Wnt7a promotes the osteogenic differentiation of human mesenchymal stem cells

Yang,Leiluo; Li,Qing; Zhang,Junhong; Li,Pengcheng; An,Pingjiang; Wang,Chunqing; Hu,Pingsheng; Zou,Xue; Dou,Xiaowei; Zhu,Lixin

doi:10.3892/ijmm.2021.4927

Wnt7a promotes the osteogenic differentiation of human mesenchymal stem cells

Authors:
- Leiluo Yang
- Qing Li
- Junhong Zhang
- Pengcheng Li
- Pingjiang An
- Chunqing Wang
- Pingsheng Hu
- Xue Zou
- Xiaowei Dou
- Lixin Zhu
View Affiliations

Affiliations: Department of Spinal Surgery, Orthopaedic Medical Center, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong 510282, P.R. China, Department of Orthopedics, The Affiliated Hospital of Guizhou Medical University, Guiyang, Guizhou 550001, P.R. China, Department of Pathology, Hebei Eye Hospital, Xingtai, Hebei 054000, P.R. China, Department of Burns and Plastic Surgery, The 8th Medical Center of Chinese PLA General Hospital, Beijing 100091, P.R. China, Clinical Research Center, The Affiliated Hospital of Guizhou Medical University, Guiyang, Guizhou 550001, P.R. China
Published online on: April 6, 2021 https://doi.org/10.3892/ijmm.2021.4927
Article Number: 94
Copyright: © Yang 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

Mesenchymal stem cells (MSCs) have the ability of differentiating into osteoblasts. Elucidating the molecular mechanisms of MSC differentiation into osteoblasts may provide novel therapeutic strategies for bone‑related diseases. Increasing evidence has confirmed that Wnt signaling plays the key role in osteoblast differentiation; however, the role of individual Wnt proteins in osteogenesis needs to be investigated. The present study thus aimed to explore the role of Wnt7a in bone formation. For this purpose, human bone‑derived MSCs were identified by flow cytometry and the cell differentiation potential, including osteogenic and adipogenic differentiation was examined. In order to explore the role of Wnt7a in MSC osteogenic differentiation, Wnt7a expression was measured at the mRNA and protein level following treatment with the osteogenic inducer, bone morphogenetic protein (BMP)4/7, and following the induction of osteogenic or adipogenic differentiation. The ectopic expression of Wnt7a in MSCs was confirmed and its influence on MSC osteogenic differentiation was detected using osteocyte markers and by Alizarin Red S staining. Mechanistically, the influence of Wnt7a on Runt‑related transcription factor 2 (RUNX2) expression was examined at the mRNA and protein level. The regulatory effects of Wnt7a on RUNX2 promoter activities were examined by promoter reporter assay, and by examining the binding of TCF1, a downstream target of Wnt, to the RUNX2 promoter by ChIP assay. The results revealed that the knockdown of Wnt7a in MSCs decreased the expression of osteocyte markers and inhibited osteogenic differentiation. In accordance, the overexpression of Wnt7a in MSCs increased the expression of osteocyte markers and promoted osteogenic differentiation. Mechanistically, the knockdown of Wnt7a in MSCs reduced RUNX2 expression and the overexpression of Wnt7a in MSCs promoted RUNX2 expression. Furthermore, it was confirmed that Wnt7a regulated RUNX2 promoter activities by promoter report assay, and by examining the binding of TCF1 to the RUNX2 promoter by ChIP assay. On the whole, the present study demonstrates that Wnt7a plays a key role in MSC differentiation into osteoblasts and the findings presented herein may provide a promising therapy target for bone‑related diseases.

