Mdm2 Regulates Dentinogenesis And Dentin-pulp Complex Homeostasis In Mice

Part I The expression and function of Mdm2 in odontoblast differentiationObjective:We want to detect the expression pattern of Mdm2 during odontoblast（-like）differentiation of m DPCs in vitro and in vivo.The effects of Mdm2 on the differentiation of m DPCs to odontoblast-like cells in vitro are also detected.The effects of Mdm2 on odontoblast differentiation and odontogenesis during mouse tooth development are studied using a conditional knockout mouse model.Materials and methods:The spatiotemporal expression pattern of Mdm2 during tooth development is detected by immunohistochemistry in mouse molars from embryonic day14.5（E14.5）to postnatal 4 weeks（4W）as well as in postnatal 2 days（PN2）mouse incisors.The m DPCs are isolated and cultured in vitro.Real-time RT-PCR and Western blot are used to detect the expression pattern of Mdm2 during the differentiation of m DPCs into odontoblast-like cells in vitro.m DPCs transfected with Mdm2 si RNA and overexpression plasmid are induced in differentiation medium（DM）.The m RNA levels of the odontoblast marker genes Dspp,Dmp1,and Collagen I,as well as ALP activity and alizarin red staining are measured.The changes of cell proliferation/toxicity are also detected using CCK-8.Dmp1-Cre and Mdm2^flox/flox mice are crossed to obtain Mdm2 conditional knockout mice(Dmp1-Cre;Mdm2^flox/flox,Mdm2 c KO).The tooth phenotypes of Mdm2 c KO mice are observed by HE staining,X-ray,micro-CT（μ-CT）,and immunostaining.Results:Mdm2 is expressed in cells of all stages of odontoblast differentiation in PN2mouse incisors.The expression of Mdm2 is low in undifferentiated mesenchymal cells and preodontoblasts.The expression levels of Mdm2 increase gradually during differentiation.Moreover,Mdm2 accumulates in the nuclei of odontoblasts.The expression pattern of Mdm2 in mouse molars at different developmental stages is consistent with that in incisors.The m RNA and protein levels of Mdm2 are increased after the differentiation induction of m DPCs in vitro.Mdm2 knockdown results in the decrease of Dspp,Dmp1,and Collagen I m RNA levels,ALP activity,and mineralized nodules formation.Mdm2 overexpression results in the increase of Dspp,Dmp1,and Collagen I m RNA levels,ALP activity,and mineralized nodules formation.However,Mdm2 knockdown or overexpression has no significant effects on the cell proliferation/toxicity of m DPCs.HE staining is used to observe the phenotype of Mdm2 c KO mouse molars.Compared with the control mice,odontoblasts of PN2 Mdm2 c KO mice have shorter processes and are less differentiated.2W and 3W Mdm2 c KO mice have thinner mineralized dentin,thicker predentin and irregular mineralized front.6W Mdm2 c KO mice show wearing of molar cusps and reparative dentin formation below the wearing.X-ray andμ-CT show that the enamel thickness of Mdm2 c KO mice is unchange,but the dentin thickness become thinner,the roots become shorter and the pulp cavity become larger.Immunostaining also showed that the protein levels of Dspp and Dmp1 are decreased.Conclusion:Mdm2 is expressed in m DPCs and odontoblasts and promotes odontoblast differentiation and dentinogenesis in vitro and in vivo.Part II Mdm2 promotes odontoblast differentiation through monoubiquitinating Dlx3 which leads to Dlx3 transactivation on the downstream gene DsppObjective:With a complex protein structure,Mdm2 could interact with different substrates in different tissues and cells.In this part,we first determine whether Mdm2 plays a role in odontoblast differentiation and dentinogenesis by regulating p53.If p53 is not the specific substrate of Mdm2 in this process,the specific substrate of Mdm2 during odontoblast differentiation will be further identified by predicting and screening on the website.According to the first part,Mdm2 accumulates in the nuclei of odontoblasts in vivo.It is necessary to study the entry mechanism of Mdm2.Materials and methods:To determine whether Mdm2 functions through regulating p53,the expression of p53 and phosphorylated p53（p-p53（Ser15）,p-p53）in odontoblasts of Mdm2 c KO mice are detected by immunostaining.The number of apoptotic odontoblasts and m RNA levels of p53 downstream genes are measured.Mice are injected with Nutlin-3a,a small molecular inhibitor that inhibits the binding of Mdm2 and p53,to observe whether the dentinogenesis is affected.The Dmp1-Cre;Mdm2^flox/flox;p53^flox/+mice are obtained by mating Mdm2 c KO mice with p53^flox/flox mice,to observe whether p53 knockdown could partially rescue the dentin formation phenotype during tooth development in Mdm2 c KO mice.Since Mdm2 does not function through regulating p53,then the ubiquitinated protein during odontoblast differentiation are identified by coimmunoprecipitations.Combining with predictive measures（website for predicting E3 ligases of substrates:http://ubibrowser.ncpsb.org.cn/ubibrowser/home/index）,Dlx3 