Raja R H, Paterson A J, Shin T H, Kudlow J E
Department of Medicine, University of Alabama, Birmingham 35294.
Mol Endocrinol. 1991 Apr;5(4):514-20. doi: 10.1210/mend-5-4-514.
We and others have previously reported that transforming growth factor-alpha (TGF alpha) expression is hormonally responsive and its expression is coregulated with that of its receptor [the epidermal growth factor (EGF) receptor]. The 5'-flanking region of the TGF alpha gene was characterized to determine whether it could confer hormone responsiveness to a reporter gene (luciferase) in human mammary carcinoma cells (MDA468). This segment of the gene is GC rich and contains an element strikingly similar to the core element of the EGF receptor gene that has been shown to mediate both basal and hormone-stimulated expression of the EGF receptor. We now report that a 313-basepair (bp) proximal element of the TGF alpha 5'-flanking region (-373 to -59 relative to the TGF alpha translation start codon) is capable of conferring responses to phorbol ester and EGF. This gene segment does not contain the EGF receptor gene homolog or potential AP-2-binding sites, suggesting that these elements are not necessary for basal and EGF- or phorbol ester-responsive TGF alpha gene expression. This 313-bp proximal element also confers proper transcriptional initiation to the chimeric TGF alpha-luciferase reporter construct, indicating it is the TGF alpha promoter. A 1.1-kilobase segment of the TGF alpha 5'-flanking region also confers retinoic acid, thyroid hormone, and glucocorticoid responsiveness despite the absence of recognizable steroid hormone receptor-binding sites. These hormones stimulate reporter expression 1.5- to 2-fold in a dose-dependent manner. Extension of the 5'-flanking region to -3500 results in marked suppression of reporter gene expression. These results indicate that the TGF alpha gene 5'-flanking sequence contains the elements responsible for hormonal responsiveness of this gene and that these elements are distinct from those that regulate the expression of the EGF receptor gene.
我们和其他研究人员之前曾报道，转化生长因子α（TGFα）的表达具有激素反应性，且其表达与受体[表皮生长因子（EGF）受体]的表达共同调节。对TGFα基因的5'侧翼区域进行了特征分析，以确定它是否能赋予人乳腺癌细胞（MDA468）中的报告基因（荧光素酶）激素反应性。该基因片段富含GC，并且包含一个与EGF受体基因的核心元件极为相似的元件，该元件已被证明可介导EGF受体的基础表达和激素刺激表达。我们现在报告，TGFα 5'侧翼区域的一个313碱基对（bp）近端元件（相对于TGFα翻译起始密码子为-373至-59）能够赋予对佛波酯和EGF的反应性。该基因片段不包含EGF受体基因同源物或潜在的AP-2结合位点，这表明这些元件对于基础的以及EGF或佛波酯反应性的TGFα基因表达并非必需。这个313-bp近端元件还赋予嵌合的TGFα-荧光素酶报告构建体正确的转录起始，表明它是TGFα启动子。尽管缺乏可识别的类固醇激素受体结合位点，但TGFα 5'侧翼区域的一个1.1千碱基片段也赋予视黄酸、甲状腺激素和糖皮质激素反应性。这些激素以剂量依赖性方式刺激报告基因表达1.5至2倍。将5'侧翼区域延伸至-3500会导致报告基因表达显著抑制。这些结果表明，TGFα基因的5'侧翼序列包含负责该基因激素反应性的元件，并且这些元件与调节EGF受体基因表达的元件不同。
