SPECIAL COMMUNICATION
Simultaneous indirect activity measurements of GH and PRL genes in
the same, living mammosomatotrope
Scott T.
Willard,
Michael D.
Amstutz,
Elizabeth J.
Abraham,
Justo P.
Castaño,
David C.
Leaumont,
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 29425-2204
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ABSTRACT |
Dynamic intracellular processes in endocrine
cells are usually controlled by the coordinated modulation of two or
more functionally related genes. Attempts to gain a more complete
understanding of these processes would be facilitated greatly by a
method enabling activity measurements of two genes at the same time.
Here we describe how we developed such a system and used it to
determine indirectly whether individual, living pituitary cells could
concurrently express both the growth hormone (GH) and prolactin (PRL)
genes. Our results demonstrate that coexpression of these genes is
indeed possible. Moreover, our findings provide a general paradigm for future "real-time" analysis of other interrelated genes involved in the regulation of endocrine processes.
luciferase; pituitary cells
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INTRODUCTION |
ALMOST A DECADE AND A HALF AGO, our group reported that
individual, living cells from normal (nontransformed) pituitary tissue could secrete growth hormone (GH) and prolactin (PRL) at the same time
(8). This novel finding, obtained through use of reverse hemolytic
plaque assays, was confirmed by others who employed either similar
methodology or different techniques to demonstrate the presence in the
same cell of both hormones [immunocytochemistry (ICC)] (10,
13) or mRNAs (in situ hybridization cytochemistry) (5, 12). Subsequent
experimentation with a broad spectrum of mammalian species revealed
that this bihormonal "mammosomatotrope" is a constituent cell
type of the pituitary gland and that it serves as a transitional cell
for the functional interconversions of monohormonal somatotropes and
mammotropes that occur during pituitary development and in response to
dramatic changes in physiological status (7, 14).
One can envision at least two reasonable explanations to account for
the existence of mammosomatotropes. The first and simplest explanation
is that a monohormonal cell expressing either the GH or PRL gene would
be induced to transcribe, for a time, both of these genes. Eventually,
one of these genes would "turn-off" as the cell
transdifferentiated to one or the other monohormonal state. In the
second scenario, the expression of both genes would be mutually
exclusive, as appears to be the case for traditional somatotropes and
mammotropes. Concurrent detection of bihormonal storage and release
might, therefore, simply reflect the fact that labeled secretory
granules can persist intracellularly for many hours or even days after
biosynthesis. Likewise, the relatively long half-lives of the GH and
PRL mRNAs would favor their dual localization long after cessation of
the corresponding transcriptional events. Thus the mammosomatotrope
could be more a perception than a reality.
The goal of the present study was to resolve this mammosomatotrope
dilemma by assessing transcriptional potential for both the GH and PRL
genes in the same living cell. To this end, we adopted an experimental
strategy used previously by our group to make real-time
measurements of PRL gene expression in single mammotropes (3). This
involved transfecting mammotropes with a PRL promoter-driven luciferase
reporter construct and at a later time quantifying photonic emissions
after exposure to the substrate luciferin. [That the rate of
these emissions provides a reliable index of endogenous gene expression
is evidenced by observations that agents known to transcriptionally
activate (epidermal growth factor) and inhibit (dopamine) the PRL gene
in entire populations of cells exert predictable effects on the rate of
photonic emisions (3)]. Pursuit of our current objective
necessitated expansion of this basic model to enable indirect activity
measurements of two different genes at roughly the same time. Here we
report how we developed such a system and used it to localize both PRL
and GH gene expression to the same, living mammosomatotrope.
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MATERIALS AND METHODS |
Plasmid cloning strategies.
Our first step was to develop a system that would enable activity
measurements of two different reporters in the same living cell. For
this purpose, we exploited the published substrate specificities of
firefly and Renilla luciferases. We
reasoned that, for developmental purposes, these constructs should be
driven not only by the same promoter, but also by one not likely to be
activated differentially by mono- or bihormonal acidophils.
Accordingly, we prepared constructs in which the coding sequences for
firefly and Renilla luciferases were
placed under the control of the same cytomegalovirus (CMV) regulatory sequences. In preliminary studies, we found that the promoterless Renilla reporter
(pRL-null vector; Promega, Madison, WI) exhibited spurious background
photonic activity that could be mistaken as a false positive.
