Progesterone Receptor (PR) Inhibits Expression of Insulin-Like Growth Factor-Binding Protein-1 (IGFBP-1) in Human Endometrial Cell Line HEC-1B: Characterization of the Inhibitory Effect of PR on the Distal Promoter Region of the IGFBP-1 Gene

Jiaguo Gao and Linda Tseng

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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Progestin has been shown to have both stimulatory and inhibitory effects on the expression of insulin-like growth factor binding protein-1 (IGFBP-1) in human endometrial cells. In this study, progestin was found to reduce levels of secreted IGFBP-1 and IGFBP-1 messenger RNA and IGFBP-1 promoter activity after stably transfecting a progesterone receptor (PR; B form) expression vector into HEC-1B cells. Deletion analysis of the IGFBP-1 promoter revealed that PR specifically inhibited promoter activity derived from a 59-bp distal BsaHI/RsaI fragment. It was concluded that PR inhibited the promoter activity through protein-protein interactions based on the facts that 1) no progesterone-responsive element was revealed by a series block mutation in the BsaHI/RsaI fragment; 2) PR bound by the antiprogesterone ZK98299 inhibited IGFBP-1 promoter activity; 3) a DNA-binding mutant of PR inhibited the IGFBP-1 promoter activity; and 4) in an in vivo competition assay, the DNA-binding domain of PR did not release the inhibitory effect of intact PR. Analysis of PR deletion mutants indicated that both transcriptional activation domains of PR (TAF-1 and TAF-2) were involved in the inhibition of IGFBP-1 expression. Thus, our data may explain the superinduction of IGFBP-1 in human endometrial cells after progestin withdrawal or progestin replacement with antiprogestin.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Insulin-like growth factor-binding protein-1 (IGFBP-1), a member of the family of IGFBPs (1, 2), is mainly expressed in the liver and endometrium (3, 4). In vivo, the expression of IGFBP-1 in human endometrium is minimum during the menstrual cycle. In decidualized human endometrium, IGFBP-1 becomes the major secretory protein during pregnancy (5, 6). Therefore, the expression of IGFBP-1 in the endometrium is correlated with serum levels of progesterone, suggesting that the expression of IGFBP-1 is induced by progesterone.

In long term primary cultures of human endometrial stromal cells, levels of IGFBP-1 and IGFBP-1 mRNA and IGFBP-1 promoter activity are induced by progestin (7, 8, 9, 10). However, in long term cultures, expression of IGFBP-1 is further increased by withdrawing progestin from the culture medium (7). Moreover, levels of IGFBP-1 mRNA and the transcription rate of IGFBP-1 gene are transiently increased (superinduction) by replacing progestin with antiprogestin (8). These observations indicate that progestin has both stimulatory and inhibitory effects on expression of the IGFBP-1 gene.

Progesterone’s effects are mediated via the nuclear progesterone receptor (PR), which belongs to a superfamily of ligand-induced transactivator (11). Upon activation by progesterone, the PR is able to bind to a specific DNA sequence [progesterone-responsive element (PRE)] in the promoter of responsive genes to activate gene transcription (12). Like other trans-activators, PR consists of a DNA-binding domain and transcriptional activation domains, one located in the amino-terminal (TAF-1) and the other in the carboxyl-terminal (TAF-2) (13, 14, 15). In humans, PR occurs as two forms, PR-B and PR-A (16, 17). The amino acid sequences of PR-B and PR-A are identical, except that PR-B contains an additional N-terminal fragment of 164 amino acids.

