Effects And Mechanism Of E3 Ligase TRIM16 On Osteogenic Differentiation In Human Periodontal Ligament Stem Cells

Background and ObjectiveRegeneration and reconstruction of the functionally periodontal tissue is an effective approach and ultimate goal for treatment of periodontal diseases,and it is also the focus of research in multidisciplinary fields such as orthodontics,oral implantology and prosthodontics.In recent years,tissue engineering and cell sheet technology based on stem cells have shown great potential in periodontal tissue regeneration.Periodontal ligaments stem cells(PDLSCs)are a group of heterogeneous stem cells with self-renewal capacity and multi-directional differentiation potential derived from periodontal ligaments.Diverse evidences have defined PDLSCs as the most direct and reliable alternative cell sources for periodontal tissue regeneration.PDLSCs can exerted their potentials to rebuild the cementum-periodontal ligament-alveolar bone-like structures after osteogenic induction in vitro;thus,the fully understand of the underlying mechanism of osteogenic differentiation in PDLSCs is important and valuable to improve the therapeutic approaches for periodontal regeneration.With the further study of the molecule regulation implicated in the osteogenic differentiation of stem cells,growing evidence indicated that the ubiquitin-proteasome system has emerged as key modulators to participate in maintaining intracellular homeostasis and governing osteogenic differentiation.Ubiquitination is a dynamic posttranslational modification involved in almost all aspects of biological processes,including cell proliferation and differentiation.The E3 ubiquitin ligases,which is the most abundant group of enzymes involved in ubiquitination,can recognize substrate specifically by serving as an adaptor between the ubiquitin-protein conjugation machinery and the target molecule;thus,E3 ubiquitin ligases are of major significance in controlling bone formation.The TRIM(Tripartite motif)family is a kind of E3 ubiquitin ligases characterized by zinc finger domain(RING domain),B-Box domain and coiled-coil domain(Coiled-coil domain),which participates in cell biology processes such as cell cycle regulation,differentiation,cell migration and so on.TRIM16 belongs to the tripartite motif(TRIM)family of proteins,is widely expressed in many tissues,which is involved in many cellular processes such as cell cycle,differentiation and migration.It plays important roles in innate immune response and tumor,and it also serves as a key molecule in mounting responses to oxidative or proteotoxic stress by autophagy.Recently,the different expression of TRIM16 in periodontal tissues of patients with periodontitis and healthy individuals has been showed,and several clinical periodontal parameters were closely related to the expression of TRIM16.Previous studies in our group have found that TRIM16 protected PDLSCs from oxidative damage via activation of PICOT.What's more,TRIM 16 promotes the osteogenic differentiation of bone marrow mesenchymal stem cells.Considering that TRIM 16 may be involved in the osteogenic differentiation of PDLSCs,TRIM16 could be a new target for promoting periodontal tissue regeneration based on PDLSCs combined with its protective effect under the oxidative stress.In the study,we aimed to clarify the function of TRIM 16 in osteogenic differentiation of PDLSCs and the molecular mechanism.Our results revealed a novel role of TRIM16 in PDLSCs and provided more insights into mechanism underlying in osteogenic differentiation of PDLSCs,which could serve as an evidence for PDLSCs-oriented therapeutic strategies in the field of periodontal tissue regeneration.Methods1.Isolation and identification of PDLSCsThe classical tissue block-enzyme digestion method was used to isolate human periodontal ligament stem cells in vitro.Colony formation assay and CCK-8 experiment were performed to evaluate the self-renewal capacity of PDLSCs.Flow cytometry was used to identify the stem cell surface markers.The potential of osteogenic,adipogenic and chondrogenic differentiation of PDLSCs was examined by Alizarin red staining(ARS),Oil red O staining and Alcian blue staining.2.Expression of TRIM16 in osteogenic differentiation of PDLSCsRT-qPCR and Western Blot were performed to detect the mRNA expression and protein expression of TRIM 16 and osteogenesis-related markers RUNX2 and COL1A1 respectively.3.Constuction of the stably transfected with overexpressing TRM16 and knockdown of TRIM16To stably express TRIM16,the full-length open reading frame(ORF)of TRIM16(GenBank NM₀01348120)was amplified and then cloned into pLVX-puro at the Xhol and EcoRI sites.To knock down TRIM16,3 shRNA targeting TRIM16 and a scramble sequence were synthesized by Invitrogen(Beijing,China).Three sequences of shRNA against TRIM 16 and a negative control were then inserted into the pLKO.1 vector and named sh-TRIM16#1,sh-TRIM16#2,sh-TRIM16#3 and sh-NC,respectively.HEK293T cells were utilized for lentivirus packaging,the supernatant was collected for infection of PDLSCs.RT-qPCR and Western Blot were used to verify the transfection efficacy.4.Effect of TRIM16 on osteogenic differentiation of PDLSCs in vitroPDLSCs were divided into four groups by using corresponding lentivirus