| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Divisions of Reproductive Science and Neuroscience (C.L.B., A.A.W.), Oregon Regional Primate Research Center, Beaverton, Oregon 97006; and Department of Physiology and Pharmacology (C.L.B.), Oregon Health Sciences University, Portland, Oregon 97210
Address all correspondence and requests for reprints to: Dr. Cynthia L. Bethea, Oregon Regional Primate Research Center, 505 Northwest 185th Avenue, Beaverton, Oregon 97006. E-mail: betheac{at}ohsu.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Many studies have shown that P down-regulates the expression of PR in breast cancer cell lines and rat uterus (7, 8, 9, 10). However, there is differential regulation of PR by P within different cell types of the primate reproductive tract. During the progestin-dominated human or nonhuman primate luteal phase, PR levels decline in the glandular epithelial elements of the functionalis, but PR continues to be expressed in the glandular cells of the basalis region of the endometrium and in the stromal cells (11, 12). PR also remain in the myometrium during the luteal phase (reviewed in Ref.13). During pregnancy with serum P levels approaching 200 ng/ml, PR is expressed in the decidua (maternal placenta derived from endometrial stroma) and in the myometrium (14). In the primate ovary, PR protein and PR mRNA appear to be stimulated by P, which is present locally in high concentrations (15, 16, 17). In the primate oviduct, the stromal cells also maintain PR during E + P treatment (18). Thus, the maintenance of PR expression in the brain when P is added to an E regimen is not completely unusual. Together, these observations support the hypothesis that there is tissue specific regulation of PR by its cognate ligand. However, little is known about the mechanism(s) by which the tissue specific regulation is exerted.
The observation that PR continued to be expressed in the brain when P was added to the E regimen differs from earlier studies with rodents (19, 20, 21, 22). Macaques have a 28-day menstrual cycle like humans. Also, during the primate menstrual cycle, E levels approach 200 pg/ml and P levels rise to 4–6 ng/ml (23). In rats, P levels may reach 60–80 ng/ml, a order of magnitude higher than in primates (24). Thus, hormone treatments that model the 4-day estrus cycle of the rat vs. the 28 day menstrual cycle may differ in the levels of E and P attained as well as the length of the treatment period employed. Nonetheless, in nonhuman primates the endometrium and pituitary exhibit a different pattern of PR regulation compared to the hypothalamus with the same hormonal treatment.
Nuclear PR protein exists as two isoforms, i.e. a short form of 94 kDa (PR-A) and a long form of 120 kDa (PR-B) (25). The PR-B isoform contains a 165 amino acid segment at the N-terminal that is not present in PR-A (25). These isoforms are thought to arise either by transcription from different promoters governing the PR gene or by alternative initiation of translation from the same mRNA (26, 27). The different promoters in the human PR gene exhibit differential regulation (27, 28, 29, 30), which can lead to different levels of isoform proteins. PR-A homodimers can act as ligand dependent transcriptional activators in a cell and promoter specific context (27, 30, 31, 32). However, in promoter and cell contexts where PR-A is inactive, it may act as a transdominant inhibitor of PR-B mediated gene transcription (32, 33). McDonnell and colleagues (34, 35) also showed that coexpression of ER with PR-A (but not with PR-B) caused a ligand dependent inhibition of ER-mediated gene transcription, but not through the formation of heterodimers nor by down-regulation of ER.
We questioned whether the ratio of PR-A to PR-B isoforms would vary in different tissues that exhibit differential regulation of PR by progestins in nonhuman primates. This information could further the hypothesis that the tissue-specific regulation of PR by P may be a consequence of the tissue-specific expression of the different PR isoforms. The E and P treatment paradigm employed with macaques is based upon previous studies of the regulation of ER and PR in the pituitary and hypothalamus as it relates to neuroendocrine function (2, 6). Animals are treated for 2–3 weeks with E alone and then treated with E + P for an additional 2 weeks. Moreover, this same treatment regimen causes proliferation and differentiation of the primate endometrium that is indistinguishable from the normal menstrual cycle (11).
