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Department of Human Physiology, School of Medicine, University of California Davis, California 95616
Address all correspondence and requests for reprints to: Judith L. Turgeon, Ph.D., Department of Human Physiology, University of California, Davis, California 95616. E-mail: jlturgeon{at}ucdavis.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pituitary PR is subject to down-regulation by progesterone, although the mechanisms involved in this regulation have not been resolved. For monkey pituitary cells, Bethea and colleagues report that progesterone can reduce pituitary PR mRNA levels (1); the down-regulatory effect of progesterone on pituitary PR protein was found to be a complex response depending on time course (9, 11). For cultured rat pituitary cells, we reported a rapid down-regulation of PR mRNA occurring within 3 h of exposure to progesterone with recovery by 12 h; there was suggestive evidence for a decrease in PR protein but with no recovery within this time frame (2). In an in vivo study, Szabo et al. (3) found no effect on pituitary PR mRNA levels at 17 h after progesterone treatment, the only time point examined.
Localization of the PR within the pituitary gland is restricted to gonadotropes in rats and monkeys (2, 9, 12, 13), and, therefore, mechanisms for regulating PR levels in females might be expected to reflect the varying temporal requirements of a reproductive cycle. The dynamic regulatory changes demanded during the preovulatory period are particularly impressive. In the rat, for example, the LH surge has a duration of about 10 h; with a slight delay in onset, serum progesterone mirrors the LH profile and has a duration of about 12 h (14). During this period, progesterone has a biphasic effect on LH secretion, starting with a severalfold augmentation of the LH release action of GnRH and ending with a suppression of the efficacy of GnRH (15, 16, 17, 18, 19, 20). One potential mechanism subserving the demands of the system during this period could relate to alterations in the levels of available PR protein in gonadotropes.
An essential consideration is that PR mRNA and protein may be regulated differentially within this physiological context in gonadotropes. For example, in other cell types there is evidence that the stability of PR protein is profoundly affected by the presence of progesterone, and this effect can be independent of changes in PR mRNA (21). There are similar reports for ligand-dependent degradation of the estrogen receptor (22, 23) and the glucocorticoid receptor (24, 25).
These reports and our previous study of PR expression in gonadotropes (2) led us to hypothesize that, within a time course consistent with its physiological actions during the preovulatory LH surge, progesterone results in a down-regulation of nuclear PR protein independent of its effect on PR mRNA. The aims, therefore, of the present study were to quantitatively establish the down-regulatory effect of progesterone on nuclear PR protein under conditions known to cause a transient down-regulation of PR mRNA in cultured gonadotropes and to evaluate mechanisms involved in this pituitary action of progesterone. To assess nuclear PR we adapted quantitative confocal microscopy techniques for the analysis of nuclear PR-immunoreactivity (PR-IR) in individual gonadotropes, identified by dual immunofluorescence. Part of this study has been presented in preliminary form (26).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For immunofluorescence, normal goat serum (NGS) was from Vector Laboratories, Inc. (Burlingame, CA), tetramethyl rhodamine isothiocyanate (TRITC)-conjugated goat antimouse IgG was from Sigma, fluorescein isothiocyanate (FITC)-labeled goat antirabbit IgG was from Sigma, TOTO-3 iodide was from Molecular Probes, Inc. (Eugene, OR), and Vectashield mounting medium was from Vector Laboratories, Inc. Mouse monoclonal antibody 518B7 to bovine LHß (27) was provided by Jan Roser (University California, Davis). The polyclonal antibody used for analysis of PR was as described previously (2); it reacts with both the A and B forms of murine PR (28).
Pituitary cell culture and experimental protocols
Protocols employed in these experiments were reviewed and
approved by the University of California Davis Animal Use and Care
Administrative Advisory Committee. Rats were ovariectomized under ether
anesthesia and maintained for two weeks before use. Pituitary glands
were removed from rats following CO2 narcosis and
decapitation. Anterior pituitary tissue was enzymatically dispersed and
prepared for cell culture as described (18). Cells
were plated at 5 x 104 on Matrigel-coated 12-mm glass
coverslips placed in 22-mm multiwell plates. Wells were flooded with
minimum essential medium containing D-valine and
supplemented with 200 µM kanamycin sulfate (MEM-K), 10%
FBS that had been charcoal treated to remove endogenous steroids
[ctFBS (29)], and plus or minus 0.2 nM
E2. Residual steroid concentrations in the ctFBS
were 3 pM for progesterone and less than 1 pM
for E2 as determined by RIA. Cells were
maintained in a humidified atmosphere (37 C) of 5% CO2 in
air; on day 2, media were replenished (day of plating = day
1).
