KDM6B/Pdk1 glycolytic pathway-driven ZEB2 lactylation promotes cellular cementum formation
Zhengkun Yang, Huiyi Wang, Junhong Xiao, Qiudong Yang, Jiahui Sun, Heyu Liu, Zhendong Huang, Li Ma, Xin Huang, Chuan Wang, Xiaoxuan Wang, Zhengguo Cao
TL;DR
This study reveals a new pathway involving KDM6B, Pdk1, and ZEB2 lactylation that promotes cementum formation, offering insights for periodontal regeneration.
Contribution
The study identifies a novel KDM6B–Pdk1–ZEB2 lactylation axis crucial for cementogenesis and periodontal regeneration.
Findings
KDM6B promotes cementoblast mineralization through glycometabolic reprogramming.
PDK1 is a direct target of KDM6B, and its activation increases lactate production.
ZEB2 lactylation enhances cementoblast mineralization and can be restored with sodium lactate.
Abstract
Periodontitis is a common chronic inflammatory disease that ultimately results in irreversible tooth loss. Cementum, a bone-like tissue surrounding tooth roots, deteriorates as periodontitis advances, ultimately causing tooth loss. Therefore, cementum regeneration is considered a key factor in periodontal regeneration. Given the shared gene expression patterns and characteristics between cementum and bone, strategies for cementum regeneration may inform approaches for bone regeneration. Cementoblasts are responsible for cementum formation. This study identified lysine demethylase 6B (KDM6B) as a positive regulatory molecule that promotes cementoblast mineralization and formation. The seahorse assay revealed that KDM6B regulates glycometabolic reprogramming during cementoblast mineralization. Chromatin Immunoprecipitation (ChIP) sequencing and bulk RNA sequencing revealed that pyruvate…
Genes, proteins, chemicals, diseases, species, mutations and cell lines named across the full text — each resolved to its canonical identifier and authoritative record.
Click any figure to enlarge with its caption.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8- —https://doi.org/10.13039/501100001809National Natural Science Foundation of China (National Science Foundation of China)
- —本研究得到了国家对曹正国国家重点研发计划（No.2023YFC2506300）和武汉大学口腔医学院和医院对曹正国（ZW202402）科研项目的部分支持。
Peer Reviews
No public reviews on file for this paper yet. If you reviewed it on a platform where reviews are public (OpenReview, ICLR, NeurIPS, ICML), you can paste yours below so the community can read it here.
Videos
No videos yet. Explain this paper in a talk, walkthrough, or lecture? Add one.
Taxonomy
TopicsPeriodontal Regeneration and Treatments · Epigenetics and DNA Methylation · Oral microbiology and periodontitis research
Introduction
Periodontitis causes the degradation of periodontal support tissue and is the leading cause of tooth loss.^1^ It is a chronic infectious disease associated with various systemic disorders, including cardiovascular disease, diabetes and rheumatoid arthritis.^2^ Cementum, a thin mineralized layer covering the tooth root, is critical to periodontal attachment.^3^ Cementoblasts, which are essential for maintaining cementum homeostasis, share morphological and molecular similarities with osteoblasts and play a crucial role in periodontal health.^4^ Understanding the molecular mechanisms of cementoblast mineralization and cementogenesis is vital for periodontal regeneration. Given the similarities between cementum and bone tissue, strategies for cementum regeneration can inform approaches to bone regeneration.
Epigenetic modifications influence cell differentiation, development, disease progression and mineralization.^5–7^ Histone methylation and demethylation serve as important pathways for regulating gene expression.^8^ Histone demethylation is controlled by arginine-specific and lysine (K) -specific demethylases (KDMs).^9^ Lysine-specific demethylases are crucial for osteogenic differentiation and bone regeneration.^10^ For instance, enhancing lysine (K)-specific demethylase 6A (KDM6A) activates osteogenic genes like runt-related transcription factor 2 (RUNX2) and osteoblast-specific transcription factor (OSX), promoting bone regeneration.^11^ Lysine (K)-specific demethylase 6B (KDM6B) is also essential for osteoblast differentiation^12^ and plays a significant role in periodontal tissue homeostasis.^13^ However, research on the role of histone demethylation in cementum regeneration is limited.
KDM6B, a lysine-specific demethylase, contains a JmjC domain that requires α-ketoglutarate for histone demethylation.^14^ α-Ketoglutarate is a key product of glucose metabolism,^15^ highlighting a connection between KDM6B and glucose metabolism. KDM6B-mediated demethylation of trimethylated histone H3 at lysine 27 (H3K27me3) can act as a metabolic switch, influencing metabolic reprogramming.^16^ Our previous study noted glycometabolic reprogramming in cementoblasts during mineralization.^17^ As a pivotal glycolytic enzyme, pyruvate dehydrogenase kinase 1 (PDK1) may alter glucose metabolism phenotype,^18,19^ with its knockout in osteoblasts affecting differentiation and bone formation.^20^ However, which glycolytic enzymes histone demethylases target for metabolic reprogramming warrants further investigation.
Glycometabolic reprogramming leads to significant lactate production and accumulation.^21^ Once regarded as a byproduct of anaerobic metabolism, lactate is now recognized as a fuel in energy metabolism.^22^ Recent research has shown that lactate can form lactoyl-coenzyme A (lactoyl-CoA) and participate in the lactylation, linking it to glycometabolic reprogramming.^23^ Protein lactylation has been reported to reduce protein ubiquitination,^24,25^ thereby promoting the stability of protein function. Research has demonstrated its significant role in osteogenic differentiation.^26,27^ However, research in this area remains relatively limited.
