Abstract
Objective
Periodontitis is a chronic inflammatory disease that severely affects oral and general health. N7-methylguanosine (m7G) methylation plays critical roles in regulating gene expression and cellular functions. Methyltransferase-like 1 (METTL1), a core component of the m7G methyltransferase complex, has been implicated in various diseases, but its role in periodontitis remains unclear.
Methods
Lipopolysaccharide (LPS)-induced HPDLSCs were used in vitro experiments to evaluate the effects of METTL1 on osteogenic differentiation (ALP activity, ARS staining) and inflammation (TNF-α, IL-1β, IL-6 levels). In vivo experiments employed a mouse model of periodontitis. Alveolar bone loss was evaluated using micro-CT, and inflammatory responses were analysed histologically and through cytokine quantification. Mechanistic studies included m7G modification assays, luciferase reporter assays, RNA immunoprecipitation (RIP), and mRNA stability assays.
Results
METTL1 was upregulated in LPS-induced HPDLSCs and a mouse model of periodontitis. METTL1 knockdown enhanced osteogenic differentiation and reduced pro-inflammatory cytokine levels in HPDLSCs. In the mouse model, METTL1 knockdown alleviated alveolar bone loss, reduced inflammatory cell infiltration, and restored bone density parameters. Mechanistically, METTL1 regulated SEMA4D expression through m7G modification. SEMA4D overexpression reversed the effects of METTL1 knockdown, and mutation of the m7G site in SEMA4D attenuated the suppressive effects of METTL1 overexpression on osteogenic differentiation and inflammatory responses.
Conclusions
These findings provide novel insights into the epigenetic regulation of periodontitis and suggest that targeting the METTL1-SEMA4D axis could offer a promising strategy for disease intervention.
Key words: Periodontitis, Osteoblast differentiation, METTL1, m7G modification, SEMA4D
Introduction
Periodontitis is a chronic inflammatory disease that affects the supporting structures of the teeth, including the gingiva, periodontal ligament, and alveolar bone. It is characterised by the progressive destruction of these tissues, leading to tooth loss if left untreated.1 According to statistics, severe periodontitis affects approximately 10% to 15% of the global population, making it one of the most common chronic diseases worldwide.2 The disease is driven by a dysbiotic microbial community and the host's immune response, which results in the release of pro-inflammatory cytokines and matrix metalloproteinases, ultimately leading to tissue destruction and bone resorption.3,4 Despite advances in understanding its pathogenesis, the molecular mechanisms underlying periodontitis remain incompletely understood, particularly those related to the regulation of bone homeostasis and inflammation.
Recent studies have highlighted the role of epigenetic modifications, including RNA methylation, in the regulation of gene expression and cellular function.5 Among these modifications, N7-methylguanosine (m7G) methylation has emerged as a critical regulator of RNA stability, translation efficiency, and cellular processes.6 Methyltransferase-like 1 (METTL1), a core component of the m7G methyltransferase complex, has been implicated in various biological processes, including cell proliferation, differentiation, and tumorigenesis.7 METTL1 catalyses the addition of m7G to RNA, thereby influencing RNA metabolism and function.8 Dysregulation of METTL1 has been associated with several diseases, including cancer, where it promotes tumour progression by enhancing the translation of oncogenic mRNAs.9 However, its role in inflammatory diseases, particularly periodontitis, remains largely unexplored. In parallel, other epigenetic and epitranscriptomic regulators, such as the histone deacetylase SIRT6 and the long non-coding RNA MIR4435-2HG, have been implicated in modulating inflammation and osteogenic differentiation in other oral inflammatory contexts.10,11 This underscores the multifaceted involvement of epigenetic mechanisms in periodontal homeostasis and pathology.
In addition to its role in RNA modification, METTL1 has been linked to the regulation of genes involved in osteogenic differentiation and inflammation.12 Osteogenic differentiation is a critical process in maintaining alveolar bone homeostasis, and its dysregulation is a hallmark of periodontitis.13 Semaphorin 4D (SEMA4D), also known as CD100, is a member of the semaphorin family and plays a role in immunomodulation, neurodevelopment, angiogenesis, and bone metabolism.14 It has been shown to inhibit osteoblast differentiation and promote osteoclast activity, contributing to bone loss in inflammatory conditions.15 The relationship between METTL1 and SEMA4D, particularly in the context of m7G modification, has not been previously investigated in periodontitis.
