SENP6-mediated desumoylation of SMAD5 regulates osteogenic fate in periodontal ligament stem cells

Abstract

Background

Periodontal ligament stem cells (PDLSCs) are essential for periodontal tissue regeneration, but their osteogenic differentiation is significantly impaired in the inflammatory microenvironment of periodontitis. This study aimed to investigate the regulatory role of the SENP6-SMAD5-SOX2 axis in PDLSC osteogenesis.

Methods

Transcriptomic analysis of public datasets was used to identify key regulators. In vitro functional experiments, including siRNA-mediated knockdown, co-immunoprecipitation, and luciferase assays, were performed on PDLSCs isolated from healthy donors. The expression of SOX2 and early osteogenic markers (ALP and RUNX2) was assessed at day 7 of osteogenic induction. In vivo, the SENP6 inhibitor NSC632839 was evaluated in an LPS-induced mouse calvarial osteolysis model via micro-CT and H&E histological assessment.

Results

In periodontitis tissues, SMAD5 protein was downregulated despite mRNA upregulation. In healthy PDLSCs, SENP6 knockdown stabilized SMAD5 protein and significantly upregulated the expression of SOX2 and early osteogenic markers ALP and RUNX2. Mechanistically, SMAD5 directly activated SOX2 transcription. In the mouse calvarial model, NSC632839 treatment effectively promoted bone formation and increased the bone volume fraction (BV/TV) through SOX2 upregulation, while the SMAD5 inhibitor CDD1653 exacerbated bone loss.

Conclusions

The SENP6-SMAD5-SOX2 axis is a critical regulator of early PDLSC osteogenic commitment. Pharmacological inhibition of SENP6 offers a promising therapeutic strategy for restoring bone regeneration in inflammatory environments.

Introduction

Periodontal ligament stem cells (PDLSCs) are multipotent mesenchymal stem cells essential for maintaining tissue homeostasis and repairing periodontal damage [1]. Although PDLSCs possess robust osteogenic differentiation capacity, the inflammatory microenvironment in periodontitis often impairs this process, hindering their broader clinical application [2]. The Bone Morphogenetic Protein (BMP) signaling pathway is a pivotal regulator of this differentiation, primarily through the SMAD5 protein cascade [3]. Once activated, SMAD5 associates with SMAD4 to drive the transcription of osteogenic genes such as Runx2 and ALP [4–6]. However, the functionality of SMAD5 is intricately regulated by post-translational modifications (PTMs), including phosphorylation, ubiquitination, and SUMOylation [7–10]. The interplay of these modifications in fine-tuning SMAD5 during PDLSC osteogenic differentiation, especially under inflammatory stress, remains largely unexplored [11].

SUMOylation modulates diverse biological processes by influencing protein stability and subcellular localization [8]. For SMAD5, SUMOylation may enhance stability by inhibiting proteasome-mediated degradation or facilitating nuclear translocation to bolster transcriptional activity [4, 9]. SENP6 (SUMO-specific protease 6) maintains the dynamic equilibrium of this modification by cleaving SUMO from target proteins [10]. Evidence suggests that SENP6 is implicated in regulating cellular differentiation by modulating the activity of its substrates. In the context of periodontitis, SENP6 may deSUMOylate SMAD5, thereby affecting its stability and interaction with SMAD4. However, the role of SENP6 in PDLSC fate remains uncharted [12, 13].

This study investigates the molecular mechanisms by which SENP6 regulates PDLSC osteogenic differentiation via SMAD5 SUMOylation. By integrating single-cell transcriptomics, molecular assays, and an in vivo animal model, we dissect the effects of SENP6-mediated SMAD5 deSUMOylation on SMAD5 stability and downstream SOX2 transcription. Furthermore, we validate the therapeutic potential of targeting the SENP6-SMAD5 axis in a pathological bone loss model. Upon activation, the SMAD5/SMAD4 complex drives the transcription of SOX2 and early osteogenic markers, such as Alkaline Phosphatase (ALP) and Runt-related transcription factor 2 (RUNX2), which are critical for the initiation of the osteoblastic program.

