Abstract
Periodontal tissue regeneration engineering based on human periodontal ligament stem cells (hPDLSCs) provides a broad prospect for the treatment of periodontal disease. N-Acetyltransferase 10 (NAT10)-catalyzed non-histone acetylation is widely involved in physiological or pathophysiological processes. However, its function in hPDLSCs is still missing. hPDLSCs were isolated, purified, and cultured from extracted teeth. Surface markers were detected by flow cytometry. Osteogenic, adipogenic, and chondrogenic differentiation potential was detected by alizarin red staining (ARS), oil red O staining, and Alcian blue staining. Alkaline phosphatase (ALP) activity was assessed by ALP assay. Quantitative real-time PCR (qRT-PCR) and western blot were used to detect the expression of key molecules, such as NAT10, Vascular endothelial growth factor A (VEGFA), PI3K/AKT pathway, as well as bone markers (RUNX2, OCN, OPN). RNA-Binding Protein Immunoprecipitation PCR (RIP-PCR) was used to detect the N4-acetylcytidine (ac4C) mRNA level. Genes related to VEGFA were identified by bioinformatics analysis. NAT10 was highly expressed in the osteogenic differentiation process with enhanced ALP activity and osteogenic capability, and elevated expression of osteogenesis-related markers. The ac4C level and expression of VEGFA were obviously regulated by NAT10 and overexpression of VEGFA also had similar effects to NAT10. The phosphorylation level of PI3K and AKT was also elevated by overexpression of VEGFA. VEGFA could reverse the effects of NAT10 in hPDLSCs. NAT10 enhances the osteogenic development of hPDLSCs via regulation of the VEGFA-mediated PI3K/AKT signaling pathway by ac4C alteration.
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Abbreviations
- hPDLSCs:
-
Human periodontal ligament stem cells
- NAT10:
-
N-Acetyltransferase 10
- ALP:
-
Alkaline phosphatase
- qRT-PCR:
-
Quantitative real-time PCR
- VEGFA:
-
Vascular endothelial growth factor A
- RIP-PCR:
-
RNA-Binding Protein Immunoprecipitation PCR
- ac4C:
-
N4-Acetylcytidine
- MSCs:
-
Mesenchymal stem cells
- ARS:
-
Alizarin red staining
- SD:
-
Standard deviation
- ANOVA:
-
One-way analysis of variance
References
Arango D, Sturgill D, Alhusaini N, Dillman AA, Sweet TJ, Hanson G, Hosogane M, Sinclair WR, Nanan KK, Mandler MD, Fox SD, Zengeya TT, Andresson T, Meier JL, Coller J, Oberdoerffer S. Acetylation of cytidine in mRNA promotes translation efficiency. Cell. 2018;175(7):1872–86. https://doi.org/10.1016/j.cell.2018.10.030.
Balmus G, Larrieu D, Barros AC, Collins C, Abrudan M, Demir M, Geisler NJ, Lelliott CJ, White JK, Karp NA, Atkinson J, Kirton A, Jacobsen M, Clift D, Rodriguez R, Adams DJ, Jackson SP. Targeting of NAT10 enhances healthspan in a mouse model of human accelerated aging syndrome. Nat Commun. 2018;9(1):1700. https://doi.org/10.1038/s41467-018-03770-3.
Cai S, Liu X, Zhang C, Xing B, Du X. Autoacetylation of NAT10 is critical for its function in rRNA transcription activation. Biochem Biophys Res Commun. 2017;483(1):624–9. https://doi.org/10.1016/j.bbrc.2016.12.092.
Chen Q, Liu X, Wang D, Zheng J, Chen L, Xie Q, Liu X, Niu S, Qu G, Lan J, Li J, Yang C, Zou D. Periodontal inflammation-triggered by periodontal ligament stem cell pyroptosis exacerbates periodontitis. Front Cell Dev Biol. 2021;9:663037. https://doi.org/10.3389/fcell.2021.663037.
Cheng BF, Feng X, Gao YX, Jian SQ, Liu SR, Wang M, Xie YF, Wang L, Feng ZW, Yang HJ. Neural cell adhesion molecule regulates osteoblastic differentiation through Wnt/beta-catenin and PI3K-Akt signaling pathways in MC3T3-E1 Cells. Front Endocrinol. 2021;12:657953. https://doi.org/10.3389/fendo.2021.657953.
