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TWEAK modulates the characteristics of periodontal ligament stem cells via the Fn14/NF‑κB pathway

  • Authors:
    • Linlin Xiao
    • Man Qu
    • Lulu Chen
    • Mingli Xiang
    • Qian Long
    • Jianguo Liu
    • Xiaoyan Guan
    • Chengcheng Liao
  • View Affiliations / Copyright

    Affiliations: Department of Orthodontics II, Affiliated Stomatological Hospital of Zunyi Medical University, Zunyi, Guizhou 563000, P.R. China, Oral Disease Research Key Laboratory of Guizhou Tertiary Institution, School of Stomatology, Zunyi Medical University, Zunyi, Guizhou 563000, P.R. China
    Copyright: © Xiao et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 8
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    Published online on: October 27, 2025
       https://doi.org/10.3892/ijmm.2025.5679
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Abstract

Tumor necrosis factor‑like weak inducer of apoptosis (TWEAK)/fibroblast growth factor‑inducible 14 (Fn14) signaling represents a critical regulatory axis in tissue repair and the inflammatory response. However, the impact of TWEAK on the characteristics of periodontal ligament stem cells (PDLSCs), which subsequently influence periodontal homeostasis, remains inadequately understood. To address this, PDLSCs were isolated from human periodontitis tissue and cultured to investigate the effects of TWEAK on PDLSC proliferation, migration and osteogenic differentiation using Cell Counting Kit‑8, TUNEL, Transwell and scratch assays, and alizarin red and alkaline phosphatase staining. Transcriptome sequencing and western blot analysis were used to explore the underlying molecular mechanisms. Additionally, the potential of targeting TWEAK in periodontitis treatment was evaluated using inflammatory PDLSCs (iPDLSCs) and a rat periodontitis model. The present study demonstrated that low levels (1, 5 and 20 ng/ml) of TWEAK enhanced the proliferation and osteogenic differentiation of PDLSCs, with 1 and 5 ng/ml further enhancing their ability to promote M2 macrophage polarization. By contrast, elevated levels (100 ng/ml) of TWEAK impaired PDLSC proliferation, migration and osteogenic potential, activated the RANKL/osteoprotegerin (OPG) system, and promoted the M1 polarization of macrophages induced by PDLSCs, with the Fn14/NF‑κB pathway serving a pivotal role in this regulatory process. The expression levels of TWEAK, Fn14 and NF‑κB were significantly higher in iPDLSCs than in healthy donor‑derived PDLSCs, and these iPDLSCs exhibited reduced proliferation, migration and osteogenic potential, along with increased RANKL/OPG activation and M1 macrophage polarization. In iPDLSCs, inhibition of the TWEAK/Fn14/NF‑κB pathway enhanced cell proliferation, migration and osteogenic differentiation potential, and reversed the activation of the RANKL/OPG system and macrophage M1 polarization induced by iPDLSCs. Furthermore, high TWEAK levels were shown to accelerate the progression of rat periodontitis, while inhibition of the TWEAK/Fn14 pathway mitigated periodontitis‑induced periodontal tissue destruction in rats. Collectively, the present findings revealed the role of the TWEAK‑PDLSCs axis in the maintenance and disruption of periodontal homeostasis, and identified targeting of the TWEAK/Fn14/NF‑κB pathway in iPDLSCs during periodontitis as a promising therapeutic strategy.
View Figures

Figure 1

Identification of PDLSCs. (A)
Observation of P0 and P1 PDLSCs under a light microscope. Scale
bar, 500 μm. (B) PDLSC colonies stained with crystal violet
dye. Scale bar, 200 μm. (C) Expression profile of surface
markers in PDLSCs quantified using flow cytometry. (D) Mineralized
nodules formed by PDLSCs after 14 days of osteogenic induction
(stained with Alizarin Red S). Scale bar, 200 μm. (E)
Alkaline phosphatase staining of PDLSCs following 7 days of
osteogenic induction. Scale bar, 200 μm. (F) Oil red O
staining revealed the adipogenic differentiation potential of
PDLSCs. Scale bar, 100 μm. P0, primary; P1, first-passage;
PDLSC, periodontal ligament stem cell.

