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Subgroup of Reproductive Functions of Progesterone Mediated by Progesterone Receptor-B Isoform

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Science  08 Sep 2000:
Vol. 289, Issue 5485, pp. 1751-1754
DOI: 10.1126/science.289.5485.1751

Abstract

Progesterone regulates reproductive function through two intracellular receptors, progesterone receptor–A (PR-A) and progesterone receptor–B (PR-B), that arise from a single gene and function as transcriptional regulators of progesterone-responsive genes. Although in vitro studies show that PR isoforms can display different transcriptional regulatory activities, their physiological significance is unknown. By selective ablation of PR-A in mice, we show that the PR-B isoform modulates a subset of reproductive functions of progesterone by regulation of a subset of progesterone-responsive target genes. Thus, PR-A and PR-B are functionally distinct mediators of progesterone action in vivo and should provide suitable targets for generation of tissue-selective progestins.

The steroid hormone progesterone (P) functions in establishment and maintenance of pregnancy. The physiological effects of P are mediated by interaction with specific intracellular progesterone receptors (PRs) that are members of the nuclear receptor superfamily of transcription factors (1, 2). PRs are expressed as two protein isoforms, PR-A and PR-B, that are produced from a single gene by transcription at two distinct promoters and by translation initiation at two alternative AUG signals (3, 4). Mice lacking a functional PR gene display pleiotropic reproductive abnormalities including inability to ovulate, uterine hyperplasia and inflammation, severely limited mammary gland development, and impaired thymic function and sexual behavior (2).

The production of two PR isoforms from the PR gene is conserved in a number of vertebrate species (3, 5,6), and the ratios of the individual isoforms vary in reproductive tissues as a consequence of developmental (6) and hormonal status (7) and during carcinogenesis (8). The PR-A and PR-B isoforms differ only in that PR-B contains an additional NH2-terminal stretch of about 165 amino acids. This region encodes a transactivation function that is specific to PR-B and is required to specify target genes that can be activated by PR-B but not PR-A (9).

When expressed individually in cultured cells, PR-A and PR-B display different transactivation properties that are specific to both cell type and target gene promoter used (10). PR-B functions as a strong activator of transcription of several PR-dependent promoters and in a variety of cell types in which PR-A is inactive. Under these conditions, agonist-bound PR-A can repress transcriptional activity of PR-B and other steroid receptors including estrogen receptor α (ERα) (11). Finally, when bound to some progestin antagonists, PR-B, but not PR-A, can be converted to a strongly active transcription factor by modulating intracellular phosphorylation pathways (12).

To determine whether the functional differences between the PR isoforms observed in vitro are reflected in a differential physiological capacity to mediate the diverse reproductive functions of P, we have selectively ablated expression of PR-A in PR-A knockout (PRAKO) mice.

Previous studies have shown that mutation of an internal ATG codon at which translation of PR-A is initiated (ATGA) selectively abolishes expression of PR-A, but not of PR-B, when the full-length cDNA encoding PR is expressed in cultured cells (3,13). Thus, we have used a CRE/loxP-based gene targeting strategy (14) to introduce a conservative amino acid substitution of the ATGA codon (Met166→Ala) in the murine PR (mPR) gene in embryonic stem (ES) cells (15). Male chimeras generated from three independent clones carrying the ATGA mutation transferred the mutation to the next generation, and litters born from PRAKO+/−intercrosses resulted in normal Mendelian inheritance of the mutation.

The absence of PR-A in PRAKO−/− female mice was confirmed by protein immunoblot analysis of uterine extracts from estrogen (E)–treated animals (16, 17). A strong immunoreactive band corresponding to the PR-A protein was detected in wild-type (WT) mice, but this band was absent from uterine extracts of PRAKO−/− animals (Fig. 1). The PR-B protein was present in similar amounts in both WT and PRAKO−/− mice and was typically detected as a doublet, presumably as a result of phosphorylation (18).

Figure 1

Ablation of PR-A expression in PRAKO mice. Protein immunoblot analysis of PR-A and PR-B in WT and PRAKO−/−uterine extracts.

All animals appeared normal except that PRAKO−/−females were infertile, a phenotype that was similar to that previously observed in PRKO mice in which both PR isoforms were ablated (2). To determine whether the infertility of the PRAKO−/− mice was due to an inability to ovulate, we administered pregnant mare serum gonadotropin and human chorionic gonadotropin to 21-day-old mice to induce superovulation (2). Normal superovulation occurred in WT and PRAKO+/− mice, with comparable numbers of oocytes produced in both cases (Table 1). PRAKO−/− mice produced reduced numbers of oocytes, whereas PRKO mice produced no oocytes. Crosses between superovulated PRAKO−/− females and WT males also failed to result in successful pregnancies despite the release of a small number of oocytes from PRAKO−/− females. Uteri of these females failed to show decidualization of stromal cells in response to traumal stimulation (19), indicating that infertility was also associated with defective uterine implantation. Consistent with this finding, analysis of the expression of several implantation-specific uterine epithelial target genes indicated that PR-B regulated the expression of only a subset of these genes (Fig. 2, A to D). Expression of calcitonin (CT), histidine decarboxylase (HDC), and amphiregulin (AR) is increased in the WT uterine epithelium in response to P (in association with uterine receptivity) (20). Expression of lactoferrin (LF) is increased by E and decreased in response to P (21). Ovariectomized mice were administered a single dose of either vehicle (control), E, P, or E plus P, and uterine extracts were prepared for RNA and protein analysis (16). Ablation of PR-A resulted in a complete loss of regulation of CT, whereas the regulation of HDC was retained (Fig. 2, A and B). P-induced expression of AR was also lost in PRAKO−/− mice (Fig. 2C). PR-B alone completely inhibited the E-dependent induction of epithelial LF (Fig. 2D). These data indicate that defective implantation in PRAKO−/−uteri is associated with loss of P-regulated expression of a subset of genes associated with receptivity. This differential target gene regulation by PR-B was not due to differences in spatiotemporal expression of PR-B relative to that of PR-A. Expression of PR-B in the uterine epithelium of untreated PRAKO−/− mice was similar to that observed in WT uteri (Fig. 2E). E-treatment of PRAKO−/−mice resulted in decreased expression of PR-B in the epithelium and increased expression in the stromal and myometrial compartments.

