Hypoxia Prevents Induction of Aromatase Expression in Human Trophoblast Cells in Culture: Potential Inhibitory Role of the Hypoxia-Inducible Transcription Factor Mash-2 (Mammalian Achaete-Scute Homologous Protein-2)

Bing Jiang, Amrita Kamat and Carole R. Mendelson

Departments of Biochemistry (B.J., A.K., C.R.M.) and Obstetrics & Gynecology (C.R.M.) The University of Texas Southwestern Medical Center at Dallas Dallas, Texas 75390-9038


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The human placenta has a remarkable capacity to aromatize C19-steroids, produced by the fetal adrenals, to estrogens. This reaction is catalyzed by aromatase P450 (P450arom), encoded by the CYP19 gene. In placenta, CYP19 gene expression is restricted to the syncytiotrophoblast layer. Cytotrophoblasts isolated from human placenta, when placed in monolayer culture in 20% O2, spontaneously fuse to form syncytiotrophoblast. These morphological changes are associated with a marked induction of aromatase activity and CYP19 gene expression. When cytotrophoblasts are cultured in an atmosphere containing 2% O2, they manifest increased rates of DNA synthesis and fail to fuse and form syncytiotrophoblast. The objective of the present study was to utilize cytotrophoblasts isolated from midterm human placenta to analyze the effects of O2 on CYP19 gene expression and the molecular mechanisms that mediate these effects. We observed that when trophoblast cells were maintained in 2% O2, there was only a modest induction of CYP19 expression as a function of time in culture, and aromatase activity was barely detectable. However, when cytotrophoblasts that had been maintained in 2% O2 for 3 days were placed in a 20% O2 environment, there was a rapid onset of cell fusion and induction of P450arom mRNA and aromatase activity. In addition, mRNAs for the helix-loop-helix factors Mash-2 (mammalian achaete-scute homologous protein-2) and Id1 (inhibitor of differentiation 1) were readily detectable in freshly isolated cytotrophoblasts and were markedly decreased upon differentiation to syncytiotrophoblast in 20% O2. By contrast, when cytotrophoblasts were cultured in 2% O2, mRNA levels for Mash-2 and Id1 remained elevated. Interestingly, overexpression of Mash-2 in primary cultures of human trophoblast cells markedly inhibited cell fusion and the spontaneous induction of P450arom mRNA levels and caused a marked decrease in expression of cotransfected fusion gene constructs containing either 125, 201, 246, or 501 bp of DNA flanking the 5'-end of the placenta-specific exon (exon I.1) of the human CYP19 gene linked to the human GH (hGH) structural gene, as reporter. In studies using BeWo, a human choriocarcinoma cell line, overexpression of Mash-2 also inhibited expression of cotransfected CYP19I.1:hGH fusion gene constructs. The findings that Mash-2 had no effect on the expression of a CYP19I.1-42:hGH fusion gene in primary cultures of human trophoblast and BeWo cells suggest that Mash-2 exerts its inhibitory effects directly or indirectly though CYP19I.1 5'-flanking sequences that lie between -42 and -125 bp. By contrast, neither Id1 nor Id2 had an effect on CYP19I.1 promoter activity in the transfected BeWo cells. These findings suggest that Mash-2 may serve as a hypoxia-induced transcription factor that prevents differentiation to syncytiotrophoblast and aromatase induction in human trophoblast cultured under low O2 conditions.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the human placenta, cytotrophoblasts play a critical role in implantation and in trophoblast development. Growth of the placenta is driven by the replication of mononuclear cytotrophoblasts, which is likely regulated by autocrine and paracrine factors (1). As the cytotrophoblasts mature, they stop dividing and fuse to form the terminally differentiated syncytiotrophoblast layer, which covers the chorionic villi and is bathed by maternal blood that perfuses the intervillous spaces (2). Throughout pregnancy, the syncytiotrophoblast layer is the major site for many placental functions required for maintenance of pregnancy and for fetal growth and development, including nutrient and gas exchange, and biosynthesis of steroid and polypeptide hormones (2).

