Accessory elements, flanking DNA sequence, and promoter context play key roles in determining the efficacy of insulin and phorbol ester signaling through the malic enzyme and collagenase-1 AP-1 motifs
Accessory elements, flanking DNA sequence, and promoter context play key roles in determining the efficacy of insulin and phorbol ester signaling through the malic enzyme and collagenase-1 AP-1 motifs
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DOI：
10.1074/jbc.m203682200
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发表时间：
2002-08-02
影响因子：
4.8
通讯作者：
O'Brien, RM
中科院分区：
文献类型：
--
作者：
Ayala, JE;Streeper, RS;O'Brien, RM
胰岛素通过相同的激活蛋白-1（AP-1）基序刺激H4 IIE细胞中苹果酸酶（ME）-氯霉素乙酰转移酶（CAT）和胶原酶-I-CAT融合基因的表达。相比之下，胰岛素和佛波醇酯仅刺激胶原酶-I-CAT而非ME-CAT融合基因在HeLa细胞中的表达。本文中的实验旨在探索这种差异细胞类型和基因特异性调控的分子基础。结果突出了三个变量的影响，即启动子上下文，AP-1侧翼序列，以及通过AP-1基序调节胰岛素和佛波酯信号传导的辅助元件。因此，融合基因转染和蛋白水解剪切凝胶阻滞试验表明，AP-1侧翼序列影响AP-1与胶原酶-1和MEAP-1基序结合的构象，使得其在完全活化状态下选择性地结合后者。然而，MEAP-1侧翼序列的这种影响依赖于启动子环境。因此，当将MEAP-1基序引入胶原酶-1启动子或特异性异源启动子中时，MEAP-1基序将在HeLa细胞中介导胰岛素和佛波醇酯反应。但是，即使在胶原酶-1启动子的情况下，胰岛素和佛波醇酯的影响，通过MEAP-1基序介导的依赖于辅助因子。
Insulin stimulates malic enzyme (ME)-chloramphenicol acetyltransferase (CAT) and collagenase-l-CAT fusion gene expression in H4IIE cells through identical activator protein-1 (AP-1) motifs. In contrast, insulin and phorbol esters only stimulate collagenase-l-CAT and not ME-CAT fusion gene expression in HeLa cells. The experiments in this article were designed to explore the molecular basis for this differential cell type- and gene-specific regulation. The results highlight the influence of three variables, namely promoter context, AP-1 flanking sequence, and accessory elements that modulate insulin and phorbol ester signaling through the AP-1 motif. Thus, fusion gene transfection and proteolytic clipping gel retardation assays suggest that the AP-1 flanking sequence affects the conformation of AP-1 binding to the collagenase-1 and ME AP-1 motifs such that it selectively binds the latter in a fully activated state. However, this influence of ME AP-1 flanking sequence is dependent on promoter context. Thus, the ME AP-1 motif will mediate both an insulin and phorbol ester response in HeLa cells when introduced into either the collagenase-1 promoter or a specific heterologous promoter. But even in the context of the collagenase-1 promoter, the effects of both insulin and phorbol esters, mediated through the ME AP-1 motif are dependent on accessory factors.