Parathyroid hormone is an essential regulator of calcium homeostasis and also has a role as an anabolic hormone for bone. The hormone has multiple actions, including indirect activation of the osteoclast resulting in increased bone resorption, as well as many direct changes in the functions of the osteoblast. The latter involve a switch in the pheontype of the osteoblast from one of matrix synthesis to one of matrix degradation and active participation in the resorption process. An example of this response to PTH occurs in the rat osteoblastic cell line, UMR 106-01, where the hormone induces collagenase-3 gene transcription through a cAMP-dependent pathway requiring de novo protein synthesis. Thus, this is a secondary effect that we previously hypothesized involves the induction and activation of specific transcription factors acting on this gene. We identified the PTH-response elements as being the activator protein-I (AP-1) and the runt domain (RD) binding sites in the collagenase-3 promoter. In addition, we demonstrated a PTH-dependent cooperative interaction between the sites and the proteins binding to them. We also showed that PTH stimulated c-Fos and c-Jun protein abundance but no significant change in the level of Cbfal. These transcription factors are able to interact both in vitro and in vivo. Supporting our earlier work, the PKA pathway was shown to be the only pathway regulating the collagenase-3 promoter as a mediator of PTH action. The importance of this pathway was demonstrated by the fact that PTH stimulates the transactivation of activation domain-3 (AD3) in Cbfal through its PKA site. Thus, PTH regulates both transcription factors through this pathway, either by increasing their expression or altering their phosphorylation. Our hypothesis of the functions of these proteins is that they interact physically in a nucleosomal structure, recruiting other proteins such as coactivators, modifiers of the nucleosome and the general transcription factors. The long-term goals of this work are to delineate the mechanisms conveying PTH action to regulation of transcription of the collagenase-3 gene in osteoblasts. Consequently, the specific aims of this revised competing continuation proposal are to, 1) examine the in vivo phosphorylation of Cbfal following PTH treatment. 2) assess the PTH-regulated interaction of Fos and Jun and Cbfal, and identify the domains involved, 3) identify other PTH-regulated proteins interacting with Fos and Jun and Cbfal, 4) investigate the role of the AP-1 and RD sites and their binding proteins in the structure of the promoter and how PTH affects this. 5) use transgenic animals to verify that Cbfal regulates the collagenase-3 promoter in vivo. The results of this work will continue to contribute 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.
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