Zinc finger E-box binding homeobox 2 (ZEB2) is a transcription factor involved in critical processes such as epithelial-mesenchymal transition,^28^ neural development,^29^ and osteogenic differentiation.^30^ Previous studies have shown that ZEB2 promotes cementoblast mineralization.^31^ However, research on its specific role in this context remains limited.
In conclusion, our study explored the regulation of glucose metabolic reprogramming during cementogenesis by KDM6B through the H3K27me3-Pdk1 axis, and further clarified the role of lactylation in this process. This research provides an initial investigation into the epigenetic-metabolic crosstalk mechanisms underlying cellular cementum formation.
Results
KDM6B is upregulated during cementogenesis and cementoblast mineralization
During cementoblast mineralization, Gene Ontology (GO) enrichment analysis revealed significant enrichment in the 2-oxoglutarate-dependent dioxygenase and histone lysine demethylation pathways (Fig. 1a), with key genes in these pathways including histone lysine demethylases (KDMs). Heatmap analysis revealed increased expression of these genes during mineralization (Fig. 1b). Previous studies indicated that KDM6B enhances osteogenic differentiation in periodontal ligament stem cells (PDLSC),^32^ leading us to select KDM6B for further mechanistic exploration. To assess KDM6B expression in the cementum region, we performed immunohistochemistry (IHC) staining on mandibular specimens collected from mice at different developmental stages. Compared to the 3-week-old group, KDM6B expression in cementoblasts and the periodontal ligament (PDL) was significantly higher at 6 weeks (the rapid growth phase of cementum,^33^) but decreased by 6 months (Fig. 1c). Conversely, H3K27me3 level showed an inverse relationship with KDM6B during cementogenesis (Fig. 1c). These results suggest that KDM6B may play a key role in regulating cementogenesis. In vitro experiments further corroborated these findings, demonstrating a significant upregulation of KDM6B expression at day 7 compared to day 0 following treatment with osteogenic induction medium (OIM) (Fig. 1d, e), in agreement with RNA-seq data. Additionally, we observed a reduction in H3K27me3 levels during cementoblast mineralization (Fig. 1f). This indicates that KDM6B may regulate cementogenesis through the demethylation of H3K27me3.Fig. 1KDM6B is upregulated during cementogenesis and cementoblast mineralization**. a** Gene Ontology (GO) enrichment analysis during the cementoblast mineralization process. b Heatmap of histone lysine demethylases (KDMs) gene expression during cementoblast mineralization process. c Immunohistochemical (IHC) analysis of lysine demethylase 6B (KDM6B) and trimethylated histone H3 at lysine 27 (H3K27me3) expression in cementoblasts of 3-week-old, 6-week-old, and 6-month-old mice (n = 5). Red arrows indicate cementoblasts. CC cellular cementum, PDL periodontal ligament, D dentin. d, e Quantitative polymerase chain reaction (qRT-PCR) and western blotting (WB) results of KDM6B expression levels in cementoblasts at day 0 and day 7 with cementogenic induction. (n = 3) f WB results of H3K27me3 levels during cementoblast mineralization process. Data are presented as mean ± SD. **P < 0.01. ***P < 0.001
KDM6B positively governs cellular cementum formation and cementoblast mineralization
To further explore how KDM6B regulates cementoblast mineralization, we created stable KDM6B-knockdown OCCM-30 cells (sh-Kdm6b) and performed RNA-seq. GO enrichment analysis showed significant pathways related to H3K27me3, glucose metabolism, and cementum mineralization (Fig. 2a). Heatmap analysis revealed downregulation of osteogenic differentiation genes following KDM6B knockdown (Fig. 2b). Following Kdm6b knockdown, reduced Kdm6b expression and upregulated global H3K27me3 levels were observed (Fig. S1a, b). Subsequent quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blotting, alkaline phosphatase (ALP) and alizarin red S (ARS) staining after 7 days of mineralization induction demonstrated reduced KDM6B expression and mineralization indicators in the sh-Kdm6b group compared to controls (Fig. S1c–f). To evaluate the effect of KDM6B inhibition on cementogenesis, we administered GSK-J4, a potent KDM6B inhibitor, in vivo. After 4 weeks of intraperitoneal injection, the root thickness in the GSK-J4 group was slightly reduced compared to controls (Fig. 2c). Hematoxylin and eosin (H&E) staining showed no significant difference in acellular cementum thickness, but the area of cellular cementum was smaller in the GSK-J4 group (Fig. 2c, d). Micro-CT analysis revealed thinner cementum-dentin complexes in the first molar apical root of the GSK-J4 group and a wider root canal (Fig. 2c). Double labeling assay indicated a significantly slower calcium deposition rate in the cementum area of GSK-J4-treated mice (Fig. 2c, d). Immunofluorescence (IF) staining confirmed decreased expression of KDM6B and mineralization-related markers in the cementoblasts of the GSK-J4 group (Fig. 2e, f). In summary, these results clarify that KDM6B may positively regulate both cellular cementum formation and cementoblast mineralization.Fig. 2KDM6B positively governs cellular cementum formation and cementoblast mineralization. a, b GO enrichment and heatmap results of cementogenesis-related gene expression from RNA sequencing analysis of cementoblasts following KDM6B inhibition. c, d Microphotography, hematoxylin and eosin staining (H&E) staining (n = 4), micro-CT and double labeling (n = 4) results of apical cementum in control and GSK-J4 groups of mice. CC, cellular cementum; AC, acellular cementum; PDL, periodontal ligament. (The area of AC and CC displayed in H&E staining originates from the same specimen within each group.) e, f Immunofluorescence (IF) analysis of KDM6B, OSX, and OCN expression levels in cementoblasts of control and GSK-J4 groups (n = 5). CC cellular cementum. Data are presented as mean ± SD. **P < 0.01. ***P < 0.001. ****P < 0.000 1