To study the effects of PR on IGFBP-1 gene expression, we stably transfected a PR (B form) expression vector into the human endometrial cell line HEC-1B. We report that progestin reduced the expression of endogenous IGFBP-1 in HEC-1B cells expressing PR. Furthermore, PR specifically inhibited IGFBP-1 promoter activity derived from a distal 59-bp BsaHI/RsaI fragment (-2686 to -2628). Finally, both activation domains of PR were shown to be involved in the inhibitory effect through protein-protein interactions.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PR Inhibits the Expression of Endogenous IGFBP-1
Although HEC-1B cells express IGFBP-1 (18, 19), they lack endogenous PR (20). To investigate the effects of PR on IGFBP-1 expression, an expression vector for PR-B was stably transfected into HEC-1B cells to establish the cell line HEC-1B-PR. Expression of PR in HEC-1B-PR cells was verified by histochemistry using PR antibody (data not shown). In a transient transfection assay with a mouse mammary tumor virus (MMTV) promoter construct (pMMTVCAT) that contains PREs (21), medroxyprogesterone acetate (MPA) dramatically increased chloramphenicol acetyltransferase (CAT) activity 90-fold in HEC-1B-PR cells, but had no effect on CAT activity in parental HEC-1B cells (Table 1Go). These results demonstrated that PR were expressed and activated by MPA in HEC-1B-PR cells.


View this table:
[in this window]
[in a new window]
 
Table 1. MPA Activates the MMTV Promoter and Inhibits the IGFBP-1 Promoter in HEC-1B-PR Cells

 
When the IGFBP-1 promoter construct pBRPL was used in transfection assays, MPA inhibited 77% of IGFBP-1 promoter activity in HEC-1B-PR cells (Table 1Go), but had no effect on promoter activity in parental HEC-1B cells (Table 1Go). To determine the effect of PR on the expression of endogenous IGFBP-1, HEC-1B-PR and HEC-1B cells were cultured with or without MPA for 48 h, and the level of IGFBP-1 was measured in conditioned medium by enzyme-linked immunosorbent assay (ELISA). MPA reduced the production of IGFBP-1 by 3-fold in HEC-1B-PR cells, but did not affect the production of IGFBP-1 in HEC-1B cells (Fig. 1Go). In parallel, IGFBP-1 mRNA levels were reduced approximately 3-fold by MPA in HEC-1B-PR cells, but were unchanged by MPA in parental cells (Fig. 2Go). These results indicated that ligand-activated PR inhibits the expression of IGFBP-1 in HEC-1B cells.



View larger version (21K):
[in this window]
[in a new window]
 
Figure 1. MPA Inhibits the Secretion of IGFBP-1 Protein in HEC-1B-PR Cells

HEC-1B and HEC-1B-PR cells (1 x 106) were cultured in 60-mm dishes. Cells were treated with or without MPA in serum-free medium for 48 h. The level of IGFBP-1 in the conditioned medium was measured by ELISA as described in Materials and Methods. Results are the mean ± SD calculated from triplicate dishes.

 


View larger version (56K):
[in this window]
[in a new window]
 
Figure 2. Administration of MPA Reduces the Level of Endogenous IGFBP-1 mRNA in HEC-1B-PR Cells

Total RNAs were isolated from MPA-treated or untreated HEC-1B and HEC-1B-PR cells as described in Materials and Methods. The levels of endogenous IGFBP-1 mRNA were measured by nuclease protection assay. The riboprobe was transcribed from the 370-bp PstI/BamHI fragment of IGFBP-1 complementary DNA. 18S RNA probe was used as an internal control.

