Molecular Mechanism Of Transcription Factor USF1 During Mouse Tooth Development

Upstream stimulatory factor 1(USF1) is a member of evolutionarily conserved basic-helix-loop-helix-leucine zipper (bHLH-zip) transcription factors that interact with the DNA at symmetrical E boxes with the consensus sequence 5'CACGTG 3. It shares with the Myc oncoproteins both a similar polypeptide structure and a similar DNA-binding specificity. Yet, USF1 and Myc play antagonistic roles in the control of mammalian cell proliferation and transformatioa For example, Overexpression of USF1 can specifically abolish the transforming ability of c-Myc and inhibit the growth of a number of cancer cell lines due to c-Myc overexpression. In mammals, USF1 plays an important role during embryonic development and also has many target genes essential for tooth development. Among the proposed USF targets, it is noteworthy that many, including osteopontin, bone morphogenetic protein 4, transforming growth factor 2, cyclin B1, and TGF-P type II receptor are genes involved in proliferation, secretion or cell cycle control. Moreover, it was reported that USF1 acted to upregulate nAChR7 expression by binding to an E-box at -116 of the promoter.Nicotine is an important pathogenetic factor that leads to the dysplasia of tooth development. For many years, the detrimental effects of nicotine on tooth development have been confirmed via epidemiological investigations and animal experiments. Ourrecent work also revealed that nicotine could interfere the maturation of dental papilla cells and their physiological functions, and that it could upregulate the expression of nAChR7 in tooth germ. However, the molecular mechanism of nicotine on tooth development in details, which is the key to the prevention from nicotine's detrimental effects, remains unknown.In order to elucidate the regulatory roles of USFl in tooth development and identify its possible molecular mechanism that mediate the deleterious effect of nicotine, the following experiments were carried out.1 .Expression of USF1 during mouse tooth developmentIn this part, we first confirmed the expression of USF1 in mouse tooth germ by reverse transcription-polymerase chain reaction and Western blot, and then detected the temporal-spatial expression patterns of USFl in mouse tooth development Partial sequencing of the PCR products revealed that we successfully cloned the USFl cDNA segment that was consistent with those presented in Genebank, and that we identified another isoform of USF1, the mini USFl, which lacks 31bp at the amino terminal and has been accepted by Genebank with the accession number AF479773. Western blot assay confirmed the existence of USFl in tooth germ, and it localized specifically in the secreting ameloblasts and odontoblasts via immunohistochemistry and in situ hybridization staining. This specific temporal-spatial expression pattern of USFl during tooth development indicates that USF1 is likely to play important roles in the differentiation and maturation of ameloblasts and odontoblasts.2. Effect of USF1 on protein production of odontoblastsTo demonstrate the function of USFl in tooth development, the odontoblast cell line MDPC-23 was cultured and stably transfected with USF1or A-USF expression plasmids, then the localization of USF1 and the expression of osteopontin and TGF-2 were assayed.Immunocytochemically, USFl localized in the cytoplasm of odontoblasts but in the nucleus of Hela cells. After USFl transfection, the expression of osteopontin and TGF-2 were upregulated while they were downregulated after A-USF transfection. Thus it reveals that USFl could regulate the production of osteopontin and TGF-2 in odontblasts, and that USFl is implicated in the dentin matrix secretion and mineralization during tooth development.3. Roles of USF1 in the abnormal tooth development caused by nicotine To exploring the roles of USF 1 in the abnormal tooth development caused by nicotine, we first observed the detrimental effects of nicotine on tooth development of postnatal mouse, and then evaluated the effects of nicotine on USFl tr...