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FOXJ3 drives mesenchymal stem cell osteogenic differentiation via the Wnt/β‑catenin pathway: A novel regulator implicated in osteoporosis

  • Authors:
    • Hongwei Xiao
    • Jianfeng Li
    • Wei Huang
    • Yi Qin
  • View Affiliations / Copyright

    Affiliations: Department of Orthopedics, Zhuhai People's Hospital (The Affiliated Hospital of Beijing Institute of Technology, Zhuhai Clinical Medical College of Jinan University), Zhuhai, Guangdong 519000, P.R. China
    Copyright: © Xiao et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 50
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    Published online on: November 21, 2025
       https://doi.org/10.3892/mmr.2025.13760
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Abstract

Osteoporosis involves impaired osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). The present study identified the transcription factor forkhead box (FOXJ3) as a novel regulator of this process. During in vitro osteogenic differentiation of BMSCs, FOXJ3 expression progressively increased, and was positively correlated with osteogenic markers Runt‑related transcription factor 2 (RUNX2) and osteocalcin (OCN). Functional studies confirmed the essential role of FOXJ3: Small interfering RNA‑mediated knockdown markedly impaired differentiation, as evidenced by reduced alkaline phosphatase (ALP) activity, diminished mineralized nodule formation, and downregulation of RUNX2 and OCN. Conversely, lentivirus‑induced FOXJ3 overexpression enhanced these osteogenic markers and outcomes. Mechanistically, FOXJ3 knockdown suppressed active β‑catenin expression, indicating Wnt/β‑catenin pathway involvement. Crucially, the Wnt/β‑catenin agonist SB216763 rescued the inhibitory effects of FOXJ3 knockdown on ALP activity and mineralization. Conversely, the pro‑osteogenic effects of FOXJ3 overexpression were abrogated by the Wnt inhibitor XAV939. These findings establish FOXJ3 as a positive regulator of BMSC osteogenic differentiation acting primarily through the Wnt/β‑catenin pathway, presenting a novel potential therapeutic target for osteoporosis.
View Figures

Figure 1

FOXJ3 expression is positively
associated with the osteogenic differentiation of BMSCs. (A)
Alizarin Red S staining demonstrated that BMSCs were effectively
differentiated into osteoblasts. (B) RT-qPCR detection of FOXJ3
expression changes during the osteogenic differentiation of BMSCs
(n=5 biologically independent samples). (C) Correlation between the
expression levels of FOXJ3 and the osteogenesis-related gene RUNX2,
detected by RT-qPCR (n=9 biologically independent samples). (D)
Correlation between FOXJ3 and the osteogenesis-related gene OCN,
detected by RT-qPCR (n=9). (E) Western blotting results
demonstrated a progressive elevation in FOXJ3 protein expression
with extended osteogenic induction. (F) Semi-quantitative analysis
of western blotting (n=3). *P<0.05, **P<0.01, ***P<0.001.
BMSC, bone marrow mesenchymal stem cell; FOXJ3, forkhead box J3;
OCN, osteocalcin; RT-qPCR, reverse transcription-quantitative
polymerase chain reaction; RUNX2, Runt-related transcription factor
2.

Figure 2

In vitro knockdown of FOXJ3
inhibits osteogenic differentiation of BMSCs. (A) Knockdown
efficiency of siFOXJ3 was detected at the transcriptome level using
RT-qPCR (n=3 biologically independent samples). (B) Protein
knockdown efficiency of siFOXJ3 was evaluated through western
blotting (n=3 biologically independent samples). (C)
Semi-quantitative analysis of western blotting (n=3 biologically
independent samples). (D) ALP activity detection revealed decreased
ALP activity in BMSCs after FOXJ3 knockdown (n=5 biologically
independent samples). (E) Alizarin Red S staining demonstrated
reduced osteogenic nodule formation in BMSCs following FOXJ3
knockdown. (F) Quantitative analysis of Alizarin Red S staining
indicated impaired osteogenic differentiation capacity in BMSCs
with FOXJ3 knockdown. (G) RT-qPCR analysis showed osteogenic
differentiation-associated genes RUNX2 and OCN were downregulated
in BMSCs with FOXJ3 knockdown (n=5). *P<0.05, **P<0.01. ALP,
alkaline phosphatase; BMSC, bone marrow mesenchymal stem cell;
FOXJ3, forkhead box J3; OCN, osteocalcin; RT-qPCR, reverse
transcription-quantitative polymerase chain reaction; RUNX2,
Runt-related transcription factor 2; si, small interfering.

