The long-term objective of this research is to improve therapy for bone diseases through an understanding of the signaling pathways that lead to bone formation and bone loss. Previous studies have provided evidence that parathyroid hormone (PTH) activation of phospholipid/protein kinase C (PKC) signaling leads to increased bone resorption. The studies in the current proposal would elucidate the mechanisms of these pro-resorptive effects of PTH on phospholipid metabolism and protein kinase C in osteoblasts. The model is based on the following hypotheses: 1) PTH activates phospholipase D (PLD) through a signaling pathway that involves translocation of the small G proteins Rho A and Arf and increased intracellular calcium; 2) the diacylglycerols (DAGs) produced by the PLD action, together with the increased intracellular calcium, lead to activation of specific PKC isozymes, especially PKC-beta; 3) the downstream consequences are increased expression of interleukin-6 (IL-6) and osteoclast differentiating factor (ODF), and subsequent inhibition of osteoblast apoptosis and the promotion of osteoclastogenesis and bone resorption. Preliminary studies have demonstrated the presence of all of these proposed signaling components in the osteoblastic cell model to be used, and have shown PTH responsiveness by PLD, Rho A, Arf, PKC-alpha and -beta, IL-6 and ODF in these cells. The model will be tested by determining the effects of the small G-proteins and calcium on PLD activity both in vivo and in vitro and by determining their effects and those of the DAGs on PKC isozyme translocation and downstream responses. The studies will demonstrate whether antagonizing the actions of the small G-proteins with mutants and specific inhibitors disrupts the sequence of events and prevents the downstream responses. In addition, the impact on this pathway of PTH analogs that selectively or preferentially affect other signaling pathways will be determined. The findings will provide new understanding of parathyroid hormone signaling and actions in bone.
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