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Surgery and treatments
Ovariectomies and hysterectomies were performed by the surgical
team of the Division of Primate Medicine at the ORPRC. One to twelve
months following surgery, each steroid-treated animal received a
SILASTIC brand (Dow Corning, Midland, MI) implant, sc, in the
periscapular area under Ketamine anesthesia (Ketamine HCL, 5 mg/kg, im;
Parke-Davis, Morris Plains, NJ). The first Ovx-control animal received
an empty capsule, and the remaining spayed controls were untreated
before necropsy. The E-treated animals received a SILASTIC brand
capsule (average length of 4.5 cm; ID = 0.132in; OD = 0.183
in) filled with crystalline estradiol (Steroloids, Wilton, NH) to
achieve 100–200 pg/ml of serum E over a 28–30 day period (2, 6).
Fourteen days after initiation of E treatment, the E + P-treated group
received (under Ketamine anesthesia), one 6.0 cm capsule filled with
crystalline P (Steroloids). This treatment has been shown to achieve
serum P levels between 4 and 8 ng/ml (2, 6). A blood sample was
obtained before or during necropsy for verification of the E and P
levels. After clotting and centrifugation at 4 C, serum was harvested
and stored at -20 C until assayed for E and P by RIA in the P30
Hormone Assay Core.
Before autopsy, each animal was initially tranquilized in the home cage
with Ketamine, followed with heavy pentobarbitol sedation and
exsanguination. The brain was removed after retraction of the
calvarium. The brain was placed with the dorsal aspects upward, and the
hypothalamus was immediately dissected with a scalpel. The pituitary
was simultaneously removed from the sella turcica with rongeurs. The
uterus was obtained if present. This is a standard procedure conducted
with the aide of the center veterinary pathologist and a pathology
laboratory technician. Time from opening the body cavity to brain and
pituitary collection is approximately 10 min. Collection of the uterus,
cervix, and vagina requires pelvic dissection, which takes another 10
min. All of the animals used, including a brief experimental history
before assignment to this project, the final treatment in this project,
the presence or absence of the uterus, and the tissues used for Western
analysis are noted in Table 1
.
|
During necropsy, the pituitary gland was dissected free of meninges, wrapped in foil, and dropped in liquid nitrogen. The pituitary was then stored at -80 C until Western analysis. The posterior lobe and the pituitary stalk were removed before Western analysis. Only the anterior lobe was processed further.
After dissection, the uterus was longitudinally sliced into four quarters. The endometrium appears full and white in steroid-treated monkeys, but it is a thin white sliver of tissue in Ovx monkeys. No blood was present in the lumen of the uterus of any treated animal. The endometrium was cut from the myometrium with a pair of delicate dissecting scissors with the aid of a dissecting microscope, wrapped in foil, placed in liquid nitrogen, and stored at -80 C until Western analysis. The procedure employed in collection of the endometrium has been used extensively for the study of ER and PR (11).
Tissue preparation for Western analysis
Unless otherwise stated, all chemicals and reagents were from
Sigma Chemical Co. (St. Louis, MO). A high concentration of salt was
employed to extract both cytosolic and nuclear PR. The homogenizing
buffer for PR extraction from all tissue (pituitary, MBH, and
endometrium) consisted of 50 mM
HK2PO4, 50 mM NaF, 1 mM
EDTA, 1 mM EGTA, 12 mM glycerol, 0.5
M NaCl (pH 7.4) according to Beck et al. (37).
Protease inhibitors (Pepstatin A at 1 µg/ml, Leupeptin at 47 µg/ml,
Bacitracin at 100 µg/ml, Aprotinin at 77 µg/ml), and 5 mg/ml (0.02
M) NaMo were also added. The pituitary and MBH were
homogenized in a volume of 2 ml. Endometrial tissue was homogenized on
wt/vol basis (80 mg/ml). Homogenates were centrifuged at 4 C for 30 min
at 14,000 x g to remove insoluble protein. The
supernatant was harvested and the pellet discarded. The supernatant was
transferred to a slide-a-lyzer cassette (Pierce Chemical Co., Rockford,
IL) and dialyzed in buffer (same as homogenizing buffer minus NaCl,
protease inhibitors and NaMo) for 2 h to remove salt. All samples
were recovered from the dialysis cassettes and centrifuged for 5 min at
14,000 x g to remove any remaining insoluble protein.
