(PTH), a potent stimulator of bone resorption, is the most important regulator of serum calcium concentration in humans. PTH also affects bone formation, being anabolic or catabolic depending on the mode of administration. PTH affects a broad spectrum of genes in osteoblastic cells, but the precise mechanisms by which these genes are regulated are not fully understood. The focus of this application is to identify the molecular mechanisms by which PTH regulates gene expression in bone. Transgenic approaches will be used to demonstrate the function of PTH-responsive elements in vivo. The first goal is to study mechanisms by which PTH regulates expression of the inducible prostaglandin G/H synthase (PGHS-2). A PTH-responsive PGHS-2 promoter element will be identified by deletion mapping in stably transfected MC3T3-E1 cells. Protein-DNA interactions will be identified using electrophoretic mobility shift assays (EMSAs). Transcription factor expression and phosphorylation will be examined. Mechanisms by which PTH attenuates PGHS-2 expression will be examined. Putative PTH response elements will be altered by site-directed mutagenesis; mutant constructs will be transfected into MC3T3-E1 cells and introduced into transgenic mice lines to test functionality. The second goal is to identify DNA sequences downstream of -1683 bp which mediate PTH repression of the COL1A1 promoter in transgenic mice. EMSAs will be performed to identify protein-binding factors in the COL1A1 promoter regions of interest. Once a sequence is found, it will be mutated in the context of a full length COL1A1 promoter and tested in transgenic mice.
The final aim i s to determine the mechanisms by which PTH regulates expression of the cytokine interleukin-6 (IL-6) and the transcription factor NF-IL6 in bone and to delineate the role of NF-IL6 in PTH-mediated gene expression. Transcriptional and post transcriptional regulation will be determined. The effect of PTH on NF-IL6 protein levels will be assessed using Western immunoblotting and metabolic labeling studies. Antisense oligonucleotides and overexpression of wild-type and dominant negative NF-IL6 proteins will be used to examine the role of NF-IL6 in PTH mediated gene expression. These studies will provide fundamental knowledge about PTH-mediated gene expression in bone. If PTH is to be used as an treatment for osteoporosis, these studies will be important for understanding the changes that may occur in bone with long-term PTH therapy.
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