Vitamin D is important for skeletal growth but the mechanism by which it affects this process is poorly understood. We have obtained preliminary in vitro evidence to suggest that the hormonal form of vitamin D, 1.,25 dihydroxyvitamin D3 (1,25(OH)2D3) plays an important role in chondrogenesis. Mesenchymal cells from embryonic chick limb bud can differentiate in vitro to chondrocytes when cultured at high cell density, whereas, low density cultures fail to differentiate. This in vitro phenomenon is perhaps reflective of the prechondrogenic cell condensation that is required for cartilage formation in vivo. We have found that 1,25(OH)2D3 added into low density cultures of chicken limb bud mesenchymal cells induces chondrogenesis, while it promotes this process in the high density cultures as evidenced by morphologic changes including formation of cartilage nodules and the appearance of proteoglycans and by the synthesis of the cartilage specific collagen Type II and core protein mRNAs. The effect of 1,25(OH)2D3 is probably mediated via the receptor protein which is undetectable (by ligand binding studies and immunohistochemistry) in the mesenchymal cells, but it appears to be induced following exposure of the cells to 1,25(OH)2D3. This evidence suggests a previously unappreciated role of 1,25(OH)2D3 in development and might help to explain the so far unknown mechanisms by which vitamin D exerts its potent effects on skeletal growth. We propose here to identify 1,25(OH)2D3-responsive genes necessary for the initiation of cartilage differentiation. To achieve this goal we will construct a cDNA library of 1,25(OH)2D3 treated limb bud mesenchymal cells, and screen with subtracted probes for 1,25(OH)2D3 induced genes involved in the differentiation of these cells to chondrocytes. The spatial and temporal pattern of expression of the positive clones will be studied in relation to vitamin D receptor (VDR) during limb bud development in vivo and during mesenchymal cell differentiation in vitro, by means of in situ and Northern hybridization. In addition, we propose to determine the nucleotide sequences of the selected clones and to examine their function by transfecting them into limb bud mesenchymal cells. We expect that knowledge gained from these studies will be important in understanding skeletal development and perhaps be applicable to mammal and human physiology and pathophysiology.
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