The osteoblast, in addition to forming bone, plays a central role in bone resorption. Although the precise mechanism by which osteoblasts mediate osteoclastic bone resorption is unclear, one widely held hypothesis is that in response to bone resorbing agents, osteoblasts secrete cytokines which activate osteoclasts or their precursors. While the nature of these cytokines is unknown, one potential candidate is colony stimulating factor-1 (CSF-1). There is substantial in vivo and in vitro evidence that CSF-1 is critically important for both the proliferation and differentiation of osteoclast progenitors: (1) deficiency of CSF-1 in vivo causes osteopetrosis in the op/op mouse; (2) CSF-1 stimulates osteoclast formation and bone resorption in vitro; and (3) CSF-1 is the principal colony stimulating activity secreted by osteoblasts in response to parathyroid hormone (PTH). Although there is mounting evidence for osteoblast-derived CSF-1's role in osteoclast development, little is known regarding the regulation of CSF-1 gene expression in osteoblasts, or the isoforms of CSF-1 which are synthesized and secreted by osteoblasts. In addition, the precise physiologic role of CSF-1 in normal bone remodeling is poorly understood. The applicants have recently demonstrated that tumor necrosis factor (TNF) and PTH increase CSF-1 gene expression and protein release in osteoblasts by a transcriptional mechanism. They plan to pursue these observations by characterizing cis acting elements by which PTH and TNF induce CSF-1 gene expression, and examining potential transcription factors mediating this response, using a combination of deletional analysis and nuclear protein-DNA binding studies. They have also demonstrated by flow cytometry and by PCR analysis of reverse- transcribed RNA that osteoblasts express a cell surface form of CSF-1, which is increased by TNF treatment. They will, therefore, extend this observation by examining the relative expression, regulation and biological significance of the soluble and cell surface forms of CSF-1. For these studies they will characterize the mRNA and protein species of CSF-1 which are synthesized, processed and released by untreated and TNF-treated osteoblasts, and will examine the effects of the soluble and cell-surface forms of CSF-1 on osteoclast formation. Finally, they will examine the role of CSF-1 in bone remodeling in vivo by developing and analyzing a transgenic mouse with targeted over-expression of CSF-1 in osteoblasts. Taken together, these studies should elucidate CSF-1's regulation and role in bone and may improve the understanding of basic mechanisms of bone resorption.
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