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The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 3 926-930
Copyright © 1998 by The Endocrine Society

Original Studies

Transcriptional Regulation of the Tissue Factor Gene by Progestins in Human Endometrial Stromal Cells1

Graciela Krikun, Frederick Schatz, Nigel Mackman, Seth Guller and Charles J. Lockwood

Departments of Obstetrics and Gynecology (G.K., S.G., C.J.L.) and Biochemistry (S.G.), New York University Medical Center, New York, New York 10016; and Departments of Immunology and Vascular Biology, The Scripps Research Institute (N.M.), La Jolla, California 92037

Address all correspondence and requests for reprints to: Dr. Graciela Krikun, New York University Medical Center, Tisch Hospital, Room 533, 550 First Avenue, New York, New York 10016.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Decidualization of estradiol (E2)-primed human endometrial cells (HESCs) by progesterone is associated with elevated levels of tissue factor (TF), the primary initiator of hemostasis. Similarly, in cultured human HESCs, the synthetic progestin, medroxyprogesterone acetate (MPA), enhances TF protein and messenger ribonucleic acid (mRNA) levels. Although ineffective alone, E2 potentiates this progestin enhancement of TF expression by HESCs. The current study examines mechanisms underlying MPA enhancement of TF mRNA expression in HESCs. In the presence of the transcription-blocking agent dichlororibofuranosylbenzimidazole, no significant differences were noted in the half-lives of TF mRNA isolated from HESCs treated with E2 alone or with E2 plus MPA. This indicates that MPA-enhanced TF mRNA levels do not reflect changes in the stability of the TF message. To test the effect of progestin on TF promoter activity and to ascertain the mechanism of promoter regulation, primary or first passaged HESCs were transfected with TF promoter constructs spanning the regions -2106 to +121 (TFp-2106), -278 to +121 (TFp-278), and -111 to +14 (TFp-111) bp upstream of the transcription start site. MPA was found to enhance TF transcription by 20-fold in HESCs transfected with TFp-2106 after correcting for transfection efficiencies with a ß-galactosidase reporter plasmid. Interestingly, levels of E2- plus MPA-stimulated transcription were significantly increased using TFp-278 compared to TFp-2106, suggesting that the region between -2106 and -278 bp may contain an inhibitory element. In addition, rates of MPA-stimulated transcription using TFp-111 were significantly reduced compared to values obtained using TFp-2106 and were even further reduced compared to values obtained using TFp-278. This suggests that regulatory elements in the -111 bp region of the TF promoter are necessary for progestin-mediated regulation of the TF gene in HESCs, but are not sufficient to account for maximal rates of TF gene transcription.

Our results also demonstrated that induction of steady state TF mRNA by MPA was abolished by treating cells with E2 plus MPA in conjunction with the protein synthesis inhibitor cycloheximide. In light of the absence of a complete progesterone or estrogen response element in the published 5'-sequence of the TF promoter, our results suggest that progestin-enhanced transcription of TF mRNA in stromal cells may be mediated by an uncharacterized protein intermediate(s).


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TISSUE FACTOR (TF) is a cell membrane-bound glycoprotein that initiates hemostasis by complexing with factor VII(a) to activate the intrinsic and extrinsic coagulation factors, IX and X, respectively (1). Cytokines and growth and serum factors have been shown to evoke a rapid and transient elevation in TF expression in several cell types (2, 3, 4, 5), prompting the TF gene to be designated an immediate early response gene [6–8; for a recent review, see Mackman (9)]. In human endothelial and/or monocytic cells, induction of TF messenger ribonucleic acid (mRNA) expression by bacterial endotoxin lipopolysaccharide, tumor necrosis factor-{alpha}, or phorbol myristate 13-acetate (PMA) has been shown to be mediated by cis-acting regulatory elements on the promoter region of the TF gene, such as activating protein-1 (AP-1), Sp-1, and a nuclear factor-{kappa}B (NF{kappa}B)-like site (2, 3, 4, 10). In the endothelial cells, an NF{kappa}B-like nuclear protein was induced by cytokines or endotoxin, whereas AP-1 nuclear binding proteins were constitutively expressed (4, 3). Similar results were found with monocytes (11). In contrast, serum-enhanced TF mRNA in cultured fibroblasts was mediated by increased AP-1 binding, whereas NF{kappa}B binding was shown to be constitutive (5). In cultured human epithelial cells, serum- or PMA-enhanced TF transcription was shown to be mediated by constitutively expressed Sp-1 and by inducibly expressed Egr-1-binding protein (12).