The human placenta is unusual in its capacity to produce very large quantities of estrogen. Estrogens are formed by aromatization of C19-steroids secreted by the fetal adrenals (3). The aromatase reaction is catalyzed by an enzyme complex containing two polypeptides, the ubiquitous flavoprotein NADPHcytochrome P450 reductase, and a unique form of cytochrome P450 (P450arom), the product of the CYP19 gene, which is expressed exclusively in estrogen-producing cells (4, 5, 6).

Human CYP19 is a single-copy gene (7, 8, 9); the protein coding sequence is contained within nine exons (exons II–X) which span approximately 35 kb of DNA (7). CYP19 gene expression occurs at relatively high levels in the ovaries (10), testes (11, 12, 13), and in discrete nuclei within the brain (14) of vertebrate species from fish to man. On the other hand, humans also manifest relatively high levels of aromatase expression in stromal cells of adipose tissue (15), in fetal liver (16, 17), and in placenta, exclusively in the syncytiotrophoblast layer (18). Human P450arom mRNAs in ovary/testis, adipose, and placental cells have distinct 5'-termini encoded by alternative first exons that are spliced onto a common site that lies 36 bp upstream of the translation initiation codon (19). In placenta, the majority (>95%) of the P450arom mRNA transcripts contain sequences encoded by exon I.1 (20), which lies >=35,000 bp upstream of the translation initiation site in exon II.

Isolated human mononuclear cytotrophoblasts, when placed in culture in a 20% O2 environment, spontaneously aggregate and fuse to form nonproliferative, multinucleated syncytiotrophoblast, which produce a variety of polypeptide hormones and steroid metabolizing enzymes (1). We have observed that syncytiotrophoblast differentiation is associated with a rapid and dramatic induction of CYP19 gene expression (21). It is apparent that the differentiation of cytotrophoblasts to syncytiotrophoblast is associated with the generation of a cascade of regulatory signals; however, the molecular events that accompany this differentiation process are poorly understood. It has been suggested that biochemical differentiation can occur only after syncytial formation (22). On the other hand, others have reported that syncytial formation is not prerequisite to cellular differentiation, but is one of the consequences of the differentiation process (23).

Basic-helix-loop-helix (bHLH) transcription factors have been suggested to serve a role in placental differentiation (24). Interestingly, expression of the HLH factor, Id (inhibitor of differentiation), which lacks a basic DNA-binding domain (25), is elevated in trophoblast stem cells and declines during cell differentiation (26, 27). Similarly, expression of the gene encoding the bHLH transcription factor Mash-2 (mammalian achaete-scute homologous protein-2) (28), which is expressed at high levels in mouse trophoblasts, is diminished as trophoblasts differentiate into giant cells (29). It is not known, however, what role these HLH transcription factors may play in the regulation of CYP19 gene expression in the human placenta.

It has been reported that hypoxia stimulates cytotrophoblast proliferation (30, 31), impairs cell fusion (32), and decreases placental polypeptide hormone production (32, 33). Exposure of human placental villous explants to a low (2%) O2 environment was found to increase cytotrophoblast proliferation, decrease differentiation into invasive cells (34), and mimic the placental defect associated with preeclampsia (34, 35, 36). Preeclampsia, a disease characterized by placental hypoxia (37), is associated with reduced serum estradiol and increased progesterone levels (38, 39).

In the present study, we used midgestation human cytotrophoblasts in primary culture to analyze effects of atmospheric O2 on CYP19 gene expression. We observed that culture of cytotrophoblasts in 2% O2 blocked the induction of P450arom mRNA levels; this effect of low O2 was rapidly reversible. Additionally, differentiation of cytotrophoblasts to syncytiotrophoblast and induction of CYP19 gene expression in 20% O2 was accompanied by a marked decline in expression of the bHLH transcription factor Mash-2 and of Id1, which was prevented by culture of cytotrophoblasts in 2% O2. The finding that overexpression of Mash-2 in human trophoblast cells in primary culture markedly reduced syncytia formation and the spontaneous induction of CYP19 expression, as well as expression of cotransfected reporter gene constructs containing 5'-flanking sequences of CYP19 exon I.1, suggests that Mash-2 may serve as a hypoxia-inducible transcription factor that promotes human cytotrophoblast proliferation at the expense of differentiation.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Effects of Oxygen Tension on Differentiation and DNA Synthesis in Cultured Trophoblast Cells
When freshly isolated mononuclear cytotrophoblasts isolated from midgestation human placenta were placed in monolayer culture in a 20% O2 environment for 3 days, the cells aggregated and fused to form multinuclear syncytiotrophoblast (Fig. 1AGo). By contrast, when parallel dishes of cells were cultured in an environment of 2% O2, they aggregated, but failed to fuse (Fig. 1BGo). On the other hand, when cells that had been maintained in 2% O2 for 3 days were shifted to a 20% O2 environment, they fused to form a multinuclear syncytiotrophoblast (Fig. 1CGo). These findings support previous observations using term placental cells (32) and further indicate that the inhibitory effects of hypoxia on trophoblast fusion are reversible.