Accordingly, we sought first to eliminate this activity by cloning the
Renilla-LUC coding sequence (and
associated promoters) into the promoterless pGL2-Basic expression vector (Promega), shown previously to lack such background activity. Briefly, the CMV-promoter and
Renilla-LUC were PCR amplified from the pRL-CMV-Renilla expression vector
(Promega) with primers encoding Kpn I
and Xho I ends. The
CMV-Renilla PCR product was then
subcloned (Kpn I,
Xho I) into the pGL2-Basic vector
after the removal (Hind III,
EcoN I, Klenow fill-in and religation)
of the firefly-LUC gene. This construct will be referred to throughout
as CMV-Renilla-LUC. As a promoterless
control, the CMV promoter was removed from
CMV-Renilla-LUC with
Sma I,
Nhe I restriction endonucleases, and
the remaining construct was blunt-ended and religated (i.e.,
null-pGL2-Renilla-LUC). The
CMV-firefly-LUC vector was kindly provided by D. Kurtz (Medical Univ.
of South Carolina, Charleston, SC). A promoterless control for the
CMV-firefly-LUC was made by Sma I,
Nhe I cuts, followed by blunt-ending
of the Nhe I site and religation
(i.e., null-firefly-LUC). Experiments using the promoterless
null-firefly-LUC and
null-pGL2-Renilla-LUC reporters
revealed no photonic activity above background levels (data not shown).
A PRL-Renilla-LUC expression vector
was then generated by substituting a 2.5-kbp PRL promoter (pPRL-LUC; R. Maurer, Oregon Health Sciences University, Portland, OR) for the CMV
promoter in CMV-Renilla-LUC. The
237-bp GH-firefly-LUC reporter was constructed as reported previously
(9).
Development of a dual gene expression system in single, living
cells.
Monodispersed anterior pituitary cells from primiparous lactating rats
(days 6-10 of lactation) were
plated on photoengraved, gridded,
poly-L-lysine (GIBCO, Grand
Island, NY)-coated, glass coverslips (70,000 cells/coverslip) and
cultured in DMEM (GIBCO) + 10% fetal bovine serum (GIBCO) for 48 h.
Cells within a grid on a coverslip were then transfected by
microinjection with the CMV-firefly-LUC or
CMV-Renilla-LUC (1 µg/µl each) or
a combination thereof. After microinjection, cells were cultured for an
additional 24 h before quantification of photonic activity by
use of a VIM photon-counting camera and Argus-50 Image processor
(Hamamatsu Photonics Systems, Bridgewater, NJ).
On the day of imaging, cells transfected with CMV-firefly-LUC were
placed in a Sykes-Moore chamber (Bellco Glass, Vineland, NJ) containing
0.1 mM luciferin (Sigma Chemical, St. Louis, MO) in DMEM, and photons
were accumulated in 2-min bins over a 10-min period. The imaging medium
was then replaced with one lacking luciferin. This was accomplished by
perifusion (1 ml/min; Harvard Apparatus, Holliston, MA) over a 10-min
period. Photonic emissions were again monitored continuously in 2-min
bins. Then, coelenterazine (5 µM; Molecular Probes, Eugene, OR) was
perifused into the chamber, and photonic emissions were accumulated
every 2 min for 10 min. This was followed by perifusion once again with
luciferin-containing medium, and photon counting was repeated. A
similar reciprocal experiment was conducted with cells transfected with
CMV-Renilla-LUC, in which measurement
in the presence of 0.1 mM luciferin was followed sequentially by
measurements with medium alone or with medium containing
coelenterazine. It should be noted that the dual expression paradigm
employed here was most efficient when measurements of firefly-LUC were
followed by Renilla-LUC. The reason
for this is that photonic emissions disappeared within 10 min of
luciferin removal, whereas more than an hour was required after
coelenterazine was flushed out.
Analysis of GH and PRL gene expression in the same, living
pituitary cell.
