TRANSCRIPTIONAL REGULATION OF THE TARTRATE-RESISTANT ACID-PHOSPHATASE(TRAP) GENE BY IRON

Tartrate-resistant acid phosphatase (TRAP) was first identified in cel ls from patients with hairy cell leukaemia. Subsequently, it has been found in other leukaemias, B-lymphoblastoid cell lines, osteoclasts an d subsets of normal lymphocytes, macrophages, and granulocytes. Recent data indicate that TRAP and porcine uteroferrin, a placental iron-tra nsport protein, represent a single gene product. However, the intracel lular role of TRAP is unknown. We used a full-length human placental T RAP cDNA probe to examine TRAP expression in human peripheral mononucl ear cells (PMCs). TRAP mRNA increased 50-75-fold after 24 h in unstimu lated PMC cultures. Cell-fractionation experiments indicated that mono cytes were the main cell population accounting far increased TRAP mRNA transcripts, and this was confirmed by histochemical staining for TRA P enzyme activity. Because expression of other iron-binding and -trans port proteins is controlled by iron availability, we examined the role of iron in regulating TRAP expression. Increase of TRAP mRNA transcri pts in PMCs was inhibited by 50 mu M desferrioxamine, a potent iron ch elator. The 5' flanking region of the TRAP gene was cloned from a mous e genomic library. In preliminary transient transfection experiments, it was determined that the 5'-flanking region of the TRAP gene contain ed iron-responsive elements. Therefore, a series of stably transfected HRE H9 cell lines was developed bearing genetic constructs containing various segments of the murine TRAP 5' promoter region driving a luci ferase reporter gene. Treatment of transfectants with 100 mu g/ml iron -saturated human transferrin (FeTF) was performed to assess iron respo nsiveness of the constructs. Constructs containing a full-length TRAP promoter (comprising base pairs -1846 to +2) responded to FeTF with a 4-5-fold increase of luciferase activity whereas constructs containing only base pairs -363 to +2 of the TRAP promoter did not respond. Cons tructs containing 1240 or 881 bp of the TRAP promoter gave only a 1.5- to 2-fold increase of luciferase activity with FeTF. In all cases, in crease of luciferase activity was blocked by desferrioxamine. Cells tr ansfected with another luciferase construct driven by a simian virus 4 0 promoter did not show any increase of luciferase activity with FeTF. These data indicate that expression of TRAP is regulated by iron and that this regulation is exerted at the level of gene transcription. Th e transfection experiments also suggest that the region of the TRAP 5' -flanking sequence between base pairs - 1846 and - 1240 contains an ir on regulatory element.