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Persistent Synchronized Oscillations in Prolactin Gene Promoter Activity in Living Pituitary Cells¹

School of Biological Sciences (D.W.M., Z.C.S., M.R.H.W.), Life Sciences Building, University of Liverpool, Liverpool, L69 7ZB; Endocrine Sciences Research Group (D.W.M., A.J.N., R.A.K., N.T., M.S.G., W.R.R., J.R.E.D.), Faculty of Medicine, University of Manchester, Manchester, M13 9PT; and School of Biological Sciences (J.A.S., A.S.I.L.), University of Manchester, Manchester, M13 9PT, United Kingdom

Address all correspondence and requests for reprints to: M. R. H. White, School of Biological Sciences, Life Sciences Building, University of Liverpool, Crown Street, Liverpool, L69 7ZB, United Kingdom ( E-mail: mwhite{at}liv.ac.uk) or to J. R. E.

	Abstract

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PRL gene expression in the anterior pituitary gland responds rapidly to different hormonal signals. We have investigated the long-term timing of transcriptional activation from the PRL, GH, and cytomegalovirus promoters in response to different stimulus duration, using real-time imaging of luciferase expression in living stably transfected GH3 cells. Long-term stimulation of serum-starved cells with 50% serum induced a homogeneous rise in PRL promoter activity, with subsequent heterogeneous fluctuations in luciferase activity in individual cells. When cells were subjected to a 2-h pulse of 50% serum, followed by serum-free medium, there were long-term (approximately 50 h) synchronized, homogeneous oscillations in PRL promoter activity. This response was PRL-specific, because in GH3 cells expressing luciferase from the GH or cytomegalovirus promoters, a serum pulse elicited no oscillations in luciferase expression after an initial transient response to serum. The PRL promoter may therefore be a template for an unstable transcription complex subject to stochastic regulation, allowing an oscillatory transcriptional response to physiological signals. This suggests that precise timing and coordination of cell responses to different signal-duration may represent a novel mechanism for coordinating long-term dynamic changes in transcription in cell populations.

	Introduction

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A KEY QUESTION in cell biology is how single cells in a population change their transcription phenotype in an ordered way that is coordinated between cells in the overall cell population. Many cells exhibit both immediate and sustained increases in gene expression in response to physiological stimuli. An important example is the anterior pituitary gland, which represents one of the most complex systemic signaling systems in the vertebrate body and is centrally involved in a wide range of physiological responses. It consists of a mixed population of cells, which respond to a range of different stimuli to express a set of physiologically important hormones (1). Expression of the PRL gene in the anterior pituitary gland responds rapidly to a range of hormonal and nonhormonal signals (2). It remains unclear how this variety of stimuli gives rise to the long-term complex dynamic control of the timing and level of PRL expression.

In cells that undergo long-term changes in gene expression, one model predicts that single cells are each subject to a stable change in transcription status, such that a specific gene is permanently activated in a given cell, generating a stable phenotype in an ordered manner in each cell in the cell population. An alternative model involves stochastic changes in gene expression, with changes in the overall level of gene expression in a cell population determined by the relative probability of gene activation in individual cells at a specific time (3). For example, in situ hybridization studies on cells undergoing the switch between fetal and adult globin gene expression have previously suggested that transcription flips backwards and forwards between the different globin genes (4). These and other studies (5, 6, 7) suggest that transcription complexes may be inherently unstable, permitting rapid and stochastic changes in transcription phenotype in single cells.

We have developed stably transfected GH3 pituitary cell lines (hPRL-Luc-GH3/D44) (8) expressing the luciferase reporter gene under the control of the human PRL (hPRL) 5000-bp 5'-flanking region promoter (9, 10, 11, 12). Using real-time imaging of luciferase expression (6, 13), we found that unstimulated promoter activity in gene expression was unstable, showing rapid and apparently random fluctuations in gene expression (8). These and other studies (14) demonstrate that the PRL transcription complex is unstable. The physiological significance and mechanisms underlying this dynamic heterogeneity are unclear.

In the present study, we report long-term analysis of transcription dynamics of the PRL gene promoter in single cells. We have used long-term imaging of gene expression to study the effect of stimulatory signals on PRL-promoter-directed transcription in single cells within a pituitary cell population. We show that in the stable pituitary cell lines, a short pulse (but not the continuous presence) of a high concentration of serum gives rise to homogeneous long-term oscillations in PRL promoter-directed transcription. Such oscillations did not occur when stably transfected cells expressing luciferase under the control of the human GH (hGH) or human cytomegalovirus (hCMV) promoters were subjected to the same stimuli, showing that this dynamic behavior is PRL promoter-specific. These novel data imply that long-term coordinated changes in transcription phenotype may be a necessary feature of the normal physiology of cells, allowing adaptive responses to complex patterns of stimuli in vivo.