Figure 2

Effects of TWEAK on the proliferation
and apoptosis of PDLSCs. (A) Line chart depicting the effects of
various concentrations of TWEAK (0, 1, 5, 20, 50 and 100 ng/ml) on
PDLSC proliferation over 5 days, as assessed using the CCK-8 assay.
(B) Statistical analysis of the CCK-8 data from day 5 of TWEAK
stimulation shown in (A). (C) Statistical analysis of the average
fluorescence intensity of TUNEL (green) following treatment with
varying concentrations of TWEAK (0, 1, 5, 20, 50 and 100 ng/ml).
(D) TUNEL assay detecting the effects of various concentrations (0,
1, 5, 20, 50 and 100 ng/ml) of TWEAK on PDLSC apoptosis. Scale bar,
200 μm. Statistical analysis was performed using a one-way
ANOVA. *P<0.05; ****P<0.0001. Data are
presented as the mean ± SD (n=5 or 6). CCK-8, Cell Counting Kit-8;
OD450, optical density at 450 nm; PDLSC, periodontal ligament stem
cell; TWEAK, tumor necrosis factor-like weak inducer of
apoptosis.

Figure 3

Effects of TWEAK on the migration and
osteogenic differentiation of PDLSCs. (A) Effects of various
concentrations of TWEAK (0, 1, 5, 20, 50 and 100 ng/ml) on the
number of migrating PDLSCs, and (B) quantitative analysis of the
number of migrating cells (n=6). Scale bar, 400 μm. (C)
Effects of various concentrations of TWEAK on PDLSC migration
toward scratch wounds over 24 h, and (D) quantitative analysis of
the percentage of wound area reduction (n=12). Scale bar, 1 mm. (E)
Effects of various concentrations of TWEAK on ALP staining in
PDLSCs, and (F) quantitative analysis of grayscale values from ALP
staining (n=6). Scale bar, 200 μm. (G) Alizarin Red staining
revealed the effects of various concentrations of TWEAK on PDLSC
mineralization, and (H) quantitative analysis of grayscale values
from Alizarin Red staining (n=6). Scale bar, 200 μm. (I)
Reverse transcription-quantitative PCR was used to assess the mRNA
expression levels of RUNX2, SP7, ALP and
OPG in PDLSCs after TWEAK stimulation (n=4), with
β-actin serving as the internal control. (J) Western blot
analysis of RUNX2, SP7, ALP and OPG protein expression in PDLSCs
after TWEAK induction, and (K) semi-quantitative analysis of the
gel band intensity, using β-actin as the internal control.
Statistical analysis was performed using a one-way ANOVA.
*P<0.05; **P<0.01;
***P<0.001; ****P<0.0001. Data are
presented as the mean ± SD. ALP, alkaline phosphatase; IOD,
integral optical density; ns, not significant; OPG,
osteoprotegerin; PDLSC, periodontal ligament stem cell; RUNX2,
runt-related transcription factor 2; SP7, Sp7 transcription factor;
TWEAK, tumor necrosis factor-like weak inducer of apoptosis.

Figure 4

Effects of tumor necrosis factor-like
weak inducer of apoptosis on RANKL and OPG expression in PDLSCs and
PDLSC-induced macrophage polarization. (A) RANKL (red) and OPG
(green) expression in PDLSCs was detected by immunofluorescence
staining. Scale bar, 200 μm. Statistical analysis of the MOD
values for (B) RANKL and (C) OPG, and (D) their ratio based on (A)
(E) CD68 and CD163 expression in RAW264.7 macrophages was assessed
by immunofluorescence staining. Scale bar, 200 μm.
Statistical analysis of the MOD values for (F) CD68 and (G) CD163
based on (E). Statistical analysis was performed using a one-way
ANOVA. ***P<0.001; ****P<0.0001. Data
are presented as the mean ± SD (n=5). MOD, mean optical density;
ns, not significant; OPG, osteoprotegerin; PDLSC, periodontal
ligament stem cell; RANKL, receptor activator of nuclear factor-κB
ligand.

