TRANSCRIPTIONAL REGULATION OF THE TARTRATE-RESISTANT ACID-PHOSPHATASE (TRAP) GENE BY IRON
TRANSCRIPTIONAL REGULATION OF THE TARTRATE-RESISTANT ACID-PHOSPHATASE (TRAP) GENE BY IRON
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DOI：
10.1042/bj2980421
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发表时间：
1994-03-01
影响因子：
4.1
通讯作者：
BOLDT, DH
中科院分区：
文献类型：
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作者：
ALCANTARA, O;REDDY, SV;BOLDT, DH
抗酒石酸酸性磷酸酶(TRAP)首先在毛细胞白血病患者的细胞中被发现。随后，在其他白血病、B淋巴母细胞系、破骨细胞和正常淋巴细胞、巨噬细胞和粒细胞亚群中也发现了它。最近的数据表明，TRAP和猪子宫铁蛋白是一种胎盘铁转运蛋白，是单基因产物。然而，TRAP在细胞内的作用尚不清楚。我们使用全长的人胎盘TRAP cDNA探针来检测TRAP在人外周单个核细胞(PMC)中的表达。未经刺激的PMC培养24 h后，TRAP mRNA表达增加50~75倍。细胞分裂实验表明，单核细胞是主要的细胞群体，TRAP mRNA转录量大大增加，TRAP酶活性的组织化学染色证实了这一点。由于其他铁结合和转运蛋白的表达受铁的可获得性控制，我们研究了铁在调节TRAP表达中的作用。50 mU M去铁胺可抑制PMCs TRAP基因转录的增加。TRAP基因的5‘侧翼区是从小鼠基因组文库中克隆的。在初步的瞬时转染实验中，确定TRAP基因的5‘侧翼区含有铁反应元件。因此，我们建立了一系列稳定转基因的HRE H9细胞系，它们含有不同片段的小鼠TRAP 5‘启动子区域驱动荧光素酶报告基因的基因结构。用100微克/毫升铁饱和的人转铁蛋白(FeTF)处理转染体，以评估构建体的铁响应性。含有全长TRAP启动子(包括碱基对-1846到+2)的构建体对FeTF的荧光素酶活性有4-5倍的反应，而只包含-363到+2碱基对的TRAP启动子的构建体没有反应。含有1240或881bp的TRAP启动子的构建体仅使荧光素酶活性增加1.5~2倍。在所有情况下，荧光素酶活性的增加都被去铁胺阻断。由猴病毒40启动子驱动的另一种荧光素酶构建体转染的细胞没有表现出FeTf对荧光素酶活性的任何增加。这些数据表明，TRAP的表达受铁的调控，这种调控是在基因转录水平上进行的。转染实验还表明，在碱基对-1846和-1240之间的TRAP 5‘-侧翼序列区域含有铁调节元件。
Tartrate-resistant acid phosphatase (TRAP) was first identified in cells from patients with hairy cell leukaemia. Subsequently, it has been found in other leukaemias, B-lymphoblastoid cell lines, osteoclasts and subsets of normal lymphocytes, macrophages, and granulocytes. Recent data indicate that TRAP and porcine uteroferrin, a placental iron-transport protein, represent a single gene product. However, the intracellular role of TRAP is unknown. We used a full-length human placental TRAP cDNA probe to examine TRAP expression in human peripheral mononuclear cells (PMCs). TRAP mRNA increased 50-75-fold after 24 h in unstimulated PMC cultures. Cell-fractionation experiments indicated that monocytes were the main cell population accounting far increased TRAP mRNA transcripts, and this was confirmed by histochemical staining for TRAP enzyme activity. Because expression of other iron-binding and -transport proteins is controlled by iron availability, we examined the role of iron in regulating TRAP expression. Increase of TRAP mRNA transcripts in PMCs was inhibited by 50 mu M desferrioxamine, a potent iron chelator. The 5' flanking region of the TRAP gene was cloned from a mouse genomic library. In preliminary transient transfection experiments, it was determined that the 5'-flanking region of the TRAP gene contained 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 luciferase reporter gene. Treatment of transfectants with 100 mu g/ml iron-saturated human transferrin (FeTF) was performed to assess iron responsiveness 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. Constructs 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, increase of luciferase activity was blocked by desferrioxamine. Cells transfected with another luciferase construct driven by a simian virus 40 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. The transfection experiments also suggest that the region of the TRAP 5'-flanking sequence between base pairs - 1846 and - 1240 contains an iron regulatory element.