TRIM21 silencing inhibits the apoptosis and expedites the osteogenic differentiation of dexamethasone‑induced MC3T3‑E1 cells by activating the Keap1/Nrf2 pathway
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- Published online on: March 20, 2024 https://doi.org/10.3892/etm.2024.12502
- Article Number: 213
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Copyright: © Shi et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Steroid-induced osteonecrosis of the femoral head (ONFH), a serious orthopedic disease caused by long-term or excessive use of glucocorticoids, is a bone disorder primarily presenting femoral head collapse, resulting in hip joint dysfunction and eventually total hip arthroplasty (1,2). The prevalence rate of ONFH in Japan is ~0.0182%, while in South Korea it is ~0.0289% (3). Considering that, there is a pressing need to understand the specific molecular mechanism behind ONFH and ascertain potential therapeutic biomarkers.
The tripartite motif (TRIM) family of proteins belonging to the subfamily of E3 ubiquitin ligases has been shown to be implicated in diversified human diseases, such as tumors, inflammatory, infectious, neuropsychiatric disorders, chromosomal abnormalities as well as developmental diseases (4). As a member of the TRIM family, tripartite motif-containing protein 21 (TRIM21) can function as an E3 ligase dependent on its RING domain (5). Initially, TRIM21 was identified to act as a regulator of immune responses and participate in autoimmune diseases (5-7). A recent study revealed that TRIM21 has low expression during the osteogenic process of mesenchymal stem cells and TRIM21 negatively regulates the osteogenic capacity of mesenchymal stem cells both in vitro and in vivo (8). Nevertheless, the effects of TRIM21 on the process of ONFH remain to be elucidated.
Nuclear factor erythroid 2-related factor 2 (Nrf2) which is modulated by Kelch-like Ech-associated protein-1 (Keap1) is regarded as a principal component of the cellular defense system responding to various types of endogenous and exogenous insults (9). Numerous studies showed that the Keap1/Nrf2 pathway participates in a multitude of biological events including metabolism, cell proliferation and cell death (10,11). Notably, dysregulation of the Nrf2/Keap1 pathway has been shown to be involved in the process of ONFH (12). Concurrently, TRIM21 was reported to block the Keap1/Nrf2 pathway in hepatocarcinogenesis (13).
The present study sought to unravel the regulatory role of TRIM21 in the development of steroid-induced ONFH and to identify whether its action mechanism was associated with the Keap1/Nrf2 pathway.
Materials and methods
Cell culture and treatment
Minimal essential medium α (MEMα; Corning, Inc.) supplemented with 10% fetal bovine serum (FBS; Wuhan Saios Biotechnology Co., Ltd.) was adopted for the incubation of murine preosteoblast MC3T3-E1 (Wuhan Saios Biotechnology Co., Ltd.) cells at 37˚C with 5% CO2. Dexamethasone (Dex; Shanghai Macklin Biochemical Co., Ltd.) at the concentration of 1 µM was used to treat MC3T3-E1 cells for 24 h at 37˚C (14). Additionally, cells were treated with 10 µM Nrf2 inhibitor ML385 (Shanghai Macklin Biochemical Co., Ltd.) for 24 h at 37˚C (15) before treatment with Dex. To stimulate osteogenic differentiation, the osteogenesis-inducing medium containing 10% FBS, 5 mM L-glycerophosphate, 100 nM Dex and 50 mg/ml ascorbic acid was applied to the culture of MC3T3-E1 cells at 80% confluence and incubated for 7 days at 37˚C.
Transfection protocol
MC3T3-E1 cells at the logarithmic phase were seeded in a 6-well plate (1x105 cells/well) and were incubated at 37˚C until they reached 80% confluence. Using Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.), MC3T3-E1 cells were established with a stable knockdown of TRIM21 using 50 nmol/l short hairpin RNAs (shRNAs) for TRIM21 (sh-TRIM21#1, sense 5'-GAACCTGGACACGTTAGATAT-3', antisense 5'-ATATCTAACGTGTCCAGGTTC-3'; sh-TRIM21#2, sense 5'-TTGTCTCCTTCTACAACATAA-3', antisense 5'-TTATGTTGTAGAAGGAGACAA-3') or 50 nmol/l control shRNA (sh-NC, sense 5'-TACGGAGGACTCGATCTAG-3', antisense 5'-CTAGATCGAGTCCTCCGTA-3') ordered from Shanghai GenePharma Co., Ltd., according to the manufacturer's protocol at 37˚C for 48 h. Following 48 h of culture at 37˚C, cells were harvested for the subsequent experiments.