Figure 5

Transcriptome analysis of the effect
of TWEAK treatment on PDLSCs. (A) Venn diagram illustrating
differentially expressed genes in PDLSCs following TWEAK treatment.
(B) Multipoint differential scatter plot showing differentially
expressed genes in PDLSCs following TWEAK treatment. (C) GO
enrichment analysis comparing PDLSCs and PDLSCs treated with 50
ng/ml TWEAK. (D) KEGG enrichment analysis comparing PDLSCs and
PDLSCs treated with 50 ng/ml TWEAK. (E) GO enrichment analysis
comparing PDLSCs and PDLSCs treated with 100 ng/ml TWEAK. (F) KEGG
enrichment analysis comparing PDLSCs and PDLSCs treated with 100
ng/ml TWEAK. (G) Western blot analysis was conducted to detect the
levels of Fn14, NF-κB, P-NF-κB and NLRP3 in PDLSCs stimulated with
50 and 100 ng/ml TWEAK. (H) Statistical analysis of protein band
intensities from (G) (n=3). (I) Western blot analysis of the levels
of Fn14, NF-κB, P-NF-κB and NLRP3 in PDLSCs after Fn14 was silenced
using an shRNA. (J) Statistical analysis of protein band
intensities from (I) (n=3). Statistical analysis was performed
using (G and H) one-way ANOVA or (I and J) a two-tailed Student's
t-test. *P<0.05; **P<0.01;
***P<0.001. Data are presented as the mean ± SD. FC,
fold change; Fn14, fibroblast growth factor-inducible 14; GO, Gene
Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; NLRP3,
NOD-like receptor thermal protein domain-associated protein 3; ns,
not significant; P-, phosphorylated; PDLSC, periodontal ligament
stem cell; shRNA/sh, short hairpin RNA; TWEAK, tumor necrosis
factor-like weak inducer of apoptosis.

Figure 6

Inhibition of Fn14 and NF-κB
effectively blocks TWEAK-induced alterations in PDLSC
characteristics. (A) Western blot analysis was conducted to assess
the levels of Fn14, NF-κB, P-NF-κB and NLRP3 in PDLSCs. (B)
Statistical analysis of the intensities of the protein bands shown
in (A) (n=3). (C) A CCK-8 assay was performed to generate the
proliferation curve of PDLSCs. (D) Statistical analysis of the
OD450 values of cells from each group on day 5 of the CCK-8 assay,
as presented in (C) (n=6). (G) Results of ALP staining and (E) the
corresponding statistical analysis of PDLSCs after osteogenic
induction (n=6). Scale bar, 400 μm. (H) Results of Alizarin
Red staining and (F) the corresponding statistical analysis of
PDLSCs following osteogenic induction (n=6). Scale bar, 400
μm. (I) Reverse transcription-quantitative PCR was used to
assess the mRNA expression levels of RUNX2, SP7,
ALP and OPG in PDLSCs, with β-actin serving as
an internal reference (n=4). (J) Western blot analysis was
performed to detect the protein expression levels of RUNX2, SP7,
ALP and OPG in PDLSCs, and (K) the grayscale values of the gel
images were semi-quantitatively analyzed, with β-actin used as an
internal reference (n=3). Statistical analysis was performed using
a one-way ANOVA. *P<0.05; **P<0.01;
***P<0.001; ****P<0.0001. Data are
presented as the mean ± SD. ALP, alkaline phosphatase; CCK-8, Cell
Counting Kit-8; Fn14, fibroblast growth factor-inducible 14; IOD,
integral optical density; NLRP3, NOD-like receptor thermal protein
domain-associated protein 3; ns, not significant; OD450, optical
density at 450 nm; OPG, osteoprotegerin; P-, phosphorylated; PDLSC,
periodontal ligament stem cell; RUNX2, runt-related transcription
factor 2; sh, short hairpin RNA; SP7, Sp7 transcription factor;
TWEAK, tumor necrosis factor-like weak inducer of apoptosis.