Endometrial samples were complete at this point and placed on ice until
protein determination. Pituitary and MBH samples were further
concentrated by centrifuging the supernatant at 5,000 x
g for approximately 2 h (final volume of approximately
0.5 ml), using Centricon microconcentrators (Amicon, Inc., Beverly, MA)
with a 50-kDa cutoff. This cutoff would remove PR-C if present (38).
The pituitary and MBH samples were harvested from the concentrators,
and the protein levels were determined. Following protein
determinations, the samples were aliquoted, stored overnight at 4 C,
and subjected to PAGE the following day.
Protein assay
Protein levels were determined with the Bio-Rad (Hercules, CA)
Protein Determination Reagent according to the method of Bradford
(39).
T47D preparation
T47D cells were provided by the Cell Culture Core of the
P30 Population Center. T47D cells were grown in T150 culture flasks
(Corning Costar Corp., Cambridge, MA) in RPMI 1640 containing 10% calf
serum, 10% FCS, and 0.2 IU/ml insulin until confluent (
10 days).
For harvest, the medium was removed and 10 ml of sterile PBS (0.15
M, pH 7.4) was added to each flask. The cells were removed
from the bottom of the flask with a spatula. Aliquots of 1 ml were
stored at -80 C until needed for assay. The day of gel
electrophoresis, one vial of frozen T47D cells was removed from -80 C
and placed in liquid nitrogen until homogenization. A dounce and pestle
was prechilled on ice and equilibrated with cold homogenization buffer
(10 mM Tris base, 1.5 mM EDTA, 1.0
mM dithiothreitol, 0.5 M KCl, pH 7.4) to which
protease inhibitors (phenylmethylsulfonyl fluoride at 35 µg/ml,
Pepstatin A at 0.7 µg/ml, Leupeptin at 0.5 µg/ml, and Aprotinin at
1 µg/ml) were added. To the vial containing the frozen T47D cells, 1
ml of homogenization buffer was added, mixed thoroughly with a 1 ml
pipettor, and then decanted into the glass dounce. All homogenization
was done on ice for approximately 2 min. Appropriate amounts were
aliquoted and further prepared in the same manner as the tissue samples
for loading on the gel.
Electrophoresis and Western blotting
A standard gel apparatus was used to enable the loading of large
amounts of protein from the hypothalamus, which was necessary to detect
the much lower levels of PR per mg protein in that tissue. Sample
buffer (25–50 µl; 2x Laemmli) was added to each vial containing
pituitary, MBH, endometrial, T47D, and molecular weight markers (MWM,
Bio-Rad), boiled for 5 min, and loaded on a freshly poured 7% sodium
dodecyl sulfate (SDS) polyacrylamide gel with a 5% stacking gel.
Approximately 300 µg of endometrial protein, 800 µg of pituitary
protein, and 1000–1500 µg of MBH protein were loaded on the gel. The
gel was placed in an electrophoresis unit (SE 600 series, Hoefer, San
Francisco, CA) that was filled with running buffer (0.25 M
Tris Base, 1.92 M glycine, and 1% SDS) and connected to a
Bio-Rad power supply (model 200/2.0 A). The gel was run for
approximately 2 h at 75 V and 2 h at 150 V. The gel was then
stored at 4 C overnight.
The following morning, the gel, Biorad sponges, filter paper, and nitrocellulose (Schleicher and Schuell, Keene, NH) were soaked in blotting buffer (25 mM Tris, 192 mM Glycine, 20% Methanol, and 50 mM NaCl, pH 8.3) for approximately 20 min. A Bio-Rad transblot cell attached to a bath and circulator (model 2006, Forma Scientific, Marietta, OH) was used to transfer protein from the gel to the nitrocellulose. To increase transfer potential, an aliquot of SDS solution was added to the cold blotting buffer that filled the transblot cell (final concentration 0.05% SDS). Time of transfer was approximately 2 h at 1.6 A.
