USP36 regulates the proliferation, survival, and differentiation of hFOB1.19 osteoblast.
HFOB1.19 cells
Osteoblast differentiation
USP36
Ubiquitin-specific proteases (USPs)
WDR5
Journal
Journal of orthopaedic surgery and research
ISSN: 1749-799X
Titre abrégé: J Orthop Surg Res
Pays: England
ID NLM: 101265112
Informations de publication
Date de publication:
17 Aug 2024
17 Aug 2024
Historique:
received:
21
05
2024
accepted:
02
07
2024
medline:
17
8
2024
pubmed:
17
8
2024
entrez:
16
8
2024
Statut:
epublish
Résumé
Effective bone formation relies on osteoblast differentiation, a process subject to intricate post-translational regulation. Ubiquitin-specific proteases (USPs) repress protein degradation mediated by the ubiquitin-proteasome pathway. Several USPs have been documented to regulate osteoblast differentiation, but whether other USPs are involved in this process remains elusive. In this study, we conducted a comparative analysis of 48 USPs in differentiated and undifferentiated hFOB1.19 osteoblasts, identifying significantly upregulated USPs. Subsequently, we generated USP knockdown hFOB1.19 cells and evaluated their osteogenic differentiation using Alizarin red staining. We also assessed cell viability, cell cycle progression, and apoptosis through MTT, 7-aminoactinomycin D staining, and Annexin V/PI staining assays, respectively. Quantitative PCR and Western blotting were employed to measure the expression levels of osteogenic differentiation markers. Additionally, we investigated the interaction between the USP and its target protein using co-immunoprecipitation (co-IP). Furthermore, we depleted the USP in hFOB1.19 cells to examine its effect on the ubiquitination and stability of the target protein using immunoprecipitation (IP) and Western blotting. Finally, we overexpressed the target protein in USP-deficient hFOB1.19 cells and evaluated its impact on their osteogenic differentiation using Alizarin red staining. USP36 is the most markedly upregulated USP in differentiated hFOB1.19 osteoblasts. Knockdown of USP36 leads to reduced viability, cell cycle arrest, heightened apoptosis, and impaired osteogenic differentiation in hFOB1.19 cells. USP36 interacts with WD repeat-containing protein 5 (WDR5), and the knockdown of USP36 causes an increased level of WDR5 ubiquitination and accelerated degradation of WDR5. Excessive WDR5 improved the impaired osteogenic differentiation of USP36-deficient hFOB1.19 cells. These observations suggested that USP36 may function as a key regulator of osteoblast differentiation, and its regulatory mechanism may be related to the stabilization of WDR5.
Sections du résumé
BACKGROUND
BACKGROUND
Effective bone formation relies on osteoblast differentiation, a process subject to intricate post-translational regulation. Ubiquitin-specific proteases (USPs) repress protein degradation mediated by the ubiquitin-proteasome pathway. Several USPs have been documented to regulate osteoblast differentiation, but whether other USPs are involved in this process remains elusive.
METHODS
METHODS
In this study, we conducted a comparative analysis of 48 USPs in differentiated and undifferentiated hFOB1.19 osteoblasts, identifying significantly upregulated USPs. Subsequently, we generated USP knockdown hFOB1.19 cells and evaluated their osteogenic differentiation using Alizarin red staining. We also assessed cell viability, cell cycle progression, and apoptosis through MTT, 7-aminoactinomycin D staining, and Annexin V/PI staining assays, respectively. Quantitative PCR and Western blotting were employed to measure the expression levels of osteogenic differentiation markers. Additionally, we investigated the interaction between the USP and its target protein using co-immunoprecipitation (co-IP). Furthermore, we depleted the USP in hFOB1.19 cells to examine its effect on the ubiquitination and stability of the target protein using immunoprecipitation (IP) and Western blotting. Finally, we overexpressed the target protein in USP-deficient hFOB1.19 cells and evaluated its impact on their osteogenic differentiation using Alizarin red staining.
RESULTS
RESULTS
USP36 is the most markedly upregulated USP in differentiated hFOB1.19 osteoblasts. Knockdown of USP36 leads to reduced viability, cell cycle arrest, heightened apoptosis, and impaired osteogenic differentiation in hFOB1.19 cells. USP36 interacts with WD repeat-containing protein 5 (WDR5), and the knockdown of USP36 causes an increased level of WDR5 ubiquitination and accelerated degradation of WDR5. Excessive WDR5 improved the impaired osteogenic differentiation of USP36-deficient hFOB1.19 cells.
CONCLUSIONS
CONCLUSIONS
These observations suggested that USP36 may function as a key regulator of osteoblast differentiation, and its regulatory mechanism may be related to the stabilization of WDR5.