This study aims to investigate the expression and functional role of METTL1 in periodontitis, with a focus on its regulatory effects on SEMA4D through m7G modification. By elucidating the molecular mechanisms underlying METTL1′s actions, we hope to provide new insights into the epigenetic regulation of periodontitis and identify potential therapeutic targets for mitigating disease progression.
Methods
Cell culture
Human Periodontal Ligament Stem Cells (HPDLSCs) were purchased from Lonza (Basel, Switzerland) and cultured Alpha-Minimum Essential Medium (α-MEM) supplemented with 10% foetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin (all from Gibco, Grand Island, NY, USA) at 37 °C with 5% CO2. Lipopolysaccharide (LPS) from Porphyromonas gingivalis (PgLPS) was purchased from InvivoGen (San Diego, CA, USA). HPDLSCs were treated with 10 µg/mL PgLPS for 24 hours to induce an inflammatory response mimicking periodontitis.
HPDLSCs were cultured in α-MEM supplemented with 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 10 mM β-glycerophosphate (Sigma-Aldrich, St. Louis, MO, USA), 50 µg/mL ascorbic acid (Sigma-Aldrich), and 100 nM dexamethasone (Sigma-Aldrich) for 21 days to induce osteoblast differentiation.
Cell transfection
The shRNA targeting METTL1 (shMETTL1), shRNA negative control (shNC) SEMA4D overexpression plasmids, METTL1 overexpression plasmids, and empty vector was purchased from GenePharma Company (Shanghai, China). These plasmids were transfected into HPDLSCs using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA).
Quantitative real-time PCR (qPCR)
Total RNA was extracted from HPDLSCs using the RNeasy Mini Kit (Qiagen, Hilden, Germany). cDNA was synthesized from 1 µg of total RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). qPCR was performed using the PowerUp SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA) on the QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The relative mRNA expression levels of METTL1 and SEMA4D were calculated using the 2−∆∆CT method, with GAPDH as the internal control.
Alkaline phosphatase (ALP) activity assay
ALP activity was measured using the Alkaline Phosphatase Assay Kit (Colorimetric) (Abcam, Cambridge, UK). Briefly, 50 µL of cell lysate was mixed with 50 µL of p-nitrophenyl phosphate (pNPP) substrate solution and incubated at 37 °C for 60 minutes. The reaction was stopped by adding 50 µL of stop solution, and the absorbance was measured at 405 nm using a SpectraMax M5 Microplate Reader (Molecular Devices, San Jose, CA, USA).
Alizarin red S (ARS) staining
HPDLSCs were fixed with 4% paraformaldehyde for 15 minutes. Fixed cells were washed twice with distilled water and stained with Alizarin Red S solution (ScienCell Research Laboratories, Carlsbad, CA, USA) for 20 minutes at room temperature. For quantification, the stained calcium deposits were dissolved in 10% cetylpyridinium chloride (Sigma-Aldrich) for 1 hour at room temperature. The absorbance of the dissolved dye was measured at 562 nm using a SpectraMax M5 Microplate Reader (Molecular Devices).
Enzyme linked immunosorbent assay (ELISA)
The levels of TNF-α, IL-1β, IL-6, IL-10, and TGF-β in the culture supernatant and serum were measured using the respective human or mouse quantikine ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. The absorbance was measured at 450 nm using a SpectraMax M5 Microplate Reader (Molecular Devices).
Western blot analysis
Total protein was extracted from HPDLSCs or tissues using RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with protease inhibitors. Protein concentration was determined with a BCA assay kit (Thermo Fisher Scientific). Equal amounts of protein (30 µg) were separated by SDS-PAGE on 10% gels and subsequently transferred onto PVDF membranes (MilliporeSigma, Burlington, MA, USA). After blocking with 5% non-fat milk in TBST for 1 hour at room temperature, the membranes were incubated overnight at 4 °C with the following primary antibodies: rabbit anti-METTL1 (1:1000, 14975, CST, Danvers, MA, USA), rabbit anti-SEMA4D (1:1000, sc-373769, Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-Runx2 (1:800, sc-390351, Santa Cruz Biotechnology), rabbit anti-Osterix (1:1000, sc-393325, Santa Cruz Biotechnology), rabbit anti-OPN (1:1000, sc-21742, Santa Cruz Biotechnology), and mouse anti-GAPDH (1:5000, MA5-15738, Invitrogen, Carlsbad, CA, USA). Following three washes with TBST, membranes were incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-rabbit or anti-mouse, 1:5000, CST) for 1 hour at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) substrate (Thermo Fisher Scientific) and imaged with a chemiluminescence detection system.
