EZH2 Promotes Extracellular Matrix Degradation via Nuclear Factor-κB(NF-κB) and p38 signaling Pathways in pulpitis

Background: Pulpitis is a complicated chronic inammatory process which in a dynamic balance between damage and repair. Extracellular matrix plays an important regulatory role in wound healing and tissue repair. The aim of this study was to explore role of the epigenetic mark, enhancer of zeste homolog 2 (EZH2) on the degradation of extracellular matrix during pulpitis. Methods : Quantitative polymerase chain reaction was used to assess the expression of matrix metalloproteinases (MMPs) and type (cid:0) collagen in HDPCs upon EZH2 and EI1 stimulation. The mechanism of EZH2 affecting extracellular matrix was explored through quantitative polymerase chain reaction and Western blot. A rat model of dental pulp inammation was established, and the expression of type (cid:0) collagen in dental pulp under EZH2 stimulation was detected by immunohistochemical staining. Results :EZH2 upregulated the expression of MMP-1, MMP-3, MMP-8 and MMP-10 and decreased the production of type (cid:0) collagen in HDPCs, while EI1 had the opposite effect .EZH2 activated the Nuclear Factor-κB(NF-κB)and p38 signaling Pathways in HDPCs, the inhibition of which reversed the induction of MMPs and the suppression of type (cid:0) collagen .EZH2 can downregulated the type (cid:0) collagen levels in an experimental model of dental pulpitis in rats. Conclusion: EZH2 promotes extracellular matrix degradation via Nuclear Factor-κB(NF-κB)and P38 signaling pathways in pulpitis.EZH2 can decrease the type (cid:0) collagen levels in vivo and vitro.


Introduction
Dental pulp is derived from ectodermal stroma and is a loose connective tissue rich in extracellular matrix (ECM). Type and type III collagen is the primary constituent of the ECM of dental pulp [1]. Pulpitis is one of the common dental disorders associated with tooth pulp in ammation [2]. In pulpitis tissue, HDPCs, the main dental pulp component, can interact with immune cells to secrete signi cant amounts of in ammatory cytokines and chemokines locally. These cytokines and chemokines may promote pulpitis progression, tissue destruction, or regulate in ammatory responses to eliminate pathogens [3,4]. Recent studies have proved that ECM plays an important regulatory role in cell proliferation, differentiation, wound healing and tissue repair [5]. ECM is closely related to the repair function of dental pulp tissue, and participates in and regulates this process, such as participating in and mediating the migration and differentiation of dental pulp cells, becoming a solid support for newly generated dentin cells and taking part in the initiation of dentin mineralization [6]. As extracellular matrix plays an important role in the progress and repair of pulpitis, studying the dynamic changes of extracellular matrix in pulpitis will help reveal the mechanism of pulpitis and provide theoretical basis for the treatment of pulpitis.
Previous studies have demonstrated that the endopeptidase activity of matrix metalloproteinase (MMP) mediates the degradation of ECM, which may promote the progress of pulpitis [7]. The activity of MMP against extracellular matrix substrates is regulated at 4 "gates": 1) by transcriptional regulation of MMP genes; 2) by precursor activation; 3) by differences in substrate speci city; and 4) by MMP inhibitors [8].
Existing research attempts to reveal the characteristics of transcription factors that control the active promoter of the MMP gene, but the transcription mechanism that regulates MMP during the progression of in ammatory diseases is unclear, such as epigenetic regulation [9]. In in ammatory diseases such as rheumatoid arthritis, tuberculosis, and diabetes, epigenetics (including DNA methylation, histone modi cations, and non-coding RNA) can regulate the expression of MMP and affect the progress of in ammatory diseases [10] .
At present, it has been reported that the histone methylation of H3K27 may be involved in dental pulp in ammation and repair process [11]. Studies have shown that the reduced expression of H3K27me3 in in ammatory dental pulp tissues and dental pulp cells may induce the regeneration process of dental pulp [11]. In addition, Jumonji domain protein 3 (JMJD3) is an enzyme that eliminates the histone H3K27 methylation marker and can regulate the expression and transcriptional activity of H3K27me3. Recently, it was discovered that Jmjd3 can regulate the differentiation and cell characteristics of macrophages, suggesting that it may provide a link between in ammation and epigenome reprogramming [12].
In addition, the methyltransferase of H3K27, Enhancer of zeste homolog 2 (EZH2 ) can be used as a regulator of pulp in ammation [11]. Our previous research found that during the development of dental pulp in ammation, the expression of EZH2 increased [11]. Existing studies suggest that EZH2 is involved in the repair process of dental pulp damage, but whether this process involves extracellular matrix is unclear, and whether EZH2 can affect dental pulp in ammation by regulating MMPs remains to be studied.
It is known that in some circumstances nuclear factor kappa B (NF-κB) and MAPK signaling pathways are required for the induction of histone modi ers [13]. These NF-κB and MAPK signaling pathways are also known to be major vehicles for producing in ammation [14]. In this study, we addressed the following hypothesis: EZH2 affects the expression of MMP in dental pulp through the NF-κB pathway or MAPK pathway, thereby mediating the degradation of extracellular matrix and affecting the progress of dental pulp in ammation such as the collagen degradation.

