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.
Highlights
Among temporally regulated processes, the circadian clock is unique in that it operates precisely on a cycle of approximately 24 h to regulate time-dependent processes such as sleep–wake cycles and body temperature fluctuations
The present study demonstrates that the various treatments used by previous researchers to initiate rhythms in fibroblasts can lead to a variety of oscillatory amplitudes and phases (Figure 3)
As assessed by relative amplitude error (RAE) values, rhythmicity was initiated by all ten compounds presently considered
Summary
The circadian clock is unique in that it operates precisely on a cycle of approximately 24 h to regulate time-dependent processes such as sleep–wake cycles and body temperature fluctuations. Whereas SCN tissue is difficult to obtain and manipulate, cell cultures have important advantages: they are easy to maintain, accessible to molecular genetic tools, and can produce the large amount of material that is necessary for biochemical assays. Because of these advantages, cultured cells have provided an excellent alternative to the SCN for the study of the molecular and biochemical mechanisms of mammalian circadian systems in vitro [8,9,10,11,12,13,14,15,16,17,18,19,20]
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