Molecular Control of Enamelin during Amelogenesis

Enamelin is an extracellular enamel protein with unique roles during enamel formation. Enamelin expression is mainly restricted in ameloblasts during the secretory stage of amelogenesis. No enamelin expression has been clearly detected in other cells. This restricted spatial and temporal pattern of expression is critical for the completion of amelogenesis, and perturbation of this expression results in abnormal enamel in mice and humans. Objectives: To characterize the enamelin 5' cis-regulatory promoter region and to identify trans-acting factors that control cell- and developmental stage- specific expression of enamelin. Methods: DNA constructs covering different parts of the enamelin 5' regulatory region have been generated by PCR and subcloned into a luciferase reporter gene vector for in vitro promoter assays. The final constructs were verified by DNA sequencing and tested for promoter activity in LS8 ameloblast-like cells (Malcolm Snead, University of Southern California). Transient transfection experiments and gel shift assays were undertaken followed by real time PCR, western blots and measurement of promoter activity to elucidate the role of potential trans-acting factors on enamelin expression. The developmental pattern of transcription factors that may control enamelin expression was also investigated by immunohistochemistry. Results: Analysis of enamelin promoter driven reporter activity resulted in identification of regions of the enamelin promoter that may control cell-specific expression. Co-transfection assays suggested that Runx2 and Dlx/Msx transcription factors might play a role in regulating enamelin expression. Dlx3 showed to activate enamelin expression in contrast to Runx2 that showed to inhibit enamelin RNA steady state levels and also enamelin promoter activity. Conclusion: Our study provides novel insights into factors influencing the developmental and tissue-specific expression of enamelin within the enamel-forming ameloblasts. This study is supported by NIDCR grant DE011301.