DNMT3A transcriptionally downregulated by KLF5 alleviates LPS-induced inflammatory response and promotes osteogenic differentiation in hPDLSCs

Abstract

Background and objective  Periodontitis is an inflammatory disease typically characterized by the destruction of periodontal tissues and complicated etiology. DNA methyltransferase 3A (DNMT3A) has been implicated in possessing pro-inflammatory properties. This study sought to explore the role of DNMT3A in periodontitis and its relevant mechanism.

Methodology  Lipopolysaccharide (LPS) was used to induce inflammation in human periodontal ligament stem cells (hPDLSCs). DNMT3A and KLF5 expressions were detected using RT-qPCR and western blot. The levels of inflammatory cytokines and inflammation-related proteins were detected using ELISA and western blot. NF-κB p65 expression was detected using immunofluorescence (IF) assay, while osteogenic differentiation was assessed using ALP assay and ARS staining. Western blot was used to measure the protein contents associated with osteogenic differentiation. DNMT3A activity was detected using luciferase report assay and chromatin immunoprecipitation (ChIP) was used to verify the interaction between KLF5 and DNMT3A.

Results  DNMT3A expression increased in LPS-induced hPDLSCs. Silencing DNMT3A suppressed the LPS-induced inflammation in hPDLSCs, while promoting osteogenic differentiation. It was also found that transcriptional factor KLF5 could bind to DNMT3A promoters and regulate DNMT3A expression. Rescue experiments showed that KLF5 interference partially counteracted the inhibitory impacts of DNMT3A deficiency on inflammation and the promotive effects on osteogenic differentiation in LPS-induced hPDLSCs.

Conclusion  DNMT3A, when transcriptionally downregulated by KLF5, could alleviate LPS-challenged inflammatory responses and facilitate osteogenic differentiation in hPDLSCs.

Periodontitis; DNMT3A; KLF5; Inflammation; Osteogenic differentiation

Introduction

Periodontitis is typically characterized by the destruction of periodontal supporting tissue, which can result in tooth loss or extraction.¹ Periodontal ligament stem cells (PDLSCs) can differentiate into either periodontal nerve cells and periodontal ligaments.²,³ Additionally, PDLSCs have been reported to play important roles in the regeneration of different dental tissues due to their immunomodulatory properties.⁴ Previous studies suggest that PDLSCs often act as a double-edged swords due to their anti-inflammatory and pro-inflammatory properties.⁵,⁶ Moreover, the suppression of osteogenesis of PDLSCs within periodontal inflammatory microenvironment impairs alveolar bone regeneration, which remains an ongoing challenge for periodontitis therapy.⁷ As such, inhibiting inflammatory damage while promoting osteogenic differentiation in human PDLSCs (hPDLSCs) might be a promising strategy for treating periodontitis.

DNA methylation is often regulated by DNA methyltransferases (DNMTs) with three canonical isoforms identified in humans: DNMT1, DNMT3A and DNMT3B.⁸ A previous study has demonstrated that the inhibition of DNA methylation can promote the osteogenic differentiation of high glucose-induced hPDLSCs via the Wnt signaling pathway.⁹ DNMT1 expression is increased in PDLCs and the inhibition of DNMT1 increases the expressions of osteogenic markers and promotes mineralization, thereby enhancing osteogenic program.¹⁰ DNMT3A expression has been reported to be increased in PDLCs with low osteogenic potential.¹¹ Moreover, when compared to normal samples, DNMT3A expression is increased in periodontitis samples.¹² However, the specific role and relevant mechanism of DNMT3A in periodontitis remain unclear.

As a transcription factor, kruppel‐like factor 5 (KLF5) is highly expressed in various cell types and has been implicated in cell differentiation, pluripotency, and development.¹³,¹⁴ A previous study demonstrates that silencing KLF5 inhibits osteogenic differentiation and promotes inflammation in LPS-induced PDLSCs.¹⁵ Notably, the UCSC database (https://genome.ucsc.edu/) argues that KLF5 can regulate DNMT3A expression. Additionally, the JASPAR database (https://jaspar.elixir.no/) describes the binding sites for KLF5 in the DNMT3A promoter region. Thus, it is reasonable to speculate that DNMT3A may be regulated by the transcription factor KLF5 and play a role in periodontitis.

This study aims to explore the expression of DNMT3A and its relevant mechanism in periodontitis, potentially highlighting DNMT3A as a therapeutic target for periodontitis.

Methodology

Cell culture and treatment

Human periodontal ligament stem cells (hPDLSCs, cat no. HUM-iCell-m002; iCell Bioscience Inc; Shanghai) were incubated in α-modified Eagle’s minimum essential medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO₂. To induce inflammation in hPDLSCs, P. gingivalis LPS (1 μg/mL; cat. no. SMB00610; Sigma) was used for stimulation for 24 h ¹⁶.

