Periodontal disease afflicts millions of Americans, often resulting in bone destruction and tooth loss. The ultimate goal of therapy is regeneration of periodontal tissues, which requires the differentiated cells to produce specific extracellular matrices, in a precise temporal and spatial manner. Bone sialoprotein (BSP) gene is a unique marker for mineralization and the differentiation of bone- and tooth4orming cells. However, the mechanisms regulating the expression of this important protein are unknown. Core binding factor 1 (Cbfa1) is a """"""""master gene"""""""" for osteogenic differentiation. Cbfa1 knock out mice display a complete absence of bone. Moreover, our in vitro studies have shown that Cbfa1 regulates BSP expression. My overall goal is to determine the roles of BSP in bone and tooth development. The objective of this continuation application is to determine the mechanisms regulating BSP gene expression, particularly by Cbfa1 in vivo. The central hypothesis is that tissue-specific expression of BSP is regulated by transcription factor(s) through multiple sites in gene promoter.
Aim 1 will identify the regions of the mouse BSP promoter that confer cell- and tissue-specific expression. Transgenic mice will be generated using a 16kb BSP promoter and 5' deletion reporter constructs. This will be the first to identify regulatory elements in this longest promoter available to date of the BSP gene.
Aim 2 will determine the molecular mechanisms of tissue-specific regulation of BSP gene expression by Cbfa1. Transgenic mice expressing truncated and mutated BSP promoter will be crossed with Cbfa1-deficient mice to determine Cbfa1 regulation on BSP expression and to characterize the Cbfa1 response elements within the BSP promoter. DNA transfection using Cbfa1-deficient cells will also be performed.
Aim 3 will determine the role of Cbfa1 in controlling bone and tooth formation by regulation of the BSP gene. Using a powerful TVA receptor model, mice harboring an avian retroviral receptor driven by the BSP promoter will be generated in which BSP-expressing cells bearing viral receptor will be targeted with viral vectors carrying various regulatory genes. The effects of Cbfa1 on BSP expression and the associated bone and tooth formation will be determined. Results of these studies should provide novel and important insights into the molecular control of the transcriptional regulation of BSP gene, osteodifferentiation, as well as differentiation associated with the formation of teeth and the regeneration of associated periodontal tissues.
