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`PH: S0960-0760(97)00077-0
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`_7. Steroid Biochem. Molec. Biol. Vol. 63, No. 4-6, pp. 309-316, 1997
`© 1997 Elsevier Science Ltd. All rights reserved
`Printed in Great Britain
`0960-0760/97 $17.00 + 0.00
`
`Progesterone Receptor Repression by
`Estrogens in Rat Uterine Epithelial Cells
`
`Karsten Parczyk,‘* Robert Madino,‘ Horst Michna,‘
`Yukishige Nishino‘ and Martin R. Schneider‘
`
`‘Research Laboratories of Schering AG, D-13342 Berlin, Germany and 2Department of Morphology and Tumor
`Research, DSHS, 50933 Cologne, Germany
`
`Measurements performed using cell lines or animal tissues have shown that the progesterone recep-
`tor (PR) can be induced by estrogens. By use of immunohistochemistry we studied the effects of es-
`trogens on the PR levels in the individual cell types of the target organs uterus and breast. In the
`uteri of rats, ovariectomy induced a decrease in PR immunoreactivity within the myometrium and
`outer stromal cell layers. In contrast, in the uterine luminal and glandular epithelium and sur-
`rounding stromal cell layers the PR immunoreactivity was significantly enhanced. The same picture
`emerged when intact: rats were treated with the pure estrogen receptor antagonist, ZM 182780
`(10 mglkgld). Treatment of ovariectomized rats with estradiol resulted in high PR levels in the myo-
`metrium and stroma cells but low PR immunoreactivity in the epithelial cells. The ER-mediated
`repression of the PR immunoreactivity was evidently restricted to the uterine epithelium, as we
`found that in the epithelial cells of the mammary gland and in cells of N-nitrosomethylurea-induced
`mammary carcinomas the PR expression was induced by estrogens and was blocked by the pure
`antiestrogen ZM 182780. These results clearly show that in the rat the activated ER induces diver-
`ging effects on PR expression in different cell types even within the same organ. © 1997 Elsevier
`Science Ltd. All rights reserved
`
`j‘. Steroid Biochem. Moles. Biol, Vol. 63, No. 4-6, pp. 309-316, 1997
`
`INTRODUCTION
`
`The estrogen receptor (ER) is a nuclear transcription
`factor that - after activation by its ligand, estradiol
`(E2) — induces the expression of certain target
`genes [1,2]. Estrogen receptor antagonists that bind
`to the ER and prevent:
`its activation by the natural
`agonist have been described [3]. Pure antiestrogens
`such as ZM 182780 specifically and completely block
`the aetiVifY Of
`the ER in many SYStemS (= Pure
`
`of selective ER blockade by a pure antiestrogen and
`ER activation by estradiol on the PR levels in differ-
`ent cell types of the rat. We provide evidence that in
`contrast to other cell types the PR is repressed by es-
`trogens in the uterine epithelium.
`
`MATERIALS AND METHODS
`
`The rats were ovariectomized or treated with com-
`
`the period indicated. Analysis of the
`pounds for
`3f1'EieStF0gef1S)[4l-
`estrous cycle was done by vaginal smears.
`l7fi-estra-
`One well known estrogen inducible target gene is
`diol and ZM 182780 (701-[9—(4,4,5,5,5,-pentafluoro-
`the progesterone receptor
`(PR). From assays per-
`pentylsulfinyl)-nonyl]-estra-1,3,5,(lO)-triene—3,17/3-
`formed with whole tissue extracts the general picture
`di0i: Synthesized in the laboratories Of Sehefiflg AG:
`emerged that PR levels are enhanced by estrogens
`and reduced by progestins [5-8]. Subsequent immu— Berlin) were dissolved in Castor oil containing 20%
`nohistochemical studies revealed species and cell type
`benzyi beilzoate and administered 6 days Pei‘ Week by
`Specific
`differences
`in hormonal pR regulation
`subcutaneous injection. At the end of the experiments
`(reviewed in [9]). In our study we compared the effect
`the inguirfal mammery glands and uteri were exeiSed-
`The uteri were weighed and one half of the organ
`was directly Snap-frozen In hquld nltrogen (for hgand
`binding assay)- The Other Part Of the Orgaii a5_We11
`as the mammary glands were embedded in Tissue
`
`*Correspondence to K. Parczyk. Tel: +49-30—46817752; Fax: +49-
`30-46818069; E-mail: Karsten.Parczyk@Schering.de.
