The overall objective of this research proposal is to establish the functional significance of dual signal transduction initiated by occupancy of the parathyroid hormone receptor (PTHR) in bone cells. A single type of cloned PTHR can couple to both the cAMP and inositol lipid pathways. While many of the physiological actions of PTH have been shown to be mediated solely or primarily by cAMP, the functional role of phosphoinositide-derived second messengers is unclear, especially in bone cells. Nevertheless, we and others have identified certain cAMP-independent actions of PTH in osteoblastic cells. The mediators of these cAMP-independent actions are not known. In the new experiments proposed two hypotheses will be tested: 1. certain specific physiological actions of PTH on human osteoblasts, which are independent of protein kinase A activation, are mediated by products of the inositol lipid pathway; and 2. the PTHR, which does not couple to the phosphoinositide pathway in certain human osteoblast-like cells (e.g., SaOS-2 cells), can couple biochemically and functionally to this transduction system in appropriate heterologous systems, demonstrating that specific cellular factors distal to the receptor play crucial roles in signal transduction via the PTHR.
Two specific aims are proposed. 1. Mechanistic studies on cytosolic Ca2+ homeostasis in osteoblastic cells. Investigations will focus on: feedback regulation between the cAMP and inositol lipid pathways; specific G proteins; PTH-regulated phospholipase C; intracellular Ca2+ sequestration and release compartments; and heterologous expression of the PTHR in GH4C1 rat pituitary cells and Xenopus oocytes. 2. Functional studies on the role of the inositol lipid transduction pathway in osteoblasts. These experiments are designed to determine whether certain physiological responses to PTH in osteoblasts are or can be regulated via products of phosphoinositide hydrolysis. Results of the experiments proposed will answer two sharply focused questions: 1. why does the PTHR, which can couple to two transduction pathways, frequently not do so in osteoblasts; and 2. are certain important physiological actions of PTH on osteoblasts mediated by the inositol lipid pathway? Because PTH has both catabolic and anabolic actions, understanding the pathways regulating these opposing effects will offer the opportunity to develop new mechanism-based approaches to control excessive skeletal catabolism or deficient bone formation in such important clinical disorders as osteoporosis, hyperparathyroidism, and in some of the hypercalcemias of malignancy.