After transfer was complete, the nitrocellulose blot was trimmed and subsequently stained with 2% Ponceau S in 3% TCA for approximately 10 min or until MWM bands appeared. The MWM lane was cut off and the nitrocellulose blot was incubated in 5% milk (nonfat, powdered) for 20 min to block nonspecific binding. A 5-min wash with saline/Tween (10 mM Tris, 0.9% NaCl, 0.05% Tween 20, pH 7.4) followed. The primary antibody B39 (monoclonal rat antihuman PR), a gift from Dr. Geoffrey Greene (Ben-May Institute, University of Chicago, Chicago, IL), was diluted in Tris-buffered saline (50 mM Tris base, 154 mM NaCl, pH 7.5) at 1–2 µg/ml for endometrium and pituitary lanes and at 4 µg/ml for MBH lanes. The nitrocellulose blot was incubated in primary antibody at 4 C overnight. The primary antibody, B39, binds to the 3' hormone binding domain of human and primate PR. Hence, it recognizes PR-A, PR-B and presumably PR-C. This antibody has been extensively characterized both biochemically and immunohistochemically (2, 40, 41, 42). It binds to PR in the presence or absence of ligand.
Following overnight incubation, the nitrocellulose was washed with saline-Tween at room temperature (6x at 5 min each) and then incubated with an antirat IgG second antibody that was conjugated to HRP. The second antibody-HRP conjugate was diluted in 5% milk at a dilution of 1:25,000 and incubated with the blot for 1 h at room temperature. The nitrocellulose was again washed for 30 min using saline/Tween and developed for 1 min in the enhanced chemiluminescence (ECL) reagents (Amersham, Arlington Heights, IL). All antibody incubations and rinses used a rocker platform (BellCo, Vineland, NJ). The nitrocellulose was placed on a clean glass plate, covered with a piece of transparency, and taken to the darkroom. Kodak X-OMAT AR5 (Eastman-Kodak, Rochester, NY) developing film was placed on top of the nitrocellulose-transparency complex and allowed to expose for various times depending on the samples.
In most experiments, the pituitary and hypothalamus from spay, E-treated and E + P-treated animals were run on the same gel. The nitrocellulose blot was incubated with 2 µg/ml of B39 and developed to visualize the bands. If the PR in the hypothalamic lanes was not apparent, then the blot was cut down to contain only the hypothalamic sample lanes and the T47D control lane, lightly stripped, and then reincubated with 4 µg/ml of B39 and redeveloped. In latter experiments, the pituitary and hypothalamic lanes were cut apart and immediately developed in 2 µg/ml and 4 µg/ml of B39, respectively.
Densitometric analysis
ECL films of the Western blots were captured with a CCD XC77
camera (Sony, Toyohashi, Japan), and the NIH Image software program
using a gel analysis macro subroutine. The area of the lane to be
scanned contained the bands corresponding to PR-A and PR-B. This area
was selected by the operator. The NIH gel macro then scanned each
selected area and plotted the intensity. The baseline reading of the
film (no bands) was considered background. The operator sealed the
peaks corresponding to PR-A and PR-B on each plot. The seal is placed
from the bottom point at the start of the peak to the bottom point at
the end of the peak. This automatically subtracts the baseline. The
software program then calculates the area within the sealed peak. The
area of the PR-A peak divided by the area of the PR-B peak yields the A
to B ratio. The images were finally imported to the Freehand
(Macromedia, San Francisco, CA) software program, labeled, and then
printed on a Tektronics Phaser II printer.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
lists the animals that were used in the Western blot
analysis of the isoforms of PR. A brief experimental history of each
animal and the last treatment before necropsy is shown. The presence or
absence of the uterus during the final treatment and the tissues that
were used in the analysis from each animal are indicated. The figures
in which the tissue samples appear are also noted.
Western analysis
Protein bands with mol wt of 120K and 94K corresponding to the
long (B) and short (A) isoforms of PR, respectively, were readily
detectable in the T47D cells. Protein bands were detected in the
endometrium, pituitary, and hypothalamic samples, which comigrated with
those in T47D cells. These bands were present only when the
nitrocellulose blot was incubated with antihuman PR monoclonal
antibody, B39. Neither isoform band was observed with omission or
suboptimal concentrations of anti-PR monoclonal antibody B39.
