Mechanism of ring finger protein 11 regulating Akt signaling pathway to promote osteogenic differentiation of bone marrow mesenchymal stem cells_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

DENG Wen ¹ , LONG Ting ² ,  DU Ying ³

1. Biotherapy Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou Guangdong, 510120, P. R. China;
2. Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou Guangdong, 510080, P. R. China;
3. Department of Microbiology and Immunology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou Henan, 450001, P. R. China;

Corresponding author：

DU Ying, Email: duying@zzu.edu.cn

Keywords：

Bone marrow mesenchymal stem cells; osteogenesis; ring finger protein 11; Akt signaling pathway; tissue engineered bone; bone defect repair

DOI：

10.7507/1002-1892.202107108

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Objective To investigate the role and regulatory mechanism of ring finger protein 11 (RNF11) on Akt signaling pathway in the process of osteogenesis of bone marrow mesenchymal stem cells (BMSCs) to provide ideas for further clarifying its osteogenesis mechanism and its use in clinical treatment in the future. Methods BMSCs were isolated and cultured from fresh bone marrow of healthy donors and subcultured. The 4th generation cells were used in experiments after identification by flow cytometry, and osteogenic, chondrogenic, and adipogenic induction. BMSCs were cultured in osteogenic differentiation medium for 0-14 days. The degree of osteogenic differentiation was detected by Alizarin red staining and alkaline phosphatase (ALP) staining, and the protein expression of RNF11 was detected by Western blot. The 4th generation BMSCs were divided into blank control group (group A), empty lentivirus (Lv-NC) group (group B), and knockdown RNF11 (Lv-ShRNF11) group (group C). Osteogenesis was induced and cultured for 0-14 days. The expression of RNF11 protein was detected by Western blot, the degree of osteogenic differentiation was detected by Alizarin red staining and ALP staining, and the relative mRNA expressions of Runx2, osteocalcin (OCN), and osteopontin (OPN) were detected by real-time fluorescence quantitative PCR (qRT-PCR). The protein relative expressions of Akt, Smad1/5/8, and β-catenin signaling pathway were detected by Western blot, expressed as the ratio before and after phosphorylation. In order to study the effect mechanism of RNF11 on Akt signaling pathway, the 4th generation BMSCs were divided into Lv-NC transfection group (group A1), Lv-ShRNF11 transfection group (group B1), and Lv-ShRNF11 transfection supplemented with Akt signaling pathway activator SC79 group (group C1). The protein relative expressions of RNF11 and Akt signaling pathway were detected by Western blot, the related osteogenesis indexes were detected by Alizarin red staining, ALP staining, and qRT-PCR. Results The flow cytometry, and osteogenic, chondrogenic, adipogenic induction culture identification showed that the isolated and cultured cells were BMSCs. The protein relative expression of RNF11 increased gradually with the extension of osteogenic differentiation time (P<0.05); after knockdown RNF11, Alizarin red and ALP stainings showed that the degree of osteogenic differentiation of BMSCs in group C were significantly lower than those in groups A and B, and qRT-PCR detection showed that the relative expression of Runx2, OCN, and OPN mRNA significantly decreased (P<0.05). The protein relative expressions of RNF11 and Akt signaling pathway significantly increased with the extensions of osteogenic differentiation time (P<0.05). After knockdown RNF11, the protein relative expression of Akt signaling pathway in group C was significantly lower than that in groups A and B (P<0.05), while Smad1/5/8 and β-catenin signaling pathway had no significant effect (P>0.05). Compared with group A1, the protein relative expression of RNF11 in groups B1 and C1 significantly decreased (P<0.05). Compared with groups A1 and C1, the protein relative expression of Akt signaling pathway in group B1 was significantly lower (P<0.05); Alizarin red and ALP stainings showed that the degree of osteogenic differentiation of BMSCs in group C1 were slightly lower than that of group A1 (P>0.05), but significantly higher than that of group B1 (P<0.05); qRT-PCR detection showed that the relative expressions of Runx2, OCN, and OPN mRNA in group C1 were slightly lower than those of group A1 (P>0.05), but were significantly higher than those of group B1 (P<0.05). Conclusion RNF11 promotes the differentiation of BMSCs into osteoblasts by positively regulating the activation level of Akt signaling pathway. RNF11 can be used as a potential target to improve the bone repair efficacy of BMSCs and treat bone metabolic diseases.