In contrast to the immediate, but transient, expression of TF in the cell types described above, prior work from our laboratory established that TF expression was gradually, but chronically, enhanced by progestins in human endometrial stromal cells undergoing decidualization in vivo and in vitro (13, 14). Immunohistochemistry of human endometrium demonstrated increased staining for TF in decidualized stromal cells of late secretory phase and gestational endometrium (14). Medroxyprogesterone acetate (MPA), but not estradiol (E2), enhanced TF protein and mRNA levels in cultured human endometrial stromal cells (HESCs) (13). Consistent with E2 enhancement of progesterone receptor levels, addition of E2 further augmented the MPA-modulated increase in TF protein and mRNA levels. Elevated TF mRNA and protein levels were observed after 48–72 h of MPA treatment and persisted beyond 21 days, consistent with its putative role as a mediator of hemostasis during implantation and placentation (13, 14).

In the current study we used message stability, transient transfections, and cycloheximide (CHX) studies to elucidate a mechanism through which progestins chronically induce TF expression in HESCs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of primary endometrial stromal cultures

Freshly isolated stromal cells were isolated and grown to confluence in a basal medium, as previously described (13). The experimental period was initiated by the addition of 10-8 mol/L E2, 5 x 10-7 mol/L MPA, E2 plus MPA, or 0.1% ethanol as vehicle control. The medium was changed every 2–4 days. The cells were kept in culture for approximately 10 days.

For message stability studies, cells were pretreated with E2 or E2 plus MPA as described above, then dichlororibofuranosylbenzimidazole (DRB) was added at a final concentration of 300 µmol/L for a time course ranging from 0–7 h (where 0 reflects the addition of DRB). Total RNA was extracted at the appropriate time points, and Northern blots were conducted, probed with TF complementary DNA (14), and corrected for loading efficiencies using a glyceraldehyde-3-phosphate complementary DNA, as previously described (15).

Experiments conducted in the presence of CHX were carried out as follows. Cultures were treated with E2 or E2 plus MPA, as described above, in the presence of 0, 10, 100, or 500 ng/mL CHX. The medium was changed every 48 h, and cells were treated for 8 days. Total RNA was extracted within 16 h of the last medium change, and Northern blots were conducted as described above.

Promoter constructs

TF promoter-plasmid constructs contain the 5' flanking sequence of the TF promoter spanning -2106 to +121, -278 to +121, and -111 to +14 bp cloned upstream of VP16/TET transcription factors inserted between the PstI and BamHI site in pBSKSII (Stratagene, La Jolla, CA) (16). The TF promoter-containing plasmids were cotransfected into target cells with the plasmid pUHG10.3CAT containing the chloramphenicol acetyltransferase (CAT) reporter (17). Enhanced expression of the TF promoter drives VP16/TET expression, which, in turn, drives expression of the reporter-containing plasmid (see Fig. 1Go).



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  		Figure 1. Schematic of TF promoter constructs. The -2106 promoter (top two diagrams) includes a 56-bp segment spanning the region -227 to -172 bp containing two AP-1 sites and a NF{kappa}B-like site; two Sp-1 sites between -132 and -170 bp, and a region between -111 to +14 bp containing three overlapping Egr-1 and Sp-1 sites. Truncated promoters (bottom two diagrams) include deletions of areas 5' to either -278 or -111 bp. The latter construct (-111) is devoid of two Sp-1, two AP-1, and a {kappa}B element, but retains three overlapping Egr-1 and Sp-1 elements. The TATA box exists 22 bp 5' to the transcription start site.

 
The pSV-ß-galactosidase construct was obtained from Promega (Madison, WI).

