Functional characterization of human osteoclast inhibitory peptide-1 (OIP-1/hSca) gene promoter

We have recently identified and characterized the human osteoclast (OCL) inhibitory peptide-1 (OIP-1/hSca), a member of Ly-6 gene family. OIP-1 is an important physiologic regulator of OCL development and bone resorption activity. To determine the molecular mechanisms that regulate OIP-1 gene expression in OCL precursor cells, we isolated and characterized the OIP-1/hSca gene (2 Kb) promoter sequence. IFN-γ (50 ng/ml) treatment of RAW 264.7 macrophage cells transfected with OIP-1 gene (- 1 to - 1988 bp relative to transcription start site) promoter-luciferase reporter plasmid demonstrated a significant (4 fold) increase in OIP-1 gene promoter activity. Sequence analysis of OIP-1 gene promoter region further identified a potential Stat-1 binding motif at - 1629 to - 1639 bp position. Stat-1 specific inhibitor, fludarabine (50 μM) abolished IFN-γ stimulated OIP-1 gene promoter activity. Electrophoretic mobility shift assay (EMSA) further confirmed activated Stat-1 binding to the OIP-1 gene promoter sequence suggesting that IFN-γ regulates OIP-1 gene expression in OCL precursor cells through a Stat-1 dependent signaling pathway. We further show that knock-down of TRADD enhances IFN-γ signaling to increase OIP-1 gene expression in OCL precursor cells. These results should provide insights into the molecular control of OIP-1 gene expression and inhibition of OCL activity in the bone microenvironment.