Cell transfection

hPDLSCs were harvested in the logarithmic growth phase and then injected into 6-well plates (2.5 ml/well) at a density of 1×10⁵ cells/well. Short hairpin RNA (sh-RNA) specific to DNMT3A (sh-DNMT3A-1/2) or KLF5 (sh-KLF5-1/2), the corresponding negative control (sh-NC), pcDNA3.1 plasmid overexpressing KLF5 (oe-KLF5) and the empty vector (oe-NC) were provided by Shanghai GenePharma Co., Ltd. Transfection of 1.25 μg/mL plasmid or short hairpin RNA into hPDLSCs was performed using Lipofectamine 2000 reagent according to the manufacturer’s instructions. Following 48 h of transfection, hPDLSCs were collected for follow-up studies, and transfection efficacy was detected using RT-qPCR and western blot.

RT-qPCR

The RNA was extracted from sample hPDLSCs using Trizol reagent (Invitrogen) and reverse transcribed into cDNA using a PrimeScript RT reagent kit (Takara Bio, Inc.) according to the manufacturer’s instructions. The templates were amplified using TB Green Premix Ex Taq II on the ABI PRISM 7900 Sequence Detection System (Applied Biosystems) according to the manufacturer’s instructions. The thermocycling conditions were as follows: initial denaturation at 95°C for 3 min; followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 30 s. GAPDH was used as an internal reference and the 2^CT method was used to determine the relative gene expression.¹⁷ The sequences of primers were shown as follows: DNMT3A forward (F), 5’-CGGCCATACGGTGGAGCC-3’ and reverse (R), 5’-CAGACCTTTAGCCACGACCC-3’, KLF5 F, 5’-ACGCTTGGCCTATAACTTGGT-3’ and reverse R, 5’-CGGTCTGGTGTGAGCTGAAT-3’ or GAPDH F, 5’-TGTGGGCATCAATGGATTTGG-3’ and reverse R, 5’-ACACCATGTATTCCGGGTCAAT-3’.

Western blot

hPDLSCs were harvested in the logarithmic growth phase and then injected into 6-well plates (2.5 mL/well) at a density of 1×10⁵ cells/mL. The hPDLSCs were lysed in RIPA lysis buffer (Biosharp) and the lysates were centrifuged at 12,000 g for 10 min at 4°C to obtain proteins. Protein concentration was quantified using BCA protein assay kits according to the manufacturer’s instructions. Equal amounts of proteins (40 μg/lane) were separated with 8% SDS-PAGE and then transferred to PVDF membranes. The membranes were inhibited by 5% BSA at room temperature for 2 h, and then successively introduced overnight to primary antibodies specific to DNMT3A at 4°C (cat. no. ab307503; 1:1,000; Abcam), Cox2 (cat. no. ab179800; 1:1,000; Abcam), iNOS (cat. no. ab178945; 1:1,000; Abcam), BMP2 (cat. no. ab284387; 1:1,000; Abcam), OCN (cat. no. ab133612; 1:1,000; Abcam), Runx2 (cat. no. ab92336; 1:5,000; Abcam), KLF5 (cat. no. ab137676; 1:1,000; Abcam) or GAPDH (cat. no. ab181602; 1:1,000; Abcam). This was followed by exposure to HRP-labeled goat anti-rabbit secondary antibody (cat. no. ab6721; 1:2000, Abcam) for 2 h. The protein bands were visualized with ECL kit and protein density analysis was performed using Image-J software (Version 1.49).

Appraisement of inflammatory cytokines

The hPDLSCs were initially centrifuged at 2,000 g for 5 min at 4°C. The levels of TNF-α, IL-1β and IL-6 in cell supernatants were measured using ELISA assay kits for TNF-α (cat. no. H052-1-2; Nanjing Jiancheng Bioengineering Institute), IL-1β (cat. no. H002-1-2; Nanjing Jiancheng Bioengineering Institute) and IL-6 (cat. no. H007-1-1; Nanjing Jiancheng Bioengineering Institute) according to the manufacturer’s instructions. The OD values at a wavelength of 450 nm were measured utilizing the xMark Microplate absorbance spectrophotometer.

Immunofluorescence assay

The hPDLSCs were fixed with 4% paraformaldehyde for 30 min at 4°C and permeated with 0.1% Triton X-100 for 20 min at room temperature. Following inhibition by 2% BSA for 1 h at room temperature, hPDLSCs were incubated overnight at 4°C with primary antibodies targeting NF-κB p65 (cat. no. 10745-1-AP; 1:50; Proteintech) . The next day, hPDLSCs were incubated for 1 h at room temperature in darkness with Alexa Fluor-594-conjugated secondary antibody (cat. no. 8889S; 1:500; Cell Signaling Technology), counterstained with DAPI, and observed under a fluorescence microscope.

Appraisement of ALP activity

Initially, the hPDLSCs were maintained in osteogenic-inducing media containing 5 mmol/L sodium glycerophosphate, 50 g/mL vitamin C, 100 mmol/L dexamethasone and 10% FBS for 7 d. The hPDLSCs were cultured on 24-pore plates (1 ml/well) at a density of 2.5×10⁵ cells/mL and fixed in 4% formaldehyde for 30 min. Then, hPDLSCs were stained with a BCIP/NBT Alkaline Phosphatase Color Development Kit (cat. no. C3206; Beyotime Institute of Biotechnology; Shanghai, China) for 15 min at room temperature in the dark. ALP activity was detected using ALP activity kit according to the manufacturer’s instructions.

