LGR5 enhances the osteoblastic differentiation of MC3T3‑E1 cells through the Wnt/β‑catenin pathway

Yu,Wei; Xie,Chao-Ran; Chen,Fan-Cheng; Cheng,Pei; Yang,Lei; Pan,Xiao-Yun

doi:10.3892/etm.2021.10321

LGR5 enhances the osteoblastic differentiation of MC3T3‑E1 cells through the Wnt/β‑catenin pathway

Authors:
- Wei Yu
- Chao-Ran Xie
- Fan-Cheng Chen
- Pei Cheng
- Lei Yang
- Xiao-Yun Pan
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Affiliations: Department of Orthopedic Surgery, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325000, P.R. China, Institute of Advanced Materials for Nano‑Bio Applications, School of Ophthalmology and Optometry, Wenzhou Medical University, Wenzhou, Zhejiang 325000, P.R. China, Department of Orthopedic Surgery, Zhongshan Hospital, Fudan University, Shanghai 200433, P.R. China
Published online on: June 17, 2021 https://doi.org/10.3892/etm.2021.10321
Article Number: 889
Copyright: © Yu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Leucine‑rich repeat‑containing G‑protein coupled receptor 5 (LGR5) is a Wnt‑associated gene that contributes to cell proliferation and self‑renewal in various organs. LGR5 is expressed in Ewing sarcoma, and LGR5‑overexpressing mesenchymal stem cells promote fracture healing. However, the effects of LGR5 on osteoblastic differentiation remain unclear. The aim of the present study was to explore the function of LGR5 in osteoblastic differentiation. LGR5 was overexpressed or knocked down in the MC3T3‑E1 pre‑osteoblastic cell line via lentiviral transfection and its function in osteoblastic differentiation was investigated. The mRNA expression levels of the osteoblast differentiation markers alkaline phosphatase (ALP), osteocalcin and collagen type I a1 were determined, and ALP and Alizarin red staining were performed. In addition, the effects of LGR5 modulation on β‑catenin and the expression of target genes in the Wnt pathway were investigated. The results revealed that the overexpression of LGR5 promoted osteoblastic differentiation. This was associated with enhancement of the stability of β‑catenin and its levels in the cell nucleus, which enabled it to activate Wnt signaling. By contrast, the inhibition of LGR5 decreased the osteogenic capacity of MC3T3‑E1 cells. These results indicate that LGR5 is a positive regulator of osteoblastic differentiation, whose effects are mediated through the Wnt/β‑catenin signaling pathway. This suggests suggesting that the regulation of LGR5/Wnt/β‑catenin signaling has potential as a therapy for osteoporosis.

