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Department of Anatomy (T.K., P.Y., J.R.B., G.R.C.), University of California, San Francisco, California 94143; Department of Cell Biology (J.P.L., B.W.O.), Baylor College of Medicine, Houston, Texas 77030
Address all correspondence and requests for reprints to: Dr. Gerald R. Cunha, Cancer Research Building, UCSF Mt. Zion Cancer Center, University of California, San Francisco, California. E-mail: gcunha{at}itsa.ucsf
| Abstract |
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| Introduction |
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Both E2 and P elicit their effects via members of the
steroid hormone receptor superfamily (ER and PR, respectively) (8). Two
isoforms of the ER have been described, ER
, the classical ER, and a
recently described second form, ERß (9, 10). Although both isoforms
are expressed in the uterus (11, 12), E2 treatment of
estrogen receptor-
knockout (ERKO) mice does not stimulate uterine
epithelial proliferation or transcription of estrogen-responsive genes
(13); thus, ER
is essential to mediate E2 signaling in
the uterus. The PR is composed of two ligand-binding forms (PR-A and
PR-B) differing in size but both derived from one gene (14, 15). Both
PR isoforms have been detected in the rodent uterus (16). Steroid
autoradiography and immunohistochemistry (IHC) have revealed ER
and
PR in both uterine epithelial and stromal cells (17, 18, 19). This raises
the possibility that effects of E2 and/or P on UtE could be
elicited directly via epithelial ER
or PR, respectively, or
alternatively via paracrine mechanisms employing ER
and/or PR,
respectively, in stromal cells. Recent analysis of tissue recombinants
prepared with uteri of wild-type and ERKO mice has shown that
E2-induced epithelial mitogenesis is mediated by stromal
ER
; epithelial ER
does not participate in this process (20).
In this study, PR knockout (PRKO) mice have provided the opportunity to analyze the cellular mechanism of P inhibition of uterine epithelial DNA synthesis. PRKO mice lack functional PR as a result of disruption of the PR gene, and therefore reproductive organs in adult female PRKO mice are functionally impaired, leading to infertility (21). PRKO and wild-type mice were used to produce uterine tissue recombinants that lack PR in their epithelium, stroma, or both by using tissue separation and recombination techniques. The objective of this study was to determine whether P-inhibition of E2-induced uterine epithelial DNA synthesis is mediated via epithelial or stromal PR.
| Materials and Methods |
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Tissue separation/recombination and grafting
Procedures for separation and recombination of UtE and stroma
have been described (22). Briefly, uteri were dissected free of
adherent connective tissue and fat from neonatal (314 days) PRKO and
Balb/c mice, placed into HBSS, and cut into small pieces. In each
experiment, uteri from two to four mice of PRKO and wild-type at the
approximate same age were used. Pieces of uteri were enzymatically
dissociated by placing them in a solution of 1% trypsin in calcium-
and magnesium-free HBSS for 90 min at 4 C. Uteri were cut open, and
then stroma and epithelium were physically separated by using fine
surgical instruments. Stroma and epithelium were recombined on agar
plates and allowed to adhere during overnight culture. In each
experiment, at least 12 tissue recombinants were prepared for each
category of tissue combinations. After overnight culture, the tissue
recombinants were grafted under the renal capsules of female nude mice.
In each experiment, at least six nude mice were used as hosts, and each
kidney carried three to five tissue recombinants.
Hormone treatment
Renal capsular grafts of tissue recombinants were grown for
approximately 1 month, and then all hosts were ovariectomized. Two
weeks later, hosts received P and/or E2, or oil vehicle.
All hormone injections were given ip in 0.5 ml peanut oil daily at the
following doses: P, 0.5 mg; E2, 125 ng; E2 + P,
125 ng E2 + 0.5 mg P (see Fig. 1
). Sixteen and 17 h after the last
hormone injection, all hosts were given [3H]-thymidine
(1.5 µCi/g body weight in PBS). One hour after the last
[3H]-thymidine injection, grafts were removed and fixed
in 10% formalin.
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has been described (20). An
antigen retrieval method was used to immunohistochemically detect PR on
formalin-fixed paraffin sections using anti-PR (Dako
Corp., Carpinteria, CA) followed by biotinylated donkey
antirabbit-Ig antibody (Amersham). Signal was visualized with
streptavidin conjugated to horseradish peroxidase (Dako
Corp.) and diaminobenzidine (Sigma Chemical Co.,
St. Louis, MO) as the chromagen.
For 3H-thymidine autoradiography, tissue sections were
dipped in Kodak NTB-2 nuclear emulsion (Rochester, NY) and stored at
-80 C. Autoradiograms were exposed for 4 weeks to achieve saturation
of nuclear labeling and were developed by standard techniques. Slides
were stained with hematoxylin and eosin. Epithelial
3H-thymidine labeling index (LI) was measured as labeled
cells per total epithelial cells as described previously (20). Each
point is based on analysis of at least nine specimens (
10,000
epithelial cells per group) from five independent experiments. Data on
epithelial DNA synthesis in various groups were analyzed by Students
t test.
