Pergamon
`
`PII: SO960-0760(97)00077-0
`
`j. Steroid Biochem. Molec. Biol. Vol. 63, No. 4—6, pp. 309—316, 1997
`© 1997 Elsevier Science Ltd. All rights reserved
`Printed in Great Britain
`0960-0750/97 $17.00 + 0.00
`
`Progesterone Receptor Repression by
`Estrogens in Rat Uterine Epithelial Cells
`
`Karsten Parczyk,‘* Robert Madino,1 Horst Michna,2
`Yukishige Nishino1 and Martin R. Schneider‘
`
`1Research Laboratories of Schering AG, D-I3342 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 imrnunoreactivity 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
`
`3‘. 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 aCtiVity 0f 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
`antieStrogenS) [41-
`estrous cycle was done by vaginal smears. 17fi-estra-
`One well known estrogen inducible target gene is
`the progesterone receptor
`(PR). From assays per— diol and ZM 182780 (70c—[9-(4,4,5,5,5,—pentafluoro-
`formed with whole tissue extracts the general picture
`pentylsulfinyl)-nonyl]-estra-l,3,5,(10)-triene—3,l7[3-
`emerged that PR levels are enhanced by estrogens
`diOL synthesized in the laboratories 0f SChel'ing AG:
`and reduced by progestins [5—8]. Subsequent immu— Berlin) were dissolved in castor oil containing 20%
`nohistochemical studies revealed species and cell type
`benzyl benzoate and adminiSteYed 6 days Per week by
`Specific
`differences
`in hormonal
`13R regulation
`subcutaneous injection. At the end of the experiments
`(reviewed in [9]). In our study we compared the effect
`the ingulljlal mammtary glands and men were excused-
`
`The uterl were welghed and one half of the organ
`was direct]
`sna —frozen in li uid nitro en for 1i and
`p
`q
`g
`(
`g
`y
`binding aSSaY)‘ The Other part Of the Organ as.well
`as the mammary glands were embedded 1n 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 Jun. 1997.
`
`309
`
`
`
`
`
`InnoPharma Exhibit 1048.0001
`
`

`

`500
`
`400
`
`PR-level[fmol/mgprOteinl 200
`
`100
`
`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 Na2M004,
`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 (l 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 ZOO—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 pm 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, MAI—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 MAI-410 antibody was
`further verified by western—blot analysis of rat uterine
`extracts (not shown).
`
`
`
`310
`
`Karsten Parczyk et al.
`
`800
`
`700
`
`600
`
`
`
`
`
`300
`
`Fig. 1. Effect of ovariectomy or treatment with estrogensl
`antiestrogens on the PR level in homogenates of whole uteri:
`(a) Effect of ovariectomy or treatment of intact animals with
`ZM 182780 (10 mglkgld) for 21 days.
`(1)) Treatment of ovari-
`ectomized animals with estradiol
`(0.1 uglanimalld)
`for
`28 days. The mean valuesiS.E.M. are given. *p<0.01 vs.
`control; **p<0.05 vs. control; ***p<0.01 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:
`i=4
`
`Receptor score = ZPU) x 1'
`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 ixnmunoreactivity in the different uterine cell types of intact and ovariectomized
`rats.
`
`(a,b) PR staining pattern in intact rats (estrous phase). (a) Myometrium: nuclei of the myometrial inner and
`outer smooth muscle cell layers are prominently stained. Nuclei of blood vessel cells are also PR positive.
`(b) Endometrium: 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 glandqu 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 M. M = myometrium; S = stroma; E = epithelium.
`
`
`
`InnoPharma Exhibit 1048.0002
`
`

`

`PR Repression by Estrogens in Uterin Epithelium
`
`311
`
`
`
`Figure 2—caption opposite
`
`
`
`
`
`InnoPharma Exhibit 1048.0003
`
`

`

`312
`
`Karsten Parczyk et al.
`
`
`
`Figure 3—captz'on opposite
`
`
`
`
`
`InnoPharma Exhibit 1048.0004
`
`

`

`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.
`
`RESULTS
`
`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 ovariectomy [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. 1(b)].
`The fact that the PR level declined by only 50%
`after ovariectomy or treatment with the pure anties-
`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 function 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 anties—
`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 luminal 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 uglanimaIIday 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; barESO I‘M. M=myometrium; S=stroma;
`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 mglkgld) for 21 days; (d) ovariectomy and treatment with estradiol (0.1uglanimalld for 28 days).
`Magnification: 200 x ; bar E 50 M.
`
`
`
`
`
`InnoPharma Exhibit 1048.0005
`
`

`

`314
`
`Karsten Parczyk er al.
`
`
`
`Figure 4—caption on page 313
`
`
`
`
`
`InnoPharma Exhibit 1048.0006
`
`

