Dynamics of Stimulus-Expression Coupling as Revealed by Monitoring of Prolactin Promoter-Driven Reporter Activity in Individual, Living Mammotropes
Carlos Villalobos,
William J. Faught and
L. Stephen Frawley
Laboratory of Molecular Dynamics Department of Cell Biology and
Anatomy Medical University of South Carolina Charleston, South
Carolina 29425
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ABSTRACT
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Single-cell paradigms have greatly expanded
our knowledge about stimulus-secretion coupling, but the understanding
of stimulus-gene expression coupling has lagged behind for lack of a
dynamic model sufficiently sensitive to provide single-cell resolution.
In the present study, we made continuous indirect measurements within
individual, living cells of expression dynamics both before and after
treatment with a gene-activating secretagogue. This was accomplished by
transfecting (via microinjection) individual, primary mammotropes with
a PRL promoter-driven luciferase reporter plasmid, and then quantifying
the rate of photonic emissions (reflective of endogenous gene
activity). We found that individual cells exhibit spontaneous, random,
short-term fluctuations of basal reporter activity and are extremely
heterogeneous in terms of responses to a stimulatory agent (TRH). In
addition, we found that responses are affected by several factors
including the secretory status of the pituitary donor, the manner in
which the stimulus is presented, and by the initial level of reporter
activity. Moreover, the responsiveness of an individual cell can
fluctuate dramatically over time. These results invite speculation that
a given cell can "sense" its gene activation state and regulate its
response accordingly to satisfy requirements for the corresponding
secretory product.
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INTRODUCTION
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Cellular responses induced by physiological and/or pharmacological
agonists are generally manifested as both short- and long-term changes
that range in duration from milliseconds to hours or days. Whereas the
former mode of response is mediated by modulation of ligand- or second
messenger-activated effector proteins, the latter requires the
expression of new genes or changes in the transcriptional rate of
previously activated genes. The secretion of hormones and neuropeptides
is not an exception in this regard, and releasing factors and
neurotransmitters that activate exocytosis or synaptic transmission
usually evoke changes in the expression of genes corresponding to the
secretory products (1). The use of dynamic approaches, especially those
at the single-cell level, has greatly improved our knowledge about
stimulus-secretion coupling (2, 3). However, our understanding of
the coupling between stimulus and gene expression is much less
developed, especially in those cases (such as the pituitary gland or
the central nervous system) where functional complexity and
heterogeneity of the tissue warrant a single-cell approach.
The PRL-secreting mammotrope, one of five major hormone-producing cell
types within the adenohypophysis, is rapidly becoming a model of choice
for dynamic analysis of gene expression at the single-cell level (4).
This view is supported by three considerations. First, the PRL gene is
extremely well characterized in terms of its 5'-regulatory sequences
and how second messenger systems indirectly impinge upon them to effect
modulation of gene expression (5, 6, 7). Second, flow through the PRL
biosynthetic pathway has been the subject of intense investigation for
almost four decades, and the mechanistic relationship between hormonal
gene expression and secretion is becoming increasingly clear (8, 9).
Third, and perhaps most important, the mammotropes high level of
basal PRL transcription provides the potential for monitoring hormonal
gene expression at the single-cell level (9). Indeed, we have exploited
this potential by developing a paradigm for making multiple
measurements of PRL gene expression from the same, living mammotrope.
This is accomplished by exposing mammotropes to luciferin after
transfecting them with a PRL promoter-driven luciferase construct, and
then quantifying photonic emissions [reflective of PRL promoter-driven
gene expression (4, 10)] with an extremely sensitive photon capture
system. Because our method of transfection (microinjection) allows
delivery of a predetermined amount of reporter plasmid to every cell,
this strategy also enables dynamic analysis of gene expression in
normal (primary) as opposed to transformed mammotropes.
Dynamic analysis of PRL gene expression has provided a number of
insights about gene activity in single cells that were unattainable
previously because of technical constraints. These include, but are not
restricted to, the finding that mammotropes injected with the same
amount of plasmid can differ from one another by more than 100-fold in
the basal level of gene expression (4). Moreover, individual
mammotropes photonically sampled in narrow (10 min) windows at 24-h
intervals were found to exhibit striking, random, day-to-day changes in
their level of gene expression (4). In addition, not all transfected
mammotropes exhibited predictable changes of photonic emissions when
treated for 24 h with either dopamine or epidermal growth factor
(agents reported by others to inhibit or stimulate, respectively, PRL
gene expression within entire cultures of pituitary cells) (4).