On day 4, the cells were changed to serum-free MEM-K containing 1 mg BSA/ml (MEM/BSA) with or without experimental treatments. In experiments to determine the effect of E2 on the appearance of the PR in the nucleus, cells that had been incubating without estrogen were exposed to 0.2 nM E2 for varying periods from 0–36 h. For experiments to determine the effect of progesterone on nuclear PR-IR, cells that had been incubating in E2-containing medium were changed into MEM/BSA ± 40 nM progesterone, ± RU486 (40 or 200 nM), ± 0.2 nM E2, and ± MG132 (1 or 10 µM) for varying periods for up to 36 h. To assess nuclear PR recovery, for some wells in these groups the media were changed after 12 h, the cells rinsed 2x in MEM/BSA, and incubation was continued in the absence of progesterone ± E2 and ± 5 µM cycloheximide for 12 or 24 h. For all treatment groups, termination was accomplished by rinsing the cells in PBS at room temperature followed by fixation with precooled methanol:acetone (1:1) for 7 min at -20 C; fixed cells were stored in PBS at 4 C until subjected to immunofluorescence staining.
Immunocytochemistry
Immunofluorescence staining was carried out at room temperature
by successive incubation of the coverslips in the following solutions:
1) 5% NGS, 1 h; 2) anti-LH (1:500) and anti-PR (1:750), 2 h;
3) PBS-0.1% BSA-0.1% tween 20 (PBSA) washes; 4) 5% NGS, 30 min; 5)
TRITC conjugated goat antimouse IgG (1:160), 1 h; 6) PBSA washes;
7) FITC labeled goat antirabbit IgG (1:160) and 2.4 nM
TOTO-3 nuclear DNA stain, 1 h; 8) PBSA washes; 9) distilled water
wash and mount using Vectashield mounting medium for fluorescence. As
controls, either or both of the primary antibodies were omitted for
some coverslip incubations. All antibodies were diluted in 5% NGS.
Confocal microscopy
Microscopy data were obtained with a Carl Zeiss LSM
510 laser scanning confocal microscope (Carl Zeiss, Inc.,
Thornwood, NY) using a 40x/1.3 N.A. lens. Fluorescence data were
collected in three separate channels: channel 1 was set to collect
fluorescence of the nuclear stain TOTO-3 (excitation: 633 nm; emission:
650 nm Long Pass filter); channel 2 collected FITC fluorescence, PR-IR,
(excitation: 488 nm; emission: 505–530 nm Band Pass filter); channel 3
collected TRITC fluorescence, LH, (excitation: 543 nm; emission:
560–615 nm Band Pass filter). For each channel and for all
collections, the pinhole was set to 250 µm, giving an optical slice
of approximately 3 µm. Additionally, a transmitted light image was
obtained in channel 4. Overlapping optical slices (22–28; step size
approximately 1 µm) were obtained through the entire cell. Each slice
was 1024 x 1024 pixels and had a pixel depth of 8 bits.
Subsequently, from the data set of each cell, a single optical slice
was selected for analysis as described below.
Data analysis
As we reported previously for the ovariectomized rat anterior
pituitary cells in primary culture, LH immunoreactive cells constitute
7–8% of the cell population and PR-IR and LH colocalize in 98–100%
of these cells (26). For the present study, only cells
that stained positively for LH were selected for analysis. The optical
slices for each gonadotrope were examined and the slice containing the
maximum nuclear diameter was used for analysis of PR-IR. The selected
images were exported in TIFF format and analyzed using ImageJ
(available from the NIH: http://rsb.info.nih.gov/ij/applet/). Each
exported TIFF file was converted to an 8-bit RGB stack. Using the RGB
stack the net mean nuclear PR-IR was determined as the gray-scale
intensity in the G (Green; channel 2) channel minus the corresponding
background value (relative intensity units).