Figure 7

Inhibition of Fn14 and NF-κB
effectively blocks TWEAK-induced alterations in the
microenvironmental regulatory potential of PDLSCs. (A) Expression
levels of OPG (green) and RANKL (red) in PDLSCs were detected by
immunofluorescence staining, followed by quantitative analysis of
the MOD values for (B) RANKL and (C) OPG, and (D) the MOD ratio of
RANKL/OPG (n=5). Scale bar, 200 μm. (E) Expression levels of
CD68 and CD163 in RAW264.7 macrophages were detected by
immunofluorescence staining, followed by quantitative analysis of
the MOD values for (F) CD68 and (G) CD163 (n=5). Scale bar, 200
μm. Statistical analysis was performed using a one-way
ANOVA. **P<0.01; ***P<0.001;
****P<0.0001. Data are presented as the mean ± SD.
Fn14, fibroblast growth factor-inducible 14; MOD, mean optical
density; ns, not significant; OPGa, osteoprotegerin; PDLSC,
periodontal ligament stem cell; RANKL, receptor activator of
nuclear factor-κB ligand; sh, short hairpin RNA; TWEAK, tumor
necrosis factor-like weak inducer of apoptosis.

Figure 8

Inhibition of the
TWEAK/Fn14/NF-κB/NLRP3 pathway enhances the functional properties
of iPDLSCs. (A) Expression profile of surface markers in iPDLSCs
quantified using flow cytometry. (B) Levels of TWEAK, Fn14, NF-κB,
P-NF-κB and NLRP3 in PDLSCs, iPDLSCs, and iPDLSCs after the
downregulation of Fn14, NF-κB and NLRP3, and (C) statistical
analysis of the band density values (n=3). (D) Apoptosis levels in
PDLSCs, iPDLSCs, and iPDLSCs after downregulation of Fn14, NF-κB
and NLRP3 were detected using the TUNEL assay, and (E) statistical
analysis of the average fluorescence intensity of TUNEL was
performed (n=5). Scale bar, 200 μm. (F) A Cell Counting
Kit-8 assay was used to assess the proliferative potential of
PDLSCs, iPDLSCs, and iPDLSCs after the downregulation of Fn14,
NF-κB and NLRP3, and (G) statistical analysis of the OD450 values
on day 5 of the experiment was performed (n=6). (H) Transwell
migration assay evaluating the migratory potential of PDLSCs,
iPDLSCs, and iPDLSCs after Fn14, NF-κB or NLRP3 downregulation,
with (I) quantification of the number of migrated cells (n=6).
Scale bar, 400 μm. (J) Wound healing assay evaluating the
migratory potential of PDLSCs, iPDLSCs, and iPDLSCs after Fn14,
NF-κB or NLRP3 downregulation, with (K) quantification of the
percentage of wound closure (%) (n=16). Scale bar, 1 mm. (L) ALP
staining was used to evaluate the mineralization potential of
PDLSCs, iPDLSCs, and iPDLSCs after downregulation of Fn14, NF-κB or
NLRP3, with (M) quantification of the integral optical density of
the ALP-stained images (n=6). Scale bar, 400 μm. (N)
Alizarin Red staining was used to evaluate the mineralization
potential of PDLSCs, iPDLSCs, and iPDLSCs after downregulation of
Fn14, NF-κB or NLRP3, with (O) quantification of the integral
optical density of the Alizarin Red-stained images (n=6). Scale
bar, 400 μm. (P) Reverse transcription-quantitative PCR was
used to evaluate the mRNA expression levels of RUNX2,
SP7, ALP and OPG in PDLSCs, iPDLSCs, and
iPDLSCs after the downregulation of Fn14, NF-κB and NLRP3 (n=4).
(Q) Western blotting was used to detect the expression levels of
RUNX2, SP7, ALP and OPG in PDLSCs, iPDLSCs, and iPDLSCs after the
downregulation of Fn14, NF-κB and NLRP3, and (R) statistical
analysis of the band density values was performed (n=3).
Statistical analysis was performed using a one-way ANOVA.
*P<0.05; **P<0.01;
***P<0.001; ****P<0.0001. Data are
presented as the mean ± SD. ALP, alkaline phosphatase; Fn14,
fibroblast growth factor-inducible 14; IOD, integral optical
density; iPDLSC, inflammatory PDLSC; NLRP3, NOD-like receptor
thermal protein domain-associated protein 3; ns, not significant;
OD450, optical density at 450 nm; OPG, osteoprotegerin; P-,
phosphorylated; PDLSC, periodontal ligament stem cell; RUNX2,
runt-related transcription factor 2; sh, short hairpin RNA; SP7,
Sp7 transcription factor; TWEAK, tumor necrosis factor-like weak
inducer of apoptosis.

