 0.1% TB solution for 2 minutes. Finally, the sections were dehydrated in gradient ethanol, cleared in xylene, and sealed with neutral resin. 2.5 Saffron O-fast green Staining and Sirius red staining For saffron O-fast green staining, the procedures of nuclear staining and neutral resin sealing were the same as H&E staining. In the special steps, the sections were first immersed in fast green dye for 10 minutes, washed in pure water three times, stained with saffron O dye for 1 minute, washed in pure water three times, soaked in 2% acetic acid for several seconds to remove excess dye, and stained in fast green for another two minutes. For sirius red staining, the sections were soaked in picrate-sirius red solution for 1 hour before the nuclei were stained. Notably, saffron O stained and sirius red stained samples were not dehydrated in low concentration ethanol. 2.6 Immunohistochemistry (IHC) staining After being dewaxed in xylene and hydrated in gradient alcohol, the sections were treated with 0.125% trypsin (Xi'an GuoAn Biological Technology Co.) for antigen retrieval at 37°C for 30 minutes. The subsequent steps were performed according to the instructions of the IHC kit (Beijing Zhong Shan Gold Bridge Biological Technology Co., SP9001). The samples were incubated with primary antibodies against MMP3 (1:50, 66338-1-Ig, Proteintech), MMP13 (1:100, 18165-1-AP, Proteintech), MMP19 (1:100, 14244-1-AP, Proteintech), and COL Ⅱ (1:80, A1560, ABclonal) at 4°C overnight. DAB solution (Beijing Zhong Shan Gold Bridge Biological Technology Co., ZLI-9018) was used for color reactions, followed by hematoxylin staining, dehydration, and sealing. 2.7 Total protein extraction and western blotting Total protein was extracted using RIPA lysis buffer (Beyotime, P0013B). The protein concentration was determined using a BCA protein assay kit (Shanghai Epizyme Biomedical Technology Co., Ltd, ZJ101). Equal masses of protein were loaded into each well for SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins with different molecular weights were separated and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, ISEQ00010). The PVDF membranes were blocked with 5% skim milk for 2 hours and incubated with primary antibodies against SELS (1:1000, 15591-1-AP, Proteintech), COL Ⅱ (1:500, A1560, ABclonal), MMP13 (1:500, 18165-1-AP, Proteintech), MMP19 (1:1000, 14244-1-AP, Proteintech), and GAPDH (1:10000, 60004-1-Ig, Proteintech) at 4°C overnight. The next day, after incubation with goat anti-mouse IgG/HRP (1:20000, CWBio, China) or goat anti-rabbit IgG/HRP (1:10000, Jackson, USA), the PVDF membranes were treated with an enhanced chemiluminescence kit (Millipore, Billerica, MA) for chemiluminescence, and the protein bands were analyzed using ImageJ software. 2.8 RNA extraction and real-time PCR Total RNA was isolated according to the TRIzol protocol and reverse-transcribed to cDNA via a RevertAid First Stand cDNA Synthesis Kit (K1622, Thermo Fisher Scientific). Relative gene expression was quantified using an iQ5 Cycler (Bio-Rad, Munich, Germany) under the following conditions: initial denaturation at 95°C for 5 min; 40 cycles of 95°C for 10 s, 60°C for 30 s, and 72°C for 20 s, and melting curve analysis at 95°C for 15 s, 60°C for 60 s, and 97°C for 1 s. Primer pairs are shown in Table 1 . The data were calculated using the formula: experimental group/control group = 2 −ΔΔCT . Table 1 List of primers for RT-qPCR Gene Forward Reverse Sels 5′-AAATCTGACAAAAAGCCTTTGC-3′ 5′-TCCAGGAGCAGGTTCCAC-3′ Col2a1 5′-AACACTGCCAACGTCCAGAT-3′ 5′-CTGCAGCACGGTATAGGTGA-3′ Mmp3 5′-TAGCAGGTTATCCTAAAAGCA-3′ 5′-CCAGCTATTGCTCTTCAAT-3′ Mmp10 5′-CCCAGCTAACTTCCACCTTTC-3′ 5′-AGCAGGATCACATTTGTCTGG-3′ Mmp13 5′-CTTCTTCTTGTTGAGCTGGACTC-3′ 5′-CTGTGGAGGTCACTGTAGACT-3′ Timp2 5′-TCAGAGCCAAAGCAGTGAGC-3′ 5′-GCCGTGTAGATAAACTCGATGTC-3′ Gapdh 5′-GGGCTCATGACCACAGTCCATG-3′ 5′-CCTTGCCCACAGCCTTGGCA-3′ 2.9 Statistical analysis Statistical analysis was conducted using SPSS 19.0. One-way analysis of variance (ANOVA) and independent samples t -test were employed to compare the results. A significance level of P < 0.05 was considered statistically significant. 3 Results 3.1 SelS deficiency disrupts ECM homeostasis in hypertrophic chondrocytes To investigate the relationship between reduced SelS expression and cartilage matrix degradation in patients with KBD, we assessed the mRNA and protein levels of type II collagen (COL II), a major component of the cartilage matrix, as well as matrix metalloproteinases (MMPs) using qRT-PCR and western blotting after knocking down SelS gene in hypertrophic chondrocytes. Compared to the sh-NC group, the mRNA and protein levels of COL Ⅱ were decreased in the sh-Sels group (Fig. 1 a, f and j). The expression levels of Mmp3 , Mmp10 and Mmp13 were increased, while the metallopeptidase inhibitor Timp2 was downregulated in the sh-Sels group (Fig. 1 b ~ e). Notably, the sh-Sels group exhibited a two-fold elevation in MMP19 protein levels compared to the sh-NC group ( P < 0.05) (Fig. 1 h and l). The results demonstrate that SelS silencing disrupts ECM homeostasis in hypertrophic chondrocytes, leading to the degradation of ECM components. 3.2 Remedial effects of SelS overexpression on ECM degradation caused by SelS knockdown To further validate the relationship between SelS and ECM degradation in chondrocytes, we added SelS overexpression plasmids into the cells of sh-NC and sh-Sels groups. The results showed that the mRNA expression of SelS increased approximately 400-fold in sh-NC cells transfected with the SelS-overexpressing plasmid, confirming the effectiveness of the plasmid (Fig. 2 a). The mRNA levels of Col2a1 , Mmp13 , and Mmp19 were measured using RT-qPCR. Compared to the empty vector group (sh-Sels+pCDNA3.1), the mRNA expression of Col2a1 was significantly increased in SelS overexpression group (sh-Sels+pCDNA3.1-SelS), indicating that the decrease of Col2a1 expression caused by SelS knockdown was remedied (Fig. 2 b). Additionally, SelS overexpression partially reversed the elevated expression of Mmp13 and Mmp19 in the sh-Sels group (Fig. 2 c and d). These results suggest that SelS overexpression can partially rescue the ECM degradation caused by SelS knockdown. 3.3 Effects of SelS gene knockout on cartilage matrix degradation in mice Homozygous SelS gene knockout mice were obtained through heterozygous mating. At 10 days of age, the mice were toe-clipped for labeling and subsequently used for DNA extraction and genotype identification. Genotyping was performed via agarose gel electrophoresis after PCR amplification of DNA (Fig. 3 a). Additionally, western blotting of costal cartilage protein extracts confirmed the successful construction of SelS gene knockout mice (Fig. 3 b). Measurements of body length and weight at 7 weeks of age revealed no significant differences between the two groups (Fig. 3 c and d). After the mice were sacrificed, the knee joints were collected for paraffin embedding and sectioning, followed by staining. The results of TB staining and saffron O-fast green staining revealed that the articular cartilage exhibited lighter staining compared to the epiphyseal plate. TB staining revealed that the articular cartilage in Sels −/− group showed decreased staining intensity compared to wild type mice, while the changes in the epiphyseal plate cartilage were not significant. These results suggest a decrease in proteoglycan content of the articular cartilage in Sels −/− mice (Fig. 3 e). Similar results were observed with saffron O-fast green staining (Fig. 3 f). Compared to wild type mice, SelS gene knockout mice displayed lighter red staining of articular cartilage. Both staining results indicate an imbalance in cartilage matrix homeostasis, but they do not clarify whether this imbalance is due to decreased synthesis, increased catabolism, or both. 3.4 The collagen content of articular cartilage was decreased in Sels −/− mice COL II is a major component of the cartilage matrix, forming a fibrous network that contributes to its mechanical properties, providing strength and structural integrity to the cartilage under compression forces. To visualize the collagen fibers, sirius red staining was employed and observed under a polarized light microscope. The intensity of the red color indicates the amount of collagen. It can be observed that the red color in cartilage slices from Sels −/− mice was weaker than that from wild type mice, indicating a reduced collagen content in the samples. It was further supported by the results of both IHC staining and western blotting, which revealed lower levels of COL II in the cartilage of Sels −/− mice compared to the control group (Fig. 4 ). 3.5 SelS gene knockout enhanced the catabolism of cartilage in mice In vitro experiments, SelS knockdown increased the expression of MMPs, but what about in vivo? We detected the expression of MMP3, MMP13 and MMP19 by IHC staining and western blotting. In the articular cartilage of wild type mice, only a few cells presented positive staining for MMP3 and MMP13, while a larger number of cells showed positive staining for MMP19. Compared to wild type mice, MMP3, MMP13 and MMP19 positive staining were significantly increased in the cartilage of Sels −/− mice, especially MMP19, which was more secreted to ECM in the deep layer of articular cartilage (Fig. 5 ). However, the results of western blotting did not indicate a significant difference in the expression of MMP13 and MMP19 in the proteins extracted from the costal cartilage between the two groups, possibly because the samples came from different tissues. 