	Materials and Methods

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Cell lines and reagents
The construction of the hPRL-Luc-GH3/D44 cell line was described previously (8). GH3 cells expressing the firefly luciferase reporter gene under the control of the hCMV promoter [pMW16 (6)] or the hGH promoter [-496/+1, kindly provided by Dr. P. Cattini, Manitoba, Canada (15),] were stably selected after cotransfection with a neomycin phosphotransferase expression vector and selection with G418 followed by ring cloning of stable transfectants. For each cell line, cells were normally grown in DMEM (containing phenol red) with the addition of pyruvate/glutamine and 10% FCS (Life Technologies, Inc., Paisley, UK).

Preparation of cells for imaging
Before each experiment, cells were plated out at 10⁵ cells/ml onto 35-mm coverslip dishes (MatTek Corp., Ashland, MA). They were incubated throughout each luminescence imaging experiment on the heated stage of an Axiovert 135 TV microscope (Carl Zeiss, Welwyn Garden City, UK) in a humidified chamber in 5% CO₂. Ten hours after plating onto the coverslip dishes, the cells were cultured in serum-free medium for a further 24 h. Luciferin (Bio-Synth Inc., Staad, Switzerland) was then added to the medium to a final concentration of 1 mM, and the cells were incubated for a further 12 h before each experiment.

Serum shock protocol
To investigate the response of the PRL promoter to transient stimulation, we studied its activity in serum-starved cells after a brief serum shock. The serum shock protocol used was based on that described by Balsalobre et al. (16), who showed that a 50% serum shock in Rat-1 fibroblasts caused oscillations in the levels of messenger RNAs encoding circadian clock or circadian output proteins. In all experiments, the serum-free medium was replaced with either fresh serum-free medium or medium containing 50% horse serum (Life Technologies, Inc.). The medium was then either left on the cells for the course of the experiment; or after 2 h, the cells were washed in serum-free medium and then exposed for the remaining experimental period to serum-free medium alone. Throughout these medium changes, the luciferin concentration was maintained at 1 mM.

Luminescence imaging
Luminescence imaging was carried out using a Hamamatsu VIM photon-counting CCD camera (C2400–40) attached to the base port of the microscope (17). Images were obtained using a 10x, 0.5 numerical aperture objective and a 0.63x C-mount adapter. Sequential images were acquired andanalyzed using the Hamamatsu Argus 50 photon-counting software. The images obtained were an integration of the photons counted during a 30-min sampling period. Collection of each image was automatically started every 3 h. Images used for display were slice images, processed using the Hamamatsu Photonics software with a smoothing function. The smoothing function used a 3x3 neighborhood matrix (weighted 2 for the central pixel and 1 for surrounding pixels) to provide a moving average over the image. This smoothes the image by suppressing sharp changes and is effective in eliminating noise. Before display, background in these images was reduced by removing 1 count from each pixel (offset = -1). Center of gravity images were used for quantification, either using total photon counts from the whole field of view or photon counts derived from defined cellular areas (477 pixels). Typically, a total field of 100–200 cells was visualized. An average background noise level of 2,788 (±58) counts was observed from the whole field of 262,144 pixels per 30-min integration.

Quantification and statistical analysis
Data were subjected to mathematical analysis to assess the significance of the oscillations observed, using time series analysis (18) to determine the dominant frequency. The Cluster peak detection algorithm (19) and analysis of approximate entropy (20) were used to evaluate the statistical significance of fluctuations in luciferase activity.

Cell cycle analysis
Cells were detached from triplicate Petri dishes by trypsinization at 4-h intervals after exposure to serum. Propidium iodide, 50 mg/ml (Sigma, Poole, UK), in PBS containing 0.15% Triton X-100 (vol/vol) and 0.15 mg/ml ribonuclease A was then added to the cell suspension. DNA content was estimated by measurement of propidium iodide fluorescence from cell samples (21) using an Ortho Cyteron absolute flow cytometer (Ortho Diagnostic Systems Inc., Raritan, NJ).

	Results

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Population response of pituitary cells to serum stimulation
To investigate the response of the PRL promoter to transient stimulation, we studied its activity in serum-starved cells after a brief serum shock. Subconfluent hPRL-Luc-GH3/D44 cells were transferred to serum-free conditions for 36 h (cell cycle analysis by flow cytometry indicated that 15% of the serum-starved cells were in G2/M phase, which was as low as could be achieved; data not shown). Cells were then exposed to a 2-h pulse of 50% horse serum, and then placed in serum-free medium for 100 h. Luminescence images, integrated over 30-min periods, were collected before and after serum addition and then at 3-h intervals. The hPRL-Luc-GH3/D44 cells displayed synchronized and persistent oscillations after the serum pulse (Figs. 1A and 2A). An initial peak of activity lasted for up to 8 h after the serum stimulation, after which the photon counts returned to baseline levels and remained steady for over 24 h. A second broader peak of luciferase activity started 33 h after the serum pulse, reaching a peak at 48 h, before again declining to baseline. In the experiment shown in Fig. 1A, a third peak was seen starting at approximately 90 h (Figs. 1A and 2A).

