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PRL Gene Expression in Individual Living Mammotropes Displays Distinct Functional Pulses That Oscillate in a Noncircadian Temporal Pattern

Laboratory of Molecular Dynamics, Department of Cell Biology and Anatomy, Medical University of South Carolina (G.M.L., R.V.-M., D.C.L., W.J.F., L.S.F., F.R.B.), Charleston, South Carolina 29425

Address all correspondence and requests for reprints to: Dr. Fredric R. Boockfor, Laboratory of Molecular Dynamics, Department of Cell Biology and Anatomy, Medical University of South Carolina, 173 Ashley Avenue, Charleston, South Carolina 29425. E-mail: . boockfor{at}musc.edu

	Abstract

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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.

	Introduction

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THE PRECISE TEMPORAL dynamics by which gene promoters regulate gene expression in single living cells is currently a key question in cell biology, especially in cellular endocrinology, because temporal regulation in endocrine tissues is often a critical factor for correct physiological hormone function. It is therefore of great interest that one currently emerging view encompasses the idea that gene expression inside individual cells may occur in temporally episodic bursts or pulses (1, 2, 3, 4, 5). This idea of pulsatile gene expression has a strong theoretical basis. However, there are currently few biological examples due in part to the fact that in single cells [where such pulsatile dynamics should be predicted to manifest most profoundly (6)] measurement of gene expression is technically difficult to achieve. Fortunately, recent advances in imaging-microscopy have brought about the means to address these fundamental issues. Notably, several investigations employing such novel techniques have focused on the study of endocrine cell paradigms. Of these models, PRL gene expression is especially useful because it exemplifies a regulated gene expression activity mediated by RNA polymerase II (7) that is completely cell specific, being restricted to pituitary mammotropes (8). To date, this single cell analysis of mammotropes has generated some preliminary evidence to indicate that PRL gene expression may occur in a pulsatile manner. For example, one group, using neoplastic rat pituitary cells that expressed a human PRL promoter-driven luciferase reporter gene, found distinct episodes of intermittent PRL promoter expression in single cells occurring every 2–3 d (9, 10). The results of this study clearly demonstrated the presence of pulses, but no determinant analysis of this activity was presented. Moreover, its relationship to PRL gene expression in the pituitary, especially the dynamics, remained open to question, inasmuch as the cells were stably transfected with a heterologous promoter (human), and pulse modification was only achieved using extreme serum concentrations (50%). By contrast, our own investigations of PRL gene expression have employed nondividing primary cultures of rat pituitary mammotropes in which a rat PRL promoter (in a luciferase reporter construct) was microinjected, and the cells were exposed to much lower serum concentrations (10%) for culture. With this paradigm, we recently demonstrated that individual mammotropes displayed what we termed random fluctuations in PRL promoter-driven luciferase expression (PRL-GE) at a rate of one to several times a day, but a quantitative characterization again proved elusive (11). However, when taken together, it appears that intermittent bursts or pulses of PRL-GE occur in individual PRL cells.

In the current study we sought to define clearly the dynamics of these PRL gene expression fluctuations in single mammotropes. To achieve this, we first developed a modified PRL-GE luciferase reporter vector, which provided an enhanced dynamic resolution of PRL-GE. Using this new probe we have for the first time been able to clearly visualize and quantify the temporal dynamics of PRL-GE pulses. Moreover, we have begun to establish the functional relationship between these pulses and overall gene expression in single cells by evaluating alterations in the pulse shape dynamic that occur in response to increased calcium entry through L-type, voltage-gated, calcium channels, a treatment known to enhance PRL gene transcription directly (12, 13, 14). The results of our study indicate that pulse activity is a fundamental property of both basal and stimulated PRL gene expression in individual mammotropes.

