CLONING AND CHARACTERIZATION OF THE 5'-FLANKING REGION OF THE MOUSE TARTRATE-RESISTANT ACID-PHOSPHATASE GENE

Little information is available on the molecular mechanisms controllin g osteoclastic bone resorption. We used tartrate-resistant acid phosph atase (TRAP) to begin to investigate the regulation of bone resorption at the molecular level. TRAP is expressed at high levels in osteoclas ts and may play an important role in the bone resorptive process. Ther efore, we isolated the murine TRAP gene from a mouse spleen genomic li brary and characterized its promoter. A restriction map was generated for the 17 kb TRAP insert. A 2 kb SmaI fragment, containing the 5'-fla nking region, was subcloned and the nucleotide sequence determined. Se quence analysis of the SmaI fragment revealed the presence of numerous candidate transcription factor binding sequences, including those for AP1 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 confir med by PCR analysis of RNA isolated from murine osteoclasts. Primer ex tension analysis indicated the presence of a transcription initiation site at -552 bp from the ATG. The region from -1846 to 2 bp relative t o the ATG initiation codon drove the transient expression of a lucifer ase reporter gene when transfected into HRE H9 rabbit endometrial cell s. PMA treatment of HRE H9 cells enhanced luciferase transcription app roximately threefold. These data suggest that the TRAP promoter is com plex and contains multiple regulatory elements. The availability of th e TRAP promoter may also permit production of transgenic mice, which c an be used to develop previously unavailable osteoclast cell lines.