Identifiants
pubmed: 39152465
doi: 10.1186/s13018-024-04893-8
pii: 10.1186/s13018-024-04893-8
doi:
Substances chimiques
USP36 protein, human
0
Ubiquitin Thiolesterase
EC 3.4.19.12
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
483Subventions
Organisme : Xiamen Humanity Hospital
ID : No. 3502Z20214ZD1094
Informations de copyright
© 2024. The Author(s).
Références
Henry JP, Bordoni B, Histology. Osteoblasts. StatPearls. Treasure Island (FL)2023.
Marie PJ. Osteoblast dysfunctions in bone diseases: from cellular and molecular mechanisms to therapeutic strategies. Cell Mol Life Sci. 2015;72(7):1347–61. https://doi.org/10.1007/s00018-014-1801-2 . Epub 20141209.
doi: 10.1007/s00018-014-1801-2
pubmed: 25487608
Rutkovskiy A, Stenslokken KO, Vaage IJ. Osteoblast differentiation at a glance. Med Sci Monit Basic Res. 2016;22:95–106. Epub 20160926. doi: 10.12659/msmbr.901142. PubMed PMID: 27667570; PubMed Central PMCID: PMCPMC5040224.
doi: 10.12659/MSMBR.901142
pubmed: 27667570
pmcid: 5040224
Gaur T, Lengner CJ, Hovhannisyan H, Bhat RA, Bodine PV, Komm BS, et al. Canonical WNT signaling promotes osteogenesis by directly stimulating Runx2 gene expression. J Biol Chem. 2005;280(39):33132–40. https://doi.org/10.1074/jbc.M500608200 . Epub 20050725.
doi: 10.1074/jbc.M500608200
pubmed: 16043491
Phimphilai M, Zhao Z, Boules H, Roca H, Franceschi RT. BMP signaling is required for RUNX2-dependent induction of the osteoblast phenotype. J Bone Min Res. 2006;21(4):637–46. https://doi.org/10.1359/jbmr.060109 . Epub 20060405.
doi: 10.1359/jbmr.060109
Shimoyama A, Wada M, Ikeda F, Hata K, Matsubara T, Nifuji A, et al. Ihh/Gli2 signaling promotes osteoblast differentiation by regulating Runx2 expression and function. Mol Biol Cell. 2007;18(7):2411–8. https://doi.org/10.1091/mbc.e06-08-0743 . Epub 20070418.
doi: 10.1091/mbc.e06-08-0743
pubmed: 17442891
pmcid: 1924839
Wang BL, Dai CL, Quan JX, Zhu ZF, Zheng F, Zhang HX, et al. Parathyroid hormone regulates osterix and Runx2 mRNA expression predominantly through protein kinase A signaling in osteoblast-like cells. J Endocrinol Invest. 2006;29(2):101–8. https://doi.org/10.1007/BF03344081 . PubMed PMID: 16610234.
doi: 10.1007/BF03344081
pubmed: 16610234
Battaglino R, Kim D, Fu J, Vaage B, Fu XY, Stashenko P. c-myc is required for osteoclast differentiation. J Bone Min Res. 2002;17(5):763–73. .763. PubMed PMID: 12009006.
doi: 10.1359/jbmr.2002.17.5.763
Zhang C, Cho K, Huang Y, Lyons JP, Zhou X, Sinha K, et al. Inhibition of wnt signaling by the osteoblast-specific transcription factor Osterix. Proc Natl Acad Sci U S A. 2008;105(19):6936–41. https://doi.org/10.1073/pnas.0710831105 . Epub 20080505.
doi: 10.1073/pnas.0710831105
pubmed: 18458345
pmcid: 2383965
Mulder MP, Witting K, Berlin I, Pruneda JN, Wu KP, Chang JG, et al. A cascading activity-based probe sequentially targets E1-E2-E3 ubiquitin enzymes. Nat Chem Biol. 2016;12(7):523–30. https://doi.org/10.1038/nchembio.2084 . Epub 2016/05/18.
doi: 10.1038/nchembio.2084
pubmed: 27182664
pmcid: 5108872
Nijman SM, Luna-Vargas MP, Velds A, Brummelkamp TR, Dirac AM, Sixma TK, et al. A genomic and functional inventory of deubiquitinating enzymes. Cell. 2005;123(5):773–86. PubMed PMID: 16325574.
doi: 10.1016/j.cell.2005.11.007
pubmed: 16325574
Scheffner M, Nuber U, Huibregtse JM. Protein ubiquitination involving an E1-E2-E3 enzyme ubiquitin thioester cascade. Nature. 1995;373(6509):81–3. https://doi.org/10.1038/373081a0 . Epub 1995/01/05.