KDM6B positively governs the glycolytic process of cementoblasts
After KDM6B knockdown, glycolysis-related genes were downregulated, particularly lactate dehydrogenase a (LDHA) and Pdk1, while tricarboxylic acid (TCA) cycle genes showed mixed regulation (Fig. 3a, b). This suggests that aerobic glycolysis may no longer be the primary glucose metabolism pathway in cementoblasts. QRT-PCR and Western blotting analyses confirmed a reduction in key glycolytic markers, including LDHA, PDK1, glucose transporter 1 (GLUT1) and pyruvate kinase m2 (PKM2), along with an increase in oxidative phosphorylation markers such as succinate dehydrogenase complex subunit a (SDHA) and PDH (pyruvate dehydrogenase) (Fig. S2a–c). This indicates a metabolic shift toward oxidative phosphorylation following KDM6B knockdown. Glucose uptake significantly increased in the sh-Kdm6b group compared to the sh-NC group, while adenosine triphosphate (ATP) content decreased (Fig. S2d), likely due to higher glucose uptake and increased ATP consumption to adapt to metabolic changes. Seahorse assays revealed reduced extracellular acidification rate (ECAR) (Fig. 3c), basal glycolysis rate, compensatory glycolysis rate, and glycoATP production (Fig. S2e) in the sh-Kdm6b group. Mitochondrial stress tests showed increased oxygen consumption rate (OCR) (Fig. 3d), maximum respiration, and spare respiratory capacity (Fig. S2f) in the sh-Kdm6b group, indicating enhanced oxidative phosphorylation function. IF results further demonstrated reduced expression of LDHA and PDK1 in cementoblasts of the first molar from the control and GSK-J4 groups (Fig. 3e), supporting the conclusion that KDM6B inhibition leads to decreased glycolytic indicators in vivo.Fig. 3KDM6B positively governs the glycolytic process of cementoblasts. a, b Heatmap of glycolysis and TCA cycle-related genes expression of cementoblasts following KDM6B inhibition. c Seahorse examinations of extracellular acidification rate (ECAR). d Seahorse examinations of oxygen consumption rate (OCR). e IF analysis of LDHA and PDK1 expression levels in cementoblasts of control and GSK-J4 groups (n = 5). Data are presented as mean ± SD. *P < 0.05. **P < 0.01. ***P < 0.001. ****P < 0.000 1
KDM6B regulates cementoblast mineralization by targeting Pdk1
To identify the key glycolysis genes targeted by KDM6B, we conducted ChIP sequencing analysis. Peak visualization results revealed that H3K27me3 was enriched within 1 kb upstream of the transcription start site (TSS) of Pdk1, Ldha, Glut1 and Pkm2. The H3K27me3 peaks in the sh-Kdm6b group (KD-IP group) increased, among which the peaks of Ldha and Pdk1 increased significantly (Fig. 4a). Based on the significant changes in Pdk1 observed following KDM6B inhibition in our preliminary validation (Figs. 3a and S2a, b), we selected Pdk1 as a candidate target gene for further investigation. The 1 kb region upstream of the Pdk1 TSS were divided into four sites (Fig. 4b). ChIP-qPCR confirmed the regulatory role of KDM6B at sites 1, 2, and 4 (Fig. 4b). Following Pdk1 knockdown with siRNA (Fig. S3a), decreased protein and mRNA expression of PDK1, OSX, BSP and OCN was observed by western blotting and qRT-PCR (Fig. 4c, d). Additionally, ALP activity also decreased in si-Pdk1 (Fig. S3b). However, after overexpressing Pdk1 following knockdown of Kdm6b (Fig. S3c), both expression levels of PDK1 and mineralization-related indicators were restored (Fig. 4e, f). Furthermore, ALP activity increased in cells with overexpressed Pdk1 following knockdown of Kdm6b (Fig. S3d). In conclusion, the findings suggest that the KDM6B/Pdk1 axis plays a positive role in regulating cementoblast mineralization.Fig. 4KDM6B regulates cementoblast mineralization by targeting Pdk1**. a** Chromatin Immunoprecipitation (ChIP) sequencing detection of H3K27me3 peaks at 1 000 bp upstream of the transcription start sites of glycolysis-related markers under KDM6B inhibition. b By dividing Pdk1 upstream into 4 sites, ChIP-qPCR shows that inhibiting Kdm6b could upregulate H3K27me3 in these 4 sites (n = 3). c, d QRT-PCR and WB results indicate reduced mineralization levels after transient transfection with si-Pdk1 (n = 3). e, f QRT-PCR and WB assays reveal rescue of the mineralization phenotype following Pdk1 overexpression under Kdm6b inhibition. Data are presented as mean ± SD. *P < 0.05. **P < 0.01. ***P < 0.001. ****P < 0.000 1
KDM6B-Pdk1-lactate axis promotes the cementoblast mineralization through lactylation
Lactate production increased during the mineralization process of cementoblasts. (Fig. S4). However, after Kdm6b knockdown, lactate production decreased (Fig. 5a). QRT-PCR and western blotting results indicated upregulated expression of mineralization-related indicators after lactate sodium treatment (Fig. 5b, c). Additionally, results of ALP and ARS also suggested that lactate sodium promoted cementoblast mineralization (Fig. S5). The lactate production of glycolysis is closely related to the level of lactylation.^34^ IHC results showed that lactylation levels in the cementoblasts were significantly higher at 6 weeks compared to 3 weeks, but notably lower in the 6-month-old group (Fig. 5d). It is also greatly elevated during the mineralization process (Fig. 5e). After the addition of lactate sodium, the level of lactylation significantly increases (Fig. 5f), and this level is regulated by the KDM6B-Pdk1 axis (Fig. 5g). This suggests that the KDM6B-Pdk1 axis mediates mineralization via lactylation.Fig. 5KDM6B-Pdk1-lactate axis promotes the cementoblast mineralization through lactylation**. a** Lactate production following Kdm6b knockdown (n = 3). b, c QRT-PCR and WB results for the control and lactate sodium-treated groups. d Levels of L-lactyl-lysine in cementoblasts at different growth stages in mice (n = 5). Red arrows indicate cementoblasts. CC cellular cementum, PDL periodontal ligament, D dentin. e Lactylation levels in cementoblasts during mineralization. f Lactate sodium addition increases cellular lactylation levels. g WB results of mineralization levels following Pdk1 overexpression under Kdm6b inhibition. Data are presented as mean ± SD. **P < 0.01. ***P < 0.001
