Extensive evidence suggests that osteoblastic lineage cells are required for osteoclast formation and that balanced bone remodeling is achieved, in part, by coupling the generation of these two cell types. Expression of receptor activator of NFkappaB is the key mechanism by which stromal/osteoblastic cells support osteoclastogenesis. RANKL is required for osteoclast formation in vitro and in vivo, and with M-CSF, is sufficient for osteoclast formation. Cytokines and hormones that stimulate osteoclast formation do so by stimulation RANKL in stromal/osteoblastic cells. However, the mechanisms regulating RANKL expression in these cells remain unknown. It is also unknown whether osteoclast supporting (or RANKL-expressing) cells or their progeny are ultimately involved in bone formation. The ultimate goals of this application are to elucidate the molecular mechanisms that link osteoclast and osteoblast formation and to determine whether osteoclast-supporting cells represent a precursor of matrix-synthesizing osteoblasts. In preliminary studies, the murine and human RANKL promoters were isolated, and the role of the osteoblast-specific transcription factor Cbfa1 in their regulation was determined. In addition, it was shown that IL-6 type cytokines and IL-1, but not 1,25 (OH)2D3 or PTH, utilize, the gp130/STAT3 pathway to induce RANKL expression. Further, the STAT3 and VDR transcription factors were shown to RANKL expression via an intermediate gene or genes. In the proposed studies, cis-acting sequences that regulate RANKL gene activity will be identified by creating receptor constructs that mimic the endogenous RANKL gene in cell lines and transgenic mice. The protein or proteins, stimulate by gp130 activation, that induce RANKL will be identified via an expression cloning strategy utilizing retroviral expression techniques. Finally, it will be determined whether osteoclast-supporting (or RANKL-expressing) cells progress to become matrix-synthesizing osteoblasts by inserting a Cre recombinase cDNA into the RANKL locus via homologous recombination in embryonic stem cells. The resulting mice will be crossed with mice harboring a Cre-responsive lacZ reporter gene such that lacZ will permanently mark these cells and their progeny.
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