 
PR Specifically Inhibits the Promoter Activity Derived from the 59-bp BsaHI/RsaI fragment
Previously, we have shown that activated PR inhibits IGFBP-1 promoter activity derived from a 130-bp ClaI/XbaI fragment (from -2732 to -2600 bp), which can enhance transcription about 10-fold (20). To define DNA sequences mediating the effect of PR in this ClaI/XbaI fragment, a series of IGFBP-1 promoter deletion constructs was cotransfected with the PR-B expression vector (hPR1) into HEC-1B cells. As shown in Fig. 3Go, MPA inhibited 60% of CAT activity derived from IGFBP-1 promoter (from -2732 to +68 bp, construct pCXPL). Consistent with previous results (22), deletion of ClaI/BsaHI (-2730 to -2688 bp) increased CAT activity, but promoter activity was still repressed by MPA (Fig. 3Go, construct pBXPL). Deletion of the RsaI/XbaI fragment (-2630 to -2600 bp) had no effect on promoter activity, and promoter activity was repressed by MPA (Fig. 3Go, construct pCRPL). However, when the BsaHI/RsaI fragment (-2688 to -2630 bp) that mediates the activation (22) was missing from the promoter constructs, MPA was no longer able to inhibit CAT activity (Fig. 3Go, constructs pRXPL, pCBPL, and pPL). These results indicate that ligand-activated PR specifically inhibited activation derived from the BsaHI/RsaI fragment.



View larger version (19K):
[in this window]
[in a new window]
 
Figure 3. The Activation Derived from the BsaHI/RsaI Fragment in the IGFBP-1 Promoter Is Inhibited by PR

Restriction enzyme sites ClaI (-2732), RsaI (-2630), and XbaI (-2600) were used to make deletion constructs. The BsaHI (-2688) site was created by oligo-directed mutagenesis, and this point mutation has no effect on the promoter activity (22). Eight micrograms of IGFBP-1 promoter/CAT constructs were cotransfected with 1.5 µg hPR1 into HEC-1B cells. After transfection, cells were treated with (+MPA) or without (-MPA) MPA for 48 h in serum-free medium. Normalized CAT activity derived from pCXPL (-MPA) was assigned a value of 100. Results are the mean ± SD calculated from triplicate dishes.

 
When the BsaHI/RsaI fragment was 5'-linked to the thymidine kinase (TK) promoter, it was able to enhance the activity of TK promoter 15-fold in the absence of MPA (Fig. 4Go, pBRTKCAT vs. pTKCAT). The enhanced activity of the TK promoter was repressed 80% by activated PR as well (Fig. 4Go, pBRTKCAT plus hPR1, -MPA vs. +MPA). However, activated PR had no effect on the TK promoter (Fig. 4Go, pTKCAT plus hPR1, -MPA vs. +MPA). These results indicate that the BsaHI/RsaI fragment and the basal promoter are sufficient to mediate the inhibitory effect of PR.



View larger version (20K):
[in this window]
[in a new window]
 
Figure 4. The Activation Derived from the BsaHI/RsaI Fragment Can Be Repressed by PR in a Heterologous TK Promoter Construct

The BR fragment was 5'-linked to the TK promoter. Eight micrograms of the promoter construct were cotransfected with 1.5 µg hPR1 or pSG5 (empty vector) into HEC-1B cells. After transfection, cells were treated with (+MPA) or without (-MPA) MPA for 48 h in serum-free medium. Normalized CAT activity derived from pBRTKCAT (-MPA) was assigned a value of 100. Results are the mean ± SD calculated from triplicate dishes.

 
Repression of IGFBP-1 Promoter by PR Does not Involve DNA-PR Interaction
Three activating cis-elements have been identified in the BsaHI/RsaI fragment in HEC-1B cells (22) (Fig. 5Go). However, sequence analysis did not reveal a consensus PRE. To identify potential PREs, a series of block mutants in the BsaHI/RsaI fragment was tested for their effects on PR function. Mutants 1 and 3, which have no effect on promoter activity (22), did not affect the inhibitory effect of activated PR (Fig. 5Go, pBRm1PL and pBRm3PL, -MPA vs. +MPA). The reduced activation from cis-element I mutant (pBRm2PL), cis-element II mutants (pBRm4PL pBRm5PL, and pBRm6PL), and cis-element III mutant (pBRm7PL) was still repressed by activated PR (Fig. 5Go, -MPA vs. +MPA). Even the activation from the double mutated BsaHI/RsaI fragment (pBRm2, 7PL and pBRm4, 6PL) was repressed by adding MPA (Fig. 5Go, -MPA vs. +MPA). These results indicate that there is no PRE in the BsaHI/RsaI fragment, and therefore, that PR does not repress the promoter activity via PR-promoter interaction.



