The Mechanism Of P53 Acetylation Signaling Pathway In Sodium Fluoride Induced Apoptosis In Ameloblasts

Fluorine is a widespread substance in the biological world,as well as an essential trace element and a common industrial pollutant.Although optimal concentrations of fluoride are beneficial for normal development of teeth and bones,exposure to high concentrations of fluoride can cause acute or chronic fluorosis,and result in dental fluorosis,skeletal fluorosis,damage in the urinary and reproductive system,nervous system,antioxidant system and immune system.Dental fluorosis is a developmental disorder caused by fluoride over-exposure during enamel formation,Dental fluorosis manifests hypomineralized,mottled,discolored and porous enamel that is susceptible to decay.Ameloblasts secrete enamel matrix protein and manage matrix mineralization to promote the development of enamel.Studies have shown that fluoride can induce oxidative damage and apoptosis of ameloblasts to cause dental fluorosis.Although enamel fluorosis has been studied for decades,the mechanisms underlying fluoride-induced fluorosis are still not fully understood.p53 is a tumor suppressor protein,and acetylated p53 is involved in the regulation of cell cycle and apoptosis.Therefore,this study used Elisa assay,DAPI stain,real-time quantitative PCR(q RT-PCR),Western blot,immunocytochemistry,co-immunoprecipitation,MTT assay and crystal violet assay to explore the role of p53acetylation signaling pathway in fluoride-induced apoptosis in ameloblasts.This study provides a new potential therapeutic target to prevent dental fluorosis.EXP.1 The role of p53 acetylation in NaF-induced apoptosis in ameloblastsAmelobast-derived cells(LS8)were treated with 5 mM NaF for 0.5 h,1 h,2 h,4 h,6 h,18 h,24 h.Elisa assay and DAPI staining showed that NaF induced cell apoptosis.Crystal violet assay showed that NaF decreased cell density and changed cell morphology.The q RT-PCR results showed that NaF increased Bax mRNA level at 18 h and 24 h,while decreased Bcl-2 mRNA level at 6 h,18 h and 24 h.The Bax/Bcl-2 mRNA ratio increased at 6 h,18 h and 24 h.Western blot results showed that NaF induced p53 acetylation from 2to 18 h,increased?H2AX and cleaved-caspase 9 protein expression level at 6 h,18 h and24 h.NaF induced Cyt c release into the cytosol at 6 h,18 h and 24 h,and decreased mitochondrial Cyt c and Bcl-2 protein expression levels.The Bax and cleaved-caspase 3protein expression level increased at 18 h and 24 h.The ratio of Bax/Bcl-2 protein expression was increased since 6 h.These data suggested that NaF induced mitochondrial apoptosis via p53 acetylation in ameloblasts.EXP.2 The role of p53/MDM2/p21 signaling pathway in NaF-induced apoptosis in ameloblasts.(1)LS8 cells were treated with 5 mM NaF for 0.5 h,1 h,2 h,4 h,6 h,18 h and 24 h.Results showed that NaF increased MDM2 and p21 mRNA expression levels after 6 h treatment.NaF induced p-MDM2 and p-p21 protein expression.p21 protein expression level was increased by NaF at the early phase(0.5-2 h),but decreased at the late phase(18h and 24 h).These data showed that NaF induced p53 downstream target MDM2,p21mRNA expression levels and p-MDM2,p-p21 protein expression levels.(2)LS8 cells were pretreated with MDM2 antagonist Nutlin-3a or proteasome inhibitor MG-132 for 2 h,followed by 5 mM NaF treatment for 6 h or 24 h.NaF increased MDM2-p53 binding and MDM2-p21 binding at 6 h.These bindings were inhibited by Nutlin-3a.In addition,Nutlin-3a inhibited cell apoptosis at 24 h,and increased p53,MDM2,p21 and p-p21 protein expression levels at 24 h.Nutlin-3a enhanced MDM2mRNA expression levels at 24 h,inhibited Cyt c release into the cytosol,and decreased Bax/Bcl-2 protein ratio and cleaved-caspase 9,cleaved-caspase 3,?H2AX protein expression levels at 24 h.MG-132 decreased NaF-increased cell apoptosis,inhibited p21and p-p21 degradation,decreased Bax/Bcl-2 mRNA and protein ratio,enhanced mitochondrial Cyt c protein expression level,inhibited Cyt c release into the cytosol,and decreased cleaved-caspase 9,cleaved-caspase 3 and?H2AX protein