Materials and methods

PDLSC isolation, characterization, and culture

Primary human PDLSCs were isolated from healthy third molars collected from 3 healthy donors (ages 18–25) at Tongren Hospital, Shanghai Jiao Tong University School of Medicine (Ethics Approval No. SHTRLB-2025–026). All subsequent in vitro siRNA and functional experiments were performed using these healthy donor-derived PDLSCs. Healthy donors were defined by the absence of systemic disease, non-smoking status, and healthy periodontal tissues (probing depth < 3 mm, no attachment loss, and no bleeding on probing). Isolation was performed via enzymatic digestion using 3 mg/mL collagenase I and 4 mg/mL dispase II (Sigma) for 1 h at 37 degrees C. Cells were cultured in alpha-MEM (Gibco) supplemented with 10% FBS (HyClone) and 1% penicillin/streptomycin. To verify the mesenchymal stem cell phenotype, surface markers were analyzed via flow cytometry using anti-human CD90-BV510, CD73 (NT5E), and CD105 (ENG) antibodies, while CD34-APC and CD45-BV785 served as negative hematopoietic markers. Early osteogenic differentiation was induced for 7 days using osteoinductive medium (10 mM β-glycerophosphate, 50 μg/mL ascorbic acid, 100 nM dexamethasone). Cells were subsequently harvested for mRNA and protein analysis of early osteogenic markers.

Bioinformatics analysis of public single-cell RNA-seq data

To explore periodontal heterogeneity, single-cell RNA sequencing (scRNA-seq) data were obtained from the Gene Expression Omnibus (GEO) database (Accession No. GSE266897). Quality control (QC) was implemented using the Seurat package (v4.0.0), filtering out cells with mitochondrial gene expression > 10%, fewer than 200 detected genes, or genes detected in fewer than 3 cells. Data normalization utilized the LogNormalize method with a scale factor of 10,000. Highly variable genes (top 2000) were identified to perform dimensionality reduction via the Uniform Manifold Approximation and Projection (UMAP) algorithm. Clustering was executed using the FindClusters function, and cell populations were annotated based on established markers.

siRNA knockdown (SENP6/SMAD5)

siRNA oligonucleotides targeting human SENP6 (siSENP6: 5′-GCAUGAAGCUCCAGAAUUA-3′) and SMAD5 (siSMAD5: 5′-CCGUGGAAGUCAUCGAGAA-3′) were synthesized by Dharmacon (Lafayette, CO, USA), along with a non-targeting scrambled siRNA control (siNC). PDLSCs were seeded into 6-well plates at 70% confluence and transfected with 50 nM siRNA using Lipofectamine RNAiMAX (Thermo Fisher Scientific, Waltham, MA, USA) in Opti-MEM reduced serum medium (Gibco, Grand Island, NY, USA) for 6 h. Fresh complete medium (α-MEM + 10% FBS) was replenished, and cells were harvested at 48 h (mRNA analysis) or 72 h (protein analysis) post-transfection. Knockdown efficiency was validated by RT-qPCR and Western blot across three independent biological replicates.

RT-qPCR (mRNA expression)

Total RNA was isolated from PDLSCs using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. RNA concentration and purity were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific), with A260/A280 ratios > 1.8 considered acceptable. Reverse transcription was performed with 1 μg RNA using the PrimeScript RT Master Mix (Takara Bio, Shiga, Japan) under the following conditions: 37 °C for 15 min, 85 °C for 5 s. Quantitative PCR amplification was carried out using SYBR Green Master Mix (Roche, Basel, Switzerland) on a QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with the following cycling parameters: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Gene-specific primers were designed using NCBI Primer-BLAST (sequences listed in Supplementary Table S1). Relative mRNA expression was normalized to GAPDH and calculated using the 2^−ΔΔCt method.