View larger version (22K):
[in this window]
[in a new window]
 
Figure 5. None of the Block Mutations through the BsaHI/RsaI Fragment Eliminates the Repressive Effect of PR Complex

Seven single block mutants were created by oligo-directed mutagenesis as previously described (22). The double mutants were created by recombination of the corresponding single mutant. I, II, and III regions indicate cis-elements I, II, and III, respectively. Eight micrograms of each construct were cotransfected with 1.5 µg hPR1 into HEC-1B cells. Normalized CAT activity derived from pBRPL (-MPA) was assigned a value of 100. Results are the mean ± SD calculated from triplicate dishes.

 
In the presence of PR, administration of ZK98299, whose complex with PR does not bind to DNA (23), significantly reduced the IGFBP-1 promoter activity (Fig. 6Go, pBRPL plus hPR1, control vs. ZK98299). As a control, ZK98299 failed to activate the MMTV promoter that contains functional PREs (Fig. 6Go, pMMTVCAT plus hPR1, control vs. ZK98299). When the PR mutant DBDcys that contains the Cys587 to Ala587 DNA-binding domain mutation (24) was cotransfected with pBRPL or pMMTVCAT, MPA was able to inhibit IGFBP-1 promoter activity, but was unable to increase CAT activity derived from pMMTVCAT (Table 2Go). These results provided additional evidences that PR represses promoter activity via protein-protein interactions.



View larger version (33K):
[in this window]
[in a new window]
 
Figure 6. PR Bound by Antiprogestin ZK98299 Retains Its Ability to Repress the IGFBP-1 Promoter

Eight micrograms of construct pMMTVCAT or pBRPL were cotransfected with 1.5 µg hPR1 or pSG5 (empty vector) into HEC-1B cells. After transfection, cells were treated with MPA, ZK98299, or ethanol (control) for 48 h in serum-free medium. Normalized CAT activity derived from pBRPL (pBRPL plus pSG5, control) was assigned a value of 100. Results are the mean ± SD calculated from triplicate dishes.

 

View this table:
[in this window]
[in a new window]
 
Table 2. PR Mutated in DNA-Binding Domain Remains Its Ability to Repress the IGFBP-1 Promoter

 
Repression of IGFBP-1 Promoter by PR Involves Both TAF-1 and TAF-2 Domains of PR
Deletion constructs of PR were used to locate the repressive domains in PR. When the N-terminal that contains the activation domain TAF-1 was deleted (15), administration of MPA reduced IGFBP-1 promoter activity (Fig. 7Go, hPR3). When the C-terminal of PR that contains the activation domain TAF-2 and the progesterone-binding domain was deleted (15), the repressive effect of PR became independent of progestin (Fig. 7Go, hPR5). Construct hPR5 contains about two thirds of the hinge region of PR. When both activation domains were deleted [hPR(C)] (25), no repressive effect was observed [Fig. 7Go, hPR(C)]. These results indicated that both the N- and C-terminals of PR, which contain TAF-1 and TAF-2, respectively, are involved in the repression of IGFBP-1 promoter activity and that the DNA-binding domain of PR alone is not sufficient to inhibit promoter activity.



View larger version (15K):
[in this window]
[in a new window]
 
Figure 7. Both Transcriptional Activation Function Domains of PR Are Involved in the Repression of IGFBP-1 Promoter

Eight micrograms of pBRPL were cotransfected with 1.5 µg PR expression vectors into HEC-1B cells. hPR1 expresses the B form of PR (amino acids 1–933). hPR3, hPR5, and hPR(C) express truncated PR containing amino acids 551–933, 1–673, and 556–639, respectively. TAF-1 and TAF-2 are transcriptional activation functions. The C domain is the DNA-binding domain. After transfection, cells were treated with MPA or without MPA for 48 h in serum-free medium. Normalized CAT activity derived from pBRPL cotransfected with hPR1 (-MPA) was assigned a value of 100. Results are the mean ± SD calculated from triplicate dishes.