Figure 2

Expression of P-responsive target genes in the uterine epithelium of PRAKO−/−mice. Expression of (A) CT, (B) HDC, and (C) AR in uterine total RNA derived from WT, PRAKO−/−, or PRKO animals. RNA levels were standardized to that of cyclophilin (CPH) and represented as an average fold increase in four experiments ± SEM. (D) Protein immunoblot of uterine LF expression in WT, PRAKO−/−, or PRKO mice. No hormone treatment (C), estrogen (E), or estrogen plus progesterone (EP). (E) PR immunohistochemistry of uterine sections obtained from ovariectomized untreated (control) and E-treated (E) WT, PRAKO (−/−), and PRKO animals. (Insets) Panels represent higher magnifications of areas indicated by the asterisk. Bars, 100 μm (controls) and 50 μm (plus E).

Table 1

Dependence of ovulation on PR-A.

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Progesterone is a potent antagonist of E-induced proliferation of the uterine epithelium (2). We examined mice administered daily with either vehicle, E alone, or E plus P for 4 days and injected with 5-bromo-2′-deoxyuridine (BrdU) for 2 hours (16). Histological analysis of uterine sections indicated that the epithelial morphology was similar in control WT, PRAKO−/−, and PRKO mice (Fig. 3). Treatment of all groups with E resulted in hyperplasia of the luminal epithelium (LE) and the appearance of numerous scattered BrdU-positive proliferating cells. The addition of P with E resulted in an inhibition of uterine epithelial proliferation in WT animals. However, as previously observed in PRKO mice (2), this antiproliferative effect of P was absent in PRAKO−/− mice. Indeed, treatment of PRAKO−/− mice with E plus P resulted in a P-dependent increase in proliferation over that observed with E alone. This observation was confirmed by quantitative comparison of BrdU-labeled cells in E- and E plus P–treated WT, PRAKO−/−, and PRKO uterine epithelium (15). These results indicate that selective ablation of PR-A results in a gain of P-dependent proliferative activity mediated through PR-B that is not observed in E- and P-treated WT or PRKO mice. This acquisition of a P-dependent proliferative response indicates that PR-A may diminish overall P as well as E responsiveness in the uterus. The finding that PR-B can contribute to, rather than inhibit, uterine epithelial cell proliferation may have important clinical implications for the hormonal management of uterine endometrial dysplasias. The relative expression of PR isoforms under these conditions will be an important determinant of the effectiveness of progestin therapy. Thus, generation of PR-A–selective progestin that can distinguish between different conformations of PR-A and PR-B as recently demonstrated in the case of ER (22) may be of significant clinical value.

Figure 3

(left). Abnormal proliferative responses to E and P treatment in PRAKO−/− uterus. BrdU immunolabeling of uteri from ovariectomized WT, PRAKO−/−, and PRKO mice treated with sesame oil (Control), E, or E plus P (EP). Bars, 200 μm (controls) and 50 μm (hormone- treated).

To determine whether PR-B can elicit morphogenic responses of the mammary epithelium to P, we compared the morphology of mammary glands of ovariectomized WT, PRAKO−/−, and PRKO mice treated with E and P for 3 weeks (23). Analysis of whole mounts of the thoracic mammary glands showed extensive hormone-dependent ductal branching that filled the fat pad and the appearance of multiple alveolar lobules in PRAKO−/− mice (Fig. 4A). Further, the organized expression of E-cadherin outlining the mammary epithelial cells in PRAKO−/− mice (Fig. 4B) indicated normal architecture and basal membrane integrity. Thus, PR-B is sufficient to elicit normal proliferation and differentiation of the mammary epithelium in response to P. Finally, E- and P-treated PRAKO−/− mice also displayed normal thymic involution, a process we have previously shown to be PR dependent (24)

Figure 4

(right). Independence of mammary gland alveologenesis and tertiary ductal side branching from PR-A. (A) Thoracic mammary gland whole mounts of untreated (Control) or E plus P (EP)–treated WT, PRAKO−/−, and PRKO mice. Bar, 500 μm. (B) E-cadherin immunofluorescence shows an organized expression pattern for E-cadherin in the mammary gland of both the WT and PRAKO−/− mice (green); cytokeratin-14 in myoepithelial cells (red). Bars, 20 μm.

Our data indicate that PR-B mediates reproductive responses to P in a tissue-selective manner and that the PR-A and PR-B isoforms are functionally distinct mediators of progestin action in vivo. Thus, correct relative expression of the PR-A and PR-B isoforms is likely to be of critical importance to ensure appropriate reproductive tissue responses to P. Isoform-selective modulators of PR activity may allow tissue-selective modulation of progestin activity in hormonal therapy.

  • * To whom correspondence should be addressed. E-mail: orlac{at}bcm.tmc.edu

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