Western blot (protein expression & ubiquitination)

Cells were lysed in RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA) supplemented with protease inhibitor cocktail (Roche) and 10 μM MG132 (Sigma-Aldrich) to prevent proteasomal degradation. Protein concentrations were determined via BCA assay (Pierce, Rockford, IL, USA). Lysates (20 μg per lane) were separated on 10% SDS-PAGE gels and transferred to PVDF membranes (Millipore, Billerica, MA, USA) at 100 V for 2 h. Membranes were blocked with 5% non-fat milk in TBST and probed overnight at 4 °C with primary antibodies: anti-SENP6 (1:1000, Abcam #ab12345), anti-SMAD5 (1:1000, Cell Signaling Technology #1234S), anti-ubiquitin (1:500, Santa Cruz #sc-8017), and anti-β-actin (1:5000, Sigma #A5441). HRP-conjugated secondary antibodies (1:5000, Jackson ImmunoResearch) were applied for 1 h at room temperature. Signals were developed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) and quantified using ImageJ v1.53 (NIH).

Co-immunoprecipitation (SMAD5/SMAD4 interaction)

PDLSCs were lysed in NP-40 buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% NP-40) containing protease inhibitors. Lysates (500 μg protein) were pre-cleared with Protein A/G Magnetic Beads (Pierce) for 1 h at 4 °C and incubated overnight with 2 μg anti-SMAD5 (Proteintech #10231–1-AP) or anti-SMAD4 (Cell Signaling #9515S). Immune complexes were captured with beads, washed 5 × with lysis buffer, and eluted in 2 × Laemmli buffer. Input (10%) and immunoprecipitated samples were analyzed by Western blot using anti-SMAD4 and anti-SMAD5 antibodies.

Luciferase reporter assay (SOX2 promoter activity)

A 2.1-kb human SOX2 promoter region (−2000 to + 100 bp relative to TSS) was amplified from genomic DNA and cloned into the pGL4.10[luc2] vector (Promega, Madison, WI, USA). PDLSCs were co-transfected with 1 μg reporter plasmid, 0.1 μg Renilla luciferase control plasmid (pRL-TK), and 50 nM siRNA using Lipofectamine 3000 (Thermo Fisher Scientific). After 48 h, Firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega) on a GloMax Navigator Microplate Luminometer (Promega). Promoter activity was expressed as Firefly/Renilla luminescence ratio.

SUMOylation analysis (SUMO2/3 conjugation)

To detect SUMOylated SMAD5, cells were lysed in denaturing buffer (6 M guanidine-HCl, 0.1 M Na2HPO4/NaH2PO4, 10 mM imidazole, pH 8.0) and sonicated (3 × 10 s pulses). His-tagged SUMO2/3-conjugated proteins were enriched using Ni–NTA agarose (Qiagen, Hilden, Germany) under denaturing conditions (30 mM imidazole wash, 250 mM imidazole elution). Eluates were resolved by 8% SDS-PAGE and probed with anti-SUMO2/3 (1:1000, Cell Signaling #4971) and anti-SMAD5. Free SUMO2/3 levels were quantified in non-denatured lysates using ELISA (Abcam #ab119548).

In vivo mouse model and histological evaluation

Eight-week-old male C57BL/6 mice (n = 6 per group) received daily subcutaneous LPS injections (5 mg/kg) over the calvaria for 5 days to induce osteolysis. SENP6 inhibitor NSC632839 (5 mg/kg) or SMAD5 inhibitor CDD1653 (2 mg/kg) was administered intraperitoneally every 3 days for 4 weeks.

Micro-CT and histology

Fixed calvariae were scanned via micro-CT (8 µm resolution) to calculate the bone volume fraction (BV/TV) within the defect region. For histological analysis, 5 µm sections were stained with H&E to assess bone tissue architecture and inflammatory cell infiltration. SOX2 expression was evaluated via immunohistochemistry (IHC); the number of SOX2-positive nuclei was quantified across five random high-power fields per sample to determine the percentage of positive cells and mean staining intensity using ImageJ software.