ARS staining

Initially, the hPDLSCs were maintained in osteogenic-inducing media for 21 d, cultured on 24-pore plates (1 ml/well) at a density of 2.5×10⁵ cells/mL and fixed in 4% formaldehyde for 15 min at room temperature according to the manufacturer’s instructions. Afterwards, hPDLSCs were exposed to 2% Alizarin red staining (cat. no. ST1078; Beyotime Institute of Biotechnology; Shanghai, China) and a microscope was used for observation. To elute bone nodules, 10% cetylpyridinium chloride was used and the absorbance was measured at 562 nm.

Luciferase report assay

The JASPAR database described the binding sites of KLF5 and DNMT3A promoters. To verify the interaction, a luciferase report assay was performed on the Luciferase Reporter System (cat. no. E1910; Promega Corporation; Beijing, China). Then, hPDLSCs were injected into 96-well plates (0.1 ml/well) at a density of 4×10⁵ cells/mL for 24 h at 37°C. The pGL3-DNMT3A promoter WT and pGL3-DNMT3A promoter MUT luciferase reporter vectors, along with oe-KLF5 and oe-NC, were transfected into hPDLSCs using Lipofectamine 2000 reagent according to the manufacturer’s instructions. Luciferase activity was measured and normalized to that of Renilla.

ChIP assay

Chromatin immunoprecipitation (ChIP) was performed using a commercially available kit (cat. no. P2078; Beyotime Institute of Biotechnology; Shanghai, China). The hPDLSCs were subjected to 1% formaldehyde for the crosslinking of the targeted proteins with corresponding genomic DNA. Then, hPDLSCs were centrifuged at 13,000 g for cell collection and rinsed with pre-chilled PBS. The hPDLSCs were then sonicated, producing DNA fragments ranging from 200 to 500 bp with a high intensity ultrasonic processor (cat. no. CN-04714-50; Cole-Parmer; Shanghai, China). The immunoprecipitation of equal amount of chromatin was performed. Thereafter, the lysates were immunoprecipitated with 2 µg anti-DNMT3A or IgG antibodies. Finally, the purified DNA was subjected to PCR amplification.

Statistical analysis

All experiments were duplicated three times. Data were presented as mean ± standard deviation. Statistical analysis was conducted using GraphPad Prism 8.0 software. Differences between the two groups were analyzed using Student’s t-test and the comparisons among multiple groups were demonstrated using one-way ANOVA along with Tukey’s post hoc test. P-values less than 0.05 indicated statistical significance.

Results

DNMT3A expression was upregulated in LPS-induced hPDLSCs

Initially, RT-qPCR and western blot were used to detect DNMT3 expression. Compared with the control group, the mRNA and protein levels of DNMT3 were significantly increased in LPS-stimulated hPDLSCs (Figure 1A). To reduce DNMT3 expression, sh-DNMT3A was transfected into hPDLSCs and the transfection efficiency was evaluated using RT-qPCR and western blot. As shown in Figure 1B, DNMT3 expression was significantly decreased by sh-DNMT3A, with sh-DNMT3A-1 producing the greatest reduction, thus being selected for subsequent experiments. Furthermore, the increased DNMT3 expression stimulated by LPS in hPDLSCs was reduced by sh-DNMT3 (Figure 1C).

Figure 1
DNMT3A expression was upregulated in LPS-induced hPDLSCs. (A) The mRNA and protein expression of DNMT3A was detected using RT-qPCR and western blot. (B) The transfection efficiency of sh-DNMT3A was detected with RT-qPCR and western blot. (C) The mRNA and protein expression of DNMT3A in LPS induced hPDLSCs transfected with sh-DNMT3A was detected using RT-qPCR and western blot. *P<0.05, **P<0.01 and ***P<0.001.

DNMT3A silence inhibited LPS-induced inflammation in hPDLSCs

Periodontitis is characterized by chronic inflammation.¹⁸ To investigate the role of DNMT3A in inflammation in LPS-induced hPDLSCs, the levels of inflammatory cytokines were firstly assessed. The elevated levels of TNF-α, IL-1β and IL-6 stimulated by LPS in hPDLSCs were significantly reduced after DNMT3A silencing (Figure 2A). Western blot analysis indicated the contents of inflammation-related proteins and demonstrated that LPS induction significantly increased Cox2 and iNOS levels in hPDLSCs, which were reduced by DNMT3A interference (Figure 2B). Moreover, immunofluorescence assays revealed that the elevated NF-κB p65 level in the LPS group was significantly decreased after DNMT3A interference in the LPS+sh-DNMT3A group (Figure 2C).

Figure 2
DNMT3A silencing inhibited LPS-induced inflammation in hPDLSCs. (A) The levels of inflammatory cytokines were detected using ELISA assay. (B) The expressions of inflammation-related proteins were detected using western blot. (C) The expression of NF-κB p65 was detected using IF assay. **P<0.01 and ***P<0.001.