	Materials and Methods

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Reporter constructs
The new reporter plasmid (pPLDA12, PRL-lucPEST) was constructed by subcloning a 480-bp PCR-amplified fragment bearing a proteolytic signal consisting of a proline-, glutamate-, serine-, and threonine-enriched ornithine decarboxylase region with the amino acid substitutions D433A and D434A (PEST-ODCDA) from pDA61 (15) into the EcoNI and ApaI sites of pPRL-LUC vector (16). This yielded an in-frame fusion of the PEST sequence with the luciferase COOH-terminal sequence. PCR amplification was carried out with the primers LUCODC-F1 (5'-AAAATCAGAGAGATCCTCATAAAGGCCA) and LUCODC-R10APA (5'-TTCATCGGGCCCATAGGTTGGAATC), which contain the cloning restriction sites EcoNI and ApaI, respectively (underlined). The PCR was performed with Pfu DNA polymerase (Stratagene, La Jolla, CA) using standard techniques (17). Cycling conditions were 94 C for 1 min, 60 C for 30 sec, and 72 C for 30 sec, for a total of 34 cycles. The integrity of the resulting construct was determined by dideoxynucleotide sequencing.

Animals
Primiparous lactating female rats (6–10 d postpartum; Harlan Sprague Dawley, Inc., Madison, WI) were individually housed with their litters (six pups each) and maintained under standard conditions until the day of death and removal of the anterior pituitary gland. These conditions consisted of ambient temperature ranging from 24-26 C, relative humidity of 50–60%, artificial lighting between 0500 and 1900 h, and free access to water and pelleted food. All procedures involving these animals were conducted in accordance with protocols approved by the institution and the university committee on animal care.

Cell culture and microinjection
Primary cultures of anterior pituitary cells were obtained by trypsin dispersion and cultured on 45-mm (custom grid-etched) glass coverslips coated with poly-L-lysine as described previously (18). Cultured cells were maintained (37 C, 5% CO₂-95% air) in medium (phenol red-free DMEM, Life Technologies, Inc., Grand Island, NY) supplemented with FBS (10%) and antibiotics. After 2 d in culture, cells were transfected by microinjection (Eppendorf 5242) as reported previously, except that PRL-lucPEST (0.2 µg/µl) was used. Cells were then incubated under normal culture conditions, and experiments were performed 24–48 h later.

Bioluminescent imaging
Transfected cells were placed in a temperature-controlled (37 ± 0.2 C) observation chamber (Bioptechs FCS2, Biological Optical Technologies, Inc., Butler, PA) mounted on the stage of an upright microscope (Axioskop, Carl Zeiss, Jena, Germany). The chamber (75-µl volume) was perfused (70 µl/min) by laminar flow with culture medium containing luciferin (0.1 mM) alone or with drugs. These drugs consisted of either BAYK8644 (300 nM) plus KCl (10 nM) or nimodipine (600 nM; both from Calbiochem-Novabiochem, San Diego, CA). Photon images were visualized using x20 (Fluar, Carl Zeiss) or x10 (Neofluar, Carl Zeiss) objectives that provided good signal/noise ratio and photonic image brightness. Single cell bioluminescence was measured with a photon-counting camera system (Argus 50, Hamamatsu, Bridgewater, NJ) that was set to accumulate photons during 30-min windows, which were repeated contiguously for up to 70 h.

Quantification and analysis
Photonic images were analyzed off-line as previously described (11, 18). PRL-GE pulses were considered significant when they comprised an increase lasting more than 2 h and more than twice the calculated estimate of signal/noise variation (SNV; [(signal count) + (SD of detected background²]) (9). For Fourier analysis, harmonic peaks were considered significant if the amplitude peak value was greater than the average + 2x SEM of Fourier noise (FN). FN was calculated from amplitude fluctuations detected below a periodicity of 3 h. For drug treatment experiments, the results obtained with BAYK8644 and nimodipine were analyzed by determining the area under the curves (profiles) and the means of pulse indexes such as peak amplitude (peak maxima), frequency, and duration and comparing them to controls. This was accomplished using one-way ANOVA, followed by Newman-Keuls test for multiple comparisons. Single comparisons (control vs. nimodipine only) were made using unpaired t tests. P < 0.05 was considered significant.