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1	Jiang M, Wang T, Yan X, Liu Z, Yan Y, Yang K, Qi J, Zhou H, Qian N, Zhou Q, et al: A novel rhein derivative modulates bone formation and resorption and ameliorates estrogen-dependent bone loss. J Bone Miner Res. 34:361–374. 2019.PubMed/NCBI View Article : Google Scholar
2	Yadav VK, Balaji S, Suresh PS, Liu XS, Lu X, Li Z, Guo XE, Mann JJ, Balapure AK, Gershon MD, et al: Pharmacological inhibition of gut-derived serotonin synthesis is a potential bone anabolic treatment for osteoporosis. Nat Med. 16:308–312. 2010.PubMed/NCBI View Article : Google Scholar
3	Black DM and Rosen CJ: Clinical practice. Postmenopausal osteoporosis. N Engl J Med. 374:254–262. 2016.PubMed/NCBI View Article : Google Scholar
4	Canalis E: Management of endocrine disease: Novel anabolic treatments for osteoporosis. Eur J Endocrinol. 178:R33–R44. 2018.PubMed/NCBI View Article : Google Scholar
5	Riggs BL and Hartmann LC: Selective estrogen-receptor modulators-mechanisms of action and application to clinical practice. N Engl J Med. 348:618–629. 2003.PubMed/NCBI View Article : Google Scholar
6	Leder BZ: Parathyroid hormone and parathyroid hormone-related protein analogs in osteoporosis therapy. Curr Osteoporos Rep. 15:110–119. 2017.PubMed/NCBI View Article : Google Scholar
7	Luo J, Zhou W, Zhou X, Li D, Weng J, Yi Z, Cho SG, Li C, Yi T, Wu X, et al: Regulation of bone formation and remodeling by G-protein-coupled receptor 48. Development. 136:2747–2756. 2009.PubMed/NCBI View Article : Google Scholar
8	Xu JG, Huang C, Yang Z, Jin M, Fu P, Zhang N, Luo J, Li D, Liu M, Zhou Y and Zhu Y: Crystal structure of LGR4-Rspo1 complex: Insights into the divergent mechanisms of ligand recognition by leucine-rich repeat G-protein-coupled receptors (LGRs). J Biol Chem. 290:2455–2465. 2015.PubMed/NCBI View Article : Google Scholar
9	de Lau W, Barker N, Low TY, Koo BK, Li VS, Teunissen H, Kujala P, Haegebarth A, Peters PJ, van de Wetering M, et al: Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling. Nature. 476:293–297. 2011.PubMed/NCBI View Article : Google Scholar
10	Jaks V, Barker N, Kasper M, van Es JH, Snippert HJ, Clevers H and Toftgård R: Lgr5 marks cycling, yet long-lived, hair follicle stem cells. Nat Genet. 40:1291–1299. 2008.PubMed/NCBI View Article : Google Scholar
11	Barker N, Huch M, Kujala P, van de Wetering M, Snippert HJ, van Es JH, Sato T, Stange DE, Begthel H, van den Born M, et al: Lgr5(+ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro. Cell Stem Cell. 6:25–36. 2010.PubMed/NCBI View Article : Google Scholar
12	Barker N, van Es JH, Kuipers J, Kujala P, van den Born M, Cozijnsen M, Haegebarth A, Korving J, Begthel H, Peters PJ and Clevers H: Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature. 449:1003–1007. 2007.PubMed/NCBI View Article : Google Scholar
13	Morita H, Mazerbourg S, Bouley DM, Luo CW, Kawamura K, Kuwabara Y, Baribault H, Tian H and Hsueh AJ: Neonatal lethality of LGR5 null mice is associated with ankyloglossia and gastrointestinal distension. Mol Cell Biol. 24:9736–9743. 2004.PubMed/NCBI View Article : Google Scholar
14	Kemper K, Prasetyanti PR, De Lau W, Rodermond H, Clevers H and Medema JP: Monoclonal antibodies against Lgr5 identify human colorectal cancer stem cells. Stem Cells. 30:2378–2386. 2012.PubMed/NCBI View Article : Google Scholar
15	Tanese K, Fukuma M, Yamada T, Mori T, Yoshikawa T, Watanabe W, Ishiko A, Amagai M, Nishikawa T and Sakamoto M: G-protein-coupled receptor GPR49 is up-regulated in basal cell carcinoma and promotes cell proliferation and tumor formation. Am J Pathol. 173:835–843. 2008.PubMed/NCBI View Article : Google Scholar
16	Nakata S, Campos B, Bageritz J, Bermejo JL, Becker N, Engel F, Acker T, Momma S, Herold-Mende C, Lichter P, et al: LGR5 is a marker of poor prognosis in glioblastoma and is required for survival of brain cancer stem-like cells. Brain Pathol. 23:60–72. 2013.PubMed/NCBI View Article : Google Scholar
17	Vieira GC, Chockalingam S, Melegh Z, Greenhough A, Malik S, Szemes M, Park JH, Kaidi A, Zhou L, Catchpoole D, et al: LGR5 regulates pro-survival MEK/ERK and proliferative Wnt/beta-catenin signalling in neuroblastoma. Oncotarget. 6:40053–40067. 2015.PubMed/NCBI View Article : Google Scholar