DNMT3A silencing promoted LPS-induced osteogenic differentiation in hPDLSCs

The osteogenic differentiation ability of PDLSCs in periodontitis patients is often impaired.¹⁹ Compared with the control group, LPS significantly reduced ALP activity in hPDLSCs, while DNMT3A silencing increased ALP activity in the LPS+sh-DNMT3A group (Figure 3A). Furthermore, the role of DNMT3A deficiency in the mineralization in LPS-insulted hPDLSCs was investigated through ASR staining. As shown in Figure 3B, the reduced number of mineralized nodules in hPDLSCs due to LPS stimulation was significantly increased after interfering DNMT3A. Additionally, western blot analysis indicated the contents of proteins associated with osteogenic differentiation and found that DNMT3A knockdown significantly increased the contents of BMP2, OCN and Runx2 in LPS-induced hPDLSCs compared with the LPS+sh-NC group (Figure 3C).

Figure 3
DNMT3A silencing promoted LPS-induced osteogenic differentiation in hPDLSCs. (A) ALP activity was detected using ALP assay. (B) Mineralization was detected using ARS staining. (C) The expressions of osteogenic differentiation-related proteins were detected using western blot. ***P<0.001.

Transcriptional factor KLF5 could bind to DNMT3A and regulate DNMT3A expression

To investigate the role of KLF5 in periodontitis, KLF5 expression was analyzed using RT-qPCR and western blot. Compared with the control group, LPS stimulation significantly reduced KLF5 expression in hPDLSCs (Figure 4A). The UCSC database argued that KLF5 could regulate DNMT3A expression, while the JASPAR database identified the binding sites of KLF5 and DNMT3A promoters (Figure 4B). To increase or decrease KLF5 expression, oe-KLF5 or sh-KLF5 was respectively transfected into hPDLSCs. Compared with the oe-NC group, oe-KLF5 significantly increased KLF5 expression in hPDLSCs, while sh-KLF5 significantly reduced it. Also notable is the fact that sh-KLF5-1 contributed to lower KLF5 expression, and it was thus selected for follow-up studies (Figure 4C). To explore the regulatory impacts of KLF5 on DNMT3A, DNMT3A expression in hPDLSCs transfected with oe-KLF5 or sh-KLF5 was detected. It was noted that oe-KLF5 decreased DNMT3A expression while sh-KLF5 increased DNMT3A expression in hPDLSCs, indicating that KLF5 negatively regulated DNMT3A expression (Figure 4D). Additionally, KLF5 overexpression was discovered to reduce DNMT3A activity compared with the DNMT3A-WT+oe-NC group (Figure 4E). The ChIP assay results further confirmed that DNMT3A was enriched in anti-KLF5 (Figure 4F).

Figure 4
Transcriptional factor KLF5 could bind to DNMT3A and regulate DNMT3A expression. (A) The mRNA and protein expression of KLF5 was detected using RT-qPCR and western blot. (B) The JASPAR database described the binding sites of KLF5 and DNMT3A promoters. (C) The mRNA and protein expression of KLF5 in hPDLSCs transfected with oe-KLF5 or sh-KLF5 was detected using RT-qPCR and western blot. (D) The mRNA and protein expression of DNMT3A in hPDLSCs transfected with oe-KLF5 or sh-KLF5 was detected using RT-qPCR and western blot. (E) The activity of DNMT3A promoter was detected using luciferase report assay. (F) The binding of KLF5 with DNMT3A promoters was confirmed by ChIP assay. **P<0.01 and ***P<0.001.

KLF5 silencing partially reversed the suppressive effects of DNMT3A depletion on LPS-induced inflammation in hPDLSCs

To investigate the interaction between DNMT3A and KLF5 in LPS-challenged hPDLSCs, sh-DNMT3A and sh-KLF5 were transfected into hPDLSCs and the abovementioned functional experiments were repeated. Compared with the LPS group, DNMT3A deficiency reduced DNMT3A expression, which was partially restored by sh-KLF5 (Figure 5A). In comparison with the LPS+sh-DNMT3A+sh-NC group, the reduced levels of TNF-α, IL-1β and IL-6 and decreased expressions of Cox2 and iNOS in DNMT3A-silenced hPDLSCs under LPS stimulation were significantly increased by KLF5 interference (Figure 5B-C). Moreover, KLF5 silencing also increased NF-κB p65 levels in the LPS+sh-DNMT3A+sh-KLF5 group (Figure 5D).

Figure 5
KLF5 silencing partially reversed the suppressive effects of DNMT3A depletion on LPS-induced inflammation in hPDLSCs. (A) The mRNA and protein expression of DNMT3A in hPDLSCs transfected with sh-KLF5 or sh-KLF5 was detected using RT-qPCR and western blot. (B) The levels of inflammatory cytokines were detected using ELISA assay. (C) The expressions of inflammation-related proteins were detected using western blot. (D) The expression of NF-κB p65 was detected using IF assay. *P<0.05, **P<0.01 and ***P<0.001.

