Ligand-Activated Progesterone Receptor Isoform hPR-A Is a Stronger Transactivator Than hPR-B for the Expression of IGFBP-1 (Insulin-Like Growth Factor Binding Protein-1) in Human Endometrial Stromal Cells

Jiaguo Gao, James Mazella, Meiyi Tang and Linda Tseng

Department of Obstetrics/Gynecology and Reproductive Medicine School of Medicine State University of New York at Stony Brook Stony Brook, New York 11794


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In human endometrium, the levels of progesterone receptor (PR) isoforms hPR-A and hPR-B are differentially regulated during the reproductive cycle. Progesterone significantly increases the content of hPR-A, the predominant isoform in decidualized stromal cells (1). The purpose of this study was to determine the capacity of hPR-A and hPR-B to transactivate the progestin-dependent target gene in human endometrial stromal cells. We examined the effect of cotransfection of hPR-A or hPR-B on the expression of the human insulin-like growth factor binding protein-1 (IGFBP-1) in endometrial stromal cells. The primary culture of human endometrial stromal cells was transfected with the hPR-A or hPR-B expression vector and the IGFBP-1 promoter construct p275CAT, which contains two functional progesterone response elements (PRE1 and PRE2) in decidualized stromal cells. Medroxyprogesterone acetate (MPA) increased the promoter activities ranging from 1.2- to 27-fold in cells cotransfected with hPR-A or hPR-B in eight endometrial specimens. The promoter activity increased by the hPR-A was significantly higher than hPR-B (15 ± 8 vs. 4 ± 2, mean ± SD; n = 8, P < 0.005). Site-specific mutation showed that the induced activity by hPR-A was mediated through the PRE1 and PRE2 sites. Addition of hPR-B reduced the effect of hPR-A. The high transactivation capacity of hPR-A was also activated by other ligands, progesterone, Org 2058, and norethindrone. These observations indicate that hPR-A is a stronger transactivator than hPR-B for the IGFBP-1 promoter in endometrial stromal cells.

Previous studies have shown the progestin- dependent production of IGFBP-1 correlates with its mRNA levels and transcription rate. Thus, we have determined the effect of hPR-A and hPR-B on the production of IGFBP-1 in stromal cells treated with MPA. The production rate in cells uniformly infected with AdPRA (recombinant Ad5- directed PR expression system) was significantly higher (P < 0.001) than the rate in uninfected cells and in cells infected with AdPRB or AdCMV (the Ad5 viral expression vector). This result, in concert with the promoter analysis, provides evidence that hPR-A is a strong inducer for the chromosomal IGFBP-1 gene in endometrial stromal cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Ligand-activated progesterone receptor (PR) mediates the majority of progestational effects in endometrium, although nongenomic progesterone action has been implicated. Human endometrium contains both isoforms of PR, hPR-A and hPR-B. Steroid hormones regulate the content of these two isoforms differentially in two types of endometrial cells (1, 2). In glandular cells, progestin reduces both hPR-A and hPR-B, whereas in stromal cells, it increases the content of both isoforms in cells treated with progestin for 1–5 days. Prolonged exposure of stromal cells to progestin significantly increases the content of hPR-A (1), the major isoform in decidualized endometrial stromal cells. Thus, it is generally believed that the majority of the progestational effects and antiprogestin blockade are mediated through hPR-A in endometrial decidual cells. This concept remains to be verified.

Progesterone-regulated gene expression in endometrial cells has been extensively studied in many laboratories. However, information about PR isoform-specific gene regulation is limited in this target tissue. We believe that hPR-A is active and perhaps more active than hPR-B on those genes that are extensively expressed in decidual cells. However, in several endocrine-sensitive breast cancer cell lines, hPR-B is a stronger transactivator than hPR-A. In addition, hPR-A acts as a dominant repressor of hPR-B on the progesterone response element (PRE) linked-reporter constructs or mouse mammary tumor virus (MMTV) (3, 4) as well as the endogenous target gene (5). Although hPR-A is not a universal repressor (6), additional information showed that the repression is through the structure-specific interaction with the consensus PRE (7, 8). These findings raise the question of the functional aspects of hPR-A in endometrial stromal/decidual cells. The purpose of this study was to determine the transactivation capacity of hPR-A in endometrial stromal cells by using a progestin-induced gene, human insulin-like growth factor binding protein-1 (IGFBP-1).

