The Regulatory Effects Of CXXC5 On Porphyromonas Gingivalis-related Cementoblast Differentiation And Autophagy

Part.Ⅰ: Effects of P.gingivalis on Cementoblast Differentiation and Autophagy in Apical Periodontitis Models Objective Porphyromonas gingivalis associates tightly with periodontal diseases,and it is also a dominant pathogen of periapical periodontitis.This part aims to elucidate the effects of downward-infiltrating P.gingivalis on differentiation and autophagy of tooth root surface lining cementoblasts in murine apical periodontitis(AP)models.Methods The mice were anesthetized to open the pulp chambers of the left mandibular first molars with a high-speed dental handpiece.The pulps were exposed in the oral cavity for 21 d to create an inflammatory environment for cementoblasts and establish AP models in vivo.Half of the mice were further treated with a repeated oral application of P.gingivalis(MWF/week)in a 2% carboxymethylcellulose vehicle.No surgical interventions were done on the right first molars of the mice without P.gingivalis treatment,which was maintained as the healthy control group.After animal sacrifice,X-ray photography was performed first to rule out samples with insufficient unfolding of pulp chamber roof or perforation of pulp chamber floor,and to preliminarily assess the extent of apical lesions.To clearly evaluate ranges of periapical bone destruction,the mandibles from three groups(healthy,AP,and AP plus P.gingivalis)were scanned by micro-CT.The expression levels of Il-6 and Tnf-α in periapical tissues were detected by q PCR.Inflammatory cell infiltration in apical tissues was revealed by H&E staining,and immunohistofluorescence was carried out to verify the apical invasion of P.gingivalis through root canals.Immunohistochemistry was performed to detect the expressions of osteogenic marker Osterix as well as autophagy markers LC3 and p62 in apical cementoblasts and ambient periodontal ligament cells.Results1.The murine AP model was successfully established by pulp exposure.In contrast to mandibles in the healthy group,samples in the AP group exhibited significant apical bone destruction around the first molars,and additional P.gingivalis treatment enlarged the ranges of apical bone resorption,revealed by X-ray photography and micro-CT analysis.2.The expression levels of Il-6 and Tnf-α in apical tissues of the AP group were much higher than those in the healthy group.H&E staining showed evident inflammatory cell infiltration in the AP and AP plus P.gingivalis group.Immunohistofluorescence testified that pulp exposure combined with oral application of P.gingivalis suspension enabled the bacteria to invade apical areas and further interact with tooth root lining cementoblasts.3.Immunohistochemistry results demonstrated that Osterix,LC3,and p62 were all positive in apical cementoblasts and ambient periodontal ligament cells around mandibular first molars.A sharply reduced expression of Osterix in cementoblasts was observed in the following three groups: the healthy group,the AP group,and the AP plus P.gingivalis group.Compared to the AP group,darker LC3 B staining and lighter p62 staining were observed in cementoblasts of the P.gingivalis treated AP group.This is also the first time to detect autophagy of cementoblasts in vivo.Conclusion P.gingivalis aggravated AP in mouse models,with repression of cell differentiation and activation of autophagy activities in cementoblasts.However,the molecular mechanisms concerning P.gingivalis-suppressed differentiation and P.gingivaliselicited autophagy in cementoblasts still need to be elucidated by further exploration.Part.Ⅱ: CXXC5 Mediates P.gingivalis-suppressed Cementoblast Functions Partially via MAPK Signaling Network Objective CXXC-type zinc finger protein 5(CXXC5)is a novel transcription regulator of gene expression and cell signaling.This part aims to explore the possible involvement of CXXC5 in P.gingivalis-suppressed cementoblast differentiation,and to uncover the mechanism by which CXXC5 mediates cementoblast differentiation.Methods In vitro,OCCM-30,an immortalized mouse cementoblast cell line,was treated with P.gingivalis for different time lengths or with different multiplicity of infection,and the samples were collected for detection of proinflammatory cytokines Il-6,Mcp-1,and Rantes,osteogenic markers Osterix,OCN,and BSP,as well as mineralized nodule formation.The expressions of osteogenic markers and Cxxc5 during cementoblast differentiation were analyzed by RNA-seq and testified by q PCR.Immunofluorescence was carried out to verify the intercellular distribution and expression of CXX5 upon P.gingivalis treatment.In vivo,mouse AP models with P.gingivalis invasion or not were used to examine CXXC5 expression in apical areas.Further,OCCM-30 was transfected with siRNA and overexpression plasmids of CXXC5,respectively.The knockdown,overexpression,and rescue effects were revealed in terms of associated osteogenic gene and protein expressions,as well as activities of several signaling pathways,analyzed by either q PCR or western blot analysis.Meanwhile,ALP staining and ALP activity were used to clearly determine cell mineralization.Specific inhibitors for MAPKs and PI3K-Akt signaling were employed to confirm the participation of these pathways in CXXC5-promoted cementoblast differentiation.Results1.P.gingivalis repressed the mineralization capacity of