 
The DNA-binding domain construct of PR, hPR(C), was also used in an in vivo competition assay to study the effect of the DNA-binding domain on promoter repression. MMTV promoter activity enhanced by PR was inhibited 60% when hPR(C) was cotransfected with the PR expression vector at a 12.5:1 ratio (Table 3Go). Under the same conditions, however, hPR(C) was not able to reverse the inhibitory effect of PR on IGFBP-1 promoter activity (Table 3Go).


View this table:
[in this window]
[in a new window]
 
Table 3. Overexpression of DNA-Binding Domain of PR Cannot Release the Inhibitory Effect of PR on IGFBP-1 Promoter

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the present study, we have shown that ligand-activated PR reduced levels of secreted IGFBP-1 and IGFBP-1 mRNA and inhibited IGFBP-1 promoter activity in HEC-1B-PR cells. We have also demonstrated that the inhibitory effect of PR on promoter activity is specific to the activation derived from the BsaHI/RsaI fragment and did not involve PR-promoter interaction.

At present, it is unclear how the activated PR inhibits promoter activity, but several potential explanations exist. Activation of promoters by distal elements requires interactions between distal activators and the basal transcription machinery. As PR can bind to the basal transcription factor TFIID in a DNA binding-independent manner (25), we tested whether PR binding to TFIID interfered with interactions between distal activators and the basal transcriptional machinery. Deletion analysis have shown that the DNA-binding domain of PR [hPR(C)] is responsible and sufficient for TFIID binding (25). However, hPR(C) was not able to either inhibit IGFBP-1 promoter activity or reverse the inhibitory effect of intact PR in in vivo competition assays. Therefore, this mechanism seems unlikely to explain the observed repression of IGFBP-1 promoter.

Nuclear receptors have also been proposed to inhibit activation by tethering to activator bound to DNA (26). Mutation analysis revealed that activation from each of the cis-elements in the BsaHI/RsaI fragment was inhibited by PR. We have previously shown that a number of proteins bind to various cis-elements in the BsaHI/RsaI fragment (22). However, gel mobility shift assays with the BsaHI/RsaI fragment and nuclear extracts from HEC-1B-PR cells revealed that none of the specific complexes was further retarded by activated PR (data not shown), suggesting that PR did not bind to any of the proteins bound to these cis-elements.

Therefore, we hypothesize that PR inhibits IGFBP-1 promoter activity by competing with one or more coactivators that are needed for activation derived from the BsaHI/RsaI fragment. It has been shown that PR can bind to coactivator through its activation domain (27). More experiments are needed to verify this hypothesis.

The inhibitory effect of PR on IGFBP-1 expression reported here may explain the superinduction of IGFBP-1 observed in primary cultures after progestin withdrawal or exposure to antiprogestin (7, 8), but contradicts results showing that IGFBP-1 is induced by progestin in primary culture cells. This discrepancy may be explained by the hypothesis that progestin has a direct inhibitory and an indirect stimulatory effect on IGFBP-1 expression (20). In primary cultures of human endometrial cells, the induction of IGFBP-1 by progestin was shown to be gradual, with maximum induction reached by approximately 25 days (7, 8). Therefore, it is likely that progestin initiates a signaling pathway that ultimately leads to the activation of IGFBP-1, instead of activating the promoter directly. PRL is another decidual-specific protein whose expression pattern is similar to that of IGFBP-1 in human endometrium (8, 28). Promoter analysis revealed that progesterone activation of the PRL gene does not occur via direct PR-promoter interactions (29).