Additional bands were observed in the monkey samples at higher than
120K and lower than 94K mol wt. One band at approximately 68–69K was
observed consistently in PR-positive and PR-negative tissues,
suggesting that this band resulted from nonspecific binding of the
second antibody to monkey IgG. Several bands were present between 68K
and the sample front (about 35–40K) in the endometrium samples, one of
which may represent PR-C (38). Material in this range was greatly
reduced or absent in the pituitary and hypothalamic samples due to the
use of the 50K cutoff microconcentrators. Therefore, only the bands at
120K and 94K were considered isoforms of PR and subjected to further
analysis.
When increasing amounts of T47D extract were loaded on the gel, there
was a corresponding linear increase in the signal corresponding to PR-A
and PR-B detected on the ECL film. The densitometric analysis yielded
peak areas that increased in a linear manner as well (Fig. 1).
|
illustrates the A and B
isoforms in the endometrium homogenate from animals treated with E or E
+ P. The PR-A isoform was most prominent in the endometrium from three
animals treated with E for 28 days. Both isoforms were detectable in
two out of the three animals treated with E + P and the PR-A isoform
was again most prominent. The PR-A isoform exhibited a secondary band
in the endometrium samples from E-treated monkeys. PR-B also migrated
as a doublet in two of the three E-treated samples. Based upon the
conclusions of others (10, 43), the multiple bands probably represent
different phosphorylation states of the two isoforms. With addition of
P, both isoforms migrated as singlet bands. The endometrium is
extremely reduced in Ovx animals. One endometrium from an Ovx monkey
was processed, and PR was undetectable. It seemed unnecessary to repeat
this observation.
|
. There was 5.28 times more A than
B isoform in the E-treated endometrium and 5.88 times more A than B
isoform in the E + P-treated endometrium. There is no statistical
difference in the A to B ratio between the E and E + P-treated samples
(unpaired t test). Therefore, in further comparisons the
ratios of the E and E + P-treated endometrium samples are combined.
|
with positive control T47D
samples. T47D samples were included on every gel, and the isoform bands
in each sample comigrated with the isoforms of the T47D sample that was
included on the respective gel. However, for illustration purposes, the
T47D sample that corresponds to the majority of the pituitary samples
on the same horizontal row of the figure is shown. Pituitary samples
from other gel dates are marked with an asterisk, and the
bands in these marked samples may not line up exactly with the T47D
sample shown.
|
The A and B isoforms were also detectable in the pituitaries of monkeys treated with E + P. There appears to be a modest, but incomplete, down-regulation of PR in some of the E + P-treated pituitaries. This is most evident when comparing E and E + P-treated samples run on the same gel such as 14416 (E) vs. 16843 (E + P). However, even comparison of nonmatched samples such as 15608 (E) vs. 16866 (E + P) or 16899 (E + P) indicates a modest decrease in the amount of PR detected. In the E + P-treated pituitaries, the A isoform appears more prominent than the B isoform. Both isoforms appeared to migrate as singlet bands in the presence or absence of P.
Densitometric analysis was applied to the A and B isoforms from the E
and E + P-treated pituitary samples and the results are shown in Table 2. There was 1.67 times more A than B isoform in the E-treated
pituitaries and 1.87 times more A than B isoform in the E + P-treated
pituitaries. There is no statistical difference in the A to B ratio
between the E and E + P-treated samples (unpaired t test).
Therefore, in further comparisons the ratios of the E and E + P-treated
pituitary samples are combined.
Hypothalamus
A comparison of the strength of the hypothalamic PR signal at
different concentrations of B39 and with different exposure times is
shown in Fig. 4. Pituitary and T47D
samples developed with 2 µg/ml B39 are shown for comparison. The
hypothalamic signal improved at higher concentrations of B39 and with
longer exposure to the film. At 4 µg/ml of B39 and with 20 min
exposure, a signal that can be subjected to densitometric analysis is
present. There appears to be more PR-B than PR-A, and the B isoform
migrated as a triplet, which may represent different phosphorylated
states. With the longer exposure time, the T47D and pituitary samples
are overexposed.
|
illustrates the ten hypothalamic
samples from the three treatment groups. Again, samples marked with an
asterisk were not run on the same gel as the other samples
in the row. The A and B isoforms were too faint for densitometric
analysis in the hypothalamic samples from the Ovx or spayed monkeys.
