RANK- NFATc1 signaling forms positive feedback loop on rank gene expression via functional NFATc1 responsive element in rank gene promoter

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, partly through the direct binding of NFATc1, to target gene promoters. We have previously cloned a 6-kb fragment containing the 5'-flanking region of the mouse RANK gene and have demonstrated the presence of binding elements of hematological transcription factors, such as MITF, PU.1 and AP-1. Here, we demonstrated the presence of the functional NFATc1 responsive element on the RANK gene promoter. Transfection of an NFATc1-expression vector increased RANK mRNA that was subsequently nullified by NFATc1 knockdown. With the use of electrophoretic mobility shift assay (EMSA), an oligonucleotide (-388/-353) showed specific protein-DNA binding that was blockshifted with an anti-NFATc1 antibody and washed out with excess amounts of the cold consensus sequence. Co-transfection studies with the use of an NFATc1-expression vector and RANK promoter-reporter constructs showed that NFATc1 increased promoter activity 2-fold in RAW264.7 cells that was again nullified as disclosed by mutagenesis studies. Taken together, these results indicate that RANK transcription is positively regulated by the RANKL signal through the direct binding of NFATc1 to its specific binding site of the RANK gene promoter, and suggest the presence of a crucial positive feedback mechanism of gene expression that promotes accelerated terminal differentiation of RANK-positive committed precursors to mature osteoclasts.