The IGFBP-1 gene is activated by progestin, and its gene product is the major secretory protein in decidualized endometrial stromal cells (9, 10, 11, 12, 13). Previous studies have shown that cis-elements PRE1 and PRE2 in the IGFBP-1 promoter are the sites responsible to increase the promoter activity in decidualized endometrial stromal cells (13). However, it is not clear whether the activation is mediated from the predominant hPR-A or from the less dominant hPR-B or both. To determine the capacity of transactivation by the PR isoforms, we have measured the effect of cotransfection of hPR-A or hPR-B expression vector on the IGFBP-1 promoter activity in undifferentiated stromal cells in which PRE1/PRE2 have little activity (13). In addition, the production of IGFBP-1 was measured in stromal cells uniformly transfected with AdPRA or AdPRB. Our results indicate that hPR-A is a stronger transactivator than hPR-B to increase the promoter activity in transient transfection assay and production of IGFBP-1 from the endogenous IGFBP-1 gene in endometrial stromal cells.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
hPR-A Is a Stronger Activator Than hPR-B For The IGFBP-1 Promoter Activity in Human Endometrial Stromal Cells
The transactivation capacity of hPR-A and hPR-B on the IGFBP-1 promoter was determined using the promoter construct p275CAT (-275 to +68 bp, Fig. 1Go) for most of the experiments presented in this paper (Table 1Go and Figs. 2 to 5GoGoGoGo). Only three experiments were carried out using p246CAT and p1.2CAT (Table 1Go). The promoter activity was low in cells not treated with MPA ( Figs. 2–5GoGoGoGo) in agreement with the previous observation (13). MPA had no significant effect on the promoter activity in cells cotransfected with the empty vector (Fig. 4Go, lane 1). MPA increased the promoter activity derived from p246CAT (n = 2), p275CAT (n = 5), and p1.2CAT (n = 1). The degree of activation varied from 1.2- to 27-fold compared with cells not exposed to MPA (Table 1Go) and had no clear correlation with the stage of menstrual cycle nor with the length of the promoter. However, the capacity of transactivation by hPR-A was significantly higher than that of hPR-B determined in eight specimens (Table 1Go; 15- vs. 4-fold increase respectively, n = 8, mean ± SD p < 0.005).



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Figure 1. IGFBP-1 Promoter Construct

The location of the functional PRE1 (-193 to -179 bp) and PRE2 (-102 to -88 bp) sites are shown in the diagram. Also shown are the sequences of wild-type PRE1 and PRE2, and the nucleotides mutated for the construction of mutants p275PRE1.2 m, p275PRE1m, and p275PRE2m.

 

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Table 1. Activation of IGFBP-1 Promoter Activity Cotransfected with hPR-A or hPR-B in Human Endometrial Stromal Cells

 


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Figure 2. Promoter Activity of IGFBP-1 (2A) and MMTV (2B) Induced by hPR-A or hPR-B in Human Endometrial Stromal Cells Treated with MPA

Eight micrograms of plasmid p275CAT or pMMTV-CAT, 1 µg of hPR-A or hPR-B, and 1 µg of pRSV-luc were transfected into human stromal cells (early secretory phase) and then cultured with or without 0.1 µM MPA for 2 days. CAT activities were normalized to the luciferase activities determined in the same cell lysate. Results are mean ± SD of triplicate dishes. Promoter activity in cells treated with MPA was significantly higher than untreated cells, MPA+ vs. MPA-, P < 0.0001. The difference between hPR-A vs. hPR-B in MPA+ samples was also significant (Fig. 2AGo, P < 0.0001 and Fig. 2BGo, P < 0.0006).

 


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Figure 3. Effect of hPR-A on the IGFBP-1 Promoter Mutant Constructs of PRE1 and PRE2

Stromal cells (early-midsecretory) were transfected with p275CAT, p275PRE1m, p275PRE2m, or p275PRE1,2 m and cotransfected with hPR-A and pRSV-Luc and then treated without or with MPA for 2 days. Results are means ± SD of triplicate dishes. Reductions of the promoter activity by p275 PRE1m, p275 PRE2m, and p275 PRE1.2 m were significant, P < 0.01, 0.001, and 0.0002.