There was a marked induction of the PR-A and PR-B isoforms in the
hypothalamus of the monkeys treated with E for 28 days. The A isoform
appears nearly equimolar to the B isoform upon visual inspection. The
PR-A and PR-B isoforms were also detectable in the hypothalamus of
monkeys treated with E + P. Both isoforms migrated as single bands in
the presence or absence of P with one exception. An E-treated animal
(15608) exhibited PR-B in a doublet.
|
. The average amount of A and B isoforms were similar in the
E-treated hypothalami (A/B = 0.62) and in the E + P-treated
hypothalami (A/B = 1.05). However, the ratio in the hypothalami
ranged from 0.15 to 2.08. In four of the eight steroid-treated animals,
there was between 2- and 10-fold more B than A. In three of the eight
steroid-treated animals, A and B were equimolar, and in one animal
there was 2-fold more A than B. Upon statistical comparison, there was
no difference in the A to B ratio between the E and E + P-treated
hypothalamic samples (unpaired t test). In the hypothalamus
from the ovarian intact monkey, there was 4 times more B than A
(A/B = 0.23). In further comparisons, the ratios of all of the
hypothalamic samples are combined.
Overall comparison
To determine if there was a significant difference between the A/B
ratio of the different tissues, the E and E + P treatment groups were
combined and compared with each other and to the T47D cells with
unpaired ANOVA followed by Student-Newman-Keul’s (SNK) pairwise
comparison. As illustrated in Fig. 6, there was a significant difference in the A/B ratio between the various
tissues (P < 0.001, ANOVA). Pairwise comparison
revealed that the A/B ratio in the endometrium was significantly higher
than in the pituitary (P < 0.01, SNK) and hypothalamus
(P < 0.01, SNK). In addition, the A/B ratio in the
pituitary was significantly higher than in the hypothalamus
(P < 0.05, SNK). The T47D cells differed from the
endometrium but not the pituitary or hypothalamus.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Interpretations
PR-A homodimers may mediate gene transcription independently of
PR-B in a cell and promoter-specific manner, and there are reports of a
few genes that are preferentially induced by PR-A (27, 30, 31).
Molecular transfection experiments have also suggested that when PR-A
is unable to activate a specific promoter, then PR-A may act as a
dominant negative inhibitor of PR-B, again in a promoter specific
manner (32, 33). PR-A also decreases the transcriptional efficiency of
ER (34, 35), and PR-A mediated repression of human ER transcriptional
activity depends largely on the absolute expression level of PR-A (35).
Thus, different ratios of PR-A and PR-B could result in different
outcomes when P is present. For example, in the endometrium P would
bind to 5 times more A than B, resulting in more A/A or A/B dimers in
which PR-A is dominant (44). The greater amount of PR-A could decrease
the transcriptional efficiency of B, decrease the transcriptional
efficiency of ER, or activate specific genes that prefer PR-A
homodimers. In the hypothalamus, P would bind to more or equal amounts
of B vs. A. In this context, there is a greater chance of
B/B homodimers forming, which could maintain transcriptional activity
of PR-B regulated genes. ER could also maintain transcriptional
activity. Hence, the action of E and P could continue in the
hypothalamus when P is added to an E regimen. This is consistent with
several of the known physiological actions of E and P in the
hypothalamus (2, 45, 46, 47, 48). The ratio of A to B in the pituitary was
intermediate between that observed in the hypothalamus and endometrium.
In previous studies, we and others observed that addition of P to an E
regimen decreased PR in the pituitary (2, 49). Perhaps the 2-fold
higher level of PR-A than B contributes to the down-regulation of PR by
P in the pituitary.
The ratio of A/B was the same in the E-treated and in the E + P-treated groups in all the tissues examined. Thus, P did not appear to differentially affect the expression of one isoform or the other in the hypothalamus, pituitary or endometrium. The similarity in the A/B ratio in E or E + P-treated monkey endometrium is in agreement with previous human data obtained during the E dominated follicular phase vs. the P dominanted luteal phase (50). The lack of a change in the A/B ratio in hypothalamus and pituitary upon addition of P is consistent with the endometrium. The mechanism by which P regulates expression of its cognate receptor has been the focus of many studies, but the identification of a classical canonical PRE in the promoter region of the PR gene has been elusive. An intragenic (+698/+723) E-responsive element has been located in rabbit PR, which mediates E and tamoxifien induction. Repression by PR, retinoic acid receptors and AP-1, was mediated by the same response element without direct contact of these proteins with the DNA (51). These data indicate that protein-protein interactions may play a significant role in the action of P on the expression of PR and the proteins involved could vary in a cell or tissue specific manner. However, rabbits exhibit predominantly PR-B, so whether this mechanism is operative in primates is unknown.