expression levels.These data showed that Nutlin-3a and MG-132 alleviated NaF-induced apoptosis in ameloblasts.The p53/MDM2/p21 signaling pathway promoted NaF-induced apoptosis in LS8 cells.EXP.3 The role of Histone Acetytrasferases(HATs)in NaF-induced apoptosis in ameloblasts(1)LS8 cells were treated with 5 mM NaF for 0.5 h,1 h,2 h,4 h,6 h,18 h and 24 h.Results showed that CBP mRNA expression level increased at 6 h and 18 h,while P300mRNA expression level decreased at 24 h.Tip60 mRNA expression level was increased by NaF at 6 h.NaF induced Acetyl-CBP/P300 expression level from 1 h to 18 h,and p-Tip60protein expression level were enhanced from 2 h to 24 h.In addition,NaF increased CBP-p53 binding at 2 h.These data suggested that NaF increased HATs activity and the binding between HATs and p53.(2)LS8 cells were treated with HATs inhibitors:curcumin or MG-149(Tip60 specific inhibitor)followed by 5 mM NaF treatment for 6 h,18 h and 24 h.Results showed that curcumin and MG-149 attenuated NaF-induced cell proliferation suppression,inhibited cell apoptosis at 18 h and 24 h.Curcumin decreased NaF-increased Acetyl-CBP/P300protein expression level at 6 h and 18 h.Curcumin and MG-149 also inhibited NaF-mediated Tip60 phosphorylation at 6 h,18 h and 24 h,inhibited p53 acetylation at 6 h,18 h and 24 h,decreased Cyt c(cytosol),cleaved-caspase 9,cleaved-caspase 3 and?H2AX protein expression levels at 18 h and 24 h,and suppressed Bax/Bcl-2 mRNA and protein ratio at 18 h and 24 h.These data showed that inhibition of HATs activity restrained NaF-induced p53 acetylation to alleviate NaF apoptosis in ameloblasts.EXP.4 The role of SIRT1-p53 signaling pathway in NaF-induced apoptosis in ameloblasts(1)LS8 cells were treated with 5 mM NaF for 0.5 h,1 h,2 h,4 h,6 h,18 h and 24 h.Results showed that NaF increased SIRT1 mRNA expression level and induced p-SIRT1protein expression level,which showed that NaF increased SIRT1 activity.(2)Parental wild type LS8 cells(LS8^WT),SIRT1 overexpressing LS8 cells(LS8^SIRT1/Over)and negative control cells(LS8^{Lac Z})were treated with 5 mM NaF for 6 h.The Immunoprecipitation results showed that NaF decreased SIRT1-Tip60 binding and SIRT1-p53 binding in WT cells,but did not change the binding in LS8^SIRT1/Over cells.These data showed SIRT1 over expressor increased SIRT1-Tip60 binding and SIRT1-p53binding.(3)LS8^WT,LS8^SIRT1/Over and LS8^{Lac Z} cells were treated with 5 mM NaF for 6 h,18 h and 24 h.Compared to LS8^WT or LS8^{Lac Z} cells,apoptosis were significantly inhibited in LS8^SIRT1/Over cells at 18 h and 24 h.Tip60 mRNA expression,p-Tip60,Tip60 protein expression and p53 acetylation were suppressed in LS8^SIRT1/Over cells at 6 h,18 h and 24 h.Bax/Bcl-2 mRNA and protein expression ratio,cleaved-caspase 9,cleaved-caspase 3 and?H2AX protein levels were decreased at 18 h and 24 h in NaF-treated LS8^SIRT1/Over cells.NaF induced Cyt c release into the cytosol at 18 h in WT cells and LS8^{Lac Z} cells,while NaF inhibited Cyt c release in LS8^SIRT1/Over cells.These results suggested that SIRT1 over expression inhibited NaF-induced p53 acetylation to prevent NaF-induced apoptosis in ameloblasts.Above-mentioned results in the present study verified that NaF enhanced HAT activity,and induced p53 acetylation to promote apoptosis in ameloblasts.NaF also induced p53downstream target MDM2 and p21 mRNA expression level.MDM2 phosphorylation promoted MDM2-p21 binding and induced p21 degradation that resulted in apoptosis in LS8 cells.SIRT1 deacetylated p53 through directly binding with p53 or Tip60,and inhibited Tip60 phosphorylation against NaF-induced apoptosis.p53 acetylation plays an important role in NaF-induced apoptosis.The p53 acetylation signaling pathway may be a potential therapeutic target for dental fluorosis.This study verified NaF-induced ameloblast apoptosis via p53 acetylation.It reported the relationship between p53/MDM2/p21 signaling pathway and apoptosis induced by NaF in ameloblasts.And discussed the role of histone acetyltransferase and SIRT1 in NaF-induced p53 acetylation to promote apoptosis in ameoblast.