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Figure 1. Effects of Oxygen on Differentiation of Human Trophoblast Cells in Culture

Freshly isolated cytotrophoblasts were suspended in DMEM containing 2% FCS and plated at a density of 2 x 106 cells per dish. Cells were maintained either in an atmosphere of 20% O2 (panel A) or 2% O2 (panel B) for 3 days. After 3 days, some dishes were shifted from the 2% to the 20% O2 environment and cultured for an additional 3 days (panel C). In the studies shown in the lower panels, the trophoblast cells were incubated for 3 days in either 20% (panel D) or 2% (panel E) O2; BrdU (1 µM) was added during the last 24 h of culture. The cells were then fixed and incubated with a fluorescein-labeled antibody to BrdU, followed by fluorescence microscopy.

 
To analyze the effects of oxygen tension on DNA synthesis by cultured trophoblast, freshly isolated cytotrophoblast cells from midgestation human placenta were incubated for 72 h in a 20% or 2% O2-containing environment; bromodeoxyuridine (BrdU; 1 µM) was added to the medium during the last 24 h of culture. As can be seen in Fig. 1DGo, essentially no BrdU incorporation was detected in trophoblast cultured in 20% O2. By contrast, BrdU incorporation was readily detectable in cells cultured in a 2% O2 environment (Fig. 1EGo). When cells that had been maintained in 2% O2 for 3 days were shifted to 20% O2 and cultured for another 3 days, incorporation of BrdU added during the last 24 h of culture (between days 5 and 6) was essentially undetectable (not shown). These findings are correlated with those of [3H]thymidine incorporation, in which it was observed that incorporation of radiolabeled thymidine into DNA of trophoblast cells maintained in 2% O2 was 2.5-fold greater than in cells cultured in 20% O2 (data not shown). Our observations corroborate previous reports (35), which indicate that under hypoxic conditions, cytotrophoblast DNA synthesis and cell proliferation are increased.

Effects of Oxygen on Aromatase Activity and on P450arom mRNA levels in Human Trophoblast Cells in Culture
To evaluate the effects of O2 tension on aromatase expression, cytotrophoblasts from midgestation human placenta were cultured for up to 6 days in 2% or 20% O2. As observed previously, the midgestation cytotrophoblasts manifest barely detectable levels of aromatase activity (Fig. 2Go) and low or undetectable levels of P450arom mRNA (Fig. 3Go) (21). When the cells are cultured in a 20% O2 environment, aromatase activity (Fig. 2Go) and P450arom mRNA levels (Fig. 3Go) are rapidly induced, reaching peak levels within 3–4 days of culture (21). By contrast, when cytotrophoblasts were maintained in a 2% O2 environment, aromatase activity (Fig. 2Go) and P450arom mRNA levels (Fig. 3Go) remained low. On the other hand, when cells that had been cultured in 2% O2 for 3 days were shifted to a 20% O2 environment, aromatase activity was rapidly increased (Fig. 2Go); this was associated with an increase in levels of P450arom mRNA (Fig. 3Go). Conversely, when trophoblasts that had been cultured in 20% O2 for 3 days were transferred to a 2% O2 environment, aromatase activity declined rapidly (data not shown). It should be noted that the aromatization reaction requires molecular O2. To ensure that the assayed aromatase activity was a reflection of the effects of atmospheric O2 on CYP19 gene expression and on levels of P450arom protein and not merely due to availability of molecular O2, the 1-h incubations with [3H]androstenedione substrate were carried out in a 20% O2 environment regardless of the O2 tension of the prior incubation. Since both aromatase activity and P450arom mRNA levels were markedly reduced under hypoxic conditions, we consider it likely that the low aromatase activity was due to effects of hypoxia on CYP19 gene expression.



