ETV2 promotes osteogenic differentiation of human dental pulp stem cells through the ERK/MAPK and PI3K-Akt signaling pathways

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Abstract

Background

The repair of cranio-maxillofacial bone defects remains a formidable clinical challenge. The Ets variant 2 (ETV2) transcription factor, which belongs to the E26 transformation-specific (ETS) family, has been reported to play a key role in neovascularization. However, the role of ETV2 in the osteogenesis of human dental pulp stem cells (hDPSCs) remains unexplored.

Methods

Transgenic overexpression of ETV2 was achieved using a lentiviral vector, based on a Dox-inducible system. The effects of Dox-induced overexpression of ETV2 on the osteogenesis of hDPSCs were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR), western blot, immunofluorescence staining, alkaline phosphatase (ALP) staining, and Alizarin Red S (ARS) staining. Additionally, RNA-sequencing (RNA-Seq) analysis was performed to analyze the underlying mechanisms of ETV2-induced osteogenesis. Additionally, the role of ETV2 overexpression in bone formation in vivo was validated by animal studies with a rat calvarial defect model and a nude mice model.

Results

Our results demonstrated that ETV2 overexpression significantly upregulated the mRNA and protein expression levels of osteogenic markers, markedly enhanced ALP activity, and promoted matrix mineralization of hDPSCs. Moreover, the results of RNA-Seq analysis and western blot showed that the ERK/MAPK and PI3K-Akt signaling pathways were activated upon transgenic overexpression of ETV2. The enhanced osteogenic differentiation of hDPSCs due to ETV2 overexpression was partially reversed by treatment with inhibitors of ERK/MAPK or PI3K-AKT signaling. Furthermore, the results of in vivo studies demonstrated that ETV2 overexpression improved bone healing in a rat calvarial defect model and increased ectopic bone formation in nude mice.

Conclusions

Collectively, our results indicated that ETV2 overexpression exerted positive effects on the osteogenesis of hDPSCs, at least partially via the ERK/MAPK and PI3K/AKT signaling pathways.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13287-022-03052-2.

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Most cited references 44

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Comparing the Osteogenic Potentials and Bone Regeneration Capacities of Bone Marrow and Dental Pulp Mesenchymal Stem Cells in a Rabbit Calvarial Bone Defect Model

Yu-Chieh Lee, Ya-Hui Chan, Sung-Chih Hsieh … (2019)

The bone regeneration efficiency of bone marrow mesenchymal stem cells (BMSCs) and dental pulp mesenchymal stem cells (DPSCs) combined with xenografts in the craniofacial region remains unclear. Accordingly, this study commenced by comparing the cell morphology, cell proliferation, trilineage differentiation, mineral synthesis, and osteogenic gene expression of BMSCs and DPSCs in vitro. Four experimental groups (empty control, Bio-Oss only, Bio-Oss+BMSCs, and Bio-Oss+DPSCs) were then designed and implanted in rabbit calvarial defects. The BMSCs and DPSCs showed a similar morphology, proliferative ability, surface marker profile, and trilineage-differentiation potential in vitro. However, the BMSCs exhibited a higher mineral deposition and expression levels of osteogenic marker genes, including alkaline phosphatase (ALP), runt related transcription factor 2 (RUNX2), and osteocalcin (OCN). In the in vivo studies, the bone volume density in both MSC groups was significantly greater than that in the empty control or Bio-Oss only group. Moreover, the new bone formation and Collagen I / osteoprotegerin protein expressions of the scaffold+MSC groups were higher than those of the Bio-Oss only group. Finally, the Bio-Oss+BMSC and Bio-Oss+DPSC groups had a similar bone mineral density, new bone formation, and osteogenesis-related protein expression. Overall, the DPSCs seeded on Bio-Oss matched the bone regeneration efficacy of BMSCs in vivo and hence appear to be a promising strategy for craniofacial defect repair in future clinical applications.

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Regulation and Role of Transcription Factors in Osteogenesis

Wilson Cheuk Wing Chan, Zhijia Tan, Michael To … (2021)

Bone is a dynamic tissue constantly responding to environmental changes such as nutritional and mechanical stress. Bone homeostasis in adult life is maintained through bone remodeling, a controlled and balanced process between bone-resorbing osteoclasts and bone-forming osteoblasts. Osteoblasts secrete matrix, with some being buried within the newly formed bone, and differentiate to osteocytes. During embryogenesis, bones are formed through intramembraneous or endochondral ossification. The former involves a direct differentiation of mesenchymal progenitor to osteoblasts, and the latter is through a cartilage template that is subsequently converted to bone. Advances in lineage tracing, cell sorting, and single-cell transcriptome studies have enabled new discoveries of gene regulation, and new populations of skeletal stem cells in multiple niches, including the cartilage growth plate, chondro-osseous junction, bone, and bone marrow, in embryonic development and postnatal life. Osteoblast differentiation is regulated by a master transcription factor RUNX2 and other factors such as OSX/SP7 and ATF4. Developmental and environmental cues affect the transcriptional activities of osteoblasts from lineage commitment to differentiation at multiple levels, fine-tuned with the involvement of co-factors, microRNAs, epigenetics, systemic factors, circadian rhythm, and the microenvironments. In this review, we will discuss these topics in relation to transcriptional controls in osteogenesis.

