Osteoporosis, the debilitating loss of bone, affects over 25 million people in the United States alone. While much recent osteoporosis research has focused on postmenopausal (Type I) osteoporosis, relatively little has focused on age-related (Type II) osteoporosis. To better understand the pathophysiology of osteoporosis, and thereby provide direction for its treatment and prevention, it is necessary to examine the cellular mechanisms responsible for this age-related bone loss. We propose that age-related bone loss may partly be a result of the diminished capacity of bone cells to adapt to changes in the extracellular environment. Our central hypothesis is that bone cell signal transduction is compromised by the aging process and that this results in a decrease in osteoblast sensitivity to hormonal stimuli. Consequently, bone formation rates diminish with age. The objective of this five year FIRST award is to characterize and compare hormonal regulation of signal transduction in osteoblasts isolated from the periosteum of young (4 month), mature (12 month) and old (28 month) rats. These goals will be accomplished through the completion of four specific aims: 1) examination of basal and hormone-stimulated adenylate cyclase activity in osteoblasts isolated from young, mature and old rats; 2) quantification of basal and hormone-stimulated cytosolic calcium mobilization in osteoblasts from young, mature and old rats; 3) evaluation of the effect of direct activators of adenylate cyclase (e.g. forskolin), or G-protein mediated activators of adenylate cyclase (e.g. cholera toxin), on adenylate cyclase activity in osteoblasts isolated from young, mature and old rats; 4) comparison of the hormonal regulation of calcium currents in osteoblasts isolated from young, mature and old rats. Osteoblasts will be isolated using collagenase digestion and the osteoblast phenotype monitored by examining akaline phosphatase activity, parathyroid hormone-stimulated adenylate cyclase activity and expression of MRNA for bone matrix proteins. Adenylate cyclase activity will be determined by measuring CAMP accumulation while cytosolic calcium dynamics monitored utilizing the photoprotein aequorin and fluctuation analysis of the calcium signal. The whole cell variant of patch clamp technique will be used to characterize calcium currents. While age-related changes in signal transduction have been reported of several cell types, we are unaware of reports of such changes in bone cells. The result of this project will not only provide new insight into the pathogenesis of age-related bone loss, but will also provide a unique perspective toward treatment prophylaxis.