Figure 9

Effect of inhibition of the tumor
necrosis factor-like weak inducer of apoptosis/Fn14/NF-κB/NLRP3
pathway on the microenvironmental regulatory ability of iPDLSCs.
(A) Immunofluorescence staining was used to detect the expression
levels of RANKL and OPG in PDLSCs, iPDLSCs, and iPDLSCs after the
downregulation of Fn14, NF-κB and NLRP3, followed by statistical
analysis of the average optical density values of (B) RANKL and (C)
OPG, and (D) the RANKL/OPG ratio (n=5). Scale bar, 200 μm.
(E) Immunofluorescence staining was used to detect the expression
levels of CD68 and CD163 in macrophages cocultured with PDLSCs,
iPDLSCs, and iPDLSCs after the downregulation of Fn14, NF-κB and
NLRP3, followed by statistical analysis of the average optical
density values of (F) CD68 and (G) CD163 (n=5). Scale bar, 200
μm. Statistical analysis was performed using a one-way
ANOVA. *P<0.05; **P<0.01;
****P<0.0001. Data are presented as the mean ± SD.
Fn14, fibroblast growth factor-inducible 14; iPDLSC, inflammatory
PDLSC; MOD, mean optical density; NLRP3, NOD-like receptor thermal
protein domain-associated protein 3; ns, not significant; OPG,
osteoprotegerin; PDLSC, periodontal ligament stem cell; RANKL,
receptor activator of nuclear factor-κB ligand; sh, short hairpin
RNA.

