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

The receptor activator of NF-kappaB ligand induces the expression of tartra te-resistant acid phosphatase (TRAP) and transcription factor, PU.1-interac ting protein (Pip), during osteoclastogenesis. In this paper, we have exami ned the role of transcription factors in the regulation of TRAP gene expres sion employing reporter constructs containing the promoter region of TRAP g ene. Transient transfection of RAW264 cells with sequential 5'-deletions of mouse TRAP gene promoter-luciferase fusion constructs indicated that at le ast two sites are required for the full promoter activity. Deletion and sit e-directed mutation studies revealed that M-box and interferon regulatory f actor element sites are critical for TRAP gene expression in the cell, sugg esting that microphthalmia transcription factor (MITF) and Pip could induce the gene expression independently. Moreover, the overexpression of MITF an d Pip functionally stimulated TRAP promoter activity in a synergistic manne r. Analysis of the deletion mutants of Pip protein indicated that both N-te rminal DNA-binding and C-terminal regulatory domains are indispensable to t he promoter-enhancing activity. Subcellular localization of green fluoresce nce protein-fused Pip and its mutant proteins indicated that the C-terminal domain is required for the translocation of Pip into the nucleus. These re sults suggest that Pip regulates and acts synergistically with MITF to indu ce the promoter activity of TRAP gene.