In the present study, we have concentrated on the B form of the PR because we were unable to select cell clones that express functional PR-A. It has been reported that the B and A forms have either similar or opposite effects on transcription (17, 30). However, when the IGFBP-1 promoter (-3.6 kilobases to +68 bp) construct was cotransfected with PR-A expression vector, administration of MPA did not increase promoter activity (data not shown). Transfection data suggested that PR-A also inhibited activation from the BsaHI/RsaI fragment (data not shown), which agrees with the finding that hPR3 (deletion of N-terminal) repressed IGFBP-1 promoter activity.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Establishment of the Cell Line HEC-1B-PR
HEC-1B cells (American Type Culture Collection, Rockville, MD) were maintained in MEM in Earle’s salt solution supplemented with 10% bovine serum. HEC-1B cells (1 x 106) were cotransfected with 9 µg hPR1 (15) and 1 µg pSV2neo (31) by the method of Chen and Okayama (32). Forty-eight hours after transfection, cells were subcultured, and clones were selected with Geneticin (G418 sulfate; Life Technologies, Gaithersburg, MD) at a concentration of 400 µg/ml. Well separated clones were isolated and cultured with 200 µg/ml Geneticin. Expression of PR was verified by immunohistochemistry as described previously (20). A construct containing the mouse mammary virus promoter was transfected into viable clones to test PR function.

Detection of IGFBP-1 Protein and mRNA
HEC-1B and HEC-1B-PR cells (1 x 106) were seeded onto 60-mm culture dishes in MEM with 10% bovine serum. Sixteen hours later, the cells were cultured in serum-free medium with or without MPA for 48 h. Conditioned medium were collected, and cells were lysed by an acid guanidinium thiocyanate solution.

Levels of IGFBP-1 in conditioned medium were measured by ELISA as described previously (33), using IGFBP-1 (Upstate Biotechnology, Lake Placid, NY) as a standard.

Total RNA was isolated in acid guanidinium thiocyanate solution and further purified by CsCl centrifugation. A PstI/BamHI fragment (from 512–881 bp) of IGFBP-1 complementary DNA was cloned into pSP6/T7–19, and an antisense riboprobe was made by transcription with SP6 RNA polymerase. Levels of IGFBP-1 RNA were measured by solution hybridization/nuclease protection assays using the RPAII kit (Ambion, Austin, TX). 18S RNA (Ambion) was measured as an internal control.

Transient Transfection Assays
The deletion constructs of IGFBP-1 promoter, pCXPL, pBXPL, pCRPL, pBRPL, pRXPL, and pPL, were described previously (22). Mutations in the BsaHI/RsaI were generated by oligo-directed mutagenesis as previously described (22). Plasmids hPR1, hPR3, and hPR5 (15) were gifts from Pierre Chambon (Institute de Chimie Biologique, Strasbourg, France). hPR(C) (25) was a gift from Ludger Klein-Hitpass (Universitatsklinikum Essen, Germany), and DBDcys (24) was a gift from K. B. Horwitz (University of Colorado, Denver, CO). All plasmids were purified twice by CsCl centrifugation.

A total of 10 µg DNA including 0.5 µg pRSV-luc (34) were transfected into 1 x 106 cells in a 60-mm dish as described previously (20, 22, 32). CAT and luciferase assays were carried out as previously described (20, 22). CAT activities were normalized to luciferase activities.


    ACKNOWLEDGMENTS
 
We thank Dr. John Bruno for his critical comments on the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Dr. Jiaguo Gao, Department of Obstetrics and Gynecology, School of Medicine, State University of New York, Stony Brook, New York 11794.

This work was supported by NIH Grant HD-19247.

