The objectives of this grant application are to determine whether 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) alters bone turnover and osteoblast growth by altering transforming growth factor beta (TGF beta) signaling and immediate early gene expression in bone. 1alpha,25(OH)2D3 and TGF beta play an important and interactive role in bone. It is, however, not known whether changes in the osseous expression of TGF beta in vivo are necessary for the action of 1alpha,25(OH)2D3 in bone, and it is unknown as to how 1alpha,25(OH)2D3 alters TGF beta signaling pathways. The hypotheses of the application are: 1) 1alpha,25(OH)2D3 requires and regulates TGF beta and TGF beta signaling pathways to exert its biological effect in osteoblasts and bone, and 2) 1alpha,25(OH)2D3 and TGF beta effects in osteoblasts are mediated, in part, by changes in the expression of a novel immediate early gene, IEX-1. Our specific aims are: 1) To determine whether 1alpha,25(OH)2D3 requires TGF beta and TGF beta signaling pathways to exert its biological effects in bone. We will determine the responses of mice that express a truncated, dominant negative, TGF beta type II receptor in osteoblasts to 1alpha,25(OH)2D3. 2) To identify the mechanisms by which 1alpha,25(OH)2D3 alters the expression of TGF beta type I and II receptors in osteoblasts. We will determine whether 1alpha,25(OH)2D3 increases cell surface TGF beta type I and II receptors. If transcriptional mechanisms are found to be important in controlling TGF beta type I and II receptor expression, we will identify 1alpha,25(OH)2D3-response elements in the receptor genes. 3) To determine if 1alpha,25(OH)2D3 alters Smad phosphorylation and synthesis in human osteoblasts. We will treat osteoblasts with 1 1alpha,25(OH)2D3 and assess changes in Smad 2 and 3 phosphorylation and Smad 2, 3, 4, and 7 protein and mRNA concentrations. 4) To determine whether the effects of 1alpha,25(OH)2D3 and TGF beta in osteoblasts require regulation of the immediate early gene, IEX-1. We will determine if IEX-1 is regulated by 1alpha,25(OH)2D3 and TGF beta; the mechanisms of IEX-1 regulation will be assessed; and the effects of reduced or enhanced IEX-1 expression on 1alpha,25(OH)2D3 and TGF beta action will be studied. By combining in vivo and in vitro methods, we will be able to address mechanistic questions regarding the dependence of 1alpha,25(OH)2D3 on TGF beta in osteoblasts, and we will be able to elucidate the role of IEX-1 in 1alpha,25(OH)2D3 and TGF beta signaling in osteoblasts. Results of this investigation could lead to a better understanding of the pathogenesis of bone diseases and the formulation of new therapeutic strategies in disorders such as hyperparathyroidism and osteoporosis.
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