Dentin Sialoprotein Is Processed By Gelatinases In The Odontoblasts

Dentin sialoprotein(DSP),the NH2-terminal fragment of dentin sialophosphoprotein(DSPP),is further processed into small molecular fragments during dentin formation,and its COOH-and NH2-terminal fragments displayed different expression pattern in tooth.Gelatinases,a subfamily of matrix metalloproteinases(MMPs),participate in various physiological and pathological events by degrading extracellular substrates.But recently,it has reported that gelatinases are able to cleave some intracellular protein substrates.So we hypothesized that activated gelatinases may participate in the cleavage of DSP in the cytoplasm of odontoblast cells.In this study,coexpression of DSP and gelatinases in the odontoblasts was proved by double Immunofluorescence.Binding and interactions between DSP and gelatinases in the odontoblasts were detected by In Situ PLA.Catalytic activity of gelatinases was measured by zymography and In Situ zymography,and Results showed the presence of gelatinases activities(pro-and active forms)in the odontoblasts.DSP protein profiles in odontoblastic-induced Wild-type(WT)dental papilla cells(DPCs)treated with or without MMP inhibitors and in odontoblastic-induced Mmp9-/-DPCs were analyzed by Western blotting.DSP protein pattern in Mmp9-/-odontoblastic cells and in the DPCs treated with matrix metalloproteinase 9(MMP9)inhibitor showed additional high molecular weight(HMW)DSP bands and higher ratio of HMW DSP to low molecular weight(LMW)fragments,compared with that in the WT odontoblastic cell without MMP inhibitor treatment.Rising ratio of HMW DSP to LMW fragment was also observed in odontoblastic cells treated with matrix metalloproteinase 2(MMP2)and/or MMPs inhibitor.FURIN is co-localized and interacted with gelatinases in the odontoblasts.Expression levels of activated MMP9 inside the odontoblasts were decreased when function of endogenous FURIN was inhibited.Overall,these results indicated the FURIN may involves with the activation of MMP9 in the odontoblasts intracellularly,and activated MMP9 and MMP2 cleaves DSP into small fragments,which might be an activation step of DSP biological function.Part I: Dentin sialoprotein is processed by gelatinases in the odontoblasts.Objective:To demonstrate that dentin sialoprotein is processed by activated gelatinases in the odontoblast cells.Materials and Methods:1.DPCs extracted from neonatal mice were maintained in DMEM containing 20% fetal bovine serum and when the cells confluent up to 80%,culture condition was replaced with mineralization-inducing media.2.Mice of postnatal day(PN)1,3 and 7 were sacrificed and mandible tissues were dissected.All specimens were fixed,demineralized and dehydrated.Paraffin-embedded sections were cut and prepared for further research.Immunofluorescence experiments were carried out on the odontoblasticinduced DPCs plated on coverslips and tissue sections to determine the expression patterns of dentin sialoprotein and gelatinase respectively in the odontoblast cells,and to explore the co-localization of the two proteins.3.Interaction of DSP with either MMP9 or MMP2 was detected by the In situ PLA.4.In order to evaluate the enzymatic activity of gelatinases in the odontoblasts,Zymography and in situ Zymography were performed in the odontoblasticinduced DPCs and tooth frozen sections.5.Odontoblasts with the same confluence were randomly divided into four groups,and DMSO,MMP9 inhibitor,MMP2 inhibitor and broad spectrum matrix metalloproteinase inhibitor were added into the culture medium for 8h.The odontoblast cells of Mmp9-/-mice with the same confluence were cultured for the same time,and proteins in all the above cells were collected,and western blot analysis was used to measure the changes of DSP protein pattern inside the cells.Results:1.Gelatinase was widely expressed in the tissue sections of mouse molar embryo,and was expressed in the odontoblast layer,the dental papilla,the surrounding alveolar bone and other tissues.2.DSP was highly localized in the pre-and secretory odontoblasts of mouse molars at PN 1,3 and 7.Co-distribution of DSP and gelatinases was detected in the cytoplasm of odontoblast cells.3.Interaction between gelatinase and DSP was observed in the odontoblasts of mouse mandibular molars and in the odontoblastic-induced DPCs.4.For in situ Zymography,enzymatic cell number and fluorescence intensity were both decreased significantly in all three groups treated with MMP inhibitors compared with control group.For Zymography,multiple forms of gelatinolytic enzymes were detected in cell lysates and supernatants,including the pro MMP2,activated MMP2 and latent and activated forms of MMP9.5.Intracellular expression patterns of DSP in the odontoblastic-induced DPCs from Mmp9-/-and WT mice were different.Without inhibitor treatment,several HMW DSP bands(ranging from 100 to 250 k Da)were observed in the Mmp9-/-cellular proteins but not in the WT cellular proteins,and several LMW DSP bands(lower than 70 k Da)were seen in WT group but not in the Mmp9-/-group.When odontoblastic-induced WT DPCs treated with MMP2 or MMPs inhibitor,several HMW DSP bands also appeared in the cytoplasmic proteins.The ratio of the HMW DSP to the LMW fragments were significantly higher in the Mmp9-/-odontoblast cells,and in the odontoblast cells treated with either MMP9 inhibitor,MMP2 inhibitor or MMPs inhibitor.Conclusion:DSP is processed by activated MMP9 and MMP2 into small molecular fragments in the odontoblasts.Part II: study on the mechanism of activated MMP9 in the odontoblast cellsObjective:To investigate the mechanism of activation of MMP9 in the odontoblast cellsMaterials and Methods:1.Immunohistochemical assay was used to detect FURIN protein expression in the WT mouse mandible tissue sections.2.Co-localization of FURIN and MMP9 in the odontoblast cells was detected by immunofluorescence.3.The binding of FURIN and MMP9 in the odontoblast cells was detected by immunoprecipitation.4.To investigate the role of Furin in processing gelatinases inside the odontoblast cells,a specific FURIN inhibitor Decanoyl-RVKR-CMK was applied to inhibit function of FURIN intracellularly.The expression of MMP9 in the odontoblasts was detected by Western Blotting after treated with FURIN inhibitor.Results:1.FURIN is widely expressed in the odontoblasts,dental papilla cells and bone.2.FURIN co-localized with MMP9 in the DPCs of WT mouse.3.FURIN could be efficiently immunoprecipitated with pro-MMP9 and scarcely immunoprecipitated with mature MMP9 enzyme in the odontoblasts.4.Compared with group without addition of Decanoyl-RVKR-CMK,the amount of activated MMP9 and the ratio of latent MMP9 to activated-MMP9 in the odontoblasts treated with Furin inhibitor are significantly increased.Conclusion:FURIN may involves with the activation of MMP9 in the odontoblasts...