Received for publication December 23, 1996. Revision received February 21, 1997. Accepted for publication February 25, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Czech MP 1989 Signal transmission by the insulin-like growth factors. Cell 59:235–238[Medline]
  2. Clemmons DR 1993 IGF binding proteins and their functions. Mol Reprod Dev 35:368–375[Medline]
  3. Lee PD, Conover CA, Powell DR 1993 Regulation and function of insulin-like growth factor-binding protein-1. Proc Soc Exp Biol Med 204:4–29[Abstract]
  4. Rutanen EM, Seppala M 1992 Insulin-like growth factor binding protein-1 in female reproductive functions. Int J Gynaecol Obstet 39:3–9[Medline]
  5. Rutanen EM, Koistinen R, Wahlstrom T, Sjoberg J, Stenman UH, Seppala M 1984 Placental protein 12 (PP12) in the human endometrium: tissue concentration in relation to histology and serum levels of PP12, progesterone and oestradiol. Br J Obstet Gynaecol 91:377–381[Medline]
  6. Bryant-Greenwood GD Rutanen EM, Partanen S, Coelho TK, Yamamoto SY 1993 Sequential appearance of relaxin, prolactin and IGFBP-1 during growth and differentiation of the human endometrium. Mol Cell Endocrinol 95:23–29[CrossRef][Medline]
  7. Bell SC, Jackson JA, Ashmore J, Zhu HH, Tseng L 1991 Regulation of insulin-like growth factor-binding protein-1 synthesis and secretion by progestin and relaxin in long term cultures of human endometrial stromal cells. J Clin Endocrinol Metab 72:1014–1024[Abstract]
  8. Tseng L, Gao JG, Chen R, Zhu HH, Mazella J, Powell DR 1992 Effect of progestin, antiprogestin, and relaxin on the accumulation of prolactin and insulin-like growth factor-binding protein-1 messenger ribonucleic acid in human endometrial stromal cells. Biol Reprod 47:441–450[Abstract]
  9. Gao JG, Mazella J, Tseng L 1994 Activation of the human IGFBP-1 gene promoter by progestin and relaxin in primary culture of human endometrial stromal cells. Mol Cell Endocrinol 104:39–46[CrossRef][Medline]
  10. Gao JG, Tseng L 1996 Distal Sp3 binding sites in the hIGFBP-1 gene promoter suppress transcriptional repression in decidualized human endometrial stromal cells: identification of a novel Sp3 form in decidual cells. Mol Endocrinol 10:613–621[Abstract]
  11. Evans RM 1988 The steroid and thyroid receptor superfamily. Science 240:889–895[Medline]
  12. Klein-Hitpass L, Tsai SY, Weigel NL, Allan GF, Riley D, Rodriguez R, Schrader WT, Tsai MJ, O’Malley BW 1990 The progesterone receptor stimulates cell-free transcription by enhancing the formation of a stable preinitiation complex. Cell 60:247–257[Medline]
  13. Gronemeyer H 1991 Transcription activation by estrogen and progesterone receptors. Annu Rev Genet 25:89–123[CrossRef][Medline]
  14. Bocquel MT, Kumar V, Stricker C, Chambon P, Gronemeyer H 1989 The contribution of the N- and C-terminal regions of steroid receptors to activation of transcription is both receptor and cell-specific. Nucleic Acids Res 17:2581–2595[Abstract]
  15. Meyer ME, Pornon A, Ji JW, Bocquel MT, Chambon P, Gronemeyer H 1990 Agonistic and antagonistic activities of RU486 on the functions of the human progesterone receptor. EMBO J 9:3923–3932[Abstract]
  16. Horwitz KB, Alexander PS 1983 In situ photolinked nuclear progesterone receptors of human breast cancer cells: subunit molecular weights after transformation and translocation. Endocrinology 113:2195–2201[Abstract]
  17. Kastner P, Krust A, Turcotte B, Stropp U, Tora L, Gronemeyer H, Chambon P 1990 Two distinct estrogen-regulated promoters generate transcripts encoding the two functionally different human progesterone receptor forms A and B. EMBO J 9:1603–1614[Abstract]
