application) Osteocytes comprise over 90% of all bone cells, yet little is known of their function(s) or of the involvement of systemic hormones in regulating their activity. These cells occupy lacunae deep within the mineralized matrix of bone and communicate with one another and with osteoblasts via gap junctions located at the ends of long cytoplasmic processes that course through tunnels (cannalicula) in the bone. Current evidence suggests that osteocytes play a major """"""""mechanosensory"""""""" role, whereby they are stimulated by sheer and stretch forces to produce local mediators such as NO and PGE2 and to increase expression of c-fos and IGF-1. Parathyroid hormone (PTH) is known to influence the output of this mechanosensory response and to delay osteocyte apoptosis, both of which may alter bone turnover. Carboxyl(C)-fragments of PTH accumulate in blood during renal failure, which is associated with severe bone disease, and considerable evidence now points to the presence on bone cells of novel receptors with selectivity for C-terminal portions of intact PTH(1-84) ( """"""""CPTHRs""""""""). In preliminary studies with genetically altered, conditionally transformed clonal bone cell lines (osteoblasts, osteocytes, chondrocytes and marrow stromal cells) lacking endogenous PTH/PTHrP receptors (""""""""PTH1Rs""""""""), it has been shown that intact hPTH(1-84) and certain PTH C-fragments can displace binding of a CPTHR-specific radioligand and can elicit numerous biological responses, including effects upon cell proliferation, c-fos mRNA expression, 45Ca influx, cytosolic free Ca++ and protein kinase C, in the absence of PTH1Rs. Among such clonal bone-derived cell lines genetically the highest levels of CPTHR s were found on osteocytes. This project will address the ligand recognition, signal transduction and cellular responses (including gene regulation) of CPTHRs in clonal, conditionally transformed murine osteocytes that lack endogenous PTH1R expression and therefore can respond to intact PTH(l-84) only via CPTHRs. Biological and gene-regulatory responses to PTH1R activation, with and without mechanical loading, then will be assessed after introduction of recombinant PTH1Rs into these osteocytes by stable transfection of PTH1R cDNA. Possible functional interactions of CPTHRs and PTH1Rs within osteocytes will be sought. These studies will provide new understanding of osteocyte function and of the role of PTH, acting through two distinct receptor systems, in regulating osteocyte behavior. These results could have significant implications for therapy of bone disorders such as hyperparathyroidism, osteoporosis and renal osteodystrophy.