`Received 20 Feb. 1997; accepted 16 ]un. 1997.
`
`309
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`Karsten Parczyk er al.
`
`800
`
`700
`
`600
`
`
`
`
`
`300
`
`500
`
`400
`
`PR-level[fmol/mgprotein] 200
`
`frozen in isopentane precooled with liquid
`Tek@,
`nitrogen and stored at —80°C (for PR immunocyto-
`chemistry).
`
`Measurement of PR from homogenates via ligand binding
`assay
`
`For the extraction of whole PR the frozen tissues
`
`were pulverized and homogenized in high salt buffer
`(20 mM Tris,
`10 mM Na2MoO4,
`10% glycerol,
`1.5 mM EDTA, and 400 mM KCI, pH 7.5) contain-
`ing a protease cocktail. After centrifugation of the
`homogenates at 100 000 g (1 h, 4°C) and after deter-
`mination of protein levels the extracts were diluted to
`a final KCl concentration of 50 mM for the ligand
`binding assay. This assay was performed using [3H]—
`ORG-2058 with or without a 200-fold excess of un-
`
`labelled ORG-2058 to differentiate between unspeci-
`fic and specific binding. Incubation was carried out at
`4°C for 16 h. After separation of unbound steroid via
`the dextran—coated charcoal method the specific bind-
`ing and thus the PR content was calculated as
`described [10] .
`
`Immunocytochemical detection of the PR in the rat
`
`A minimum of three 5 ,urn cryosections of each spe-
`cimen were performed. The sections were fixed for
`10 min in 3.7% formaldehyde—PBS, 4 min in metha-
`nol
`(—20°C) and 2min in acetone (—20°C). After
`blocking in rat serum (122 in PBS)
`for 30 min a
`monoclonal antibody against the PR (reacting with
`the PR—A and PR-B form, MA1—410, Dianova,
`Hamburg) [11] was applied at 20 ,ug/ml in PBS with
`2% BSA for 18 h at 4°C. After blocking of endogen-
`ous peroxidases the second antibody-biotinylated rat
`anti-mouse IgG (Dianova) — was applied as a 1:600
`dilution. The biotinylated secondary antibody was
`detected by the ABC technique (Vector) using diami-
`nobenzidine. To check for unspecific binding all spe-
`cimens were also incubated in parallel with a mouse
`IgG at 20 ug/ml
`instead of the first antibody. The
`resulting staining intensity was always very low. In
`addition, the specificity of the MA1-410 antibody was
`further verified by western-blot analysis of rat uterine
`extracts (not shown).
`
`100
`
`Fig. 1. Effect of ovariectomy or treatment with estrogensl
`antiestrogens on the PR level in homogenates of whole uteri:
`(a) Effect of ovariectomy or treamient of intact animals with
`ZM 182780 (10 mglkgld) for 21 days.
`(1)) Treatrnent of ovari-
`ectomized animals with estradiol
`(0.1 nglanimalld)
`for
`28 days. The mean valuesiS.E.M. are given. *p<0.01 vs.
`control; **p<0.05 vs. control; ***p<0.0l vs. ovariectomy;
`Dunnet test (11 2 7).
`
`The intensity and distribution of specific staining
`were evaluated visually as described by Snijders er
`al. [12]. A receptor score was calculated as follows:
`{:4
`
`Receptor score = ZP(z') x 2'
`i=0
`
`where i is intensity of staining from 0 (no staining) to
`4 (very intense staining) and P(z) is the percentage of
`stained cells in category 2' (0—100%). From each spe-
`cimen three cross sections were scored independently
`
`Fig. 2 (facing page). PR immunoreactivity in the different uterine cell types of intact and ovariectomized
`rats.