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Figure 2. Induction of Aromatase Activity in Human Trophoblast Cells in Culture Is Prevented by Incubation in a 2% O2 Environment

Freshly isolated cytotrophoblasts plated at a density of 2 x 106 cells per dish were maintained in either 2% or 20% O2 environment for up to 6 days. After 3 days, some dishes that had been maintained in 2% O2 were shifted to 20% O2 and cultured for 3 additional days. Aromatase activity (picomoles of androgen metabolized to estrogen/mg/min) was analyzed every 24 h by assaying the incorporation of tritium from [1ß-3H]androstenedione into water. Shown are the means ± SEM of data from three independent experiments each conducted in triplicate.

 


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Figure 3. Hypoxia Prevents Induction of CYP19 Gene Expression in Trophoblast Cells in Culture

Freshly isolated cytotrophoblasts were cultured in 2% or 20% O2 for up to 6 days. After 3 days, some dishes cultured in 2% O2 were shifted to the 20% O2 environment and cultured for another 3 days. Aliquots (20 µg) of total RNA obtained from cytotrophoblasts before and after culture under the different conditions were analyzed for P450arom mRNA transcripts by Northern blotting using a 32P-labeled human P450arom cDNA probe. This experiment was repeated three times with similar results. Shown is an autoradiogram of Northern blot from a representative experiment. The blot was reprobed for 28S rRNA as a control for RNA loading and transfer. In cells cultured for 3 and 6 days in 2% O2, P450arom mRNA levels were reduced by >85% as compared with normalized values for cells cultured for those periods in 20% O2.

 
Expression of the HLH Transcription Factors, Mash-2 and Id1 and Id2, Is Elevated in Freshly Isolated Cytotrophoblasts and Declines in Association with Syncytiotrophoblast Differentiation: Evidence for Induction by Hypoxia
To analyze changes in expression of the HLH transcription factors, Mash-2, Id1, and Id2, during trophoblast differentiation, as well as the effects of oxygen, RNA was isolated from cytotrophoblasts before culture and after 3 days of monolayer culture either in a 2% or 20% O2 environment. The levels of Mash-2 and Id2 mRNA transcripts were analyzed by Northern analysis of 3 µg poly(A)+ RNA, whereas the levels of Id1 mRNA were analyzed by Northern analysis of 20 µg of total RNA because of its relative abundance (Fig. 4Go). As can be seen, mRNA transcripts for Mash-2, Id1, and Id2 were detectable in cytotrophoblasts before culture. After 3 days of culture in a 20% O2 environment, the mRNA levels for all three HLH proteins declined, as compared with those in the cells before culture. On the other hand, when trophoblast cells were maintained in a 2% O2 environment, the levels of Mash-2 and Id1 were increased as compared with cells cultured in 20% O2, while Id2 mRNA declined to levels even lower than those observed in cells cultured in 20% O2 (Fig. 4Go). These findings suggest that expression of Mash-2 and Id1 are induced by hypoxia and may mediate the inhibitory effects of low oxygen on CYP19 gene expression.



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Figure 4. Effects of Low Oxygen on Expression of bHLH Transcription Factors

In the Northern blots shown on the left, poly(A)+ RNA (3 µg/lane) obtained from freshly isolated cytotrophoblasts (Cyto) and trophoblast cells cultured in atmospheres containing either 2% O2 or 20% O2 for 3 days were probed using 32P-labeled cDNAs encoding Mash-2, Id2, and ß-actin (as a control for loading and transfer). In cells cultured in 2% O2, the levels of Mash-2 mRNA were increased by 7.5-fold over the normalized values for cells cultured in 20% O2. In the Northern blots shown on the right, total RNA (20 µg/lane) obtained from freshly isolated cytotrophoblasts and trophoblast cells cultured in atmospheres containing either 2% O2 or 20% O2 for 3 days were analyzed using 32P-labeled probes for Id1 and 28S rRNA (control for RNA loading and transfer). This experiment was repeated twice with similar results. Shown is a representative Northern blot.