Data are presented as mean ± SEM. Three to 17 cells were analyzed on each coverslip and averaged; therefore, n refers to the number of coverslips examined from separate experiments and reflects a minimum of three separate cell dispersions per experimental group. All statistical analyses were done using SigmaStat (SPSS, Inc., Chicago, IL). For multiple comparisons, differences between groups were determined by ANOVA and the Student’s-Neuman-Keuls method; where differences are indicated as being significant, P < 0.05. For regression analysis, the slope of the regression line was tested for significance.
In immunocytochemical studies, there are several potential sources of variation in fluorescence intensity in addition to those attributable to treatment effects. For this study, the sources of noise in the PR-IR signal include inherent biological variation in the cell to cell expression of PR. Additionally, there is the experimentally introduced variability inherent in the fixation and immunostaining procedures, even though these methods were standardized and unchanged throughout these experiments. To aid in assessment of the variability introduced by these factors, independent of treatment effects, the E2 continued-12 h experimental group was included in each experiment. For this group, we assessed the variation between cells on the same coverslip as a measure of biological variability, and we examined the results between these independently immunostained coverslips for the combined effects of immunostaining and fixation. The objective was to estimate the magnitude of the random variation and, further, to assess the presence of systematic errors. We found that biological variation and variation in the fixation and immunostaining procedures each contribute significantly to the observed variation in PR-IR; however, these variations were shown statistically to be random and did not exhibit systematic variation over the course of these experiments.
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Results |
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; regression analysis, P
< 0.001). For all subsequent studies described below, pituitary cells
were cultured in the presence of 0.2 nM
E2 for 3 days before the experimental
manipulations were initiated. Shown in Fig. 2 (A and B) are examples of
immunofluorescence staining of pituitary cells on day 4 of culture in
E2; gonadotropes, identified by cytoplasmic LH
immunoreactivity (red), show nuclear PR immunoreactivity
(green).
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, for the first 12 h after
E2 removal there was no significant difference in
PR-IR compared with that found in gonadotropes that continued to
incubate in E2-containing medium. However, over
36 h there was a significant decrease in nuclear PR-IR for the
E2-removed cells (regression analysis,
P < 0.01).
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for
gonadotropes cultured for 3 days in E2, a
significant decrease in PR-IR was detected by 9 h after the
addition of 40 nM progesterone to serum-free medium
(26 ± 8% of control). By 24 h of exposure to progesterone,
there is a near disappearance of nuclear PR-IR in gonadotropes (3
± 0.4% of control) (Fig. 2C
). As our earlier studies had shown that
under these conditions PR-mRNA in gonadotropes decreased following
progesterone exposure but returned to control levels by 12 h even
in the continued presence of progesterone (2), we next
asked if removal of progesterone would allow nuclear PR protein to
recover. For these experiments, cells exposed to progesterone and
E2 for 12 h were rinsed and incubation was
continued in the presence of E2 alone for 12 or
24 h. As shown in Fig. 2D and Fig. 3, removal of the progesterone
resulted in a significant recovery of nuclear PR-IR. By 12 h,
there is an 8-fold increase over that which is found if progesterone
had remained in the incubation medium (Fig. 3 and Table 2; compare prog
24 h vs. prog 12 h/prog removed 12 h groups). The
PR-IR showed a tendency to increase with time after progesterone
removal but the increase was not significantly greater at 24 h
than that found 12 h after progesterone removal (Fig. 3).
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, if E2 is removed
concurrent with progesterone addition (E2
discontinued + prog group), the decrease in nuclear PR-IR in
gonadotropes after 12 h is not significantly different than that
found when E2 is continued. This suggests that
the acute down-regulation of PR protein by progesterone in gonadotropes
does not involve interference with the E2
receptor under these conditions. However, during the subsequent 12 when
progesterone is removed from the incubation medium, the recovery of
nuclear PR is abolished unless E2 is present
(Table 2). To further define the conditions necessary for nuclear PR
recovery after progesterone removal, we examined the effect of the
translation inhibitor, cycloheximide. If the inhibitor is added
concurrent with the removal of progesterone, the recovery of nuclear
PR-IR in the subsequent 12 h is prevented (Table 2). Thus, under
these conditions, once gonadotropes are exposed to progesterone and PR
protein is down regulated, the reappearance of nuclear PR-IR requires
the absence of progesterone, the presence of E2,
and protein synthesis.