Transient transfections

Primary or once passaged HESCs were seeded at a density of 2 x 105 cells/3-cm polystyrene well and allowed to attach and proliferate to approximately 60% subconfluence for 24 h. HESCs were then treated with E2 (10-8 mol/L) or E2 plus MPA (10-6 mol/L) for 6 days. Subsequent to this treatment, the cultures were transiently transfected in OPTI-MEM medium (Life Technologies, Grand Island, NY) with 1 µg of the various TF promoters and 1 µg of the CAT reporter construct, using 10 µL Lipofectamine (Life Technologies) for 6 h. The medium was then changed back to basal medium containing either E2 or E2 plus MPA as described above for an additional 48 h. Transfection efficiency was determined in parallel wells using 3 µg ß-galactosidase control vector. Cells were lysed after 48 h according to the manufacturer’s instructions (Promega). The lysates to be used for CAT analysis were heated to 60 C. All samples were centrifuged, and the supernatant was stored at -80 C.

Transfection reporter assays

CAT assay. One hundred microliters of cell extract was mixed with [14C]chloramphenicol (0.05 mCi/mL; New England Nuclear, Boston, MA) and n-butyryl coenyzme A (Promega) and incubated for 3 h at 37 C, and the reaction was terminated by the addition of 300 µL xylene. The xylene phase was extracted twice with Tris-HCl (0.25 mol/L; pH 8.0), and the final xylene phase containing only the butyrylated [14C]chloramphenicol was added to a scintillant and counted in a Beckman LS 5000TD scintillation counter (Palo Alto, CA). All experiments were conducted in triplicate.

ß-Galactosidase assay. Fifty microliters of cell extract were added to 50 µL 2 x assay buffer (Promega) containing o-nitrophenyl-ß-D-galactopyranoside, placed in a Microlite 96-well plate, and allowed to incubate at 37 C until a faint yellow color was visible. The reaction was stopped by the addition of 150 µL sodium carbonate (1 mol/L) and was immediately read at 405 nm with an automatic microplate reader (Molecular Devices, Menlo Park, CA).

Statistical analysis

All data were analyzed with a Mann-Whitney rank sum test, Sigma Stat computer program (Jandell Scientific, San Rafael, CA). P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TF mRNA stability

The effects of steroids on levels of steady state TF mRNA in cultured primary endometrial stromal cells were determined by Northern analysis (Fig. 2Go). E2 treatment alone did not affect levels of TF mRNA compared with those in the vehicle control group. The addition of E2 plus MPA, however, dramatically increased TF mRNA levels. The content of TF protein and its functional activity were similarly regulated by steroids in HESCs (14). As E2 is present at significant levels throughout the proliferative and secretory phases of the menstrual cycle, these studies employed E2-treated cells as controls.



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  		Figure 2. Progestational effect on TF mRNA in HESCs. Northern blot of TF mRNA levels from confluent HESCs derived from the secretory phase and maintained in culture medium with or without steroids. Cells were incubated in vehicle control, 10-8 mol/L E2, or E2 plus MPA at either 10-7 or 10-8 mol/L. Levels of TF mRNA were normalized to levels of glyceraldehyde-6-phosphate dehydrogenase mRNA.

 
Figure 3Go examines the effect of MPA treatment on TF mRNA stability. Cultured stromal cells were treated with either E2 or E2 plus MPA and then exposed to the transcriptional inhibitor DRB, which causes premature nucleotide chain termination. Northern analysis was conducted to measure the half-life of TF mRNA. The results depicted in Fig. 3Go demonstrate that the half-life of TF mRNA was approximately 3 h in both E2-treated and E2- plus MPA-treated cells. Similar results were observed in four independent preparations of HESCs obtained from the proliferative to the late secretory phases of the cycle and in cells passaged one or two times (not shown). These findings indicate that progestins do not affect the stability of TF mRNA.



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  		Figure 3. Effect of progestin on TF mRNA stability in HESCs. HESCs were treated with E2 or E2 plus MPA as described in Materials and Methods, and the level of TF mRNA was analyzed by Northern blotting 0–7 h after the addition of 300 µmol/L DRB.

 
MPA activation of TF gene transcription

As MPA enhancement of steady state TF mRNA levels cannot be accounted for by changes in message stability, we examined the possibility that increased TF mRNA occurs through transcriptional activation. Thus, HESCs were transiently transfected with constructs containing -2106 to +121 bp of the TF promoter (TFp-2106) and a CAT reporter gene. Figure 4Go (left panel) shows that CAT activity is augmented by MPA treatment. When these results are corrected for transfection efficiencies by parallel transfections with a ß-galactosidase reporter plasmid (Fig. 4Go, right panel), E2 plus MPA was found to enhance TF transcription rates 20-fold compared to treatment with E2 alone (P < 0.05). Parallel transfections, rather than cotransfections, with ß-galactosidase were carried out to avoid compromising cell viability by introducing excess DNA. Figure 4Go depicts results obtained with a MPA concentration of 10-6 mol/L. Identical results were observed at 10-7 mol/L.