3.6 TGF-β pathway was involved in cartilage matrix degradation caused by SelS silencing RNA sequencing was performed after SelS knockdown in hypertrophic chondrocytes, the data presented in our previous article [ 24 ] . GO enrichment analysis of the differentially expressed genes, revealed that in the cellular component (CC) category, these genes were significantly enriched in extracellular matrix proteins, with a total of 49 differentially expressed genes identified. Protein interaction analysis of the 49 genes was conducted using the STRING database, as illustrated in Fig. 6 a. It suggested that low expression of SelS may activate enzymes such as MMPs and ADAMTS via the TGF-β pathway, leading to the degradation of cartilage matrix. For further verification, ATDC5 cells were treated with TGF-β pathway inhibitor GW788388 for 24h. A CCK-8 assay was used to determine the maximum nonlethal concentration of the inhibitor in ATDC5 cells, which was found to be 10 µM (Fig. 6 b). RT-qPCR results indicated that GW788388 increased Sels expression in sh-NC and sh-Sels cells (Fig. 6 c). There was no significant difference in mRNA expression of Mmp13 between sh-Sels and sh-Sels+GW788388 groups( P = 0.349), while the mRNA expression of Mmp19 was significantly decreased ( P = 0.018) in sh-Sels+GW788388 group. The results indicated that TGF-β pathway inhibitor could not reverse the increase of Mmp13 caused by SelS deficiency, but partially reversed the increase of Mmp19 caused by SelS deficiency (Fig. 6 . d and e). Additionally, GW788388 increased the expression of Col2a1 , and had a remedial effect on the decrease of Col2a1 expression caused by SelS deficiency (Fig. 6 f). 4 Discussion The major components of cartilage ECM include water, collagen and proteoglycan. Collagen constitutes the most abundant structural macromolecule in ECM, accounting for approximately 60% of the dry weight of cartilage, with COL II comprising 90–95% of the total collagen content [26] . In this study, SelS deficiency suppressed COL II synthesis and enhanced MMPs expression through activation of the TGF-β signaling pathway, ultimately resulting in cartilage matrix degradation. These findings suggest that SelS may play a protective role in cartilage degenerative diseases, such as OA, by regulating the activity of the TGF-β pathway. However, selenium-deficient regions often present low selenium content in food and water, leading to insufficient SelS expression in the local population. This accelerates cartilage matrix degradation and, when combined with other pathogenic factors such as T-2 toxin, promotes the onset and progression of KBD. Our previous studies have demonstrated that the positive expression rate of TGF-β1 in the cartilage of KBD patients is significantly higher than that in the control group, and the expression levels of TGF-β receptors I and II are upregulated in the cartilage of both KBD children and rat models, suggesting that the TGF-β signaling pathway may play a critical role in the pathogenesis and progression of KBD [27] . In vitro experiments revealed that GW788388 can partially alleviate the upregulation of MMP-13 induced by T-2 toxin, indicating that MMP-13 expression in T-2 toxin-treated chondrocytes is regulated via the TGF-β signaling pathway [28] . Therefore, modulating the TGF-β signaling pathway represents a promising therapeutic strategy for KBD. The degradation of the cartilage matrix caused by SelS deficiency might be associated with its function in ERAD and oxidative stress regulation. SelS is an important member of the ERAD complex, and its deficiency may affect the ERAD function of chondrocytes. A previous study has shown that ERAD-related genes are upregulated during cartilage formation, and disruption of ERAD function leads to collagen accumulation in the ER, inhibiting its secretion into the extracellular space while enhancing cartilage catabolism. This results in chondrodysplasia in developing embryos and cartilage loss in adult joints [29] . If SelS deficiency induces ERAD dysfunction, it would theoretically trigger ER stress. However, surprisingly, knocking down SelS expression in chondrocytes does not induce ER stress [24] . There may be unknown compensatory or regulatory mechanisms involved in this process. In addition, a recent study demonstrated that the absence of SelS induces oxidative stress in cartilage, while reactive oxygen species (ROS) can activate the TGF-β signaling pathway [30, 31] .Numerous studies have shown that TGF-β regulates the expression of MMPs and components of the extracellular matrix, thereby significantly influencing the cellular microenvironment [28, 32, 33] . For instance, TGF-β induces the expression of MMP-2 and MMP-9 through the p38 MAPK signaling pathway [33] . Additionally, TGF-β1 elevates the levels of MMP-13 and type X collagen via NF-κB activation in chondrocytes [34] . The interaction between SelS and TGF-β warrants further investigation. This study provides a new perspective on the molecular mechanism of cartilage degeneration: SelS is not only a regulator of oxidative stress but also may serve as a key regulatory node in cartilage metabolism through the TGF-β pathway. This discovery offers potential targets for the treatment of OA and KBD. For instance, upregulating SelS expression through drugs or gene editing may inhibit the excessive activation of TGF-β, thereby delaying the process of cartilage degradation. Furthermore, the association between SelS and the TGF-β pathway may have implications for the study of other fibrotic diseases, such as liver fibrosis and pulmonary fibrosis. Declarations Author Contribution All authors contributed to the study conception and design. Material preparation ,data collection and analysis were performed by Hui Wang, Yinan Liu and Meng Zhang. YawenShi optimized the experimental methods. The SelS gene knockout mice were provided by Jian Sun , and the funds were provided by Jinghong Chen, Ying Zhang and Mengying Wang. The first draft of the manuscript was written by Hui Wang , and Chen Chen provided guidance for the writing of this manuscript. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Funding Declaration ： This research was funded by the National Natural Science Foundation of China (No.82473748 and No.82204172), Natural Science Basic Research Program of Shaanxi Province (No. 2023-JC-QN-0902) and the Fundamental Research Funds for the Central Universities, Xi’an Jiaotong University (No. xzy012022109). Institutional Review Board Statement: All procedures performed in studies involving human participants followed the 1964 Helsinki Declaration and its later amendments or com-parable ethical standards, and were approved by the Human and Ethical Committee for Medical Research at Xi’an Jiaotong University, School of Medicine (Protocol NO.3063058). All procedures involving animals complied with the Guidance on the operation of the Animals Act 1986 and followed the ARRIVE guidelines. Ethical approval was granted by the Animal Ethics Committee, Medical School of Xi’an Jiaotong University (Protocol NO.0074). References Ghelichkhani, F., F.A. Gonzalez, M.A. Kapitonova, et al., Selenoprotein S: A versatile disordered protein. Archives of Biochemistry and Biophysics, 2022. 731 . Ken Walder, Lakshmi Kantham, Janine S. McMillan, et al., Tanis: A Link Between Type 2 Diabetes and Inflammation? DIABETES, 2002. 51 (6): p. 1859-1866. Lee, J.H., K.J. Park, J.K. Jang, et al., Selenoprotein S-dependent Selenoprotein K Binding to p97(VCP) Protein Is Essential for Endoplasmic Reticulum-associated Degradation. Journal of Biological Chemistry, 2015. 290 (50): p. 29941-29952. Ye, Y., W. Bian, F. Fu, et al., Selenoprotein S inhibits inflammation-induced vascular smooth muscle cell calcification. JBIC Journal of Biological Inorganic Chemistry, 2018. 23 (5): p. 739-751. Zhuang, C., G. Liu, H.W. Barkema, et al., Selenomethionine Suppressed TLR4/NF-κB Pathway by Activating Selenoprotein S to Alleviate ESBL Escherichia coli-Induced Inflammation in Bovine Mammary Epithelial Cells and Macrophages. Frontiers in Microbiology, 2020. 11 . Li, F., A. Mao, X. Fu, et al., Correlation between SEPS1 gene polymorphism and type 2 diabetes mellitus: A preliminary study. Journal of Clinical Laboratory Analysis, 2019. 33 (8). Li, X.-X., H.-J. Guan, J.-P. Liu, et al., Association of selenoprotein S gene polymorphism with ischemic stroke in a Chinese case–control study. Blood Coagulation & Fibrinolysis, 2015. 26 (2): p. 131-135. Rueli, R.H.L.H., D.J. Torres, A.S.T. Dewing, et al., Selenoprotein S Reduces Endoplasmic Reticulum Stress-Induced Phosphorylation of Tau: Potential Role in Selenate Mitigation of Tau Pathology. Journal of Alzheimer's Disease, 2016. 55 (2): p. 749-762. Sutherland, A., D.-H. Kim, C. Relton, et al., Polymorphisms in the selenoprotein S and 15-kDa selenoprotein genes are associated with altered susceptibility to colorectal cancer. Genes & Nutrition, 2010. 5 (3): p. 215-223. Deng, X. and Y. Tan, A national cross-sectional analysis of selenium intake and risk of osteoarthritis: NHANES 2003–2016. Frontiers in Public Health, 2023. 10 . Wang, Y., D. Xie, J. Li, et al., Association between dietary selenium intake and the prevalence of osteoporosis: a cross-sectional study. BMC Musculoskeletal Disorders, 2019. 20 (1). Zhang, B., L. Yang, W. Wang, et al., Environmental selenium in the Kaschin–Beck disease area, Tibetan Plateau, China. Environmental Geochemistry and Health, 2010. 33 (5): p. 495-501. Yu, N., F. Han, X. Lin, et al., The Association Between Serum Selenium Levels with Rheumatoid Arthritis. Biological Trace Element Research, 2015. 172 (1): p. 46-52. Ren, F.L., X. Guo, R.J. Zhang, et al., Effects of selenium and iodine deficiency on bone, cartilage growth plate and chondrocyte differentiation in two generations of rats. Osteoarthritis and Cartilage, 2007. 15 (10): p. 1171-1177. Cao, J.J., B.R. Gregoire, and H. Zeng, Selenium Deficiency Decreases Antioxidative Capacity and Is Detrimental to Bone Microarchitecture in Mice. The Journal of Nutrition, 2012. 142 (8): p. 1526-1531. Min, Z., W. Zhao, N. Zhong, et al., Abnormality of epiphyseal plate induced by selenium deficiency diet in two generation DA rats. Apmis, 2015. 