PDK1-ZEB2 lactylation enhances cementoblast mineralization
We further investigated the proteins involved in lactylation. ZEB2 has been previously identified as a protein that promotes cementoblast mineralization,^31^ and molecular docking results suggest the presence of potential lactylation modification sites (Fig. 6a). Through lactylation proteomics analysis, we discovered lactylation modifications at lysine (K) residues K485, K762, and K993 (Figs. S6–S8). Mutating these sites to arginine (R) to inhibit lactylation confirmed their occurrence, as demonstrated by co-immunoprecipitation (Co-IP) results (Fig. 6b). Western blot, qRT-PCR (Fig. 6c, d) and ALP staining (Fig. S9) analyses revealed that lactylation at K485 significantly suppresses the expression of mineralization-related genes and mineralization levels, indicating that it serves as a key regulatory site for mineralization. The site is species-conserved (Fig. S10). Furthermore, we verified that ZEB2 lactylation is positively regulated by Pdk1 (Fig. 6e), and supplementation with lactate sodium can restore the reduced levels of mineralization-related indicators and ZEB2 lactylation resulting from Pdk1 knockdown (Fig. S11a–c). The data demonstrate that PDK1 promotes ZEB2 lactylation, thereby promoting mineralization.Fig. 6PDK1-ZEB2 lactylation enhances cementoblast mineralization. a Docking Results of ZEB2 and L-Lactate. b Co-immunoprecipitation (Co-IP) confirms the occurrence of lactylation modifications. c, d Mineralization levels detected by qRT-PCR (n = 3) and WB after mutating lysine (K) to arginine (R). e IF results reveal that ZEB2 lactylation levels were rescued by lactate sodium addition under Pdk1 inhibition. Nala, lactate sodium. Data are presented as mean ± SD. Scale bars: 30 μm, **P < 0.01. ***P < 0.001. ****P < 0.000 1
Elevated lactylation recovers KDM6B inhibition-mediated cementogenesis downregulation in vivo
To explore the in vivo effects of lactylation further, we constructed a subcutaneous ectopic mineralization model using cementoblasts. The modeling process is shown (Fig. 7a). The diameter of the sh-Kdm6b + lactate sodium group was larger than that of the sh-Kdm6b group (Fig. S12). IF assays indicated increased expression of OCN and BSP in the sh-Kdm6b + lactate sodium group (Fig. 7b). H&E and Masson staining revealed a substantial increase in collagen fibers in the sh-Kdm6b + lactate sodium group, whereas the sh-Kdm6b group showed minimal collagen fibers (Fig. 7c). Alizarin red staining revealed more and larger mineralized nodules in the sh-Kdm6b + lactate sodium group compared to the sh-Kdm6b group (Fig. 7c). Next, we further probed whether application of lactate sodium in vivo could enhance the level of lactylation. Results indicated a decrease in lactylation levels in the sh-Kdm6b group compared to the control, with an increase observed in the sh-Kdm6b + lactate sodium group. (Fig. 7d). This finding suggests that application of lactate sodium in vivo can rescue the reduction in lactylation level resulting from KDM6B knockdown, thus promoting cementogenesis.Fig. 7. Elevated lactylation recovers KDM6B inhibition-mediated cementogenesis downregulation in vivo. a Flow chart for the subcutaneous transplantation model in nude mice. b BSP and OCN levels in ectopic tissues of sh-NC, sh-Kdm6b, and sh-Kdm6b+lactate sodium groups (n = 4). c H&E, Masson and ARS staining results for different groups. d Lactylation levels in ectopic tissues of the sh-NC, sh-Kdm6b, and sh-Kdm6b+Nala groups (n = 4). Nala, lactate sodium. Data are presented as mean ± SD. **P < 0.01. Scale bars: 100 μm, ***P < 0.001
In conclusion, our study demonstrates that KDM6B targets Pdk1 via histone demethylation to enhance glycolysis during cementoblast mineralization and cellular cementum formation, resulting in elevated lactate levels. This increase in lactate facilitates the lactylation of ZEB2, thereby promoting cellular mineralization (Fig. 8).Fig. 8. Schematic illustration of the study. During cementoblast mineralization, KDM6B expression is upregulated and promotes glycolysis by positively regulating Pdk1 through histone demethylation. The elevated glycolytic lactate production enhances the lactylation of the downstream ZEB2 protein, thereby facilitating mineralization
Discussion
Cementum, the avascular mineralized tissue enveloping the tooth root, is essential for anchoring periodontal ligament fibers and preventing bacterial invasion and gingival epithelium growth toward the root surface.^35^ This study is the first to investigate the role of protein post-translational modification in cellular cementum formation and cementoblast mineralization through glucose metabolic reprogramming. Given the similarity between cementoblasts and osteoblasts, this research also offers new insights into osteoblast mineralization and bone regeneration.