`
`(a,b) PR staining pattern in intact rats (estrous phase). (a) Myometriurn: nuclei of the myometrial inner and
`outer smooth muscle cell layers are prominently stained. Nuclei of blood vessel cells are also PR positive.
`(b) Endometriurn: The fibroblast-like stromal cell nuclei are PR positive. The luminal epithelium shows the
`typical increase in epithelial cell height. These cells show virtually no PR positive staining (arrow).
`(c,d) PR staining 21 days after ovariectomy: (c) Myometrium: No or only marginal PR immunoreactivity is
`detectable. (cl) Endometrium: High PR expression in the luminal and glandular epithelial cells (arrow) and in
`the surrounding stromal cell layers. Toward the outer stromal cell layers the PR expression is gradually
`reduced. The low epithelial cell height is typical for uterine histology after ovariectomy. Magnification: 200 X ;
`bar E 50 ;4M. M = myometrium; S = stroma; E = epithelium.
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`PR Repression by Estrogens in Uterin Epithelium
`
`313
`
`by two investigators. The scoring results of the indi-
`vidual
`investigators correlated with an r2 value of
`0.79.
`
`RJESULTS
`
`Studies on the effects of ovariectomy, the antiestro-
`gen ZM 182780 or treatment with estradiol (E2) on
`the PR expression in the rat uterus by means of the
`classical ligand binding assay using extracts of whole
`tissue homogenates revealed that the PR level started
`to decline 3 days after ovariectomy and after 14 days
`had reached 50% of the untreated controls
`(not
`shown). The PR levels had not declined any further 3
`or 4 weeks post ovariec1:omy [Fig. 1(a)]. Treatment of
`intact animals with the pure antiestrogen ZM 182780
`(10 mg/kg/d for 21 days) resulted in a PR level com-
`parable to ovariectomy. When ovariectomized animals
`were treated with E2 (0.1 yg/animal/d for 28 days)
`the PR levels were restored to the level of intact ani-
`
`mals [Fig. l(b)].
`The fact that the PR level declined by only 50%
`after ovariectomy or treatment with the pure antics-
`trogen ZM 182780 raised the question of whether PR
`expression in the uterus is,
`in general, not
`fully
`dependent on estrogens or whether the PR is differen-
`tially regulated by estrogens in the various uterine cell
`types.
`The immunohistochemical analysis to study the cell
`type specific PR expression revealed that
`the PR
`staining was localized exclusively in the nuclei of all
`cell
`types looked at and after all treatment regimes
`carried out. In the uteri of intact rats changes in PR
`immunoreactivity during the
`estrous
`cycle were
`obvious. We found high PR expression in the myo-
`metrial smooth muscle cells, blood vessels and in
`stromal cells during the estrous (and proestrus) phase
`where the serum E2 levels were high [Fig. 2(a and b);
`Fig. 5 for quantification]. After ovariectomy, the PR
`expression significantly declined in the myometrium
`
`and in the outer stromal cells, whereas in the luminal
`and glandular epithelium and the surrounding cell
`layers
`the PR immunoreactivity was
`significantly
`enhanced [Fig. 2(c and d); Fig. 5].
`The specific blockade of the ER ftmction in intact
`animals by use of the pure ER antagonist
`(ZM
`182780; 10 mg/kg for 21 days) also resulted in an
`ovariectomy-like PR expression pattern — significant
`PR repression in the outer uterine cell layers and PR
`induction in the uterine epithelium [Fig. 3(a and b);
`Fig. 5]. Treatment of ovariectomized animals with E2
`(0.3 ug/animal/day for 14 days) resulted in a high PR
`expression in all cell
`layers except the uterine epi-
`thelium where its expression was repressed [Fig. 3(c
`and d); Fig. 5].