Material And Methods
Cell culture and treatment Primary HDPCs were collected from the caries-free third molars and the premolars that need to be extracted for orthodontics of healthy donors in maxillofacial surgery clinic of Peking University School of Stomatology (ethics approval number: PKUSSIRB-201732003). Cells were maintained in α-MEM medium (Gibco, USA) with 10% fetal calf serum (FBS) (Excell Bio, Australia) and 100U/ml penicillin and 100μg/ml streptomycin sulfate, and passages 3-5 were used . Cells were treated with recombinant EZH2(20ng/mL,abnova,USA) or the inhibitor of EZH2 EI1 (2uMol/L APExBIO,abnova,USA) for the given times. In the speci ed experiments, cells were pretreated with one of the following speci c pathway inhibitors: BAY11-7082(an Iκ B phosphorylation inhibitor, 1 uM , Selleckchem , Houston, TX) or SB203580(a p38 MAPK inhibitor,20 uM, Cell Signaling Technology, Danvers, MA).

Characterization of HDPCs
The fourth-generation HDPC was inoculated in a 12-well plate and grown in slides. After the cells grew close to con uence, they were xed with 4% paraformaldehyde. Primary antibodies included vimentin(1:1000; proteintech , USA) and cytokeratin 14 (1:1000; proteintech ,USA ). A standard immunohistochemistry kit (Zhongshanjinqiao , Beijing, China) was used for immunohistochemistry. After counterstaining with hematoxylin and dehydration, the expression of related antibodies was observed.

Western Blot Analysis
The cells were lysed using RIPA buffer(Solarbio,China) supplemented with protease inhibitors (Solarbio,China). Cell homogenates were obtained by gently scraping the cells from each well, and the protein concentrations from the lysates were determined by using the BCA Protein Assay Kit (ThermoScienti c, Rockford,IL). Prior to loading, total protein samples were denatured by heating at 95°C for 10min in 5x SDS-PAGE sample loading buffer (Applygen, China). 30μg of the protein sample were separated by SDS-polyacrylamide gel electrophoresis at 110V for 90min and transferred to 0.45-μm polyvinylidene fluoride (PVDF) membranes at a constant current of 300mA for 60min. After blocking in 5% nonfat dry milk (Bioruler, China) at room temperature, the membranes were incubated with rabbit antiphospho-p65 (catalogno.3033, Cell Signaling Technolog, Danvers,MA), rabbit anti -p65 (catalogno.8242, Cell Signaling Technolog, Danvers,MA), rabbit anti -phospho-p38 (catalogno. 4511, Cell Signaling Technolog, Danvers,MA), rabbit anti-p38(catalogno.9212, Cell Signaling Technolog, Danvers,MA) overnight at 4°C. The membrane was incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin(Proteintech,USA) based on the source of the corresponding primary antibody, and the immunoblots were detected using a Western enhanced chemiluminescence blotting kit (ECL, SOLIBRO, China).

