The goals of this application are to study the effect of growth factors on chondrocyte maturation. Growth plate chondrocytes will be compared with articular chondrocytes. The cells will be obtained by sequential enzymatic digestion of chick tissues and will be maintained in short term cultures. Countercurrent centrifugal elutriation will be used to produce populations of growth plate chondrocytes in differing stages of maturation. The effects, production, and regulatory interactions of a mitogenic autocrine factor (PTH/PTHrP), and a group of differentiative factors (bone morphogenetic proteins, BMPs) will be evaluated. These two classes of molecules are unique in the sense that PTH/PTHrP is the most potent growth plate chondrocyte mitogen we have ever investigated, and one of the few growth factors which has effects on chondrocytes derived from the growth plate but not on articular cells. The BMPs (especially BMP7) are the only protein growth factors known to induce differentiation in growth plate chondrocytes. Thus, we will be working with two prototype regulators with opposing actions: one responsible for expanding cell number and the other responsible for controlling differentiation. With such a system we intend to investigate some of the developmental signals that control the chondrocyte phenotype pathways.
The specific aims i nvolve (l) demonstration of autocrine production of PTHrP and expression of its receptors by growth plate chondrocytes with specific localization within the zones of the growth plate, (2) study of the differentiative effects of BMPs, with particular focus on BMP7, on growth plate and articular chondrocytes, with evaluation of receptor expression, autocrine production, and the possibility that BMP7 can confer a calcifying (growth plate-like) phenotype on articular cells and (3) differential screening of gene expression between articular cells and growth plate cells, with identification and characterization of some novel differentially expressed genes, one of which we have recently cloned and sequenced. Cloning of the chick PTH receptor will be another aspect of the project, along with production of fusion proteins for production of antibodies for this protein as well as PTHrP and BMP7. In addition the functional role of PTHrP in chondrocytes well be evaluated by inhibition of expression of the growth factor or the receptor using several strategies.
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