18	Zhu C, Zheng XF, Yang YH, Li B, Wang YR, Jiang SD and Jiang LS: LGR4 acts as a key receptor for R-spondin 2 to promote osteogenesis through Wnt signaling pathway. Cell Signal. 28:989–1000. 2016.PubMed/NCBI View Article : Google Scholar
19	Liu SL, Zhou YM, Tang DB, Zhou N, Zheng WW, Tang ZH, Duan CW, Zheng L and Chen J: LGR6 promotes osteogenesis by activating the Wnt/β-catenin signaling pathway. Biochem Biophys Res Commun. 519:1–7. 2019.PubMed/NCBI View Article : Google Scholar
20	Scannell CA, Pedersen EA, Mosher JT, Krook MA, Nicholls LA, Wilky BA, Loeb DM and Lawlor ER: LGR5 is expressed by ewing sarcoma and potentiates Wnt/β-catenin signaling. Front Oncol. 3(81)2013.PubMed/NCBI View Article : Google Scholar
21	Lin W, Xu L, Pan Q, Lin S, Feng L, Wang B, Chen S, Li Y, Wang H, Li Y, et al: Lgr5-overexpressing mesenchymal stem cells augment fracture healing through regulation of Wnt/ERK signaling pathways and mitochondrial dynamics. FASEB J. 33:8565–8577. 2019.PubMed/NCBI View Article : Google Scholar
22	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
23	Krishnan V, Bryant HU and Macdougald OA: Regulation of bone mass by Wnt signaling. J Clin Invest. 116:1202–1209. 2006.PubMed/NCBI View Article : Google Scholar
24	Carmon KS, Gong X, Lin Q, Thomas A and Liu Q: R-spondins function as ligands of the orphan receptors LGR4 and LGR5 to regulate Wnt/beta-catenin signaling. Proc Natl Acad Sci USA. 108:11452–11457. 2011.PubMed/NCBI View Article : Google Scholar
25	Glinka A, Dolde C, Kirsch N, Huang YL, Kazanskaya O, Ingelfinger D, Boutros M, Cruciat CM and Niehrs C: LGR4 and LGR5 are R-spondin receptors mediating Wnt/β-catenin and Wnt/PCP signalling. EMBO Rep. 12:1055–1061. 2011.PubMed/NCBI View Article : Google Scholar
26	Gong X, Carmon KS, Lin Q, Thomas A, Yi J and Liu Q: LGR6 is a high affinity receptor of R-spondins and potentially functions as a tumor suppressor. PLoS One. 7(e37137)2012.PubMed/NCBI View Article : Google Scholar
27	Yoon JK and Lee JS: Cellular signaling and biological functions of R-spondins. Cell Signal. 24:369–377. 2012.PubMed/NCBI View Article : Google Scholar
28	Yamada W, Nagao K, Horikoshi K, Fujikura A, Ikeda E, Inagaki Y, Kakitani M, Tomizuka K, Miyazaki H, Suda T and Takubo K: Craniofacial malformation in R-spondin2 knockout mice. Biochem Biophys Res Commun. 381:453–458. 2009.PubMed/NCBI View Article : Google Scholar
29	Zhang H, Han X, Wei B, Fang J, Hou X, Lan T and Wei H: RSPO2 enhances cell invasion and migration via the WNT/β-catenin pathway in human gastric cancer. J Cell Biochem. 120:5813–5824. 2019.PubMed/NCBI View Article : Google Scholar
30	Friedman MS, Oyserman SM and Hankenson KD: Wnt11 promotes osteoblast maturation and mineralization through R-spondin 2. J Biol Chem. 284:14117–14125. 2009.PubMed/NCBI View Article : Google Scholar
31	Leucht P, Minear S, Ten Berge D, Nusse R and Helms JA: Translating insights from development into regenerative medicine: The function of Wnts in bone biology. Semin Cell Dev Biol. 19:434–443. 2008.PubMed/NCBI View Article : Google Scholar
32	Baron R and Kneissel M: WNT signaling in bone homeostasis and disease: From human mutations to treatments. Nat Med. 19:179–192. 2013.PubMed/NCBI View Article : Google Scholar
33	Barker N and Clevers H: Leucine-rich repeat-containing G-protein-coupled receptors as markers of adult stem cells. Gastroenterology. 138:1681–1696. 2010.PubMed/NCBI View Article : Google Scholar
34	Liu D, He XC, Qian P, Barker N, Trainor PA, Clevers H, Liu H and Li L: Leucine-rich repeat-containing G-protein-coupled receptor 5 marks short-term hematopoietic stem and progenitor cells during mouse embryonic development. J Biol Chem. 289:23809–23816. 2014.PubMed/NCBI View Article : Google Scholar
35	Plaks V, Brenot A, Lawson DA, Linnemann JR, Van Kappel EC, Wong KC, de Sauvage F, Klein OD and Werb Z: Lgr5-expressing cells are sufficient and necessary for postnatal mammary gland organogenesis. Cell Rep. 3:70–78. 2013.PubMed/NCBI View Article : Google Scholar
36	Xu L, Lin W, Wen L and Li G: Lgr5 in cancer biology: Functional identification of Lgr5 in cancer progression and potential opportunities for novel therapy. Stem Cell Res Ther. 10(219)2019.PubMed/NCBI View Article : Google Scholar
37	Luo J, Yang Z, Ma Y, Yue Z, Lin H, Qu G, Huang J, Dai W, Li C, Zheng C, et al: LGR4 is a receptor for RANKL and negatively regulates osteoclast differentiation and bone resorption. Nat Med. 22:539–546. 2016.PubMed/NCBI View Article : Google Scholar