Cell Counting Kit-8 (CCK-8) assay
MC3T3-E1 cells subjected to transfection and indicated treatment were plated into 96-well plates (3,000 cells/well). After being incubated for 2 h with 10 µl CCK-8 solution (Beyotime Institute of Technology), the absorbance was recorded at 450 nm by using a microplate reader (BMG-Labtech, Ltd.).
Lactate dehydrogenase (LDH) release assay
The cytotoxicity in the supernatants of MC3T3-E1 cells subjected to centrifugation at 8,000 x g for 10 min at 4˚C was examined with an LDH assay kit (Nanjing Jiancheng Bioengineering Institute). Absorbance was reco.
Evaluation of reactive oxygen species (ROS) production
A 2 µM 2,7-dichloro-dihydrofluorescein diacetate (DCFH-DA; Shanghai Aladdin Biochemical Technology Co., Ltd.) solution was added to the MC3T3-E1 cells, previously subject to transfection and indicated treatment, for 30 min of incubation at 37˚C in the dark. Following washing with PBS, the fluorescence intensity was observed under a fluorescence microscope (Zeiss GmbH).
Detection of oxidative stress indexes
Following centrifugation at 8,000 x g for 10 min at 4˚C, superoxide dismutase (SOD; cat. no. S930985), glutathione peroxidase (GSH-Px; cat. no. G930918) and malonaldehyde (MDA; cat. no. M930417) contents were evaluated using specific assay kits from Shanghai Macklin Biochemical Co., Ltd. The absorbance values were recorded using a microplate reader.
Flow cytometry analysis
An Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (Nanjing KeyGen Biotech Co., Ltd.) was used to assess the apoptosis of MC3T3-E1 cells subjected to transfection and indicated treatment. Cells were plated into 6-well plates and incubated at 37˚C. MC3T3-E1 cells were suspended in the binding buffer containing Annexin-V-FITC and PI. The apoptosis rate was detected using flow cytometry (BD FACSCalibur; BD Biosciences). Data were analyzed using FlowJo software v7.6.1 (Tree Star, Inc.). The apoptotic rate was calculated as the percentage of early + late apoptotic cells.
Measurement of caspase 3 activity
Caspase 3 activity was examined with a caspase-3 activity assay kit (cat. no. ab252897; Abcam) according to the manufacturer's instructions. Cell lysates were incubated with caspase-3 substrate DEVD-AFC for 2 h at 37˚C in the dark. The fluorescence was observed using a fluorescence plate reader (excitation at 400 nm and emission at 505 nm).
Alkaline phosphatase (ALP) staining and Alizarin red S (ARS) staining
Following osteogenic differentiation, MC3T3-E1 cells previously subjected to transfection and indicated treatment were fixed in 4% paraformaldehyde for 15 min at 37˚C, before being incubated with ALP staining solution (MK BioScience Co., Inc.) for 4 h or ARS solution (Shanghai Macklin Biochemical Co., Ltd.) for 30 min at 37˚C. The images were captured under an inverted light microscope (Zeiss GmbH).
Reverse transcription-quantitative (RT-q) PCR
Extraction of total RNA from 1x104 MC3T3-E1 cells was conducted using TRIzol® reagent (Thermo Fisher Scientific, Inc.). The cDNA was synthesized using the PrimeScript RT reagent kit (Takara Bio, Inc.) according to the instructions provided by the manufacturer. qPCR was performed on the ABI 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) with cDNA as templates using SYBR Green PCR Master Mix Reagents (Takara Bio, Inc.) according to the manufacturer's instructions. The following thermocycling conditions were used for qPCR: Initial denaturation for 2 min at 94˚C; followed by 35 cycles for 30 sec at 94˚C and 45 sec at 55˚C. The calculation of relative mRNA levels was performed based on the 2-∆∆Cq method (16). GAPDH was employed as the internal reference. Specific primer sequences were: TRIM21 forward, 5'-CCTGGTTAGATTCCACGGCA-3' and reverse, 5'-TGAACTGCCCCCATTCTTCC-3'; and GAPDH forward, 5'-GCCTCCTCCAATTCAACCCT-3' and reverse, 5'-CTCGTGGTTCACACCCATCA-3'. All experiments were replicated three times.
