The objective of this competitive continuation is to examine the role of 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) and the 1alpha,25-dihydroxyvitamin D3 receptor (VDR) in bone development. The hypothesis of this proposal is that 1,25(OH)2D3, via its receptor, plays an important role in bone formation by directly influencing the expression of osteoinductive growth factors, and early and late response genes in osteoblasts and osteoblast precursors. The application's Specific Aims are to: 1) examine in vivo, in the vitamin D-deficient and the vitamin D-replete state, the time and pattern of expression of the VDR, osteoinductive growth factors and the vitamin D-dependent proteins in limb, vertebral and calvarial tissues from fetal, neonatal, young and adult rats; and to correlate these patterns with bone development. Morphological and histomorphometric patterns occurring as a result of vitamin D deficiency will be compared with those found in the vitamin D-sufficient state; 2) examine the ontogeny of the VDR, osteoinductive growth factors, and vitamin D-dependent proteins in rat demineralized allogeneic bone matrix implanted into vitamin D-deficient or vitamin D-replete rats. The influence of 1,25(OH)2D3 on the expression of osteoinductive growth factors will be assessed in the implanted matrix following the infusion of 1,25(OH)2D3. The role of the VDR will be assessed by blocking the expression of the receptor in the implanted matrix with anti-sense oligonucleotides; 3) examine the mechanism by which 1,25(OH)2D3 influences the development of osteoblasts and early osteoblast precursor cells. Planned experimentation will examine the effect of 1,25(OH)2D3 on fetal human osteoblast cells with respect to the expression of osteoinductive growth factors, the expression of """"""""early response genes,"""""""" the induction of novel """"""""late response genes"""""""" and the development of a mature osteoblast phenotype. VDR expression will be blocked with antisense oligonucleotides in fetal human osteoblast cells and the functional consequences of receptor deficiency determined. Methods to be used include cell culture, immunohistology, in situ hybridization, subtractive hybridization and differential display PCR techniques. The applicant suggests that these studies will provide an understanding of how 1,25(OH)2D3 and its receptor affect the development of bone. Because peak bone mass is an important variable in determining the risk for osteoporosis in later life, an appreciation of the factors affecting the formation of bone could potentially lead to the development of strategies to increase bone mass. These findings would also be applicable to fracture healing, to post-transplantation bone disease, and renal osteodystrophy.
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