`'
`Our studies of the mammary gland revealed that
`the PR was exclusively expressed in the epithelial
`cells of the glandular buds (Fig. 4). In contrast to the
`uterine epithelium, the PR was fully down—regulated
`after ovariectomy or treatment with the pure antics-
`trogen ZM 182780 [Fig. 4(b and C); Fig. 5 for
`quantification]. E2 treatment
`(0.1 ,ug/animal/d) of
`ovariectomized animals again resulted in high PR
`immunoreactivity [Fig. 4(d); Fig. 5]. In accordance
`with this, we found in homogenates of N—nitro-
`somethylurea (NMU)—induced mammary carcinomas
`of the rat
`that after
`treatment with ZM 182780
`
`(25 mg/kg/d) the PR was reduced nearly to the limit
`of detection (not shown).
`
`DISCUSSION
`
`In this study, we describe the effects of ovari-
`ectomy, treatment with estradiol (E2) or a pure anti-
`estrogen on the PR immunoreactivity pattern in the
`different cell types of the uterus and mammary gland.
`All studies were carried out with adult rats and long
`term treatment was performed to look at the effects
`when steady—state levels are achieved.
`
`Fig. 3 (facing page). Impact of the antiestrogen ZM 182780 or estradiol on the PR immunoreactivity in the
`rat uterus.
`
`(a,b) PR distribution in the uterus of intact animals treated with the pure antiestrogen ZM 182780 (10 mglkgld
`for 21 days).
`(a) Myometrium: Nuclei of smooth muscle and blood vessel cells are only weakly stained.
`(b) Endometrium: High PR staining intensity in the lurninal and glandular epithelial cells (arrow). The
`adiacent stromal cells are also PR positive. The PR expression declines towards the outer stromal
`cell layers.
`(c,d) PR staining of ovariectomized animals treated with E2 (0.3 uglanimallday for 14 days): (c) myometrium,
`(d) endometrium: The PR taining pattern and intensity is similar to intact animals in proestrus or estrus
`[Fig. 2(a, b)]. The nuclei are deeply stained in the smooth muscle and stromal cells. The epithelial PR
`immunoreactivity is
`low (arrow). Magnification: 200x; bar-E50 p.M. M=myometrium; S=stt-oma;
`E = epithelium.
`
`Fig. 4 (overleaf). IEfi‘ect of ovariectomy andlor treatment with estradiol or the antiestrogen ZM 182780 on PR
`expression in the mammary gland: (a) intact control; (b) ovariectomy; (c) intact animal treated with ZM
`182780 (10 mglltgld) for 21 days; (cl) ovariectomy and treatrnent with estradiol (0.1 pglanimalld for 28 days).
`Magnification: 200 x ; bar 2 50 uM.
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`PR Repression by Estrogens in Uterin Epithelium
`
`315
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`tribution. Our finding that the selective blockade of
`the estrogen receptor by the pure antiestrogen ZM
`182780 [4] in intact animals also resulted in a strong
`PR up-regulation in the uterine epithelium and that
`treatment of ovariectomized animals with estradiol
`
`resulted in a suppression of epithelial PR provides
`strong evidence that this is an ER mediated effect.
`A similar decline of PR immunoreactivity after
`estradiol treatment was described for the isthmus epi-
`thelium of the rabbit oviduct [18]. In the uteri of
`estradiol treated immature rabbits the epithelial PR
`expression was lower and more heterogenous as com-
`pared to the stromal and myometrial cells. In con-
`trast, in uteri of estradiol treated guinea pigs high PR
`expression was observed in all uterine cell
`types,
`including epithelial cells [14]. Furthermore,
`in the
`uteri of ovariectomized monkeys high PR levels were
`found in the glandular but not
`in the luminal
`epithelium [19]. These data suggest species,
`tissue
`and cell type specific differences in uterine PR regu-
`lation.
`
`It has been reported by Nephew er al. [20] and by
`Bigsby and Li [21]
`that
`in rats estrogens can also
`increase the c-jun mRNA level in the uterine myome-
`trium and repress it in the uterine luminal epithelium.
