This research will examine the basic mechanisms underlying the role of transforming growth factor beta-1 (TGFb) in endochondral bone formation by focusing on the interrelationship between TGFb and 1,25(OH)2D3 (1,25D) and 24,25(OH)D3 (24,25D), using a well-characterized cell culture model that allows comparison of cells at two distinct stages of maturation: resting zone (RC) and growth zone (GC) chondrocytes. The following hypotheses will be addressed: TGFb production and activation are regulated by vitamin D metabolites via genomic and non-genomic mechanisms; matrix vesicle enzymes activate TGFb in the extracellular matrix; TGFb regulates production of 1,25D and 24,25D in a cell maturation-dependent manner and some of TGFb's effects are due to TGFb-dependent production of these hormones; TGFb-dependent regulation of chondrocyte maturation is mediated by protein kinase C (PKC); and, TGFb regulates endochondral development via signaling pathways that are distinct from those used by 1,25D and 24,25D. To test these hypotheses, the following Specific Aims will be pursued: (1) to characterize the regulation of active TGFb production by 1,25D and 24,25D. The investigators will determine if 1, 25D and 24,25D regulate TGFb production by modulating LTGFb gene expression, protein synthesis, or activation, or by changing LTGFb binding protein (LTBP) mRNA levels, protein synthesis, or incorporation into the extracellular matrix. Whether vitamin D metabolites act via traditional vitamin D receptors (VDR) or rapid membrane-mediated effects will be assessed using specific analogues of vitamin D with low affinity for the VDR. The mechanisms by which matrix vesicles activate LTGFb will be assessed by determining which enzymes are responsible, and whether they are regulated by vitamin D metabolites through genomic or non-genomic pathways. Once the LTGFb activator is identified, they will initiate experiments to purify and sequence the protein and clone its gene; (2) to characterize the extrarenal regulation of vitamin D metabolite production by TGFb. The applicants will examine the direct regulation of 25D hydroxylation by measuring both 1alpha- and 24- hydroxylase mRNA levels and activity in the TGFb-treated RC chondrocytes. Whether TGFb exerts its effects via regulation of the hydroxylase co-factor, cytochrome P450, will be determined using specific inhibitors. The investigators will also examine whether the effects of TGFb are mediated through TGFb receptor binding, PKC and the MAP kinase pathway; (3) to determine the mechanisms by which TGFb acts synergistically with 24,25D to regulate chondrocyte maturation. The applicants will characterize the mechanisms by which TGFb exerts its effects on RC chondrocytes by measuring alkaline phosphatase mRNA production and activity and the acquisition of a 1,25D-responsive phenotype characteristic of the more mature growth zone cells. How these TGFb-mediated effects are modulated by 24,25D will be determined, including changes in TGFb type I and type II receptors. It is suggested that these results will provide insight into the role these factors play in bone development and repair.
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