Although valuable and provocative, these previous findings suffered in
terms of temporal resolution and interpretability owing to the fact
that multiple measurements of gene expression on the same cells were
made at 24-h intervals rather than continuously. Accordingly, we
decided to adopt a strategy that involves continuous monitoring of gene
expression (11) both before and after exposure to a secretagogue. We
chose TRH as our prototypic secretagogue for the present study because
it is not only the consensus PRL-releasing factor in mammals but is
also a well documented, physiological stimulator of PRL gene
transcription (12, 13). Armed with this dynamic analytical tool, we
pursued at the single cell level the following objectives: 1) to
establish the dynamics (time course) and demographics of the PRL gene
response to TRH; 2) to assess whether gender of the mammotrope donor,
which greatly influences PRL release, affects responsiveness of the PRL
gene; and 3) to explore the relationship between initial state of gene
expression and the capacity of a given cell to mount a response after
stimulation.
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RESULTS
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The major objective of this study was to assess expression
responses of the PRL gene in individual, living mammotropes. To this
end, we measured photonic emissions from pituitary cells transfected
with a luciferase reporter plasmid (rPRL-LUC) under the control of
5'-regulatory sequences of the rat PRL gene (-2430 to +39). These
measurements were made in a spatial and time-resolved manner. A
reasonable degree of temporal resolution was provided by the short
functional half-life (1 h) of the reporter enzyme in rat mammotropes
(4). Figure 1
illustrates by
representative example the experimental procedure. Here, a
microscopic field containing transfected anterior pituitary
cells was subjected to photon counting measurements for several hours
before and after continuously perifusing the cells with TRH-containing
medium. Photonic emissions were accumulated in 30-min bins, quantified,
and converted into a pseudocolor image that corresponded to the
ascending scale shown on the right. All cells that exhibited
photonic emissions were considered mammotropes for reasons provided in
detail later (see Discussion). In the first series of
studies, we analyzed the responses of 152 individual mammotropes in
primary cultures obtained from male (n = 47) and lactating female
(n = 105) rats in 21 independent experiments. Representative
profiles of individual cells treated continuously with TRH (n =
96) or vehicle (n = 56) are shown in Fig. 2
. Consistent with our previous
observations in cells from lactating animals (4, 14), the initial level
of photonic activity reflective of PRL gene expression varied
greatly from cell to cell in cultures derived from both male and
lactating female rats. After TRH addition, some cells (blue
and red traces) exhibited clear increases in photonic
emissions whereas others (green traces) did not show any
apparent change after stimulation or exhibited a paradoxical decrease
in their rate of photonic emissions. In addition, the dynamics of the
changes evoked by TRH differed considerably from cell to cell. For
example, some mammotropes (see blue traces in B, F, and G,
in Fig. 2
) exhibited striking increases of photonic activity that
tended to decline toward basal values before the end of the sampling
period. In contrast, other cells (blue trace in A and
red traces in F and G, Fig. 2
) responded in a more slow and
sustained manner. Finally, in control experiments, we did not observe
increases in photonic emissions reflective of PRL gene expression in
cells perifused with vehicle instead TRH (panels D and H in Fig. 2
).
These data indicate that TRH-induced increases of reporter activity
were not attributable to possible artifacts of the experimental
protocol but rather to specific interactions with mammotropes that
likely involved TRH receptors.

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Figure 1. Continuous Measurements of Reporter Activity from
Individual Mammotropes in Primary Culture
Single pituitary cells were transfected by microinjection with a
luciferase construct under control of the PRL promoter. One or two days
later, cells were incubated for 4 h in luciferin-containing
medium, transferred to a photon capture system, and perifused
continuously with the same medium. After obtaining a bright field image
of transfected cells for reference purposes (A), we then accumulated
photonic emissions (reflective of PRL gene expression) in 30-min bins
for several hours before and after treatment. This representative
example shows accumulated, specific photonic emissions
(signal-background) for the sampling period just before (B), 3 h
after (C), or 6 h after (D) TRH (1 µM) addition. The
rate of photonic activity is presented here as pseudocolors that
progress in accordance with the ascending color scale shown. Note that
mammotropes with the lowest basal level of reporter activity were the
most responsive to TRH treatment.