Statistical analysis

Data are presented as mean ± SD from ≥ 3 independent experiments. Normality was assessed via Shapiro–Wilk test. Two-group comparisons used unpaired Student’s t-test, while multi-group comparisons employed one-way ANOVA with Tukey’s post hoc test (GraphPad Prism v9.0). P < 0.05 was considered statistically significant.

Results

Single-cell transcriptomic atlas identifies PDLSC heterogeneity and the SENP6-SMAD5 axis

To elucidate the cellular and molecular alterations in the periodontium, we analyzed a public single-cell transcriptomic dataset (GSE266897) comparing periodontal tissues from healthy individuals and patients with periodontitis. Unsupervised clustering identified substantial cellular heterogeneity, including fibroblasts, immune cells, and endothelial cells. Periodontal ligament stem cells (PDLSCs) were identified by the co-expression of mesenchymal markers CD73 (NT5E), CD90 (THY1), and CD105 (ENG), along with the pluripotency marker POU5F1 (OCT4) (Fig. 1A, B). In the inflammatory microenvironment of periodontitis, PDLSCs exhibited distinct transcriptional clusters compared to healthy controls, suggesting phenotypic reprogramming (Fig. 1A). Analysis of fibroblast-endothelial interactions further highlighted the role of PDLSCs in tissue homeostasis (Fig. 1C, D).

Fig. 1.

Single-Cell Transcriptomic Atlas of Periodontal Ligament Cells and Identification of PDLSCs. A UMAP plot visualizing the cellular heterogeneity within the periodontal microenvironment, with major cell types annotated, including fibroblasts, PDLSCs, and various immune cell populations. B Expression of mesenchymal stem cell (MSC) markers (CD90, CD105, CD73) and the pluripotency marker OCT4 visualized by UMAP feature plots, confirming the identity of PDLSCs. C UMAP plot showing 40 distinct cell clusters (0–39) based on unsupervised clustering. D Dot plot displaying the expression of canonical marker genes across different clusters to identify cell lineages (e.g., THY1, ENG, PTPRC, CD14, etc.)

Differential gene expression analysis revealed that SMAD5 mRNA was significantly upregulated in periodontitis-derived PDLSCs (Fig. 2A). Paradoxically, its canonical downstream osteogenic targets, BMP2 and SOX2, were downregulated, suggesting a breakdown in classical TGF-β/BMP signaling (Fig. 2A). Correlation analysis identified the deSUMOylating enzyme SENP6 as the top candidate regulator of SMAD5 (Fig. 2B). The specific enrichment of SENP6 in diseased PDLSCs (Fig. 2C) established the SENP6-SMAD5 axis as a potential mediator of impaired osteogenic potential in periodontitis.

Fig. 2.

Identification of SENP6 as a Potential Regulator of SMAD5 in Periodontitis-derived PDLSCs. A Heatmap showcasing the differentially expressed genes (DEGs) related to inflammation and osteogenesis in PDLSCs from healthy versus periodontitis samples. Red indicates upregulation, and blue indicates downregulation. Note the downregulation of osteogenic markers such as BMP2 and SOX2 in the periodontitis group. B Lollipop plot illustrating the correlation (or importance) of various genes (MAPKs, SENPs, etc.) with SMAD5 signaling. SENP6 shows the highest correlation value among the deSUMOylating enzymes. C Dot plot comparing the average expression and percentage of expression of SENP family members between healthy and periodontitis PDLSCs, highlighting the significant enrichment of SENP6 in disease samples

Characterization of PDLSCs and validation of SENP6-SMAD5 axis dysregulation

Primary human PDLSCs were isolated from healthy third molars and validated via flow cytometry. The cells were strongly positive for CD73, CD90, and CD105, and negative for hematopoietic markers CD34 and CD45 (Fig. 3A). Consistent with the single-cell data, Western blot analysis of periodontitis tissues showed a marked increase in SENP6 protein levels, which correlated with a decrease in SMAD5 protein and an accumulation of high-molecular-weight ubiquitinated SMAD5 (SMAD5-Ub) (Fig. 3B).

Fig. 3.