Western blotting
After the preparation of cellular lysates using RIPA buffer (Beyotime Institute of Biotechnology), the quantification of protein concentration was performed using the BCA method (Beyotime Institute of Biotechnology). Total protein (40 µg protein per lane) was separated on 10% gels using SDS-PAGE and then transferred onto the polyvinylidene difluoride membranes. After blocking with 5% BSA (Beyotime Institute of Biotechnology) for 1.5 h at room temperature, the membranes were then immunoblotted with primary antibodies including TRIM21 (cat. no. ab207728; 1:1,000; Abcam), B-cell lymphoma 2 (Bcl-2; cat. no. ab196495; 1:2,000; Abcam), Bcl-2 associated X (Bax; cat. no. ab3191; 1:1,000; Abcam), osterix (Osx; cat. no. ab209484; 1:1,000; Abcam), runt-related transcription factor 2 (RUNX2; cat. no. ab236639; 1:1,000; Abcam), bone morphogenetic protein 2 (BMP2; cat. no. ab284387; 1:1,000; Abcam), Nrf2 (cat. no. 12721; 1:1,000; Cell Signaling Technology, Inc.), Keap1 (cat. no. ab119403; 1:1,000; Abcam), heme oxygenase-1 (HO-1; cat. no. ab189491; 1:2,000; Abcam), NAD(P)H:quinone oxidoreductase 1 (NQO1; cat. no. ab80588; 1:10,000; Abcam) and GAPDH (cat. no. ab181603; 1:10,000; Abcam) at 4˚C overnight, before being probed with HRP-conjugated secondary antibody (cat. no. ab6721; 1:2,000; Abcam) for 1 h at room temperature. Protein signals were visualized using chemiluminescence reagents (MilliporeSigma). The western blotting images of proteins were processed using ImageJ software (version 1.8.0; National Institutes of Health) with GAPDH as the loading control.
Statistical analysis
Data are presented as mean ± standard deviation of three independent experiments and analyzed using GraphPad Prism 8 software (GraphPad Software, Inc.; Dotmatics). Statistical comparison between two groups was made using an unpaired student's t-test. One-way analysis of variance followed by Tukey's post hoc test was used to compare the effects of more than two groups. P<0.05 was considered to indicate a statistically significant difference.
Results
TRIM21 knockdown increases viability and reduces cytotoxicity in Dex-treated MC3T3-E1 cells
To investigate the role of TRIM21 in ONFH, Dex was initially used for the establishment of a cell model of ONFH in MC3T3-E1 cells. Subsequently, TRIM21 mRNA levels and protein expression were evaluated using RT-qPCR and western blotting, respectively, and TRIM21 expression was noticeably increased in MC3T3-E1 cells administered with Dex (Fig. 1A and B). Moreover, TRIM21 expression was distinctly decreased after transfection of sh-TRIM21#1/2 (Fig. 1C and D). Therefore, sh-TRIM21#2 was chosen for the follow-up assays as TRIM21 exhibited lower expression in the sh-TRIM21#2 group. Based on the data from the CCK-8 assay, the viability was diminished in MC3T3-E1 cells exposed to Dex and the viability of Dex-treated MC3T3-E1 cells was improved through the knockdown of TRIM21 (Fig. 1E). In addition, the results from the LDH assay showed that the stimulated LDH release in Dex-challenged MC3T3-E1 cells was decreased when TRIM21 was silenced (Fig. 1F). In conclusion, TRIM21 knockdown suppressed Dex-stimulated viability loss and LDH release in MC3T3-E1 cells.
TRIM21 knockdown mitigates oxidative stress and apoptosis of Dex-challenged MC3T3-E1 cells
At the same time, as elucidated using DCFH-DA staining, TRIM21 silencing decreased Dex-induced ROS production in MC3T3-E1 cells (Fig. 2A). Dex exposure diminished SOD and GSH-Px activities whilst upregulating MDA content in MC3T3-E1 cells, which were all partially reversed by silencing of TRIM21 (Fig. 2B-D). The apoptosis of MC3T3-E1 cells was evaluated upon exposure to Dex. As shown in Fig. 2E and F, Dex treatment markedly increased the apoptotic rate of MC3T3-E1 cells, which was then decreased following TRIM21 knockdown. The increased caspase 3 activity that was found in MC3T3-E1 cells challenged with Dex was decreased when TRIM21 was knocked down (Fig. 2G). Western blotting also implied that Dex treatment resulted in the upregulation of Bax expression and the downregulation of Bcl-2 expression in MC3T3-E1 cells (Fig. 2H). However, in MC3T3-E1 cells treated with Dex, transfection of sh-TRIM21#2 clearly reduced Bax expression although is strengthened Bcl-2 expression. Overall, TRIM21 knockdown impeded Dex-stimulated oxidative stress and apoptosis in MC3T3-E1 cells.