 
Effects of Mash-2, Id1, and Id2 on CYP19 Promoter Activity in Transfected Choriocarcinoma Cells
To analyze effects of Mash-2, Id1, and Id2 on CYP19 promoter activity, BeWo choriocarcinoma cells were transfected with fusion genes containing either 42 (basal promoter), 125, 201, 246, or 501 bp of DNA flanking the 5'-end of placenta-specific exon I.1 of the human CYP19 gene, linked to the hGH structural gene, as reporter. In previous studies, we observed that the 125, 246, and 501 bp-containing fusion genes were expressed at relatively high levels in human trophoblast cells in primary culture, whereas levels of expression of the 42 bp-containing construct were barely detectable (21). In the present study, the choriocarcinoma cells were cotransfected either with empty pCMV expression vectors, or with pCMV expression vectors containing Mash-2, Id1, or Id2. Mash-2 markedly inhibited expression of the -125, -201, -246, and -501 bp-containing fusion genes after 72 h of incubation, as well as at earlier time points (data not shown), whereas, no effects on the basal promoter construct (Fig. 5Go) or on the promoterless hGH-containing plasmid (pACsk2.0GH) (data not shown) were observed. Furthermore, Id1 and Id2 had only a modest effect to inhibit expression of the CYP19I.1-501:hGH fusion gene (Fig. 5Go). These findings suggest that the increased levels of Mash-2 expression observed under hypoxic conditions may block induction of CYP19I.1 promoter activity in trophoblast cells either directly or indirectly through exon I.1 5'-flanking sequences between -42 and -125 bp.



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Figure 5. Effects of Overexpression of the HLH Factors Mash-2, Id1, and Id2 on Expression of Cotransfected Human CYP19I.1:hGH Fusion Genes in Human BeWo Choriocarcinoma Cells

BeWo cells were transfected with either CYP19I.1-42:hGH, CYP19I.1-125:hGH, CYP19I.1–201:hGH, CYP19I.1-246:hGH, or CYP19I.1-501:hGH fusion gene constructs (4.5 µg each). The cells were cotransfected either with empty expression plasmid or with expression plasmid containing a cDNA encoding Mash-2 (2.0 µg). In parallel, BeWo cells transfected with the CYP19I.1-501:hGH fusion gene construct (4.5 µg) were cotransfected either with Id1, or Id2 cDNAs (2.0 µg each). Each dish also was transfected with ß-galactosidase expression vector (0.5 µg). After 72 h of incubation, the media were collected for assay of hGH and of ß-galactosidase activity. ß-Galactosidase values were used to correct for transfection efficiency. Shown are the corrected means ± SEM of data from three independent experiments each conducted in triplicate.

 
Effects of Mash-2 on Syncytiotrophoblast Differentiation and CYP19 Gene Expression in Human Trophoblast in Primary Culture
To determine the effects of overexpression of Mash-2 on trophoblast differentiation and CYP19 gene expression, we generated recombinant adenoviruses containing CMV/Mash-2 and used these to infect human cytotrophoblast cells maintained in a 20% O2 environment. Freshly isolated cytotrophoblasts, plated at a density of 2 x 106 cells per 35-mm dish in 2% FBS, were infected with the recombinant adenoviruses containing either CMV/Mash-2 or CMV/ß-gal at a multiplicity of infection (m.o.i.) of 5.0. After 72 h of culture, the cells were stained with hematoxylin and eosin and viewed by light microscopy. When cells were incubated either in the absence of recombinant adenovirus (Fig. 6AGo) or in the presence of adenovirus containing CMV/ß-gal at a m.o.i. of 5.0 (Fig. 6BGo), there was clear evidence of syncytia formation. By contrast, when the cells were incubated with adenovirus containing CMV/Mash-2 (m.o.i. = 5.0), syncytia formation was markedly inhibited (Fig. 6CGo).