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  		Figure 4. Transcriptional regulation of TF expression in HESCs by progestin. HESCs were treated with E2 or E2 plus MPA and transfected with a TF promoter construct spanning the regions -2106 to +121 bp, and levels of CAT activity were determined. CAT activity was expressed before (left) and after (right) normalization for transfection efficiency with a plasmid containing the ß-galactosidase gene (mean ± SEM; n = 5 separate experiments; tissues were obtained from the proliferative (n = 3) and secretory (n = 2) phases of the cycle. *, P < 0.05; **, P < 0.01.

 
To identify potential progestin-responsive regions in the TF gene, TF promoter constructs truncated at -278 bp (TFp-278) and -111 bp (TFp-111) were also transiently transfected into HESCs treated with E2 or E2 plus MPA (Fig. 1Go). As shown in Fig. 5Go, significantly greater CAT activities were observed using TFp-278 compared to TFp-2106 in response to both E2 and E2 plus MPA (P < 0.01), suggesting that the region between -2106 and -278 bp may contain a transcriptional inhibitory element. Similar findings were described by Mackman et al. with COS-7 cells after induction by serum (18). Although MPA enhanced the rates of transcription by about 10-fold in cells transfected with TFp-111, overall CAT activity was significantly decreased in both E2-treated and E2- plus MPA-treated cells compared to rates observed with TFp-2106 (P < 0.01). An even further decrease was observed compared to rates with TFp-278 (P < 0.01). Hence, although the presence of cis-acting regulatory elements within the -111 bp promoter (see Fig. 1Go) appears to be necessary for progestin regulation of the TF gene in HESCs, elements within the region -111 to +14 bp are not sufficient to account for all of the MPA-dependent effects on TF gene transcription (Fig. 5Go).



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  		Figure 5. Effect of truncation on TF promoter activities in HESCs. CAT activity was determined in HESCs transfected with TF promoters spanning -2106 to +121 (TFp -2106), -278 to +121 (TFp -278), and -111 to +14 (TFp -111) bp. CAT activities were corrected for transfection efficiency by parallel transfection with a plasmid containing the ß-galactosidase gene. Results were obtained from five experiments. All points were determined in triplicate, and results are expressed as the mean ± SEM. *, P < 0.01, for differences between E2 vs E2+MPA for each individual promoter construct.

 
Effects of CHX on TF mRNA content

As no complete progesterone or estrogen response elements have been reported in the TF promoter, we determined whether a protein intermediate(s) is required for the observed induction of TF mRNA by MPA. Cultured HESCs were treated with either E2 or E2 plus MPA with or without the protein synthesis inhibitor CHX and analyzed by Northern analysis. Figure 6Go shows that CHX treatment strongly inhibited the induction of TF mRNA in E2- plus MPA-treated HESCs. This inhibitory response was evident at 10 ng/mL CHX and was maximal at 500 ng/mL CHX. The results indicate that a protein intermediate(s) is involved in the progestational induction of TF mRNA. Moreover, the increased expression of TF mRNA by cells concomitantly treated with E2 and CHX may reveal the existence of an inhibitor of E2 action. It is interesting to note that CHX treatment also inhibited morphological decidualization of HESCs induced by MPA (not shown).



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  		Figure 6. Effect of CHX on progestational regulation of TF mRNA levels in HESCs. Cells were treated with E2 or E2 plus MPA, and the effect of CHX (0–500 ng/mL) on levels of TF mRNA was examined after Northern blotting. Results are representative of four independent experiments. E, E2; P, E2 plus MPA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study investigated the mechanism(s) by which MPA chronically elevates steady state levels of TF mRNA and protein in HESCs. Crossman et al. (19) reported that the rapid accumulation of TF mRNA in human endothelial cells exposed to lipopolysaccharide is largely due to an increase in mRNA stability, rather than to an increased rate of TF gene transcription. In this study, results with the transcriptional inhibitor DRB indicate that progestins do not regulate TF expression through changes in the stability of TF mRNA.

