Our overall goal is to understand the basic mechanisms regulating endochondral ossification. this project examines how bone-derived factors influence cell differentiation and cell function.
The aims address the mechanisms of transforming growth factor beta1 (TGFbeta) action in endochondral bone formation by focusing on the interrelationship of TGFbeta and 1,25-(OH)2D3 and 24,25-(OH)2D3. We will use a well-characterized model which compares chondrocytes derived from the resting zone and growth zone of rat costochondral cartilage to test the following hypotheses: (1) Response to TGFbeta differs as a function of chondrocyte maturation; (2) synthesis and activation of TGFbeta differs as a function of chondrocyte maturation; (3) Some of the effects of TGFbeta are due to TGFbeta-dependent production of 1,25-(OH)2D3 and 24,25-(OH)2D3; (4) Matrix vesicle enzymes are involved in the activation of TGFbeta in the extracellular matrix; and (5) There is a synergistic interaction of TGFbeta and vitamin D metabolites. To test these hypotheses, we propose the following specific aims.
Aim I. To characterize regulation of active TGFbeta production and its binding protein by vitamin D metabolites and TGFbeta. The effects of 1,25- (OH)2D3, 24,25-(OH)2D3, and TGFbeta, itself, on TGFbeta mRNA levels, TGFbeta-binding protein mRNA levels, latent TGFbeta and active TGFbeta will be quantitated as a function of dose and time.
Aim II. To characterize TGFbeta-dependent metabolism of 25-OH-D3. The dose- dependence and time course of 1,25-(OH)2D3 and 24,25-(OH)2D3 synthesis from labeled 25-OH-D3 by RC and GC chondrocytes stimulated with TGFbeta will be measured. Regulation of the hydroxylase system will be examined.
Aim III. To examine the role of matrix vesicles in activation of latent TGFbeta. We will examine (1) whether matrix vesicles contain an enzyme which can convert TGFbeta from its latent to its active form; (2) whether chondrocytes can regulate the activity of matrix processing enzymes and the TGFbeta activating enzyme by the production of activators (e.g., plasminogen activator) or inhibitors (e.g., plasminogen activator inhibitor or TIMP); (3) whether these events are regulated by TGFbeta or vitamin D metabolites; and (4) whether both genomic and nongenomic control is involved.
Aim I V. To characterize relationship between TGFbeta and vitamin D metabolites. We will characterize the relative contributions of TGFbeta and the vitamin D metabolites in terms of arachidonic acid turnover, prostaglandin production, and protein kinase C activity with respect to dose and time. These studies will provide new information concerning the interaction of steroid hormones and the growth factors in genomic and nongenomic regulation of cell function and new insights into the roles these factors may play in bone development and repair.
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