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Figure 2. Photonic Profiles of Individual Mammotropes
Challenged with TRH
Individual transfected mammotropes from males (left
panels) and lactating females (right panels)
were continuously monitored, and specific photonic emissions were
accumulated in 30-min bins for several hours before and after long-term
exposure to TRH (1 µM) or vehicle. Profiles in each panel
correspond to photonic emissions from selected cells present in the
same microscopic field that were either responsive to TRH (dark
blue and red traces) or not responsive
(green traces). Each panel contains representative
profiles from a single experiment.
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It is well established that mammotropes from lactating animals exhibit
a much higher rate of PRL secretion than do their counterparts obtained
from other pituitary donors, particularly males. To explore a possible
relationship between PRL secretory status of the donor and gene
expression, we averaged and compared photonic emission values for
transfected mammotropes measured under resting conditions (just before
stimulation with TRH). Surprisingly, we found that mammotropes derived
from lactating females and males did not differ (on average) in the
level of reporter activity measured under resting conditions (Fig. 3
). Next, we evaluated, within the same
set of data, possible gender-specific differences in responsiveness to
TRH by assigning the cells treated with the secretagogue into one of
two groups: those that were responsive to TRH and those that were not.
[Responsive cells were defined as those that exhibited a clear
increase in photonic emissions after TRH stimulation (e.g.
blue and red traces in Fig. 2
). Although
seemingly arbitrary, such decisions were surprisingly clear-cut.] We
found that 69% (n = 36) of mammotropes from males exhibited
unequivocal increases of photonic emissions after TRH addition. For
lactating females, however, the percentage of mammotrope responders was
significantly lower (40%, n = 60, P < 0.05). To
compare both the kinetics and magnitudes of the responses, we expressed
each value obtained during the monitoring period as a percentage of the
values obtained before TRH addition. The resulting normalized values
were each pooled into three different subgroups: TRH-responsive cells,
TRH-nonresponsive cells, and vehicle-treated cells (Fig. 4
). On average, the stimulation of PRL
promoter-driven reporter activity by TRH was considerably greater for
female-derived mammotropes than for their male counterparts (6.4-fold
vs. 3.3-fold, respectively, P < 0.05). The
larger SEM observed for female mammotropes reflects the
higher variability in responses. Taken together, these results
demonstrate that although the endocrine (secretory) status of the
pituitary donor does not influence the basal level of gene expression
dramatically (estimated by reporter activity), it impacts greatly on
both the capacity (proportional abundance) of mammotropes to mount a
response after an evocative stimulus and their responsiveness
(fold-increase).

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Figure 3. Basal Levels of PRL Promoter-Driven Reporter
Activity within Mammotropes Derived from Male and Lactating Female Rats
Illustrated here are the mean (±SEM) levels of specific
photonic emissions measured during the 30 min immediately preceding
stimulation with TRH. Data reflect measurements on 96 cells (36 for
males, 60 for females) studied in 13 independent experiments.
Differences between treatment groups were not significant
(P > 0.05).
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Figure 4. Dynamic Analysis of Responsiveness to TRH
Photonic values for individual cells were normalized to the three
baseline measurements obtained just before TRH (1 µM)
addition. Mean (±SEM) values were then calculated for
cells assigned to one of three specific groups on the basis of their
response to TRH or vehicle. , TRH-responsive cells (n = 24
lactating, n = 25 male). , Cells not responsive to TRH (n
= 36 lactating, n = 11 male). , Cells treated with vehicle
(n = 45 lactating, n = 11 male). The arrow
shows the time at which the perifusion medium was switched to one
containing either TRH or vehicle alone.
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Having established gender-specific differences in the dynamics and
demographics of mammotrope responsiveness, we questioned whether the
initial, resting level of expression might also influence
responsiveness. To address this question, we used raw data from the
same set of experiments and compared pretreatment levels of photonic
emissions for TRH-responsive cells and TRH-nonresponsive cells (Fig. 5A
). Interestingly, those mammotropes
that proved to be TRH nonresponsive exhibited pretreatment levels of
reporter activity that were 4- to 6-fold higher than the corresponding
values for TRH-responsive cells (Fig. 5A
), and this was true regardless
of the gender of the pituitary donor. Thus, only those mammotropes with
low to moderate levels of PRL promoter-driven reporter activity at the
time of challenge were capable of responding to an evocative stimulus.