 


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Figure 4. Induction of the IGFBP-1 Promoter Activity by Various Doses of hPR-A, hPR-B, and Mixtures of hPR-A and hPR-B in Endometrial Stromal Cells

Stromal cells (early secretory phase) were transfected with p275CAT (8 µg), various concentrations of hPR-A, hPR-B, mixtures of hPR-A/hPR-B, or hPRB-DBDcys, and pRSV-Luc. The vector (plasmid without PR insert) was included in control sample (2 µg) and in samples transfected with PR constructs to yield 2 µg DNA. The promoter activities (normalized to pRSVluc) were measured in cells after 2 days treatment with or without MPA. Results are mean ± SD of triplicate dishes.

 


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Figure 5. Effects of MPA, Progesterone (P), ORG2058 (ORG), Norethindrone (NOR), RU-486 (Ru) and Cortisol (F) on the IGFBP-1 Promoter Activity Induced by hPR-A or hPR-B in Endometrial Stromal Cells

Stromal cells (midsecretory phase) were transfected with p275CAT, hPR-A, or hPR-B and pRSV-luc. Cells were treated with 0.1 µM of steroids and cultured for 2 days. Results are mean ± SD of triplicate dishes. Promoter activity was significantly higher in cells cotransfected with hPR-A than hPR-B when treated with MPA, P, ORG, or Nor, P < 0.002.

 
Differential Effects of hPR-A and hPR-B on the Promoter Activity of IGFBP-1 and MMTV in Endometrial Stromal Cells
To demonstrate that the relative capacity of transactivation by hPR-A or hPR-B is target gene specific, the promoters of IGFBP-1 and MMTV were separately transfected into stromal cells. The promoter activity derived from p275CAT cotransfected with hPR-A was significantly higher than that of hPR-B (Fig. 2AGo; P < 0.001). In contrast, we found that hPR-B was a stronger transactivator than hPR-A for the promoter of MMTV in endometrial stromal cells (Fig. 2BGo), which is similar to data observed in cancer cell lines (4). This result shows that the ligand-activated PR isoforms display a spectrum of transactivation capacity in different target genes in endometrial stromal cells. In addition, we found that the promoter activities derived from p275CAT were similarly induced by hPR-A or hPR-B in HeLa or HepG2 cells (data not shown). This result showed that the capacity of PR transactivation on the target gene also varies in different types of cells.

PRE1 and PRE2 Sites in the IGFBP-1 Promoter Mediate the hPR-A Function
The PRE1 and PRE2 sites are located in the proximal promoter region shown in Fig. 1Go. Previously, we have shown that PRE1 and PRE2 are functional in decidualized stromal cells (13). To determine whether the action of hPR-A is mediated through these two sites, stromal cells were transfected with hPR-A and p275CAT and three PRE mutants, respectively (Fig. 1Go). Figure 3Go shows the promoter activities derived from the wild-type plasmid p275CAT and three mutants, mutation of PRE1, PRE2, or both. p275PRE1m or p275PRE2m reduced the promoter activity to 75% and 45%, respectively, of the p275CAT (P < 0.05 and <0.001). p275PRE1,2 m, double mutation at PRE1 and PRE2 sites, decreased the promoter activity to 15% of the wild type (P < 0.0001) (Fig. 3Go). These findings indicate that the transactivation of hPR-A is mediated by both PRE1 and PRE2 sites in the IGFBP-1 promoter.