View Figures

View References

1	Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S and Marshak DR: Multilineage potential of adult human mesenchymal stem cells. Science. 284:143–147. 1999. View Article : Google Scholar : PubMed/NCBI
2	Lin RZ, Moreno-Luna R, Li D, Jaminet SC, Greene AK and Melero-Martin JM: Human endothelial colony-forming cells serve as trophic mediators for mesenchymal stem cell engraftment via paracrine signaling. Proc Natl Acad Sci USA. 111:10137–10142. 2014. View Article : Google Scholar : PubMed/NCBI
3	Caplan AI: Mesenchymal stem cells. J Orthop Res. 9:641–650. 1991. View Article : Google Scholar : PubMed/NCBI
4	Caplan AI and Bruder SP: Mesenchymal stem cells: Building blocks for molecular medicine in the 21st century. Trends Mol Med. 7:259–264. 2001. View Article : Google Scholar : PubMed/NCBI
5	Yang N, Wang G, Hu C, Shi Y, Liao L, Shi S, Cai Y, Cheng S, Wang X, Liu Y, et al: Tumor necrosis factor α suppresses the mesenchymal stem cell osteogenesis promoter miR-21 in estrogen deficiency–induced osteoporosis. J Bone Miner Res. 28:559–573. 2013. View Article : Google Scholar
6	Alford AI, Kozloff KM and Hankenson KD: Extracellular matrix networks in bone remodeling. Int J Biochem Cell Biol. 65:20–31. 2015. View Article : Google Scholar : PubMed/NCBI
7	Beck GR Jr, Khazai NB, Bouloux GF, Camalier CE, Lin Y, Garneys LM, Siqueira J, Peng L, Pasquel F, Umpierrez D, et al: The effects of thiazolidinediones on human bone marrow stromal cell differentiation in vitro and in thiazolidinedione-treated patients with type 2 diabetes. Transl Res. 161:145–155. 2013. View Article : Google Scholar
8	Tang Y, Xie H, Chen J, Geng L, Chen H, Li X, Hou Y, Lu L, Shi S, Zeng X and Sun L: Activated NF-κB in bone marrow mesenchymal stem cells from systemic lupus erythematosus patients inhibits osteogenic differentiation through downregulating Smad signaling. Stem Cells Dev. 22:668–678. 2012. View Article : Google Scholar
9	D'Amelio P, Tamone C, Sassi F, D'Amico L, Roato I, Patanè S, Ravazzoli M, Veneziano L, Ferracini R, Pescarmona GP and Isaia GC: Teriparatide increases the maturation of circulating osteoblast precursors. Osteoporos Int. 23:1245–1253. 2012. View Article : Google Scholar
10	Zhang S, Chen X, Hu Y, Wu J, Cao Q, Chen S and Gao Y: All-trans retinoic acid modulates Wnt3A-induced osteogenic differentiation of mesenchymal stem cells via activating the PI3K/AKT/GSK3β signalling pathway. Mol Cell Endocrinol. 422:243–253. 2016. View Article : Google Scholar : PubMed/NCBI
11	Noronha-Matos JB and Correia-de-Sá P: Mesenchymal stem cells ageing: Targeting the 'Purinome' to promote osteogenic differentiation and bone repair. J Cell Physiol. 231:1852–1861. 2016. View Article : Google Scholar : PubMed/NCBI
12	Liu H, Xia X and Li B: Mesenchymal stem cell aging: Mechanisms and influences on skeletal and non-skeletal tissues. Exp Biol Med (Maywood). 240:1099–1106. 2015. View Article : Google Scholar
13	Anastas JN and Moon RT: WNT signalling pathways as therapeutic targets in cancer. Nat Rev Cancer. 13:11–26. 2013. View Article : Google Scholar
14	Zhou L and Liu Y: Wnt/beta-catenin signalling and podocyte dysfunction in proteinuric kidney disease. Nat Rev Nephrol. 11:535–545. 2015. View Article : Google Scholar : PubMed/NCBI
15	Hynes NE, Ingham PW, Lim WA, Marshall CJ, Massagué J and Pawson T: Signalling change: Signal transduction through the decades. Nat Rev Mol Cell Biol. 14:393–398. 2013. View Article : Google Scholar : PubMed/NCBI
16	Clevers H and Nusse R: Wnt/β-catenin signaling and disease. Cell. 149:1192–1205. 2012. View Article : Google Scholar : PubMed/NCBI