In addition, the basal level of reporter activity served as an
excellent predictor of the magnitude of the TRH response. More
specifically, we found that the fold-increase of reporter activity
evoked by TRH in those responsive mammotropes was inversely
proportional to the pretreatment level of such activity (Fig. 5B
). As
before, gender of the pituitary donor had no influence on this inverse
relationship between the resting level of reporter activity and
fold-induction of the response. When viewed as a whole, these findings
indicate that the initial level of PRL gene expression has a striking
impact on both the ability of an individual mammotrope to respond to a
transcriptional stimulus and the magnitude of the response.

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Figure 5. Relationship between Initial PRL Promoter-Driven
Reporter Activity within Individual Mammotropes and Responsiveness to
TRH
A, The average (mean ± SEM) level of specific
photonic emissions just before TRH stimulation is shown for
TRH-responsive cells ( ) and those not responsive to the secretagogue
( ). Other details as in Fig. 4 . *, P < 0.05
vs. nonresponsive cells. B, Distribution of responses as
a function of basal reporter activity. Values for individual cells were
assigned to one of the three subgroups according to the mammotropes
basal level of specific photonic emissions. <100 (n = 20
lactating, 14 male); 100500, (n = 22 lactating, 11 male); >500
(n = 18 lactating, 11 male). The maximum fold-increase in photonic
emissions induced by TRH was then calculated for each responsive cell
only and averaged for each group. The numbers adjusted for only the
responsive cells are as follows: <100, 12 female, 10 male; 100500,
11 female, 9 male; >500, 1 female, 6 male. Within each gender,
subgroups with different letters are significantly different at
P < 0.05.
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In a previous study, we subjected the same transfected mammotropes to
photonic measurements in 10-min windows on 2 consecutive days and found
that the resting level of reporter activity changed spontaneously,
sometimes dramatically, from one day to the next (4). This observation,
coupled with our aforementioned discovery of an inverse relationship
between the basal state of PRL gene expression and responsiveness,
invited speculation that a particular cells response to a
transcriptional challenge should also fluctuate over time. As a first
step toward testing this line of reasoning, we subjected transfected
cells to continuous measurements of photonic emissions for 24 h
and found (Fig. 6
) that a majority of
mammotropes did indeed exhibit spontaneous, random changes in the rate
of photonic emissions reflective of PRL gene expression. In fact, 58%
of the mammotropes studied (n = 24; four independent experiments)
exhibited at least a 2-fold change (ranging up to 50-fold) in the
photonic emissions rate over the course of the day-long measurement
sessions.

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Figure 6. Individual Mammotropes Exhibit Spontaneous
Fluctuations in the Resting Level of Photonic Emissions
Transfected mammotropes from lactating rats were subjected to
continuous measurement of photonic emissions in 30-min bins for 24
h. Shown here are the profiles of individual cells that were in the
same microscopic fields (A, B, and C). Data are representative of 24
individual mammotropes studied in four independent experiments.
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After demonstrating that individual mammotropes do undergo dynamic,
short-term changes of basal PRL promoter-driven reporter activity, we
next focused on the issue of whether responsiveness might likewise
change over time. Our experimental strategy was to expose single,
transfected mammotropes to successive, transient TRH challenges. The
concentration of secretagogue was the same as that used in the
continuous perifusion experiments, and challenges (of 10 min duration)
were separated by 8 h. The rationale here was that if the basal
rate of expression dictates the direction as well as the magnitude of
response, then those cells in which expression was elevated by an
initial TRH challenge should exhibit a diminished response when
presented with a second challenge 8 h later. Figure 7
provides representative examples of
cells that were subjected to single (left and
right columns) or double (center column) TRH
challenges. As shown, the initial stimulus evoked a spectrum of
responses consistent with our findings illustrated earlier (Fig. 2
).
Individual mammotropes were then pooled into two subpopulations on the
basis of whether or not they responded to the initial, transient
presentation of TRH. To facilitate comparisons between these groups,
all photonic values were then normalized to those obtained before
stimulation (Fig. 8
).