`Thus, the inverse regulation of E2—dependent genes
`in the uterine epithelium could be a phenomenon
`also true for factors other than PR.
`
`In summary, we could show that in the rat uterine
`luminal and glandular epithelium progesterone recep-
`tor expression is repressed by estrogens whereas in
`the other cell types of the same organ, and also in the
`mammary gland epithelial and mammary carcinoma
`cells, PR expression can be induced by estrogens.
`Thus, our findings provide a biological system to elu-
`cidate the mechanism(s) of how the same transcrip-
`tion factor
`(the
`activated ER)
`can induce
`the
`expression of a protein/gene in one cell type and at
`the same time repress it
`in another cell
`type even
`within the same organ.
`
`REFERENCES
`
`1. Gronemeyer H., Transcriptional activation by estrogen recep-
`tor and progesterone receptors. Annu. Rev. Genet. 25 (1991)
`89-123.
`2. Green S. and Chambon P., The oestrogen receptor: from per-
`ception to mechanism. In Nuclear Hormone receptors, ed. M.
`Parker, Academic Press, New York, 1991, pp. 15-39.
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`_7. Steroid Biochem. Molec. Biol. 43
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`5. Milgrom E., Luu Thi M. T., Atger M. and Baulieu E.-E.,
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`and
`6. Nardulli A. M., Greene G. L., O’Malley B. W.
`Katzenellenbogen B. S., Regulation of progesterone receptor
`
`|nnoPharma Exhibit 1048.000?
`
`Uterus Stroma
`
`Mammary Gland
`Epithelium
`
`250
`
`200
`150
`
`mo
`
`so
`
`o
`
`200
`
`150
`
`100
`
`so
`
`o
`
`Uterus Epithelium
`‘k
`*
`
`250
`
`2 zoo
`‘E 150
`
`2 3
`
`3 100
`I
`so
`
`a
`
`250
`
`Uterus Myometrium
`
`2 §
`
`200
`§ 150
`«ii 100
`
`50
`
`0
`
`..
`
`*
`
`\?c}
`9
`o~
`'3’
`
`'$ Q6‘.
`x
`0
`av
`0*
`
`X6],
`
`xfbc}
`0
`6‘
`‘
`‘\°
`
`’& 4%.].
`x
`0
`at
`o“
`
`<8’
`*
`
`Fig. 5. Immunohistochemical progesterone receptor scores
`(meaniS.E., n=5) in the various uterine compartrnents
`(glandular and luminal epithelium, stroma, myometrium)
`and the mammary gland epithelium after the treatments
`indicated. *1) < 0.05 vs. intact control animals; Durmet test.
`
`from the immunocytochemical
`The key result
`analysis of the PR expression is that in the glandular
`and luminal epithelial cells of the uterus the PR
`immunoreactivity is repressed by estrogens and that
`PR expression can be induced by treating intact ani-
`mals with the pure antiestrogen ZM 182780. This is
`in contrast to all other uterine cell types, the mam-
`mary epithelial cells and the NMU induced mam-
`mary carcinoma cells, where the PR staining intensity
`was estrogen—inducible as could be expected from the
`classical studies in the literature [6, 13].
`Since
`the PR is
`located
`exclusively in the
`nucleus [14] and the size of the nuclei did not change
`during the treatment the changes in PR immunoreac-
`tivity should reflect the changes in the amount of this
`protein.
`The high PR levels in the epithelium after estrogen
`ablation are likely to be the cause for the remaining
`PR levels that we and others found in whole uterine
`
`homogenates of adult rats after ovariectomy[15,16].
`In a study on PR regulation during pregnancy in rats,
`Ohta er al. [17] also mentioned an increase of the
`uterine epithelial PR after ovariectomy. The authors
`could not exclude the possibility that progesterone
`depletion or other consequences of ovariectomy
`beside estrogen depletion affected the PR level or dis-
`
`
`
`316
`
`Karsten Parczyk er al.
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`
`|nnoPharma Exhibit 1048.0008
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