  18. Gong Y, Ballejo G, Alkhalaf B, Molnar P, Murphy LC, Murphy LJ 1992 Phorbol esters differentially regulate the expression of insulin-like growth factor-binding proteins in endometrial carcinoma cells. Endocrinology 131:2747–2754[Abstract]
  19. Pekonen F, Nyman T, Rutanen EM 1991 Human endometrial adenocarcinoma cell lines HEC 1B and KLE secrete insulin-like growth factor binding protein-1 and contain IGF-I receptors. Mol Cell Endocrinol 75:81–87[CrossRef][Medline]
  20. Gao JG, Mazella J, Powell DR, Tseng L 1994 Identification of a distal regulatory sequence of the human IGFBP-1 gene promoter and regulation by the progesterone receptor in a human endometrial adenocarcinoma cell line. DNA Cell Biol 13:829–837[Medline]
  21. Cato ACB, Miksicek R, Schutz G, Arnemann J, Beato M 1986 The hormone regulatory element of mouse mammary tumor virus mediates progesterone induction. EMBO J 5:2237–2240[Abstract]
  22. Gao JG, Tseng L 1995 Activation of human insulin-like growth factor binding protein-1 gene promoter by a distal regulatory sequence in a human endometrial adenocarcinoma cell line. Mol Endocrinol 9:1405–1412[Abstract]
  23. Klein-Hitpass L, Cato ACB, Henderson D, Ryffel GU 1991 Two types of antiprogestins identified by their differential action in transcriptionally active extracts from T47D cells. Nucleic Acids Res 19:1227–1234[Abstract]
  24. Takimoto GS, Tasset DM, Eppert AC, Horwitz KB 1992 Hormone-induced progesterone receptor phosphorylation consists of sequential DNA-independent and DNA-dependent stages: analysis with zinc finger mutants and the progesterone antagonist ZK98299. Proc Natl Acad Sci USA 89:3050–3054[Abstract]
  25. Schwerk C, Klotzbucher M, Sachs M, Ulber V, Klein-Hitpass L 1995 Identification of a transactivation function in the progesterone receptor that interacts with TAFII110 subunit of the TFIID complex. J Biol Chem 270:21331–21338[Abstract/Free Full Text]
  26. Starr DB, Thomas JR, Yamamoto KR 1995 A single amino acid residue governs a switch between transcription activation, repression by the glucocorticoid receptor. In: Herr W, Tjian R, Yamamoto K (eds) Mechanisms of Eukaryotic Transcription. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p 218 (Abstract)
  27. Onate SA, Tsai SY, Tsai MJ, O’Malley BW 1995 Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science 270:1354–1357[Abstract]
  28. Huang JR, Tseng L, Bischof P, Janne OA 1987 Regulation of prolactin production by progestin, estrogen, and relaxin in human endometrial stromal cells. Endocrinology 121:2011–2017[Abstract]
  29. Gellersen B, Kempf R, Telgmann R, DiMattia G 1994 Nonpituitary human prolactin gene transcription is independent of Pit-1 and differentially controlled in lymphocytes and in endometrial stroma. Mol Endocrinol 8:356–373[Abstract]
  30. Vegeto E, Shahbaz MM, Wen DX, Goldman ME, O’Malley BW, McDonnell DP 1993 Human progesterone receptor A form is a cell- and promoter-specific repressor of human progesterone receptor B function. Mol Endocrinol 7:1244–1255[Abstract]
  31. Southern PJ, Berg P 1982 Transformation of mammalian cells to antibiotic resistance with a bacterial gene under the control of the SV40 early region promoter. J Mol Appl Genet 1:327–341[Medline]
  32. Chen C, Okayama H 1987 High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 7:2745–2752[Medline]
  33. Frost RA, Mazella J, Tseng L 1993 Insulin-like growth factor binding protein-1 inhibits the insulin-like growth factor and progestin stimulated growth factor of human endometrial stromal cells. Biol Reprod 49:104–111[Abstract]
  34. de Wet JR, Wood KV, DeLuca M, Helinski DR, Subramani S 1987 Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol 7:725–737[Medline]