This study confirms that KDM6B acts as an activator that promotes cellular cementum formation. Studies have shown that histone demethylases can mediate osteogenic differentiation. KDM6B can target H3K27me3 in the Osx promoter to regulate osteoblast differentiation.^36^ KDM6A and KDM6B are essential in oral regeneration. KDM6A activates the transcription of mineralization-related genes via demethylation in the Alp and Runx2 promoter regions, thereby facilitating the osteogenic differentiation in human PDLSC.^37^ Similarly, KDM6B activates bone morphogenetic protein 2 (BMP2) transcription and elicits the odontoblastic differentiation of dental pulp stem cells.^38^ Additionally, KDM6B can boost the osteogenic differentiation and anti-inflammatory function of PDLSC.^39^ However, researches on the regulation of histone methylation during cementum formation remain limited. The results of in vivo and in vitro experiments indicate that KDM6B positively regulates cellular cementum formation and the cementoblast mineralization. Notably, our investigation revealed that KDM6B inhibition specifically impaired cellular cementum formation (Fig. 2c), while leaving acellular cementum unaffected. This selective effect may be explained by several factors: First, the developmental chronologies of acellular and cellular cementum demonstrate distinct temporal patterns.^33^ Second, the ontological origin of cementoblasts remains controversial (derive either through direct differentiation from dental mesenchymal cells or via epithelial-mesenchymal transformation of Hertwig’s epithelial root sheath),^40^ with both cementoblast types capable of forming either cementum variety.^41^ Additionally, cellular cementum contains embedded cementocytes that establish intricate regulatory networks with cementoblasts and osteoclasts,^42^ thereby modulating cellular cementum formation through multiple signaling pathways. Future studies should employ genetic mouse models to investigate cementoblast progenitors and their contributions to specific cementum types during different developmental stages.
This research shows that KDM6B promotes cementoblast mineralization by mediating glycometabolic reprogramming. Our results are consistent with previous studies. Research has revealed that lactate sodium can facilitate the multi-directional differentiation of human mesenchymal stem cells via KDM6B-mediated glycolysis.^43^ KDM6B targets Ldha to enhance glycolysis and lactate production, thereby promoting osteosarcoma lung metastasis.^44^ The KDM6B/6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 2 axis promotes tumor growth and metastasis through metabolic reprogramming.^45^ It is thereby evident that histone demethylases can enhance aerobic glycolysis via glycometabolic reprogramming. We have noted that following the knockdown of KDM6B, the ATP content declined. This is likely because cementoblasts primarily utilize aerobic glycolysis during mineralization. After the knockdown of KDM6B, the function of aerobic glycolysis was downregulated, the rate of ATP generation from glycolysis decreased, and consequently, the total amount of remaining ATP within the cells decreased. Our data has demonstrated that KDM6B can target the key gene Pdk1, which drives glycometabolic reprogramming,^46^ thereby promoting cementoblast mineralization. Our study is the first to confirm the targeted regulatory relationship between KDM6B and Pdk1, offering new insights into the KDM6B-mediated metabolic reprogramming network. Notably, our in vitro functional rescue experiments revealed that despite higher Pdk1 expression in the knockdown group under overexpression conditions, multiple cementogenic markers showed lower expression compared to the sh-NC group. We hypothesize this discrepancy may be associated with KDM6B inhibition: Firstly, KDM6B may regulate cementoblast mineralization through additional pathways. As evidenced by previous studies, it promotes bone formation via PKCδ/MAPKs pathway^47^ and enhances osteogenic differentiation of PDLSCs through Wnt signaling pathway.^48^ Moreover, as demonstrated in Fig. 4a, KDM6B targets multiple key glycolytic enzymes. Its inhibition consequently leads to systemic metabolic reduction. Given that glycolysis serves as a critical metabolic pathway for bone formation,^49^ this may ultimately affect downstream mineralization marker expression. Therefore, the alternative pathways through which KDM6B regulates cementoblast mineralization warrant further investigation.