Methylated RNA immunoprecipitation (MeRIP)
The m7G methylation status of SEMA4D was assessed utilizing the GenSeq® m7G MeRIP kit (Cloudseq, Shanghai, China). Total RNA was isolated from 2×107 cells and fragmented to approximately 200 nt. Magnetic beads were conjugated with 2 μL of either m7G or IgG antibody for 1 hour on a rotator, followed by washing with IP buffer. Subsequently, 200 μL of nuclease-free water containing the fragmented RNA was introduced to the prepared beads and incubated for 1 hour at 4 °C on a rotator, after which the beads were washed again with IP buffer. The enriched RNA was then purified, and gene expression levels were quantified via qPCR.
RNA immunoprecipitation (RIP)
RIP assays were performed using the EZ-Magna RIP Kit (Millipore, Shanghai, China). HPDLSCs were rinsed twice with PBS and centrifuged. Cell lysis was carried out in 1 mL of RIP lysis buffer on ice for 10 minutes. The lysates were then incubated overnight at 4 °C with magnetic beads coated with either anti-METTL1 antibody (ab271063, Abcam, CA, USA) or anti-IgG (Sigma-Aldrich). Following purification, RNA was isolated, and SEMA4D expression levels were quantified via qPCR.
Luciferase reporter assay
The wild-type (WT) and mutant-type (MUT) SEMA4D sequences were cloned into the pmirGLO reporter plasmid (Promega, Madison, WI, USA). Following METTL1 knockdown, firefly luciferase activity of the reporter constructs was assessed using the Dual-Luciferase Reporter Kit (Promega), with Renilla luciferase activity serving as the internal control for normalization.
Detection of RNA stability
The stability of SEMA4D mRNA was evaluated by qPCR in HPDLSCs following treatment with 5 μg/mL actinomycin D (Merck, Darmstadt, Germany) at 0, 4, 8, and 12-hours intervals.
Animal study
C57BL/6 mice (8-10 weeks old, male) were purchased from Jackson Laboratory (Bar Harbor, ME, USA), housed in a pathogen-free facility with a 12-hours light/dark cycle and access to standard rodent chow and water ad libitum. After 1 week, mice were anesthetized by inhalation of 3% isoflurane (Sigma-Aldrich).
The mouse was placed in a supine position, and the mouth was gently opened using a sterile gauze pad. The maxillary gingiva surrounding the second molar was gently injected with 10 µL of lentiviral suspension (1 × 106 infectious units) using a 33-gauge needle. The contralateral side was injected with the control lentivirus. After 3 days, a 5-0 silk suture (Ethicon, Somerville, NJ, USA) was ligated around the maxillary left second molar, ensuring the ligature was placed subgingivally to induce periodontal inflammation. The contralateral side (right maxillary second molar) was left unligated as a control. The ligature was secured with a surgical knot, and excess suture material was trimmed using fine surgical scissors. The ligature was left in place for 14 days to induce chronic periodontitis. After 14 days, mice were euthanized by inhalation of 5% isoflurane, and gingival tissues surrounding the maxillary second molars were collected.
Hematoxylin and eosin (H&E) staining
Periodontal tissue samples were collected and immediately fixed in 10% Neutral Buffered Formalin. The tissue was dehydrated, paraffin-embedded, and finally cut into 5 μm thick sections. Sections were stained with haematoxylin solution for 10 minutes and eosin solution for 2 minutes. Sections were mounted with DPX Mountant, covered with No. 1.5 Coverslips and dried. Stained sections were examined under a light microscope (Nikon, Tokyo, Japan).
