The biological actions of 1,25(OH)2 D3, the hormonal form of vitamin D, are mediated via the vitamin D receptor (VDR), a member of the large steroid receptor superfamily. VDR, which acts as a transcriptional factor to activate the expression of target genes, is found in many cell types and tissues that are not involved in calcium metabolism, in addition to being present in bone cells. In hematolymphopoietic tissue VDR is expressed in lymphocytes and monocytes/macrophages. In vitro, 1,25(OH)2 D3 can modulate cell growth and differentiation of these cell lineages and it is also recognized as an immunomodulator of macrophage and lymphocyte activities. Clinically, Vitamin D deficiency has been associated with defects in cell-mediated immunity and susceptibility to infections. However, it is unclear if VDR activation is essential for normal immune system development and function in vivo. The research proposed will determine the role of VDR in lymphocyte and monocyte differentiation and activation. Based on preliminary in vitro findings, we propose the following hypothesis: VDR is involved in immune and inflammatory responses by regulating differentiation and function of T lymphocytes and monocytes/macrophages. The specific aims of the proposed research are to: 1) generate VDR-deficient embryonic stem (ES) cells, and determine in vitro whether these cells can differentiate to lymphocytes, monocytes/macrophages and osteoclasts, given the appropriate conditions; 2) generate VDR knockout mice by germ line transmission and establish the phenotype in regard to immune and hematopoietic system development and function; and 3) generate transgenic mice expressing VDR in the thymus, to establish if VDR expression affects the development, differentiation and function of T lymphocytes. Preliminary studies performed include: a) isolation and interim characterization of the gene encoding the murine VDR from an embryonic stem cell library; b) construction of a targeting vector for the murine VDR gene: c) destruction of the VDR gene in ES cells; d) demonstration of VDR transcripts in ES cells; and e) evidence that 1,25(OH)2 D3 can induce differentiation of ES cells to osteoclast-like cells. We believe that the new studies proposed will elucidate the biological significance of VDR in vivo, specifically in regard to the role of VDR in lymphohematopoiesis.