Dynamic Changes in Prolactin Promoter Activation in Individual Living Lactotrophic Cells

Abstract

The firefly luciferase gene has become widely used as a convenient reporter for studies of gene promoter regulation. Very recently, the development of ultralow-light imaging cameras has enabled the quantitative digital imaging of light signals resulting from luciferase activation in the presence of luciferin substrate. We have applied this technology to the study of PRL promoter activation in individual pituitary tumor cells to study the temporal and spatial characteristics of the expression of a well-characterized pituitary hormone gene. Rat pituitary GH3 cells were transfected by lipofection with a luciferase reporter gene linked to 5000 bp from the human PRL gene 5'-flanking region. A series of stably transfected cell clones were generated, and one of these was chosen for detailed study on the basis of appropriate regulation of high-level luciferase expression by a series of known stimuli including TRH, forskolin, the calcium channel agonist Bay K8644, and basic fibroblast growth factor (bFGF). These cells were subjected to direct imaging of luciferase activity using a Hamamatsu photon-counting camera linked to a Zeiss Axiovert microscope with an Argus-50 image processor. Cells were exposed to 1 mM luciferin, and images were integrated over 30-min periods for up to 72 h. The total photon count over a given field settled to steady levels within 10 h and then remained constant for over 55 h. Addition of forskolin, TRH, or bFGF increased the total photon count of fields of 20-100 cells by 2- to 4-fold consistent with previous data from transient expression assays using the human PRL promoter. Individual cells, on the other hand, showed marked marked temporal and spatial heterogeneity and variability of luciferase expression when studied at 3-h intervals. Unstimulated cells showed variable luciferase expression with up to 40-fold excursions in photon counts per single cell area within 12-h periods. Stimulation of cells with either TRH, forskolin, or bFGF resulted in smooth increases in photon output over fields of 20-100 cells, but again individual cell responses differed widely, with some cells showing slow progressive rises in photon output, others showing phasic or transient responses, and yet others showing no response. In conclusion, we found a surprising degree of heterogeneity and temporal variability in the level of gene expression in individual living pituitary tumor cells over long periods of time, with markedly divergent responses to hormonal or intracellular stimulation. The use of stably transfected clonal cell lines with extended periods of reporter gene imaging offers a valuable insight into control of gene expression in living cells in real time.

Real-time imaging of gene promoter activity using an adenoviral reporter construct demonstrates transcriptional dynamics in normal anterior pituitary cells

“…The firefly luciferase reporter gene has become widely used for assays of promoter function, and its activity can be monitored by microscopic luminescence imaging (White et al 1990, 1995, Craig et al 1991, Rutter et al 1998. Using real-time microscopic imaging of a stably transfected rat pituitary cell line, we have recently demonstrated that prolactin (PRL) promoter activity in individual living cells is not stable and exhibits unexpected 40-fold fluctuations from hour to hour (Takasuka et al 1998). Although different individual cells display markedly different mRNA content and peptide secretion rates as determined by in situ hybridization and haemolytic plaque assays (Hofland et al 1991), our data indicated that this heterogeneity is subject to rapid dynamic change (Takasuka et al 1998).…”

“…Using real-time microscopic imaging of a stably transfected rat pituitary cell line, we have recently demonstrated that prolactin (PRL) promoter activity in individual living cells is not stable and exhibits unexpected 40-fold fluctuations from hour to hour (Takasuka et al 1998). Although different individual cells display markedly different mRNA content and peptide secretion rates as determined by in situ hybridization and haemolytic plaque assays (Hofland et al 1991), our data indicated that this heterogeneity is subject to rapid dynamic change (Takasuka et al 1998). The mechanisms for the instability of PRL gene transcription are unknown, but recent imaging and biochemical data indicate that nuclear hormone receptors are recruited to and released from chromatin very rapidly in response to hormonal signals (McNally et al 2000, Shang et al 2000.…”

“…To date our studies have relied on stably transfected cell lines (Takasuka et al 1998, McFerran et al 2001, Norris et al 2003, but it is vital that this work be extended to normal cells, to allow better understanding of physiological processes, and applied to other species. Pioneering studies using microinjection of individual pituitary cells have revealed important information about pituitary gene regulation in relation to endocrine signals (Castano et al 1996, Villalobos et al 1998, but the microinjection procedure is technically difficult and inevitably entails the selection of a small number of cells for any given experiment.…”

Novel approaches to in vitro transgenesis

“…Furthermore, these assays measure gene expression by extracting RNA from a population of cells, thus averaging gene expression across an entire population. Single-cell analysis using sophisticated microscopy has revealed startling heterogeneity of gene transcription between cells even within clonal populations (Takasuka et al 1998, Ashall et al 2009, Eldar & Elowitz 2010.† What kind of delivery system would be most appropriate (plasmid, viral, episomal, stably integrated or transient expression).…”

Calcium influx and DREAM protein are required for GnRH gene expression pulse activity

“…A pulse was defined as a significant rise in photonic activity corresponding to a value greater than 5 % of the photonic signal count at the beginning of the pulse plus two times the total signal-noise variation (SNV). SNV was calculated by the square root of each value plus the standard deviation (SD) of the corresponding background values squared (SNV = √[(signal count) + (SD of detected background 2 )]) (Takasuka et al, 1998). All the cells analyzed expressed a detectable level of luciferase activity (generating photonic patterns with or without pulses) that was significantly higher than the background level indicating that the cells remained viable during the course of the experiment.…”