| Abstract |
Receptor activator of NF-κB (RANK) expressed on osteoclasts and their precursors is a receptor for RANK ligand (RANKL). Signals transduced by RANKL-RANK interaction induce genes essential for the differentiation and function of osteoclasts. We have cloned a basic promoter region of the mouse RANK gene and have analyzed the transcription machinery by transcription factors such as PU.1 (-480), and MITF (-100). Here, we examined the regulatory mechanisms of RANK gene transcription through AP-1 binding site, agagctca (-240). RANK mRNA expression in pre-osteoclastic RAW264.7 cells was induced by Phorbol12-myristate13-acetate (PMA) and suppressed by protein kinase C (PKC) inhibitor calphostin C. In RAW264.7 cells, Fos knockdown by siRNA blocked the inducible effect of PMA on RANK expression. By EMSA, an oligonucleotide (-246/-238) showed DNA protein binding, the specificity of which was confirmed by block-shift assay with an anti-Fos antibody and by the addition of the excess of a cold consensus probe. Co-transfection with a Fos expression vector showed that Fos increased RANK promoter activity 6-fold in RAW264.7 cells, and the addition of PU.1 and MITF superinduced the activity more than twenty-fold by the addition of PU.1 and MITF. Mutagenesis of the putative AP-1 site (-240) blocked the inducible effect of Fos on promoter activity. Taken together, these results indicate that during the differentiation of bone marrow mono-nucleated cells into osteoclast precursors, RANK transcription is positively regulated by Fos/AP-1 through the binding element of its gene promoter, supporting the concept that Fos activation by continuous CSF-1 stimulation on macrophages triggers initial expression of RANK and, later, a positive feedback loop by RANKL-RANK interaction.
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