infection:MOCK(Control of TRIM 16 overexpression),TRIM 16(TRIM 16 overexpression),shNC(Control of shTRIM16),shTRIM16(Knockdown of TRIM16).After treatment with osteogenic medium,ALP activity detection,ALP staining were performed to evaluate the osteogenic differentiation in early stage;ARS and its quantification by CPC were performed to evaluate the mineralization of PDLSCs.Besides,the mRNA levels of RUNX2,SP7 and COL1A1 were detected by RT-qPCR,the protein levels of RUNX2 and COL1A1 were detected by Western Blot,the expression of OCN was detected by ELISA.5.Effect of TRIM16 on osteogenic differentiation of PDLSCs in vivoHeterotopic bone formation assay and micro-CT analysis were performed to further verify the effect of TRIM 16 on osteogenic differentiation of PDLSCs in vivo.Animal procedures got approval by the Animal Care and Use Committee of Shandong University.PDLSCs with TRIM16 overexpression and the controls were harvested after treatment with osteogenic medium for 1 w,PDLSCs cells incubated with Bio-Oss Collagen(Geistlich,Germany)scaffolds for 1d at 37? were implanted into the dorsal sides of the subcutaneous tissues in nude mice BALB/c homozygous nude mice(6 weeks old,12 mice).Six weeks later,implants were harvested and fixed in 4%PFA.Micro-CT analysis was performed using Micro-CT with Quantum FX system.The software Minics 20.0 was utilized for reconstructing image slices.The ratio of new bone volume to existing tissue volume(BV/TV)was calculated by using CTAn software.Besides,H&E staining,Masson staining were performed for histological analysis.6.The pathway in which RUNX2 was regulated by TRIM16 and the ubiquitination of RUNX2Due to the results that TRIM 16 enhanced the expression of RUNX2 at protein level without change at mRNA level,RUNX2 expression was detected by Western Blot after transfected with different dose of TRIM16 plasmids.Then correlation analysis was performed according to the brands relatively quantified by Image J.Then the protein biosynthesis inhibitor cycloheximide(CHX),the proteasome inhibitor MG 132 and autophagy inhibitor 3-MA were selected to explore the pathway in which RUNX2 was regulated by TRIM 16.After treatment with these inhibitors respectively,RUNX2 expression was detected in cells with/without overexpression of TRIM 16 by Western Blot.After HA-Ubiquitin-WT/K48/K63,Myc-RUNX2 with or without TRIM16 were co-transfected,Co-IP were performed to detect the ubiquitination of RUNX2.7.Predicting the target molecule involved in TRIM16-mediated stability of RUNX2The protein mixture binding RUNX2 was collected by Co-IP,then was identified by LC-MS/MS,and the underlying molecule involved in TRIM16-mediated stability of RUNX2 was predicted subsequently after literature review and Co-IP verification.RT-qPCR and Western Blot were performed to explore the effect of TRIM16 on the target protein.8.Exploring the role of the identified protein in TRIM16-mediated stability of RUNX2Plasmid construction,lentivirus packaging and transfection were performed to manipulate the expression of the identified protein.After transfection with or without TRIM 16 and the identified protein,ALP activity and AR staining were performed to evaluate the osteogenic differentiation of PDLSCs,the expression of related proteins was detected by Western Blot;The interaction between proteins was observed by Co-IP,immunofluorescence staining and co-localization analysis.Besides,the ubiquitination of RUNX2 was detected by Co-IP.Results1.Isolation and identification of PDLSCsPrimary PDLSCs derived from periodontal ligament explants had a long spindle-like morphology and were arranged in whirlpool formations.The passaged cells were in good condition and entered the logarithmic phase of proliferation after 24 hours,colony formation was observed after seeded for 10d.The surface markers of mesenchymal stem cells CD90-FITC,CD 146-PE were positively expressed,and the leukocyte surface markers CD45-PE,HLA-DR-PE were negatively expressed.The results of Alizarin red staining,Oil red O staining and Alcian blue staining were all positive,suggesting the potential of multi-directional differentiation of PDLSCs.2.Expression of TRIM16 in osteogenic differentiation of PDLSCsAfter osteogenic induction for 3d,the mRNA level of TRIM16,RUNX2 and COL1A1 was increased,then decreased gradually after 7d,while the levels were higher than the initial expression;The protein levels of TRIM16,RUNX2 and COL1A1 showed a gradually increasing trend,which providing evidence that TRIM16 is involved in the regulation of osteogenic differentiation of PDLSCs.3.TRIM16 promotes osteogenic differentiation of PDLSCs in vitroThe transfection efficiency of the TRIM16-overexpressing lentiviral vector and TRIM16 specific shRNA was examined by RT-qPCR and Western Blot.Subsequently treated with osteogenic medium,TRIM16-overexpressing PDLSCs showed increased intracellular alkaline phosphatase activity and the mineralization of extracellular matrix.In assessment of the levels of several osteogenic-related markers,TRIM16 increased mRNA expression of SP7 and COL1A1 and upregulated protein expression of COL1A1,RUNX2 and OCN,indicating that TRIM16 promotes the osteogenic differentiation of PDLSCs.Conversely,knockdown of TRIM16 has a significant inhibitory effect on the osteogenic differentiation of