A considerable amount of lactate is produced during cementoblast mineralization, and this production significantly decreases following KDM6B knockdown. These findings signify that lactate may be instrumental in cementum formation and cementoblast mineralization. In our in vivo experiments, we established a subcutaneous ectopic tissue mineralization model based on previous studies^3,50^ and found that subcutaneous injection of lactate sodium rescued the reduced mineralization levels caused by KDM6B inhibition. Collagen formation is essential for mineralization,^51^ suggesting that lactate sodium may enhance mineralization via lactylation. While there were differences in collagen formation, mineralization markers, and mineralized nodules across groups, this model is ectopic, and validation through an in situ model is needed. In conclusion, our results indicate that lactylation positively regulates cementum formation and cementoblast mineralization.
This study firstly reports ZEB2 lactylation and its role in cementoblast mineralization. Lactylation levels are influenced by glycolytic activity, and protein lactylation can prevent ubiquitination,^24^ stabilizing protein function. Previous research has shown that ZEB2 promotes cementoblast mineralization,^31^ potentially via the NF-κB pathway^52^ or interaction with smad proteins.^53,54^ Therefore, we hypothesize that ZEB2 lactylation may contribute to its stability, enhancing its role in boosting cementoblast mineralization. Data from this study confirmed the importance of the K485 site for mineralization, but further investigation and drug development targeting this site require additional validation.
However, this study does have certain limitations. First, we employed the KDM6B inhibitor GSK-J4 during early-stage cementogenesis in mice without establishing Kdm6b conditional knockout mouse models. Previous studies have demonstrated that early pharmacological intervention can influence cementogenesis, as evidenced by increased cellular cementum area following fibroblast growth factor 23 antibody injection in 4-week-old X-linked hypophosphatemia mice.^55^ Systemically administered GSK-J4 via intraperitoneal injection has been shown to effectively suppress KDM6B expression. Notably, intraperitoneal GSK-J4 administration (10 mg/kg, three times weekly for 10 weeks) was reported to ameliorate glomerular disease through H3K27me3 upregulation without significant toxicity,^56^ supporting its utility for in vivo cementogenesis studies. The observed differences between acellular and cellular cementum, which develop sequentially,^33^ may reflect stage-specific regulatory roles of KDM6B during cementum development. Conditional knockout models would better elucidate these temporal effects. Second, both in vivo and in vitro sample sizes were limited, and increased numbers would enhance statistical power. Third, while we used subcutaneous cementogenesis models in nude mice for functional rescue experiments, this single-model approach could be complemented by organoid systems—powerful tools for studying craniofacial development and regeneration.^57,58^ Finally, our Fig. 2e revealed significant OSX downregulation in periodontal ligament cells following GSK-J4 treatment. Gli1^+^ progenitor cells within the PDL are reported to contribute to both acellular and cellular cementum.^59^ Although KDM6B is known to promote osteogenic differentiation in PDLSCs,^48^ its role in cementogenic differentiation remains unexplored, suggesting an important future research direction.
In conclusion, our study confirms that KDM6B targets Pdk1 through histone demethylation, driving glycometabolic reprogramming during cementoblast mineralization and promoting increased lactate production. The elevated lactate levels regulate cell mineralization through ZEB2 lactylation. The results validated the crucial role of KDM6B and lactylation in promoting cementogenesis, highlighting new therapeutic targets for cementum and bone regeneration.
MATERIALS AND METHODS
Cell culture
The cementoblast cell line (OCCM-30) originated from murine cementoblasts and was provided by Dr. Martha J. Somerman. Cells were kept in DMEM medium (Hyclone, USA) with 10% FBS (Every Green, China). For mineralization induction, cells were cultured in OIM with 5% FBS, ascorbic acid (50 μg/mL; Sigma-Aldrich, USA), and Naβ-glycerophosphate (10 nmol/L; Sigma-Aldrich, USA) for 0, 4, and 7 days.
RNA sequencing and data analysis
After culturing the cells in plates, TRIzol (Takara, Japan) was added to extract RNA. The Beijing Genomics Institute (BGI; China) conducted the sequencing and analysis of the RNA production. Differentially expressed genes (DEGs) were screened by the criteria |log_2_(FC)| > 1 and FDR < 0.01. The pheatmap package was used to generate heat maps. Differential genes were annotated through NCBI, Uniprot, Gene Ontology (GO), and other databases, with GO enrichment conducted using the ClusterProfiler R package.^60^
qRT-PCR
Use PrimeScript™ RT Master Mix (Takara, Japan) reagent to reverse transcribe RNA into cDNA. qRT-PCR was conducted in triplicate on an Applied Biosystems QuantStudio 6 using SYBR qRT-PCR Master Mix (Vazyme, China). The protocol process included: initial denaturation (95 °C, 30 s), followed by 40 cycles (95 °C, 10 s) and 62 °C for 34 s. Primers were provided by Sangon Biotech (China, see Table S1). Gene expression fold changes were normalized to β-actin using the 2^–ΔΔCT^ method.
Western blotting
Proteins from cells were obtained using M-PER lysis buffer (Merck, Germany) and quantified with BCA kit (Beyotime, China). Samples were mixed with SDS loading buffer (Epizyme, China). Protein samples of 20–40 µg were applied onto SDS-PAGE gels (ACE, China) for electrophoresis. After electrophoresis, samples were transferred onto PVDF membranes (Millipore, USA), after which 5% skim milk was applied for blocking. Incubated with primary antibodies (refer to Table S2) overnight at 4 °C and secondary antibodies for 1 h at room temperature, samples were visualized with super-enhanced chemiluminescence substrate (Biopmk, China) and imaged on an Odyssey LI-COR scanner (USA).
