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| Author(s): |
Cristiane Pereira Borges Saito
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Piracicaba, SP. |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Faculdade de Odontologia de Piracicaba |
| Defense date: | 2009-02-18 |
| Examining board members: |
Sergio Roberto Peres Line;
Raquel Mantuaneli Scarel Caminaga;
Helena Javiel Alves;
Ana Paula de Souza;
Lília Freire Rodrigues D'Souza Li
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| Advisor: | Sergio Roberto Peres Line |
| Abstract | |
PAX9 is a key regulator during tooth development and plays an essential role in the patterning of murine and human dentition. In humans, mutations in PAX9 are associated with unique phenotypes of familial tooth agenesis that mainly involve posterior teeth. The objectives of this study were to gain new insights into the transcriptional activity and DNA methylation within the promoter region of human PAX9 gene in vitro. The methylation pattern was examined by studying PAX9 gene promoter in Dental Papilla of human Embryos through methylation-sensitive restriction enzyme and PCR amplified with specific oligos. DNA extractions from paraffin-embedded tissue sections were well established in Morphology Laboratory, Piracicaba Dental School. DNA samples from Buccal Epithelial Cells were used as control. In the present study, we have PCR amplified cDNAs encoding Rat Pax9 and Mouse Pax9 from Primary embryonic cell culture obtained from 13 day-old Wistar Rat and Mouse odontoblast cell-like culture-23 (MDPC-23) respectively and quantified by Semi-quantitative PCR technique. Furthermore, we examined the transcriptional activity of human Pax9 gene promoter: (-947 - +251) full Pax9 promoter and the promoter lacking 507bp (-485 - +22) through recombination into pGL3Basic expression vector and transfection in primary rat embryonic cells. Cell cultures were all submitted to selective regulation of both drugs: Retinoic Acid (RA) and Dexamethasone (Dex). Relative luciferase expression units were obtained by dual luciferase assay kit (Promega). Our results showed that: (1) human PAX9 promoter region is methylated in vitro; (2) RA inhibited Pax9 mRNA synthesis in Primary Rat embryonic cells while PAX9 promoter lacking 507bp (-485 - +22) was not activated by RA receptors. Human PAX9 full promoter activation was improved by RA treatment at the greater concentration; (3) Dex stimulated Pax9 mRNA activity in MDPC-23 and influenced positively both PAX9 promoter versions with 0.1µM and 1nM concentrations. The present experiments suggest that a 507bp region in PAX9 promoter may contain binding sites for RA receptors and that Dex and RA steroid hormone receptors may be directly bind to the sequences of murine Pax9 gene. The methylation pattern finding give rise to new perspectives on PAX9 patterning in normal cells phisiology. (AU) | |