Micro-computed tomography (μCT) imaging
Fixed maxillae were mounted in paraffin wax (Leica Biosystems, Buffalo Grove, IL, USA) and scanned using a SkyScan 1272 High-Resolution Micro-CT System (Bruker MicroCT, Kontich, Belgium). The distance from the cemento-enamel junction (CEJ) to the alveolar bone crest (ABC) was measured on both the palatal and mesiobuccal aspects of the maxillary second molar using CTAn software (Bruker MicroCT, Kontich, Belgium). A region of interest (ROI) was defined around the maxillary second molar, extending from the CEJ to the apex of the roots. The bone volume (BV) and tissue volume (TV) within the ROI were quantified using CTAn software (Bruker MicroCT, Kontich, Belgium).
Bioinformatics analysis
The gene expression dataset GSE10334 was downloaded from the NCBI GEO database for analysis. (https://www.ncbi.nlm.nih.gov/). Differential expression analysis and visualization (heatmap and volcano plot) were performed using the R packages limma and ggplot2, respectively.
Statistical analysis
All data are presented as mean ± standard deviation (SD) and were analysed using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA). Comparisons between two groups were performed using an unpaired Student’s t-test, while comparisons among multiple groups were conducted using one-way or two-way ANOVA followed by Tukey’s post hoc test for pairwise comparisons. A P-value < .05 was considered statistically significant.
Results
METTL1 is highly expressed in periodontitis
Initially, bioinformatics analysis of the periodontitis dataset GSE10334 (downloaded from the NCBI) was employed to identify differentially expressed genes, resulting in the generation of a heatmap (Figure 1A) and a volcano plot (Figure 1B). Among the upregulated differentially expressed genes, METTL1 was prominently identified. Subsequent in vitro and in vivo experiments were conducted to validate the expression profile of METTL1 in periodontitis. We observed a significant increase in METTL1 expression in LPS-induced HPDLSCs (Figure 1C). Similarly, the mRNA levels of METTL1 were markedly elevated in a mouse model of periodontitis (Figure 1D).
Fig. 1.
METTL1 is highly expressed in periodontitis. (A-B) Differentially expressed genes between periodontitis and healthy controls in the GSE10334 dataset are shown as a heatmap and volcano plot. (C) The mRNA expression of METTL1 in LPS-induced hPDLSCs was detected by qPCR. (n = 3). (D) The mRNA expression of METTL1 in mouse model of periodontitis was detected by qPCR. (n = 6). All data are expressed as the means ± SD. ⁎⁎P < .01.
Knockdown of METTL1 enhances osteogenic differentiation in LPS-induced HPDLSCs
To investigate the role of METTL1 in the differentiation capacity of LPS-induced HPDLSCs, we knocked down METTL1 expression in HPDLSCs and confirmed its effective knockdown by examining both mRNA and protein levels (Figure 2A and B). As shown in Figure 2C, LPS stimulation significantly suppressed ALP activity in HPDLSCs; however, METTL1 knockdown restored ALP activity. Similarly, the staining area of ARS was markedly reduced in LPS-induced HPDLSCs, but downregulation of METTL1 expression abolished the inhibitory effect of LPS on ARS staining (Fig. 2, Fig. 2). Furthermore, LPS induction elevated the levels of TNF-α, IL-1β, and IL-6 in HPDLSCs, while silencing METTL1 reversed these effects (Figures 2F-H). Consistent with the functional assays, Western blot analysis revealed that the protein expression of key osteogenic regulators, including Runx2, Osterix, and OPN was significantly downregulated upon LPS challenge. Importantly, METTL1 knockdown effectively rescued the expression of these osteogenic markers in LPS-induced HPDLSCs (Figures 2I-L). The results indicated that METTL1 knockdown mitigates the inhibitory effects of LPS on osteogenic differentiation in HPDLSCs.
Fig. 2.
Knockdown of METTL1 enhances osteogenic differentiation in LPS-induced HPDLSCs. (A-B) The transfection efficiency was detected by qPCR and Western blot. (C) ALP activity was detected in LPS-induced hPDLSCs after METTL1 knockdown. (D-E) The area of Alizarin red staining was detected in LPS-induced hPDLSCs after METTL1 knockdown. (F-H) The levels of TNF-α, IL-1β, and IL-6 were evaluated by ELISA. (I) The protein osteogenic regulatory factors, including Runx2, Osterix, and OPN, were detected by Western blot. (n = 3). All data are expressed as the means ± SD. ⁎⁎P < .01.