KLF5 silence partially reversed the promotive effects of DNMT3A depletion on LPS-induced osteogenic differentiation in hPDLSCs

Compared with the LPS group, sh-DNMT3A significantly increased ALP activity and the number of mineralized nodules in hPDLSCs, while sh-KLF5 reduced ALP activity and the number of mineralized nodules in the LPS+sh-DNMT3A+sh-KLF5 group (Figure 6A-B). Moreover, compared with the LPS+sh-DNMT3A+sh-NC group, KLF5 interference reduced the protein contents of BMP2, OCN and Runx2 in the LPS+sh-DNMT3A+sh-KLF5 group (Figure 6C).

Figure 6
KLF5 silencing partially reversed the promotive effects of DNMT3A depletion on LPS-induced osteogenic differentiation in hPDLSCs. (A) ALP activity was detected using ALP assay. (B) Mineralization was detected using ARS staining. (C) The expressions of osteogenic differentiation-related proteins were detected using western blot. *P<0.05, **P<0.01 and ***P<0.001.

Discussion

Periodontitis, which is an infectious disease that leads to the progressive destruction of periodontal ligament tissue, resulting in bone and tooth loss.²⁰ hPDLSCs have immunomodulatory properties that help protect against infections.²⁰PDLSCs can differentiate into osteoblasts and odontogenic osteocytes, and possess multipotent and proliferative properties.²¹,²² An in vivo research has demonstrated that PDLSCs can form the alveolar bone, periodontal membrane, and osteoid-like tissues to repair periodontal defects.²³ Additionally, PDLSCs have strong self-renewal and osteogenic differentiation potentials,²⁴ making them ideal candidates for periodontal defect restoration.²⁵

Numerous inflammatory cytokines accumulate in periodontal tissue following stimulation by periodontal pathogenic bacteria, triggering inflammation and periodontal bone destruction.²⁶ Chronic inflammation hampers periodontal regeneration, as proinflammatory factors can exacerbate damage to periodontal tissue.²⁷,²⁸ Moreover, multiple pro-inflammatory cytokines such as IL-6, TNF-α and IL-1β are involved in periodontitis.²⁹ Specifically, TNF-α participates in osteoclastogenesis and interferes with alveolar bone restoration.³⁰ Considering this, targeting inflammation might be a curative modality for periodontitis. DNMT3A has been proved to modulate CSE-induced inflammation in MH-S cells,³¹ and its deficiency can obstruct inflammation in alcohol-induced liver injury.³² Our results demonstrated that DNMT3A interference reduced LPS-induced levels of IL-6, TNF-α and IL-1β in hPDLSCs, alongside reduced expressions of Cox2 and iNOS.

The key to treating periodontitis lies in the repair and reconstruction of periodontal bone tissue.³³ The osteogenic capability of PDLSCs is critical for restoring alveolar bone damage caused by periodontitis .³⁴ ATP is an indicator of cell osteogenic activity and serves as a pivotal regulator in cell mineralization.³⁵ Moreover, ARS staining is used to estimate mineralization, particularly in the evaluation of advanced osteoblast differentiation.³⁶ Hara, et al.³⁷(2013) have corroborated that DNMT3A transfection increases the odontogenic differentiation of dental pulp cells. Additionally, the changes of DNMT3A-mediated DNA methylation can regulate osteogenic differentiation of hMSCs cultivated in the 3D scaffolds under oxidative stress.³⁸ Our findings indicated that DNMT3A interference could promote osteogenic differentiation in hPDLSCs, evidenced by increased ALP activity, mineralized nodules, and contents of BMP2, OCN and Runx2.

Bioinformatics analysis indicated that KLF5 can regulate DNMT3A and their binding sites. Our results showed that KLF5 could bind to DNMT3A promoters and negatively regulate DNMT3A expression. As demonstrated, KLF5 plays a role in cell proliferation during tooth development and is closely associated with enamel and dentin matrix mineralization.³⁹ In vivo research has proved that KLF5 promotes odontoblastic differentiation and facilitates the shaping of the dental papilla mesenchymal cells in mice.⁴⁰ KLF5 is also involved in periodontitis through gene regulation. For instance, the targeting of KLF5 by miR-143-3p can obstruct osteogenic differentiation in hPDLCs.⁴¹ KLF5-mediated SIRT6 can block inflammation while promoting osteogenic differentiation in LPS-induced PDLSCs.¹⁵ In this work, KLF5 depletion could facilitate inflammation while inhibiting osteogenic differentiation in LPS-induced hPDLSCs transfected with sh-DNMT3A.

Conclusion

This study revealed the modulatory role of DNMT3A in LPS-induced inflammation and osteogenic differentiation in hPDLSCs, and demonstrated that KLF5 can bind to DNMT3A. This work is the first to discuss the protective mechanism of DNMT3A in periodontitis and may provide a preclinical rationale for considering DNMT3A as a prospective therapeutic modality for periodontitis. However, there are limitations to this paper, such as the fact that role of DNMT3A in periodontitis in clinic setting is not yet determined, as well as the need for further in vivo experiments to corroborate our findings.