123 (8): p. 697-705. Horwitz, M.S., C.M. Downey, C.R. Horton, et al., Osteo-Chondroprogenitor–Specific Deletion of the Selenocysteine tRNA Gene, Trsp, Leads to Chondronecrosis and Abnormal Skeletal Development: A Putative Model for Kashin-Beck Disease. PLoS Genetics, 2009. 5 (8). Hamajima, T., Y. Mushimoto, H. Kobayashi, et al., Novel compound heterozygous mutations in the SBP2 gene: characteristic clinical manifestations and the implications of GH and triiodothyronine in longitudinal bone growth and maturation. European Journal of Endocrinology, 2012. 166 (4): p. 757-764. Zhang, Z., J. Zhang, and J. Xiao, Selenoproteins and selenium status in bone physiology and pathology. Biochimica et Biophysica Acta (BBA) - General Subjects, 2014. 1840 (11): p. 3246-3256. Chi, Q., Y. Luan, Y. Zhang, et al., The regulatory effects of miR-138-5p on selenium deficiency-induced chondrocyte apoptosis are mediated by targeting SelM. Metallomics, 2019. 11 (4): p. 845-857. Gong, Y., Y. Wu, Y. Liu, et al., Detection of selenoprotein transcriptome in chondrocytes of patients with Kashin–Beck disease. Frontiers in Cell and Developmental Biology, 2023. 11 . Yan, J., Y. Guo, Y. Fei, et al., GPx1 knockdown suppresses chondrogenic differentiation of ATDC5 cells through induction of reductive stress. Acta Biochimica et Biophysica Sinica, 2017. 49 (2): p. 110-118. Yan, J., Y. Fei, Y. Han, et al., Selenoprotein O deficiencies suppress chondrogenic differentiation of ATDC5 cells. Cell Biology International, 2016. 40 (10): p. 1033-1040. Wang, H., Z. Li, Y. Liu, et al., Effects of Selenoprotein S Knockdown on Endoplasmic Reticulum Stress in ATDC5 Cells and Gene Expression Profiles in Hypertrophic Chondrocytes. Biological Trace Element Research, 2022. 201 (4): p. 1965-1976. Chen, J., M. Luo, W. Wang, et al., Altered proteolytic activity and expression of MMPs and aggrecanases and their inhibitors in Kashin–Beck disease. Journal of Orthopaedic Research, 2014. 33 (1): p. 47-55. Sophia Fox, A.J., A. Bedi, and S.A. Rodeo, The Basic Science of Articular Cartilage: Structure, Composition, and Function. Sports Health: A Multidisciplinary Approach, 2009. 1 (6): p. 461-468. Zhang, Y., Y. Mu, Y. He, et al., Upregulated expression of transforming growth factor-β receptor I/II in an endemic Osteoarthropathy in China. BMC Musculoskeletal Disorders, 2021. 22 (1). Zhang, Y., Z. Li, Y. He, et al., T-2 toxin induces articular cartilage damage by increasing the expression of MMP-13 via the TGF-β receptor pathway. Human & Experimental Toxicology, 2022. 41 . Hyo Jung Sim, C.C., Ha Eun Kim, Ju Yeon Hong, et al., Augmented ERAD (ER-associated degradation) activity in chondrocytes is necessary for cartilage development and maintenance. Science Advances, 2022. 8 (3): p. eabl4222. Cui, Y., Y. Liao, Y. Chen, et al., Low expression of selenoprotein S induces oxidative damage in cartilages. Journal of Trace Elements in Medicine and Biology, 2024. 85 . Zhang, H., Z.-W. Wang, H.-B. Wu, et al., Transforming growth factor-β1 induces matrix metalloproteinase-9 expression in rat vascular smooth muscle cells via ROS-dependent ERK–NF-κB pathways. Molecular and Cellular Biochemistry, 2012. Wolfgang Wick, M.P.M.W., Glioma cell invasion: regulation of metalloproteinase activity by TGF-β. Journal of Neuro-Oncology, 2001. 53 : p. 177-185. Kim，, E.-S., Mi-Sung Kim, and A. Moon, TGF-beta-induced upregulation of MMP-2 and MMP-9 depends on p38 MAPK, but not ERK signaling in MCF10A human breast epithelial cells. International Journal of Oncology, 2004. 25 (5): p. 1375-1382. Park, J.-Y., H.C. Bae, S.H. Pyo, et al., TGFβ1-Induced Transglutaminase-2 Triggers Catabolic Response in Osteoarthritic Chondrocytes by Modulating MMP-13. Tissue Engineering and Regenerative Medicine, 2021. 18 (5): p. 831-840. Additional Declarations No competing interests reported. Supplementary Files westernblotoriginalimagePDF.zip FigureS1.jpg Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 18 May, 2026 Reviewers agreed at journal 18 May, 2026 Reviewers agreed at journal 16 May, 2026 Reviewers agreed at journal 04 May, 2026 Reviewers invited by journal 18 Apr, 2026 Editor assigned by journal 17 Apr, 2026 Submission checks completed at journal 17 Apr, 2026 First submitted to journal 30 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9267022","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":626743542,"identity":"e6b7b589-d3d1-449c-86d3-e5932592a862","order_by":0,"name":"Hui Wang","email":"","orcid":"","institution":"Xi'an Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Wang","suffix":""},{"id":626743545,"identity":"46ed984a-8c46-4604-9170-f09e8b7e279b","order_by":1,"name":"Yinan Liu","email":"","orcid":"","institution":"Xi'an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Yinan","middleName":"","lastName":"Liu","suffix":""},{"id":626743549,"identity":"a502e3cb-12eb-471c-9c2b-cde7534dc285","order_by":2,"name":"Meng Zhang","email":"","orcid":"","institution":"Xi'an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Meng","middleName":"","lastName":"Zhang","suffix":""},{"id":626743557,"identity":"16eb8452-ee96-4f81-b337-213123ad34cc","order_by":3,"name":"Mengying Wang","email":"","orcid":"","institution":"Xi'an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Mengying","middleName":"","lastName":"Wang","suffix":""},{"id":626743563,"identity":"bb439f7a-a785-4c58-a44d-05feeaea6893","order_by":4,"name":"Yawen Shi","email":"","orcid":"","institution":"Xi'an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Yawen","middleName":"","lastName":"Shi","suffix":""},{"id":626743565,"identity":"24b98982-942a-4f14-b70d-f9c22f783c1e","order_by":5,"name":"Jian Sun","email":"","orcid":"","institution":"Xi'an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Jian","middleName":"","lastName":"Sun","suffix":""},{"id":626743566,"identity":"ac59b88c-a242-465b-b9a5-58dedd4f9f51","order_by":6,"name":"Chen Chen","email":"","orcid":"","institution":"University of Queensland","correspondingAuthor":false,"prefix":"","firstName":"Chen","middleName":"","lastName":"Chen","suffix":""},{"id":626743573,"identity":"79e0468a-8dc8-4aa8-8cfe-dc7d45081958","order_by":7,"name":"Ying Zhang","email":"","orcid":"","institution":"Xi'an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Zhang","suffix":""},{"id":626743575,"identity":"34ae89aa-7735-4918-a73a-5ac5c35ca73d","order_by":8,"name":"Jinghong Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYBACxmYGhgNAmpmfvbHx4QdStLBL9hxuNpYgxTZ+gxnpbQI8xChlbucxPPBzR620geTDNgYJBjs53QaCDmNLONh75rixuXRi24MChmRjswMEtTAfOMDbdizZcnZiu4EEw4HEbYS1MDYc/Nt2rH7DzYNtEjzEaWE+cJi3rYbZ4AYj0VrYEg7Lth1gluxJBAayARF+Mew/Y/zxbVsdMCqPP3z4ocJOjrCWBjB1GMo1IKAcBOQhVB0RSkfBKBgFo2DEAgASlUUEwnCwowAAAABJRU5ErkJggg==","orcid":"","institution":"Xi'an Jiaotong University","correspondingAuthor":true,"prefix":"","firstName":"Jinghong","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2026-03-30 12:41:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9267022/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9267022/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107886391,"identity":"d6983da4-9463-4d1e-ae88-58ba7819f565","added_by":"auto","created_at":"2026-04-27 09:28:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":255545,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of SelS knockdown on matrix homeostasis in hypertrophic chondrocytes. a-e: mRNA levels of \u003cem\u003eCol2a1\u003c/em\u003e, \u003cem\u003eMmp3\u003c/em\u003e, \u003cem\u003eMmp10\u003c/em\u003e, \u003cem\u003eMmp13\u003c/em\u003e and \u003cem\u003eTimp2\u003c/em\u003e. f-g: Representative images of western blotting results of SELS, type II collagen, MMP13 and MMP19. i-l: Statistical analysis of western blotting results. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026gt;0.05, n=3.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9267022/v1/be0bc52d35465762c637502d.png"},{"id":108803450,"identity":"ebd694e5-864d-4c6e-b6a6-f7cb728d6aa4","added_by":"auto","created_at":"2026-05-08 14:54:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":150960,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of SelS overexpression on ECM degradation caused by SelS knockdown in chondrocytes. a-d: The mRNA levels of \u003cem\u003eSels, Col2a1\u003c/em\u003e, \u003cem\u003eMmp13\u003c/em\u003e, and \u003cem\u003eMmp19 \u003c/em\u003ein sh-NC and sh-Sels cells after adding SelS overexpression plasmid for 48h, respectively. n=3, *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9267022/v1/6588786d40286c7beee1c445.png"},{"id":108006447,"identity":"fe4015c3-0e3d-478f-8428-d447f7d5067d","added_by":"auto","created_at":"2026-04-28 12:55:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1044689,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of SelS knockout mice and staining results of knee joint. a: Agarose gel electrophoresis of PCR amplified products for genotype identification. b: Western blot analysis of SELS levels of in proteins extracted from mice costal cartilage, n=5. c: Statistical analysis of mice body length. d: Statistical analysis of mice body weight. e and f: Pictures of TB staining and saffron O-fast green staining.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9267022/v1/8c5ddc14f88466b464e60fbe.png"},{"id":107886392,"identity":"2d4a8bff-0eb2-480a-acb5-33219268448e","added_by":"auto","created_at":"2026-04-27 09:28:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":714498,"visible":true,"origin":"","legend":"\u003cp\u003eResults of collagen content detection in mice cartilages. Left: Pictures of sirius red staining and IHC staining of COL II. Right: The results of western blotting and statistical analysis of COL II levels. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, n=3.