	Results

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Pulsatile oscillations of PRL-GE in individual primary mammotropes
Wild-type luciferase has a relatively long half-life that (in the context of a reporter gene) is likely to result in distorted signal dynamics, making transient signal changes difficult to resolve. For this reason we engineered our existing pPRL-LUC plasmid (19) by addition of a proteolytic signal sequence (see Materials and Methods). This modification greatly reduces the luciferase half-life inside living cells and thereby improves reporter gene signal dynamics (15). Cells transfected with the new reporter gene were monitored using single cell bioluminescence microscopy, revealing distinct pulses of reporter gene expression in 57 of 77 mammotropes (n = 7). Figure 1 shows a representative example of this behavior, where two individual cells displayed PRL-GE pulses.

View larger version (30K): [in this window] [in a new window]   Figure 1. Individual mammotropes display pulses of PRL reporter gene expression. A, The black and white panel shows a brightfield view of two mammotropes that were subsequently imaged, and selected examples taken at the times indicated are presented (pseudo-colored panels). As illustrated by the pseudo-colored bar in the image taken at 6 h, warmer-colored pixels represent higher photon count levels. In this example each cell gives a burst-like pulse of PRL reporter gene expression at different times during the experiment (18 and 33 h). Note that the actual dynamic range used to generate the colored images is represented by the pseudo-colored bar (PRL-GE = signal/SNV). B, The averaged dynamic of 47 PRL-GE pulses, which were temporally aligned according to their peak amplitudes. Data from individual pulses were first normalized by dividing (point by point) the observed photon signal by the calculated SNV (see Materials and Methods). The gray-shaded area indicates the level of deflection that was used as a criterion for pulse activity (i.e. 2 times the SNV). Vertical bars indicate the SEM.

View larger version (38K): [in this window] [in a new window]   Figure 2. PRL reporter gene pulses in single cells occur repetitively, in an oscillatory manner, and across a wide range of frequencies. A, Representative PRL-GE recordings from six individual mammotropes grouped into pairs according to the frequency at which these cells elaborated PRL-GE pulses. The photon signal is expressed as a function of the SNV (see Materials and Methods). The gray-shaded area and vertical error bars indicate the level of deflection that was used as a criterion for pulse activity (see Materials and Methods), and the asterisks indicate pulses. B, Representative examples of amplitude/periodicity plots for Fourier transformations calculated from PRL-GE recordings shown on the three uppermost axes of A, which displayed ultradian, diurnal, or infradian pulse frequencies, respectively, and harmonic peaks are indicated by asterisks. Note that the x-axis represents any 30-h period of the experiment and does not directly reflect a specific part of the profile presented in the panel above. The presence of a peak in this profile indicates a timed repeating pulse occurrence or harmonic. The higher the amplitude of the peak, the stronger the harmonic of pulses timed in this manner. Also note the absence of harmonic peaks for the cells that displayed only a single PRL-GE.

Effect of L-type voltage-gated calcium channel activation on PRL-GE
We reasoned that if PRL-GE pulses were functionally important in dictating overall levels of PRL promoter-driven gene expression, then known regulators of PRL-promoter activity should also modulate pulsatility. Previous studies have shown that pharmacological activation of L-type voltage-gated calcium channels (L-VGCC) causes an increase in PRL-GE in populations of primary mammotropes, whereas L-VGCC inhibition has the opposite effect (12, 13, 14), due to calcium-sensitive regulatory elements in the PRL promoter (8). For this reason, we chose to test the effects of BAYK8644 (a specific L-VGCC agonist that strongly enhances calcium entry by maintaining the open state of individual channels) and nimodipine (a specific L-VGCC antagonist that abolishes calcium entry by inhibiting channel opening).