Interference with TRIM21 contributes to the osteogenic differentiation of MC3T3-E1 cells exposed to Dex
Using ALP staining, it was observed that the decreased ALP activity in MC3T3-E1 cells treated with Dex increased again following TRIM21 knockdown (Fig. 3A). Furthermore, the data from the ARS staining showed that the attenuated calcium accumulation in MC3T3-E1 cells caused by Dex treatment was aggravated by the TRIM21 knockdown (Fig. 3B). Western blotting analysis of osteogenic differentiation-related markers also indicated that Dex exposure decreased Osx, RUNX2 and BMP2 protein expressions in MC3T3-E1 cells, which were partly restored following knockdown of TRIM21 (Fig. 3C). In summary, TRIM21 knockdown exacerbated the differentiation of Dex-treated MC3T3-E1 cells into osteoblasts.
TRIM21 knockdown activates the anti-oxidant Keap1/Nrf2 pathway
The expression of Keap1/Nrf2 pathway-related proteins was investigated by the present study because the Keap1/Nrf2 pathway was shown to play an inhibitory role in oxidative stress (17). The decreased Nrf2, HO-1 and NQO1 expression and the increased Keap1 expression in Dex-challenged MC3T3-E1 cells were all restored after TRIM21 was knocked down (Fig. 4), suggesting that TRIM21 inhibition might activate Keap1/Nrf2 signaling in Dex-induced MC3T3-E1 cells.
TRIM21 knockdown activates Keap1/Nrf2 signaling to hamper the oxidative stress and apoptosis and drives the osteogenic differentiation in Dex-treated MC3T3-E1 cells
The present study employed the Nrf2 inhibitor ML385 to test the hypothesis that TRIM21 might participate in the biological events in the Dex-induced cellular model of ONFH by mediating Keap1/Nrf2 signaling. Compared with the Dex group, ROS and MDA levels were decreased whilst SOD and GSH-Px activities were increased in Dex + sh-TRIM21 group (Fig. 5A-D). However, ROS (detected by DCFH-DA staining) and MDA levels were upregulated while SOD and GSH-Px activities were downregulated in the Dex + sh-TRIM21 + ML385 group compared with the Dex + sh-TRIM21 group. In addition, the weakened apoptotic rate, accompanied by the declined caspase 3 activity, Bax expression and elevated Bcl-2 expression in Dex-exposed MC3T3-E1 cells transfected with TRIM21 shRNA were all partially counteracted by pretreatment with ML385 (Fig. 5E-H). Additionally, TRIM21 deletion improved ALP activity and calcium accumulation in MC3T3-E1 cells challenged with Dex, which were then partially abrogated by ML385, implying that the stimulatory role of TRIM21 knockdown in the osteogenic differentiation of Dex-exposed MC3T3-E1 cells was abolished by inactivation of Nrf2 (Fig. 6A and B). This finding was also evidenced by the decreased Osx, RUNX2 and BMP2 expressions in Dex + sh-TRIM21 + ML385 group compared with the Dex + sh-TRIM21 group (Fig. 6C). In conclusion, inhibition of Nrf2 partly counteracted the effects of TRIM21 knockdown on the oxidative stress, apoptosis and osteogenic differentiation in Dex-treated MC3T3-E1 cells.
Discussion
Osteoblasts, which are the sole bone-forming cells, are responsible for bone formation and maintenance of bone mass. Osteoblast dysfunction is one of the pivotal mechanisms leading to steroid-induced ONFH (18) and autologous osteoblast cell implantation may be regarded as an effective therapy for ONFH (19). Administration with glucocorticoids, extensively applied for the treatment of autoimmune and inflammatory diseases as immunosuppressive and anti-inflammatory drugs, is the most common non-traumatic cause of ONFH (20). As an artificially synthetic glucocorticoid, Dex has also been implicated in the process of ONFH. In particular, Dex has been proposed to suppress the differentiation and stimulate apoptosis, autophagy, ferroptosis and oxidative stress in osteoblasts (21-23). Osteoblast cells caused by stimulation with 1 µM Dex for 24 h have been widely used as a cell model to explore the mechanisms involving in steroid-related ONFH (14,24,25). Thus, the mechanism underlying Dex-induced ONFH was investigated in the present study after murine MC3T3-E1 preosteoblast cells were exposed to Dex.