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9267022/v1/2065ee2fccc93ffda71d9142.png"},{"id":108007101,"identity":"87537364-dba6-4fcb-8165-eb98fa446a13","added_by":"auto","created_at":"2026-04-28 12:58:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":614651,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression levels of MMP3, MMP13 and MMP19 were evaluated using IHC staining and western blotting. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026gt;0.05, n=3.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9267022/v1/e44e3f307587c69c9e22c0c4.png"},{"id":108490651,"identity":"f178ad5e-3b11-4883-bfe6-8feb68d9f5e8","added_by":"auto","created_at":"2026-05-05 09:46:00","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":340707,"visible":true,"origin":"","legend":"\u003cp\u003eTGF-β pathway was involved in the degradation of cartilage matrix caused by SelS silencing. (a) Diagram of protein interaction analysis of differentially expressed genes related to extracellular matrix homeostasis. (b) Effect of different concentrations of a TGF-β inhibitor (GW788388) on ATDC5 cells viability, n=4. (c) Effect of TGF-β inhibitor (GW788388) on \u003cem\u003eSels\u003c/em\u003e expression in sh-NC and sh-Sels cells. (d~f) Effect of a TGF-β inhibitor (GW788388) on \u003cem\u003eMmp13\u003c/em\u003e, \u003cem\u003eMmp19\u003c/em\u003e, and \u003cem\u003eCol2a1\u003c/em\u003e expression in sh-NC and sh-Sels cells. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026gt;0.05, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, n=3.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9267022/v1/7aed88ffef8f7dc19b55b0df.png"},{"id":108976790,"identity":"1c41515a-665e-4193-8f20-f22acbd5b5f1","added_by":"auto","created_at":"2026-05-11 11:28:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3496636,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9267022/v1/13500260-b28c-41ab-990c-64fd983aba7e.pdf"},{"id":107886390,"identity":"4b390e69-6d9a-457a-922f-b2c9e7310483","added_by":"auto","created_at":"2026-04-27 09:28:41","extension":"zip","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":493101,"visible":true,"origin":"","legend":"","description":"","filename":"westernblotoriginalimagePDF.zip","url":"https://assets-eu.researchsquare.com/files/rs-9267022/v1/97cb2c728ea0617555cefe5a.zip"},{"id":107886394,"identity":"88d5a174-0f13-4098-95a2-db9ad3c8e00b","added_by":"auto","created_at":"2026-04-27 09:28:41","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":434456,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9267022/v1/de907633797d78335753949c.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Selenoprotein S deficiency induces matrix degradation via TGF-β pathway leading to cartilage damage","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eSelenoprotein S (SelS), also known as SEPS1 or VIMP, is a member of the selenoprotein family. The SelS gene is located on chromosome 15 in humans. SelS contains a selenocysteine (Sec) residue at position 188 of its amino acid sequence, which is encoded by the UGA codon, typically a stop signal in translation. However, selenoproteins, including SelS, possess specialized translational machinery that recognizes the UGA codon as a Sec incorporation signal. The presence of Sec is crucial for SelS function, enabling it to participate in redox reactions and contributing to unique biological activities\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Initially, SelS was identified as a glucose-regulating protein because of its inverse correlation with blood glucose and insulin levels\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Subsequent studies demonstrated that SelS, as a component of the ER-associated protein degradation (ERAD) complex, facilitates the transfer of misfolded proteins from the ER to the cytosol for degradation\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Moreover, SelS has been shown to regulate inflammation by inhibiting NF-κB activity and reducing the production of proinflammatory cytokines\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Given its roles in glucose regulation, oxidative stress management, ERAD, and inflammation modulation, SelS has garnered significant attention in research related to diabetes, Alzheimer's disease, cardiovascular disease, and cancer\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. The expression of SelS is regulated by various factors, including cellular stress, inflammation, and selenium intake, and exhibits tissue-specific patterns, with higher levels observed in secretory tissues such as the liver, pancreas, colon, and small intestine\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn recent years, the role of selenium in bone and cartilage diseases has received increasing attention. Selenium deficiency has been associated with the onset and progression of diseases such as Kashin-Beck disease (KBD), osteoarthritis (OA), rheumatoid arthritis (RA), and osteoporosis (OP)\u003csup\u003e[\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Feeding mice a low-selenium diet induced the formation of fibrocartilage on the joint surface, ultimately resulting in degenerative changes in articular cartilage\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Selenium deficiency diet decreased femoral trabecular bone volume/total volume and trabecular number while increasing trabecular separation in mice, suggesting that selenium deficiency enhances bone resorption and damages bone microstructure\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. In a rat model, a two-generation selenium-deficient diet caused growth retardation, epiphyseal plate lesions, and reduced expression of GPx1 and COL II in chondrocytes, indicating diminished anabolic and antioxidant capacity in chondrocytes\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSelenium exerts its biological functions in the body primarily through selenoproteins. After absorption and transformation, selenium is transported via the liver to various organs and tissues for the synthesis of selenoproteins. The most direct consequence of low selenium diet is insufficient expression of selenoproteins. Extensive researches have demonstrated the critical role of selenoproteins in bone and cartilage development. Selenoprotein synthesis depends on the Trsp gene, which encodes selenocysteine tRNA\u003csup\u003e[Ser]Sec\u003c/sup\u003e, which is essential for incorporating Sec residues into selenoproteins. Mice with osteo-chondroprogenitor-specific deletion of Trsp exhibited growth retardation, abnormal epiphyseal growth plates, delayed skeletal ossification, and significant chondronecrosis in articular, auricular, and tracheal cartilages\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. The relationship between selenoproteins and bone development was further supported by observations of growth retardation and delayed bone maturation in patients with SBP2 deficiency, a key trans-acting factor for Sec insertion into selenoproteins\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Knockout of SelP, which is responsible for selenium transport, leads to bone loss\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Selenium deficiency induces chondrocyte apoptosis associated with SelM\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Transcriptomic analysis of selenoproteins in chondrocytes from patients with KBD indicates that abnormal expression of GPX and DIO family selenoproteins may contribute to the pathogenesis of KBD\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Knockdown of GPx1 or SelO has been shown to reduce the expression of Sox9, Col II, and aggrecan, inhibit glycosaminoglycans (GAGs) accumulation and chondrocyte proliferation, and induce cell apoptosis\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e.The effects of abnormal SelS expression on bone and cartilage formation and development remain poorly understood.\u003c/p\u003e \u003cp\u003eKBD, an endemic osteoarthritis, is prevalent in low-selenium areas. KBD patients have lower levels of blood selenium, hair selenium, and urine selenium compared to people from non-KBD areas. Therefore, selenium deficiency is widely recognized as a major contributing factor to KBD. Decreased expression of SelS has been observed in the articular cartilage of KBD patients. Initially, it was hypothesized that reduced SelS expression would induce ER stress and subsequent chondrocyte death. However, experimental evidence suggests that SelS knockdown does not trigger ER stress in chondrocytes\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. To further elucidate the impact of SelS knockdown on hypertrophic chondrocytes, which are the primary damaged cells in KBD, RNA sequencing was performed, and differentially expressed genes were identified. The results demonstrated that SelS knockdown significantly upregulated the expression of matrix metalloproteinases (\u003cem\u003eMmp3\u003c/em\u003e, \u003cem\u003eMmp10\u003c/em\u003e, \u003cem\u003eMmp13\u003c/em\u003e, and \u003cem\u003eMmp19\u003c/em\u003e) while downregulating the expression of type II collagen (\u003cem\u003eCol2a1\u003c/em\u003e) in hypertrophic chondrocytes. Degradation of extracellular matrix (ECM) in cartilage, characterized by elevated MMPs expression and reduced type II collagen (COL II) and proteoglycan levels, represents a critical pathological feature of KBD\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Therefore, we hypothesized that decreased SelS expression might contribute to cartilage damage via ECM degradation. Thus, the objective of this study was to verify the relationship between SelS and cartilage matrix degradation and investigate its underlying mechanism.