References

• ¹ - Slots J. Periodontitis: facts, fallacies and the future. Periodontology 2000. 2017;75:7-23. doi: 10.1111/prd.12221
  » https://doi.org/10.1111/prd.12221
• ² - Tomokiyo A, Wada N, Maeda H. Periodontal ligament stem cells: regenerative potency in periodontium. stem cells and development. 2019;28:974-85. doi: 10.1089/scd.2019.0031
  » https://doi.org/10.1089/scd.2019.0031
• ³ - Mohebichamkhorami F, Fattahi R, Niknam Z, Aliashrafi M, Khakpour Naeimi S, et al. Periodontal ligament stem cells as a promising therapeutic target for neural damage. Stem Cell Res Ther. 2022;13(1):273. doi: 10.1186/s13287-022-02942-9
  » https://doi.org/10.1186/s13287-022-02942-9
• ⁴ - Andrukhov O, Behm C, Blufstein A, Rausch-Fan X. Immunomodulatory properties of dental tissue-derived mesenchymal stem cells: implication in disease and tissue regeneration. World journal of stem cells. 2019;11(9):604-17. doi: 10.4252/wjsc.v11.i9.604
  » https://doi.org/10.4252/wjsc.v11.i9.604
• ⁵ - Cheng M, Zhou Q. Targeting EZH2 ameliorates the LPS-inhibited PDLSC osteogenesis via Wnt/ß-catenin pathway. Cells Tissues Organs. 2020;209(4-6):227-35. doi: 10.1159/000511702
  » https://doi.org/10.1159/000511702
• ⁶ - Misawa MY, Silvério Ruiz KG, Nociti FH Jr, Albiero ML, Saito MT, Nóbrega Stipp R, et al. Periodontal ligament-derived mesenchymal stem cells modulate neutrophil responses via paracrine mechanisms. J Periodontol. 2019;90(7):747-55. doi: 10.1002/jper.18-0220
  » https://doi.org/10.1002/jper.18-0220
• ⁷ - Lin L, Li S, Hu S, Yu W, Jiang B, Mao C,et al. UCHL1 impairs periodontal ligament stem cell osteogenesis in periodontitis. J Dent Res. 2023;102(1):61-71. doi: 10.1177/00220345221116031
  » https://doi.org/10.1177/00220345221116031
• ⁸ - Man X, Li Q, Wang B, Zhang H, Zhang S, Li Z. DNMT3A and DNMT3B in breast tumorigenesis and potential therapy. Front Cell Dev Biol. 2022;10:916725. doi:10.3389/fcell.2022.916725
  » https://doi.org/10.3389/fcell.2022.916725
• ⁹ - Liu Z, Chen T, Sun W, Yuan Z, Yu M, Chen G, et al. DNA demethylation rescues the impaired osteogenic differentiation ability of human periodontal ligament stem cells in high glucose. Sci Rep. 2016;6:27447. doi: 10.1038/srep27447
  » https://doi.org/10.1038/srep27447
• ¹⁰ - Assis RI, Schmidt AG, Racca F, Silva RA, Zambuzzi WF, Silvério KG, et al. DNMT1 inhibitor restores RUNX2 expression and mineralization in periodontal ligament cells. DNA Cell Biol. 2021;40(5):662-74. doi: 10.1089/dna.2020.6239
  » https://doi.org/10.1089/dna.2020.6239
• ¹¹ - Ferreira RS, Assis RI, Feltran GD, Rosário Palma IC, Françoso BG, Zambuzzi WF, et al. Genome-wide DNA (hydroxy) methylation reveals the individual epigenetic landscape importance on osteogenic phenotype acquisition in periodontal ligament cells. J Periodontol. 2022;93(3):435-48. doi: 10.1002/jper.21-0218
  » https://doi.org/10.1002/jper.21-0218
• ¹² - Wang P, Wang B, Zhang Z, Wang Z. Identification of inflammation-related DNA methylation biomarkers in periodontitis patients based on weighted co-expression analysis. Aging (Albany NY). 2021;13(15):19678-95. doi: 10.18632/aging.203378
  » https://doi.org/10.18632/aging.203378
• ¹³ - Zeng L, Zhu Y, Moreno CS, Wan Y. New insights into KLFs and SOXs in cancer pathogenesis, stemness, and therapy. Semin Cancer Biol. 2023;90:29-44. doi: 10.1016/j.semcancer.2023.02.003
  » https://doi.org/10.1016/j.semcancer.2023.02.003
• ¹⁴ - Ma JB, Bai JY, Zhang HB, Jia J, Shi Q, Yang C, et al. KLF5 inhibits STAT3 activity and tumor metastasis in prostate cancer by suppressing IGF1 transcription cooperatively with HDAC1. Cell Death Dis. 2020;11(6):466. doi: 10.1038/s41419-020-2671-1
  » https://doi.org/10.1038/s41419-020-2671-1
• ¹⁵ - Li C, Xiao F, Wen Y, Wu J, Huang N. Krüppel-like factor 5 -mediated Sirtuin6 promotes osteogenic differentiation and inhibits inflammatory injury of lipopolysaccharide-induced periodontal membrane stem cells by inhibiting nuclear fac-or kappa-B pathway. Bioengineered. 2022;13(3):6966-77. doi: 10.1080/21655979.2022.2036915