Knockdown of METTL1 suppresses alveolar bone loss and inflammatory responses in a mouse model of periodontitis
Next, we investigated the role of METTL1 in an in vivo model of periodontitis. METTL1 expression was downregulated by local injection of a lentivirus carrying METTL1-specific shRNA into the gingival tissues surrounding the maxillary second molar, and the knockdown efficiency was subsequently confirmed at both mRNA and protein levels in the harvested gingival samples (Figure 3A and B). Compared to control mice, periodontitis mice exhibited significant inflammatory cell infiltration, tissue edema, loose connective tissue, collagen fibre degradation, and disorganized periodontal ligament fibres. However, silencing METTL1 expression markedly reduced inflammatory cell infiltration, restored connective tissue density, and improved collagen fibre organization (Figure 3C). Micro-CT analysis of alveolar bone loss revealed a significant increase in the distance between the cementoenamel junction (CEJ) and alveolar bone crest (ABC) in periodontitis mice. In contrast, METTL1 knockdown significantly reduced the CEJ-ABC distance, indicating suppression of alveolar bone loss (Fig. 3, Fig. 3). Similarly, bone density parameters, such as bone volume/total volume (BV/TV), were restored in METTL1-knockdown mice (Figure 3E). Additionally, silencing METTL1 significantly suppressed the expression of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6, in periodontitis mice (Figures 3F-H). Conversely, the levels of anti-inflammatory cytokines IL-10 and TGF-β, which were reduced in the periodontitis model, were notably restored following METTL1 knockdown (Fig. 3, Fig. 3). These findings demonstrated that METTL1 knockdown alleviates alveolar bone loss and inflammatory responses in a mouse model of periodontitis, highlighting that METTL1 as a potential therapeutic target for mitigating periodontitis progression.
Fig. 3.
Knockdown of METTL1 suppresses alveolar bone loss and inflammatory responses in a mouse model of periodontitis. (A-B) The transfection efficiency was detected by qPCR and Western blot. (C) Histological analysis of the periodontium using haematoxylin and eosin (H&E). Micro-CT (computed tomography) analysis of bone resorption state. (D-E) Quantification analysis of distance between cementum-enamel junction (CEJ) and BV/TV. (F-J) The levels of TNF-α, IL-1β, IL-6, IL-10, and TGF-β were evaluated by ELISA. (n = 6). All data are expressed as the means ± SD. ⁎⁎P < .01.
METTL1 regulates SEMA4D expression through m7G modification
To elucidate the regulatory mechanism of METTL1, we analysed its correlated genes in periodontitis, identifying 1902 positively correlated genes and 2445 negatively correlated genes (Figure 4A). Subsequent KEGG pathway enrichment analysis of the positively correlated genes revealed their significant enrichment in pathways related to osteogenic differentiation (Figure 4B). Further analysis focused on osteogenic differentiation-associated genes linked to METTL1 in disease contexts, visualized in a bubble plot (Figure 4C). This analysis identified several candidates, including CDK6, SEMA4D, and HOXA2, which showed positive correlation with METTL1 expression. Among these, we selected SEMA4D for further investigation due to its documented role in inhibiting osteogenic differentiation.16 Pearson correlation analysis confirmed a positive correlation between METTL1 and SEMA4D expression (Figure 4D).
Fig. 4.
METTL1 regulates SEMA4D expression through m7G modification. Volcano plots were used to display genes associated with METTL1 in the disease context. (B) KEGG pathway analysis was performed on genes positively correlated with METTL1 in the disease. (C) Bubble plots were utilized to visualize METTL1-associated genes related to osteogenic differentiation in the disease. (D) The correlation between METTL1 and SEMA4D expression was confirmed by Pearson correlation analysis. (E) The m7G levels of SEMA4D was detected by MeRIP. (F) m7G modification sites of SEMA4D. (G-H) The binding between NUDT1 and GPX4 was measured by Luciferase reporter assays and RIP. (I) The stability of SEMA4D mRNA was measured by qPCR after hPDLSCs were treated with actinomycin D at 0, 4, 8, and 12 h. (n = 3). (J) The protein of SEMA4D were detected by Western blot. All data are expressed as the means ± SD. *P < .05 and ⁎⁎P < .01.