Fei X, Cai Y, Lin F, Huang Y, Liu T, Liu Y. Amniotic fluid mesenchymal stem cells repair mouse corneal cold injury by promoting mRNA N4-acetylcytidine modification and ETV4/JUN/CCND2 signal axis activation. Hum Cell. 2021;34(1):86–98. https://doi.org/10.1007/s13577-020-00442-7.
Froger N, Matonti F, Roubeix C, Forster V, Ivkovic I, Brunel N, Baudouin C, Sahel JA, Picaud S. VEGF is an autocrine/paracrine neuroprotective factor for injured retinal ganglion neurons. Sci Rep. 2020;10(1):12409. https://doi.org/10.1038/s41598-020-68488-z.
Fruman DA, Chiu H, Hopkins BD, Bagrodia S, Cantley LC, Abraham RT. The PI3K pathway in human disease. Cell. 2017;170(4):605–35. https://doi.org/10.1016/j.cell.2017.07.029.
Fu D, Collins K. Purification of human telomerase complexes identifies factors involved in telomerase biogenesis and telomere length regulation. Mol Cell. 2007;28(5):773–85. https://doi.org/10.1016/j.molcel.2007.09.023.
Gibon E, Lu L, Goodman SB. Aging, inflammation, stem cells, and bone healing. Stem Cell Res Ther. 2016;7:44. https://doi.org/10.1186/s13287-016-0300-9.
Han Y, Wang X, Ma D, Wu X, Yang P, Zhang J. Ipriflavone promotes proliferation and osteogenic differentiation of periodontal ligament cells by activating GPR30/PI3K/AKT signaling pathway. Drug Des Devel Ther. 2018;12:137–48. https://doi.org/10.2147/DDDT.S148457.
Hernandez-Monjaraz B, Santiago-Osorio E, Monroy-Garcia A, Ledesma-Martinez E, Mendoza-Nunez VM. Mesenchymal stem cells of dental origin for inducing tissue regeneration in periodontitis: a mini-review. Int J Mol Sci. 2018. https://doi.org/10.3390/ijms19040944.
Hu K, Olsen BR. Osteoblast-derived VEGF regulates osteoblast differentiation and bone formation during bone repair. J Clin Invest. 2016;126(2):509–26. https://doi.org/10.1172/JCI82585.
Ito S, Horikawa S, Suzuki T, Kawauchi H, Tanaka Y, Suzuki T, Suzuki T. Human NAT10 is an ATP-dependent RNA acetyltransferase responsible for N4-acetylcytidine formation in 18 S ribosomal RNA (rRNA). J Biol Chem. 2014;289(52):35724–30. https://doi.org/10.1074/jbc.C114.602698.
Kai D, Prabhakaran MP, Jin G, Tian L, Ramakrishna S. Potential of VEGF-encapsulated electrospun nanofibers for in vitro cardiomyogenic differentiation of human mesenchymal stem cells. J Tissue Eng Regen Med. 2017;11(4):1002–10. https://doi.org/10.1002/term.1999.
Kim BS, Yang SS, You HK, Shin HI, Lee J. Fucoidan-induced osteogenic differentiation promotes angiogenesis by inducing vascular endothelial growth factor secretion and accelerates bone repair. J Tissue Eng Regen Med. 2018;12(3):e1311–24. https://doi.org/10.1002/term.2509.
Li L, Liu F, Huang W, Wang J, Wan Y, Li M, Pang Y, Yin Z. Ricolinostat (ACY-1215) inhibits VEGF expression via PI3K/AKT pathway and promotes apoptosis in osteoarthritic osteoblasts. Biomed Pharmacother. 2019;118:109357. https://doi.org/10.1016/j.biopha.2019.109357.
Liu X, Tan Y, Zhang C, Zhang Y, Zhang L, Ren P, Deng H, Luo J, Ke Y, Du X. NAT10 regulates p53 activation through acetylating p53 at K120 and ubiquitinating Mdm2. EMBO Rep. 2016;17(3):349–66. https://doi.org/10.15252/embr.201540505.