A growing number of studies have underlined the pivotal roles of TRIM21 in tumorigenesis (26), immune response (5) and autophagy (27). TRIM21 is downregulated during the osteogenic process of mesenchymal stem cells (8). In addition, Liu et al (28) demonstrated that TRIM21 expression is elevated in bone specimens from osteoporosis patients and ovariectomy-induced osteoporotic mice. Their findings also supported the hypothesis that TRIM21 depletion promotes bone formation by enhancing the osteogenic differentiation of bone marrow mesenchymal stem cells and elevating the activity of osteoblast. In the present study, TRIM21 expression was raised in MC3T3-E1 cells challenged with Dex and the viability loss and LDH release in Dex-treated MC3T3-E1 cells were both reversed by silencing of TRIM21. Additionally, enhanced osteoblast apoptosis has been documented to play a crucial role in the development and maintenance of bones and to further the progression of glucocorticoids-induced ONFH (29,30). By contrast, in Dex-exposed MC3T3-E1 cells, knockdown of TRIM21 attenuated the apoptotic ability of MC3T3-E1 cells, reduced caspase 3 activity and pro-apoptotic Bax expression and enhanced anti-apoptotic Bcl-2 expression. Comparing the results of cell activity and apoptosis of Dex-treated MC3T3-E1, it was found that the cell viability of Dex and Dex + shNC groups was ~50%, but the cell apoptosis rate in that two groups was only ~20%. The reason for this result may be that there are other forms of cell death involved in this process, such as necrosis and ferroptosis (31,32). Oxidative stress, resulting in ROS accumulation, is the leading cause of osteoblast injury and death (33). Altered ROS generation can facilitate osteoblast apoptosis and deplete the expression of antioxidant enzymes leading to ONFH (34). Meanwhile, TRIM21 reduction has been reported to inactivate oxidative stress in intervertebral disc degeneration (35) and atrial remodeling (36). Consistent with this, the present study showed that TRIM21 knockdown decreased Dex-induced ROS production, decreased SOD and GSH-Px activities and increased MDA activity in MC3T3-E1 cells.
Osteogenic differentiation is deemed a pivotal determinant in bone regeneration (37). Abnormal bone remodeling and osteoblastic bone formation have been shown to participate in the occurrence and progression of ONFH (38). Belonging to the BMP family, BMP2, which plays a major role in bone development and remodeling, is a well-established promoter of osteoblastic differentiation and bone formation (39). Runx2 is the main downstream regulator of the BMP signaling pathway that regulates the expression of several osteogenic genes (40). Osx is indispensable for osteogenic differentiation and its overexpression is related to the enhancement of osteogenic differentiation (41). Notably, the absence of TRIM21 may potentiate osteogenic differentiation and decrease RUNX2 expression in mesenchymal stem cells (8). In the current study, after TRIM21 was knocked down in MC3T3-E1 cells challenged with Dex, the ALP activity was increased, calcium salt deposition was strengthened and Osx, RUNX2 and BMP2 protein expressions were all increased.
Furthermore, an emerging study has underlined that TRIM21 may serve an inhibitor role in Keap1/Nrf2 signaling in hepatocarcinogenesis (13). Nrf2, a member of the Cap-n-Collar family of basic leucine zipper proteins, is regarded as a major anti-oxidative modulator by contributing to the transcription of antioxidant genes (42). As a main repressor protein of Nrf2, Keap1 facilitates the polyubiquitination of the Nrf2 protein and drives proteasome-dependent Nrf2 degradation (42). Under unstressed conditions, Keap1 binds to Nrf2 and promotes its degradation. Once the cells are damaged, Nrf2 will dissociate from Keap1, translocate into the nucleus and subsequently activate various genes, including HO-1(42). Abundant evidence has demonstrated that activation of Keap1/Nrf2 signaling can mitigate oxidative stress in the process of steroid-induced ONFH (18). Li et al (43) showed that the Nrf2 knockdown exacerbates apoptosis, oxidative stress and inflammation while inhibiting the osteogenic differentiation of high glucose-stimulated MC3T3-E1 cells. The present data also demonstrated that Nrf2 inhibitor ML385 partially counteracted the effects of TRIM21 deletion on apoptosis, oxidative stress as well as osteogenic differentiation in Dex-treated MC3T3-E1 cells.
