Regulation of receptor activator of NF-kappaB ligand-induced tartrate-resistant acid phosphatase gene expression by PU.1-interacting protein/interferon regulatory factor-4. Synergism with microphthalmia transcription factor.

ABSTRACT

The receptor activator of NF-kappaB ligand induces the expression of tartrate-resistant acid phosphatase (TRAP) and transcription factor, PU.1-interacting protein (Pip), during osteoclastogenesis. In this paper, we have examined the role of transcription factors in the regulation of TRAP gene expression employing reporter constructs containing the promoter region of TRAP gene. Transient transfection of RAW264 cells with sequential 5'-deletions of mouse TRAP gene promoter-luciferase fusion constructs indicated that at least two sites are required for the full promoter activity. Deletion and site-directed mutation studies revealed that M-box and interferon regulatory factor element sites are critical for TPAP gene expression in the cell, suggesting that microphthalmia transcription factor (MITF) and Pip could induce the gene expression independently. Moreover, the overexpression of MITF and Pip functionally stimulated TRAP promoter activity in a synergistic manner. Analysis of the deletion mutants of Pip protein indicated that both N-terminal DNA-binding and C-terminal regulatory domains are indispensable to the promoter-enhancing activity. Subcellular localization of green fluorescence protein-fused Pip and its mutant proteins indicated that the C-terminal domain is required for the translocation of Pip into the nucleus. These results suggest that Pip regulates and acts synergistically with MITF to induce the promoter activity of TRAP gene.