The osteoblast or an osteoblast-like cell is the principal to which parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) bind in bone. We hypothesize that osteoblast-derived factors are therefore critically important local mediators of PTH-and PTHrP-induced bone resorption. A corollary to this hypothesis is that differences in the actions of PTH and PTHrP on osteoblasts may help explain the different in vivo effects of these two hormones. To pursue this hypothesis, the effects of these two agonists on osteoblasts-derived factors which may 1) activate mature osteoclasts or 2) stimulate recruitment of pre-osteoclasts will be examined. We have observed that PTH and PTHrP-treated osteoblasts release a soluble bone-resorbing activity. This activity has been partially purified and can directly stimulate resorption by isolated osteoclasts (under conditions where PTHrP is inactive). This material will be purified to homogeneity to obtain primary structural information. Amino-acid sequence information will be used to synthesize oligonucleotide probes for screening an Saos-2cDNA library. Based on our previous work, demonstrating PTH-induce GM-CSF release from osteoblasts, this molecule will be studied as an example of an osteoblast-derived factor which may regulate osteoclast recruitment. The molecular mechanisms underlying PTH/PTHrP-stimulated release of GM-CSF from osteoclast will be examining the effects of neutralizing antisera to GM-CSF on PTH/PTHrP-induced resorption in the fetal rat long-bone and mouse metatarsal assays. We have very recently identified CSF-1 receptors (c-fms) in mature rat osteoclasts. Further, in preliminary studies we have identified down- regulation of this receptor by GM-CSF suggesting an important interaction between these two colony-stimulating factors in regulating osteoclast biology. These observations will be pursued using immunocytochemical techniques and in situ hybridization. Specifically, we will study whether the effect of GM-CSF on c-fms occurs at the level of transcription or translation. Finally, a mammalian expression system employing rat insulinoma cells has been developed using the three available cDNA clones for PTHrP. The effects of amino-terminal purified recombinant PTHrP on signal transduction mechanisms in, and release of resorptive cytokines from, osteoblasts will be studied. In the aggregate, these studies should allow a more precise picture of the cellular mechanisms by which PTH and PTHrP induce resorption in bone and should lead to a clearer picture of how these compounds act under physiologic conditions as well as those of disturbed mineral homeostasis.
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