As METTL1 is a core component of the m7G methyltransferase complex, it regulates RNA stability and translation efficiency by catalysing m7G modifications, thereby influencing cellular functions. We measured the m7G levels of SEMA4D and observed a significant reduction in m7G modification upon METTL1 knockdown in HPDLSCs (Figure 4E). Figure 4F illustrates two sequences of SEMA4D containing m7G modification sites. Luciferase reporter assays revealed that silencing METTL1 decreased the luciferase activity of SEMA4D-wt specifically at the 3601 site, while the activity of SEMA4D-mut remained unchanged (Figure 4G). RIP experiments confirmed the direct interaction between METTL1 and SEMA4D (Figure 4H). Additionally, METTL1 knockdown reduced the mRNA stability of SEMA4D at 4, 8, and 12 hours after actinomycin D treatment (Figure 4I). Corroborating this finding, the protein expression level of SEMA4D was also significantly decreased upon METTL1 knockdown (Figure 4J). These findings demonstrated that METTL1 regulates SEMA4D expression through m7G modification, influencing its mRNA stability and interaction.
The overexpression of SEMA4D counteracts the inhibitory effects of METTL1 knockdown on osteogenic differentiation and inflammatory responses in LPS-induced HPDLSCs
To investigate whether the biological functions of METTL1 are mediated by SEMA4D, we conducted rescue experiments. First, SEMA4D was overexpressed in HPDLSCs by transfection with a SEMA4D plasmid, which successfully elevated both mRNA and protein expression levels of SEMA4D (Figure 5A and B). We then observed that the increased ALP activity and ARS staining area induced by METTL1 knockdown in LPS-stimulated HPDLSCs were reversed by SEMA4D overexpression (Figures 5C-E). Additionally, the downregulation of METTL1 reduced the expression of pro-inflammatory cytokines in LPS-induced HPDLSCs, including TNF-α, IL-1β, and IL-6, while SEMA4D overexpression abolished this effect (Figures 5F-H). Furthermore, the recovery of key osteogenic protein markers (Runx2, Osterix, OPN) observed upon METTL1 knockdown was similarly negated by the concurrent overexpression of SEMA4D (Figures 5I). This suggested that the biological functions of METTL1, particularly its role in regulating osteogenesis and inflammation, are mediated through SEMA4D.
Fig. 5.
The overexpression of SEMA4D counteracts the inhibitory effects of METTL1 knockdown on osteogenic differentiation and inflammatory responses in LPS-induced HPDLSCs. (A-B) The transfection efficiency was detected by qPCR and Western blot. (C) ALP activity was detected in LPS-induced hPDLSCs after METTL1 knockdown. (D-E) The area of Alizarin red staining was detected in LPS-induced hPDLSCs after METTL1 knockdown. (F-H) The levels of TNF-α, IL-1β, and IL-6 were evaluated by ELISA. (I) The protein osteogenic regulatory factors, including Runx2, Osterix, and OPN, were detected by Western blot. (n = 3). All data are expressed as the means ± SD. *P < .05 and ⁎⁎P < .01.
Mutation of the m7G modification site in SEMA4D abrogates the functional effects of METTL1 overexpression
To determine whether the regulatory effects of METTL1 on SEMA4D are dependent on its m7G modification, we performed a site-directed mutagenesis rescue experiment. We constructed a SEMA4D mutant (SEMA4D MUT) where the predicted m7G modification site was disrupted. Successful METTL1 overexpression was confirmed (Figures 6A and B). Consistent with prior findings, METTL1 overexpression in the context of wild-type SEMA4D significantly inhibited ALP activity and ARS staining (Figures 6C-E). However, this METTL1-mediated suppression of osteogenic differentiation was significantly attenuated when the m7G site on SEMA4D was mutated, as shown by the partial restoration of both ALP activity and mineralization (Figures 6C-E). Similarly, METTL1 overexpression in the SEMA4D WT background markedly enhanced the LPS-induced secretion of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) (Figures 6E-G). In contrast, this pro-inflammatory effect was largely abolished upon mutation of the SEMA4D m7G site (Figures 6F-H). Finally, Western blot analysis demonstrated that the METTL1-driven downregulation of key osteogenic proteins (Runx2, Osterix, and OPN) observed in the SEMA4D WT+METTL1 group was notably reversed when the m7G modification site on SEMA4D was mutated (Figure 6I). Collectively, these results demonstrate that the m7G modification site on SEMA4D is essential for METTL1 to exert its effects on promoting osteogenic differentiation and mitigating inflammatory responses in HPDLSCs.