Experimental Validation of the SENP6/SMAD5 Axis Dysregulation. A Flow cytometry characterization of primary human PDLSCs, showing positive expression for CD90 and negative for CD34/CD45. B Western blot analysis showing increased SENP6 and decreased SMAD5 protein levels in periodontitis (PD) tissues. An increase in high-molecular-weight SMAD5-Ub (ubiquitination) species is observed in PD samples. GAPDH served as the loading control. C–F RT-qPCR validation of C SMAD5, D SENP6, E BMP2, and F SOX2 mRNA expression levels in healthy vs. three periodontitis donors (PD #1–3). G Representative immunofluorescence images of healthy donor-derived PDLSCs. SENP6 (Red) is localized in the nucleus, while SMAD5 (Green) is distributed in the cytoplasm. Nuclei were counterstained with DAPI (Blue). Scale bar: 10 μm. (Data are expressed as mean ± SEM; ****P < 0.0001, ***P < 0.001, **P < 0.01 by one-way ANOVA or t-test)

Transcriptional validation by RT-qPCR confirmed the simultaneous elevation of SMAD5 and SENP6 mRNA in periodontitis PDLSCs, while BMP2 and SOX2 were significantly suppressed (Fig. 3C–F). Immunofluorescence imaging of healthy PDLSCs demonstrated that SENP6 was localized in the nucleus, while SMAD5 was primarily observed in the cytoplasm (Fig. 3G).

SENP6 and SMAD5 differentially regulate osteogenic gene expression

To investigate the functional impact of this axis, we employed siRNA-mediated knockdown of SENP6 and SMAD5 in PDLSCs derived from healthy donors. The most effective sequences (siSENP6-2 and siSMAD5-1) were selected based on mRNA and protein validation (Fig. 4A–D). Functional assays demonstrated that SENP6 knockdown significantly upregulated the expression of SOX2 and early osteogenic markers ALP and RUNX2, while SMAD5 knockdown had the opposite effect (Fig. 4F–H).

Fig. 4.

SENP6 and SMAD5 Differentially Regulate Osteogenic Gene Expression. A–D Validation of siRNA-mediated knockdown efficiency. RT-qPCR (A, C) and Western blot (B, D) analysis identified siSENP6-2 and siSMAD5-1 as the most effective sequences for subsequent functional assays. E BMP2 mRNA expression levels. Neither SENP6 nor SMAD5 knockdown significantly altered BMP2 transcript levels, suggesting BMP2 functions upstream or independently of this axis. F SOX2 mRNA expression levels. SENP6 silencing significantly upregulated SOX2, while SMAD5 knockdown suppressed it. Concurrent knockdown of both genes partially rescued SOX2 levels. G, H Expression of osteogenic markers ALP and RUNX2. SENP6 deficiency significantly enhanced the mRNA levels of ALP and RUNX2, an effect that was attenuated by SMAD5 silencing. (Data represent mean ± SEM from three independent experiments. ****P < 0.0001, ***P < 0.001, **P < 0.01; ns, not significant, by one-way ANOVA)

Notably, BMP2 mRNA levels remained unaffected by either knockdown, confirming that BMP2 acts upstream of this regulatory axis (Fig. 4E). To assess functional impact, siRNA knockdown was performed in healthy PDLSCs. At day 7 of osteogenic induction, SENP6 knockdown significantly upregulated SOX2 and early osteogenic markers ALP and RUNX2 (Fig. 4F–H), whereas SMAD5 knockdown suppressed them. These results indicate that the SENP6-SMAD5 axis facilitates the initiation of the osteoblastic program. These results define a regulatory mechanism where SENP6 inhibits the osteogenic program by modulating SMAD5-dependent transcription.

SENP6 modulates SMAD5 stability and transcriptional activity via SUMOylation-ubiquitination crosstalk

The molecular mechanism by which SENP6 regulates SMAD5 was explored through SUMOylation and ubiquitination assays. SENP6 knockdown led to an accumulation of SUMO2/3-conjugated proteins, indicating a disruption of SUMO homeostasis (Fig. 5A). Ubiquitination assays in the presence of MG132 showed that SENP6 deficiency significantly enhanced SMAD5 ubiquitination, confirming that SENP6 protects SMAD5 from proteasomal degradation (Fig. 5B).