Interference with progesterone’s action
In the next series we asked whether interfering with progesterone
binding to its nuclear receptor could block the progesterone-induced
down-regulation of the PR. When gonadotropes were incubated in
equimolar concentrations (40 nM) of RU486 and progesterone,
the decrease in nuclear PR-IR usually seen at 12 h of progesterone
exposure was prevented by RU486, although there was considerable
variation on a cell to cell basis as is reflected by the large
SEM (Fig. 4). Increasing the
concentration of RU486 5-fold resulted in a complete block of the
progesterone-induced PR down-regulation; RU486 by itself over 12 h
had no significant effect on nuclear PR-IR in gonadotropes (Fig. 4).
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). Preincubating gonadotropes in 1
µM MG132 for 3 h before the addition of progesterone
resulted in PR-IR that was about 70% of the zero-progesterone control,
but the pretreatment regimen did not significantly improve the
inhibitor’s effectiveness compared with simultaneous addition (Fig. 5). Representative confocal microscopy images are shown in Fig. 2
(E–F). MG132 (1 µM) by itself for 12 h had no
significant effect on nuclear PR-IR (Fig. 5). In an attempt to more
completely reverse the progesterone-induced decrease in nuclear PR-IR,
we increased the concentration of the proteasome inhibitor 10-fold.
However, when gonadotropes were exposed to 10 µM MG132
for 12 h, the pattern of PR-IR was different than found for any
other treatment groups in that PR-IR exhibited one or two focal points
or "hot spots" of saturated fluorescence in the nucleus instead of
the usual relatively homogeneous nuclear distribution. This occurred
whether or not progesterone was present. Because this unusual spatial
phenomenon was exclusively found with the higher concentration of
MG132 and also presented problems with fluorescence intensity analysis,
we have not included the data in the final comparisons.
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Discussion |
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In the absence of E2, nuclear PR is barely detectable with confocal microscopy, and this dependence of PR protein on estrogen stimulation is consistent with previous reports for pituitary cells (2, 4, 5, 6, 7, 8, 9). It is possible that the low level of PR we observed represents estrogen-independent activity involving, e.g. serum growth factors, but an alternative explanation to account for the presence of minimal PR could be residual estrogen in the charcoal-treated serum (final E2 concentration estimated in the femtomolar range). It is clear, however, that the addition of E2 in the picomolar range led within 24 h to a clear increase in nuclear PR in gonadotropes that had been cultured without added E2 for several days. This increase in PR protein is in line with the E2-induced increase in PR mRNA in rat gonadotropes under similar culture conditions (2) or in vivo (3).
In contrast to estrogen’s positive action on gonadotrope PR, progesterone exhibited a clear down-regulation of PR protein within 9 h, reaching barely detectable levels by 24 h of exposure. Functionally, this decrease in PR could account for the observed abrogation of progesterone’s acute positive action on GnRH-stimulated LH secretion that occurs on the downslope of the preovulatory LH surge (16, 17, 18, 19, 20) and may be involved in the extinction of the surge (15). In the rat, the LH surge occurs over 8–10 h and is accompanied by a progesterone surge (15), which initially serves to substantially augment the LH release action of GnRH (16, 17, 18, 30, 31). The striking down-regulation of nuclear PRs within 9 h of exposure to progesterone could account for the observed loss of the positive action of progesterone on GnRH-stimulated LH secretion and could affect the duration of the preovulatory LH surge. Indeed, it has been shown in cyclic rats in vivo that PRs fall from a high on proestrus to low levels on estrus (6, 32). In the current study we narrow the time frame to show that the decrease in gonadotrope PR can occur within the temporal extent of the LH surge, and we provide support for the hypothesis that the loss of gonadotrope responsiveness to progesterone contributes to the termination of the preovulatory surge.
Mechanistically, the progesterone-induced sustained decrease in PR
protein cannot be accounted for completely by changes in PR mRNA
levels. As we reported for pituitary cells cultured under similar
conditions in the presence of estrogen, progesterone causes a rapid but
transient down-regulation of PR mRNA. Steady-state PR mRNA levels
decreased by 3 h of exposure to progesterone but had recovered by
12 h even in the continual presence of progesterone
(2). For PR protein in contrast, the decline is sustained
for the duration of progesterone exposure; nuclear PR is barely
detectable by 24 h (Fig. 3). To recover nuclear PR protein, three
conditions were required: removal of progesterone, the presence of
estrogen, and ongoing translation. In the aggregate, this suggested
that progesterone was directly affecting the stability of PR protein in
gonadotropes.