View larger version (17K): [in this window] [in a new window]   Figure 1. Quantification of the dynamics of firefly luciferase activity in stably transfected pituitary cells after transient and long-term serum treatment of resting cells. A, Total luminescence counts emitted from a field of serum-starved hPRL-Luc-GH3/D44 cells (5 ) that were treated with 50% horse serum for 2 h, followed by return to serum-free conditions. Cells were analyzed by luminescence imaging for 102 h from the time of serum addition. B, Total luminescence counts emitted from a field of serum-starved hPRL-Luc-GH3/D44 cells treated with 50% horse serum for 88 h and analyzed by luminescence imaging. C, Total luminescence counts emitted from a field of serum-starved hPRL-Luc-GH3/D44 cells treated with serum-free medium (in place of the serum in A and B above) and then analyzed by luminescence imaging for 93 h. D, Total luminescence counts emitted from a field of serum-starved hCMV-Luc-GH3/D5 cells exposed to 50% horse serum for 2 h, followed by return to serum-free conditions. Cells were monitored for 102 h by luminescence imaging. E, Total luminescence counts emitted from a field of serum hGH-Luc-GH3/YX30 cells exposed to 50% horse serum for 2 h, followed by return to serum-free conditions. Cells were monitored for 86 h by luminescence imaging.

View larger version (86K): [in this window] [in a new window]   Figure 2. Luminescence images obtained from pituitary cells expressing luciferase under the control of the PRL promoter, compared with the hCMV promoter at different time intervals after a 2-h serum shock. A, hPRL-Luc-GH3/D44 cells, exposed to 50% horse serum for 2 h, as described above (see Fig. 1A). The inset shows an enlarged single-cell area to illustrate the behavior of a typical individual cell through the experimental period. B, Images of hCMV-Luc-GH3/D5 cells after exposure to 50% horse serum for 2 h (see Fig. 1D). In each case, the time intervals marked on each image represent the time between the addition of serum to the cells (time 0 h) and the time that the 30-min image integration was started.

To investigate whether this oscillation was PRL promoter-specific, we constructed stable transfectant cell lines with the luciferase reporter gene under the control of an alternative pituitary hormone promoter and a control viral promoter. Expression vectors with either a 500-bp hGH promoter or an hCMV-luciferase expression vector in front of the luciferase reporter gene were stably transfected into the parental GH3 pituitary cell line (the resulting cell lines were referred to as hGH-Luc-GH3/YX30 or hCMV-Luc-GH3/D5 cells, respectively). In both these cell lines, an initial transient response of luciferase expression was seen after serum shock, with luciferase activity falling rapidly back to baseline values for the remainder of the observation period (Fig. 1, D and E). In marked contrast to the hPRL-Luc-GH3/D44 cells, there was no evidence for subsequent oscillations with either the hGH-Luc-GH3/YX30 or hCMV-Luc-GH3/D5 cells.

Imaging and analysis of single-cell responses
Figure 2 shows images of the data shown graphically in Fig. 1, A and D, representing fields of hPRL-Luc-GH3/D44 cells and hCMV-Luc-GH3/D5 cells at different time intervals after the 2-h serum shock. Initial observation of these images suggested that individual hPRL-Luc-GH3/D44 cells within the population responded in a homogeneous manner. The insets in Fig. 2A are a magnified image showing the oscillations from a typical single cell within the field.

To quantify the variation in gene expression in individual single cells, we measured the time-course of luminescence from a series of single-cell areas (Fig. 3). Analysis of luciferase activity in the serum-pulsed hPRL-Luc-GH3/D44 cells (Fig. 3A) indicated that the major oscillations in PRL promoter activity were synchronous in the cell population, being seen in over 90% of cells (36 cells analyzed). Analysis of individual cells subjected to the mock serum shock showed no evidence of significant changes in the low basal level of gene expression at the single-cell level (data not shown).

View larger version (26K): [in this window] [in a new window]   Figure 3. Analysis of the dynamics of gene expression in single cells within the cell populations. A, Profile of the luminescence from 8 hPRL-Luc-GH3/D44 cells after 2-h serum shock; B, luminescence counts from 8 hPRL-Luc-GH3/D44 cells after continuous serum addition to resting cells; C, 8 representative cell areas of hCMV-Luc-GH3/D5 cells after 2-h serum shock; D, 8 representative cell areas of hGH-Luc-GH3/YX30 cells after 2-h serum shock. Counts were taken from representative cell areas of 477 pixels at different time intervals after serum addition to the cells.