Collectively, the present study showed that TRIM21 interference activated the Keap1/Nrf2 pathway to antagonize Dex-triggered apoptosis, oxidative stress and osteogenic differentiation decrease in MC3T3-E1 cells, thereby relieving the progression of steroid-induced ONFH. All these outcomes emphasize that TRIM21 may be valued as a potential target for steroid-induced ONFH. Nevertheless, this study only explored the effect of TRIM21 on mouse preosteoblast cells. Further experiments associated with osteoclast will be analyzed in the future. In addition, the other signaling pathways downstream of TRIM21, such as the YAP1/β-catenin signaling pathway (28), the intervention timing and possible screening models for high-risk patients will also be investigated in the next studies.
Acknowledgements
Not applicable.
Funding
Funding: No funding was received.
Availability of data and materials
The data generated in the present study may be requested from the corresponding author.
Authors' contributions
JS and XM conceived and designed the study. JS, LC, XW and XM conducted the experiments. LC and XW performed the literature search and data extraction. JS drafted the manuscript and XM revised it. All authors read and approved the final version of the manuscript. JS and XM confirm the authenticity of all the raw data.
Ethics approval and consent to participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
References
|
Pan J, Lu L, Wang X, Liu D, Tian J, Liu H, Zhang M, Xu F and An F: AIM2 regulates vascular smooth muscle cell migration in atherosclerosis. Biochem Biophys Res Commun. 497:401–409. 2018.PubMed/NCBI View Article : Google Scholar | |
|
Hines JT, Jo WL, Cui Q, Mont MA, Koo KH, Cheng EY, Goodman SB, Ha YC, Hernigou P, Jones LC, et al: Osteonecrosis of the femoral head: An updated review of ARCO on pathogenesis, staging and treatment. J Korean Med Sci. 36(e177)2021.PubMed/NCBI View Article : Google Scholar | |
|
Ando W, Sakai T, Fukushima W, Kaneuji A, Ueshima K, Yamasaki T, Yamamoto T and Nishii T: Working group for ONFH guidelines and Sugano N. Japanese orthopaedic association 2019 guidelines for osteonecrosis of the femoral head. J Orthop Sci. 26:46–68. 2021.PubMed/NCBI View Article : Google Scholar | |
|
Watanabe M and Hatakeyama S: TRIM proteins and diseases. J Biochem. 161:135–144. 2017.PubMed/NCBI View Article : Google Scholar | |
|
Jones EL, Laidlaw SM and Dustin LB: TRIM21/Ro52-roles in innate immunity and autoimmune disease. Front Immunol. 12(738473)2021.PubMed/NCBI View Article : Google Scholar | |
|
Ben-Chetrit E, Chan EK, Sullivan KF and Tan EM: A 52-kD protein is a novel component of the SS-A/Ro antigenic particle. J Exp Med. 167:1560–1571. 1988.PubMed/NCBI View Article : Google Scholar | |
|
Ben-Chetrit E, Fox RI and Tan EM: Dissociation of immune responses to the SS-A (Ro) 52 and 60-kd polypeptides in systemic lupus erythematosus and Sjögren's syndrome. Arthritis Rheum. 33:349–355. 1990.PubMed/NCBI View Article : Google Scholar | |
|
Xian J, Liang D, Zhao C, Chen Y and Zhu Q: TRIM21 inhibits the osteogenic differentiation of mesenchymal stem cells by facilitating K48 ubiquitination-mediated degradation of Akt. Exp Cell Res. 412(113034)2022.PubMed/NCBI View Article : Google Scholar | |
|
Ulasov AV, Rosenkranz AA, Georgiev GP and Sobolev AS: Nrf2/Keap1/ARE signaling: Towards specific regulation. Life Sci. 291(120111)2022.PubMed/NCBI View Article : Google Scholar | |
|
Fan Z, Wirth AK, Chen D, Wruck CJ, Rauh M, Buchfelder M and Savaskan N: Nrf2-Keap1 pathway promotes cell proliferation and diminishes ferroptosis. Oncogenesis. 6(e371)2017.PubMed/NCBI View Article : Google Scholar | |
|
Song MY, Lee DY, Chun KS and Kim EH: The role of NRF2/KEAP1 signaling pathway in cancer metabolism. Int J Mol Sci. 22(4376)2021.PubMed/NCBI View Article : Google Scholar | |
