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Starvation‑induced NEDD4‑mediated autophagy in BMSCs

  • Authors:
    • Chengyi Liu
    • Shuang Zhang
    • Xiaoxian Yun
    • Bo Li
    • Xiaomei Xu
    • Fuwei Lin
  • View Affiliations / Copyright

    Affiliations: Luzhou Key Laboratory of Oral and Maxillofacial Reconstruction and Regeneration, The Affiliated Stomatological Hospital of Southwest Medical University, Luzhou, Sichuan 646000, P.R. China
    Copyright: © Liu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 32
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    Published online on: January 14, 2026
       https://doi.org/10.3892/br.2026.2105
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Abstract

Autophagy regulates the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and mediates BMSC‑mediated bone remodeling under pathological conditions (such as osteoporosis and infectious bone defects). However, the regulatory mechanisms controlling autophagy in BMSCs remain unclear. Neural precursor cell‑expressed developmentally down‑regulated protein 4 (NEDD4) has been implicated in autophagy regulation. In the present study, autophagy was induced in BMSCs through serum starvation and NEDD4 expression was silenced using lentiviral short hairpin RNA. Quantitative real‑time polymerase chain reaction, western blotting, and immunofluorescence were used to examine the expression of the autophagy‑related molecules, microtubule‑associated protein 1 light chain 3 (LC3) and Beclin‑1, to explore the underlying molecular mechanisms. It was determined that BMSCs exhibited significant autophagy activation 30 min after starvation, accompanied by marked increases in NEDD4, LC3, and Beclin‑1 at both mRNA and protein levels. NEDD4 knockdown significantly attenuated the upregulation of LC3 and Beclin‑1 and prevented the starvation‑induced decrease in phosphorylated mTOR. These results indicated that NEDD4 positively regulates autophagy in BMSCs, likely through the mTOR signaling pathway. In conclusion, the present study demonstrated that NEDD4 is essential for BMSC autophagy and may serve as an important target for therapies aimed at modulating BMSC function.

View Figures

Figure 1

Culture and identification of BMSCs.
(A) Representative light microscopy image showing the
fibroblast-like morphology of third-generation (P3) BMSCs. (B)
Alizarin Red S staining indicates mineralized calcium nodules after
21 days of osteogenic induction. (C) Oil Red O staining displays
red lipid droplets in BMSCs after 14 days of adipogenic induction.
(D) Flow cytometric analysis confirms BMSC identity based on high
expression of CD29 and CD90, and low expression of CD34 and CD45.
BMSCs, bone marrow mesenchymal stem cells.

Figure 2

Time-course of autophagy induction in
BMSCs following EBSS starvation. (A) RT-qPCR analysis of NEDD4,
LC3, and Beclin-1 mRNA levels at 0, 15, 30 min, and 1, 2, 4 h
post-starvation. (B and C) A representative western blot image and
quantification of autophagy-related proteins NEDD4, LC3-I and -II,
and Beclin-1 over time. (D) Representative immunofluorescent images
showing LC3 (red) localization in the cytoplasm of BMSCs after
starvation. Nuclei were counterstained with DAPI (blue). Scale bar,
100 µm. Data are presented as the mean ± SD (n=3).
*P≤0.05, **P≤0.01, ***P≤0.001 and
****P≤0.0001. BMSCs, bone marrow mesenchymal stem cells;
EBSS, Earle s balanced salt solution; RT-qPCR, reverse
transcription-quantitative polymerase chain reaction; NEDD4, neural
precursor cell-expressed developmentally down-regulated protein 4;
LC3, light chain 3; DAPI, 4 ,6-diamidino-2-phenylindole; ns,
not significant.

Figure 3

Ultrastructural observation of
autophagy in BMSCs using TEM. (A) Representative TEM images of
cellular autophagy at different time points. (B) Quantitative
analysis of cellular autophagic vesicles at different time points.
Data are presented as mean ± SD (n=3). *P≤0.01 and
****P≤0.0001. BMSCs, bone marrow mesenchymal stem cells;
TEM, transmission electron microscopy; ns, not significant.