Construction of Rat Experimental Pulp Infection Model
12 male 6-week-old SD rats (Weitong Lihua Company, China) were subdivided into 4 groups of 3 rats each.Rats were classi ed according to the sealing medicine: LPS(10mg/ml, Sigma, USA) for group1, LPS (10mg/ml) +EZH2 (20ng/mL) for group2, LPS (10mg/ml) +EI1(20ng/mL) for group3 and group4 was not operated on as the control . Animals were sacri ced 1, 3 days after surgery and compared to a control group. Under general intraperitoneal anaesthesia, all animals except the control group underwent pulp exposure on the occlusal face of the mandibular rst molar using a 1/4-size spherical bur at high speed.
The gelatin sponge with sealing medicine is placed in the perforated hole, and the cavity was sealed with glass-ionomer (Fuji, GC Corporation, Tokyo, Japan). Upon decapitation, the jaws were immersed promptly in 4% formaldehyde, embedded in para n, and sectioned at 5-mm thickness.

Immunohistochemical Staining
Immunostaining was performed on formalin-xed, para n-embedded tissue. For immunohistochemistry, para n sections were dewaxed in xylene, rehydrated with distilled water, and then subjected to antigen retrieval for 30 min at 95°C. The slides were subsequently incubated overnight at 4°C with rabbit anti-Collagen I antibody (1:400,bioss,Beijing,China).Slides were then treated with an anti-rabbit secondary antibody(Zhongshanjinqiao , Beijing, China)and developed using avidin-conjugated HRP with diaminobenzidine as a substrate (Zhongshanjinqiao , Beijing, China),followed by hematoxylin counter staining.

Statistical Analysis
Results are presented as means ± SD of at least 3 independent biological experiments. For each experiment, we have at least 3 technical repeats. Signi cance was determined via one-way analysis of variance test. The difference was considered statistically signi cant at the P < 0.05 level.

EZH2 influenced ECM balance of HDPCs
After EZH2 stimulated HDPC for 2h, 4h, and 24h, we quantified the mRNA levels of the MMPs and type collagen. The expression of MMP-1 MMP-3 MMP-8 and MMP-10 were increased than the control group (P<0.05) (Figure 2A-2D). While the expression of MMP-2 and MMP-13 both had a decrease (P<0.001) ( Figure 2E-F); There was a signi cant decrease in the expression of type collagen (P<0.01) ( Figure 2H).
To examine Col-1 change during pulpitis in vivo, we established a pulpitis model based on experiments with rats. Pulp tissue samples were stained with hematoxylin-eosin stain to verify the infection. We detected in ammatory cell in ltration . Immunohistochemistry staining of Col-1 further revealed that EZH2 can reduce the expression of Col-1 in in ammatory dental pulp tissue(4J,K,M and N). However, EI1did not increase the expression of Col-1 signi cantly (4L and O).