|
Yang N, Sun H, Xue Y, Zhang W, Wang H, Tao H, Liang X, Li M, Xu Y, Chen L, et al: Inhibition of MAGL activates the Keap1/Nrf2 pathway to attenuate glucocorticoid-induced osteonecrosis of the femoral head. Clin Transl Med. 11(e447)2021.PubMed/NCBI View Article : Google Scholar | |
|
Wang F, Zhang Y, Shen J, Yang B, Dai W, Yan J, Maimouni S, Daguplo HQ, Coppola S, Gao Y, et al: The ubiquitin E3 ligase TRIM21 promotes hepatocarcinogenesis by suppressing the p62-Keap1-Nrf2 antioxidant pathway. Cell Mol Gastroenterol Hepatol. 11:1369–1385. 2021.PubMed/NCBI View Article : Google Scholar | |
|
Han D, Gu X, Gao J, Wang Z, Liu G, Barkema HW and Han B: Chlorogenic acid promotes the Nrf2/HO-1 anti-oxidative pathway by activating p21Waf1/Cip1 to resist dexamethasone-induced apoptosis in osteoblastic cells. Free Radic Biol Med. 137:1–12. 2019.PubMed/NCBI View Article : Google Scholar | |
|
Hu Q, Zuo T, Deng L, Chen S, Yu W, Liu S, Liu J, Wang X, Fan X and Dong Z: β-Caryophyllene suppresses ferroptosis induced by cerebral ischemia reperfusion via activation of the NRF2/HO-1 signaling pathway in MCAO/R rats. Phytomedicine. 102(154112)2022.PubMed/NCBI View Article : Google Scholar | |
|
Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001.PubMed/NCBI View Article : Google Scholar | |
|
Suzuki T, Takahashi J and Yamamoto M: Molecular basis of the KEAP1-NRF2 signaling pathway. Mol Cells. 46:133–141. 2023.PubMed/NCBI View Article : Google Scholar | |
|
Lu Z and Han K: SMAD4 transcriptionally activates GCN5 to inhibit apoptosis and promote osteogenic differentiation in dexamethasone-induced human bone marrow mesenchymal stem cells. Steroids. 179(108969)2022.PubMed/NCBI View Article : Google Scholar | |
|
Palekar G: Hip preservation with autologous osteoblast cell-based treatment in osteonecrosis of the femoral head. Orthopedics. 44:e183–e189. 2021.PubMed/NCBI View Article : Google Scholar | |
|
Kerachian MA, Séguin C and Harvey EJ: Glucocorticoids in osteonecrosis of the femoral head: A new understanding of the mechanisms of action. J Steroid Biochem Mol Biol. 114:121–128. 2009.PubMed/NCBI View Article : Google Scholar | |
|
Fang L, Zhang G, Wu Y, Li Z, Gao S and Zhou L: SIRT6 prevents glucocorticoid-induced osteonecrosis of the femoral head in rats. Oxid Med Cell Longev. 2022(6360133)2022.PubMed/NCBI View Article : Google Scholar | |
|
Sun F, Zhou JL, Wei SX, Jiang ZW and Peng H: Glucocorticoids induce osteonecrosis of the femoral head in rats via PI3K/AKT/FOXO1 signaling pathway. PeerJ. 10(e13319)2022.PubMed/NCBI View Article : Google Scholar | |
|
Peng P, Nie Z, Sun F and Peng H: Glucocorticoids induce femoral head necrosis in rats through the ROS/JNK/c-Jun pathway. FEBS Open Bio. 11:312–321. 2021.PubMed/NCBI View Article : Google Scholar | |
|
Deng S, Dai G, Chen S, Nie Z, Zhou J, Fang H and Peng H: Dexamethasone induces osteoblast apoptosis through ROS-PI3K/AKT/GSK3β signaling pathway. Biomed Pharmacother. 110:602–608. 2019.PubMed/NCBI View Article : Google Scholar | |
|
Zhou M, Liu L, Xu Y, Jiang J, Liu G and Zhai C: Effects of osteoblast autophagy on glucocorticoid-induced femoral head necrosis. Jt Dis Relat Surg. 31:411–418. 2020.PubMed/NCBI View Article : Google Scholar | |
|
Alomari M: TRIM21-a potential novel therapeutic target in cancer. Pharmacol Res. 165(105443)2021.PubMed/NCBI View Article : Google Scholar | |
|
Kimura T, Jain A, Choi SW, Mandell MA, Schroder K, Johansen T and Deretic V: TRIM-mediated precision autophagy targets cytoplasmic regulators of innate immunity. J Cell Biol. 210:973–989. 2015.PubMed/NCBI View Article : Google Scholar | |
|
Liu RX, Gu RH, Li ZP, Hao ZQ, Hu QX, Li ZY, Wang XG, Tang W, Wang XH, Zeng YK, et al: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling. Bone Res. 11(56)2023.PubMed/NCBI View Article : Google Scholar | |
