In the anterior pytuitary gland, corticotroph cells secrete ACTH in response to Corticotrophin Releasing Hormone (CRH), a process that is controlled in large part by an increase in the cytosolic free calcium concentration. At the same time, the frequency and amplitude of Ca²⁺ oscillations also modulates gene transcription, indicating that the spatio-temporal pattern of the Ca²⁺ signals (oscillations or sustained elevations) is critical for the final cellular response. In a corticotroph cell line, the nuclear or cytosolic Ca²⁺ elevation modulate, by distinct pathways, the expression of the immediate early gene (IEG) c-fos, a transcription factor controlling the expression of a variety of genes (Hardigham, nature vol 385, 260 1997). With other IEG, c-fos is the first gene product of the cellular response to an external signal and its expression in neuroendocrine cells is mostly controlled by Ca²⁺ signals. The aim of our works was to establish a direct link between the Ca²⁺ signals and the expression of the IEG c-fos by studying the mechanism of the transcriptional activation at a single cell level.
For this purpose, we quantified the expression of the c-fos gene using a reporter gene based on the enzyme beta-lactamase. This enzyme cuts a bi-fluorescent substrate, CCF-2, which emits green light when intact due to FRET and blue light when cut. This method allows us to study the expression of the c-fos gene in intact living cells in a time-based fashion. We produce a stable corticotroph AtT20 cell line expressing the beta-lactamase gene under the control of the c-fos promoter. The c-fos transcription, measured in single cells, increased by 2-3 fold upon stimulation with KCl, serum, or with the physiological agonists EGF or PACAP. The responses were strongly reduced by nuclear injection of BAPTA-D70, which prevented the agonist-induced rise of the nuclear Ca²⁺ concentration. In contrast, the nuclear calcium clamp did not affect the basal expression of c-fos, which reflected the spontaneous electrical activity of AtT-20 cells. These spontaneous Ca²⁺ oscillations were detected in the cytosolic, but not in the nuclear compartment, and were abolished by the L-type calcium channels blocker nifedipine, which also strongly reduced the basal expression of c-fos. The deletion of the CREB response element (CRE) of the c-fos promoter had no effect on the basal expression, whereas the deletion of the Serum Response Element (SRE) had a marked inhibitory effect. In conclusion, these experiments suggest that in AtT20 cells the spontaneous cytosolic Ca²⁺ activity maintain a basal c-fos protein expression by acting on the SRE element of the c-fos promoter. Further induction of c-fos expression in response to physiological agonists require an increase in nuclear Ca2+, a process that has been shown to be mediated by the CRE element.