\u003c/p\u003e"},{"header":"2 Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Cell culture and treatment\u003c/h2\u003e \u003cp\u003eThe origin of ATDC5 cells and construction process of SelS knockdown cells were described in our previous paper\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. The negative control group and the SelS gene knockdown group were expressed as sh-NC and sh-Sels, respectively. The cells were cultured in DMEM/F-12 medium (HyClone, Logan, UT, USA) containing 5% fetal bovine serum (FBS) (Gibco, Gaithersburg, PA, USA) at 37℃ and 5% CO\u003csub\u003e2\u003c/sub\u003e. The lentivirus used for SelS knockdown was purchased from Shanghai Obio Technology Company (Shanghai, China) and the overexpression plasmid was purchased from Hanbio Biotechnology (Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Origin and breeding of mice\u003c/h2\u003e \u003cp\u003e\u003cem\u003eC57BL/6 Sels\u003c/em\u003e\u003csup\u003e+/\u0026minus;\u003c/sup\u003emice were purchased from GemPharmatech LLC(Nanjing, China). All the mice were housed in the pathogen-free Animal Center of Xi\u0026rsquo;an jiaotong University under controlled temperature conditions (23\u0026deg;C) with ad libitum access to food and water. Wild type and homozygous mice were generated by crossing heterozygous mice. Neonatal mice were toe-clipped and genotyped on the 10th day after birth. The mice were sacrificed at 7 weeks of age, and the knee joint and costal cartilage were collected for subsequent experiments. All animal experiments were approved by the Animal Experimental Ethics Committee of Xi\u0026rsquo;an Jiaotong University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 DNA extraction and genotype identification\u003c/h2\u003e \u003cp\u003eGenomic DNA was extracted using the TIANamp Genomic DNA kit (DP304, TIANGEN BIOTECH, Beijing, China). After the DNA was extracted, PCR amplification was performed. The genotyping strategy and primer information are shown in Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The amplified DNA was separated by 1% agarose gel electrophoresis. In addition to DNA identification, RIPA lysate was used to extract costal cartilage proteins, and the protein level of SelS was detected by western blotting. The genotype identification and western blotting results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Hematoxylin \u0026amp; eosin (H\u0026amp;E) staining and toluidine blue (TB) staining\u003c/h2\u003e \u003cp\u003eThe knee joints of the mice were embedded in paraffin wax and cut into 5\u0026micro;m sections. After being dewaxed in xylene and hydrated in gradient ethanol, the samples were stained with hematoxylin for 5 minutes to dye the nuclei. Next, the sections were treated with hydrochloric ethanol for a few seconds to remove excess dye and further treated with ammonia liquor. For eosin staining, the sections were stained with eosin for 1 minute. For TB staining, the sections were stained with 0.1% TB solution for 2 minutes. Finally, the sections were dehydrated in gradient ethanol, cleared in xylene, and sealed with neutral resin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Saffron O-fast green Staining and Sirius red staining\u003c/h2\u003e \u003cp\u003eFor saffron O-fast green staining, the procedures of nuclear staining and neutral resin sealing were the same as H\u0026amp;E staining. In the special steps, the sections were first immersed in fast green dye for 10 minutes, washed in pure water three times, stained with saffron O dye for 1 minute, washed in pure water three times, soaked in 2% acetic acid for several seconds to remove excess dye, and stained in fast green for another two minutes. For sirius red staining, the sections were soaked in picrate-sirius red solution for 1 hour before the nuclei were stained. Notably, saffron O stained and sirius red stained samples were not dehydrated in low concentration ethanol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Immunohistochemistry (IHC) staining\u003c/h2\u003e \u003cp\u003eAfter being dewaxed in xylene and hydrated in gradient alcohol, the sections were treated with 0.125% trypsin (Xi'an GuoAn Biological Technology Co.) for antigen retrieval at 37\u0026deg;C for 30 minutes. The subsequent steps were performed according to the instructions of the IHC kit (Beijing Zhong Shan Gold Bridge Biological Technology Co., SP9001). The samples were incubated with primary antibodies against MMP3 (1:50, 66338-1-Ig, Proteintech), MMP13 (1:100, 18165-1-AP, Proteintech), MMP19 (1:100, 14244-1-AP, Proteintech), and COL Ⅱ (1:80, A1560, ABclonal) at 4\u0026deg;C overnight. DAB solution (Beijing Zhong Shan Gold Bridge Biological Technology Co., ZLI-9018) was used for color reactions, followed by hematoxylin staining, dehydration, and sealing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Total protein extraction and western blotting\u003c/h2\u003e \u003cp\u003eTotal protein was extracted using RIPA lysis buffer (Beyotime, P0013B). The protein concentration was determined using a BCA protein assay kit (Shanghai Epizyme Biomedical Technology Co., Ltd, ZJ101). Equal masses of protein were loaded into each well for SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins with different molecular weights were separated and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, ISEQ00010). The PVDF membranes were blocked with 5% skim milk for 2 hours and incubated with primary antibodies against SELS (1:1000, 15591-1-AP, Proteintech), COL Ⅱ (1:500, A1560, ABclonal), MMP13 (1:500, 18165-1-AP, Proteintech), MMP19 (1:1000, 14244-1-AP, Proteintech), and GAPDH (1:10000, 60004-1-Ig, Proteintech) at 4\u0026deg;C overnight. The next day, after incubation with goat anti-mouse IgG/HRP (1:20000, CWBio, China) or goat anti-rabbit IgG/HRP (1:10000, Jackson, USA), the PVDF membranes were treated with an enhanced chemiluminescence kit (Millipore, Billerica, MA) for chemiluminescence, and the protein bands were analyzed using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 RNA extraction and real-time PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated according to the TRIzol protocol and reverse-transcribed to cDNA via a RevertAid First Stand cDNA Synthesis Kit (K1622, Thermo Fisher Scientific). Relative gene expression was quantified using an iQ5 Cycler (Bio-Rad, Munich, Germany) under the following conditions: initial denaturation at 95\u0026deg;C for 5 min; 40 cycles of 95\u0026deg;C for 10 s, 60\u0026deg;C for 30 s, and 72\u0026deg;C for 20 s, and melting curve analysis at 95\u0026deg;C for 15 s, 60\u0026deg;C for 60 s, and 97\u0026deg;C for 1 s. Primer pairs are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The data were calculated using the formula: experimental group/control group\u0026thinsp;=\u0026thinsp;2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eList of primers for RT-qPCR\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSels\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-AAATCTGACAAAAAGCCTTTGC-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026prime;-TCCAGGAGCAGGTTCCAC-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCol2a1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-AACACTGCCAACGTCCAGAT-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026prime;-CTGCAGCACGGTATAGGTGA-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMmp3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-TAGCAGGTTATCCTAAAAGCA-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026prime;-CCAGCTATTGCTCTTCAAT-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMmp10\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-CCCAGCTAACTTCCACCTTTC-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026prime;-AGCAGGATCACATTTGTCTGG-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMmp13\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-CTTCTTCTTGTTGAGCTGGACTC-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026prime;-CTGTGGAGGTCACTGTAGACT-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTimp2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-TCAGAGCCAAAGCAGTGAGC-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026prime;-GCCGTGTAGATAAACTCGATGTC-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eGapdh\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-GGGCTCATGACCACAGTCCATG-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026prime;-CCTTGCCCACAGCCTTGGCA-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was conducted using SPSS 19.0. One-way analysis of variance (ANOVA) and independent samples \u003cem\u003et\u003c/em\u003e-test were employed to compare the results. A significance level of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1 SelS deficiency disrupts ECM homeostasis in hypertrophic chondrocytes\u003c/h2\u003e \u003cp\u003eTo investigate the relationship between reduced SelS expression and cartilage matrix degradation in patients with KBD, we assessed the mRNA and protein levels of type II collagen (COL II), a major component of the cartilage matrix, as well as matrix metalloproteinases (MMPs) using qRT-PCR and western blotting after knocking down SelS gene in hypertrophic chondrocytes. Compared to the sh-NC group, the mRNA and protein levels of COL Ⅱ were decreased in the sh-Sels group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, f and j). The expression levels of \u003cem\u003eMmp3\u003c/em\u003e, \u003cem\u003eMmp10\u003c/em\u003e and \u003cem\u003eMmp13\u003c/em\u003e were increased, while the metallopeptidase inhibitor \u003cem\u003eTimp2\u003c/em\u003e was downregulated in the sh-Sels group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb\u0026thinsp;~\u0026thinsp;e). Notably, the sh-Sels group exhibited a two-fold elevation in MMP19 protein levels compared to the sh-NC group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh and l). The results demonstrate that SelS silencing disrupts ECM homeostasis in hypertrophic chondrocytes, leading to the degradation of ECM components.