Citation： DENG Wen, LONG Ting, DU Ying. Mechanism of ring finger protein 11 regulating Akt signaling pathway to promote osteogenic differentiation of bone marrow mesenchymal stem cells. Chinese Journal of Reparative and Reconstructive Surgery, 2022, 36(1): 102-110. doi: 10.7507/1002-1892.202107108 Copy

1.	Oryan A, Kamali A, Moshiri A, et al. Role of mesenchymal stem cells in bone regenerative medicine: What is the evidence? Cells Tissues Organs, 2017, 204(2): 59-83.
2.	Aicher WK, Bühring HJ, Hart M, et al. Regeneration of cartilage and bone by defined subsets of mesenchymal stromal cells-potential and pitfalls. Adv Drug Deliv Rev, 2011, 63(4-5): 342-351.
3.	Marie PJ, Fromigué O. Osteogenic differentiation of human marrow-derived mesenchymal stem cells. Regen Med, 2006, 1(4): 539-548.
4.	Shih YR, Chen CN, Tsai SW, et al. Growth of mesenchymal stem cells on electrospun type Ⅰ collagen nanofibers. Stem Cells, 2006, 24(11): 2391-2397.
5.	Bruder SP, Kraus KH, Goldberg VM, et al. The effect of implants loaded with autologous mesenchymal stem cells on the healing of canine segmental bone defects. J Bone Joint Surg (Am), 1998, 80(7): 985-996.
6.	袁宇, 徐林. 骨髓间充质干细胞联合3D生物打印技术治疗骨缺损的研究进展. 中国医学物理学杂志, 2021, 38(1): 110-126.
7.	Tse WT, Pendleton JD, Beyer WM, et al. Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation. Transplantation, 2003, 75(3): 389-397.
8.	Maruyama M, Moeinzadeh S, Guzman RA, et al. The efficacy of lapine preconditioned or genetically modified IL4 over-expressing bone marrow-derived mesenchymal stromal cells in corticosteroid-associated osteonecrosis of the femoral head in rabbits. Biomaterials, 2021, 275: 120972. doi: 10.1016/j.biomaterials.2021.120972.
9.	Maruyama M, Rhee C, Utsunomiya T, et al. Modulation of the inflammatory response and bone healing. Front Endocrinol (Lausanne), 2020, 11: 386. doi: 10.3389/fendo.2020.00386.
10.	Fujita T, Azuma Y, Fukuyama R, et al. Runx2 induces osteoblast and chondrocyte differentiation and enhances their migration by coupling with PI3K-Akt signaling. J Cell Biol, 2004, 166(1): 85-95.
11.	史东梅, 董明, 陆颖, 等. PI3K/Akt信号通路与骨破坏: 问题与机制. 中国组织工程研究, 2020, 24(23): 3716-3722.
12.	Peng XD, Xu PZ, Chen ML, et al. Dwarfism, impaired skin development, skeletal muscle atrophy, delayed bone development, and impeded adipogenesis in mice lacking Akt1 and Akt2. Genes Dev, 2003, 17(11): 1352-1365.
13.	Kitching R, Wong MJ, Koehler D, et al. The RING-H2 protein RNF11 is differentially expressed in breast tumours and interacts with HECT-type E3 ligases. Biochim Biophys Acta, 2003, 1639(2): 104-112.
14.	Azmi P, Seth A. RNF11 is a multifunctional modulator of growth factor receptor signalling and transcriptional regulation. Eur J Cancer, 2005, 41(16): 2549-2560.