|
Nie Z, Chen S and Peng H: Glucocorticoid induces osteonecrosis of the femoral head in rats through GSK3β-mediated osteoblast apoptosis. Biochem. Biophys Res Commun. 511:693–699. 2019.PubMed/NCBI View Article : Google Scholar | |
|
Mutijima E, De Maertelaer V, Deprez M, Malaise M and Hauzeur JP: The apoptosis of osteoblasts and osteocytes in femoral head osteonecrosis: Its specificity and its distribution. Clin Rheumatol. 33:1791–1795. 2014.PubMed/NCBI View Article : Google Scholar | |
|
Guo S, Mao L, Ji F, Wang S, Xie Y, Fei H and Wang XD: Activating AMP-activated protein kinase by an α1 selective activator compound 13 attenuates dexamethasone-induced osteoblast cell death. Biochem Biophys Res Commun. 471:545–552. 2016.PubMed/NCBI View Article : Google Scholar | |
|
Sun F, Zhou JL, Liu ZL, Jiang ZW and Peng H: Dexamethasone induces ferroptosis via P53/SLC7A11/GPX4 pathway in glucocorticoid-induced osteonecrosis of the femoral head. Biochem Biophys Res Commun. 602:149–155. 2022.PubMed/NCBI View Article : Google Scholar | |
|
Yang YH, Li B, Zheng XF, Chen JW, Chen K, Jiang SD and Jiang LS: Oxidative damage to osteoblasts can be alleviated by early autophagy through the endoplasmic reticulum stress pathway-implications for the treatment of osteoporosis. Free Radic Biol Med. 77:10–20. 2014.PubMed/NCBI View Article : Google Scholar | |
|
Deng S, Zhou JL, Fang HS, Nie ZG, Chen S and Peng H: Sesamin protects the femoral head from osteonecrosis by inhibiting ROS-induced osteoblast apoptosis in rat model. Front Physiol. 9(1787)2018.PubMed/NCBI View Article : Google Scholar | |
|
Zheng J, Chang L, Bao X, Zhang X, Li C and Deng L: TRIM21 drives intervertebral disc degeneration induced by oxidative stress via mediating HIF-1α degradation. Biochem Biophys Res Commun. 555:46–53. 2021.PubMed/NCBI View Article : Google Scholar | |
|
Liu X, Zhang W, Luo J, Shi W, Zhang X, Li Z, Qin X, Liu B and Wei Y: TRIM21 deficiency protects against atrial inflammation and remodeling post myocardial infarction by attenuating oxidative stress. Redox Biol. 62(102679)2023.PubMed/NCBI View Article : Google Scholar | |
|
Bharadwaz A and Jayasuriya AC: Osteogenic differentiation cues of the bone morphogenetic protein-9 (BMP-9) and its recent advances in bone tissue regeneration. Mater Sci Eng C Mater Biol Appl. 120(111748)2021.PubMed/NCBI View Article : Google Scholar | |
|
Tingart M, Beckmann J, Opolka A, Matsuura M, Wiech O, Grifka J and Grässel S: Influence of factors regulating bone formation and remodeling on bone quality in osteonecrosis of the femoral head. Calcif Tissue Int. 82:300–308. 2008.PubMed/NCBI View Article : Google Scholar | |
|
Halloran D, Durbano HW and Nohe A: Bone morphogenetic protein-2 in development and bone homeostasis. J Dev Biol. 8(19)2020.PubMed/NCBI View Article : Google Scholar | |
|
Liu DD, Zhang CY, Liu Y, Li J, Wang YX and Zheng SG: RUNX2 regulates osteoblast differentiation via the BMP4 signaling pathway. J Dent Res. 101:1227–1237. 2022.PubMed/NCBI View Article : Google Scholar | |
|
Han Y, Kim YM, Kim HS and Lee KY: Melatonin promotes osteoblast differentiation by regulating osterix protein stability and expression. Sci Rep. 7(5716)2017.PubMed/NCBI View Article : Google Scholar | |
|
Baird L and Yamamoto M: The molecular mechanisms regulating the KEAP1-NRF2 pathway. Mol Cell Biol. 40:e00099–20. 2020.PubMed/NCBI View Article : Google Scholar | |
|
Li X, Yu X, He S and Li J: Dipeptidyl peptidase 3 is essential for maintaining osteoblastic differentiation under a high-glucose environment by inhibiting apoptosis, oxidative stress and inflammation through the modulation of the Keap1-Nrf2 pathway. Int Immunopharmacol. 120(110404)2023.PubMed/NCBI View Article : Google Scholar |