could be the specific substrate of Mdm2.Then,whether Dlx3 could interact with Mdm2 and their subcellular locoliazation are determined by coimmunoprecipitations.And the type of Dlx3ubiquitination is determined.Whether Mdm2 could interate with and ubiquitinate Dlx3 are measured by in situ PLA.Since Mdm2 is mainly located in the nucleus of odontoblasts,the Mdm2 overexpression plasmids without nuclear localization signal（NLS）or nuclear export signal（NES）are constructed and verified.In order to determine whether Mdm2 is recruited into the nucleus by Dlx3,the subcellular localization of Mdm2 is detected by Western blot and immunofluorescence after Dlx3 knockdown or overexpression.Finally,to investigate through which region Dlx3 interacts with Mdm2,HEK293E cells are transfected with truncated Dlx3 plasmids and immunoprecipitations are performed to determine the binding region.Dlx3 without binding region is overexpressed to observe whether this could rescue the odontoblast differentiation phenotype of Mdm2overexpression.Results:The protein levels of p53 and p-p53 in odontoblasts of Mdm2 c KO mice are slightly elevated.However,the m RNA levels of downstream target gene of p53 and the number of apoptotic odontoblasts in vivo are not significantly changed.Dmp1-Cre;Mdm2^flox/flox;p53^flox/+mice could not rescue the phenotype of Mdm2 c KO mice.The results show that Dlx3 is ubiquitinated during in vitro differentiation induction,and the possible ubiquitin ligase of Dlx3 is Mdm2.In vitro and in vivo experiments show that Mdm2 could interact with and ubiquitinate Dlx3 in the nucleus.However,Dlx3ubiquitination does not lead to degradation,but promotes the transcriptional activation of downstream gene Dspp.The results show that nuclear Mdm2 could monoubiquitinate Dlx3and promote the differentiation of m DPCs into odontoblast-like cells.At the same time,overexpression of Dlx3 could promote the nuclear translocation of Mdm2.Dlx3 binds to Mdm2 via the C-terminal domain.Dlx3 without C-terminal domain could rescue the phenotype of Mdm2-induced odontoblast differentiation.Conclusion:The function Mdm2 in odontoblast differentiation is p53 independent.Mdm2 is recruited into the nucleus by Dlx3.Mdm2 interacts with and monoubiquitinates Dlx3 in the nucleus,leading to the enhanced transcriptional activity of Dlx3 target gene Dspp,thus promotes odontoblast differentiation.Part III Mdm2 promotes differentiation of odontoblast-like cells by promoting the transactivation of Dlx3 on TfamObjective:The expression of Dspp in Mdm2 c KO mice is significantly decreased but not completely disappears.So,the dentin formation defects phenotype of Mdm2 c KO mice should not be worse than that of Dspp^-/-mice.However,Mdm2 c KO mice have severer tooth phenotypes than Dspp^-/-mice,so the reduced expression of Dspp could only partially explain the tooth phenotypes of Mdm2 c KO mice.Studies have shown that Mdm2 negatively regulates mitochondrial function by NDUFS1 and MT-ND6,which are important components of mitochondrial respiratory chain complex I.Activation of mitochondrial function could promote odontoblast differentiation.We therefore attempt to investigate whether Mdm2 also promotes odontoblast differentiation by regulating mitochondria.Materials and methods:The changes of mitochondria in odontoblasts of Mdm2 c KO mice are observed by transmission electron microscope（TEM）to observe the regulation of Mdm2 on the mitochondria in odontoblasts.Using m DPCs in vitro culture and induced differentiation model,mitochondrial DNA（mt DNA）content and m RNA levels of mitochondrial genes（ND-1,ND-6,Cox-1）are detected by Mito-Tracker staining and real-time RT-PCR.The mitochondrial function is observed by measuring the ATP content.The mitochondrial membrane potential is detected by JC-1 staining,and the redox state is observed by NAD（+）/NADH detection.Studies show that Mdm2 could promote mitochondrial biogenesis and function during differentiation of odontoblast-like cells.Real-time RT-PCR is used to detect the expression changes of the key genes in regulating mitochondrial biogenesis.Tfam is found to be one of the significantly downregulated genes in Mdm2 knockdown odontoblast-like cells.Tfam is encoded by genomic DNA,translated,and synthesized in the cytoplasm and transported to mitochondria.Western blot and immunofluorescence are used to detect the expression of Tfam in vitro and in vivo after Mdm2 knockdown or knockout.To test whether Dlx3 could also regulate Tfam,Dlx3 is knocked down or overexpressed in vitro and Tfam expression and mitochondrial biosynthesis are observed.Whether Tfam is the downstream target gene of Dlx3 is determined by dual-luciferase reporter assay.Finally,Mdm2 knockdown and Tfam overexpression in m DPCs in vitro are performed to test if this could rescue the phenotypes of impaired mitochondrial biogenesis and function as well as odontoblast-like differentiation.Results:TEM shows that the number of mitochondria in PN5 Mdm2 c KO odontoblasts and Mdm2 knockout odontoblast-like cells are both decreased significantly.m DPCs from PN2 Mdm2 c KO mice are cultured in differentiation medium for 7 days.m DPCs with Mdm2overexpression in vitro are cultured in differentiation medium for 7 days.