`

`PR Repression by Estrogens in Uterin Epithelium
`
`315
`
`Uterus Epithelium
`*
`*
`
`250
`
`2 200
`<3 150
`2D.
`
`§ 100
`[I
`
`50
`
`300
`
`250
`
`e
`‘3 200
`'2; 150
`all 100
`
`50
`
`0
`
`Uterus Myometrium
`
`‘
`
`t
`
`*
`
`Uterus Stroma
`
`250
`
`200
`150
`
`100
`
`50 I
`
`Mammary Gland
`Epithelium
`
`
`
`250
`
`200
`
`150
`100
`
`so
`
`o
`
`—
`
`a? 0*
`0
`\0
`\x
`\0
`
`‘6”
`a"
`Q;
`if
`0
`0A
`
`«3‘
`«9‘
`C:
`\x
`\e
`\9
`
`4w“ 0
`Q
`0
`+x
`04
`
`Fig. 5. Immunohistochemical progesterone receptor scores
`(meaniS.E., n=5) in the various uterine compartments
`(glandular and luminal epithelium, Stroma, myometrium)
`and the mammary gland epithelium after the treatments
`indicated. *1) < 0.05 vs. intact control animals; Dunnet 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-
`
`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—
`Iation.
`
`It has been reported by Nephew et 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
`
`l. 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.
`3. Gronemeyer H., Benhamou B., Berry M., Bocquel M. T.,
`Gofflo D., Garcia T., Lerouge T., Metzger D., Meyer M. E.,
`Tora L., Vergezac A. and Chambon P., Mechanisms of anti—
`hormone action. 5‘. Steroid Biochem. Molec. Biol. 41 (1992)
`217—221.
`4. Wakeling A. E. and Bowler J., ICI 182780, a new antiestrogen
`with clinical potential. I Steroid Biochem. Molec. Biol. 43
`(1992) 173~177.
`5. Milgrom E., Luu Thi M. T., Atger M. and Baulieu E.-E.,
`Mechanism regulating the concentration and conformation of
`progesterone receptor(s)
`in the uterus. 3‘. Biol. Chem. 248
`(1973) 6366—6373.
`and
`6. Nardulli A. M., Greene G. L., O’Malley B. W.
`Katzenellenbogen B. S., Regulation of progesterone receptor
`
`
`
`
`
`InnoPharma Exhibit 1048.0007
`
`

`

`316
`
`Karsten Parczyk er al.
`
`messenger ribonucleic acid and protein levels in MCF-7 cells
`by estradiol: Analysis of estrogen’s efl'ect on progesterone
`receptor synthesis and degradation. Endocrinology 122 (1988)
`935—944.
`. Read L. D., Snider C. E., Miller J. S., Greene G. L. and
`Katzenellenbogen B. 8., Ligand—modulated regulation of pro-
`gesterone receptor messenger ribonucleic acid and protein in
`human breast cancer cell lines. Mol. Endocrinol. 2 (1988) 263—
`271.
`. Wei L. L., Krett N. L., Francis M. D., Gordon D. F., Wood
`W. M., O’Malley B. W. and Horwitz K. B., Multiple human
`progesterone receptor messenger ribonucleic acids and their
`autoregulation by progestin agonists and antagonists in breast
`cancer cells. Mol. Endocrinol. 2 (1988) 62—72.
`Clarke C. L., Cell-specific regulation of progesterone receptor
`in the female reproductive system. Mol. Cell. Endocrinol. 70
`(1990) C29—C33.
`Leake, R. E. and Habib, F., Steroid hormone receptors: assays
`and characterization. In Steroid hormone receptors — a practical
`approach, eds. B. Green and R. E. Leake, IRL Press, Oxford,
`1987, pp. 67—92.
`Traish A. and Wotiz H., Monoclonal and polyclonal antibodies
`to human progesterone receptor peptide — (533—547) recog-
`nize a specific site in unactivated (SS) and activated (48) pro-
`gesterone
`receptor
`and distinguish between
`intact
`and
`proteolwed receptors. Endorrinology 127 (1990) 1167—1175.
`Sniiiders M. P. M. L., deGoeij A. F. P. M., Debets-Te Baerts
`M. J. C., Rousch M. I. M., Koudstaal J. and Bosman F. T.,
`Immunocytochemical analysis of estrogen receptors and pro-
`gesterone receptors in the human uterus throughout the men-
`
`12.
`
`10.
`
`11.
`
`l3.
`
`14.
`
`15.
`
`16.
`
`17.
`
`18.
`
`19.
`
`20.
`
`21.
`
`strual cycle and after the menopause. ]. Reprod. Fertil. 94
`(1992) 363—371.
`Horwitz K. B. and McGuire W. L., Estrogen control of pro-
`gesterone receptor in human breast cancer. 5’. Biol. Chem. 253
`(1978) 2223—2228.
`Perrot-Applanat M., Logeat F., Groyer—Picard M. T. and
`Milgrom E., Immunocytochemical study of mammalian pro-
`gesterone receptor using monoclonal antibodies. Endocrinology
`116 (1985) 1473—1484.
`Manni A., Baker R., Arafah B. M. and Pearson O. H., Uterine
`oestrogen and progesterone receptors in the ovariectomized
`rat. }. Endocr. 91 (1981) 281—287.
`Wakeling A. E. and Bowler J., Biology and mode of action of
`pure antiestrogens. ]. Steroid Biochem. 30 (1989) 141—147.
`Ohta Y., Sato S. and Iguchi T., Immunocytochcmical localiz-
`ation of the progesterone receptor in the reproductive tract of
`adult female rats. Biol. Reprod. 48 (1993) 205—213.
`Hyde B. A., Blaustein I. D. and Black D. L., Differential regu-
`lation of progestin receptor immunoreactivity in the rabbit ovi—
`duct. Endocrinology 125 (1989) 1479—1483.
`Okulicz W. C., Savasta A. M., Hoberg L. M. and Longcope
`C., Immunofluorescent analysis of estrogen induction of pro-
`gesteroone receptor in the rhesus uterus. Endocrinology 125
`(1989) 930-934.
`Nephew K. P., Tang M. and Khan 8. A., Estrogen differen—
`tially affects c—jun expression in uterine tissue compartments.
`Endocrinology 134 (1994) 1827—1833.
`Bigsby M. B. and Li A., Differentially regulated immediate
`early genes in the rat uterus. Endocrinology 134 (1994) 1820—
`1826.
`
`
`
`
`
`InnoPharma Exhibit 1048.0008
`
`