The long range goal of this renewal application is to understand the regulation of interstitial collagenase (MMP-1) in the bone remodeling process. Bone remodeling is necessary for growth and fracture healing. In addition, this process serves to maintain calcium homeostasis in the body. MMP-1, a matrix metalloprotease produced by the osteoblast, is thought to initiate the breakdown of bone. Strong evidence has been accumulating that MMP-1 protein is associated with areas in which bone remodeling is occurring. Utilizing the rat clonal cell line UMR 106-01 as an osteoblastic model, it has been determined that secretion of MMP-1 protein is initiated in response to bone remodeling agents such as parathyroid hormone (PTH). PTH induction of MMP-1 secretion in UMR 106-01 cells is mediated mainly by cAMP. This induction is in part transcriptionally regulated. The regulation of MMP-1 transcription occurs through binding of factors to sequences in the upstream region of the gene. CREB protein activates transcription through interaction with sequences in the regulatory region of genes responsive to cAMP. Thus it follows that interaction of CREB with sequences in the regulatory region of MMP-1 is necessary for PTH stimulation of transcription. Therefore, to understand the regulation of MMP-1 by bone remodeling agents, the goals of this project are to: 1) determine the role of CREB in the PTH induction of MMP-1 mRNA, using (a) gel supershift, (b) shift- Western blotting, and (c) immunoprecipitation of phosphoproteins; 2) characterize protein(s) interacting with CREB for induction of MMP-1 mRNA by PTH, by (a) screening an expression library, (b) gel shift and DNase I footprinting, and (c) DNA sequencing; 3) affirm the effects of PTH on MMP-1 mRNA expression in primary rat osteoblasts in culture, through (a) Northern blot analysis, and (b) DNase I footprinting and gel shift; and 4) decipher the role of transcription factors that bind to the AP-2 and PEA-3 consensus sequence sites in the 5' regulatory region of MMP-1 for PTH activation of MMP-1 mRNA by (a) gel supershift, (b) shift-Western blotting, and (c) characterization of proteins. These studies are intended to contribute to our understanding of the regulation of the critical balance between bone resorption and formation in the animal and, thus, lead to therapies in the future to treat metabolic bone disorders, musculoskeletal neoplasms and delays in fracture healing.
