The ERK/MAPK Pathway and Circadian Clock Entrainment

描述（由申请人提供）：在哺乳动物中，下丘脑的视交叉上核（SCN）是主要的生物钟。最近的研究表明，内源性SCN计时需要一个节律性转录调节程序，而光诱导的昼夜节律变化是由该转录程序的改变引起的。考虑到可诱导基因表达的核心作用，细胞内信号通路和下游转录因子参与生物计时和携带的表征对于理解昼夜节律钟的功能特性至关重要。在上一轮融资中，我们收集了大量数据，支持MARK通路将光输入与时钟时序过程耦合的假设。缺乏的是对MARK通路调节生物钟的精确途径的理解。我们假设MARK信号级联在转录和翻译水平上调节时间，并且我们已经组装了一系列新的工具来验证这一假设。在Aim 1中，我们将确定MARK通路调节的激酶MSK1/2和RSK2是否作为光与时钟夹带耦合的细胞信号传导中间体。我们发表的报告显示，这两种激酶在SCN中都被光激活，初步数据表明MSK1和RSK2调节时钟携带。行为学研究将通过研究MSK和RSK缺失小鼠的染色质结构和基因表达而得到补充。在Aim 2中，我们研究了CREB/CRE-转录途径（MARK途径的靶标）在时钟夹带和时钟节律振幅中的作用。CREB/CRE通路被认为是夹带信号影响核心转录时序过程的主要途径。然而，目前还没有一套有效的工具来在体内测试这一想法。为此，我们开发了一套新的CREB抑制因子和CREB激活因子转基因小鼠，将用于在行为，生化和遗传水平上研究这个问题。在Aim 3中，我们研究了MARK通路作为SCN中光依赖性和时钟依赖性mRNA翻译的调节剂的作用。鉴于最近的研究表明，在肥胖、心血管疾病和癌症等疾病中存在昼夜节律时序/夹带失调，因此明确需要确定在起搏器夹带中起基本作用的细胞内信号事件。

DESCRIPTION (provided by applicant): In mammals, the suprachiasmatic nuclei (SCN) of the hypothalamus function as the major biological clock. Recent work has revealed that a program of rhythmic transcriptional regulation is required for endogenous SCN timekeeping and that light-induced changes in circadian timing result from alterations in this transcriptional program. Given this central role of inducible gene expression, a characterization of the intracellular signaling pathways and downstream transcription factors involved in biological timing and entrainment is critical for understanding the functional properties of the circadian clock. In our prior round of funding we collected a wealth of data supporting the hypothesis that the MARK pathway couples photic input to the clock timing process. What is lacking is an understanding of the precise routes by which the MARK pathway regulates the clock. We hypothesize that the MARK signaling cascade regulates timing at both a transcriptional and translational level, and we have assembled an array of novel tools to test this hypothesis. In Aim 1 we will determine whether the MARK pathway-regulated kinases MSK1/2 and RSK2 function as cellular signaling intermediates that couple light to clock entrainment. Our published reports reveal that both kinases are activated by light in the SCN, and preliminary data indicate that MSK1 and RSK2 regulate clock entrainment. Behavioral studies will be complimented by studies that examine chromatin structure and gene expression in MSK and RSK null mice. In Aim 2 we investigate the role of the CREB/CRE- transcription pathway (a target of the MARK pathway) in clock entrainment and clock rhythm amplitude. The CREB/CRE pathway has been implicated as a principal route by which entrainment cues impinge upon the core transcriptional timing process. However, there has yet to be an effective set of tools to test this idea in vivo. To this end, we have developed a novel set of CREB repressor and CREB activator transgenic mice that will be used to examine this question at a behavioral, biochemical and genetic level. In Aim 3, we investigate the role of the MARK pathway as a regulator of light- and clock- dependent mRNA translation in the SCN. Given recent work implicating dysregulation of circadian timing/entrainment in disorders such as obesity, cardiovascular disease and cancer, there is a clear need to identify the intracellular signaling events that play a fundamental role in pacemaker entrainment.