Contrast to other studies
The two isoforms of monkey PR identified with antibody B39 had
molecular weights similar to those in the T47D human breast cancer cell
line and consistent with earlier reports describing human PR in
endometrial samples (52). Based upon the amount of protein needed to
detect PR, there is at least 3-fold less PR per mg soluble protein in
the pituitary than in the endometrium, and there is probably another 3-
to 5-fold less PR per mg soluble protein in the MBH than in the
pituitary. Although it was important to work in a linear range of PR
detection on the blots, it was not necessary to quantitate the mass of
the PR to examine the ratio of A to B in these experiments.
In a recent characterization of the ratio of PR-A to PR-B in human breast cancer samples, the ratio ranged from 0.04 to 179.3 with a median ratio of 1.26. However, tumors containing an A/B ratio greater than 4 were overexpressed in the group (50). This observation led the authors to suggest that the subpopulation of tumors with A/B ratios greater than 4 may exhibit a different response to hormone therapy than those with an A/B ratio closer to unity. It follows that normal tissues with a different ratio of A/B may exhibit a different response to P.
Our observation that PR-B is equimolar or greater than PR-A in the hypothalamus is consistent with previous studies examining PR isoforms in rat brain and in monkey corpus luteum. In rats, the B form was predominantly expressed first around birth and PR-B mRNA existed predominantly in the neonatal rat brain cortex (53). Duffy et al. (54) recently reported that PR-B exceeds PR-A in the monkey corpus luteum, another tissue in which PR expression continues in the presence of high local concentrations of P.
Multiple bands
Hormone binding to steroid receptors causes an increase in
phosphorylation (10, 55, 56) and phosphorylation state may alter the
transcriptional activity of the receptor (37, 57, 58, 59). The presence of
multiple bands in the absence of P is believed to represent
constitutive basal phosphorylation of PR at one or more sites. When a
progestin is present, full phosphorylation occurs and the PR isoform
migrates as one band with slower mobility (37, 43). A change from
doublet to singlet bands with an increase in total 32P
incorporation upon addition of a progestin was described in the human
T47D breast cancer cells (37, 43). In the E-treated monkey endometrium,
we generally observed PR-A and PR-B as doublets. With addition of P,
PR-A and PR-B migrated as singlets, although a marked change in the
mobility was not observed. Previous examination of the isoforms of PR
in the human endometrium indicated that PR-B migrated as a triplet and
that PR-A migrated as a singlet during the E dominated follicular
phase. With exposure to P during the luteal phase, PR-B migrated as a
doublet (52). Thus, the loss of multiple bands in the endometrium with
addition of P appears consistent between humans and monkeys.
Conclusions regarding phosphorylation must be reserved until
32P incorporation studies, but the Western analysis shown
here is consistent with a change in phosphorylation status in the
presence of P in the monkey endometrium.
Multiple bands were not generally observed in the pituitary or hypothalamus. In the E or E + P-treated pituitary, both PR-A and PR-B appeared as single bands. In the hypothalamus, only the intact monkey and one E-treated monkey exhibited multiple forms of PR-B, which is consistent with multiple basal phosphorylation states in the absence of P. The PR in the hypothalmus was at the limit of detection in this assay, so we cannot rule out the possibility that lesser phosphorylated forms may be present but not always visible. Also, with the discovery of nonligand mediated phosphorylation mechanisms (46, 60), it is reasonable to speculate that other agents may be contributing to basal phosphorylation of PR in the hypothalamus and pituitary in the absence of P. However, it is interesting to note that PR-B appeared as a triplet in the hypothalamus of the intact monkey who had undetectable levels of E or P and appeared to be in the menstrual phase of the cycle. This suggests that in future studies it may be possible to determine the isoforms of PR in the hypothalamus if the animal has been deprived of steroids for a short time instead of months.
