Differential utilization of the promoter of peripheral-type benzodiazepine receptor by steroidogenic versus nonsteroidogenic cell lines and the role of Sp1 and Sp3 in the regulation of basal activity
Differential utilization of the promoter of peripheral-type benzodiazepine receptor by steroidogenic versus nonsteroidogenic cell lines and the role of Sp1 and Sp3 in the regulation of basal activity
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DOI：
10.1210/en.2003-1330
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发表时间：
2004-03-01
期刊：
影响因子：
4.8
通讯作者：
Papadopoulos, V
中科院分区：
文献类型：
--
作者：
Giatzakis, C;Papadopoulos, V
外周型苯二氮卓受体 (PBR) 参与许多细胞功能，包括类固醇生成、氧化过程、细胞增殖和凋亡。分泌组织和腺体组织，尤其是类固醇激素产生细胞，PBR 含量特别丰富。为了了解 PBR 表达和调节的机制，我们在小鼠组织和细胞系中建立了 mRNA 表达谱，随后绘制了转录起始位点并表征了该基因的启动子。我们的研究结果表明，肾、脾、肌肉、肺、肾上腺、胸腺和胃中 PBR 组织 mRNA 水平相对较高；胰腺、子宫、前列腺、心脏和睾丸处于中间状态；并且大脑和肝脏的含量很低。与肾上腺皮质 Y1 小鼠细胞和非类固醇生成 NIH-3T3 小鼠成纤维细胞相比，在合成类固醇的 MA-10 小鼠 Leydig 肿瘤细胞中也观察到相对较高水平的 PBR mRNA，尽管与成纤维细胞相比，这两种类固醇生成细胞中的 PBR 蛋白水平要高得多。转录主要在翻译起始位点上游 61 个核苷酸的腺嘌呤核苷酸处启动，但也观察到内部启动。克隆并测序了小鼠 PBR 启动子的 2.7 kb 片段。序列分析显示不存在 TATA 或 CCAAT 盒，但存在许多假定的转录因子结合位点，包括 Sp1/Sp3、AP2、Ik2、AP1、SOX、GATA 和 SRY。功能表征表明，近端启动子中的两个 Sp1/Sp3 位点对于所有测试的细胞系的基础活性都很重要，并且与非类固醇生成 NIH-3T3 细胞相比，类固醇生成 MA-10 和 Y1 细胞使用不同的启动子区域。
The peripheral-type benzodiazepine receptor (PBR) is involved in many cellular functions, including steroidogenesis, oxidative processes, cellular proliferation, and apoptosis. Secretory and glandular tissues, especially steroid hormone-producing cells, are particularly rich in PBR. To understand the mechanisms of PBR expression and regulation, we established an mRNA expression profile in mouse tissues and cell lines and subsequently mapped the transcription start site and characterized the promoter of the gene. Our findings indicate that PBR tissue mRNA levels are relatively high in kidney, spleen, muscle, lung, adrenal gland, thymus, and stomach; are intermediate in pancreas, uterus, prostate, heart, and testis; and are low in brain and liver. Relatively high levels of PBR mRNA were also observed in the steroid-synthesizing MA-10 mouse Leydig tumor cells compared with adrenocortical Y1 mouse cells and nonsteroidogenic NIH-3T3 mouse fibroblasts, although PBR protein levels were much higher in both steroidogenic cells compared with fibroblasts. Transcription was initiated primarily at an adenine nucleotide 61 nucleotides upstream of the translation start site, but internal initiation was also observed. A 2.7-kb fragment of the mouse PBR promoter was cloned and sequenced. Sequence analysis revealed the absence of TATA or CCAAT boxes, but the presence of many putative transcription factor-binding sites, including Sp1/Sp3, AP2, Ik2, AP1, SOX, GATA, and SRY. Functional characterization revealed that two Sp1/Sp3 sites in the proximal promoter are important for basal activity in all cell lines tested and that the steroidogenic MA-10 and Y1 cells use different areas of the promoter compared with nonsteroidogenic NIH-3T3 cells.