The object of this proposal is to obtain a better understanding of the actions of vitamin D, a principal factor required for the development and maintenance of bone, as well as for maintenance of calcium homeostasis. This renewal application proposes in Specific Aim I to continue studies related to the functional significance of calbindin, the first known target of vitamin D action. Studies in the previous grant period provided the first evidence that calbindin-D28k protects against TNF-induced cell death in osteoblasts and that calbindin protects against cell death by binding to and inhibiting caspase 3. These findings, which have important therapeutic implications, will be continued. We will determine whether calbindin-D28k can protect against glucocorticoid, as well as TNF-induced cell death in bone cells. We will determine which region of calbindin binds to caspase 3, and whether calbindin can inhibit other caspases in the distal portion of the cell death pathway. In addition, we will determine whether calbindin can also act by affecting other calcium regulatory proteins, including the recently identified apical calcium channel, ECaC, which is colocalized in the same cells as calbindin. Besides calbindin, the other known pronounced effect of 1,25(OH)2D3 in intestine and kidney is increased synthesis of 24(OH)ase. In Specific Aim 2, in order to obtain further insight into the molecular mechanism of 1,25(OH)2D3 action, studies begun in the last grant period related to the regulation of 24(OH)ase will be continued. In preliminary studies, we have noted that CCAATT enhancer binding protein beta (C/EBPbeta) is induced by 1,25(OH)2D3 in kidney and osteoblastic cells and can enhance the transcriptional response of 24(OH)ase to 1,25(OH)2D3. Studies are proposed to examine the mechanisms involved in modulation of VDR-mediated 24(OH)ase transcription by C/EBPbeta, as well as the role of C/EBPbeta in the cross-talk we have observed between the PKA signaling pathway and 1,25(OH)2D3. These studies would establish C/EBPbeta as a novel 1,25(OH)2D3 target gene and would indicate for the first time a role for C/EBPbeta in 24(OH)ase transcription. The proposed studies complement the studies begun in the previous grant period related to the factors that affect 24(OH)ase transcription (YY1, TFIIB, DRIP205, CBP) and the effect of signaling pathways on 24(OH)ase transcription and cofactor recruitment. This proposal, which combines studies related to an understanding of the functional significance of target proteins, with studies related to the molecular mechanism of 1,25(OH)2D3 action, will provide new insight into the mechanisms by which vitamin D mediates its biological effects and how aberrant regulation may be involved in diseases such as osteoporosis.