As hypothesized, those mammotropes that responded positively to the
first TRH challenge exhibited a pronounced diminution of reporter
activity when presented with a second challenge. [Specifically, only
12% of the cells exhibited a diminished response after the first
challenge whereas the value rose to 82% for the second challenge.]
This failure to respond cannot be attributed to the possibility that
the cells were already at a maximal level of activity. This is
evidenced by comparison of the data in panels A and B of Fig. 8
, which
reveals that the stimulatory response to the first challenge would have
continued to rise were if not for the second TRH pulse. Moreover, the
relative expression values (fold-increases) achieved 19 h after a
TRH pulse were roughly half the maximal values measured after
continuous TRH infusion (compare the responders in Fig. 8A
to those in
Fig. 4
, upper panel). These results show that when a TRH
challenge is superimposed upon an elevated baseline, the response is
not just attenuated, but reversed. As such, they provide experimental
evidence that a mammotropes capacity to respond to a transcriptional
stimulus is influenced largely by its initial level of expression.

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Figure 7. Representative Profiles of Mammotropes Subjected to
Successive TRH Challenges
Transfected mammotropes from lactating rats were subjected to photonic
emission measurements before and after a transient (10 min) stimulation
with TRH or vehicle. After 8 h, the same cells were subjected to a
second challenge with TRH or vehicle. Profiles in each panel represent
measurements of photonic emissions made on representative, individual
mammotropes present in the same microscopic field. Data are
representative of 34 (left panels), 66 (central
panels), and 33 (right panels) individual
mammotropes studied in 4, 8, and 3 independent experiments,
respectively. Red and blue traces
represent those cells that exhibited clear increases of PRL
promoter-driven reporter activity after the first stimulation with TRH.
The selected cells that exhibited no change or a reduction in the rate
of photonic emissions after TRH are presented as green
traces.
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Figure 8. Elevation of Basal Reporter Activity by an Initial
TRH Challenge Renders Mammotropes Unresponsive to a Subsequent
Challenge
Photonic emission values for each individual mammotrope studied in the
experiment described in Fig. 7 were normalized to baseline values
obtained before the first TRH or vehicle challenge. After
normalization, cells were assigned to one of two subgroups: those that
responded positively to the initial TRH challenge and those that did
not. It is noteworthy that the fraction of positive responders to a
transient (10-min) TRH challenge was only 29% vs. 40%
for cells continuously perifused with the peptide. Data for
TRH-responsive cells are the mean (±SEM) of 10 (panel A),
17 (B), and 11 (C) individual mammotropes studied in 4, 8, and 3
independent experiments, respectively. Data for cells not responsive to
TRH are presented as the mean of 24 (A), 49 (B), and 22 (C) individual
mammotropes studied in the same sets of experiments. As shown, a
single, transient presentation of TRH evoked, in responsive cells, a
long-term elevation of reporter activity (A), and this effect was
independent of the time at which the transient challenge was applied
(compare panels A and B to C). However, presentation of a second TRH
pulse (B), elicited on average a clear reduction in the rate of
photonic emissions reflective of gene activity.
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DISCUSSION
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In this study, we demonstrated the feasibility and utility of
continuously monitoring the dynamics of gene expression within
individual, living mammotropes in primary culture. This goal was
achieved by quantifying photonic emissions, reflective of endogenous
gene expression, from anterior pituitary cells transfected with a PRL
promoter-driven luciferase reporter plasmid. By using this approach, we
were able to record, in a time-resolved manner, an indirect measure of
gene expression under basal conditions and stimulus-expression coupling
in response to TRH. The validity of this single-cell approach as a
paradigm for monitoring expression dynamics is evidenced by our earlier
observation that the vast majority of transfected mammotropes exhibited
predictable photonic responses to secretagogues known to either
increase (epidermal growth factor) or decrease (dopamine) PRL gene
transcription within entire populations of pituitary cells (4).
Additional supportive evidence is that expression of the same reporter
construct was highly specific for rat mammotropes. This was revealed by
coupling photonic analysis with measurements of PRL storage
(immunocytochemistry), release (reverse hemolytic plaque assay),
and mRNA content (in situ hybridization cytochemistry) (4).