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Figure 6. Mash-2 Inhibits Differentiation of Human Trophoblasts in Primary Culture

Freshly isolated human cytotrophoblasts plated on 35-mm dishes at a density of 2 x 106 cells per dish were infected with recombinant adenoviruses containing either CMV/Mash-2 or CMV/ß-gal at a m.o.i. of 5.0. Some dishes were not infected. After 72 h of incubation, the cells were stained with hematoxylin and eosin and viewed by light microscopy. Shown are micrographs (x200) of uninfected cells (panel A), cells infected with CMV/ß-gal (panel B) or CMV/Mash-2 (panel C). This experiment was repeated three times with similar results.

 
When CYP19 gene expression in the virus-infected primary human trophoblast cells was analyzed by Northern analysis of P450arom mRNA, we observed that CMV/Mash-2 caused a dose-dependent inhibition of P450arom mRNA induction. An inhibitory effect of CMV/Mash-2 was observed at a m.o.i. as low as 0.5. On the other hand, no effect on P450arom mRNA induction was observed in cells infected with CMV/ß-gal at comparable m.o.i. (Fig. 7Go). It should be noted that the levels of P450arom mRNA in trophoblast cells infected with Mash-2-expressing adenovirus were similar to those of cells cultured for 72 h in a 2% O2-containing environment.



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Figure 7. Overexpression of Mash-2 Inhibits the Induction of P450arom Gene Expression in Trophoblast Cells in Culture

Freshly isolated cytotrophoblasts were infected by overnight incubation with recombinant adenoviruses expressing CMV/Mash-2 or CMV/ß-gal (m.o.i. = 0.5–5.0). After 72 h of further incubation in a 20% oxygen-containing environment, RNA was isolated and analyzed for mRNAs encoding P450arom, Mash-2, and ß-actin (as an index of loading and transfer). RNA also was isolated from an aliquot of the cytotrophoblast cells before culture (Cyto) and from uninfected trophoblast after culture for 72 h in a 20% O2- (Syn) or 2% O2- (2% O2) containing environment. Aliquots (20 µg) of total RNA obtained from cytotrophoblasts before and after culture under the different conditions were analyzed by Northern blotting using 32P-labeled cDNAs encoding human P450arom, rat Mash-2, and human ß-actin. Shown is an autoradiogram of a representative Northern blot of an experiment that was repeated three times with comparable results. In trophoblasts infected with Mash-2 expressing adenovirus at a m.o.i. = 5.0, P450arom mRNA levels were reduced by 85% as compared with corrected values in cells infected with comparable amounts of adenovirus expressing CMV/ß-gal.

 
To determine the effects of Mash-2 on CYP19I.1 promoter activity in human trophoblast cells in primary culture, freshly isolated cytotrophoblasts were coinfected with recombinant adenoviruses containing CYP19I.1-501:hGH (m.o.i. = 0.5) alone or in combination with either CMV/Mash-2 or with CMV/ß-gal (as control) at a m.o.i. of 1.0, 5.0, 10.0, or 20.0. As can be seen, coinfection with Mash-2-expressing adenovirus caused a dose-dependent inhibition of CYP19I.1 promoter activity; pronounced inhibition was found at m.o.i. values >=5.0. Only a modest inhibitory effect on CYP19I.1-501:hGH expression was observed upon infection of control CMV/ß-gal expressing adenovirus at a m.o.i. as high as 20 (Fig. 8AGo).



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Figure 8. Mash-2 Causes a Dose-Dependent Inhibition of Expression of Cotransfected CYP19I.1:hGH Fusion Genes in Human Trophoblast Cells in Primary Culture

A, Freshly isolated human trophoblast cells in primary culture were plated at a density of 2 x 106 cells per 35-mm dish and infected overnight with recombinant adenovirus containing the CYP19I.1-501:hGH fusion gene (m.o.i. = 0.5) in the absence (Control) or presence of coinfection with recombinant adenoviruses containing either CMV/ß-gal or CMV/Mash-2 at m.o.i. values of 1, 5, 10, or 20. Media were collected and changed daily for a 72-h period. Shown is the accumulation of hGH in the media between 48 and 72 h of incubation. Data are the means ± SEM of triplicate values from a single experiment. B, The freshly isolated trophoblast cells were coinfected with recombinant adenoviruses containing either CYP19I.1-42:hGH, CYP19I.1-125:hGH, CYP19I.1–201:hGH, CYP19I.1-246:hGH, or CYP19I.1-501:hGH fusion genes (m.o.i. = 0.5) and with recombinant adenoviruses containing CMV/ß-gal (m.o.i. = 10.0) or CMV/Mash-2 (m.o.i. = 10.0). Culture media were changed daily. After 72 h of incubation, the media were assayed for hGH. Shown is the accumulation of hGH in the media between 48 and 72 h of incubation. Data are the means ± SEM of triplicate values from a single experiment.