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Remedial effects of SelS overexpression on ECM degradation caused by SelS knockdown\u003c/h2\u003e \u003cp\u003eTo further validate the relationship between SelS and ECM degradation in chondrocytes, we added SelS overexpression plasmids into the cells of sh-NC and sh-Sels groups. The results showed that the mRNA expression of SelS increased approximately 400-fold in sh-NC cells transfected with the SelS-overexpressing plasmid, confirming the effectiveness of the plasmid (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The mRNA levels of \u003cem\u003eCol2a1\u003c/em\u003e, \u003cem\u003eMmp13\u003c/em\u003e, and \u003cem\u003eMmp19\u003c/em\u003e were measured using RT-qPCR. Compared to the empty vector group (sh-Sels+pCDNA3.1), the mRNA expression of \u003cem\u003eCol2a1\u003c/em\u003e was significantly increased in SelS overexpression group (sh-Sels+pCDNA3.1-SelS), indicating that the decrease of \u003cem\u003eCol2a1\u003c/em\u003e expression caused by SelS knockdown was remedied (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Additionally, SelS overexpression partially reversed the elevated expression of \u003cem\u003eMmp13\u003c/em\u003e and \u003cem\u003eMmp19\u003c/em\u003e in the sh-Sels group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec and d). These results suggest that SelS overexpression can partially rescue the ECM degradation caused by SelS knockdown.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Effects of SelS gene knockout on cartilage matrix degradation in mice\u003c/h2\u003e \u003cp\u003eHomozygous SelS gene knockout mice were obtained through heterozygous mating. At 10 days of age, the mice were toe-clipped for labeling and subsequently used for DNA extraction and genotype identification. Genotyping was performed via agarose gel electrophoresis after PCR amplification of DNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Additionally, western blotting of costal cartilage protein extracts confirmed the successful construction of SelS gene knockout mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Measurements of body length and weight at 7 weeks of age revealed no significant differences between the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec and d).\u003c/p\u003e \u003cp\u003e After the mice were sacrificed, the knee joints were collected for paraffin embedding and sectioning, followed by staining. The results of TB staining and saffron O-fast green staining revealed that the articular cartilage exhibited lighter staining compared to the epiphyseal plate. TB staining revealed that the articular cartilage in \u003cem\u003eSels\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e group showed decreased staining intensity compared to wild type mice, while the changes in the epiphyseal plate cartilage were not significant. These results suggest a decrease in proteoglycan content of the articular cartilage in \u003cem\u003eSels\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Similar results were observed with saffron O-fast green staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). Compared to wild type mice, SelS gene knockout mice displayed lighter red staining of articular cartilage. Both staining results indicate an imbalance in cartilage matrix homeostasis, but they do not clarify whether this imbalance is due to decreased synthesis, increased catabolism, or both.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4 The collagen content of articular cartilage was decreased in \u003cem\u003eSels\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice\u003c/h2\u003e \u003cp\u003eCOL II is a major component of the cartilage matrix, forming a fibrous network that contributes to its mechanical properties, providing strength and structural integrity to the cartilage under compression forces. To visualize the collagen fibers, sirius red staining was employed and observed under a polarized light microscope. The intensity of the red color indicates the amount of collagen. It can be observed that the red color in cartilage slices from \u003cem\u003eSels\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice was weaker than that from wild type mice, indicating a reduced collagen content in the samples. It was further supported by the results of both IHC staining and western blotting, which revealed lower levels of COL II in the cartilage of \u003cem\u003eSels\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.5 SelS gene knockout enhanced the catabolism of cartilage in mice\u003c/h2\u003e \u003cp\u003eIn vitro experiments, SelS knockdown increased the expression of MMPs, but what about in vivo? We detected the expression of MMP3, MMP13 and MMP19 by IHC staining and western blotting. In the articular cartilage of wild type mice, only a few cells presented positive staining for MMP3 and MMP13, while a larger number of cells showed positive staining for MMP19. Compared to wild type mice, MMP3, MMP13 and MMP19 positive staining were significantly increased in the cartilage of \u003cem\u003eSels\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, especially MMP19, which was more secreted to ECM in the deep layer of articular cartilage (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). However, the results of western blotting did not indicate a significant difference in the expression of MMP13 and MMP19 in the proteins extracted from the costal cartilage between the two groups, possibly because the samples came from different tissues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.6 TGF-β pathway was involved in cartilage matrix degradation caused by SelS silencing\u003c/h2\u003e \u003cp\u003eRNA sequencing was performed after SelS knockdown in hypertrophic chondrocytes, the data presented in our previous article\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. GO enrichment analysis of the differentially expressed genes, revealed that in the cellular component (CC) category, these genes were significantly enriched in extracellular matrix proteins, with a total of 49 differentially expressed genes identified. Protein interaction analysis of the 49 genes was conducted using the STRING database, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea. It suggested that low expression of SelS may activate enzymes such as MMPs and ADAMTS via the TGF-β pathway, leading to the degradation of cartilage matrix. For further verification, ATDC5 cells were treated with TGF-β pathway inhibitor GW788388 for 24h. A CCK-8 assay was used to determine the maximum nonlethal concentration of the inhibitor in ATDC5 cells, which was found to be 10 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). RT-qPCR results indicated that GW788388 increased \u003cem\u003eSels\u003c/em\u003e expression in sh-NC and sh-Sels cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). There was no significant difference in mRNA expression of \u003cem\u003eMmp13\u003c/em\u003e between sh-Sels and sh-Sels+GW788388 groups(\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.349), while the mRNA expression of \u003cem\u003eMmp19\u003c/em\u003e was significantly decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.018) in sh-Sels+GW788388 group. The results indicated that TGF-β pathway inhibitor could not reverse the increase of \u003cem\u003eMmp13\u003c/em\u003e caused by SelS deficiency, but partially reversed the increase of \u003cem\u003eMmp19\u003c/em\u003e caused by SelS deficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. d and e). Additionally, GW788388 increased the expression of \u003cem\u003eCol2a1\u003c/em\u003e, and had a remedial effect on the decrease of \u003cem\u003eCol2a1\u003c/em\u003e expression caused by SelS deficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eThe major components of cartilage ECM include water, collagen and proteoglycan. Collagen constitutes the most abundant structural macromolecule in ECM, accounting for approximately 60% of the dry weight of cartilage, with COL II comprising 90\u0026ndash;95% of the total collagen content\u003csup\u003e[26]\u003c/sup\u003e. In this study, SelS deficiency suppressed COL II synthesis and enhanced MMPs expression through activation of the TGF-\u0026beta; signaling pathway,\u0026nbsp;ultimately resulting in cartilage matrix degradation. These findings suggest that SelS may play a protective role in cartilage degenerative diseases, such as OA, by regulating the activity of the TGF-\u0026beta; pathway.\u0026nbsp;However,\u0026nbsp;selenium-deficient regions often present low selenium content in food and water, leading to insufficient SelS expression in the local population. This accelerates cartilage matrix degradation and, when combined with other pathogenic factors such as T-2 toxin, promotes the onset and progression of KBD. Our previous studies have demonstrated that the positive expression rate of TGF-\u0026beta;1 in the cartilage of KBD patients is significantly higher than that in the control group, and the expression levels of TGF-\u0026beta; receptors I and II are upregulated in the cartilage of both KBD children and rat models, suggesting that the TGF-\u0026beta; signaling pathway may play a critical role in the pathogenesis and progression of KBD\u003csup\u003e[27]\u003c/sup\u003e. In vitro experiments revealed that GW788388 can partially alleviate the upregulation of MMP-13 induced by T-2 toxin, indicating that MMP-13 expression in T-2 toxin-treated chondrocytes is regulated via the TGF-\u0026beta; signaling pathway\u003csup\u003e[28]\u003c/sup\u003e. Therefore, modulating the TGF-\u0026beta; signaling pathway represents a promising therapeutic strategy for KBD.