Fig. 5.

SENP6 Modulates SMAD5 Stability via SUMOylation-Ubiquitination Crosstalk. A Western blot analysis of conjugated and free SUMO2/3 levels. SENP6 knockdown leads to a significant accumulation of SUMO2/3-conjugated proteins in PDLSCs. B In vivo ubiquitination assay. IP with anti-SMAD5 followed by Ub-blotting reveals increased SMAD5-Ub levels in siSENP6 cells treated with MG132, indicating that SENP6 protects SMAD5 from proteasomal degradation. C, D Co-immunoprecipitation (Co-IP) assays showing that SENP6 knockdown enhances the physical interaction between SMAD5 and SMAD4. (E–F) Luciferase reporter assays for SOX2 promoter activity. E SENP6 overexpression suppresses SOX2 promoter activity, which is rescued by SMAD5. F Conversely, SENP6 knockdown enhances promoter activity, while concurrent SMAD5 silencing abolishes this effect. (Data are mean ± SEM; ****P < 0.0001, ***P < 0.001, **P < 0.01 by one-way ANOVA)

Furthermore, co-immunoprecipitation (Co-IP) experiments revealed that SENP6 knockdown promoted the assembly of the SMAD5/SMAD4 complex (Fig. 5C, D). Luciferase reporter assays for the SOX2 promoter confirmed that SENP6 overexpression significantly suppressed promoter activity, which was partially restored by SMAD5 overexpression (Fig. 5E). Conversely, SENP6 knockdown enhanced SOX2 promoter activity, an effect abolished by simultaneous SMAD5 silencing (Fig. 5F). These data demonstrate that SENP6-mediated deSUMOylation is a critical checkpoint for SMAD5 stability and its downstream transcriptional output.

Therapeutic targeting of SENP6 promotes osteogenesis in a mouse calvarial osteolysis model

To validate these findings in vivo, we utilized a mouse calvarial osteolysis model. Immunohistochemical (IHC) analysis showed that treatment with the SENP6 inhibitor NSC632839 significantly increased the number of SOX2-positive cells within the calvarial defects (Fig. 6A). In contrast, the SMAD5 inhibitor CDD1653 reduced SOX2 expression (Fig. 6A).

Fig. 6.

Therapeutic Targeting of SENP6 and SMAD5 in a Mouse Calvarial Model. A Immunohistochemical (IHC) staining of SOX2 in mouse calvarial defects. Treatment with the SENP6 inhibitor (NSC632839) significantly increased SOX2-positive cells, while the SMAD5 inhibitor (CDD1653) decreased them. Scale bar: 300 μm. B Micro-CT analysis of bone regeneration. The SENP6 inhibitor (NSC632839) significantly attenuated LPS-induced bone resorption and promoted new bone formation compared to the LPS + DMSO control. In contrast, the SMAD5 inhibitor (CDD1653) exacerbated the osteolytic defect. (Data are mean ± SEM, n = 6 per group. ****P < 0.0001 by one-way ANOVA)

Micro-CT analysis and histological assessment revealed that NSC632839 significantly attenuated LPS-induced bone resorption and promoted new bone formation (Fig. 6B). Conversely, the SMAD5 inhibitor CDD1653 exacerbated bone loss (Fig. 6B). These results demonstrate that pharmacological inhibition of SENP6 can effectively rescue osteogenic dysfunction and promote bone regeneration by stabilizing the SMAD5-SOX2 axis, identifying SENP6 as a viable therapeutic target for periodontal and bone regenerative therapies.

