The primary goal of the studies in the current proposal is to determine the functions of the Galpha12/13- RhoA/Rhq kinase-phospholipase D signaling pathway in the actions of parathyroid hormone (PTH) in osteoblasjis. Our studies during the previous funding period have characterized the PTH regulation of this pathway in UMR-106 osteoblastic cells, and have established that it mediates PTH-stimulated translocation of protein kinase C alpha and contributes to activation of interleukin-6. In other tissues, the pathway plays a prominent role in survival signaling and in cell adhesion, spreading, and cell-cell interactions. Effects of PTH to promote cell survival are likely to be critical for the anabolic effects of PTH on bone, and cell-matrix and cell-ce|j. interactions are likely to be important in bone remodeling. Therefore, the first specific aim is to examjne the role of the pathway in PTH actions on cell survival and survival signaling, including effects on MAP kinase, Akt, Runx-2 and insulin-like growth factor -1, on anabolic effects in bone organ culture, and on cytoskeleton changes in osteoblasts including stimulation of the formation of actin stress fibers and focal adhesions, antagonism of the rapid cAMP-mediated contraction and stimulation of expression of proteins critical to cytoskeletal events, i.e. focal adhesion kinase, paxillin, ICAM-1 and connexin-43. A second specific aim derives from the fact that statins and aminobisphosphonates, important bone regulating drugs, could affect the pathway through their effects to inhibit the generation of isoprenyl groups. This proposed mechanism will be tested, as will the possibility that these drugs affect osteoblast activity through their actions on the pathway. A third specific aim is to define the mechanisms of interactions within the signaling pathway, including the interactions between G12/G13 alpha subunits of heterotrimeric G proteins and RhoA and the regulation of the pathway by calcium. We will be determined whether the regulation of the pathway is similar in primary osteoblasts to what was observed in UMR-106 cells. We will determine the role of phosphatidic acid, the immediate downstream product of PLD activity, in PTH actions. The findings from the research will provide a more comprehensive understanding of the role of this newly recognized signaling pathway in osteoblasts and will test the hypothesis that it is involved in effects that are critical for the anabolic actions of PTH and that mediate PTH interactions with statins and bisphosphonates on bone.
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