Anterior pituitary tissue from lactating rats was dispersed and
cultured on glass coverslips, as described earlier. Forty-eight hours
later, cells on a grid were comicroinjected with the 237-bp GH promoter
fused to the firefly LUC coding sequence (3 µg/µl) and the 2,500-bp
PRL promoter fused to the Renilla LUC
coding sequence (0.75 µg/µl). After 24 h, cells were subjected
sequentially to image analysis of photonic activity generated in the
presence of the appropriate LUC substrate (GH-firefly-LUC = 3 mM
luciferin; PRL-Renilla-LUC = 5 µM
coelenterazine). In a typical experiment, luciferin was infused into
the Sykes-Moore chamber containing the cells, and photonic activity
indicative of GH-mediated gene transcription was recorded. Next, the
chamber was flushed with medium devoid of substrate, and this was
followed by infusion of coelenterazine for measurement of photonic
signals reflective of PRL promoter-driven
Renilla-LUC activity.
Post facto ICC was conducted for GH on coverslips
(n = 20) obtained from four different
experiments. This was done to confirm that expression of the
GH-firefly-LUC was restricted to cells containing GH, as had been
established previously for PRL (3). Moreover, these data were used to
establish whether there was a congruence between GH biosynthesis and
gene expression in the same cells. To this end, cells were fixed with
B5-buffered Formalin for 45 min immediately after photonic imaging,
rinsed, and subjected to ICC identification of GH as reported
previously (2).
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RESULTS |
Substrate specificities permit concurrent, independent measurements
of firefly and Renilla luciferases.
As shown in Fig. 1, cells microinjected
with only the CMV-firefly-LUC reporter exhibited considerable photonic
activity in the presence of luciferin. This activity decreased to
negligible levels within 10 min of luciferin removal, and subsequent
exposure to coelenterazine did not evoke an increase in photonic
activity. In contrast, reinfusion of luciferin increased the rate of
photonic emissions to a level indistinguishable from that observed in
response to the initial luciferin challenge. Similar substrate
specificity was observed when cells were transfected solely with the
CMV-Renilla-LUC construct (Fig. 1,
inset). Next, we cotransfected
individual pituitary cells (n = 80) by
microinjection with both the
CMV-Renilla-LUC and CMV-firefly-LUC
reporters and quantified photonic emissions after sequential additions
of luciferin and coelenterazine. We found that transfected cells
emitted 104.6 ± 13.4 (SE) and 444.0 ± 28.6 specific photonic
emissions/min for CMV-firefly-LUC and CMV-Renilla-LUC, respectively.
Complete separation of the firefly and
Renilla signals was evident during the
intervening wash phase, at which time only 1.5 ± 0.27 specific
photonic emissions/min were recorded (i.e., background levels) before
coelenterazine infusion. Clearly, these data demonstrate the potential
for independent monitoring of promoter activity for two different genes
within the same, single, living cell.

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Fig. 1.
Substrate specificity of firefly and
Renilla luciferases. Pituitary cells
were microinjected with either cytomegalovirus (CMV)-firefly-LUC
(main panel;
n = 20) or
CMV-Renilla-LUC
(inset;
n = 10). Twenty-four hours later,
photonic emissions were accumulated in 2-min bins and quantified in the
presence of luciferin, wash medium (DMEM), or coelenterazine. Each
trace represents a single cell, and results are representative of those
obtained from 2 other completely independent experiments.
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Use of dual reporter system reveals pituitary cells that
concurrently express the GH and PRL genes.
Having established that we could independently monitor the activity of
two different reporter constructs in the same cell, we next modified
this system to enable concurrent, independent assessments of GH and PRL
gene expression. Specifically, individual pituitary cells were
microinjected with a GH promoter-driven firefly luciferase reporter
construct or a PRL promoter-driven
Renilla luciferase construct. After
exposure to respective substrates, we found (Table
1) that we could detect GH or PRL gene
expression within individual pituitary cells in primary culture.
Activity measurements after cotransfection of both reporters revealed
cells capable of expressing both genes concurrently (Fig.
2 and Table 1). At first
glance, these dual expressers appeared to be quite rare in that they
accounted for only 15 cells out of 355 that expressed GH or PRL.