Indeed, we could not detect any pituitary cell transfected with the
rPRL-LUC reporter that emitted photons and subsequently proved to be
something other than a mammotrope. Given the rapid decay of firefly
luciferase activity in rat mammotropes (t1/2 = 1
h, Ref. 14), this system comprises a highly responsive and valid tool
for estimating (albeit indirectly) the dynamics of gene expression
within living, primary mammotropes.
Armed with this tool, we characterized first the dynamic response to
TRH, a physiologically relevant transcriptional stimulus of the PRL
gene. We found that not all mammotropes responded to TRH with a robust
augmentation of reporter activity. In fact, a significant fraction of
transfected mammotropes exhibited no stimulation whatsoever. Why did
some mammotropes from the same pituitary gland exhibit responses to TRH
whereas others did not? Although available evidence will not support an
unequivocal answer to this question, the modulation of the
concentration of free intracellular calcium
([Ca2+]i) is probably involved in the
regulatory mechanism. Indeed, cytosolic Ca2+ plays a
crucial role in the control of basal PRL gene transcription (15) and is
a requisite mediator of the transcriptional response of mammotropes to
TRH (16). Interestingly, only a subpopulation of primary mammotropes
was found to exhibit an increase of [Ca2+]i
after exposure to TRH (17, 18). Therefore, heterogeneity in the
distribution of functional TRH receptors (i.e. those linked
to Ca2+ mobilization) may very well account for the failure
of some mammotropes to mount an acute response to TRH. Another possible
explanation for the selective response to TRH is that the decay
characteristics of the luciferase protein and mRNA may have compromised
our ability to detect very rapid and transient changes of PRL gene
expression in the majority of nonresponders. Indeed, results from
nuclear run-on assays show that changes of PRL gene expression can be
detected within just a few minutes, but available evidence does not
support the idea that such changes are transient (19, 20). Therefore,
while we cannot discount this alternative possibility, we deem it
remote.
In pursuit of our second objective, we explored the relationship
between the secretory status of the pituitary donor and the level of
PRL promoter-driven gene expression. For this purpose, we compared the
resting reporter activity values of mammotropes from male and lactating
female rats because the secretory capacity of mammotropes from the
former group was reported to pale in comparison with that of the latter
group (21). Interestingly, we failed to find a gender-specific
difference in basal reporter activities suggesting that other variables
might contribute to differences in secretory capacity. On the other
hand, we found that the secretory status of the pituitary donor did
have a striking influence on the proportional abundance of
TRH-responsive mammotropes; the fraction for males was almost 2-fold
greater than that for females (69% vs. 40%, respectively).
Collectively, these results raise an interesting and provocative
question: If striking, gender-specific differences in basal expression
are not obvious, and the proportional abundance of TRH-responsive
mammotropes favors males, why do females secrete more PRL than males?
The answer might derive from at least two considerations. First, the
percentage of all pituitary cells that secrete PRL is 2-fold greater
for lactating females than for males (
55% vs. 30%,
respectively). Thus, the absolute numbers of TRH-responsive mammotropes
are very similar for both genders of pituitary donor. Second, and
perhaps equally important, the magnitude of response to TRH is greater
(again by
2-fold) for mammotropes derived from females as opposed to
males. The net result, then, is that both the dynamics and demographics
of TRH responsiveness favor a higher level of PRL output by pituitary
cells from females. Of course, this conclusion must be tempered by the
fact that we did not measure PRL secretion in parallel in these
particular experiments, and that our inability to detect a
gender-specific difference in basal expression cannot be interpreted
unequivocally to mean that one does not exist. The latter consideration
notwithstanding, our studies reveal fundamental differences in the way
mammotropes from males and females respond to an evocative stimulus of
PRL gene transcription.
Our final objective was to evaluate the intriguing possibility that the
basal level of expression might influence the capacity of a mammotrope
to mount a response after stimulation with TRH, and three types of
analysis were employed for this purpose. First, we screened the
profiles of individual mammotropes exposed to TRH and observed (Fig. 2
)
that elevated pretreatment levels of reporter activity were generally
associated with poor TRH responsiveness and vice versa. The
existence of such an inverse relationship between basal activity and
magnitude of stimulation was confirmed more quantitatively when we
plotted the averaged magnitudes of response against the resting rate of
photonic emissions (Fig. 5B
) and found a negative association between
the two parameters. Finally, we conducted an experiment in which we
were able to reverse (positive to negative) TRH responsiveness by
pharmacologically elevating the pretreatment level of PRL
promoter-driven reporter activity (Fig. 8
). The results of all three
analyses support the same conclusion: that responsiveness to TRH is
dictated largely by the cells level of expression at the time of
challenge.
