Folate deficiency has been associated with various developmental defects, heart disease, and cancer. However, the molecular basis linking folate deficiency to these health conditions is complex and not fully understood. The long-term goal of this research effort is to address this important question and increase understanding of how a nutritional deficiency in folate alters cell function and impacts human health. The major goals of this proposal are 1) to determine the mechanism by which the expression of the dickkopf-1 (Dkk-1) gene is decreased in folate deficient cells and 2) to determine the extent to which major signaling pathways are altered in folate deficient cells. Our general hypothesis is that folate deficiency is affecting cell signaling pathways, leading to gene expression changes and further modifications to cell function. A validated set of folate-sensitive genes has been generated by microarray analysis comparing folate deficient and sufficient cells. From this data set Dkk-1 was selected for analysis. Dkk-1 is a secreted protein that inhibits the Wnt pathway. Dkk-1 is important in stem cell growth and differentiation, has a critical role during embryonic development, and has been linked to cancer. Hence, understanding how Dkk-1 expression is decreased in folate deficient cells is of obvious significance. Preliminary results have implicated the Ras pathway in the response of some of the folate sensitive genes, including Dkk-1, to folate deficiency. Moreover, a number of the folate sensitive genes are associated with the Wnt pathway. The gene regulatory sequences and transcription factors responsible for the decrease in Dkk-1 gene expression in folate deficient cells and in cells treated with various cell cycle and signaling pathway inhibitors will be defined using a Dkk- 1-luciferase reporter. The basis for any changes in binding activity of the identified transcription factors will then be analyzed. Modifications in MARK and Wnt pathway components will be examined in folate deficient cells using specific kinase inhibitors and antibodies. Results from this proposal are expected to generate a molecular pathway linking folate deficiency to changes in cell signaling and gene expression and to provide support for an alternative mechanism for how folate deficiency is altering cell function and increasing the risk of certain diseases and developmental defects. [unreadable] [unreadable] [unreadable]