  » https://doi.org/10.1080/21655979.2022.2036915
• ¹⁶ - Duan Y, An W, Wu H, Wu Y. Salvianolic acid c attenuates LPS-Induced inflammation and apoptosis in human periodontal ligament stem cells via toll-like receptors 4 (TLR4)/Nuclear Factor kappa B (NF-?B) pathway. Med Sci Monit. 2019;25:9499-508. doi: 10.12659/msm.918940
  » https://doi.org/10.12659/msm.918940
• ¹⁷ - Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402-8. doi:10.1006/meth.2001.1262
  » https://doi.org/10.1006/meth.2001.1262
• ¹⁸ - Gusmão J, Fonseca KM, Ferreira BS, Freitas Alves BW, Ribeiro HL Jr, Lisboa MR, et al. Electroacupuncture reduces inflammation but not bone loss on periodontitis in arthritic rats. Inflammation. 2021;44(1):116-28. doi: 10.1007/s10753-020-01313-x
  » https://doi.org/10.1007/s10753-020-01313-x
• ¹⁹ - Lu W, Zhang L, Ji K, Ding L, Wu G. Regulatory mechanisms of GCN5 in osteogenic differentiation of MSCs in periodontitis. Clin Exp Dent Res. 2023;9(3):464-71. doi: 10.1002/cre2.695
  » https://doi.org/10.1002/cre2.695
• ²⁰ - Marconi GD, Fonticoli L, Guarnieri S, Cavalcanti MF, Franchi S, Gatta V, et al. Ascorbic acid: a new player of epigenetic regulation in lps-gingivalis treated human periodontal ligament stem cells. Oxid Med Cell Longev. 2021;2021:6679708. doi: 10.1155/2021/6679708
  » https://doi.org/10.1155/2021/6679708
• ²¹ - Queiroz A, Albuquerque-Souza E, Gasparoni LM, França BN, Pelissari C, Trierveiler M, et al. Therapeutic potential of periodontal ligament stem cells. World J Stem Cells. 2021;13(6):605-18. doi: 10.4252/wjsc.v13.i6.605
  » https://doi.org/10.4252/wjsc.v13.i6.605
• ²² - Marconi GD, Diomede F, Pizzicannella J, Fonticoli L, Merciaro I, Pierdomenico SD, et al. Enhanced VEGF/VEGF-R and RUNX2 expression in human periodontal ligament stem cells cultured on sandblasted/etched titanium disk. Front Cell Dev Biol. 2020;8:315. doi: 10.3389/fcell.2020.00315
  » https://doi.org/10.3389/fcell.2020.00315
• ²³ - Tour G, Wendel M, Moll G, Tcacencu I. Bone repair using periodontal ligament progenitor cell-seeded constructs. J Dent Res. 2012;91(8):789-94. doi: 10.1177/0022034512452430
  » https://doi.org/10.1177/0022034512452430
• ²⁴ - Zhao Z, Liu J, Weir MD, Schneider A, Ma T, Oates TW, et al. Periodontal ligament stem cell-based bioactive constructs for bone tissue engineering. Front Bioeng Biotechnol. 2022;10:1071472. doi: 10.3389/fbioe.2022.1071472
  » https://doi.org/10.3389/fbioe.2022.1071472
• ²⁵ - Wu Y, Wang X, Zhang Y, Wen Z, Li Y, Zhang K, et al. Proanthocyanidins ameliorate LPS-inhibited osteogenesis of PDLSCs by restoring lysine lactylation. Int J Mol Sci. 2024;25(5):2947. doi: 10.3390/ijms25052947
  » https://doi.org/10.3390/ijms25052947
• ²⁶ - Naruishi K, Nagata T. Biological effects of interleukin-6 on gingival fibroblasts: cytokine regulation in periodontitis. J Cell Physiol. 2018;233(9):6393-400. doi: 10.1002/jcp.26521
  » https://doi.org/10.1002/jcp.26521
• ²⁷ - Wang L, Li X, Song Y, Zhang L, Ye L, Zhou X, et al. NELL1 augments osteogenesis and inhibits inflammation of human periodontal ligament stem cells induced by BMP9. J Periodontol. 2022;93(7):977-87. doi: 10.1002/jper.20-0517
  » https://doi.org/10.1002/jper.20-0517
• ²⁸ - Schilling E, Weiss R, Grahnert A, Bitar M, Sack U, Hauschildt S. Molecular mechanism of LPS-induced TNF-a biosynthesis in polarized human macrophages. Mol Immunol. 2018;93:206-15. doi: 10.1016/j.molimm.2017.11.026
  » https://doi.org/10.1016/j.molimm.2017.11.026
• ²⁹ - Wang RP, Huang J, Chan KW, Leung WK, Goto T, Ho YS, et al. IL-1ß and TNF-a play an important role in modulating the risk of periodontitis and Alzheimer's disease. J Neuroinflammation. 2023;20(1):71. doi: 10.1186/s12974-023-02747-4
  » https://doi.org/10.1186/s12974-023-02747-4