17	Palomo T, Al-Jallad H, Moffatt P, Glorieux FH, Lentle B, Roschger P, Klaushofer K and Rauch F: Skeletal characteristics associated with homozygous and heterozygous WNT1 mutations. Bone. 67:63–70. 2014. View Article : Google Scholar : PubMed/NCBI
18	Laine CM, Joeng KS, Campeau PM, Kiviranta R, Tarkkonen K, Grover M, Lu JT, Pekkinen M, Wessman M, Heino TJ, et al: WNT1 mutations in early-onset osteoporosis and osteogenesis imperfecta. N Engl J Med. 368:1809–1816. 2013. View Article : Google Scholar : PubMed/NCBI
19	Rauch F and Glorieux FH: Osteogenesis imperfecta. Lancet. 363:1377–1385. 2004. View Article : Google Scholar : PubMed/NCBI
20	Keupp K, Beleggia F, Kayserili H, Barnes AM, Steiner M, Semler O, Fischer B, Yigit G, Janda CY, Becker J, et al: Mutations in WNT1 cause different forms of bone fragility. Am J Hum Genet. 92:565–574. 2013. View Article : Google Scholar : PubMed/NCBI
21	Keller KC, Ding H, Tieu R, Sparks NR, Ehnes DD and Zur Nieden NI: Wnt5a supports osteogenic lineage decisions in embryonic stem cells. Stem Cells Dev. 25:1020–1032. 2016. View Article : Google Scholar : PubMed/NCBI
22	Chen J, Tu X, Esen E, Joeng KS, Lin C, Arbeit JM, Rüegg MA, Hall MN, Ma L and Long F: WNT7B promotes bone formation in part through mTORC1. PLoS Genet. 10:e10041452014. View Article : Google Scholar : PubMed/NCBI
23	Tan SH, Senarath-Yapa K, Chung MT, Longaker MT, Wu JY and Nusse R: Wnts produced by Osterix-expressing osteolineage cells regulate their proliferation and differentiation. Proc Natl Acad Sci USA. 111:E5262–E5271. 2014. View Article : Google Scholar : PubMed/NCBI
24	Cawthorn WP, Bree AJ, Yao Y, Du B, Hemati N, Martinez-Santibañez G and MacDougald OA: Wnt6, Wnt10a and Wnt10b inhibit adipogenesis and stimulate osteoblastogenesis through a β-catenin-dependent mechanism. Bone. 50:477–489. 2012. View Article : Google Scholar
25	Cheng SL, Shao JS, Cai J, Sierra OL and Towler DA: Msx2 exerts bone anabolism via canonical Wnt signaling. J Biol Chem. 283:20505–20522. 2008. View Article : Google Scholar : PubMed/NCBI
26	Ye L, Fan Z, Yu B, Chang J, Al Hezaimi K, Zhou X, Park NH and Wang CY: Histone demethylases KDM4B and KDM6B promotes osteogenic differentiation of human MSCs. Cell Stem Cell. 11:50–61. 2012. View Article : Google Scholar : PubMed/NCBI
27	Najdi R, Proffitt K, Sprowl S, Kaur S, Yu J, Covey TM, Virshup DM and Waterman ML: A uniform human Wnt expression library reveals a shared secretory pathway and unique signaling activities. Differentiation. 84:203–213. 2012. View Article : Google Scholar : PubMed/NCBI
28	Cao Y, Sun Z, Liao L, Meng Y, Han Q and Zhao RC: Human adipose tissue-derived stem cells differentiate into endothelial cells in vitro and improve postnatal neovascularization in vivo. Biochem Biophys Res Commun. 332:370–379. 2005. View Article : Google Scholar : PubMed/NCBI
29	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. View Article : Google Scholar
30	Dou XW, Liang YK, Lin HY, Wei XL, Zhang YQ, Bai JW, Chen CF, Chen M, Du CW, Li YC, et al: Notch3 maintains luminal phenotype and suppresses tumorigenesis and metastasis of breast cancer via trans-activating estrogen receptor-α. Theranostics. 7:4041–4056. 2017. View Article : Google Scholar :
31	Oosterwegel MA, van de Wetering ML, Holstege FC, Prosser HM, Owen MJ and Clevers HC: TCF-1, a T cell-specific transcription factor of the HMG box family, interacts with sequence motifs in the TCRβ and TCRδ enhancers. Int Immunol. 3:1189–1192. 1991. View Article : Google Scholar : PubMed/NCBI