\u003c/p\u003e\n\u003cp\u003eThe degradation of the cartilage matrix caused by SelS deficiency might be associated with its function in ERAD and oxidative stress regulation. SelS is an important member of the ERAD complex, and its deficiency may affect the ERAD function of chondrocytes. A previous study has shown that ERAD-related genes are upregulated during cartilage formation, and disruption of ERAD function leads to collagen accumulation in the ER, inhibiting its secretion into the extracellular space while enhancing cartilage catabolism. This results in chondrodysplasia in developing embryos and cartilage loss in adult joints\u003csup\u003e[29]\u003c/sup\u003e. If SelS deficiency induces ERAD dysfunction, it would theoretically trigger ER stress. However, surprisingly,\u0026nbsp;knocking down SelS expression in chondrocytes does not induce ER stress\u003csup\u003e[24]\u003c/sup\u003e. There may be unknown compensatory or regulatory mechanisms involved in this process. In addition, a recent study demonstrated that the absence of SelS induces oxidative stress in cartilage, while reactive oxygen species (ROS) can activate the TGF-\u0026beta; signaling pathway\u003csup\u003e[30, 31]\u003c/sup\u003e.Numerous studies have shown that TGF-\u0026beta; regulates the expression of MMPs and components of the extracellular matrix, thereby significantly influencing the cellular microenvironment\u003csup\u003e[28, 32, 33]\u003c/sup\u003e. For instance, TGF-\u0026beta; induces the expression of MMP-2 and MMP-9 through the p38 MAPK signaling pathway\u003csup\u003e[33]\u003c/sup\u003e. Additionally, TGF-\u0026beta;1 elevates the levels of MMP-13 and type X collagen via NF-\u0026kappa;B activation in chondrocytes\u003csup\u003e[34]\u003c/sup\u003e. The interaction between SelS and TGF-\u0026beta; warrants further investigation.\u003c/p\u003e\n\u003cp\u003eThis study provides a new perspective on the molecular mechanism of cartilage degeneration: SelS is not only a regulator of oxidative stress but also may serve as a key regulatory node in cartilage metabolism through the TGF-\u0026beta; pathway. This discovery offers potential targets for the treatment of OA and KBD. For instance, upregulating SelS expression through drugs or gene editing may inhibit the excessive activation of TGF-\u0026beta;, thereby delaying the process of cartilage degradation. Furthermore, the association between SelS and the TGF-\u0026beta; pathway may have implications for the study of other fibrotic diseases, such as liver fibrosis and pulmonary fibrosis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors contributed to the study conception and design. Material preparation ,data collection and analysis were performed by Hui Wang, Yinan Liu and Meng Zhang. YawenShi optimized the experimental methods. The SelS gene knockout mice were provided by Jian Sun , and the funds were provided by Jinghong Chen, Ying Zhang and Mengying Wang. The first draft of the manuscript was written by Hui Wang , and Chen Chen provided guidance for the writing of this manuscript. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003e This research was funded by the National Natural Science Foundation of China (No.82473748 and No.82204172), Natural Science Basic Research Program of Shaanxi Province (No. 2023-JC-QN-0902) and the Fundamental Research Funds for the Central Universities, Xi’an Jiaotong University (No. xzy012022109).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u003c/strong\u003e All procedures performed in studies involving human participants followed the 1964 Helsinki Declaration and its later amendments or com-parable ethical standards, and were approved by the Human and Ethical Committee for Medical Research at Xi’an Jiaotong University, School of Medicine (Protocol NO.3063058). All procedures involving animals complied with the Guidance on the operation of the Animals Act 1986 and followed the ARRIVE guidelines. Ethical approval was granted by the Animal Ethics Committee, Medical School of Xi’an Jiaotong University (Protocol NO.0074).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eGhelichkhani, F., F.A. Gonzalez, M.A. Kapitonova, et al., \u003cem\u003eSelenoprotein S: A versatile disordered protein.\u003c/em\u003e Archives of Biochemistry and Biophysics, 2022. \u003cstrong\u003e731\u003c/strong\u003e.\u003c/li\u003e\n \u003cli\u003eKen Walder, Lakshmi Kantham, Janine S. McMillan, et al., \u003cem\u003eTanis: A Link Between Type 2 Diabetes and Inflammation?\u003c/em\u003e DIABETES, 2002. \u003cstrong\u003e51\u003c/strong\u003e(6): p. 1859-1866.\u003c/li\u003e\n \u003cli\u003eLee, J.H., K.J. Park, J.K. Jang, et al., \u003cem\u003eSelenoprotein S-dependent Selenoprotein K Binding to p97(VCP) Protein Is Essential for Endoplasmic Reticulum-associated Degradation.\u003c/em\u003e Journal of Biological Chemistry, 2015. \u003cstrong\u003e290\u003c/strong\u003e(50): p. 29941-29952.\u003c/li\u003e\n \u003cli\u003eYe, Y., W. Bian, F. Fu, et al., \u003cem\u003eSelenoprotein S inhibits inflammation-induced vascular smooth muscle cell calcification.\u003c/em\u003e JBIC Journal of Biological Inorganic Chemistry, 2018. \u003cstrong\u003e23\u003c/strong\u003e(5): p. 739-751.\u003c/li\u003e\n \u003cli\u003eZhuang, C., G. Liu, H.W. Barkema, et al., \u003cem\u003eSelenomethionine Suppressed TLR4/NF-\u0026kappa;B Pathway by Activating Selenoprotein S to Alleviate ESBL Escherichia coli-Induced Inflammation in Bovine Mammary Epithelial Cells and Macrophages.\u003c/em\u003e Frontiers in Microbiology, 2020. \u003cstrong\u003e11\u003c/strong\u003e.\u003c/li\u003e\n \u003cli\u003eLi, F., A. Mao, X. Fu, et al., \u003cem\u003eCorrelation between SEPS1 gene polymorphism and type 2 diabetes mellitus: A preliminary study.\u003c/em\u003e Journal of Clinical Laboratory Analysis, 2019. \u003cstrong\u003e33\u003c/strong\u003e(8).\u003c/li\u003e\n \u003cli\u003eLi, X.-X., H.-J. Guan, J.-P. Liu, et al., \u003cem\u003eAssociation of selenoprotein S gene polymorphism with ischemic stroke in a Chinese case\u0026ndash;control study.\u003c/em\u003e Blood Coagulation \u0026amp; Fibrinolysis, 2015. \u003cstrong\u003e26\u003c/strong\u003e(2): p. 131-135.\u003c/li\u003e\n \u003cli\u003eRueli, R.H.L.H., D.J. Torres, A.S.T. Dewing, et al., \u003cem\u003eSelenoprotein S Reduces Endoplasmic Reticulum Stress-Induced Phosphorylation of Tau: Potential Role in Selenate Mitigation of Tau Pathology.\u003c/em\u003e Journal of Alzheimer\u0026apos;s Disease, 2016. \u003cstrong\u003e55\u003c/strong\u003e(2): p. 749-762.\u003c/li\u003e\n \u003cli\u003eSutherland, A., D.-H. Kim, C. Relton, et al., \u003cem\u003ePolymorphisms in the selenoprotein S and 15-kDa selenoprotein genes are associated with altered susceptibility to colorectal cancer.\u003c/em\u003e Genes \u0026amp; Nutrition, 2010. \u003cstrong\u003e5\u003c/strong\u003e(3): p. 215-223.\u003c/li\u003e\n \u003cli\u003eDeng, X. and Y. Tan, \u003cem\u003eA national cross-sectional analysis of selenium intake and risk of osteoarthritis: NHANES 2003\u0026ndash;2016.\u003c/em\u003e Frontiers in Public Health, 2023. \u003cstrong\u003e10\u003c/strong\u003e.\u003c/li\u003e\n \u003cli\u003eWang, Y., D. Xie, J. Li, et al., \u003cem\u003eAssociation between dietary selenium intake and the prevalence of osteoporosis: a cross-sectional study.\u003c/em\u003e BMC Musculoskeletal Disorders, 2019. \u003cstrong\u003e20\u003c/strong\u003e(1).\u003c/li\u003e\n \u003cli\u003eZhang, B., L. Yang, W. Wang, et al., \u003cem\u003eEnvironmental selenium in the Kaschin\u0026ndash;Beck disease area, Tibetan Plateau, China.\u003c/em\u003e Environmental Geochemistry and Health, 2010. \u003cstrong\u003e33\u003c/strong\u003e(5): p. 495-501.\u003c/li\u003e\n \u003cli\u003eYu, N., F. Han, X. Lin, et al., \u003cem\u003eThe Association Between Serum Selenium Levels with Rheumatoid Arthritis.\u003c/em\u003e Biological Trace Element Research, 2015. \u003cstrong\u003e172\u003c/strong\u003e(1): p. 46-52.\u003c/li\u003e\n \u003cli\u003eRen, F.L., X. Guo, R.J. Zhang, et al., \u003cem\u003eEffects of selenium and iodine deficiency on bone, cartilage growth plate and chondrocyte differentiation in two generations of rats.\u003c/em\u003e Osteoarthritis and Cartilage, 2007. \u003cstrong\u003e15\u003c/strong\u003e(10): p. 1171-1177.\u003c/li\u003e\n \u003cli\u003eCao, J.J., B.R. Gregoire, and H. Zeng, \u003cem\u003eSelenium Deficiency Decreases Antioxidative Capacity and Is Detrimental to Bone Microarchitecture in Mice.\u003c/em\u003e The Journal of Nutrition, 2012. \u003cstrong\u003e142\u003c/strong\u003e(8): p. 1526-1531.\u003c/li\u003e\n \u003cli\u003eMin, Z., W. Zhao, N. Zhong, et al., \u003cem\u003eAbnormality of epiphyseal plate induced by selenium deficiency diet in two generation DA rats.\u003c/em\u003e Apmis, 2015. \u003cstrong\u003e123\u003c/strong\u003e(8): p. 697-705.\u003c/li\u003e\n \u003cli\u003eHorwitz, M.S., C.M. Downey, C.R. Horton, et al., \u003cem\u003eOsteo-Chondroprogenitor\u0026ndash;Specific Deletion of the Selenocysteine tRNA Gene, Trsp, Leads to Chondronecrosis and Abnormal Skeletal Development: A Putative Model for Kashin-Beck Disease.\u003c/em\u003e PLoS Genetics, 2009. \u003cstrong\u003e5\u003c/strong\u003e(8).\u003c/li\u003e\n \u003cli\u003eHamajima, T., Y. Mushimoto, H. Kobayashi, et al., \u003cem\u003eNovel compound heterozygous mutations in the SBP2 gene: characteristic clinical manifestations and the implications of GH and triiodothyronine in longitudinal bone growth and maturation.\u003c/em\u003e European Journal of Endocrinology, 2012. \u003cstrong\u003e166\u003c/strong\u003e(4): p. 757-764.\u003c/li\u003e\n \u003cli\u003eZhang, Z., J. Zhang, and J. Xiao, \u003cem\u003eSelenoproteins and selenium status in bone physiology and pathology.\u003c/em\u003e Biochimica et Biophysica Acta (BBA) - General Subjects, 2014. \u003cstrong\u003e1840\u003c/strong\u003e(11): p. 3246-3256.\u003c/li\u003e\n \u003cli\u003eChi, Q., Y. Luan, Y. Zhang, et al., \u003cem\u003eThe regulatory effects of miR-138-5p on selenium deficiency-induced chondrocyte apoptosis are mediated by targeting SelM.