Involvement of upstream stimulatory factors 1 and 2 in RANKL-induced transcription of tartrate-resistant acid phosphatase gene during osteoclast differentiation

Tartrate-resistant acid phosphatase ( TRAP) plays an important role in bone resorption. TRAP expression in osteoclasts is regulated by receptor activator of NF-kappaB ( RANKL), a potent activator of osteoclast differentiation. However, the molecular mechanism underlying the RANKL-induced TRAP expression remains unknown. Here we show that two regions in the mouse TRAP promoter (one at - 1858 to - 1239 and the other at - 1239 to - 1039, relative to the translation start site) are implicated in RANKL- induced TRAP transcription in RAW264.7 cells. A detailed characterization of the region at - 1239 to - 1039 identifies a 12-bp sequence, AGC-CACGTGGTG, that specifically binds nuclear proteins from RAW264.7 cells and primary bone marrow macrophages (BMMs) in an electrophoretic mobility shift assay ( EMSA). Moreover, the binding is significantly enhanced in EMSA with nuclear extracts from RANKL-treated RAW264.7 cells and BMMs, suggesting that the 12-bp sequence may be involved in RANKL- induced TRAP transcription. Various assays reveal that nuclear proteins binding to the 12-bp sequence are upstream stimulatory factors (USF) 1 and 2. Importantly, mutation of the USF-binding site partially blocks RANKL-induced TRAP transcription in RAW264.7 cells, confirming that USF1 and USF2 are functionally involved in RANKL- induced TRAP transcription. In summary, our data show that USF1 and USF2 play a functional role in RANKL- dependent TRAP expression during osteoclast differentiation.