Calcium dynamics and resting transcriptional activity regulates prolactin gene expression.

Research on the regulation of hormone gene expression by calcium signaling is hampered by the difficulty of monitoring both parameters within the same individual, living cells. Here we achieved concurrent, dynamic measurements of both intracellular Ca(2+) concentration ([Ca(2+)](i)) and prolactin (PRL) gene promoter activity in single, living pituitary cells. Cells were transfected with the luciferase reporter gene under control of the PRL promoter and subjected to bioluminescence and fluorescence imaging before and after presentation of TSH-releasing hormone (TRH), a prototypic regulator of PRL secretion and gene expression that induces a transient Ca(2+) release, followed by sustained Ca(2+) influx. We found that cells displaying specific photonic emissions (i.e. mammotropes) showed heterogeneous calcium and transcriptional responses to TRH. Transcriptionally responsive cells always exhibited a TRH-induced [Ca(2+)](i) increase. In addition, transcriptional responses were related to the rate of Ca(2+) entry but not Ca(2+) release. Finally, cells lacking transcriptional responses (but showing [Ca(2+)](i) rises) exhibited larger levels of resting PRL promoter activity than transcriptionally responsive cells. Thus, our results suggest that the sustained entry of Ca(2+) induced by TRH (but not the Ca(2+) release) regulates transcriptional responsiveness. Superimposed on this regulation, the previous, resting PRL promoter activity also controls transcriptional responses.

PRL gene expression in individual living mammotropes displays distinct functional pulses that oscillate in a noncircadian temporal pattern.

PRL gene expression in the anterior pituitary has been the focus of intensive investigation for many years, but very little information is available on the actual dynamics by which this process occurs in individual mammotrope cells. Here, we used single cell bioluminescent imaging microscopy and a recently refined reporter gene strategy to measure PRL promoter-driven gene expression (PRL-GE) in individual living primary mammotropes. Using this approach we report a new phenomenon involving repetitive on/off gene expression bursts that occurred in a distinctly noncircadian oscillatory pattern. Furthermore, we demonstrate a functional basis for these gene expression oscillations, inasmuch as PRL-GE pulses were sensitive to calcium-dependent modulation, which we show arose exclusively as changes in the shape of individual pulse episodes. Our results provide the first clear evidence that PRL-GE, in its homologous cell environment, displays oscillatory bursts of activity. Moreover, they strongly support the idea that these discrete on/off bursts of activity serve as an important determinant of the timing and level of PRL-GE under both basal and stimulated conditions.