Influence on cell population averages.
To confirm that each drug exerted its predicted effects, we first exposed cells constantly to either nimodipine or BAYK8644 and analyzed the areas under the PRL-GE profiles averaged from cell populations. As shown in Fig. 3A and compared with control cells (first panel), continuous L-VGCC activation by BAYK8644 caused PRL-GE to rise continuously during an initial 5-h period, whereupon the signal stabilized at a maximal level for approximately 10 h and then decayed slowly back to basal levels during the next 10 h (second panel). Overall, the PRL-GE levels in response to BAYK8644 were significantly higher than control levels [42.3 ± 6.3 vs. 98.0 ± 35.8 photonic emissions/hour (peh), control vs. treated, respectively]. In contrast, nimodipine treatment (third panel) appeared to cause a decline in PRL-GE, but analysis of controls vs. nimodipine treatment for the entire treatment period did not reveal a difference (42.3 ± 6.3 vs. 28.4 ± 3.2 peh, respectively). Other laboratories have reported an influence of nimodipine on overall PRL gene expression, but in each case the treatment was for 18 h or longer (12, 21). To determine whether nimodipine influences could be detected after a longer treatment period in these experiments, we reanalyzed the data by partitioning it into two periods, 5–20 h and 20–35 h. Although analysis revealed no differences in PRL-GE between controls and nimodipine-treated cells during 5–20 h of treatment (18.1 ± 2.1 and 15.2 ± 1.3 peh, respectively), we found that PRL-GE declined in cells that were treated with nimodipine for a longer time (20–35 h; 23.9 ± 4.4 vs. 13.3 ± 2.3 peh, control vs. treated). Additional analysis of intermittent treatment with nimodipine (5 h) followed by BAYK8644 (5 h) was accomplished by comparing the averaged levels of PRL-GE at the beginning of each intermittent treatment interval to that at the end. Using this approach, we found that the initial exposure to nimodipine followed by BAYK8644 caused a moderate decrease and then a subsequent increase in PRL-GE (fourth panel). Furthermore, readdition of nimodipine caused PRL-GE to decrease again, clearly demonstrating that PRL-GE was directly responsive to multiple exposures to these L-VGCC drugs. Although further addition of BAYK8644 did not cause a significant change, subsequent addition of nimodipine caused a small, but significant, reduction in PRL-GE. When taken together, these results demonstrated clearly that at the cell population level, both L-VGCC-specific drugs behaved conventionally in our hands, demonstrating their usefulness for single cell analysis of PRL-GE pulses.

View larger version (54K): [in this window] [in a new window]   Figure 3. L-VGCC drug-induced changes in PRL-GE measured at the single cell level do not clearly reflect changes averaged from the whole cell population. A, PRL-GE signal averaged from populations of cells that received no drug treatment (control), BAYK8644 (300 nM plus 10 mM KCl; gray bar), nimodipine (600 nM; open bar), or 5-h intermittent treatment using both drugs (two-tone bar). Differences between BAYK8644 or nimodipine and controls were determined by comparison of areas under the curves. The averaged data are normalized to the initial signal level, and the vertical bars show the SEM for 40, 28, 19, and 23 cells, respectively. B, Exact same data as in A, but this time each axis is plotted with the single cell PRL-GE recordings. Note that pulses continue unabated throughout the course of all experimental treatments.