To more clearly show the relationship between PR status and DNA synthesis, tissue recombinants were first stained for PR. Immunohistochemical images were then captured using a Leaf Lumina camera/scanner interfaced to a Macintosh computer. After imaging, coverslips were removed and slides were processed for 3H-thymidine autoradiogrphy, as above. Silver grains from 3H-thymidine autoradiograms were imaged and superimposed on the original PR immunohistochemical images as previously described (20).
| Results |
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localization was also analyzed, and ER
immunostaining was detected in both epithelium and stroma of all four
types of tissue recombinants (data not shown).
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3040%) in all four types of
tissue recombinants (Fig. 2
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| Discussion |
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(20, 27, 28). The present study clearly shows that P inhibition
of E2-induced epithelial DNA synthesis is mediated by
stromal PR. Thus, stimulation as well as inhibition of uterine
epithelial DNA synthesis by E2 and P, respectively, is
mediated via stromal ER and PR. P inhibition of estrogen-induced uterine epithelial proliferation is a general phenomenon occurring in several mammalian species including human (2) and is the rationale for hormonal therapy for proliferative lesions of the endometrium. Etiologic studies show that P is preventive for development of endometrial carcinoma, whereas estrogen promotes this disease (29, 30). P inhibits proliferation of normal human endometrial epithelium, reverses or normalizes both spontaneous and estrogen-induced endometrial hyperplasia, and inhibits growth of human endometrial carcinoma (7, 30). Our tissue recombinant studies in the mouse raise the distinct possibility in humans that paracrine mechanisms may play a role in regulating proliferation of both normal and neoplastic endometrial epithelial cells. Given the generalized alteration in epithelial differentiation during carcinogenesis and the well recognized alterations in carcinoma-associated stromal cells (31, 32, 33, 34, 35), it is likely that development of endometrial carcinoma may entail perturbation of paracrine pathways of growth regulation. Current analysis of heterospecific tissue recombinants composed of normal human or neoplastic UtE plus either wild-type or PRKO uterine stroma will define whether P inhibits proliferation of human UtE via paracrine mechanisms as is the case in the mouse.
Cooke et al. (20, 28) demonstrated the key role of stroma in
E2-induced uterine and vaginal epithelial proliferation. In
response to E2, uterine stroma produces paracrine
growth-regulating molecules that stimulate uterine epithelial
proliferation. Adapting the model of E2 effect on uterine
epithelial proliferation proposed by Cooke et al.
(20), we propose the following models of P inhibition of uterine
epithelial proliferation. Mechanistically the growth inhibitory effect
of P on uterine epithelial proliferation could result from impaired
intracellular synthesis or secretion of E2-induced
paracrine factors (pathway no. 1, Fig. 4
). Alternatively, P may elicit the
secretion of a paracrine factor that antagonizes the action of
E2-induced paracrine mediators through indirect mechanisms
(pathway no. 2, Fig. 4
, see discussion below). Another possibility is
that the P-induced paracrine mediator is a direct inhibitor of
epithelial proliferation (pathway no. 3, Fig. 4
). Given these possible
mechanistic scenarios, levels of trophic or inhibitory factor
transcripts or proteins could be one facet of regulating the
bio-availability and biological activity of paracrine mediators. The
activation of a trophic or inhibitory paracrine mediator may involve
steps such as enzymatic modification (peptide cleavage, phosphorylation
or glycosylation, etc.) or regulation of bio-availability of the factor
(secretion, binding and/or release from extracellular matrix, etc.).
Critical regulatory steps could involve the paracrine mediators
themselves, their receptors, binding proteins, or enzymes that modify
activity of the molecules involved. Given the scenario that
E2 stimulates UtE proliferation via stromal ER and that P
inhibits uterine epithelial proliferation via stromal PR, current
knowledge on expression patterns of known molecules is not yet
sufficiently detailed to explain the paracrine models suggested by our
tissue recombinant studies.
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(27). Hence, the regulation of mammary epithelial proliferation
by steroid hormones may involve both direct and paracrine pathways
mediated by epithelial PR and stromal ER, respectively. In this regard,
the mammary gland is radically different from the mouse uterus in which
growth stimulatory and inhibitory effects of E2 and P are
both paracrine events mediated by stromal ER and PR, respectively. P
regulates proliferation of many other type of cells in various organs,
like spiral arteries of rhesus endometrium (40). The mechanisms of P
regulation of proliferation (direct or indirect) could be quite
different among different cell types and in different species and needs
to be examined on a case by case basis. In this study, we have demonstrated that P inhibition of E2-induced uterine epithelial DNA synthesis is mediated by stromal PR. Effects of E2 and P on epithelial function may be elicited by either direct (mediated by epithelial receptors) or indirect (mediated by stromal receptors) mechanisms in different organs. Understanding the cellular mechanism of the actions of P on uterine function will require a determination of whether a given effect of P is mediated either by stromal or epithelial PR.
| Footnotes |
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Received April 20, 1998.
| References |
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and ß. Endocrinology 138:863870
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