Animal models
The study used male C57BL/6 mice of varying ages (3 weeks, 6 weeks, and 6 months; weights: 9–32 g; n = 8 per group), with all protocols approved by the Ethics Committee of the School and Hospital of Stomatology, Wuhan University (Approval No. S07921060E). Following euthanasia by CO₂ asphyxiation, mandibles were collected.
3-week-old male C57BL/6 mice (9–12 g) were randomly divided into a healthy control and a GSK-J4 group (12 mice per group). GSK-J4 (10 mg/kg) was administered intraperitoneally three times a week for 5 weeks. Mice were euthanized at 8 weeks, and mandibles were collected. All procedures were approved by the Ethics Committee of the School and Hospital of Stomatology, Wuhan University (Approval No. S07923050C).
Male BALB/c-nu nude mice, 6 weeks old and weighing 19–20 g, were used in the study. The study was approved by the Animal Experimentation Ethics Committee of Wuhan University (Approval No. WP20230603). Gelatin sponges with sh-NC, sh-Kdm6b, or sh-Kdm6b + lactate sodium OCCM-30 cells were implanted subcutaneously, with four implantation sites per mouse. From day 3 post-surgery, the sh-NC and sh-Kdm6b groups received subcutaneous PBS injections three times weekly, while the sh-Kdm6b + lactate sodium group received sodium lactate (2 g/kg, 50 μL) every other day. After 4 weeks, cell-tissue composites were collected post-euthanasia. All animal experiments complied with the updated ARRIVE guidelines for preclinical animal studies.
Immunohistochemistry and immunofluorescence staining
For IHC, sections were prepared using the UltraSensitive SP IHC Kit (MXB Biotech, China). Samples were treated over 12 h at 4 °C with specific primary antibodies (see Table S2). Cementoblast morphology was observed and imaged microscopically. For IF assay, samples were pre-treated with goat serum at 37 °C for 1 h. Incubate overnight with different primary antibodies (see Table S2) at 4 °C. They were subsequently treated with FITC-conjugated secondary antibody (ABclonal, China) and 594-conjugated Goat anti-Rabbit IgG (ABclonal, China) for 1 h. The working diluted ratio of secondary antibody was at 1:200. Nuclei were counterstained with DAPI dye (ZSGB-BIO, China). Image capture was conducted with fluorescence microscopy.
ALP staining, ALP activity assay, and ARS staining
ALP staining was performed using ALP Assay Kit (Beyotime, China) on days 4 and 7 with OIM induction, followed by imaging. ALP activity was assessed usinga detection kit (Nanjing Jianchen, China). Fixed cells were dyed with 1% Alizarin Red Solution (OriCell, China) to visualize mineralized nodules, which were then photographed.
Transfections with siRNA, lentivirus, and plasmid
OCCM-30 cells underwent transient siRNA transfection with si-NC (GenePharma, China) and si-Pdk1 (5’- GCUGAGUAUUUCUUUCAAGUUTT-3’; GenePharma, China) using Lipofectamine 2000 (Thermo Scientific, USA), followed by a medium change after 6 h.
Sh-NC and sh-Kdm6b lentiviruses (GenePharma, China) were introduced at multiplicity of infection of 100 with Polybrene (5 ng/mL). After 24 h, original transfection medium was substituted with puromycin-containing medium (4 μg/mL; Beyotime, China) for 2–4 days to select cells.
To overexpress target genes, NC-, Pdk1-, and Zeb2-over plasmids (Miaoling Biotech, China) were transfected using TurboFect (Thermo Scientific, USA). A lysine-to-arginine mutation was introduced into the Zeb2 plasmid using the Mutation kit (Vazyme, China). Samples were collected post-transfection for further analysis.
ATP, glucose, and lactate measurements
For ATP measurement, cementoblasts were plated in a black 96-well plate at a density of 2 × 10^4^ cells per well. After cultivating for 4 h, ATP content was then assessed using the CellTiter-Glo Luminescent Assay (Promega, USA) on a Tecan Spark microplate reader (Switzerland), with results normalized to cell count. For glucose consumption and lactate production assays, adherent cells were washed with PBS and incubated with phenol red-free DMEM (Gibco™, USA) for 24 h. The measurements of glucose levels were performed by the Glucose (HK) Assay Kit (Sigma-Aldrich, USA). Lactate production levels were assessed with an assay kit (Nanjing Jiancheng, China).
Seahorse assays
After seeding cementoblasts at a density of 5 × 10^4^ cells per well in XF24 plates (Agilent, USA), ECAR and OCR were measured using the Agilent Seahorse XF Glycolytic Rate Assay Kit (Agilent, USA) and the XF Cell Mito Stress Test Kit (Agilent, USA).
For the Glycolytic Rate Assay, the working concentrations were Rotenone/Antimycin A (Rot/AA; 0.5 μmol/L) and 2-DG (inhibitor of glycolysis; 50 mmol/L). For the Cell Mito Stress Test, concentrations used 1.5 μmol/L oligomycin (ATP synthase inhibitor), 1 μmol/L FCCP (mitochondrial membrane potential uncoupler), and 0.5 μmol/L Rot/AA.
Double fluorochrome labeling
One week before euthanasia, mice received an intraperitoneal injection of calcineurin (5 μg/g; Merck, Germany), followed by alizarin complexone (20 μg/g; Merck, Germany) 48 h prior. Mandibular specimens were collected, fixed, and dehydrated in graded ethanol (70%–100%). Then samples were embedded in methyl methacrylate (Sinopharm, China) without decalcification. Samples were sectioned (10 μm) by Servicebio (China) and scanned with a whole-slide scanner. The distance between calcineurin and alizarin fluorescent bands was measured and analyzed using ImageJ.