When considered collectively, our findings invite speculation that
mammotropes are able to sense their expression state and send a
feedback signal conveying information as to whether an individual cell
need respond to TRH, and if so, the magnitude of the response. The
physiological implications of such a servo-mechanistic model are rather
obvious: mammotropes could be induced by an appropriate stimulus to
convert between two expression states responsive and nonresponsive.
In this manner, PRL gene expression could be tightly regulated to
satisfy, but not exceed, physiological requirements for production of
the corresponding hormone. While the identity of the putative,
autocrine (or intracrine) feedback agent remains to be established,
there are already some candidate molecules to subserve such a role.
These include cytoplasmic Ca2+ (for reasons detailed
earlier) and PRL itself [owing to the well established autocrine
feedback effects of the hormone on its own secretion (22), and the
presence of PRL receptors within the nucleus (23)]. Other candidates
deserving of special consideration are the so-called lumicrine peptides
for which RESP18 is a prototype in neuroendocrine cells (24). Induction
of RESP18 expression initiates a signaling pathway that conveys a
signal from the lumen of the endoplasmic reticulum to the nucleus to
regulate expression of responsive genes.
During the course of completing the present studies on PRL gene
expression, and subsequent to our prior publication of a system for
making continuous measurements of gene expression in single, living
cells (11), Takasuka et al. (25) reported the results of a
study in which they made multiple measurements (30-min windows at 3-h
intervals) of gene expression on cells from a rat PRL-secreting cell
line stably transfected with a human PRL promoter-driven luciferase
construct. They observed with intermittent measurements on transformed
cells (transfected with a heterologous promoter), as we did in the
present study with normal mammotropes (transfected with a homologous
promoter), that the basal level of PRL gene expression is not constant
but can vary in the same cell over the course of several hours.
Collectively, these results confirm and provide an explanation for our
earlier observation (4) that individual, transfected mammotropes
sampled photonically in 10-min windows at 24-h intervals exhibited what
appeared to be spontaneous, random fluctuations of PRL gene expression.
These same investigators also made intermittent measurements of PRL
promoter-driven reporter activity before and after treatment with TRH.
On the basis of six single-cell profiles (no quantitative data were
provided), they proposed that the gene expression response to TRH was
heterogeneous, a conclusion supported by a more rigorous, quantitative
analysis in our present study.
In summary, we have developed and refined a model for monitoring, in a
time-resolved manner, an indirect measure of stimulus-expression
coupling within single, living mammotropes. We have used this paradigm
to gain several interesting and unexpected insights about the
dynamics of PRL gene activation. These include the finding
that expression responses are highly heterogeneous and are impacted
greatly by the secretory status of the pituitary donor and the
presentation of the stimulus. Moreover, we found that these responses
are entrained by the initial level of PRL gene expression. Inasmuch as
the basal level of PRL gene expression can change spontaneously over
time, it seems reasonable to propose that responsiveness of a given
cell to a transcriptional stimulus might also vary over time as a
function of its expression state.
 |
MATERIALS AND METHODS
|
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Cell Dispersion and Microinjection
Anterior pituitaries from male (250 g) and primiparous,
lactating female (days 610 postpartum) rats (Sprague-Dawley Harlan,
Madison, WI) were dispersed with trypsin as reported elsewhere (4). All
the rats used were of a comparable age at the time of pituitary
collection. Monodispersed cells were plated on gridded coverslips
previously coated with poly-L-lysine at a density of 75,000
cells/75 µl of a defined medium (phenol red-free Medium 199/Nutrient
mixture F-12 [1:1], Life Technologies, Inc.,
Gaithersburg, MD, in which L-valine had been replaced by
D-valine) and supplemented with 0.1% BSA,
insulin-transferrin-selenium Premix, and antibiotics. Cells were
allowed to attach for about 1 h, covered with 2 ml of defined
medium supplemented with 10% FBS, and incubated in a humidified
atmosphere of 5% CO2-95% air. After 2 days in culture,
cells within a particular grid were microinjected with a reporter
plasmid (rPRL-LUC, 0.2 µg/µl in 10 mM PBS) in which 2.5
kbp of the 5'-flanking region of the rat PRL gene were placed upstream
of the coding sequence for firefly luciferase. Cell microinjection was
performed as described previously (4) to ensure the delivery of the
same amount of plasmid among cells. After microinjection, cells were
washed twice and cultured for 24 or 48 h in phenol red-free DMEM
(Life Technologies, Inc.) supplemented with 10
mM HEPES, 10% FBS, 0.1% BSA, and antibiotics.