As shown in Table 1, our analysis revealed that regardless of drug treatment, pulse frequency, peak amplitude, and duration remained unaltered. This suggested strongly that the averaged changes detected at the cell population level were more likely to reflect subtle single cell changes that were not reflected by these parameters. The next most obvious possibility for single cell PRL-GE pulse changes was the empirical shape of the individual pulse profile. To examine this possibility, we analyzed the shape of PRL-GE pulses by aligning individual pulse episodes according to the first point demarcating the initiation of each pulse. These data were then averaged into a single profile for each drug treatment condition (Fig. 4A). With this approach, we found changes induced by treatment of cells with BAYK8644 and nimodipine. Most notably, the effect of BAYK8644 was clearly revealed as a strong prolongation of the period over which the highest levels of PRL-GE were maintained after pulse initiation (Fig. 4A, gray circles). For comparison, the pulse shape profiles measured in control cells reached lower levels and subsequently decayed into a shoulder phase, marked by a distinct inflection point (Fig. 4A, solid circles). When analyzed by comparison of areas under each profile, we found that PRL-GE increased, with BAYK8644 reaching almost twice that in controls (69.6 ± 5.8 vs. 136.2 ± 21.4 peh, controls vs. treated). The pulse shape profile calculated from cells treated with nimodipine exhibited no shoulder phase, but when analyzed revealed that the area under the profile was not different from the control value for pulses cumulated for the entire treatment period (69.6 ± 5.8 vs. 55.5 ± 3.4 peh, controls vs. nimodipine-treated). In light of our observations that nimodipine treatment did not cause overall changes in PRL-GE until the second half of the experiment, we compared PRL-GE pulses from nimodipine-treated and control cells that occurred from 20–35 h (Fig. 4B). Analysis of the area under the curves of these pulse profiles revealed a significant nimodipine-induced decrease compared with controls (67.3 ± 5.5 vs. 48.0 ± 3.2 peh, controls vs. nimodipine-treated).

View larger version (25K): [in this window] [in a new window]   Figure 4. L-VGCC drugs modulate single cell PRL-GE by altering the shape of individual pulses: evidence for a specific calcium-sensitive component. A, Averaged PRL-GE pulse shape profiles measured from each condition (as described in Fig. 3) calculated by alignment of pulses according to the first point preceding pulse initiation. The averaged pulse shape was calculated for cells that received no treatment (control, solid circles), BAYK8644 (gray circles) or nimodipine (open circles). Differences between BAYK8644 or nimodipine and controls were determined by analysis of areas under the profiles. Vertical bars show the SEM calculated from 47, 37, and 29 individual pulses, respectively. B, Averaged PRL-GE pulse shape profiles measured during 20–35 h of the absence (control, solid circles) or presence (open circles) of nimodipine and aligned as described above. Analysis was performed by comparison of the areas under the averaged pulse profiles for pulses measured during this time period.

	Discussion

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Our conclusion that PRL-GE occurs in periodic pulses demands that we consider what cellular mechanisms could underlie the timing of this pattern of activity. The two most obvious possibilities are not supported by our results. The first idea is that PRL-GE pulse-timing could involve intracellular feedback signaling between PRL hormone release and biosynthesis. This is suggested by the observations that individual mammotropes display episodic variations in basal PRL secretory activity (25) and from studies in Paramecium where hormone release triggers transcriptional activation of genes encoding proteins stored in secretory granules (26). From this it would be reasonable to suggest that discrete episodes of PRL release should correspond to episodes of PRL-GE, which would result in pulses at timed intervals depending upon hormone release activity. However, because basal PRL release is known to depend critically upon L-VGCC intracellular Ca²⁺ ([Ca²⁺]_i) entry, (27, 28) our finding that the frequency of PRL-GE pulse elaboration remained unaffected by long-term L-VGCC inhibition is strongly inconsistent with this possibility. The second pulse-timing/elaboration hypothesis is based on our previous suggestion that ordered patterns of spontaneous [Ca²⁺]_i oscillations in mammotropes directly trigger PRL-GE fluctuations (11, 18, 29). However, inhibition of L-VGCC by nimodipine, which has been shown to greatly reduce the frequency of [Ca²⁺]_i oscillations in clonal pituitary cells (30), had little influence on the frequency of PRL-GE pulses in our study. For this reason, it is unlikely that [Ca²⁺]_i oscillations play a direct role, if any, on the initiation or timing of PRL-GE pulses.