Fig. 6.
Mutation of the m7G modification site in SEMA4D abrogates the functional effects of METTL1 overexpression. (A-B) The transfection efficiency was detected by qPCR and Western blot. (C) ALP activity was detected in LPS-induced hPDLSCs after METTL1 knockdown. (D-E) The area of Alizarin red staining was detected in LPS-induced hPDLSCs after METTL1 knockdown. (F-H) The levels of IL-1β, IL-6, and TNF-α were evaluated by ELISA. (I) The protein osteogenic regulatory factors, including Runx2, Osterix, and OPN, were detected by Western blot. (n = 3). All data are expressed as the means ± SD. ⁎⁎P < .01.
Discussion
Periodontitis is a multifactorial inflammatory disease characterized by the progressive destruction of periodontal tissues.17 Currently, the molecular mechanisms underlying bone loss and inflammation in periodontitis remain incompletely understood. This study elucidates the role of METTL1, a key component of the m7G methyltransferase complex, in regulating osteogenic differentiation and inflammatory responses in periodontitis. Our findings demonstrate that METTL1 exacerbates periodontitis by promoting the m7G modification of SEMA4D, thereby providing new insights into the epigenetic regulation of this disease.
Most of the research on METTL1 in recent years has focused on cancer,18 while research on the role of METTL1 in inflammatory diseases is limited. METTL1 has been reported to be identified as a novel m7G-hub biomarker during osteoarthritis progression and used to construct models to predict the occurrence of osteoarthritis.19 METTL1 also regulates immune signalling pathways such as the Notch signalling pathway, the MAPK pathway, and the Toll-like receptor signalling pathway, which can regulate immune cell and inflammatory cytokine release.20, 21, 22 Such findings provide a basis for the exploration of METTL1 in inflammatory diseases. Our initial bioinformatics analysis identified METTL1 as one of the significantly upregulated genes in periodontitis, a finding validated in both LPS-induced HPDLSCs and a mouse model of periodontitis. The elevated expression of METTL1 in these models suggests its potential involvement in the cellular response to bacterial infection, a hallmark of periodontitis. These results demonstrate that METTL1 may be a key regulator in the inflammatory microenvironment of periodontitis.
Osteogenic differentiation, the process by which mesenchymal stem cells differentiate into osteoblasts, is significantly suppressed by the inflammatory microenvironment induced by periodontitis, leading to alveolar bone destruction and impaired regeneration.23 This inhibition of osteogenic differentiation further exacerbates the pathological progression of periodontitis by reducing the regenerative capacity of alveolar bone.24 Consequently, promoting osteogenic differentiation represents a critical therapeutic strategy for periodontitis, aiming to restore alveolar bone regeneration. Notably, METTL1 is markedly downregulated during osteogenic differentiation and inhibits this process through cytokine network modulation.12,25 One of the most striking findings of this study is the role of METTL1 in regulating osteogenic differentiation. LPS stimulation significantly suppressed osteogenic differentiation, as evidenced by reduced ALP activity and ARS staining, and elevated levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6). However, METTL1 knockdown restored these effects, indicating that METTL1 acts as a negative regulator of osteogenic differentiation under inflammatory conditions. The restorative effect of METTL1 knockdown was consistently reflected at the molecular level by the rescued expression of key osteogenic transcription factors and markers. Specifically, the protein levels of Runx2 (a master regulator of osteoblast lineage commitment),26 Osterix (a downstream transcription factor essential for osteoblast differentiation and bone formation),27 and OPN (an early marker of osteogenic differentiation and mineralization)28 were all significantly downregulated under LPS challenge but effectively recovered upon METTL1 silencing. This coordinated restoration underscores that METTL1 knockdown alleviates the inhibition of the core osteogenic transcriptional program, thereby promoting the functional recovery of osteoblast differentiation. In addition, METTL1 knockdown in a mouse model of periodontitis led to reduced inflammatory cell infiltration, improved tissue organization, and decreased alveolar bone loss. These in vivo results further support the notion that METTL1 is a critical regulator of periodontal tissue destruction. The suppression of alveolar bone loss and inflammation in METTL1-knockdown mice underscores its potential as a therapeutic target for mitigating periodontitis progression.