（1）The number of mitochondria labeled with Mito-Tracker in Mdm2 c KO odontoblast-like cells is significantly decreased.The number of mitochondria in Mdm2 overexpression cells is increased.（2）The mt DNA content is decreased in Mdm2 c KO odontoblast-like cells,while the m RNA levels of ND-6,Cox-1 and ND-1 are also decreased.（3）The ATP content in Mdm2 c KO odontoblast-like cells is significantly decreased,while the ATP content in Mdm2overexpression odontoblast-like cells is significantly increased.（4）The NAD（+）/NADH ratio in Mdm2 c KO odontoblast-like cells is significantly decreased.（5）The mitochondrial membrane potential of Mdm2 c KO odontoblast-like cells is decreased.Mdm2 knockdown in vitro or Mdm2 knockout in vivo lead to the decrease of Tfam m RNA and protein levels.Dlx3 overexpression also increases Tfam m RNA and protein levels and mitochondrial biosynthesis.Mdm2 promotes transcriptional activation of Tfam promoter by Dlx3.Overexpression of Tfam in vitro could rescue the mitochondrial biogenesis and function and odontoblast differentiation defects upon Mdm2 knockdown.Conclusion:Mdm2 promotes Dlx3 transactivation on the downstream gene Tfam,thus promotes the mitochondrial biogenesis and function,leading to the enhanced differentiation of odontoblast-like cells.Part IV Mdm2 maintains odontoblast longevity and dentin-pulp complex homeostasis by regulating p53Objective:Odontoblast could survive throughout the lifespan,and is an important part of the dentin-pulp complex.Mdm2 has been shown to regulate the differentiation of odontoblasts during tooth developmental stages.It is also important for odontoblasts as long-lived cells to maintain the homeostasis of dentin-pulp complex in adult mice.Mdm2 is an important E3 ubiquitin ligase involved in the formation of ubiquitin-proteasome system,which removes damaged cellular components and maintains cell homeostasis.Some studies have shown that inhibition of Mdm2 on p53 leads to p53 hyperactivation,which is related to cell apoptosis.Could Mdm2 play a role in regulating the odontoblast longevity and dentin-pulp complex homeostasis through p53 in odontoblasts of adult mice?Materials and methods:The dentin thickness and pulp cavity volume of mandibular first molars are observed byμ-CT of 3M and 5M control and Mdm2 c KO mice.The morphology and mineralization of dentine tubules are observed by SEM,CA/P content and microhardness tests.The thickness of dentin and the morphology of odontoblasts are observed by HE staining.Odontoblast apoptosis are observed by TUNEL staining.To determine whether Mdm2 knockout in odontoblasts of adult mice results in p53activation leading to odontoblast apoptosis,the protein levels of p53 and p-p53 in odontoblasts of Mdm2 c KO mice are observed by immunostaining.The tooth phenotype of5M Dmp1-Cre;Mdm2^flox/flox;p53^flox/+mice are observed byμ-CT and HE staining and compared with Mdm2 c KO and control mice to determine whether p53 knockdown could rescue the tooth phenotypes upon Mdm2 knockout in adult mice.Results:Compared with the control mice,3M and 5M Mdm2 c KO mice have increased dentin thickness,shorter root length,pulp cavity calcification and obliteration.Scanning electron microscopy（SEM）shows that the intertubular and peritubular dentin of Mdm2 c KO mice are decreased and the dentine tubules are enlarged.The contents of calcium and phosphorus,ratio of calcium and phosphorus,and microhardness of inner dentin of Mdm2c KO mice are lower,but those of outer dentin are higher than control mice.These results suggest that Mdm2 c KO mice have a lower degree of mineralization in the inner layer and a higher degree of mineralization in the outer layer of dentin.The morphology of odontoblasts in 3M Mdm2 c KO mice is abnormal compared with control.There is no odontoblasts or pulp cells in 5M Mdm2 c KO mice.Tunel staining shows that the number of apoptotic cells in the odontoblast layer of Mdm2 c KO mice at 2M and 3M is increased compared with control mice.The protein levels of p53 and p-p53 in odontoblasts of Mdm2 c KO mice are detected by immunostaining.Theμ-CT results show that 5M Dmp1-Cre;Mdm2^flox/flox;p53^flox/+mice have thinner dentin,larger pulp cavity compared with Mdm2 c KO mice.The morphology and structure of odontoblasts are also rescued to normal.Conclusion:Mdm2 deletion in odontoblasts promotes the accumulation and activation of p53 in adult mouse odontoblasts,and leads to the apoptosis of odontoblasts.This destroys the the dentin-pulp complex homeostasis,leading to the continously formation of dentin and pulp cavity calcification and obliteration.