Continuous Monitoring of PRL Gene Expression in Single, Living
Cells
For monitoring of photonic emissions, reflective of PRL gene
expression, microinjected cells were incubated in phenol-free DMEM
supplemented with 0.1% BSA, 10% FBS, 10 mM HEPES, and
antibiotics for 24 or 48 h. Four hours before measurements of
reporter activity, we incubated the cells with the same medium
containing 0.1 mM luciferin (Sigma, St. Louis,
MO). [This step was taken to ensure the equilibration of intracellular
luciferin stores before imaging and to allow stabilization of a
diminution of photonic activity that occurs in some cells during the
first few hours after exposure to low concentrations of luciferin].
Then, coverslips were assembled in Sykes-Moore chambers that were
subsequently filled with a culture medium of the same composition as
before except that it was devoid of BSA and bicarbonate, and
supplemented with 10% FBS and 0.1 mM luciferin.
[Preliminary experiments established this to be a saturating dose of
luciferin for perfusion studies. Higher concentrations did not
influence the average rate of photonic emissions]. The chamber bearing
the coverslip was next transferred to the heated (37 C) stage of a
Zeiss Axioscope (Carl Zeiss, Jena, Germany) located in a
dark room. Transfected cells were then reidentified with the help of
the numbered/lettered grid, and a bright field image was captured for
reference purposes. Photonic emissions from cells in single grids on
three separate coverslips were generally captured consecutively, and
the coverslip supporting the most photon-emitting cells was chosen for
long-term monitoring of reporter activity. This was achieved by
accumulating photonic emissions in 30-min bins for 18 h. During
this period, cells were perifused continuously with the same medium at
a very low rate (10 µl/min) to ensure replenishment of nutrients and
substrate. After the first 6 h, cells were perifused for 10 min
with the same medium containing either TRH (1 µM) or
vehicle at a rate of 1 ml/min (to accelerate exchange of chamber
contents), and then the flow was returned to the lower rate with the
same treatments. For administration of transient TRH pulses, cells were
infused (1 ml/min) with TRH (or vehicle) for 10 min after which they
were infused again with vehicle (1 ml/min) for another 10 min in order
to remove the secretagogue before returning to the normal flow
rate.
For photonic emission measurements, we used a photon capture system
comprised of a Hamamatsu VIM photonic camera and an Argus 50 image
processor (Hamamatsu Photonics, Bridgewater, NJ). To quantify reporter
activity in living cells, we accumulated images of photonic emissions
and superimposed them over the corresponding bright field image of
cells. The number of photonic events within a window of fixed area
corresponding to the position of each transfected cell was then
calculated. Photonic measurements made in at least 10 adjacent areas
devoid of cells were used to compute a background value that was
subsequently subtracted from the total accumulation to calculate
specific photonic emissions from each cell. Other details of this
procedure have been reported previously (4, 14).
Comparisons between treatment groups were made with a two-tailed,
Students t test, and results were expressed as mean
± SEM. Differences were considered significant at
P < 0.05.
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to D. C. Leaumont for technical assistance
and Dr. R. Maurer for the gift of the rPRL-LUC plasmid.
 |
FOOTNOTES
|
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Address requests for reprints to: Dr. L. Stephen Frawley, Laboratory of Molecular Dynamics, Department of Cell Biology and Anatomy, Medical University of South Carolina, 171 Ashley Avenue, Charleston, South Carolina 29425.
This work was supported by NIH Grant DK-38215 (to L.S.F.) and a
postdoctoral fellowship from the Ministerio de Educación y
Cultura of Spain (to C.V.).
Received for publication December 21, 1998.
Revision received May 26, 1999.
Accepted for publication July 8, 1999.
 |
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