Quantitative Analyses of Circadian Gene Expression in Mammalian Cell Cultures

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Abstract
The central circadian pacemaker is located in the hypothalamus of mammals, but essentially the same oscillating system operates in peripheral tissues and even in immortalized cell lines. Using luciferase reporters that allow automated monitoring of circadian gene expression in mammalian fibroblasts, we report the collection and analysis of precise rhythmic data from these cells. We use these methods to analyze signaling pathways of peripheral tissues by studying the responses of Rat-1 fibroblasts to ten different compounds. To quantify these rhythms, which show significant variation and large non-stationarities (damping and baseline drifting), we developed a new fast Fourier transform–nonlinear least squares analysis procedure that specifically optimizes the quantification of amplitude for circadian rhythm data. This enhanced analysis method successfully distinguishes among the ten signaling compounds for their rhythm-inducing properties. We pursued detailed analyses of the responses to two of these compounds that induced the highest amplitude rhythms in fibroblasts, forskolin (an activator of adenylyl cyclase), and dexamethasone (an agonist of glucocorticoid receptors). Our quantitative analyses clearly indicate that the synchronization mechanisms by the cAMP and glucocorticoid pathways are different, implying that actions of different genes stimulated by these pathways lead to distinctive programs of circadian synchronization. The circadian biological clock controls the adaptation of animals and plants to the daily environmental cycle of light and darkness. As such, this clock is responsible for jet lag and has consequences for mental health (e.g., depression), physical health (e.g., athletic performance and the timing of heart attacks), and social issues (e.g., shift work). The central circadian pacemaker is located in the hypothalamus of the mammalian brain, but essentially the same oscillating system operates in nonneural tissues. Using luciferase, an enzyme that emits light, the authors could monitor circadian gene expression in mammalian fibroblasts via luminescence emission that is controlled by the biological clock. Using this method, they report the collection and analysis of precise rhythmic data from these cells. These methods were used to analyze signaling pathways by studying the responses of fibroblasts to a variety of different treatments, including drugs, growth factors, and serum. The authors developed a new analysis procedure that specifically optimizes the quantification of amplitude for cyclic data to analyze these rhythms. This enhanced analysis method successfully distinguishes among the various signaling treatments for their rhythm inducing properties. The quantitative analyses clearly indicate that the synchronization mechanisms by the cyclic AMP and glucocorticoid pathways are different. Therefore, these pathways lead to distinctive programs of circadian synchronization.