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FOXC1 Promotes Osteoblastic Differentiation of Bone Marrow Mesenchymal Stem Cells via the Dnmt3b/CXCL12 Axis

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Abstract

Bone defects have remained a clinical problem in current orthopedics. Bone marrow mesenchymal stem cells (BM-MSCs) with multi-directional differentiation ability have become a research hotspot for repairing bone defects. In vitro and in vivo models were constructed, respectively. Alkaline phosphatase (ALP) staining and alizarin red staining were performed to detect osteogenic differentiation ability. Western blotting (WB) was used to detect the expression of osteogenic differentiation-related proteins. Serum inflammatory cytokine levels were detected by ELISA. Fracture recovery was evaluated by HE staining. The binding relationship between FOXC1 and Dnmt3b was verified by dual-luciferase reporter assay. The relationship between Dnmt3b and CXCL12 was explored by MSP and ChIP assays. FOXC1 overexpression promoted calcium nodule formation, upregulated osteogenic differentiation-related protein expression, promoted osteogenic differentiation, and decreased inflammatory factor levels in BM-MSCs, and promoted callus formation, upregulated osteogenic differentiation-related protein expression, and downregulated CXCL12 expression in the mouse model. Furthermore, FOXC1 targeted Dnmt3b, with Dnmt3b knockdown decreasing calcium nodule formation and downregulating osteogenic differentiation-related protein expression. Additionally, inhibiting Dnmt3b expression upregulated CXCL12 protein expression and inhibited CXCL12 methylation. Dnmt3b could be binded to CXCL12. CXCL12 overexpression attenuated the effects of FOXC1 overexpression and inhibited BM-MSCs osteogenic differentiation. This study confirmed that the FOXC1-mediated regulation of the Dnmt3b/CXCL12 axis had positive effects on the osteogenic differentiation of BM-MSCs.

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Abbreviations

BM-MSCs:

Bone marrow mesenchymal stem cells

FOXC1:

Forkhead Box C1

Dnmt3b:

DNA-methylated transferase 3b

CXCL12:

C-X-C chemokine ligand 12

OSX:

Osterix

OCN:

Osteocalcin

Runx2:

Runt-related transcription factor 2

BMP:

Bone morphogenetic protein

Msx2:

Muscle segment homeobox 2

Gli2:

Gli family zinc finger protein 2

ChIP:

Chromatin immunoprecipitation

FBS:

Fetal bovine serum

PBS:

Phosphate-buffered saline

WB:

Western blot analysis

qRT-PCR:

Quantitative real-time PCR

PVDF:

Poly vinylidene difluoride filter

SDS-PGE:

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

MSP:

Methylation-specific PCR

ELISA:

Enzyme-linked immunosorbent assay

HE:

Hematoxylin and eosin

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Funding

This work was supported by the Natural Science Foundation of Inner Mongolia Autonomous Region (No. 2021MS08135).

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Authors and Affiliations

  1. Department of Orthopedics, The Third Affiliated Hospital, Inner Mongolia Medical University, No. 20 Shaoxian Road, Kundulun District, Baotou, 014010, Inner Mongolia, People’s Republic of China

    Peiguang Zhang, Bo Feng, Guangming Dai, Kecheng Niu & Lan Zhang

Authors
  1. Peiguang Zhang
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  2. Bo Feng
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  3. Guangming Dai
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  4. Kecheng Niu
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  5. Lan Zhang
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Contributions

PZ: Conception of manuscript. BF: Manuscript design. GD: Manuscript writing. KN: Manuscript reviewing. LZ: Manuscript editing.

Corresponding author

Correspondence to Lan Zhang.

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Competing interests

The authors declare that there is no conflict of interest.

Ethical Approval and Consent to Participate

The animal study was reviewed and approved by the Animal Ethical Committee of the Third Affiliated Hospital, Inner Mongolia Medical University (2021MER-062).

Data Availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Zhang, P., Feng, B., Dai, G. et al. FOXC1 Promotes Osteoblastic Differentiation of Bone Marrow Mesenchymal Stem Cells via the Dnmt3b/CXCL12 Axis. Biochem Genet 62, 176–192 (2024). https://doi.org/10.1007/s10528-023-10403-y

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  • DOI: https://doi.org/10.1007/s10528-023-10403-y

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