Caveats
Earlier binding studies found that P treatment down-regulates
cytosolic PR when the entire endometrium is homogenized (61). Yet, PR
was detected in the E + P-treated endometrium in this Western analysis.
Later studies showed that within the endometrium only the epithelial
cells of the glandular elements in the functionalis exhibit a
down-regulation of PR with P treatment. The stromal cells and the
epithelial cells of the basalis maintain PR expression in the presence
of P (11, 13). Thus, homogenization of the entire endometrium would
include all of these different cell types and surely included stromal
cells which continue to express PR during P treatment. Hence, the
presence of PR in the endometrium from the E + P-treated
monkeys is understandable.
PR in the pituitary of the E + P-treated rhesus monkeys in this Western blot analysis exhibited a modest but incomplete down-regulation. In previous experiments with cynomolgus macaques, PR protein as measured with ICC or with a gradient shift assay and the same B39 antibody was markedly reduced in the pituitary when P was added to the E regimen (2). In addition, PR mRNA was down-regulated in the pituitary in E + P-treated monkeys (6). P can be antagonistic to E or synergistic with E depending on the relative level of P to E in a species specific manner (62). However, in previous experiments and in this study, the E/P ratio was similar, that is about 1/64. Another possible explanation relates to the length of time between Ovx and hormone treatment or to the long-term treatment with steroids in previous protocols. The cynomolgus macaques used in the earlier study of PR protein had been in no previous experimental protocols. They were Ovx during the early follicular phase and immediately treated with implants for the following 28 days. In this study, the use of longer-term Ovx rhesus or the use of animals with several previous artificial cycles (a constant level of E for extended periods) could have affected the pituitary response to P. Finally, the possibility that there is a species difference in the pituitary sensitivity to P cannot be completely discounted. Nonetheless, the conclusion that P always down-regulates PR in the pituitary probably requires some adjustment and further experimentation to better clarify the exact parameters. In future studies we hope to examine the levels of the PR isoforms during the menstrual cycle with normal fluctuating levels of E and P.
Conclusion
In summary, Western blot analysis of E and E + P-treated female
rhesus macaques revealed that the endometrium expresses about 5 times
the amount of PR-A than PR-B and the pituitary expresses about twice as
much PR-A than PR-B. However, the hypothalamus exhibited equimolar or
greater levels of PR-B than PR-A. The A/B ratio was significantly
higher in the endometrium than in the pituitary and hypothalamus. In
addition, the A/B ratio was significantly higher in the pituitary than
in the hypothalamus. Moreover, the presence of P did not significantly
alter the expression of PR-A relative to PR-B in any of the tissues
examined. Therefore, there appears to be tissue-specific expression in
the relative levels of PR-A and PR-B that may subserve the tissue
specific actions of P.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received August 8, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  S. Mani Progestin Receptor Subtypes in the Brain: The Known and the Unknown Endocrinology, June 1, 2008; 149(6): 2750 - 2756. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Shao, E. Egecioglu, B. Weijdegard, K. Ljungstrom, C. Ling, J. Fernandez-Rodriguez, and H. Billig Developmental and hormonal regulation of progesterone receptor A-form expression in female mouse lung in vivo: interaction with glucocorticoid receptors. J. Endocrinol., September 1, 2006; 190(3): 857 - 870. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. K. Mani, A. M. Reyna, J. Z. Chen, B. Mulac-Jericevic, and O. M. Conneely Differential Response of Progesterone Receptor Isoforms in Hormone-Dependent and -Independent Facilitation of Female Sexual Receptivity Mol. Endocrinol., June 1, 2006; 20(6): 1322 - 1332. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Shao, E. Markstrom, P. A. Friberg, M. Johansson, and H. Billig Expression of Progesterone Receptor (PR) A and B Isoforms in Mouse Granulosa Cells: Stage-Dependent PR-Mediated Regulation of Apoptosis and Cell Proliferation Biol Reprod, March 1, 2003; 68(3): 914 - 921. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Fang, S. Wong, and B. F. Mitchell Messenger RNA for progesterone receptor isoforms in the late-gestation rat uterus Am J Physiol Endocrinol Metab, December 1, 2002; 283(6): E1167 - E1172. [Abstract] [Full Text] [PDF]
|
|