Transactivation of the IGFBP-1 Promoter by hPR-A Is Dose Dependent
To test whether differential transactivation of hPR-A and hPR-B on the IGFBP-1 promoter is caused by the concentration of PR-A or PR-B, stromal cells were transfected with p275CAT and cotransfected with various amounts of hPR-A or hPR-B expression vector. The promoter activity increased by 2.5-, 8-, and 20-fold at concentrations of 0.1, 0.3, and 1 µg hPR-A, respectively (Fig. 4Go, lanes 1–4). The response to hPR-B was also dose dependent at the same concentrations (1.5-, 3-, and 4-fold increase, lanes 5–7, respectively). At a comparable dose, the potency of hPR-A was always stronger than hPR-B. No self-squelching was shown within the range tested.

hPR-B Inhibits the Effect of PR-A and Repression Does Not Require the DNA Binding Domain
To test whether hPR-B would interfere with the action of hPR-A, cells were transfected with hPR-A alone or a mixture of hPR-B and hPR-A. Addition of hPR-B reduced the activity by 50% and 60% at the ratios of hPR-B/hPR-A 0.3 and 1, respectively (Fig. 4Go, lanes 8 and 9). These results suggest that hPR-B acts as a repressor to quench the transactivation of hPR-A. Cotransfection with hPRB-DBDcys, which produces a mutant hPR-B unable to bind to the DNA, did not reverse the inhibition (lane 10). The result indicates that inhibition does not require DNA binding. Data suggest that the inhibition is mediated by protein-protein interaction, although the exact nature of the interaction is not clear and requires additional study.

Various Ligands Do Not Change the Relative Capacity of Transactivation of hPR-A and hPR-B
To test whether the high capacity of hPR-A is ligand specific, the promoter activity was determined in cells treated with MPA, progesterone (P), or the 19 nor-progestins: Org2058 and norethindrone. Although the degree of activation varied among different progestins (5- to 8-fold), hPR-A was always more potent than hPR-B (Fig. 5Go). These results show that the high capacity of transactivation from hPR-A is not caused by a specific ligand. The antiprogestin, RU-486, caused a moderate increase in cells co-transfected with hPR-A or hPR-B (1.8- or 1.6-fold increase). Cortisol had no effect on the promoter activity.

hPR-A Is a Strong Inducer For the Production of IGBFP-1 in Endometrial Stromal Cells
The findings described in the previous sections were demonstrated in transient transfection experiments. It is important to provide evidence whether hPR-A is indeed a strong transactivator for the endogenous IGFBP-1 gene in stromal cells. Previous studies have shown that rate of production and mRNA levels correlate with the rate of transcription of the IGFBP-1 gene (9, 10). Thus, we have determined the effect of hPR-A and hPR-B on the induction of the endogenous IGFBP-1 gene in stromal cells. Such experiment requires a high efficiency of transfection to achieve a uniform gene transfer. To increase the efficacy, Ad5-recombinant gene transfer technique was used for this study. Adßgal infection was used as a positive control (Fig. 6DGo). Stromal cells remained viable after infection with various Ad5-recombinant constructs. Immunohistochemical localization showed that approximately 95% of the stromal cells expressed hPR-A and hPR-B with a similar intensity of staining (Fig. 6Go, B and C) in the nuclei when the cells were infected with equal amount of AdPR-A and AdPR-B, respectively (five viral particles per cell). Twelve days after infection, the intensity was reduced approximately 50%. In stromal cells with no viral infection or infected with the vector AdCMV, no detectable PR was found under the same experimental condition (Fig. 6AGo), indicating that the endogenous hPR is relatively low compared with the cells infected with AdPRs.



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Figure 6. Immunohistochemistry Localization of hPR-A and hPR-B in Endometrial Stromal Cells Transfected with AdCMV, AdPRA, and AdPRB

Stromal cells isolated from a midsecretory endometrium were treated with 0.1 µM MPA and infected with AdCMV, AdPRA, and AdPRB, and Adßgal (10 particles per cell) for two days, respectively. Cells were processed for immunohistochemical staining in A, B, and C and ß-gal enzyme reaction in D as described in detail in Materials and Methods (A, phase contrast; B, C, and D, bright field).