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ECM compliance regulates osteogenesis by influencing MAPK signaling downstream of RhoA and ROCK.

D. Kim, Chirag Khatiwala, Russell A. Putnam … (2009)

The compliance of the extracellular matrix (ECM) regulates osteogenic differentiation by modulating extracellular signal-regulated kinase (ERK) activity. However, the molecular mechanism linking ECM compliance to the ERK-mitogen-activated protein kinase (MAPK) pathway remains unclear. Furthermore, RhoA has been widely implicated in integrin-mediated signaling and mechanotransduction. We studied the relationship between RhoA and ERK-MAPK signaling to determine their roles in the regulation of osteogenesis by ECM compliance. Inhibition of RhoA and ROCK in MC3T3-E1 pre-osteoblasts cultured on substrates of varying compliance reduced ERK activity, whereas constitutively active RhoA enhanced it. The expression of RUNX2, a potent osteogenic transcription factor, was increased on stiffer matrices and correlated with elevated ERK activity. Inhibition of RhoA, ROCK, or the MAPK pathway diminished RUNX2 activity and delayed the onset of osteogenesis as shown by altered osteocalcin (OCN) and bone sialoprotein (BSP) gene expression, alkaline phosphatase (ALP) activity, and matrix mineralization. These data establish that one possible mechanism by which ECM rigidity regulates osteogenic differentiation involves MAPK activation downstream of the RhoA-ROCK signaling pathway.

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Author and article information

Contributors

Jianguang Xu:

ORCID: http://orcid.org/0000-0001-8773-1241

xujianguang@ahmu.edu.cn

Journal

Journal ID (nlm-ta): Stem Cell Res Ther

Journal ID (iso-abbrev): Stem Cell Res Ther

Title: Stem Cell Research & Therapy

Publisher: BioMed Central (London )

ISSN (Electronic): 1757-6512

Publication date (Electronic): 4 October 2022

Publication date PMC-release: 4 October 2022

Publication date Collection: 2022

Volume: 13

Electronic Location Identifier: 495

Affiliations

[1 ]GRID grid.410587.f, School of Stomatology, , Shandong First Medical University and Shandong Academy of Medical Sciences, ; Jinan, 250000 People’s Republic of China

[2 ]GRID grid.186775.a, ISNI 0000 0000 9490 772X, Key Lab of Oral Diseases Research of Anhui Province, College and Hospital of Stomatology, , Anhui Medical University, ; 69 Meishan Road, Hefei, 230032 People’s Republic of China

[3 ]GRID grid.11135.37, ISNI 0000 0001 2256 9319, Central Laboratory, , Peking University School and Hospital of Stomatology, ; Beijing, 100081 People’s Republic of China

Author information

Jianguang Xu http://orcid.org/0000-0001-8773-1241

Article

Publisher ID: 3052

DOI: 10.1186/s13287-022-03052-2

PMC ID: 9533526

PubMed ID: 36195958

SO-VID: 76c143a3-2426-4b2b-8f91-21f6a0d49665

License:

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

History

Date received : 20 February 2022

Date accepted : 18 July 2022

Funding

Funded by: FundRef http://dx.doi.org/10.13039/501100003995, Natural Science Foundation of Anhui Province;

Award ID: 1908085MH255

Award Recipient : Jianguang Xu

Funded by: Basic and Clinical Collaborative Research of Anhui Medical University

Award ID: 2020xkjT025

Award Recipient : Jianguang Xu

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ScienceOpen disciplines: Molecular medicine

Keywords: etv2,osteogenic differentiation,human dental pulp stem cells,bone formation,rna-sequencing

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ScienceOpen disciplines: Molecular medicine

Keywords: etv2, osteogenic differentiation, human dental pulp stem cells, bone formation, rna-sequencing

ETV2 promotes osteogenic differentiation of human dental pulp stem cells through the ERK/MAPK and PI3K-Akt signaling pathways

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Neuronal Signaling

Most cited references 44

Comparing the Osteogenic Potentials and Bone Regeneration Capacities of Bone Marrow and Dental Pulp Mesenchymal Stem Cells in a Rabbit Calvarial Bone Defect Model

Regulation and Role of Transcription Factors in Osteogenesis

ECM compliance regulates osteogenesis by influencing MAPK signaling downstream of RhoA and ROCK.

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