Figure 3

FOXJ3 promotes osteogenic
differentiation of BMSCs in vitro. (A) OE efficiency of
OE-FOXJ3 was detected at the transcriptome level by RT-qPCR. (B)
Protein overexpression efficiency of OE-FOXJ3 was verified by
western blotting. (C) Semi-quantitative analysis of western
blotting (n=3). (D) ALP activity detection was performed in BMSCs
after FOXJ3 OE (n=5). (E) Alizarin Red S staining was conducted to
assess osteogenic nodules in BMSCs following FOXJ3 OE. (F)
Quantitative analysis of Alizarin Red S staining demonstrated
enhanced osteogenic differentiation capacity in BMSCs after FOXJ3
OE (n=5 biologically independent samples). (G) RT-qPCR assay
detected upregulated expression of the osteogenic
differentiation-related genes RUNX2 and OCN in BMSCs post-FOXJ3 OE
(n=5 biologically independent samples). *P<0.05, **P<0.01.
ALP, alkaline phosphatase; BMSC, bone marrow mesenchymal stem cell;
FOXJ3, forkhead box J3; OCN, osteocalcin; OE, overexpression;
RT-qPCR, reverse transcription-quantitative polymerase chain
reaction; RUNX2, Runt-related transcription factor 2.

Figure 4

FOXJ3 regulates the Wnt/β-catenin
pathway. (A) Changes in the expression levels of proteins in the
pathways associated with osteogenic differentiation after FOXJ3
knockdown were detected using western blotting. (B)
Semi-quantitative analysis of western blotting demonstrated that
FOXJ3 knockdown inhibited the Wnt/β-catenin pathway (n=3).
*P<0.05. FOXJ3, forkhead box J3; p-, phosphorylated; si, small
interfering.

Figure 5

FOXJ3 regulates BMSC osteogenic
differentiation through the Wnt/β-catenin pathway. (A) Detection of
ALP activity in BMSCs after FOXJ3 knockdown and treatment with the
Wnt/β-catenin pathway agonist SB216763 (n=3 biologically
independent samples). (B) Alizarin Red S staining and (C)
quantification of osteogenic nodules in BMSCs after FOXJ3 knockdown
and treatment with the Wnt/β-catenin pathway agonist SB216763 (n=3
biologically independent samples). (D) Detection of ALP activity in
BMSCs after FOXJ3 OE and treatment with the Wnt/β-catenin pathway
inhibitor XAV939 (n=3 biologically independent samples). (E)
Alizarin Red S staining and (F) quantification of osteogenic
nodules in BMSCs after FOXJ3 OE and treatment with the
Wnt/β-catenin pathway inhibitor XAV939 (n=3 biologically
independent samples). *P<0.05, **P<0.01. ALP, alkaline
phosphatase; BMSC, bone marrow mesenchymal stem cell; FOXJ3,
forkhead box J3; OE, overexpression; si, small interfering.
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Copy and paste a formatted citation
Spandidos Publications style
Xiao H, Li J, Huang W and Qin Y: FOXJ3 drives mesenchymal stem cell osteogenic differentiation via the Wnt/&beta;‑catenin pathway: A novel regulator implicated in osteoporosis. Mol Med Rep 33: 50, 2026.
APA
Xiao, H., Li, J., Huang, W., & Qin, Y. (2026). FOXJ3 drives mesenchymal stem cell osteogenic differentiation via the Wnt/&beta;‑catenin pathway: A novel regulator implicated in osteoporosis. Molecular Medicine Reports, 33, 50. https://doi.org/10.3892/mmr.2025.13760
MLA
Xiao, H., Li, J., Huang, W., Qin, Y."FOXJ3 drives mesenchymal stem cell osteogenic differentiation via the Wnt/&beta;‑catenin pathway: A novel regulator implicated in osteoporosis". Molecular Medicine Reports 33.1 (2026): 50.
Chicago
Xiao, H., Li, J., Huang, W., Qin, Y."FOXJ3 drives mesenchymal stem cell osteogenic differentiation via the Wnt/&beta;‑catenin pathway: A novel regulator implicated in osteoporosis". Molecular Medicine Reports 33, no. 1 (2026): 50. https://doi.org/10.3892/mmr.2025.13760
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Spandidos Publications style
Xiao H, Li J, Huang W and Qin Y: FOXJ3 drives mesenchymal stem cell osteogenic differentiation via the Wnt/&beta;‑catenin pathway: A novel regulator implicated in osteoporosis. Mol Med Rep 33: 50, 2026.
APA
Xiao, H., Li, J., Huang, W., & Qin, Y. (2026). FOXJ3 drives mesenchymal stem cell osteogenic differentiation via the Wnt/&beta;‑catenin pathway: A novel regulator implicated in osteoporosis. Molecular Medicine Reports, 33, 50. https://doi.org/10.3892/mmr.2025.13760
MLA
Xiao, H., Li, J., Huang, W., Qin, Y."FOXJ3 drives mesenchymal stem cell osteogenic differentiation via the Wnt/&beta;‑catenin pathway: A novel regulator implicated in osteoporosis". Molecular Medicine Reports 33.1 (2026): 50.
Chicago
Xiao, H., Li, J., Huang, W., Qin, Y."FOXJ3 drives mesenchymal stem cell osteogenic differentiation via the Wnt/&beta;‑catenin pathway: A novel regulator implicated in osteoporosis". Molecular Medicine Reports 33, no. 1 (2026): 50. https://doi.org/10.3892/mmr.2025.13760
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