cementoblasts and induced proinflammatory cytokine production in a dose-dependent manner.2.The expression of CXXC5 decreased in P.gingivalis-treated OCCM-30 cells and murine AP models,but gradually increased during cementoblast differentiation.3.RNA interference of CXXC5 significantly inhibited cementoblast differentiation,represented by the decline of bone-associated markers Osterix,OCN,and ALP activity.CXXC5 overexpression facilitated cell differentiation,and therefore attenuated the P.gingivalis-repressed effects on cementoblast differentiation.4.Erk1/2,p38,and PI3K-Akt were inactivated by silencing CXXC5 and activated upon CXXC5 overexpression,whereas Wnt/β-catenin exhibited an opposite trend.The employment of specific inhibitors revealed that the CXXC5-dependent promotion of cementoblast differentiation was partially abrogated by p38 and PI3 KAkt inhibitors but was exacerbated by inhibiting Erk1/2.Conclusion Our data clarified that cementoblast differentiation and CXXC5 expression were repressed by P.gingivalis stimulation,and that CXXC5 overexpression could partially attenuate P.gingivalis-repressed osteogenic effects of cementoblasts.This research indicated overexpressing CXXC5 may be conducive in the regeneration of impaired cementum caused by P.gingivalis invasion and suggested that MAPK signaling network balanced the facilitation effects of CXXC5 on cementoblast differentiation.Therefore,CXXC5 is a novel potential therapeutic target for cementum reconstruction.Part.Ⅲ: CXXC5 Orchestrates Stat3/Erk/Akt Signaling Networks toModulate P.gingivalis-elicited Autophagy in Cementoblasts Objective P.gingivalis is reported to induce inflammation and autophagy in various types of periodontal cells.The expression of transcription factor CXXC5 is significantly repressed by P.gingivalis invasion.This study aims to investigate the autophagyinducing effects of P.gingivalis,the contribution of CXXC5 in autophagy regulation,and the possible involvement of signals in cementoblasts,root surface cells pivotal in the apical areas.Methods Short-term OCCM-P.gingivalis coculture system was established in vitro.The changes in levels of proinflammatory cytokines,phosphorylated Stat3,autophagy-related LC3,and p62 were examined.Immunofluorescence staining was performed to display the internalized P.gingivalis and the distribution of LC3 signals in cementoblasts.TEM was used to observe autophagic vesicle accumulation in the P.gingivalis-stimulated samples.The downregulation of CXXC5 in cementoblasts co-cultured with P.gingivalis was validated by biochemical assays.Next,cementoblasts with loss and gain of CXXC5 were developed to investigate its effect on inflammation and autophagy modulation,and changes of potentially related signaling pathways including P65,Jak/Stat3,PI3K-Akt,and Erk1/2 were evaluated.The specific inhibitors for Jak/Stat3,PI3K-Akt,and Erk1/2 were applied,and downstream proinflammatory cytokine production and autophagy-related protein expressions were determined.RNA interference of ATG5 and Beclin1 was carried out to explore the bidirectional regulation of autophagy on inflammation.Ultimate cell fate caused by P.gingivalis invasion was determined by Ed U assay and Annexin V/PI kit.Results1.P.gingivalis elicited inflammation and autophagy in cementoblasts,represented by enhanced proinflammatory chemokine production,Stat3 phosphorylation,LC3-II accumulation,and p62 degradation.Fluorescence microscopy detected bright LC3 spots around the nucleus and internalized bacteria in the P.gingivalis-infected samples.Moreover,TEM captured evident autophagic vesicles(autophagosomes and autolysosomes)in the P.gingivalis-cocultured group.2.Short-term P.gingivalis treatment decreased CXXC5 expression.The knockdown of CXXC5 attenuated the inflammatory responses and yielded an autophagy-silent effect;thus partially restricting the excessive inflammation and autophagy induced by P.gingivalis invasion.In contrast,CXXC5 overexpression elevated the levels of inflammatory factors Il-6,Mcp-1,and Rantes,activated p-Stat3 and LC3-II,and decreased p62 expression.3.Biochemical assays detected Stat3/Akt/Erk activation during P.gingivalis treatment and CXXC5 overexpression,and the employment of their inhibitors Stattic,LY294002,and PD98059 confirmed their positive influence on autophagy and inflammation.Furthermore,P.gingivalis and CXXC5 induced autophagy through ATG5 and Beclin1 activation,given specific siRNA for them significantly suppressed LC3 conversion and p62 degradation.4.In the P.gingivalis-treated cells,the percentage of Annexin V(+)cells steadily increased while the red Ed U fluorescent signals were evidently quenched.Furthermore,ROS production was triggered by P.gingivalis infection as more OCCM-30 cells produced ROS and the total amount of ROS generation was significantly enhanced.Conclusion P.gingivalis increased autophagy activities and decreased transcription factor CXXC5 expression in cementoblasts,while CXXC5 downregulation limited the extent of excessive autophagy in turn.The autophagy-inducing effects were partially dependent on Stat3/Akt/Erk signaling and autophagy-related Beclin1 and ATG5.Furthermore,P.gingivalis was likely to affect cell proliferation and apoptosis via maintenance of high autophagic levels.Therefore,P.gingivalis can cause damage to cementoblasts not only by traditional inflammation activation,but also by autophagy induction.