 
The effect of overexpression of hPR-A or hPR-B on the endogenous IGFBP-1 gene was determined by measuring the amount of IGFBP-1 accumulated in medium by enzyme-linked immunosorbent assay (ELISA) (9). Various concentrations of viral particles (1, 5, 10, 20, and 50 particles/cell) were infected into cells to determine whether Ad5 infection would impair the production of IGFBP-1. We found that cells infected with >20 particles per cell reduced the production rate. Figure 7Go shows the production of IGFBP-1 in cells infected with five viral particles per cell and then treated with MPA. IGFBP-1 was undetectable by ELISA in long-term cell culture (11 days) without MPA treatment in agreement with previous observation (9). In cells treated with MPA, the production rate was minimal on the first 6 days of culture, which is similar to the results published previously (9). The rate was significantly increased after 7 days MPA treatment (Fig. 7Go) with no difference between uninfected cells and cells infected with AdCMV (0.04, 0.25, and 4.4; 0.05, 0.45, and 3.2 µg/1 x 106 cells/day on days 7, 9, and 11, respectively). This result indicates that infection of the Ad5 vector (five particles per cell) did not appreciably alter the MPA-induced production of IGFBP-1. The production rates in cells infected with AdPRB (five particles per cell) were also similar to the rates in uninfected cells. The production rates in cells infected with AdPRA (five particles per cell) were 0.07, 2.2 to 11 µg/1 x 106 cells/day on days 7, 9, and 11, respectively, which are significantly higher than that of uninfected cells or Ad5 or Ad5-hPR-B infected cells (P < 0.01, day 7; P < 0.001, days 9 and 11). These results indicate that hPR-A, not hPR-B, is a strong transactivator for the induction of IGFBP-1 production.



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Figure 7. Production Rate of IGFBP-1 in Endometrial Stromal Cells

Stromal cells isolated from a midsecretory endometrium were infected with AdCMV, AdPRA, and AdPRB, respectively, and then cultured with 0.1 µM MPA for 11 days. IGFBP-1 in the media was measured by ELISA every other day. The amount of IGFBP-1 in media collected before day 6 was below the detection limit of ELISA. The production rate on days 7, 9, and 11 are shown. Data represent triplicate determination, mean ± SD. Production rates on day 7 were 0.043, 0.049, 0.071, and 0.031 µg/1 x 106 cells per day in control (cells not infected by Ad) and cells infected with AdCMV, AdPRA, and AdPRB, respectively. Day 7, AdPRA vs AdPRB (P < 0.01); days 9 and 11, AdPRA vs. control, AdCMV, and AdPRB (P < 0.001).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The promoter activity of the IGFBP-1 gene has been studied in endometrial stromal cells (10, 11, 12, 13, 14, 15, 16) as well as in HepG2 cells (17, 18). The transactivators that interact with the functional cis-elements in the IGFBP-1 promoter are different in these two types of cells (10, 11, 12, 13, 14, 15, 16, 17, 18). For example, distal promoter SP1 sites are extremely active in decidualized stromal cells which account for >95% of the total induction (12, 15, 16), but they are silent in HepG2 cells (15, 16, 17). In the proximal promoter region, CCAAT (Fig. 1Go) represses the activity in endometrial cells (14) but it activates the promoter in HepG2 cells by HNF1, a liver factor not expressed in stromal cells (14). More importantly, progestin and its receptor are essential to activate the endometrial/decidual IGFBP-1 expression (10) whereas in HepG2 cells, glucocorticoids activate its expression (17, 18).

In this study, we have demonstrated that ligand-activated hPR-A is a stronger transactivator than hPR-B to increase the promoter activity as well as the induction of endogenous IGFBP-1 gene in endometrial stromal cells. The promoter analyses, using transient transfection of a short promoter fragment, have shown that active sites, PRE1/2, mediate the PR activation. Also, the response is dose dependent and can be detected after 2 days incubation with MPA. These data provide evidence that transactivation of PR is directly interacting on the IGFBP-1 promoter although indirect effect is also probable. These data imply that the high production of IGFBP-1 (Fig. 7Go), although it was slow to rise, is mediated by interaction of hPR-A with the PRE1 and PRE2 sites.

The delayed effect shown in Fig. 7Go is likely caused by the multiple regulatory elements that prevent an immediate response. For example, we have shown that CCAAT (-82 to -52 bp) represses the promoter activity mediated by NF-Ys which was down-regulated by MPA (14). The overall production of IGFBP-1 is likely resulted from the balance between activation and repression at transcription and posttranscription levels (9, 10, 11, 12, 13, 14, 15).