Micro-CT scanning
Mouse mandibles were dissected, trimmed of excess muscle and incisors, and placed in PBS. The Bruker Skyscan 1276 Micro-CT system (Belgium) was preheated for 15 min, after which the samples were positioned on the holder. Pre-scanning and flat-field correction were performed. The mandibles were scanned using a voltage of 70 kV, a current of 114 µA, and a voxel resolution of 10 µm. A region of interest (ROI) was defined for each mandible, extending from 220 µm mesial to the mesial root of the first mandibular molar to 220 µm distal to the distal root. Image reconstruction was carried out using NRecon, with angle adjustment and ROI selection performed in DATA Viewer. Sagittal and coronal images of the ROI were then exported.
H&E, Masson, and Alizarin Red Staining
Specimens were fixed in paraformaldehyde for 1 day. Samples were then rinsed, decalcified, dehydrated, embedded, and sectioned. Sections were sent to Servicebio (China) for H&E staining, Masson trichrome and Alizarin Red staining. After staining, sections were dehydrated, mounted, and scanned using a digital pathology whole-slide scanner.
ChIP-sequencing and ChIP-qPCR
Cell samples were collected, cross-linked, lysed, and digested. Immunoprecipitation was conducted with an H3K27me3 antibody (Abcam, UK), followed by elution and DNA recovery per the Pierce™ Magnetic ChIP Kit (Thermo Fisher, USA) protocol. A portion of the product was sent to the BGI for analysis, with the remainder used for ChIP-qPCR validation (primer details in Table S3).
Molecular docking and sequence alignment
The homology model for ZEB2 was built using SWISS-MODEL. The L-lactate molecular structure was obtained from PubChem, and docking was conducted using CB-DOCK2^61^ after preprocessing both molecules with MOE software to determine the small molecule-protein binding site.
ZEB2 protein sequences from different species were obtained from NCBI. Multiple sequence alignment was conducted using CLUSTALW, and the alignment results were presented using ESPript.
Lactylation proteomics
Cells were collected after 0 and 7 days of mineralization induction. After being washed thrice, the collections were centrifuged for 10 min (3 000 r/min; 4 °C). Cell pellets were snap-frozen in liquid nitrogen for 5 min after removing the supernatant. Samples were then sent to PTO BIO (China) for lactylation proteomics analysis.
Co-immunoprecipitation
Co-IP assays were conducted according to the instructions (Absin, China), using a Tag-FLAG antibody (1:50; CST, USA) for enrichment. The enriched products were then analyzed by Western blotting.
Statistical analysis
Each experiment was performed in triplicate. Data was conducted with GraphPad Prism 9 (GraphPad Software, USA) and shown as mean ± standard deviation. Statistical differences were assessed with t-tests and one-way ANOVA. The statistical significance is indicated by “” for P < 0.05, “” for P < 0.01, “” for P < 0.001, and “****” for P < 0.000 1.
Supplementary information
Supplementary Information
The reference list from the paper itself. Each links out to its DOI / PubMed record.
- 1Yamauchi, Y., Shimizu, E. & Duncan, H.F. Dynamic alterations in acetylation and modulation of histone deacetylase expression evident in the dentine-pulp complex during dentinogenesis. Int. J. Mol. Sci.25, 6569 (2024).10.3390/ijms 25126569 PMC 1120358438928274 · doi ↗ · pubmed ↗
- 2Huang, X. et al. Sirt 3 rescues porphyromonas gingivalis-impaired cementogenesis via SOD 2 deacetylation. Cell Prolif. 58, e 70022 10.1111/cpr.70022 (2025).10.1111/cpr.70022 PMC 1241463640068967 · doi ↗ · pubmed ↗
- 3Deng P. et al. Loss of KDM 4B impairs osteogenic differentiation of OMS Cs and promotes oral bone aging. Int. J. Oral Sci.14, 24 (2022).10.1038/s 41368-022-00175-3PMC 907907635525910 · doi ↗ · pubmed ↗
- 4Deng, A. et al. Innovative PDK 1-degrading PROTA Cs transform cancer aerobic glycolysis and induce immunogenic cell death in breast cancer. Exploration e 20240031 10.1002/EXP.20240031 (2025).10.1002/EXP.20240031 PMC 1238006540873633 · doi ↗ · pubmed ↗
- 5Majumder, S. et al. Shifts in podocyte histone H 3K 27me 3 regulate mouse and human glomerular disease. J. Clin. Investig.128, 483–499.10.1172/JCI 95946 PMC 574949829227285 · doi ↗ · pubmed ↗
- 6Motoike, S., Inada, Y., Toguchida, J., Kajiya, M. & Ikeya, M. Jawbone-like organoids generated from human pluripotent stem cells. Nat. Biomed. Eng. 10.1038/s 41551-025-01419-3 (2025).10.1038/s 41551-025-01419-3PMC 1262324240603745 · doi ↗ · pubmed ↗
- 7Yu, G., Wang, L.-G., Han, Y. & He, Q.-Y. cluster Profiler: an R Package for comparing biological themes among gene clusters. OMICS 16, 284–287 (2012).10.1089/omi.2011.0118 PMC 333937922455463 · doi ↗ · pubmed ↗