Werlen G, Belin D, Conne B, Roche E, Lew D P, Prentki M
Department of Medicine, University of Geneva Medical School, Switzerland.
J Biol Chem. 1993 Aug 5;268(22):16596-601.
To gain direct insight into the action of the second messenger Ca2+ on transcriptional regulation, we have developed an intact cell model in which the intracellular free Ca2+ concentration ([Ca2+]i) can be measured, set, and varied at any level within the physiological range and in which the expression of early response genes is assayed in parallel. Using promyelocytic HL-60 cells, we have observed an exquisite sensitivity to Ca2+ of c-fos, c-jun, and zif268 mRNA accumulation, since early and maximal inductions were observed at 200-300 nM [Ca2+]i. At early times (10-20 min), the [Ca2+]i dose dependence of c-fos transcription and mRNA accumulation displayed a bell shape since c-fos expression was barely modified at high (700-1,250 nM) [Ca2+]i. The threshold [Ca2+]i concentration for prolonged (60 min) c-fos mRNA accumulation was greater than 200 nM. This indicates that the quantitative effects of Ca2+ on a given gene can vary markedly as a function of both the [Ca2+]i concentration and the duration of stimulation. Strikingly, a [Ca2+]i perturbation of only 1 min was sufficient for full induction of c-fos and zif268 transcripts. This demonstrates that a transient perturbation of [Ca2+]i has long term effects on gene expression. The half-life of c-fos mRNA (16 min) was unaltered by Ca2+. Nuclear run-on analysis of the distribution of RNA polymerase II along the c-fos locus indicated that Ca2+ promotes a small increase in transcriptional initiation and a pronounced relief of a block to transcriptional elongation beyond intron 1. The extreme sensitivity to [Ca2+]i, in terms of both the length of time and the dose of [Ca2+]i required for maximal gene induction, demonstrates that Ca2+ is a major physiological regulator of early response gene expression. In addition, the results indicate that a c-fos intragenic element is the main target of Ca(2+)-regulated transcriptional activation.
为了直接深入了解第二信使Ca2+对转录调控的作用，我们建立了一个完整细胞模型，在该模型中，可以在生理范围内的任何水平测量、设定和改变细胞内游离Ca2+浓度（[Ca2+]i），并同时检测早期反应基因的表达。使用早幼粒细胞HL-60细胞，我们观察到c-fos、c-jun和zif268 mRNA积累对Ca2+具有极高的敏感性，因为在[Ca2+]i为200 - 300 nM时观察到早期和最大诱导。在早期（10 - 20分钟），c-fos转录和mRNA积累的[Ca2+]i剂量依赖性呈钟形，因为在高[Ca2+]i（700 - 1250 nM）时c-fos表达几乎没有改变。延长（60分钟）c-fos mRNA积累的阈值[Ca2+]i浓度大于200 nM。这表明Ca2+对给定基因的定量影响可根据[Ca2+]i浓度和刺激持续时间而显著变化。引人注目的是，仅1分钟的[Ca2+]i扰动就足以完全诱导c-fos和zif268转录本。这表明[Ca2+]i的短暂扰动对基因表达具有长期影响。Ca2+未改变c-fos mRNA的半衰期（16分钟）。对RNA聚合酶II沿c-fos基因座分布的核运行分析表明，Ca2+促进转录起始略有增加，并显著缓解内含子1以外转录延伸的阻滞。就最大基因诱导所需的时间长度和[Ca2+]i剂量而言，对[Ca2+]i的极端敏感性表明Ca2+是早期反应基因表达的主要生理调节因子。此外，结果表明c-fos基因内元件是Ca(2+)调节的转录激活的主要靶点。