Liu YF, Zhu JJ, Yu TX, Liu H, Zhang T, Zhang YP, Xie SA, Zheng M, Kong W, Yao WJ, Pang W, Zhao CR, Tang YJ, Zhou J. Hypermethylation of mitochondrial DNA in vascular smooth muscle cells impairs cell contractility. Cell Death Dis. 2020;11(1):35. https://doi.org/10.1038/s41419-020-2240-7.
Lui PP, Wong OT, Lee YW. Transplantation of tendon-derived stem cells pre-treated with connective tissue growth factor and ascorbic acid in vitro promoted better tendon repair in a patellar tendon window injury rat model. Cytotherapy. 2016;18(1):99–112. https://doi.org/10.1016/j.jcyt.2015.10.005.
Ma K, Zhang C, Li W. Gamabufotalin suppressed osteosarcoma stem cells through the TGF-beta/periostin/PI3K/AKT pathway. Chem Biol Interact. 2020;331:109275. https://doi.org/10.1016/j.cbi.2020.109275.
Ma Y, Ran D, Zhao H, Song R, Zou H, Gu J, Yuan Y, Bian J, Zhu J, Liu Z. Cadmium exposure triggers osteoporosis in duck via P2X7/PI3K/AKT-mediated osteoblast and osteoclast. Sci Total Enviro. 2021;750:141638. https://doi.org/10.1016/j.scitotenv.2020.141638.
Olsson AK, Dimberg A, Kreuger J, Claesson-Welsh L. VEGF receptor signaling—in control of vascular function. Nat Rev Mol Cell Biol. 2006;7(5):359–71. https://doi.org/10.1038/nrm1911.
Seo BM, Miura M, Gronthos S, Bartold PM, Batouli S, Brahim J, Young M, Robey PG, Wang CY, Shi S. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet. 2004;364(9429):149–55. https://doi.org/10.1016/S0140-6736(04)16627-0.
Shen Q, Zheng X, McNutt MA, Guang L, Sun Y, Wang J, Gong Y, Hou L, Zhang B. NAT10, a nucleolar protein, localizes to the midbody and regulates cytokinesis and acetylation of microtubules. Exp Cell Res. 2009;315(10):1653–67. https://doi.org/10.1016/j.yexcr.2009.03.007.
Shinkaruk S, Bayle M, Lain G, Deleris G. Vascular endothelial cell growth factor (VEGF), an emerging target for cancer chemotherapy. Curr Med Chem Anticancer Agents. 2003;3(2):95–117. https://doi.org/10.2174/1568011033353452.
Sui BD, Hu CH, Liu AQ, Zheng CX, Xuan K, Jin Y. Stem cell-based bone regeneration in diseased microenvironments: challenges and solutions. Biomaterials. 2019;196:18–30. https://doi.org/10.1016/j.biomaterials.2017.10.046.
Sun K, Luo J, Guo J, Yao X, Jing X, Guo F. The PI3K/AKT/mTOR signaling pathway in osteoarthritis: a narrative review. Osteoarthritis Cartilage. 2020;28(4):400–9. https://doi.org/10.1016/j.joca.2020.02.027.
Tan TZ, Miow QH, Huang RY, Wong MK, Ye J, Lau JA, Wu MC, Bin AHL, Soong R, Choolani M, Davidson B, Nesland JM, Wang LZ, Matsumura N, Mandai M, Konishi I, Goh BC, Chang JT, Thiery JP, Mori S. Functional genomics identifies five distinct molecular subtypes with clinical relevance and pathways for growth control in epithelial ovarian cancer [Journal Article]. EMBO Mol Med. 2013;5(7):1051–66. https://doi.org/10.1002/emmm.201201823.
Taylor V, Wong M, Brandts C, Reilly L, Dean NM, Cowsert LM, Moodie S, Stokoe D. 5’ phospholipid phosphatase SHIP-2 causes protein kinase B inactivation and cell cycle arrest in glioblastoma cells. Mol Cell Biol. 2000;20(18):6860–71. https://doi.org/10.1128/MCB.20.18.6860-6871.2000.
Tomokiyo A, Wada N, Maeda H. Periodontal ligament stem cells: regenerative potency in periodontium. Stem Cells Develop. 2019;28(15):974–85. https://doi.org/10.1089/scd.2019.0031.
