The Experimental Study Of Slimb/β-TrCP Regulate Mouse Tooth Development Via Key Signaling Transduction Pathway

BACKGROUND: Along with the evolution and life lengthening of mankind, tooth loss may be the important factor of low life quality. Although modern dentistry technique could provide excellent quality of prosthetic for tooth loss, such as various kinds of denture, dental implants, however, it can not prevent tooth loss due to the little knowledge of mechanisms of tooth development. Tooth development may take a very sophisticated process and background. So, the study of mechanism is very important. The previous study had acquired lots of results; Tooth morphogenesis is regulated by epithelial-mesenchymal interactions and shares a similar molecular signaling network with other epithelial appendages. In tooth, the epithelial signaling centers function in three stages of morphogenesis. The initiation of tooth germ, the formation of crown base and the formation of each cusp. And there may be lots of signaling molecular constructing network participating in this process. It is well established that Among these signaling molecular, Sonic hedgehog (SHH) , and wingless signaling transduction pathway are very important to tooth morphogenesis and tooth development. The Hedgehog (Hh) signaling pathway controls many key developmental processes during animal embryogenesis. The fundamental importance of SHH in organogenesis was demonstrated of its role in patterning the brain, spinal cord, axial skeleton, limbs and epithelial mesenchymal interactions. Epithelial organs such as teeth are defined as those organs in which epithelial-derived cells become the major component of the organ following epithelial-mesenchymal interactions. And this is the main obstacle for tooth regeneration in vitro. But up to now, However, the mechanisms of these signaling molecular were regulated are not very clear. This study focus on the mechanism of slimb interact with Shh, wnt signal transduction in normal tooth development.METHODS AND MATRIALS This study employed cell culture, gene transfection, RT-PCR, flow cytometry,RNAi, immunohistochemistry, immunofluorescence, systematically detected slimb gene expression,β-TrCP expression in tooth germs. Acquire the mouse head of embryo day 18, postnatal day 0,3,6 respectively, 4% paraformaldehyde fixation for 48 hours, Paraffin embedding, examined using LsAB (labeled streptavidin-biotin) method, then observed theβ-TrCP protein expression pattern, and the images were analyzed. And study spectrum of gene expression after slimb-RNAi. The RNAi effects were detected by RT-PCR and Western blot, respectively. RNA extraction and semi-quantitative real-time PCR (qRT-PCR); Plasmids construction for promoter assays.RESMLTS Our data demonstrated thatβ-TrCP expressed in oral epithelium, tooth bud, mesenchymal cell cytoplasm of ameloblast and odontoblast of different stage of tooth development. And these findings provide the evidence of antagonist regulatory pathways for Shh in teeth development. Andβ-TrCP were characteristically expressed in the cytoplasm of hair stem of hair follicle, hair cuticle, cuticle of root sheath, Huxley's layer of internal root sheath cell, external root sheath and mesenchymal tissue, respectively, whereasβ-TrCP were negatively expressed in connective tissue sheath, Henley's layer of internal root sheath.The study of RNAi for slimb/β-TrCP demonstrated that once the slimb/β-TrCP were silenced , GLI1,GLI3,β-catenin were down-regulated in tooth germ cells, and then promote the proliferationof odontogenic cell such as dental epithelium cells and dental mesenchymal cells. And there were many amounts of Undifferentiated cells appeared. The phenomenon is similar in epithelial cells and mesenchymal cells. The study of RNAi for slimb/β-TrCP in vivo demonstrated that in early stage of mouse development, siRNA of slimb/β-TrCP can lead the mouse to death. But, in later stage of mouse development, siRNA of slimb/β-TrCP affect tooth development slightly. If siRNA of slimb/β-TrCP were injected belly cavity before the bell stage of tooth development, the tooth development would arrest at bell stage, If it were injected after bell stage, tooth development were affected slightly. CONCLUSION Our data demonstrated that slimb regulate tooth development via key signaling pathway such as sonic hedgehog signaling pathway and wnt pathway. The mechanisms of regulation were controlled by cell cycle alteration and apoptosis of dental cells after B-TrCP RNAi. The slimb gene RNAi induce enamel enlargement and Shh RNAi result in apoptosis of immature tooth epithelium cell, as same as dental follicle cells. In conclusion, although the mechanisms of tooth development are sophisticated but slimb gene regulate different other gene expression may be the important pathway.Based on these findings,β-TrCP expressed during from early stage to later stage of murine tooth development, we conclude that the slimb/βTrCP have important role in tooth development. It regulate tooth development via sonic hedgehog, wnt signaling pathway. Andβ-TrCP expressed characteristically of developmental hair follicle tissue. It's a implication ofβ-TrCP regulate developmental hair follicle keeping normal development via mediating different signal transduction pathway.