Although overexpression of hPR-A or hPR-B was used in the present study, we consider that the results are physiologically relevant since the study was carried out in a primary culture system that depicts decidualization in vivo (16). Also, no squelching was observed (Fig. 4Go). In Fig. 4Go, we found that hPR-B reduced the hPR-A activated promoter activity (lanes 5–7, 8, and 9). These results coincide with the low production rate in the first 6 days of culture with MPA and in undifferentiated stromal cells (9) since they contain an equivalent amount of hPR-A and hPR-B (1). The high promoter activity and production rate activated by PRA (Fig. 3Go, lanes 2–4, and Fig. 7Go) correlate with the exponential increase of the IGFBP-1 production and mRNA level in decidualized stromal cells (9, 10) where hPR-A is predominant (1, 2). Taken together, the present findings illustrate, at least in part, the induction mode of IGFBP-1 gene in endometrial stromal/decidual cells, i.e. activation of the endometrial cell IGFBP-1 gene is progestin dependent and mediated by the content of the PR isoforms at different stages of stromal cell decidualization. Other factors, such as Sp3 and CCAAT binding proteins, also regulate the transcription (12, 13, 14, 15, 16), which may be either dependent or independent on the progestin regulation.

In comparison, hPR-B transactivates the pMMTV-CAT stronger than that of hPR-A in stromal cells (Fig. 2BGo). Our observations, together with data obtained in cancer cell lines (3, 4, 5, 6, 7, 8), indicate that the two PR isoforms can have dissimilar responses to different promoters. In addition, we have shown that hPR-B acts as a transdominant repressor of hPR-A in contrast to the findings in cancer cell lines (3, 4, 5). Since the repressor "domain" resides in both isoforms (6, 7, 8), the repressive effects of the two isoforms appear to be also relative depending on the target genes and cell context.

At present, the molecular mechanism of the high transactivation capacity of hPR-A for the IGFBP-1 promoter is unclear. It may be due to the differences in binding capacity or affinity of hPR-A/hPR-B to PRE1/PRE2 sites. However, our binding analysis ruled out this possibility. It is likely due to its interaction with the adjacent sequences of PRE1 and PRE2 or its interaction with other factors associated with these two regions in stromal cells.

The high capacity of hPR-A to transactivate the IGFBP-1 gene suggests that endogenous hPR-A in decidual cells may be also active on the other genes expressed in endometrial/decidual cells. Previous studies have shown that progestin induces aromatase, fibronectin, IGFs, PRL/PRL-R, hPRs, and IGFBP-1 to a moderate degree in the predecidual cells. Among these genes, only hPR-A, PRL, PRL-R, IGFBP-1, and IGF-II are extensively induced in decidual cells (1, 9, 10, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22). The continuous induction of these genes could be associated with the high transactivation capacity of hPR-A directly or indirectly acting on the target genes. Interestingly, preliminary data showed that hPR-A is also a strong transactivator for the production of PRL (our unpublished observation). Further study is needed to clarify the PR isoform specific for gene activation in this system.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture
Human endometrial specimens were obtained from premenopausal women (35 to 50 yr old) who had undergone hysterectomies for a variety of medical reasons but not related to any abnormality of the endometrium. Permission for using these human specimens was approved by the Human Subject Committee of our institution in accordance with US Department of Health regulations. Endometrial stromal cells were digested from tissue fragments with collagenase, isolated (purity >95%), and cultured in Petri dishes, as described previously (22). Stromal cells (1 x 106 cells per dish) were transfected with a total of 10 µg plasmid constructs, 8 µg IGFBP-1 promoter, 0.1 to 1 µg hPR-A or hPR-B expression vectors, and 1 µg pRSV-Luc by a modified calcium phosphate precipitation method (23).