Trubiani O, Pizzicannella J, Caputi S, Marchisio M, Mazzon E, Paganelli R, Paganelli A, Diomede F. Periodontal ligament stem cells: current knowledge and future perspectives. Stem Cells Develop. 2019;28(15):995–1003. https://doi.org/10.1089/scd.2019.0025.
Yang J, Moraga A, Xu J, Zhao Y, Luo P, Lao KH, Margariti A, Zhao Q, Ding W, Wang G, Zhang M, Zheng L, Zhang Z, Hu Y, Wang W, Shen L, Smith A, Shah AM, Wang Q, Zeng L. A histone deacetylase 7-derived peptide promotes vascular regeneration via facilitating 14–3–3gamma phosphorylation. Stem cells. 2020;38(4):556–73. https://doi.org/10.1002/stem.3122.
Yang W, Li HY, Wu YF, Mi RJ, Liu WZ, Shen X, Lu YX, Jiang YH, Ma MJ, Shen HY. ac4C acetylation of RUNX2 catalyzed by NAT10 spurs osteogenesis of BMSCs and prevents ovariectomy-induced bone loss. Mol Ther Nucleic Acids. 2021;26:135–47. https://doi.org/10.1016/j.omtn.2021.06.022.
Ying Y, Luo J. Salidroside promotes human periodontal ligament cell proliferation and osteocalcin secretion via ERK1/2 and PI3K/Akt signaling pathways. Exp Ther Med. 2018;15(6):5041–5. https://doi.org/10.3892/etm.2018.6006.
Zafari F, Shirian S, Sadeghi M, Teimourian S, Bakhtiyari M. CD93 hematopoietic stem cells improve diabetic wound healing by VEGF activation and downregulation of DAPK-1. J Cell Physiol. 2020;235(3):2366–76. https://doi.org/10.1002/jcp.29142.
Zhang Y, Xing Y, Jia L, Ji Y, Zhao B, Wen Y, Xu X. An In Vitro comparative study of multisource derived human mesenchymal stem cells for bone tissue engineering. Stem Cells Develop. 2018;27(23):1634–45. https://doi.org/10.1089/scd.2018.0119.
Zheng C, Chen J, Liu S, Jin Y. Stem cell-based bone and dental regeneration: a view of microenvironmental modulation. Int J Oral Sci. 2019;11(3):23. https://doi.org/10.1038/s41368-019-0060-3.
Zhu Z, Xing X, Huang S, Tu Y. NAT10 promotes osteogenic differentiation of mesenchymal stem cells by mediating N4-Acetylcytidine modification of Gremlin 1. Stem Cells Int. 2021;2021:8833527. https://doi.org/10.1155/2021/8833527.
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We would like to give our sincere gratitude to the reviewers for their constructive comments.
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This work was supported by the role of ETS2-NAT10 in promoting osteogenic differentiation of BMSCs by activating PRP (2022JC085).
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10266_2023_793_MOESM1_ESM.tif
Supplementary file 1: Fig. S1. Mutated NAT10 reduced ac4C level and inhibits osteogenesis. A. RIP was conducted to detect the ac4C level in the control and NAT10-KR group. B. Western blot was conducted to detect Runx2, OCN, and OPN. There were more than three of these duplicates. Mean ± SD was used to express the data. *P <0.05, **P< 0.01, and ***P< 0.001 are all considered significant.
10266_2023_793_MOESM2_ESM.tif
Supplementary file 2: Fig. S2. NAT10 stimulates the phosphorylation of PI3K and AKT. A. Western blot was performed to detect the expression of p-PI3K, PI3K, p-AKT, and AKT. There were more than three of these duplicates. Mean ± SD was used to express the data. *P <0.05, **P< 0.01, and ***P< 0.001 are all considered significant.
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Cui, Z., Xu, Y., Wu, P. et al. NAT10 promotes osteogenic differentiation of periodontal ligament stem cells by regulating VEGFA-mediated PI3K/AKT signaling pathway through ac4C modification. Odontology 111, 870–882 (2023). https://doi.org/10.1007/s10266-023-00793-1
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DOI: https://doi.org/10.1007/s10266-023-00793-1