\u003c/em\u003e Metallomics, 2019. \u003cstrong\u003e11\u003c/strong\u003e(4): p. 845-857.\u003c/li\u003e\n \u003cli\u003eGong, Y., Y. Wu, Y. Liu, et al., \u003cem\u003eDetection of selenoprotein transcriptome in chondrocytes of patients with Kashin\u0026ndash;Beck disease.\u003c/em\u003e Frontiers in Cell and Developmental Biology, 2023. \u003cstrong\u003e11\u003c/strong\u003e.\u003c/li\u003e\n \u003cli\u003eYan, J., Y. Guo, Y. Fei, et al., \u003cem\u003eGPx1 knockdown suppresses chondrogenic differentiation of ATDC5 cells through induction of reductive stress.\u003c/em\u003e Acta Biochimica et Biophysica Sinica, 2017. \u003cstrong\u003e49\u003c/strong\u003e(2): p. 110-118.\u003c/li\u003e\n \u003cli\u003eYan, J., Y. Fei, Y. Han, et al., \u003cem\u003eSelenoprotein O deficiencies suppress chondrogenic differentiation of ATDC5 cells.\u003c/em\u003e Cell Biology International, 2016. \u003cstrong\u003e40\u003c/strong\u003e(10): p. 1033-1040.\u003c/li\u003e\n \u003cli\u003eWang, H., Z. Li, Y. Liu, et al., \u003cem\u003eEffects of Selenoprotein S Knockdown on Endoplasmic Reticulum Stress in ATDC5 Cells and Gene Expression Profiles in Hypertrophic Chondrocytes.\u003c/em\u003e Biological Trace Element Research, 2022. \u003cstrong\u003e201\u003c/strong\u003e(4): p. 1965-1976.\u003c/li\u003e\n \u003cli\u003eChen, J., M. Luo, W. Wang, et al., \u003cem\u003eAltered proteolytic activity and expression of MMPs and aggrecanases and their inhibitors in Kashin\u0026ndash;Beck disease.\u003c/em\u003e Journal of Orthopaedic Research, 2014. \u003cstrong\u003e33\u003c/strong\u003e(1): p. 47-55.\u003c/li\u003e\n \u003cli\u003eSophia Fox, A.J., A. Bedi, and S.A. Rodeo, \u003cem\u003eThe Basic Science of Articular Cartilage: Structure, Composition, and Function.\u003c/em\u003e Sports Health: A Multidisciplinary Approach, 2009. \u003cstrong\u003e1\u003c/strong\u003e(6): p. 461-468.\u003c/li\u003e\n \u003cli\u003eZhang, Y., Y. Mu, Y. He, et al., \u003cem\u003eUpregulated expression of transforming growth factor-\u0026beta; receptor I/II in an endemic Osteoarthropathy in China.\u003c/em\u003e BMC Musculoskeletal Disorders, 2021. \u003cstrong\u003e22\u003c/strong\u003e(1).\u003c/li\u003e\n \u003cli\u003eZhang, Y., Z. Li, Y. He, et al., \u003cem\u003eT-2 toxin induces articular cartilage damage by increasing the expression of MMP-13 via the TGF-\u0026beta; receptor pathway.\u003c/em\u003e Human \u0026amp; Experimental Toxicology, 2022. \u003cstrong\u003e41\u003c/strong\u003e.\u003c/li\u003e\n \u003cli\u003eHyo Jung Sim, C.C., Ha Eun Kim, Ju Yeon Hong, et al., \u003cem\u003eAugmented ERAD (ER-associated degradation) activity in chondrocytes is necessary for cartilage development and maintenance.\u003c/em\u003e Science Advances, 2022. \u003cstrong\u003e8\u003c/strong\u003e(3): p. eabl4222.\u003c/li\u003e\n \u003cli\u003eCui, Y., Y. Liao, Y. Chen, et al., \u003cem\u003eLow expression of selenoprotein S induces oxidative damage in cartilages.\u003c/em\u003e Journal of Trace Elements in Medicine and Biology, 2024. \u003cstrong\u003e85\u003c/strong\u003e.\u003c/li\u003e\n \u003cli\u003eZhang, H., Z.-W. Wang, H.-B. Wu, et al., \u003cem\u003eTransforming growth factor-\u0026beta;1 induces matrix metalloproteinase-9 expression in rat vascular smooth muscle cells via ROS-dependent ERK\u0026ndash;NF-\u0026kappa;B pathways.\u003c/em\u003e Molecular and Cellular Biochemistry, 2012.\u003c/li\u003e\n \u003cli\u003eWolfgang Wick, M.P.M.W., \u003cem\u003eGlioma cell invasion: regulation of metalloproteinase activity by TGF-\u0026beta;.\u003c/em\u003e Journal of Neuro-Oncology, 2001. \u003cstrong\u003e53\u003c/strong\u003e: p. 177-185.\u003c/li\u003e\n \u003cli\u003eKim，, E.-S., Mi-Sung Kim, and A. Moon, \u003cem\u003eTGF-beta-induced upregulation of MMP-2 and MMP-9 depends on p38 MAPK, but not ERK signaling in MCF10A human breast epithelial cells.\u003c/em\u003e International Journal of Oncology, 2004. \u003cstrong\u003e25\u003c/strong\u003e(5): p. 1375-1382.\u003c/li\u003e\n \u003cli\u003ePark, J.-Y., H.C. Bae, S.H. Pyo, et al., \u003cem\u003eTGF\u0026beta;1-Induced Transglutaminase-2 Triggers Catabolic Response in Osteoarthritic Chondrocytes by Modulating MMP-13.\u003c/em\u003e Tissue Engineering and Regenerative Medicine, 2021. \u003cstrong\u003e18\u003c/strong\u003e(5): p. 831-840.\u003c/li\u003e\n\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":"biological-trace-element-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bter","sideBox":"Learn more about [Biological Trace Element Research](https://www.springer.com/journal/12011)","snPcode":"12011","submissionUrl":"https://submission.nature.com/new-submission/12011/3","title":"Biological Trace Element Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Selenoprotein S, cartilage, type Ⅱ collagen, matrix metalloproteinases, TGF-β","lastPublishedDoi":"10.21203/rs.3.rs-9267022/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9267022/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e The deficiency of trace element selenium is a global nutritional issue. Selenium exerts its biological functions in the human body through selenoproteins, which play crucial roles in bone and cartilage development. Selenoprotein S (SelS), a key selenoprotein involved in the regulation of oxidative stress and inflammation, has an unclear role in cartilage development. The purpose of this study was to investigate the effects of SelS deficiency on cartilage matrix degradation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Chondrocytes with SelS gene knockdown and mice with SelS gene knockout were constructed. The mRNA and protein levels of COL II, MMP3, MMP10, and MMP13 were measured by RT-qPCR and western blotting, respectively. Changes in cartilage morphology and matrix composition were evaluated using histological staining techniques including toluidine blue (TB), saffron O-fast green, and sirius red staining. The expression levels of MMP3, MMP13, MMP19 and COL II in the knee joints of mice were detected by immunohistochemical (IHC) staining. A TGF-β pathway inhibitor (GW788388) was applied to SelS knockdown chondrocytes to verify whether the TGF-βsignaling pathway is involved in the matrix degradation induced by SelS deficiency.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Compared with the sh-NC group, the mRNA and protein levels of COL Ⅱ were decreased in the sh-Sels group.\u003cem\u003e \u003c/em\u003eThe expression levels of \u003cem\u003eMmp3\u003c/em\u003e, \u003cem\u003eMmp10\u003c/em\u003e and \u003cem\u003eMmp13 \u003c/em\u003ewere also increased significantly, while the metallopeptidase inhibitor \u003cem\u003eTimp2\u003c/em\u003e was downregulated in the sh-Sels group. SelS gene knockout in mice did not affect body length or weight but resulted in reduced proteoglycan and collagen contents in articular cartilage. Compared with wild-type mice, the expression of COL Ⅱ was decreased, while the expressions of MMP3, MMP13, and MMP19 were increased in the articular cartilage of SelS knockout mice. Furthermore, treatment with the TGF-β pathway inhibitor (GW788388) partially rescued the reduction in \u003cem\u003eCol2a1\u003c/em\u003eexpression induced by SelS deficiency.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eSelS plays a crucial role in maintaining cartilage homeostasis, and its deficiency results in cartilage matrix degradation. The TGF-β signaling pathway is involved in the degradation of cartilage matrix caused by SelS silencing.\u003c/p\u003e","manuscriptTitle":"Selenoprotein S deficiency induces matrix degradation via TGF-β pathway leading to cartilage damage","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-27 09:28:34","doi":"10.21203/rs.3.rs-9267022/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"204986513644758239255474636999365330638","date":"2026-05-19T02:59:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"109564555689377460247789311490275665069","date":"2026-05-18T08:30:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"250315097017381900872173936327350061967","date":"2026-05-17T02:54:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"319053696969744541760070839484958576650","date":"2026-05-04T23:44:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-18T20:47:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-17T12:02:02+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-17T04:51:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biological Trace Element Research","date":"2026-03-30T12:34:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biological-trace-element-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bter","sideBox":"Learn more about [Biological Trace Element Research](https://www.springer.com/journal/12011)","snPcode":"12011","submissionUrl":"https://submission.nature.com/new-submission/12011/3","title":"Biological Trace Element Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f2773e78-8bf2-4c28-a3fe-19ff89234778","owner":[],"postedDate":"April 27th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"204986513644758239255474636999365330638","date":"2026-05-19T02:59:16+00:00","index":27,"fulltext":""},{"type":"reviewerAgreed","content":"109564555689377460247789311490275665069","date":"2026-05-18T08:30:25+00:00","index":26,"fulltext":""},{"type":"reviewerAgreed","content":"250315097017381900872173936327350061967","date":"2026-05-17T02:54:07+00:00","index":25,"fulltext":""},{"type":"reviewerAgreed","content":"319053696969744541760070839484958576650","date":"2026-05-04T23:44:09+00:00","index":20,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T09:28:35+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-27 09:28:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9267022","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9267022","identity":"rs-9267022","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}