Autologous down-regulation is common for steroid receptors. In T47D human breast cancer cells, for example, the half-life of PR protein is reported to be 21 h in control cells and only 6 h in the presence of a progesterone analog (33). Recent reports suggest that, at least in part, ligand-induced decrease in receptors is due to degradation through a ubiquitin-proteasome pathway. In this pathway, which is present in the cytoplasm and nucleus of eukaryotic cells, substrates are degraded by a large multisubunit ATP-dependent protease complex, the proteasome (34, 35, 36). In most cases, the substrate is targeted for degradation by the attachment of ubiquitin moieties, and there is evidence for ubiquitination of the PR (21) as well as the estrogen and glucocorticoid receptors (22, 25). We show in gonadotropes that, in the presence of the proteasome inhibitor MG132, the expected progesterone-dependent decrease in nuclear PR is partially prevented. This is consistent with a ligand-modulated destruction of the PR through the proteasome pathway. That the degradation of the PR was not completely inhibited could be related to the mode of action of MG132. This peptide aldehyde is a reversible, transition state analog, and at the concentration used in this study it may be less effective than the activated PR in competition for proteasome binding.
The exact mechanisms by which ligand binding promotes receptor degradation through the ubiquitin-proteasome pathway are not clear, but various strategies could be invoked depending on the cell context. For example, ligand-dependent dissociation of the steroid receptor from the hsp-90 chaperone system can destabilize the receptor, making it more accessible to the ubiquitin-proteasome system (reviewed in Ref. 37), and recent evidence shows that phosphorylation of PR activated by the progesterone analog R5020 can mark the receptor for destruction by the proteasome pathway (21). In this latter study, a unique serine residue in the human PR was reported to be responsible for targeting the receptor for degradation in T47D breast cancer cell lines. Interestingly, although both progesterone and RU486 have been shown to increase phosphorylation of this serine in the human PR, the antiprogestin reduces PR protein levels only slightly and transiently or not at all in T47D cells (38, 39), whereas in another human breast cancer cell line, MCF-7, RU486 is nearly as effective as progesterone in reducing PR levels (39). Clearly, protein degradation must have multiple control points depending on cell context and could involve, for example, the conformational modifications in the substrate for targeting, specificity in the components of the ubiquitination enzymes, and regulation of proteasome activation (22, 34, 35, 36). For degradation of nuclear hormone receptors or other transcription factors, the kinetics of DNA-receptor binding and the composition of the multicomponent transcription complex also could contribute to the cell or context variability in the timing and extent of receptor loss through the proteasome pathway (22, 40).
In our studies, not only was RU486 by itself without effect on PR down-regulation, the PR antagonist was able to completely block the progesterone-dependent loss of nuclear PR in rat gonadotropes. This finding is consistent with an in vivo study of rat uterus in which the down-regulation of PR protein and mRNA by progesterone was prevented by simultaneous administration of RU486 (41). Whether the action of the antagonist as a blocker of progesterone-dependent PR down-regulation in these studies compared with the action of RU486 on PR degradation in breast cancer cell lines (38, 39) is due to species differences in PR, receptor and coregulator levels, or primary tissue vs. cell lines is not known.
In summary, we show that progesterone leads to a clear down-regulation of nuclear PR protein in gonadotropes within 9 h of exposure. The loss of PR protein is due, at least in part, to degradation through the proteasome pathway; the appearance of new PR protein requires removal of progesterone and ongoing translation in the presence of E2. The time course for this progesterone-induced decrease in PR protein in gonadotropes coincides with the temporal extent of the preovulatory LH surge and accompanying progesterone increase in rats. We suggest that the progesterone surge, which initially serves to substantially augment the LH release action of GnRH, leads to loss of PR protein and consequently contributes to the termination of the LH surge.
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Acknowledgments |
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Footnotes |
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Received May 8, 2000.
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-activated STAT1 by the ubiquitin-proteasome pathway.
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