15.	Connor MK, Azmi PB, Subramaniam V, et al. Molecular characterization of ring finger protein 11. Mol Cancer Res, 2005, 3(8): 453-461.
16.	Mattioni A, Castagnoli L, Santonico E. RNF11 at the crossroads of protein ubiquitination. Biomolecules, 2020, 10(11): 1538. doi: 10.3390/biom10111538.
17.	Santonico E, Belleudi F, Panni S, et al. Multiple modification and protein interaction signals drive the Ring finger protein 11 (RNF11) E3 ligase to the endosomal compartment. Oncogene, 2010, 29(41): 5604-5618.
18.	Malonis RJ, Fu W, Jelcic MJ, et al. RNF11 sequestration of the E3 ligase SMURF2 on membranes antagonizes SMAD7 down-regulation of transforming growth factor β signaling. J Biol Chem, 2017, 292(18): 7435-7451.
19.	Budhidarmo R, Zhu J, Middleton AJ, et al. The RING domain of RING Finger 11 (RNF11) protein binds Ubc13 and inhibits formation of polyubiquitin chains. FEBS Lett, 2018, 592(8): 1434-1444.
20.	Gao Y, Ganss BW, Wang H, et al. The RING finger protein RNF11 is expressed in bone cells during osteogenesis and is regulated by Ets1. Exp Cell Res, 2005, 304(1): 127-135.
21.	Subramaniam V, Li H, Wong M, et al. The RING-H2 protein RNF11 is overexpressed in breast cancer and is a target of Smurf2 E3 ligase. Br J Cancer, 2003, 89(8): 1538-1544.
22.	Pranski EL, Dalal NV, Herskowitz JH, et al. Neuronal RING finger protein 11 (RNF11) regulates canonical NF-κB signaling. J Neuroinflammation, 2012, 9: 67. doi: 10.1186/1742-2094-9-67.
23.	Pranski EL, Dalal NV, Sanford CV, et al. RING finger protein 11 (RNF11) modulates susceptibility to 6-OHDA-induced nigral degeneration and behavioral deficits through NF-κB signaling in dopaminergic cells. Neurobiol Dis, 2013, 54: 264-279.
24.	池玉磊, 卜宪敏, 查玉梅, 等. 骨髓间充质干细胞复合支架材料治疗骨缺损: 研究现状及前景展望. 中国组织工程研究, 2019, 23(29): 4749-4756.
25.	Jo H, Mondal S, Tan D, et al. Small molecule-induced cytosolic activation of protein kinase Akt rescues ischemia-elicited neuronal death. Proc Natl Acad Sci U S A, 2012, 109(26): 10581-10586.
26.	Shembade N, Parvatiyar K, Harhaj NS, et al. The ubiquitin-editing enzyme A20 requires RNF11 to downregulate NF-kappaB signalling. EMBO J, 2009, 28(5): 513-522.
27.	Zhao SJ, Kong FQ, Jie J, et al. Macrophage MSR1 promotes BMSC osteogenic differentiation and M2-like polarization by activating PI3K/AKT/GSK3β/β-catenin pathway. Theranostics, 2020, 10(1): 17-35.
28.	Yang C, Liu X, Zhao K, et al. miRNA-21 promotes osteogenesis via the PTEN/PI3K/Akt/HIF-1α pathway and enhances bone regeneration in critical size defects. Stem Cell Res Ther, 2019, 10(1): 65. doi: 10.1186/s13287-019-1168-2.