 
To define the CYP19I.1 5'-flanking sequences that mediate the inhibitory effects of Mash-2 on CYP19 promoter I.1 activity in primary human trophoblast cells, freshly isolated cytotrophoblasts were coinfected with recombinant adenoviruses containing CYP19I.1:hGH fusion genes comprised of 42, 125, 201, 246, and 501 bp of exon I.1 5'-flanking sequence (m.o.i. = 0.5) and recombinant adenoviruses containing either CMV/ß-gal or CMV/Mash-2 (m.o.i. = 10.0). The relative levels of expression of the various fusion gene constructs in the trophoblast cells were similar to that reported previously (21); the CYP19I.1-246:hGH fusion gene was expressed at the highest level, while expression of the basal promoter construct (CYP19I.1-42:hGH) was barely detectable. In those studies, we had observed that, whereas expression of the CYP19I.1-501:hGH fusion gene was specific for the primary cultures of human placental cells, the CYP19I.1-125:hGH, CYP19I.1-201:hGH, and CYP19I.1-246:hGH fusion genes also were expressed at relatively high levels in lung and kidney cell lines (21). As we had observed in the transfected BeWo cells (Fig. 5Go), Mash-2 had a pronounced inhibitory effect on expression of all CYP19I.1:hGH fusion genes except the basal promoter construct (Fig. 8BGo).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
When cytotrophoblasts from midgestation or term human placenta are maintained in primary monolayer culture in a 20% O2 environment, they rapidly fuse to form syncytia (21, 40). These morphological changes are temporally associated with a marked induction of aromatase activity and CYP19 gene expression (21). On the other hand, when cytotrophoblasts are cultured in a hypoxic environment, cell fusion is prevented (32). In the present study, we observed that the induction of aromatase activity and CYP19 expression that occurs in association with spontaneous differentiation of human cytotrophoblasts to syncytiotrophoblast in culture is oxygen dependent. When trophoblasts were incubated in a 2% O2 environment, induction of aromatase activity and of CYP19 gene expression were prevented in association with an inhibition of cell fusion and increased incorporation of BrdU into DNA. These effects of hypoxia on aromatase expression, DNA synthesis, and syncytia formation were rapidly reversed when the trophoblasts were transferred to a 20% O2 environment.

Oxygen has been proposed to serve an important role in the modulation of trophoblast differentiation vs. proliferation (32, 34). During human placental development, cytotrophoblast stem cells follow two alternate pathways of differentiation (41). Along one pathway, cytotrophoblasts detach from their basement membranes and aggregate to form columns. The cells within these columns rapidly proliferate and invade the uterine endometrium, part of the myometrium, and spiral arterioles, which are remodeled and enlarged. In the other pathway of differentiation, cytotrophoblasts within the floating chorionic villi also detach from their basement membranes and fuse to form multinucleated syncytiotrophoblast, which are bathed in maternal blood. The remodeling of the spiral arterioles results in a pronounced increase in blood flow to the intervillous space and concomitant increase in O2 availability to syncytiotrophoblast cells of the floating chorionic villi, which produce polypeptide hormones, as well as steroid hormones and steroid-metabolizing enzymes, including aromatase P450 (35). Interestingly, estrogen biosynthesis by the human placenta increases markedly after the ninth week of gestation (3), coinciding with the time that cytotrophoblast invasion, remodeling, and enlargement of the uterine arterioles are initiated. The findings of the present study suggest that placental vascularization and increased O2 availability are critical for the increase in placental estrogen production during pregnancy. In the pregnancy-associated disease preeclampsia, in which the placenta is relatively hypoxic, cytotrophoblast proliferation is increased and differentiation into invasive cells is reduced. Thus, fewer arterioles are invaded and O2 availability to the hormone-producing syncytiotrophoblast is reduced (37). Such pregnancies are frequently associated with increased synthesis of progesterone, decreased synthesis of estrogens and insulin-like growth factor-1, as well as fetal growth retardation (39). In a recent epidemiological study, it was found that women who either were offspring of a preeclamptic pregnancy, or who experienced preeclampsia in their own pregnancies, have a reduced breast cancer risk (39).

