Cloning and characterization of the 5′-flanking region of the mouse tartrate-resistant acid phosphatase gene

Abstract

Little information is available on the molecular mechanisms controlling osteoclastic bone resorption. We used tartrate-resistant acid phosphatase (TRAP) to begin to investigate the regulation of bone resorption at the molecular level. TRAP is expressed at high levels in osteoclasts and may play an important role in the bone resorptive process. Therefore, we isolated the murine TRAP gene from a mouse spleen genomic library and characterized its promoter. A restriction map was generated for the 17 kb TRAP insert. A 2 kb Smal fragment, containing the 5′-flanking region, was subcloned and the nucleotide sequence determined. Sequence analysis of the Smal fragment revealed the presence of numerous candidate transcription factor binding sequences, including those for API and H-APF-1. The H-APF-1 site matches the consensus sequence for the IL-6-regulated transcription factor. An intron was identified at −1 to −393 bp relative to the ATG. The presence of an intron was confirmed by PCR analysis of RNA isolated from murine osteoclasts. Primer extension analysis indicated the presence of a transcription initiation site at −552 bp from the ATG. The region from −1846 to 2 bp relative to the ATG initiation codon drove the transient expression of a luciferase reporter gene when transfected into HRE H9 rabbit endometrial cells. PMA treatment of HRE H9 cells enhanced luciferase transcription approximately threefold. These data suggest that the TRAP promoter is complex and contains multiple regulatory elements. The availability of the TRAP promoter may also permit production of transgenic mice, which can be used to develop previously unavailable osteoclast cell lines.