OSTEOBLAST MEDIATED TURNOVER OF COLLAGENASE IN BONE

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DESCRIPTION (Adapted from the Applicant's Abstract): In some inherited disorders of the skeleton, including osteopetrosis and osteosclerosis, excess bone accumulates whereas in osteoporosis bone loss significantly exceeds bone gain. Historically, bone formation was associated with the osteoblast and resorption with the osteoclast. However, data has accumulated which indicates that the osteoblast takes an active role in the resorption process. This cell has been shown to be the primary target for a variety of resorption promoting agents, and to be capable of producing neutral proteinases, such as plasminogen activator and collagenase (MMP-1). The applicant has shown that PTH stimulates secretion of the latter enzyme by the rat osteoblastic cell line, UMR, with maximal concentrations of MMP1 appearing in the extracellular medium 12-24 h after addition of the hormone. These subsequently decline, becoming almost undetectable by 96 h. A cell-mediated binding mechanism was suggested by the rapid and saturable removal of exogenous purified rat MMP-l at 37 C. Binding studies, using 125I-MMP-l at 4 C, indicated a saturable receptor of a single class and 12000 receptors/cell. A time course revealed specific receptor-mediated binding within 10 min, and equilibrium by 60 min, while dissociation experiments demonstrated reversibility. The receptor was shown to be specific for rat MMP-l since a host of related and unrelated proteins failed to compete for binding. Internalization studies revealed maximal intracellular accumulation at 30 min and complete degradation of 125I- MMP-l by 90 min, suggesting this receptor functions in the UMR osteoblastic cells to eliminate extracellular MMP-l. Treatment of UMR cells with PTH causes a downregulation of the binding activity after 24 h, with a later rebound to twice the control binding levels. This is paralleled by coordinate changes in the rate of internalization and degradation of the ligand in PTH-treated cells. Thus, the pattern of receptor abundance and ligand degradation in PTH-treated cells correlates inversely with our observations of the extracellular concentrations of MMP-l suggesting the receptor dictates the abundance and function of the enzyme in the extracellular milieu. Thus, the aims of this revised competing continuation proposal are to further characterize and identify the receptor. This will be accomplished by: 1) determining the biochemical properties of the receptor, by 125I- labelling the receptor and purifying it on a affinity column; 2) using this purified material to obtain peptide sequence data; 3) cloning the receptor; 4) using the clone to express and examine regulation of the receptor; and 5) mutation of the receptor to determine its functional domains. The results of this work should aid in dissection of a cell- mediated pathway for regulation of elaborated MMP-l in osteoblasts and other cells. In so doing, the data should also provide some insight into those many skeletal disorders where bone remodelling (aberrant MMP1 expression/uptake) has gone awry.