Figure 4

Optimization of lentiviral infection
for NEDD4 knockdown in BMSCs. (A) Immunofluorescence imaging
showing infection efficiency (MOI) with or without HitransG
enhancers. (B) Cell morphology after puromycin selection at
different concentrations. The optimal concentration is 3.5 µg/ml.
(C) RT-qPCR results showing knockdown efficiency of three shRNA
sequences targeting NEDD4; sh3 was selected for subsequent
experiments. Data are shown as mean ± SD (n=3).
****P≤0.0001. NEDD4, neural precursor cell-expressed
developmentally down-regulated protein 4; BMSCs, bone marrow
mesenchymal stem cells; MOI, multiplicity of infection; RT-qPCR,
reverse transcription-quantitative polymerase chain reaction; shRNA
or sh-, short hairpin; NC, negative control.

Figure 5

NEDD4 knockdown impairs
starvation-induced autophagy in BMSCs. (A) RT-qPCR analysis of
NEDD4, LC3 and Beclin-1 mRNA expression under control, EBSS, sh-NC
+ EBSS, and sh-NEDD4 + EBSS conditions. (B) Representative LC3
immunofluorescent images and quantification. LC3 puncta are
markedly reduced in the NEDD4 knockdown group. Scale bar, 50 µm.
Data are expressed as mean ± SD (n=3). **P≤0.01,
***P≤0.005 and ****P≤0.0001. NEDD4, neural
precursor cell-expressed developmentally down-regulated protein 4;
BMSCs, bone marrow mesenchymal stem cells; RT-qPCR, reverse
transcription-quantitative polymerase chain reaction; LC3, light
chain 3; EBSS, Earle s balanced salt solution; sh-, short
hairpin; NC, negative control.

Figure 6

Effects of NEDD4 knockdown on
autophagy markers and mTOR signaling. A representative western blot
image and quantification of LC3-II, Beclin-1, total mTOR, and
p-mTOR in BMSCs under EBSS treatment, with and without NEDD4
knockdown. NEDD4 knockdown reverses EBSS-induced reduction in
p-mTOR, indicating mTOR reactivation. Data are shown as mean ± SD
(n=3). *P≤0.01, **P≤0.005,
***P≤0.0001. Proteins shown within the panel were
detected on the same membrane (cut by molecular weight) unless
otherwise indicated. NEDD4, neural precursor cell-expressed
developmentally down-regulated protein 4; mTOR, mammalian target of
rapamycin; p-, phosphorylated; BMSCs, bone marrow mesenchymal stem
cells; EBSS, Earle s balanced salt solution; sh-, short
hairpin; NC, negative control.
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Copy and paste a formatted citation
Spandidos Publications style
Liu C, Zhang S, Yun X, Li B, Xu X and Lin F: <p>Starvation‑induced NEDD4‑mediated autophagy in BMSCs</p>. Biomed Rep 24: 32, 2026.
APA
Liu, C., Zhang, S., Yun, X., Li, B., Xu, X., & Lin, F. (2026). <p>Starvation‑induced NEDD4‑mediated autophagy in BMSCs</p>. Biomedical Reports, 24, 32. https://doi.org/10.3892/br.2026.2105
MLA
Liu, C., Zhang, S., Yun, X., Li, B., Xu, X., Lin, F."<p>Starvation‑induced NEDD4‑mediated autophagy in BMSCs</p>". Biomedical Reports 24.3 (2026): 32.
Chicago
Liu, C., Zhang, S., Yun, X., Li, B., Xu, X., Lin, F."<p>Starvation‑induced NEDD4‑mediated autophagy in BMSCs</p>". Biomedical Reports 24, no. 3 (2026): 32. https://doi.org/10.3892/br.2026.2105
Copy and paste a formatted citation
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Spandidos Publications style
Liu C, Zhang S, Yun X, Li B, Xu X and Lin F: <p>Starvation‑induced NEDD4‑mediated autophagy in BMSCs</p>. Biomed Rep 24: 32, 2026.
APA
Liu, C., Zhang, S., Yun, X., Li, B., Xu, X., & Lin, F. (2026). <p>Starvation‑induced NEDD4‑mediated autophagy in BMSCs</p>. Biomedical Reports, 24, 32. https://doi.org/10.3892/br.2026.2105
MLA
Liu, C., Zhang, S., Yun, X., Li, B., Xu, X., Lin, F."<p>Starvation‑induced NEDD4‑mediated autophagy in BMSCs</p>". Biomedical Reports 24.3 (2026): 32.
Chicago
Liu, C., Zhang, S., Yun, X., Li, B., Xu, X., Lin, F."<p>Starvation‑induced NEDD4‑mediated autophagy in BMSCs</p>". Biomedical Reports 24, no. 3 (2026): 32. https://doi.org/10.3892/br.2026.2105
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