32	van Beest M, Dooijes D, van De Wetering M, Kjaerulff S, Bonvin A, Nielsen O and Clevers H: Sequence-specific high mobility group box factors recognize 10-12-base pair minor groove motifs. J Biol Chem. 275:27266–27273. 2000. View Article : Google Scholar : PubMed/NCBI
33	Gaur T, Lengner CJ, Hovhannisyan H, Bhat RA, Bodine PV, Komm BS, Javed A, van Wijnen AJ, Stein JL, Stein GS and Lian JB: Canonical WNT signaling promotes osteogenesis by directly stimulating Runx2 gene expression. J Biol Chem. 280:33132–33140. 2005. View Article : Google Scholar : PubMed/NCBI
34	Tseng PC, Hou SM, Chen RJ, Peng HW, Hsieh CF, Kuo ML and Yen ML: Resveratrol promotes osteogenesis of human mesenchymal stem cells by upregulating RUNX2 gene expression via the SIRT1/FOXO3A axis. J Bone Miner Res. 26:2552–2563. 2011. View Article : Google Scholar : PubMed/NCBI
35	Baglìo SR, Devescovi V, Granchi D and Baldini N: MicroRNA expression profiling of human bone marrow mesenchymal stem cells during osteogenic differentiation reveals Osterix regulation by miR-31. Gene. 527:321–331. 2013. View Article : Google Scholar : PubMed/NCBI
36	Li E, Zhang J, Yuan T and Ma B: MiR-143 suppresses osteogenic differentiation by targeting Osterix. Mol Cell Biochem. 390:69–74. 2014. View Article : Google Scholar : PubMed/NCBI
37	AlAmer N, Bondalapati A, Garcia-Godoy F and Kandalam U: Osteogenic differentiation of orofacial tissue-derived mesenchymal stem cells-A review. Curr Tissue Eng. 5:11–20. 2016. View Article : Google Scholar
38	Atashi F, Modarressi A and Pepper MS: The role of reactive oxygen species in mesenchymal stem cell adipogenic and osteogenic differentiation: A review. Stem Cells Dev. 24:1150–1163. 2015. View Article : Google Scholar : PubMed/NCBI
39	Fakhry M, Hamade E, Badran B, Buchet R and Magne D: Molecular mechanisms of mesenchymal stem cell differentiation towards osteoblasts. World J Stem Cells. 5:136–148. 2013. View Article : Google Scholar : PubMed/NCBI
40	Deng ZL, Sharff KA, Tang N, Song WX, Luo J, Luo X, Chen J, Bennett E, Reid R, Manning D, et al: Regulation of osteogenic differentiation during skeletal development. Front Biosci. 13:2001–2021. 2008. View Article : Google Scholar
41	Harada SI and Rodan GA: Control of osteoblast function and regulation of bone mass. Nature. 423:349–355. 2003. View Article : Google Scholar : PubMed/NCBI
42	Komori T: Regulation of bone development and maintenance by Runx2. Front Biosci. 13:898–903. 2007. View Article : Google Scholar : PubMed/NCBI
43	James AW: Review of signaling pathways governing MSC osteogenic and adipogenic differentiation. Scientifica (Cairo). 2013:6847362013.
44	Nakashima K, Zhou X, Kunkel G, Zhang Z, Deng JM, Behringer RR and de Crombrugghe B: The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell. 108:17–29. 2002. View Article : Google Scholar : PubMed/NCBI
45	Artigas N, Gámez B, Cubillos-Rojas M, Sánchez-de Diego C, Valer JA, Pons G, Rosa JL and Ventura F: p53 inhibits SP7/Osterix activity in the transcriptional program of osteoblast differentiation. Cell Death Differ. 24:2022–2031. 2017. View Article : Google Scholar : PubMed/NCBI
46	Leucht P and Helms JA: Wnt signaling: An emerging target for bone regeneration. J Am Acad Orthop Surg. 23:67–68. 2015. View Article : Google Scholar
47	Yin X, Li J, Salmon B, Huang L, Lim WH, Liu B, Hunter DJ, Ransom RC, Singh G, Gillette M, et al: Wnt signaling and its contribution to craniofacial tissue homeostasis. J Dental Res. 94:1487–1494. 2015. View Article : Google Scholar
48	van de Wetering M, Oosterwegel M, Dooijes D and Clevers H: Identification and cloning of TCF-1, a T lymphocyte-specific transcription factor containing a sequence-specific HMG box. EMBO J. 10:123–132. 1991. View Article : Google Scholar : PubMed/NCBI