However, a more rigorous analysis suggests that we grossly
underestimated the size of the GH-expressing population. This view is
supported by our observation that 23.7 ± 3.1% of pituitary cells
stained positively for GH in our experiments, whereas photonic
emissions could be detected in only 2.0 ± 0.43% of cells
transfected with the GH-firefly-LUC reporter plasmid. What might
account for such a discrepancy? One possibility is that the GH promoter
is much weaker than its PRL counterpart in the context of the model we
used for these experiments. Consistent with this notion is our
finding that the activity of
PRL-Renilla-LUC was more than 50-fold
higher than that of the GH-firefly-LUC reporter in coexpressing
cells (Table 1). It is also noteworthy that the photonic
potential of Renilla-LUC is
acknowledged by others to be stronger than that of firefly-LUC (1), and
our observation that
CMV-Renilla-LUC provided a
4.2-fold stronger signal than CMV-firefly-LUC is consistent with
this idea. Finally, it is possible in cells cotransfected with
both plasmids that one of them may have suppressed activity of the
other, as has been reported previously (6), thereby further affecting
our sensitivity of detection. These sensitivity considerations
notwithstanding, the majority of cells positive for GH gene expression
(83.3%) were also found to be positive for PRL gene expression.

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Fig. 2.
Dual imaging of growth hormone (GH)-firefly-LUC
(B) and prolactin
(PRL)-Renilla-LUC
(C) activity in single, living
pituitary cells. A: a captured
bright-field image of pituitary cells that were cotransfected 24 h
earlier with both the GH-firefly-LUC and
PRL-Renilla-LUC reporter plasmids.
Exposure to luciferin revealed one cell expressing the GH promoter
(B), whereas treatment with
coelenterazine revealed 3 PRL-expressing cells
(C). Note that one of the cells
(extreme left) was a dual expresser.
Warmer colors reflect higher rates of photonic emissions, consistent
with ascending color scale to right of
panels.
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|
 |
DISCUSSION |
Dual gene reporter systems for use in whole populations of cells (1) as
well as single cells (4, 11) have been developed previously.
Invariably, the second reporter (under control of a viral promoter) has
been used to assess transfectional efficiency and/or to normalize the
relative activity of the first "physiologic" reporter. Here, we
describe for the first time a method for indirectly quantifying the
activity of two physiologically relevant genes in the same living cell.
Unlike previous systems, this one can achieve complete separation of
reporter signals, thereby obviating concerns about false positive
results. Additional advantages of the current system are that the
activities of both genes can be assessed within a reasonable time frame
(20 min) and that the procedures involved do not compromise cell
viability. This latter advantage raises the possibility that two
functionally interrelated genes can be monitored, in the same cell,
multiple times over several hours or even days, as has been achieved
previously with a single gene reporter system (3, 15).
With this powerful tool in hand, we set out to answer the compelling
question posed earlier: can the GH and PRL genes be transcribed concurrently in an individual, living cell? The answer to this question
is an unequivocal yes. Although the size of the GH-expressing population was admittedly underestimated because of sensitivity considerations, we did find that the vast majority of detectable GH
expressers were also positive for PRL. Should this pattern of overlap
persist when these sensitivity problems are overcome, our findings
would suggest that many if not most acidophils transcribe both genes at
the same time. Although a particular cell might transcribe primarily
the PRL gene at a given time, the "pilot light" for GH
transcription would continue to flicker. Such a possibility was first
raised by Hashimoto et al. (10) who employed dual-labeling, colloidal
gold, electron-microscopic ICC to show that most acidophils in the
bovine pituitary were either conspicuously mammotropic or somatotropic.
However, most cells stored significant quantities of the minority
hormone as revealed by objective, quantitative criteria.
In summary, we have shown clearly that independent measurements of two
gene promoters can be achieved within the same living cell. Moreover,
we have shown that expression of the GH and PRL genes is not mutually
exclusive. Taken together with previous reports on simultaneous storage
of GH and PRL mRNAs (5, 12) and proteins (10, 13) and dual secretion
(8), our results on concurrent transcription demonstrate that the
mammosomatotrope is a bona fide cell type and not just an artifact
arising from the potentially long half-lives of stored hormones and
corresponding mRNAs.
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ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-38215 to L. S. Frawley.
