TRIM21 silencing inhibits the apoptosis and expedites the osteogenic differentiation of dexamethasone‑induced MC3T3‑E1 cells by activating the Keap1/Nrf2 pathway

Shi,Jiaqi; Chen,Li; Wang,Xu; Ma,Xin

doi:10.3892/etm.2024.12502

TRIM21 silencing inhibits the apoptosis and expedites the osteogenic differentiation of dexamethasone‑induced MC3T3‑E1 cells by activating the Keap1/Nrf2 pathway

Authors:
- Jiaqi Shi
- Li Chen
- Xu Wang
- Xin Ma
View Affiliations

Affiliations: Department of Orthopedics, Huashan Hospital Affiliated to Fudan University, Shanghai 200040, P.R. China
Published online on: March 20, 2024 https://doi.org/10.3892/etm.2024.12502
Article Number: 213
Copyright: © Shi et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Steroid‑induced osteonecrosis of the femoral head (ONFH) is a serious complication caused by long‑term or excessive use of glucocorticoids. The present study aimed to ascertain the effects of tripartite motif‑containing protein 21 (TRIM21) on the process of steroid‑induced ONFH and its hidden action mechanism. TRIM21 expression in dexamethasone (Dex)‑treated mouse MC3T3‑E1 preosteoblast cells was examined using reverse transcription‑quantitative PCR and western blotting. The Cell Counting Kit‑8 (CCK‑8) method and lactate dehydrogenase release assay were used to respectively measure cell viability and injury. Flow cytometry analysis was used to assay cell apoptosis. Caspase 3 activity was evaluated using a specific assay, while alkaline phosphatase and Alizarin red S staining were used to evaluate osteogenesis. 2,7‑dichloro‑dihydrofluorescein diacetate fluorescence probe was used to estimate reactive oxygen species generation. Specific assay kits were used to appraise oxidative stress levels. In addition, the expression of apoptosis‑, osteogenic differentiation‑ and Kelch‑like ECH‑associated protein 1 (Keap1)/nuclear factor erythroid 2‑related factor 2 (Nrf2) signaling‑associated proteins was assessed using western blotting. In Nrf2 inhibitor (ML385)‑pretreated MC3T3‑E1 cells exposed to Dex, cell apoptosis, osteogenesis and oxidative stress were detected again as aforementioned. Results revealed that TRIM21 expression was raised in Dex‑induced MC3T3‑E1 cells and TRIM21 deletion improved the viability and osteogenic differentiation, whereas it hampered the oxidative stress and apoptosis in MC3T3‑E1 cells with Dex induction. In addition, silencing of TRIM21 activated Keap1/Nrf2 signaling. Moreover, ML385 partially abrogated the effects of TRIM21 depletion on the oxidative stress, apoptosis and osteogenic differentiation in MC3T3‑E1 cells exposed to Dex. In conclusion, TRIM21 silencing might activate Keap1/Nrf2 signaling to protect against steroid‑induced ONFH.

View Figures

View References

1	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
2	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
3	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
4	Watanabe M and Hatakeyama S: TRIM proteins and diseases. J Biochem. 161:135–144. 2017.PubMed/NCBI View Article : Google Scholar
5	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
6	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
7	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
8	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
9	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
10	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
11	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
12	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
13	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
14	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 p21^Waf1^/^Cip1 to resist dexamethasone-induced apoptosis in osteoblastic cells. Free Radic Biol Med. 137:1–12. 2019.PubMed/NCBI View Article : Google Scholar
15	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
16	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
17	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
18	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
19	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
20	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
21	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
22	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
23	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
24	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
25	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
26	Alomari M: TRIM21-a potential novel therapeutic target in cancer. Pharmacol Res. 165(105443)2021.PubMed/NCBI View Article : Google Scholar
27	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
28	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
29	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
30	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
31	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
32	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
33	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
34	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
35	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
36	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
37	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
38	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
39	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
40	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
41	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
42	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
43	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