doi: 10.1038/373081a0
pubmed: 7800044
Zhou F, Li F, Fang P, Dai T, Yang B, van Dam H, et al. Ubiquitin-specific protease 4 antagonizes osteoblast differentiation through dishevelled. J Bone Min Res. 2016;31(10):1888–98. https://doi.org/10.1002/jbmr.2863 . Epub 20160520.
doi: 10.1002/jbmr.2863
Greenblatt MB, Shin DY, Oh H, Lee KY, Zhai B, Gygi SP, et al. MEKK2 mediates an alternative beta-catenin pathway that promotes bone formation. Proc Natl Acad Sci U S A. 2016;113(9):E1226–35. https://doi.org/10.1073/pnas.1600813113 . Epub 20160216.
doi: 10.1073/pnas.1600813113
pubmed: 26884171
pmcid: 4780654
Guo YC, Wang MY, Zhang SW, Wu YS, Zhou CC, Zheng RX et al. Ubiquitin-specific protease USP34 controls osteogenic differentiation and bone formation by regulating BMP2 signaling. EMBO J. 2018;37(20). Epub 20180904. https://doi.org/10.15252/embj.201899398 . PubMed PMID: 30181118; PubMed Central PMCID: PMCPMC6187217.
Kim JM, Yang YS, Park KH, Ge X, Xu R, Li N, et al. A RUNX2 stabilization pathway mediates physiologic and pathologic bone formation. Nat Commun. 2020;11(1):2289. https://doi.org/10.1038/s41467-020-16038-6 . Epub 20200508.
doi: 10.1038/s41467-020-16038-6
pubmed: 32385263
pmcid: 7210266
Ji L, Lu B, Zamponi R, Charlat O, Aversa R, Yang Z, et al. USP7 inhibits Wnt/beta-catenin signaling through promoting stabilization of Axin. Nat Commun. 2019;10(1):4184. https://doi.org/10.1038/s41467-019-12143-3 . Epub 20190913.
doi: 10.1038/s41467-019-12143-3
pubmed: 31519875
pmcid: 6744515
Li J, Hu L, Liu Y, Huang L, Mu Y, Cai X et al. DDX19A Senses Viral RNA and Mediates NLRP3-Dependent Inflammasome Activation. Journal of immunology (Baltimore, Md: 1950). 2015;195(12):5732-49. Epub 2015/11/06. https://doi.org/10.4049/jimmunol.1501606 . PubMed PMID: 26538395.
Mizerska-Kowalska M, Slawinska-Brych A, Kalawaj K, Zurek A, Pawinska B, Rzeski W et al. Betulin Promotes Differentiation of Human Osteoblasts In Vitro and Exerts an Osteoinductive Effect on the hFOB 1.19 Cell Line Through Activation of JNK, ERK1/2, and mTOR Kinases. Molecules. 2019;24(14). Epub 20190719. https://doi.org/10.3390/molecules24142637 . PubMed PMID: 31331121; PubMed Central PMCID: PMCPMC6680433.
Harris SA, Enger RJ, Riggs BL, Spelsberg TC. Development and characterization of a conditionally immortalized human fetal osteoblastic cell line. J Bone Min Res. 1995;10(2):178–86. .5650100203. PubMed PMID: 7754797.
doi: 10.1002/jbmr.5650100203
Komori T. Regulation of proliferation, differentiation and functions of osteoblasts by Runx2. Int J Mol Sci. 2019;20(7):1694. https://doi.org/10.3390/ijms20071694 . Epub 2019/04/17.
doi: 10.3390/ijms20071694
pubmed: 30987410
pmcid: 6480215
Karsenty G. Minireview: transcriptional control of osteoblast differentiation. Endocrinology. 2001;142(7):2731–3. https://doi.org/10.1210/endo.142.7.8306 . Epub 2001/06/21.
doi: 10.1210/endo.142.7.8306
pubmed: 11415989
Oughtred R, Rust J, Chang C, Breitkreutz BJ, Stark C, Willems A, et al. The BioGRID database: a comprehensive biomedical resource of curated protein, genetic, and chemical interactions. Protein Sci. 2021;30(1):187–200. Epub 20201123. doi: 10.1002/pro.3978. PubMed PMID: 33070389; PubMed Central PMCID: PMCPMC7737760.
doi: 10.1002/pro.3978
pubmed: 33070389
Gori F, Friedman LG, Demay MB. Wdr5, a WD-40 protein, regulates osteoblast differentiation during embryonic bone development. Dev Biol. 2006;295(2):498–506. https://doi.org/10.1016/j.ydbio.2006.02 . Epub 20060530.
doi: 10.1016/j.ydbio.2006.02
pubmed: 16730692
Zhu ED, Demay MB, Gori F. Wdr5 is essential for osteoblast differentiation. J Biol Chem. 2008;283(12):7361-7. Epub 20080116. https://doi.org/10.1074/jbc.M703304200 . PubMed PMID: 18201971.
