Parathyroid hormone (PTH) is an essential regulator of calcium homeostasis and also has a role as an anabolic hormone for bone. PTH induces matrix metalloproteinase-13 (MMP-13) gene transcription in osteoblastic cells through a cAMP-dependent pathway requiring de novo protein synthesis, i.e., this is a secondary event. We have identified the PTH-response elements as being the runt domain and the activator protein-1 binding sites in the MMP-13 promoter. We have demonstrated a PTH-dependent cooperative interaction between the sites and the proteins (Runx2 and Fos/Jun) binding to them. We have now established that these two sites in the MMP-13 promoter are in a single nucleosome close to the TATA box and PTH relaxes and modifies this nucleosome but does not cause its dissociation. The relaxation is the product of several steps which occur as early and late events: In the early events, first, PTH stimulates p300 (a histone acetyltransferase, HAT) activity, causes the phosphorylation of Runx2, and p300 is recruited to the promoter resulting in early site-specific acetylation of histone H4;in the late events, newly synthesized Fos/Jun associate with the promoter and then appear to interact with Runx2 followed by recruitment of CBP (CREB binding protein), another HAT, to the entire proximal promoter and acetylation of histone H3 resulting in loosening of the nucleosome. Maximal transcription of the MMP-13 gene then ensues. Our hypothesis is that the gene is in a repressed state and PTH causes the stepwise modification of the chromatin though dissociation of histone deacetylases and co-repressor proteins and association of HATs and activator proteins. The long-term goals of this work are to delineate the mechanisms conveying PTH action to regulation of transcription of the MMP-13 gene in osteoblasts. Consequently, the specific aims of this competing continuation proposal are to, 1) investigate the early events at the RD site of the MMP-13 promoter, and, 2) investigate the late events at the AP-1 site and the interaction of Fos/Jun with other proteins on the MMP-13 promoter. The results of this work will make major contributions to our knowledge of how PTH exerts its nuclear effects on osteoblast function. In so doing, the data will also provide new perspectives into treatment of disorders of calcium metabolism. This research will investigate how a protein hormone (parathyroid hormone, PTH) is able to interact with a cell's surface and transmit signals to the nucleus to cause the DMAand associated proteins to change their structure and cause expression of an enzyme involved in bone breakdown. PTH is essential for regulating serum calcium levels, and is also being used to treat osteoporosis. The results of our research could lead to new drugs being developed, in place of PTH, to treat osteoporosis.
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