Single-cell analysis of the serum-shocked hCMV-Luc-GH3/D5 cells (Fig. 3C) or hGH-Luc-GH3/YX30 cells (Fig. 3D) showed a homogeneous rise in expression in 33 out of 35 cells analyzed (hCMV) and 19 out of 19 cells analyzed (hGH), followed by a fall to basal level expression. Only 2 of 35 of the hCMV-Luc-GH3/D5 cells and 1 of the hGH-Luc-GH3/YX30 cells showed any significant subsequent change in expression level, and these changes were not maintained for a long period.

Cell cycle analysis
In parallel experiments, hPRL-Luc-GH3/D44 cells were subjected to cell cycle analysis after exposure to the 2-h pulse of 50% serum. Cells were analyzed at 4-h intervals for 72 h, by flow cytometry, and no consistent changes were seen in the proportions of cells in G1 phase or G2/M phase (Fig. 4). These data indicate no major alteration in cell cycle kinetics that could be responsible for the marked oscillations observed in PRL promoter activity.

View larger version (16K): [in this window] [in a new window]   Figure 4. Analysis of cell cycle kinetics in hPRL-Luc-GH3/D44 cells after 2-h serum shock. Cell samples were collected, every 4 h, after a 2-h exposure to 50% horse serum. Flow cytometry was used to assess the DNA content of cells. Representative DNA histograms at 24-h intervals are shown.

	Discussion

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One possibility is that our results might be related to observations by Balsalobre et al. (16), who, using a similar protocol, observed 22.5-h near-circadian oscillations in clock genes and clock output genes. In multiple experiments using the present protocol, we consistently observed subsequent homogeneous oscillations in PRL promoter activity, although, interestingly, the periodicity varied between 50 h and 75 h in different experiments after the initial peak in luciferase activity. Remarkably, we observed good homogeneity between individual cells in every experiment. This suggests, therefore, either that an endogenous timing mechanism resides in the lactotrophic cell or that a completely novel cell-cell signaling mechanism exists. The liability of pituitary cells to generate oscillatory patterns of transcription in response to extracellular signals may be related to the function of circadian clock components, though the long periods that we have observed might imply a distinct oscillatory mechanism. Clock genes are expressed in peripheral tissues (22), and oscillatory expression can be induced by activation of the cyclic AMP response element binding protein pathway (23) or by glucocorticoid treatment (24) in Rat-1 fibroblasts. Further studies will be needed to address whether similar mechanisms occur in endocrine tissues.

The alternative possibility to account for our data is that the oscillatory patterns of promoter activity are unrelated to clock phenomena, but simply represent an inherent instability in transcriptional response characteristics. It has been suggested that gene enhancers increase the probability, rather than the level, of gene transcription (7), and this may be affected by a variety of potential stimuli. Many noncircadian oscillatory phenomena have been observed in different systems, and an interaction between noise and stochastic responses of the components may moderate the overall patterns of gene expression in the intact organism.

These observations may also be important in vivo, because others have reported variations in PRL gene expression between individual primary lactotrophic cells derived from female rat pituitary glands (14). We suggest that in genes whose activity tends to change rapidly and regularly in response to a range of signals, a tendency to respond in a stochastic (4, 5, 6, 7, 8) or oscillatory manner may be important for in vivo physiology. Our approach of continuous reporter gene imaging provides a unique opportunity to study coordinated gene expression throughout a cell population over long periods of time (80–100 h). The observation of the contrast between an unstable heterogeneous system in constantly stimulated conditions and the homogeneous, sustained oscillation after brief stimulation, opens up the exciting possibility that the timing of signaling may affect the complex long-term program of gene expression changes.

	Acknowledgments

 
We are grateful to D. Spiller, M. Castro, A. Cossins, and A. McNeilly for advice and critical reading of the manuscript; and to Dr. P. A. Cattini for the gift of the hGH-luc construct.

	Footnotes

 
¹ This work is supported by a Prize Ph.D. studentship from the Society for Endocrinology (to D.W.M.), the Biotechnology and Biological Sciences Research Council, the Wellcome Trust (Fellowship to A.J.N.), Pharmacia & Upjohn, Inc. (Milton Keynes, UK), Hamamatsu Photonics (Hamamatsu City, Japan), and Carl Zeiss (Welwyn Garden City, UK).

² Present address: Department of Immunology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo, 162-8640, Japan.

³ Present address: The Conway Institute, c/o Biotechnology Centre, University College Dublin, Belfield, Dublin 4, Ireland.

Received February 8, 2001.

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This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by McFerran, D. W. Articles by White, M. R. H. Search for Related Content PubMed PubMed Citation Articles by McFerran, D. W. Articles by White, M. R. H.