Several novel findings in the current study do point toward an alternative hypothesis for the mechanism underlying pulse elaboration/timing. We have shown that PRL-GE pulses comprised discrete episodes, remarkably reminiscent of an on/off signal dynamic. This fact is made all the more striking when considered in light of abundant evidence supporting a model view that transcriptional activity (ergo gene expression) follows an on/off dynamic due to the probabilistic nature of enhancer effects on promoter activity (31, 32). Previous studies provided evidence that L-VGCC calcium entry controls overall PRL-GE (measured in mammotrope cell populations) by a transcriptional mechanism, inasmuch as it depended upon specific regions of the PRL promoter (12, 14, 33). Because our current results show that treatment with agents shown to influence L-VGCC calcium entry leads to changes in the profile of single cell PRL-GE pulses, it is logical to suggest that individual PRL-GE pulse episodes reflect, at least in part, oscillatory on/off bursts of transcriptional activity. However, our results further suggest that modifications in calcium influx, rather than controlling pulse initiation, act by shifting the dynamics of initiated pulses. Moreover, in light of our observations in whole cell populations, it appears that these individual pulse alterations, although subtle, form the basis for VGCC drug actions on PRL-GE in pituitary cell cultures.

Our findings lead us to favor the view that PRL-GE pulses arise from feedback interactions among the multiplicity of transcriptional regulatory features comprising the PRL promoter (8). We suggest that the oscillatory dynamics that we have characterized here are likely to be an inherent consequence of the molecular dynamics underlying this promoter’s activity in single cells. Similar arguments have been evoked to explain gene expression pulses in other (noncircadian) mammalian cell types (1, 2, 10), but these studies lacked evidence showing either the oscillatory nature of pulses or their functional modulation. Mathematical modeling studies based on the molecular dynamics of transcriptional regulation suggest that gene expression pulses can be elaborated and timed by transcriptional pacemaker mechanisms (3) and argue that this should be detectable at the single cell level (6). Our current observations lend new and compelling support to these fascinating ideas.

In summary, we have demonstrated that PRL gene expression in individual mammotropes occurs in pulses that follow a noncircadian oscillatory pattern. Moreover, these pulses were sensitive to regulatory influences, as evidenced by the ability of calcium modulators to change the shape of individual pulses. This distinct oscillatory process may have important implications in overall mammotrope function. It would be reasonable to anticipate that gene expression pulses could be translated into intermittent activity in the biosynthesis and/or release of PRL. The exact timing between these processes would be difficult to determine because of the large amount of stored PRL present in mammotropes and the many factors governing its release (34). Nevertheless, the possibility of a strong relationship between these processes is consistent with previous results from our laboratory revealing that individual PRL cells can shift in secretion from very large to very small relative amounts of PRL on a day to day basis (25). This, when viewed in light of the present findings of the potential for secretory modulators to shape pulse activity, raises the intriguing possibility that the presence and pattern of gene expression episodes may be a critical component in determining the functional capacity of individual mammotropes. Future experiments will be directed toward more clearly defining the role of these gene expression pulses in the control of PRL secretion.

	Acknowledgments

 
This work is dedicated to the memory of Steve Frawley, who is greatly missed by all those who worked with him. We thank R. A. Maurer for pPRL-LUC, G. Meaden for help with Fourier transformation analysis, and the MUSC Biotechnology Resource Laboratory for DNA sequence analysis.

	Footnotes

 
This work was supported by NIH Grant R01-DK-54838 (to L.S.F. and F.R.B.). Presented in part at the 83rd Annual Meeting of The Endocrine Society (Abstract P1-94).

¹ Current address: Dynamic Imaging Center, Institute Pasteur, 25 rue du Docteur Roux, Paris 75015, France.

Abbreviations: [Ca²⁺]_i, Intracellular Ca²⁺; L-VGCC, L-type voltage-gated calcium channels; peh, photonic emissions per hour; PRL-GE, PRL promoter-driven gene expression; SNV, signal/noise variation.

Received August 28, 2001.

Accepted for publication November 5, 2001.

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