To determine whether progestins modulate the rate of transcription of the TF gene in HESCs, transfection studies were carried out using a construct spanning -2106 to +121 bp of the TF promoter. These studies demonstrated that MPA enhances TF promoter activity in HESCs approximately 4-fold. Although it was evident that treating HESCs with E2 plus MPA increased the expression of CAT compared to that in cells treated with E2 alone, the differences did not seem large enough to account for the magnitude of the MPA-associated increases in steady state levels of TF mRNA. We, therefore, considered the possibility that MPA-mediated decidualization of HESCs reduced cellular uptake of exogenous DNA. Indeed, parallel transfections indicated that decidualization greatly reduced ß-galactosidase expression. This experimental problem was also noted by Gao et al. (20) in transfecting decidualized HESCs with the insulin-like growth factor-binding protein-1 promoter. We believe that this phenomenon reflects permeability changes caused by decidualization in the surrounding basal laminar component and/or the tendency of these cells to form multicellular layers in vitro (21).

In HeLa cells, basal TF promoter activity requires Sp-1 binding, whereas induction of TF gene transcription by serum and PMA required Egr-1 binding, and maximal induction required both Sp1 and Egr-1 binding (12). To investigate the role of cis-acting elements responsible for the progestational induction of TF in HESCs, we employed truncated TF-CAT promoter constructs. Deletion of -2106 to -278 bp showed increased CAT activity in response to both E2 and E2 plus MPA. In contrast, deletion of -2106 to -111 bp maintained the differential effects of E2vs. E2 plus MPA, but the relative CAT activities were reduced by 60% compared to those in cultures transfected with TFp-2106 and by 80% compared to those in cultures transfected with TFp-278. This suggests that although the presence of Egr-1, Sp-1, or yet to be identified cis-acting regulatory elements present within the -111 promoter is necessary for MPA regulation of the TF gene in HESCs, it is not sufficient to account for all of the effects on TF gene transcription. In addition, we cannot rule out changes in transport effected by MPA treatment of HESCs. To address this issue, future studies will be conducted employing site-directed mutagenesis of the promoter’s Sp-1 region, Egr-1 region, or both. As AP-1 and {kappa}B sites regulate expression of TF promoter in other cell types (9), it is possible that deletion of AP-1 and/or the NF{kappa}B-like elements between -278 and -111 bp was responsible for decreased levels of TF transcription for both E2-treated and E2- plus MPA-treated HESCs.

In contrast with the rapid induction of TF mRNA observed in endothelial, monocytic, or fibroblast cells, TF is chronically induced in HESCs. It is interesting to note that, to date, the TF promoter has not been shown to contain complete estrogen or progesterone response elements (22). We posit that MPA acting on the estrogen-primed HESCs is activating one or more intermediary agents responsible for the chronic induction of TF at the level of the gene. Results in this study using CHX are consistent with this hypothesis. Furthermore, it would appear that the same intermediate protein(s) is crucial to the process of decidualization itself, as reflected by the absence of decidualization-associated morphological changes in CHX-treated cultures. The HESC system employed in these studies is a valuable model for dissecting the regulation of hemostatic factors requisite for menstruation and implantation. These two events require that the system allow for the paradoxical maintenance of hemostasis during endovascular trophoblast invasion and the facilitation of the diffuse endometrial hemorrhage accompanying menstruation. Failure to properly regulate this system could result in infertility, spontaneous abortion, and placental abruption as well as dysfunctional uterine bleeding and breakthrough bleeding associated with hormonal contraception. Understanding the regulation of TF expression may provide insight into the development of new therapies in the attempt to target these prevalent disorders.


   Footnotes
 
1 This work was supported in part by NIH Grant R29-HD-29540–01A1 (to C.J.L.). Back

Received August 11, 1997.

Revised October 20, 1997.

Accepted November 18, 1997.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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M. E. Mendelsohn and R. H. Karas
The Protective Effects of Estrogen on the Cardiovascular System
N. Engl. J. Med., June 10, 1999; 340(23): 1801 - 1811.
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H. Okada, M. Sanezumi, T. Nakajima, S. Okada, K. Yasuda, and H. Kanzaki
Rapid down-regulation of CD63 transcription by progesterone in human endometrial stromal cells
Mol. Hum. Reprod., June 1, 1999; 5(6): 554 - 558.
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