Molecular Mechanisms of the Biological Clock in Cultured Fibroblasts

Abstract

In mammals, the central circadian pacemaker resides in the hypothalamic suprachiasmatic nucleus (SCN), but circadian oscillators also exist in peripheral tissues. Here, using wild-type and cryptochrome (mCry)-deficient cell lines derived from mCry mutant mice, we show that the peripheral oscillator in cultured fibroblasts is identical to the oscillator in the SCN in (i) temporal expression profiles of all known clock genes, (ii) the phase of the various mRNA rhythms (i.e., antiphase oscillation of Bmal1 and mPer genes), (iii) the delay between maximum mRNA levels and appearance of nuclear mPER1 and mPER2 protein, (iv) the inability to produce oscillations in the absence of functional mCry genes, and (v) the control of period length by mCRY proteins.

Assessment of circadian function in fibroblasts of patients with bipolar disorder

“…[62][63][64] Several studies have shown that the circadian oscillators in fibroblasts are similar to those operative in the SCN, and fibroblasts may serve as a valid in vitro model for molecular oscillators in the brain. 64,[70][71][72] The periods for the circadian gene expression in fibroblasts have been found to correspond well with human circadian physiology and mouse behavior. 71 Although the luminescence reporter assay has the advantage of higher throughput and being less laborious, it is not readily applicable to accurately measure amplitude and overall level of the expression of more than one gene.…”

“…It has been reported that similar components of the clock machinery underlie rhythmic gene expression in cultured fibroblasts as in the central clock in the SCN. 64,70,72 Based on this finding, we investigated properties of circadian oscillators in fibroblast cell lines from patients with bipolar disorder. We found that core genes of the circadian clockwork-BMAL1, PER1, PER2, REV-ERBa, DEC2 and DBP-show similar rhythmic expression pattern in fibroblasts from bipolar patients and healthy controls.…”

“…Validation of the fibroblast model as a readout of the core clock machinery comes from experiments showing that genetic differences can be manifested in both central and peripheral oscillators. 64,70,72 However, we cannot assume that all circadian changes detected in fibroblasts will translate to physiological and behavioral anomalies. 71 Therefore, caution is needed to extend our results from fibroblasts to CNS mechanisms and overt behavior.…”

“…[62][63][64] The circadian oscillators in cultured cells can be synchronized by treatment with 50% serum (so-called serum shock) or with various chemicals (that is, glucocorticoid, endothelin, forskolin, and so on) which elicit different signaling pathways. [65][66][67][68][69][70] This makes it possible to detect the circadian gene expression by conventional methods such as real-time quantitative PCR (Q-PCR) or luminescence reporter assays. [62][63][64] Several studies have shown that the circadian oscillators in fibroblasts are similar to those operative in the SCN, and fibroblasts may serve as a valid in vitro model for molecular oscillators in the brain.…”

Immortalized Suprachiasmatic Nucleus Cells Express Components of Multiple Circadian Regulatory Pathways

“…To provide useful applications for central mammalian pacemaker research, cell line models must exhibit persistent self-sustained oscillations in circadian gene products, restore animal circadian rhythms in SCN-lesioned hosts and exhibit time-dependent responses to stimuli through regulatory pathways that characterize SCN function. Rhythmic gene expression has been demonstrated in NIH/ 3T3 fibroblasts, Rat-1 fibroblasts and spontaneously immortalized embryonic mouse fibroblasts after synchronizing signals (1)(2)(3)(4)(5). However, there is no published evidence that these lines express spontaneous, self-sustained rhythms or rescue rhythms in SCNlesioned animals (5,6).…”