The Effect Of Proteolytic Fragments Of MEPE In Dentinogenesis

Matrix extracellular phosphoglycoprotein (MEPE), was first isolated from oncogenichypophosphatemic osteomalacia (OHO)，normal tissue expression was found in bone，teeth，bone marrow and brain with very low level expression found in lung, kidney, andhuman placenta. MEPE/OF45expression was shown in dental tissue, in particularodontoblasts, by reverse-transcription polymerase chain reaction (RT-PCR) amplificationand characterization of a partial mouse cDNA.， and should be considered as a candidategene for dentin structural diseases mapping to human chromosome4q21, such asdentinogenesis imperfecta types II and III, and dentin dysplasia type II. MEPE was presentin three sizes in the rat pulp MEPE, one full length and two cleaved fragments; it wasproposed that MEPE is activated through proteolytic processing during its secretion fromodontoblasts. The C-terminal fragment cleavage product appeared to be the active form ofMEPE. However it is still unclear that whether the different fragments have differentfunction in the dentin matrix and how they participate and control the dentinogenesis. In this study, we observed the localization of MEPE and it’s cleavage products byimmunohistochemical staining at different tooth developmental stages; and weinvestigated the effect of MEPE and it’s cleavage product on the differentiation,proliferation and induced mineralization ability of odontoblasts.1. Expression of Matrix Extracellular Phosphoglycoprotein and the cleavagefragments in mouse tooth developmentwe investigated the expression and localization of MEPE and it’s cleavage productsby immunohistochemical staining in mouse tooth germ during tooth development. In earlyage of tooth development, MEPE N-terminal fragment and C-terminal fragment wereexpressed in odontoblast and ameloblast. During the development of dental germ, theexpression was mainly increased in predentin and weakened in odontoblast and ameloblast.It suggested that MEPE possibly plays an important role in dentinogenesis in time andspatial manner.2. Cell biological properties of Matrix Extracellular Phosphoglycoprotein and thecleavage fragment over-expression stable cell linesWe compared the proliferation, mineralization capability of the MEPE full-length，N-terminal，C-terminal over-expression stable cell lines and mock stable cell line toexplicate the effect of MEPE on differentiation and mineralization of odontoblasts. Wefound that OLC-F and OLC-C over-expression stable cell lines showed lowermineralization capability. When cultured in the mineralizing medium, ALP activity in themedium secreted by the OLC-F and OLC-C was much lower than that of mock cells. Atthe same time, alizarin red S staining showed that OLC-F and OLC-C formed much lessmineralized nodules than that of mock cells. It suggested that MEPE full-length andC-terminal inhibit differentiation and mineralization of odontoblasts. 3. Effects of Matrix Extracellular Phosphoglycoprotein and the cleavage fragment onbiological properties of odontoblast in vitroWe explored the proliferation and osteogenesis differentiation capability ofodontoblasts stimulated with recombinant MEPE and the C-terminal fragment. The resultsindicated that MEPE and the C-terminal fragment appeared to play an important positiverole in proliferation of odontoblast.and had good ability to inhibit osteogenesisdifferentiation and mineralization capability of odontoblast. The expressions ofenamelin(ENAM), transforming growth factor-β2(TGFB2) and integrin α2(ITGA2) weredetermined by quantitative real-time RT-PCR. The results showed that the mRNA level ofITGA2、ENAM were up-regulated and the mRNA level of TGFB2were down-regulated.4. Effects of Matrix Extracellular Phosphoglycoprotein and the cleavage fragment onapatite mineralization in vitroWe investigated the Effects of MEPE full-length and C-terminal fragment on apatitemineralization with the dual-membrane system. The results showed that hydroapatitecrystals aggregated to form crystal sphere in MEPE full-length and C-terminal fragmentgroups while the BSA and DDW control group could not. It indicated that MEPE andC-terminal fragment affect the crystal morphology and act as the crystal nucleating agent.