Discussion
In the experiment, HDPCs were further subjected to immunocytochemical detection of cell sources. Postnatal expression of vimentin is restricted to broblasts, endothelial cells, lymphocytes, and several specialized cells of the thymus and the brain [17]. Cytokeratins (CK) are abundant in keratinized cells, particularly CK14 and CK19, which are expressed in strati ed squamous epithelial cells [18]. In our experiment, we showed that the cells were positive for vimentin and negative for cytokeratin 14, which proved that the cells were mesoderm derived and there were no epithelial cells mixed, which was consistent with the biological characteristics of HDPCs.
Our experiment veri ed that EZH2 could increase the expression of MMP-1, MMP-3,MMP-8 and MMP-10 and (Fig2). MMP-1(Collagenase 1, broblast collagenase) is an essential enzyme responsible for degrading type and type III collagen since the initial breakdown of these brillar collagen network is mediated primarily by MMP-1 [19,20]. In Su-Jung Shin's experiment, the concentrations of MMP-1 in acute-in amed pulps and chronic-in amed pulps groups were signi cantly higher than in the control [21]. According to Bergenholtz G, MMP-8 in pulpal in ammatory lesions is mainly of PMN origin [22].PMNs are cells forming pulp abscesses, and the main MMP-8 positivity was accumulated around the pulp abscess suggesting that it also contributes to the tissue destruction of pulp necrosis and abscesses. Previous studies have revealed that PMN-leucocytes, macrophages and plasma cell produced MMP-8 in pulp and periapical granulomas [23]. Evrosimovska B, et al. con rmed that degradation of the collagen of the organic matrix from pulp tissue during chronic in ammation is an enzyme process and that collagenses (MMP-1, -8) are invovlved in these destructive process [24]. Rhim EM et al. found the expression of MMP-3, -10 was up-regulated in the pulp cells after 24 hours of stimulation with TNF-α [25].In our study , EZH2 suppressd the production of type collagen in HDPCs. Type and III collagens are the most abundant collagens in dental pulp , and collagen type expression is thought to have a direct effect on osteogenic differentiation and mineralization [26]. During the process of in ammation, reduced collagen synthesis and increased degradation led to disintegration of ECM and remodeling of dental pulp [27].Our experiment suggested that EZH2 may affect the development of Pulpitis by regulating the expression of MMPs, but the speci c mechanism is not clear, so we did further experiment.
In our study, the EZH2-induced in ammation response in HDPCs appears to represent a collaboration of the NF-κB, and MAPK pathways. EZH2 stimulated the phosphorylation of MAPK and NF-κB, whereas inhibition of MAPK or NF-κB by speci c inhibitors induced a dramatic reduction in EZH2-induced MMPs production, suggesting that they all have some roles in EZH2-mediated in ammation (Fig.3). These results are in accordance with previous studies showing that ASH1L (absent, small, or homeotic 1-like, an H3K4 methyltransferase that can antagonize EZH2) signi cantly upregulate the expression of MMP-1, MMP-2 and MMP-13 through the Mitogen-activated Protein Kinase (MAPK) signaling pathway [16].In the rat experimental pulp infection model, EZH2 decreased the expression of Col-1, but EI1 could not upregulate the expression of Col-1 in rat pulpitis. EI1 could increase the expression of Col-1 in vitro, suggesting that the development of pulpitis is a multifactor regulatory process, and inhibition of EZH2 alone is not enough to promote the repair of collagen bers.
During pulpitis, pulp can be in a dynamic balance between damage and repair response [28,29]. In recent years, studies have shown that ECM plays an important regulatory role in wound healing and tissue repair [5]. Exploring the dynamic changes of extracellular matrix in dental pulp in ammation will provide a theoretical basis for the treatment of pulpitis. Early studies have found that epigenetic regulatory factor EZH2 is involved in the process of dental pulp in ammation and repair [11],but whether this process involves extracellular matrix is not clear. This study veri ed that EZH2 affects the expression of MMP in dental pulp through the NF-κB pathway or MAPK pathway (Fig.3), thereby mediating the degradation of extracellular matrix and affecting the progress of dental pulp in ammation.
In ammation can stimulate the production of MMPs with subsequent ECM remodeling [8]. Odontoblasts secrete some of the essential MMPs for both physiologic and pathologic conditions. MMPs also appear to be a participant in the process of reversible and irreversible pulpitis[6].

Conclusions
EZH2 promotes extracellular matrix degradation via Nuclear Factor-κB(NF-κB)and P38 signaling pathways in pulpitis.EZH2 can decrease the type collagen levels in vivo and vitro. The down-regulation of EZH2 promotes the development of in ammatory pulp to repair reaction, and provides new ideas for the treatment of pulpitis. Availability of data and materials: All data generated or analysed during this study are included in this published article.

Abbreviations
Competing interests: The authors declare that they have no competing interests.