To elucidate the molecular mechanisms underlying METTL1′s effects, we identified SEMA4D as a key downstream target. SEMA4D is a well-characterized gene that regulates osteoblast and osteoclast activity, inhibiting osteogenic differentiation and contributing to imbalanced bone metabolism and impaired bone regeneration.29,30 In periodontitis, SEMA4D exacerbates alveolar bone resorption and delays bone regeneration by regulating inflammatory responses and bone metabolism.31 Herein, METTL1 regulates SEMA4D expression through m7G modification, which enhances RNA stability and translation efficiency. Notably, overexpression of SEMA4D reversed the effects of METTL1 knockdown on osteogenic differentiation and inflammatory responses in LPS-induced HPDLSCs, indicating that that SEMA4D is a critical downstream effector of METTL1 in regulating osteogenesis and inflammation. Furthermore, our site-directed mutagenesis experiment provides direct evidence supporting the functional dependency of this regulatory axis on the specific m7G site within SEMA4D mRNA. Mutation of this site markedly attenuated the METTL1 overexpression-induced suppression of osteogenic differentiation and pro-inflammatory cytokine secretion, indicating that the m7G modification mediated by METTL1 is indispensable for its downstream effects. This reinforces the conclusion that the METTL1-m7G-SEMA4D pathway operates through a precise epitranscriptomic mechanism.
While our study provides significant insights into the role of METTL1 in periodontitis, several limitations should be acknowledged. First, our mechanistic investigation focused primarily on SEMA4D as a downstream effector of METTL1. While this establishes a direct link, it does not preclude the potential involvement of other m⁷G-modified transcripts in regulating osteogenesis or inflammation. A more comprehensive profiling of the METTL1-mediated m⁷G methylome (eg, via meRIP-seq) in the periodontal context could unveil additional key targets, whose individual or synergistic contributions to the observed phenotypic changes warrant future investigation. Second, the translational potential of targeting METTL1 in human periodontitis requires further validation in clinical studies. Additionally, the long-term effects of METTL1 inhibition on periodontal tissue regeneration and systemic health warrant investigation.
From a clinical perspective, our findings possess significant translational potential. First, the identification of METTL1 as a key upstream regulator provides a novel molecular framework for understanding the exacerbated osteoblast dysfunction and inflammation characteristic of progressive periodontitis. Second, the METTL1-SEMA4D axis presents a promising dual-target therapeutic strategy. Inhibiting METTL1 activity or disrupting its interaction with SEMA4D could simultaneously promote bone regeneration and suppress pathological inflammation, addressing two core pathological features of the disease. Future efforts could focus on developing small-molecule inhibitors or targeted RNA therapies against this axis. Third, METTL1 levels in gingival crevicular fluid or serum could be explored as a potential prognostic or diagnostic biomarker for disease activity and treatment response, aiding in personalized periodontitis management.
In summary, this study demonstrates that METTL1 is a critical regulator of osteogenic differentiation and inflammatory responses in periodontitis. Through its role in m7G modification, METTL1 influences the expression and stability of SEMA4D, which in turn modulates cellular processes involved in periodontitis pathogenesis. These findings provide a deeper understanding of the molecular mechanisms underlying periodontitis and highlight METTL1 as a promising therapeutic target.
Author contributions
All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Yong-Song Huang, Zi-Hao Zhang, Mei-Yun Zhou, Lin-Ya Geng and Ting-Ting Wang. The first draft of the manuscript was written by Yong-Song Huang and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Ethical approval
This study was approved by the Ethics Committee of The First Affiliated Hospital of Bengbu Medical University. All animal experiments comply with the ARRIVE guidelines. All methods were carried out in accordance with relevant guidelines and regulations.
Funding
This study was supported by the Science Research Project of Bengbu Medical University [2022byzd034] and the Natural Science Research Project of Anhui Educational Committe [2023AH051979].
Conflict of interest
None disclosed.
Data availability
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
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