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FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for correspondence and reprint requests: L. S. Frawley, Dept.
of Cell Biology and Anatomy, Medical Univ. of South Carolina, 171 Ashley Ave., BSB 621, Charleston, SC 29425-2204 (E-mail:
frawleys{at}musc.edu).
Received 18 May 1999; accepted in final form 27 July 1999.
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REFERENCES |
1.
Behre, G.,
L. T. Smith,
and
D. G. Tenen.
Use of a promoterless Renilla luciferase vector as an internal control plasmid for transient co-transfection assays of Ras-mediated transcription activation.
Biotechniques
26:
24-28,
1999[Medline].
2.
Boockfor, F. R.,
J. P. Hoeffler,
and
L. S. Frawley.
Analysis by plaque assays of GH and PRL release from individual cells in cultures of male pituitaries.
Neuroendocrinology
42:
64-70,
1986[Medline].
3.
Castaño, J. P.,
R. D. Kineman,
and
L. S. Frawley.
Dynamic monitoring and quantification of gene expression in single, living cells: a molecular basis for secretory cell heterogeneity.
Mol. Endocrinol.
10:
599-605,
1996[Abstract].
4.
Day, R. N.,
M. Kawecki,
and
D. Berry.
Dual-function reporter protein for analysis of gene expression in living cells.
Biotechniques
25:
848-856,
1998[Medline].
5.
Dollé, P.,
J. L. Castrillo,
L. E. Theill,
T. Deerinck,
M. Ellisman,
and
M. Karin.
Expression of GHF-1 protein in mouse pituitaries correlates both temporally and spatially with the onset of growth hormone gene activity.
Cell
60:
809-820,
1990[Medline].
6.
Farr, A.,
and
A. Roman.
A pitfall of using a second plasmid to determine transfection efficiency.
Nucleic Acids Res.
20:
920,
1991[Medline].
7.
Frawley, L. S.,
and
F. R. Boockfor.
Mammosomatotropes: presence and functions in normal and neoplastic pituitary tissue.
Endocr. Rev.
12:
337-355,
1991[Medline].
8.
Frawley, L. S.,
F. R. Boockfor,
and
J. P. Hoeffler.
Identification by plaque assays of a pituitary cell type that secretes both growth hormone and prolactin.
Endocrinology
116:
734-737,
1985[Abstract].
9.
Frawley, L. S.,
W. J. Faught,
J. Nicholson,
and
B. Moomaw.
Real time measurement of gene expression in living endocrine cells.
Endocrinology
135:
468-471,
1994[Abstract].
10.
Hashimoto, S.,
G. Fumagalli,
A. Zanini,
and
J. Meldolesi.
Sorting of three secretory proteins to distinct secretory granules in acidophilic cells of the cow anterior pituitary.
J. Cell Biol.
105:
1579-1586,
1987[Abstract].
11.
Kennedy, H. J.,
B. Viollet,
I. Rafiq,
A. Kahn,
and
G. A. Rutter.
Upstream stimulatory factor-2 (USF2) activity is required for glucose stimulation of L-pyruvate kinase promoter activity in single living islet
-cells.
J. Biol. Chem.
272:
20636-20640,
1997[Abstract/Free Full Text].
12.
Lloyd, R. V.,
M. Cano,
W. F. Chandler,
A. L. Barkan,
E. Horvath,
and
K. Kovacs.
Human growth hormone and prolactin secreting pituitary adenomas analyzed by in situ hybridization.
Am. J. Pathol.
134:
605-613,
1989[Abstract].
13.
Nikitovich-Winer, M. B.,
J. Atkin,
and
B. E. Maley.
Co-localization of prolactin and growth hormone within specific adenohypophyseal cells in male, female and lactating female rats.
Endocrinology
121:
625-630,
1987[Abstract].
14.
Takahashi, S.
Development and heterogeneity of prolactin cells.
Int. Rev. Cytol.
157:
33-98,
1995[Medline].
15.
Takasuka, N.,
M. R. H. White,
C. D. Wood,
W. R. Robertson,
and
J. R. Davis.
Dynamic changes in prolactin promoter activation in individual living lactotrophic cells.
Endocrinology
139:
1361-1368,
1998[Abstract/Free Full Text].
Am J Physiol Endocrinol Metab 277(6):E1150-E1153
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