Vamvakopoulos N C, Chrousos G P
Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892.
Mol Cell Endocrinol. 1993 Jul;94(1):73-8. doi: 10.1016/0303-7207(93)90053-m.
Corticotropin-releasing hormone (CRH) plays a major role in the coordination of the stress response. Its gene is expressed in multiple brain regions, the peripheral sympathetic system and the placenta, as well as in peripheral inflammatory sites where CRH acts as a pro-inflammatory cytokine. The human (h) CRH gene, in addition to its primary promoter (TATA box I), has a second distal promoter-like structure (TATA box II) and a functional cyclic adenosine monophosphate-responsive element, all of which are preserved in the rat and ovine genes. To examine the functionality of TATA II, we positioned a 881-bp-long segment of the 5' flanking region of the hCRH gene containing TATA II, but lacking TATA I, upstream from a chloramphenicol acetyltransferase (CAT) reporter gene cloned in a pUC vector. We transfected COS-7 cells with this construct and examined responsiveness of CAT activity to potential stimulants and inhibitors. Phorbol ester (TPA) and forskolin had mild but clear stimulatory effects on CAT expression (approximately 1.5- and approximately 1.3-fold, respectively), with a combined effect of approximately 1.9-fold. Dexamethasone (DEX) inhibited TPA-stimulated CAT activity by approximately 2.6-fold. In contrast, in the presence of a co-transfected glucocorticoid receptor cDNA expression plasmid, DEX augmented TPA-stimulated CAT expression by approximately 3.1-fold. The predicted secondary structures of the primary transcripts employing the distal and proximal promoters had significant differences, which could affect their stability and translatability.2
促肾上腺皮质激素释放激素（CRH）在应激反应的协调中起主要作用。其基因在多个脑区、外周交感神经系统、胎盘以及外周炎症部位表达，在这些外周炎症部位CRH作为一种促炎细胞因子发挥作用。人类（h）CRH基因除了其主要启动子（TATA框I）外，还有第二个远端启动子样结构（TATA框II）和一个功能性环磷酸腺苷反应元件，所有这些在大鼠和绵羊基因中都得以保留。为了检测TATA II的功能，我们将hCRH基因5'侧翼区包含TATA II但缺乏TATA I的一段881 bp长的片段置于克隆于pUC载体的氯霉素乙酰转移酶（CAT）报告基因上游。我们用该构建体转染COS-7细胞，并检测CAT活性对潜在刺激物和抑制剂的反应性。佛波酯（TPA）和福斯可林对CAT表达有轻微但明显的刺激作用（分别约为1.5倍和约1.3倍），联合作用约为1.9倍。地塞米松（DEX）抑制TPA刺激的CAT活性约2.6倍。相反，在共转染糖皮质激素受体cDNA表达质粒的情况下，DEX使TPA刺激的CAT表达增加约3.1倍。使用远端和近端启动子的初级转录本的预测二级结构有显著差异，这可能影响它们的稳定性和可翻译性。