Discussion

The present study provides a comprehensive exploration of the SENP6-SMAD5-SOX2 regulatory axis in PDLSCs during periodontitis. By integrating single-cell transcriptomics, molecular biological validation, and in vivo functional assays, we identified SENP6 as a critical regulator of SMAD5 stability and osteogenic transcription. This integrated workflow—transitioning from high-dimensional data screening to precise mechanistic dissection—aligns with the structural paradigms established in recent landmark studies exploring periodontitis biomarkers and therapeutic targets [14, 15].

However, this study has certain limitations. First, while transcriptomic data utilized periodontitis samples, functional assays were performed primarily on healthy PDLSCs; future studies should evaluate whether these mechanisms are fully conserved in diseased cells. Second, although SMAD5 protein levels and localization were assessed, the specific impact of SENP6 on SMAD5 phosphorylation (p-SMAD5) remains to be elucidated. Finally, while we established that SENP6 inhibition triggers the expression of early markers, further investigation is required to determine if this differentiation is sustained to late-stage extracellular matrix mineralization.

A central finding of this work is the reconciliation of the inverse relationship between SMAD5 mRNA upregulation and protein downregulation observed in periodontitis PDLSCs [16, 17]. While transcriptomic data indicated elevated SMAD5 levels, biochemical analysis revealed significant protein depletion and enhanced ubiquitination in diseased tissues. Our data demonstrate that SENP6-mediated deSUMOylation promotes SMAD5 ubiquitination and subsequent proteasomal degradation. This "SUMOylation-ubiquitination crosstalk" represents a vital post-translational regulatory layer in inflammatory bone loss [18]. Interestingly, SENP6’s destabilizing effect on SMAD5 in PDLSCs contrasts with its role in stabilizing substrates like BRCA2 in other contexts, underscoring its tissue-specific functionality [16].

A significant aspect of this study concerns the role of SOX2 in osteogenic fate. Although SOX2 is traditionally recognized as a factor for maintaining stem cell pluripotency, our findings suggest that in the pathological context of periodontitis, the SENP6-SMAD5-SOX2 axis acts as a rheostat balancing stem cell maintenance and osteogenic differentiation [17]. While SOX2 is indeed essential for stemness, our results demonstrate that its direct transcriptional activation by SMAD5 is a prerequisite for initiating early osteogenic programs in PDLSCs [19]. The restoration of SOX2 expression via SENP6 inhibition effectively promotes mineralized nodule formation and bone regeneration [20]. This suggests that SOX2 may play a dual role in PDLSCs: sustaining the progenitor pool while participating in lineage commitment upon the activation of the BMP/SMAD signaling pathway [21].

The in vivo efficacy of the SENP6 inhibitor NSC632839 in a mouse calvarial osteolysis model provides preclinical validation for targeting this axis [13, 14, 22]. Although the calvarial model does not fully replicate the complex anatomy of the periodontium, it offers a robust and standardized environment to evaluate bone regeneration under inflammatory stress [6, 23]. Furthermore, while the BMP pathway involves SMAD1 and SMAD8, our single-cell correlation analysis specifically identified SMAD5 as the most significantly dysregulated R-SMAD in relation to SENP6 expression in periodontitis[24].

In conclusion, this study delineates a novel post-translational regulatory axis governing PDLSC osteogenic fate. By bridging cellular heterogeneity with biochemical stability, we position SENP6 as a promising therapeutic target for bone regeneration in periodontal diseases. Our findings advocate for dual-pathway modulation strategies to counteract the robustness of SMAD signaling networks and restore tissue homeostasis during inflammatory bone loss.

Author contributions

H.J. conceptualized the study, secured funding, designed the methodology, conducted investigations, and drafted the manuscript. C.L. performed data curation, formal analysis, and visualization. W.W. contributed to methodology, investigation, and validation. H.X. conducted data analysis, software development, and validation. B.J. provided resources, supervised the study, and reviewed the manuscript. W.W. assisted with investigation and visualization. X.W. conceptualized the study, supervised the research, and reviewed the manuscript. All authors reviewed and approved the final manuscript.

Funding

This research was independently supported by the Natural Science Foundation of Changning District, Shanghai (Medical Project CNKW2024Y12).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.