PDLSCs.Interestingly,TRIM 16 regulated the protein expression of RUNX2,but had no effects on RUNX2 mRNA expression.4.TRIM16 promotes osteogenic differentiation of PDLSCs in vivoHeterotopic bone formation assay showed that the BV/TV increased significantly in the TRIM 16-overexpressing group compared with the control group.Histological examination corroborated the result of BV/TV.H&E and Masson staining showed more bone-like structures and collagen deposit in PDLSCs of the TRIM16-overexpressing group than the control group,demonstrating that TRIM 16 promotes the osteogenic differentiation of PDLSCs significantly both in vivo and in vitro.5.The promotion of osteogenic differentiation in PDLSCs by TRIM16 depends on its regulation of RUNX2The results mentioned above showed that TRIM 16 upregulated the RUNX2 protein level without affecting its mRNA expression.To verify the effects on RUNX2 expression of TRIM16,we used various doses of TRIM16 overexpression plasmid to examine the changes of RUNX2 protein levels in HEK293T cells.TRIM 16 consistently increased RUNX2 protein expression in a dose-dependent manner(Pearson Correlation=0.962,p<0.05),inferring that the promotion of osteogenic differentiation in PDLSCs mediated by TRIM 16 might depend on its regulation of RUNX2.6.TRIM16 reduced ubiquitination and degradation of RUNX2In order to analyze the possible pathways of TRIM 16 regulating RUNX2 protein,the protein synthesis inhibitor cycloheximide(CHX),the proteasome inhibitor MG 132,and the autophagy inhibitor 3-methyladenine(3-MA)were used to treat PDLSCs transfected with TRIM 16 overexpression plasmid and control group,then the expression changes of RUNX2 protein in each group were detected by Western Blot.The results showed that TRIM16 retarded the reduction of RUNX2 protein compared to the control group after treatment with CHX,and TRIM 16-induced RUNX2 stability compared with control group was attenuated by MG 132,which was not affected significantly by 3-MA,indicating that TRIM 16 inhibited the proteasomal degradation of RUNX2.Besides,we detected the effect of TRIM16 on ubiquitination of RUNX2.The results showed that TRIM16 reduced the ubiquitination of RUNX2,especially the K48-linked poly-ubiquitination,which mainly serves as signals for proteasome degradation,while the K63-linked poly-ubiquitination of RUNX2 was increased,which mediate various functions such as protein re-localization,proteins oligomerization,protein stability and activation of signaling pathway.7.CHIP is identified as an important modulator involved in TRIM16-mediated stability of RUNX2Liquid chromatography-mass spectrometry analysis(LC-MS/MS)was applied to identify the RUNX2 protein binding mixture in PDLSCs stably overexpressing TRIM16,then 128 potential.RUNX2 binding proteins were identified.According to literature review and immunoprecipitation verification,HSP70 chaperone protein CHIP(C terminus of Hsc70-interacting protein)/STUB1 was speculated to be involved in the regulation of RUNX2 by TRIM 16.RT-qPCR and Western Blot results revealed that the expression of CHIP decreased significantly.during the osteogenic differentiation of PDLSCs,and TRIM 16 inhibited the expression of CHIP.In order to further investigate the role of CHIP in TRIM 16-mediated osteogenic differentiation,a CHIP overexpression vector was constructed and CHIP stably overexpressing PDLSCs were established.Through ALP staining,ALP activity determination,ARS staining and quantitative analysis,it was found that CHIP could attenuate the positive effects of TRIM 16 on the osteogenic differentiation of PDLSCs,and the promoting regulation of RUNX2 by TRIM 16 is weakened after CHIP overexpression.TRIM 16 reduced CHIP-mediated K48-linked ubiquitination of RUNX2.What's more,con-focal analysis revealed the interaction between TRIM 16 and RUNX2,and TRIM 16 inhibited the combination of CHIP and RUNX2,verifying that TRIM 16 inhibits the CHIP-mediated RUNX2 proteasome degradation to stabilize the expression level of RUNX2 protein.Conclusion1.The expression of TRIM 16 is upregulated during the osteogenic differentiation.2.TRIM 16 promotes the osteogenic differentiation of PDLSCs in vitro,and enhances the ectopic bone formation of PDLSCs in vivo;3.TRIM 16 inhibits CHIP-mediated ubiquitination and degradation of RUNX2.Features and innovations1.There are still limited researches on the biological functions of TRIM 16.This study revealed a novel role of TRIM 16 in osteogenic differentiation of periodontal ligament stem cells,which enriched the understanding of TRIM 16.2.The mechanisms by which E3 ligase participates in osteogenic differentiation are complex and diverse.We found that TRIM 16 co-regulated the osteogenic differentiation of PDLSCs with another E3 ligase CHIP/STUB1,promoting the cognition of regulatory manner of E3 ligase in cellular biological processes.3.RUNX2 is a key transcription factor in bone homeostasis.In this study,we demonstrated that TRIM 16 inhibited the degradation of RUNX2 by ubiquitin-proteasome pathway,improving the understanding of the regulation of RUNX2 by post-translational modifications,which provides more theoretical basis for further researches on the application of PDLSCs-oriented tissue engineering and cell sheet technology in the field of periodontal tissue regeneration.