Abstract
Osteoclasts are large multinucleated, bone-resorbing cells derived from hematopoietic precursors in response to receptor activator of nuclear factor-kappaB ligand (RANKL). RANKL activates a number of signal transduction pathways, which stimulate, in turn, a series of specific transcription factors that initiate the process of osteoclastogenesis. Perhaps the most important of these is nuclear factor of activated T cells cytoplasmic 1 (NFATc1), a DNA-binding protein that upon activation translocates to the nucleus where it stimulates transcription. The objective of this study was to explore the process whereby RANKL induces NFATc1 and to assess the role of this factor in the activation of an additional key osteoclast target gene. We found that whereas several NFAT members are expressed in RAW264.7 cells, soluble RANKL-induced up-regulation is limited to NFATc1 through a mechanism that is largely autoregulatory. Thus, although we observed the presence of resident NFAT members at the inducible Nfatc1 P1 promoter at very early times after RANKL treatment, a selective and time-dependent increase in the binding of up-regulated NFATc1 to Nfatc1 was observed beginning at 12 h. Several additional factors that are activated by soluble RANKL and also participate in NFATc1 up-regulation include c-Fos and RNA polymerase II. Chromatin immunoprecipitation analysis also revealed a similar, time-dependent accumulation of NFATc1 at multiple sites on the Acp5 promoter, thereby highlighting a central contributing role for NFATc1 in the activation of this gene as well. Our studies provide additional molecular detail regarding the mechanisms through which RANKL induces NFATc1 in osteoclast precursors and into mechanisms by which NFATc1 induces the expression of at least one gene responsible for the osteoclast phenotype.
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