• ³⁰ - Zhou R, Shen L, Yang C, Wang L, Guo H, Yang P, et al. Periodontitis may restrain the mandibular bone healing via disturbing osteogenic and osteoclastic balance. Inflammation. 2018;41(3):972-83. doi: 10.1007/s10753-018-0751-5
  » https://doi.org/10.1007/s10753-018-0751-5
• ³¹ - Qiu J, Liu X, Yang G, Gui Z, Ding S. MiR-29b level-mediated regulation of Klotho methylation via DNMT3A targeting in chronic obstructive pulmonary disease. Cell Dev. 2023;174:203827. doi: 10.1016/j.cdev.2023.203827
  » https://doi.org/10.1016/j.cdev.2023.203827
• ³² - Xu JJ, Zhu L, Li HD, Du XS, Li JJ, Yin NN, et al. DNMT3a-mediated methylation of PSTPIP2 enhances inflammation in alcohol-induced liver injury via regulating STAT1 and NF-?B pathway. Pharmacol Res. 2022;177:106125. doi: 10.1016/j.phrs.2022.106125
  » https://doi.org/10.1016/j.phrs.2022.106125
• ³³ - Seo BM, Miura M, Gronthos S, Bartold PM, Batouli S, Brahim J, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet. 2004;364(9429):149-55. doi: 10.1016/s0140-6736(04)16627-0
  » https://doi.org/10.1016/s0140-6736(04)16627-0
• ³⁴ - Li W, Huang X, Yu W, Xu Y, Huang R, Park J, et al. Activation of functional somatic stem cells promotes endogenous tissue regeneration. J Dent Res. 2022;101(7):802-11. doi: 10.1177/00220345211070222
  » https://doi.org/10.1177/00220345211070222
• ³⁵ - Qin W, Chen JY, Guo J, Ma T, Weir MD, Guo D, et al. Novel calcium phosphate cement with metformin-loaded chitosan for odontogenic differentiation of human dental pulp cells. Stem Cells Int. 2018;2018:7173481. doi: 10.1155/2018/7173481
  » https://doi.org/10.1155/2018/7173481
• ³⁶ - Zhou Y, Fan W, Xiao Y. The effect of hypoxia on the stemness and differentiation capacity of PDLC and DPC. Biomed Res Int. 2014;2014:890675. doi: 10.1155/2014/890675
  » https://doi.org/10.1155/2014/890675
• ³⁷ - Hara ES, Ono M, Eguchi T, Kubota S, Pham HT, Sonoyama W, et al. miRNA-720 controls stem cell phenotype, proliferation and differentiation of human dental pulp cells. PLoS One. 2013;8(12):e83545. doi: 10.1371/journal.pone.0083545
  » https://doi.org/10.1371/journal.pone.0083545
• ³⁸ - Li L, Ling Z, Dong W, Chen X, Vater C, Liao H, et al. Dnmt3a-Mediated DNA methylation changes regulate osteogenic differentiation of hMSCs cultivated in the 3D Scaffolds under oxidative stress. Oxid Med Cell Longev. 2019;2019:4824209. doi: 10.1155/2019/4824209
  » https://doi.org/10.1155/2019/4824209
• ³⁹ - Chen Z, Couble ML, Mouterfi N, Magloire H, Chen Z, Bleicher F. Spatial and temporal expression of KLF4 and KLF5 during murine tooth development. Arch Oral Biol. 2009;54(5):403-11. doi: 10.1016/j.archoralbio.2009.02.003
  » https://doi.org/10.1016/j.archoralbio.2009.02.003
• ⁴⁰ - Chen Z, Zhang Q, Wang H, Li W, Wang F, Wan C, et al. Klf5 mediates odontoblastic differentiation through regulating dentin-specific extracellular matrix gene expression during mouse tooth development. Sci Rep. 2017;7:46746. doi: 10.1038/srep46746
  » https://doi.org/10.1038/srep46746
• ⁴¹ - Wangzhou K, Lai Z, Lu Z, Fu W, Liu C, Liang Z, et al. MiR-143-3p inhibits osteogenic differentiation of human periodontal ligament cells by targeting KLF5 and inactivating the Wnt/ß-catenin pathway. Front Physiol. 2020;11:606967. doi: 10.3389/fphys.2020.606967
  » https://doi.org/10.3389/fphys.2020.606967

• Data availability
  The analyzed data sets generated during the present study are available from the corresponding author on reasonable request.

Edited by

• Editor:
  Ana Carolina Magalhães

• Associate Editor:
  Ana Carolina Morandini

Data availability

The analyzed data sets generated during the present study are available from the corresponding author on reasonable request.

Publication Dates

• Publication in this collection
  28 Oct 2024
• Date of issue
  2024

History

• Received
  5 July 2024
• Reviewed
  4 Sept 2024
• Accepted
  10 Sept 2024