Plasmid Constructs and Transfection Assay
IGFBP-1 promoter-reporter constructs p275CAT and p1.2CAT [the promoter regions between -275 to +68 bp and -1.2 kb to +68 linked to the chloramphenicol acetyl transferase (CAT) reporter gene] were constructed previously (Fig. 1Go and Ref. 13). Both constructs contain the PRE1 and PRE2 sites, which are located at -193 to -179 bp and -102 to -88 bp of the IGFBP-1 promoter, respectively. Mutants (p275PRE1.2 m, p275PRE1m, and p275PRE2m) were constructed previously by double or single mutation at core sequence of the PRE1 and/or PRE2 site (Fig. 1Go). hPR-A and hPR-B expression vectors were a gift from Dr. P. Chambon (24). hPRB-DBDcys, a construct of PR-B DNA binding mutation, was provided by Dr. K. Horwitz (25). pMMTV-CAT (containing two sets of consensus PRE) and pRSV-luc were also used (26, 27, 28). Plasmids were purified twice by CsCl/ethidium bromide gradient centrifugation before use. After transfection, cells were cultured without or with MPA for 2 days and then harvested in 0.15 ml lysis buffer (Promega Corp., Madison, WI). The promoter activity (CAT) was measured in a reaction mixture of cell lysis, 0.1 µCi [3H]acetyl coenzyme A (NEN Life Science Products, Boston, MA) and chloramphenicol at 37 C in 0.2 ml buffer solution containing 0.12 M Tris, pH 6.8. The reaction was stopped by adding 25 µl of 2 M perchloric acid and counted in 3 ml of ScintiLene (Fisher Scientific, Pittsburgh, PA) by a Beckman Coulter, Inc. scintillation counter. The CAT activity was normalized to luciferase activity derived from pRSV-Luc cotransfected with the promoter construct. The Luc activity was measured by Luminometer (Fluoroskan Ascent, Fl, Labsystem OY, Helsinki, Finland). Each experimental design was confirmed in at least two independent experiments. Representative results are summarized in the present report. Data are presented as mean ± SD calculated from triplicate dishes.

Construction and Infection of AdPRA and AdPRB and IGFBP-1 Assay
Recombinant adenoviral transfer vector containing hPR-A or hPR-B under the control of the cytomegalovirus (CMV) was constructed according to the established procedures (29, 30, 31). A replication-defective human adenovirus, Ad5 [lacking E1A and E1B, replication incompetent in normal mammalian cells (29, 30)], was used for the construction of AdPRA and AdPRB. Briefly, hPR-A and hPR-B cDNAs were separately cloned into plasmid [pACsk2CMV5 (31)], containing CMV-1 promoter and adenovirus sequences to yield pACsk2CMV-PRA, and pACsk2CMV-PRB, respectively. The recombinant plasmids and pJM17 (29) were transfected into human embryo kidney cells (293 cells) by lipofectamine (Life Technologies, Inc., Gaithersburg, MD). The two plasmids were recombined at the overlapping viral DNA sequence to yield a recombinant packageable viral genome. Positive viral plaques with hPR-A or hPR-B insert were identified and then propagated to produce large quantities of the recombinant AdPRA and AdPRB, respectively. Titered viral stocks were used to infect the endometrial stromal cells. Cells (1 x 106 cells per dish) were incubated with recombinant viral particles (1, 5, 10, or 50 particles per cell) to achieve an optimal infection. Ninety five percent of the cell population was infected by Ad5 viral particle as shown in Fig. 6Go.

To identify recombinant PR, stromal cells infected with AdPRA and AdPRB were incubated with MPA 2 to 12 days. Cells were then processed for immunohistochemistry using Mab hPRa3 provided by Dr. P. Satyaswaroop (32) and the Histostain SP-kit detection system (Zymed Laboratories, Inc. South San Francisco, CA) described previously (1). Cells infected with Adßgal were stained with substrate X-gal solution (United States Biochemical Corp., Cleveland, OH). The IGFBP-1 in culture medium was measured by ELISA (9) from stromal cells infected with equal amount of viral particles, Ad5, AdPRA, and AdPRB, respectively.


    ACKNOWLEDGMENTS
 
We are grateful to the late Dr. Satyaswaroop who provided us the Mab hPRa3 to identify the PR in stromal cells. We thank the clinicians in the Department of Obstetrics/Gynecology and Reproductive Medicine and Department of Pathology and SUNY-Stony Brook for providing us with viable endometrial specimens.


    FOOTNOTES
 
Address requests for reprints to: Linda Tseng, Ph.D, Department of Obstetrics/Gynecology and Reproductive Medicine, State University of New York at Stony Brook School of Medicine, Stony Brook, New York 11794.

Supported by NIH Grant HD-19247.

Received for publication May 25, 2000. Revision received August 15, 2000. Accepted for publication August 23, 2000.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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