1. Oryan A, Kamali A, Moshiri A, et al. Role of mesenchymal stem cells in bone regenerative medicine: What is the evidence? Cells Tissues Organs, 2017, 204(2): 59-83.
2. Aicher WK, Bühring HJ, Hart M, et al. Regeneration of cartilage and bone by defined subsets of mesenchymal stromal cells-potential and pitfalls. Adv Drug Deliv Rev, 2011, 63(4-5): 342-351.
3. Marie PJ, Fromigué O. Osteogenic differentiation of human marrow-derived mesenchymal stem cells. Regen Med, 2006, 1(4): 539-548.
4. Shih YR, Chen CN, Tsai SW, et al. Growth of mesenchymal stem cells on electrospun type Ⅰ collagen nanofibers. Stem Cells, 2006, 24(11): 2391-2397.
5. Bruder SP, Kraus KH, Goldberg VM, et al. The effect of implants loaded with autologous mesenchymal stem cells on the healing of canine segmental bone defects. J Bone Joint Surg (Am), 1998, 80(7): 985-996.
6. 袁宇, 徐林. 骨髓间充质干细胞联合3D生物打印技术治疗骨缺损的研究进展. 中国医学物理学杂志, 2021, 38(1): 110-126.
7. Tse WT, Pendleton JD, Beyer WM, et al. Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation. Transplantation, 2003, 75(3): 389-397.
8. Maruyama M, Moeinzadeh S, Guzman RA, et al. The efficacy of lapine preconditioned or genetically modified IL4 over-expressing bone marrow-derived mesenchymal stromal cells in corticosteroid-associated osteonecrosis of the femoral head in rabbits. Biomaterials, 2021, 275: 120972. doi: 10.1016/j.biomaterials.2021.120972.
9. Maruyama M, Rhee C, Utsunomiya T, et al. Modulation of the inflammatory response and bone healing. Front Endocrinol (Lausanne), 2020, 11: 386. doi: 10.3389/fendo.2020.00386.
10. Fujita T, Azuma Y, Fukuyama R, et al. Runx2 induces osteoblast and chondrocyte differentiation and enhances their migration by coupling with PI3K-Akt signaling. J Cell Biol, 2004, 166(1): 85-95.
11. 史东梅, 董明, 陆颖, 等. PI3K/Akt信号通路与骨破坏: 问题与机制. 中国组织工程研究, 2020, 24(23): 3716-3722.
12. Peng XD, Xu PZ, Chen ML, et al. Dwarfism, impaired skin development, skeletal muscle atrophy, delayed bone development, and impeded adipogenesis in mice lacking Akt1 and Akt2. Genes Dev, 2003, 17(11): 1352-1365.
13. Kitching R, Wong MJ, Koehler D, et al. The RING-H2 protein RNF11 is differentially expressed in breast tumours and interacts with HECT-type E3 ligases. Biochim Biophys Acta, 2003, 1639(2): 104-112.
14. Azmi P, Seth A. RNF11 is a multifunctional modulator of growth factor receptor signalling and transcriptional regulation. Eur J Cancer, 2005, 41(16): 2549-2560.
15. Connor MK, Azmi PB, Subramaniam V, et al. Molecular characterization of ring finger protein 11. Mol Cancer Res, 2005, 3(8): 453-461.
16. Mattioni A, Castagnoli L, Santonico E. RNF11 at the crossroads of protein ubiquitination. Biomolecules, 2020, 10(11): 1538. doi: 10.3390/biom10111538.
17. Santonico E, Belleudi F, Panni S, et al. Multiple modification and protein interaction signals drive the Ring finger protein 11 (RNF11) E3 ligase to the endosomal compartment. Oncogene, 2010, 29(41): 5604-5618.
18. Malonis RJ, Fu W, Jelcic MJ, et al. RNF11 sequestration of the E3 ligase SMURF2 on membranes antagonizes SMAD7 down-regulation of transforming growth factor β signaling. J Biol Chem, 2017, 292(18): 7435-7451.
19. Budhidarmo R, Zhu J, Middleton AJ, et al. The RING domain of RING Finger 11 (RNF11) protein binds Ubc13 and inhibits formation of polyubiquitin chains. FEBS Lett, 2018, 592(8): 1434-1444.
20. Gao Y, Ganss BW, Wang H, et al. The RING finger protein RNF11 is expressed in bone cells during osteogenesis and is regulated by Ets1. Exp Cell Res, 2005, 304(1): 127-135.
21. Subramaniam V, Li H, Wong M, et al. The RING-H2 protein RNF11 is overexpressed in breast cancer and is a target of Smurf2 E3 ligase. Br J Cancer, 2003, 89(8): 1538-1544.
22. Pranski EL, Dalal NV, Herskowitz JH, et al. Neuronal RING finger protein 11 (RNF11) regulates canonical NF-κB signaling. J Neuroinflammation, 2012, 9: 67. doi: 10.1186/1742-2094-9-67.
23. Pranski EL, Dalal NV, Sanford CV, et al. RING finger protein 11 (RNF11) modulates susceptibility to 6-OHDA-induced nigral degeneration and behavioral deficits through NF-κB signaling in dopaminergic cells. Neurobiol Dis, 2013, 54: 264-279.
24. 池玉磊, 卜宪敏, 查玉梅, 等. 骨髓间充质干细胞复合支架材料治疗骨缺损: 研究现状及前景展望. 中国组织工程研究, 2019, 23(29): 4749-4756.
25. Jo H, Mondal S, Tan D, et al. Small molecule-induced cytosolic activation of protein kinase Akt rescues ischemia-elicited neuronal death. Proc Natl Acad Sci U S A, 2012, 109(26): 10581-10586.
26. Shembade N, Parvatiyar K, Harhaj NS, et al. The ubiquitin-editing enzyme A20 requires RNF11 to downregulate NF-kappaB signalling. EMBO J, 2009, 28(5): 513-522.
27. Zhao SJ, Kong FQ, Jie J, et al. Macrophage MSR1 promotes BMSC osteogenic differentiation and M2-like polarization by activating PI3K/AKT/GSK3β/β-catenin pathway. Theranostics, 2020, 10(1): 17-35.
28. Yang C, Liu X, Zhao K, et al. miRNA-21 promotes osteogenesis via the PTEN/PI3K/Akt/HIF-1α pathway and enhances bone regeneration in critical size defects. Stem Cell Res Ther, 2019, 10(1): 65. doi: 10.1186/s13287-019-1168-2.

Chinese Journal of Reparative and Reconstructive Surgery

Mechanism of ring finger protein 11 regulating Akt signaling pathway to promote osteogenic differentiation of bone marrow mesenchymal stem cells

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