Functional role of a novel cis-acting element (GAGA box) in human type-1 angiotensin II receptor gene transcription.
Functional role of a novel cis-acting element (GAGA box) in human type-1 angiotensin II receptor gene transcription.
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DOI：
10.1677/jme.0.0250097
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发表时间：
2000-08
影响因子：
3.5
通讯作者：
BD Wyse;SL Linas;TJ Thekkumkara
中科院分区：
文献类型：
--
作者：
BD Wyse;SL Linas;TJ Thekkumkara
生长激素/生长因子已被证明可增加血管紧张素1型受体的表达。在本研究中，我们确定了控制gh诱导的人类1型血管紧张素受体（hAT(1)）转录的顺式调控区域。在人近端小管细胞中转染了在hAT(1)启动子控制下的氯霉素乙酰转移酶（CAT）报告质粒，GH诱导了CAT活性。hAT(1)启动子区域的连续缺失表明，cDNA序列5'端上游-314 bp至-70 bp之间的区域是发生这种激活所必需的。虽然序列分析在该区域发现了假定的多个核蛋白结合位点，但我们确定位于-161 bp和-149 bp之间的12 bp序列（5'- gagagggagag -3', GAGA box）对gh介导的激活很重要。通过迁移率转移实验，我们发现在GH处理过的核提取物中，标记的GAGA盒的DNA结合活性增加，这表明该序列是GH响应元件。西南分析鉴定出一个18 kDa的GAGA盒结合蛋白（GAGA- bp）。gh诱导的GAGA-BP活性在2.5 min内出现，在5 min时达到最大。激活不需要从头合成蛋白质。移除GAGA盒可以消除gh诱导的转录以及hAT(1)基因的基础转录。另有研究表明，表皮生长因子、血小板源性生长因子和胰岛素可激活ga - bp，提示这些生长因子也可通过GAGA盒调控hAT(1)基因的转录。我们的数据表明，GAGA-BP作为反式作用因子与hAT(1)启动子中的顺式作用调控元件结合，这是基础因子和生长因子介导的hAT(1)基因转录激活所必需的。
GH/growth factors have been shown to increase angiotensin type 1 receptor expression. In the present study we determined the cis-acting regulatory region controlling GH-induced transcription of the human type-1 angiotensin receptor (hAT(1)). In human proximal tubule cells transfected with a chloramphenicol acetyl transferase (CAT) reporter plasmid under the control of the hAT(1) promoter, GH induced CAT activity. Serial deletions of the hAT(1) promoter region indicated that an area between -314 bp and -70 bp upstream of the 5'-end of the cDNA sequence was essential for this activation to occur. Although sequence analysis identified putative multiple nuclear protein binding sites in this region, we determined that a 12 bp sequence (5'-GAGAGGGAGGAG-3', GAGA box) located between -161 bp and -149 bp was important for GH-mediated activation. Using mobility shift assays we demonstrated increased DNA binding activity to the labeled GAGA box in nuclear extracts treated with GH, suggesting this sequence is a GH response element. Southwestern analysis identified an 18 kDa GAGA box-binding protein (GAGA-BP). GH-induced activity of the GAGA-BP occurred within 2.5 min and reached a maximum at 5 min. Activation did not require de novo protein synthesis. Removal of the GAGA box abolished GH-induced transcription as well as basal transcription of the hAT(1) gene. Additional studies demonstrated that epidermal growth factor, platelet-derived growth factor and insulin activate the GAGA-BP, suggesting these growth factors can also regulate the transcription of the hAT(1) gene through the GAGA box. Our data show that the GAGA-BP acts as a trans-acting factor binding to the cis-acting regulatory element in the hAT(1) promoter, which is necessary for the basal and growth factor(s)-mediated transcriptional activation of the hAT(1) gene.