Figure 10

Effects of TWEAK and TWEAK-Fn14-IN-1
on the progression of rat periodontitis. (A) Micro-CT images of the
rat maxilla, with the distance between the two red short lines
representing the CEJ-ABC distance on the buccal side, and with
statistical analysis of the (B) distance of CEJ-ABC (n=6), and (C)
BV/TV at the root bifurcation of the maxillary second molar (n=6).
Scale bar, 1 mm. Images of (D) H&E and (E) Masson's trichrome
staining of rat periodontal tissues. Scale bar, 1 mm (top) or 100
μm (bottom). (F) Representative images of TRAP/alkaline
phosphatase double staining in the periodontal tissue of the rat
maxillary second molar, with (G) quantification and statistical
analysis of osteoclast numbers (TRAP-positive, multinucleated cells
located in the bone resorption lacunae) at the mesial root (n=6).
Red triangles indicate osteoclasts. Scale bar, 50 μm. (H)
Immunofluorescence staining of CD163 (green) and CD68 (red) in
periodontal tissues, with (J) quantification of the mean
fluorescence intensity of CD163 and (K) quantification of the mean
fluorescence intensity of CD68 (n=6). Scale bar, 100 μm. (I)
Immunofluorescence staining of RUNX2 (green) and Periostin (red) in
periodontal tissues, with (L) quantification of the mean
fluorescence intensity of RUNX2 and (M) quantification of the mean
fluorescence intensity of Periostin (n=6). Scale bar, 100
μm. Blank represents the unmodeled group, PBS refers to the
control group where PBS was used instead of TWEAK or
TWEAK-Fn14-IN-1 during modeling, and TWEAK and TWEAK-Fn14-IN-1
represent experimental groups where the recombinant TWEAK protein
or TWEAK-Fn14-IN-1 inhibitor was applied, respectively. Statistical
analysis was performed using a one-way ANOVA.
*P<0.05; **P<0.01;
****P<0.0001. Data are presented as the mean ± SD.
ABC, alveolar bone crest; BV/TV, bone volume to total volume; CEJ,
cementoenamel junction; Fn14, fibroblast growth factor-inducible
14; MOD, mean optical density; ns, not significant; RUNX2,
runt-related transcription factor 2; TRAP, tartrate-resistant acid
phosphatase; TWEAK, tumor necrosis factor-like weak inducer of
apoptosis.
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Copy and paste a formatted citation
Spandidos Publications style
Xiao L, Qu M, Chen L, Xiang M, Long Q, Liu J, Guan X and Liao C: TWEAK modulates the characteristics of periodontal ligament stem cells via the Fn14/NF‑&kappa;B pathway. Int J Mol Med 57: 8, 2026.
APA
Xiao, L., Qu, M., Chen, L., Xiang, M., Long, Q., Liu, J. ... Liao, C. (2026). TWEAK modulates the characteristics of periodontal ligament stem cells via the Fn14/NF‑&kappa;B pathway. International Journal of Molecular Medicine, 57, 8. https://doi.org/10.3892/ijmm.2025.5679
MLA
Xiao, L., Qu, M., Chen, L., Xiang, M., Long, Q., Liu, J., Guan, X., Liao, C."TWEAK modulates the characteristics of periodontal ligament stem cells via the Fn14/NF‑&kappa;B pathway". International Journal of Molecular Medicine 57.1 (2026): 8.
Chicago
Xiao, L., Qu, M., Chen, L., Xiang, M., Long, Q., Liu, J., Guan, X., Liao, C."TWEAK modulates the characteristics of periodontal ligament stem cells via the Fn14/NF‑&kappa;B pathway". International Journal of Molecular Medicine 57, no. 1 (2026): 8. https://doi.org/10.3892/ijmm.2025.5679
Copy and paste a formatted citation
x
Spandidos Publications style
Xiao L, Qu M, Chen L, Xiang M, Long Q, Liu J, Guan X and Liao C: TWEAK modulates the characteristics of periodontal ligament stem cells via the Fn14/NF‑&kappa;B pathway. Int J Mol Med 57: 8, 2026.
APA
Xiao, L., Qu, M., Chen, L., Xiang, M., Long, Q., Liu, J. ... Liao, C. (2026). TWEAK modulates the characteristics of periodontal ligament stem cells via the Fn14/NF‑&kappa;B pathway. International Journal of Molecular Medicine, 57, 8. https://doi.org/10.3892/ijmm.2025.5679
MLA
Xiao, L., Qu, M., Chen, L., Xiang, M., Long, Q., Liu, J., Guan, X., Liao, C."TWEAK modulates the characteristics of periodontal ligament stem cells via the Fn14/NF‑&kappa;B pathway". International Journal of Molecular Medicine 57.1 (2026): 8.
Chicago
Xiao, L., Qu, M., Chen, L., Xiang, M., Long, Q., Liu, J., Guan, X., Liao, C."TWEAK modulates the characteristics of periodontal ligament stem cells via the Fn14/NF‑&kappa;B pathway". International Journal of Molecular Medicine 57, no. 1 (2026): 8. https://doi.org/10.3892/ijmm.2025.5679
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