Young MJ, Hsu KC, Lin TE, Chang WC, Hung JJ. The role of ubiquitin-specific peptidases in cancer progression. J Biomed Sci. 2019;26(1):42. https://doi.org/10.1186/s12929-019-0522-0 . Epub 20190527.
doi: 10.1186/s12929-019-0522-0
pubmed: 31133011
pmcid: 6537419
Li Y, Reverter D. Molecular mechanisms of DUBs Regulation in Signaling and Disease. Int J Mol Sci. 2021;22(3). Epub 20210120. doi: 10.3390/ijms22030986. PubMed PMID: 33498168; PubMed Central PMCID: PMCPMC7863924.
Hariri H, St-Arnaud R. Expression and role of ubiquitin-specific peptidases in Osteoblasts. Int J Mol Sci. 2021;22(14). Epub 20210720. doi: 10.3390/ijms22147746. PubMed PMID: 34299363; PubMed Central PMCID: PMCPMC8304380.
Kim MS, Kim YK, Kim YS, Seong M, Choi JK, Baek KH. Deubiquitinating enzyme USP36 contains the PEST motif and is polyubiquitinated. Biochem Biophys Res Commun. 2005;330(3):797–804. https://doi.org/10.1016/j.bbrc.2005.03.051 . Epub 2005/04/06.
doi: 10.1016/j.bbrc.2005.03.051
pubmed: 15809067
Chang G, Xie GS, Ma L, Li P, Li L, Richard HT. USP36 promotes tumorigenesis and drug sensitivity of glioblastoma by deubiquitinating and stabilizing ALKBH5. Neurooncology. 2023;25(5):841–53. https://doi.org/10.1093/neuonc/noac238 . Epub 2022/10/15.
doi: 10.1093/neuonc/noac238
Kim MS, Ramakrishna S, Lim KH, Kim JH, Baek KH. Protein stability of mitochondrial superoxide dismutase SOD2 is regulated by USP36. J Cell Biochem. 2011;112(2):498–508. https://doi.org/10.1002/jcb.22940 . Epub 2011/01/27.
doi: 10.1002/jcb.22940
pubmed: 21268071
Sun XX, He X, Yin L, Komada M, Sears RC, Dai MS. The nucleolar ubiquitin-specific protease USP36 deubiquitinates and stabilizes c-Myc. Proc Natl Acad Sci U S A. 2015;112(12):3734–9. https://doi.org/10.1073/pnas.1411713112 . Epub 2015/03/17.
doi: 10.1073/pnas.1411713112
pubmed: 25775507
pmcid: 4378440
Zhang W, Luo J, Xiao Z, Zang Y, Li X, Zhou Y, et al. USP36 facilitates esophageal squamous carcinoma progression via stabilizing YAP. Cell Death Dis. 2022;13(12):1021. https://doi.org/10.1038/s41419-022-05474-5 . Epub 2022/12/06.
doi: 10.1038/s41419-022-05474-5
pubmed: 36470870
pmcid: 9722938
Fraile JM, Campos-Iglesias D, Rodríguez F, Astudillo A, Vilarrasa-Blasi R, Verdaguer-Dot N, et al. Loss of the deubiquitinase USP36 destabilizes the RNA helicase DHX33 and causes preimplantation lethality in mice. J Biol Chem. 2018;293(6):2183–94. https://doi.org/10.1074/jbc.M117.788430 . Epub 2017/12/24.
doi: 10.1074/jbc.M117.788430
pubmed: 29273634
Hanle-Kreidler S, Richter KT, Hoffmann I. The SCF-FBXW7 E3 ubiquitin ligase triggers degradation of histone 3 lysine 4 methyltransferase complex component WDR5 to prevent mitotic slippage. J Biol Chem. 2022;298(12):102703. https://doi.org/10.1016/j.jbc.2022.102703 . Epub 20221114.
doi: 10.1016/j.jbc.2022.102703
pubmed: 36395886
pmcid: 9764181
Wang H, Liu X, Liu Y, Yang C, Ye Y, Sheng N, et al. The E3 ubiquitin ligase RNF220 maintains hindbrain < i > hox expression patterns through regulation of WDR5 stability. Cold Spring Harbor Laboratory; 2023.
Chi Z, Zhang B, Sun R, Wang Y, Zhang L, Xu G. USP44 accelerates the growth of T-cell acute lymphoblastic leukemia through interacting with WDR5 and repressing its ubiquitination. Int J Med Sci. 2022;19(14):2022–32. https://doi.org/10.7150/ijms.74535 . Epub 20221114.
doi: 10.7150/ijms.74535
pubmed: 36483601
pmcid: 9724245