Bral C M, Ramos K S
Department of Physiology and Pharmacology, College of Veterinary Medicine, Texas A & M University, College Station 77843, USA.
Mol Pharmacol. 1997 Dec;52(6):974-82. doi: 10.1124/mol.52.6.974.
Previous studies in this laboratory have demonstrated that transcriptional deregulation of c-Ha-ras expression is associated with the induction and maintenance of proliferative vascular smooth muscle cell (SMC) phenotypes by benzo[a]pyrene (BaP). We examined previously undescribed cis-acting elements within the proximal 5' regulatory region of c-Ha-ras (-550 to +220) for their ability to influence BaP-induced transcription in murine SMCs. BaP-inducible DNA binding activity was demonstrated at a site located -30 relative to the major start site cluster at +1 that exhibits extensive homology to a consensus aryl hydrocarbon response element (AHRE), as well as a site located at -543 that contains a consensus electrophile response element (EpRE). In vitro cross-linking studies revealed the specific interaction of 104- and 96-kDa proteins with the putative AHRE and of an 80-kDa protein with the EpRE. The use of monoclonal antibodies to the aryl hydrocarbon receptor transcription factor in competition electrophoretic mobility shift assays indicated this protein is specifically induced by BaP to interact at the AHRE within the c-Ha-ras 5' regulatory region. Transient transfection with an Ha-ras promoter construct containing the putative AHRE but lacking the EpRE linked to the chloramphenicol acetyl transferase reporter gene, followed by challenge with BaP (0.3, 3.0, and 30 microM), revealed transcriptional activation that was not statistically significant. However, insertion of an oligonucleotide composed of the EpRE immediately upstream of basal sequences at -330 was associated with strong activation of transcription by BaP. These data indicate that c-Ha-ras gene expression is modulated by BaP via a complex mechanism that likely involves interactions among multiple regulatory elements. We conclude that c-Ha-ras expression is regulated by BaP at the transcriptional level, a response that may constitute an epigenetic basis of atherogenesis.
本实验室先前的研究表明，c-Ha-ras表达的转录失调与苯并[a]芘（BaP）诱导和维持增殖性血管平滑肌细胞（SMC）表型有关。我们研究了c-Ha-ras近端5'调控区（-550至+220）内以前未描述的顺式作用元件影响BaP诱导的小鼠SMC转录的能力。在相对于主要起始位点簇（位于+1）-30处的一个位点显示出BaP诱导的DNA结合活性，该位点与共有芳基烃反应元件（AHRE）具有广泛的同源性，以及位于-543处的一个位点，该位点含有共有亲电试剂反应元件（EpRE）。体外交联研究揭示了104 kDa和96 kDa蛋白与推定的AHRE的特异性相互作用以及80 kDa蛋白与EpRE的特异性相互作用。在竞争电泳迁移率变动分析中使用针对芳基烃受体转录因子的单克隆抗体表明，该蛋白被BaP特异性诱导在c-Ha-ras 5'调控区内的AHRE处相互作用。用含有推定的AHRE但缺少与氯霉素乙酰转移酶报告基因相连的EpRE的Ha-ras启动子构建体进行瞬时转染，随后用BaP（0.3、3.0和30 microM）刺激，结果显示转录激活无统计学意义。然而，在-330处的基础序列上游紧邻插入由EpRE组成的寡核苷酸与BaP强烈激活转录有关。这些数据表明，BaP通过一种可能涉及多个调控元件之间相互作用的复杂机制调节c-Ha-ras基因表达。我们得出结论，c-Ha-ras表达在转录水平受BaP调控，这种反应可能构成动脉粥样硬化发生的表观遗传基础。