HLH transcription factors have been suggested to serve important roles in trophoblast differentiation (27). This is of particular interest in consideration of the role of bHLH transcription factors, including MyoD, myogenin, myf-5, and mrf-4, in the differentiation of skeletal myoblasts to form differentiated multinucleated myotubes (42). These skeletal muscle-specific transcription factors bind to so-called E-box sequences in DNA as obligate heterodimers with the ubiquitously expressed E-factors E12 or E47 (43, 44). On the other hand, the HLH factor Id (inhibitor of differentiation), which lacks a basic DNA-binding domain, acts as a negative regulator of differentiation because of its ability to sequester E12 and E47, preventing their interaction with and DNA binding of muscle-specific bHLH proteins (25). In the present study, we observed that mRNAs for the bHLH factors Mash-2, Id1, and Id2 were readily detectable in freshly isolated cytotrophoblasts from midgestation human placenta and were markedly decreased upon differentiation to syncytiotrophoblast in 20% O2. By contrast, when cytotrophoblasts were cultured in 2% O2, mRNA levels for Mash-2 and Id1 remained elevated.

Intriguingly, in the present study we observed that overexpression of Mash-2 in primary cultures of human trophoblast cells mimicked the effects of culture in low (2%) O2, in that Mash-2 prevented cell fusion and markedly inhibited the induction of expression of the endogenous CYP19 gene. Both in BeWo choriocarcinoma cells and in human trophoblast cells in primary culture transfected with CYP19I.1:hGH reporter gene constructs, cotransfection of CMV/Mash-2 markedly reduced expression of fusion genes containing 125, 201, 246, or 501 bp of CYP19 exon I.1 5'-flanking sequences; however, no effect of Mash-2 was observed on expression of a basal promoter construct (CYP19I.1-42:hGH). Expression levels of CYP19I.1:hGH fusion genes containing 125–501 bp of CYP19 exon I.1 5'-flanking DNA also were reduced upon culture in 2%, as compared with 20% O2 (our unpublished observations). In contrast to our findings with Mash-2, overexpression of Id1 or Id2 in the cotransfected BeWo cells had little effect on CYP19 promoter I.1 activity. The lack of effect of Id1 and Id2 on CYP19 promoter expression may possibly be explained by the recent finding that E12 and E47 mRNAs are undetectable in human trophoblasts before or after differentiation in culture (41).

Mash-2 protein was reported to bind to an E-box of a muscle-specific enhancer as a heterodimer with E12 (45). Our findings in deletion mapping studies using human trophoblast in primary culture, as well as BeWo cells, suggest that a Mash-2-responsive element(s) may be localized between -42 and -125 bp. This 83-bp region contains an E-box consensus sequence that could possibly serve as a binding site for Mash-2 or another bHLH transcription factor with which Mash-2 interacts. However, mutation of this E-box in the context of a CYP19I.1:hGH fusion gene containing 246 bp of exon I.1 5'-flanking region had no effect on basal expression or on the action of Mash-2 to inhibit fusion gene expression. Additionally, in electrophoretic mobility shift assays utilizing in vitro transcribed/translated Mash-2 in the absence or presence of coexpressed E12, we were unable to demonstrate direct binding of Mash-2 to this site (our unpublished observations). As noted above, E12 and E47 are not expressed in human trophoblast; thus, Mash-2 may interact with and prevent the binding and/or activity of a stimulatory transcription factor(s) that binds to this region. On the other hand, Mash-2 may not bind to a response element within the 5'-flanking sequence of the CYP19 gene, but may rather block the program of trophoblast differentiation and prevent the expression of one or more stimulatory transcription factors that bind to response elements upstream of promoter I.1 and are essential for developmental induction of CYP19 gene expression.

In addition to its effects on CYP19 gene expression in the human trophoblast, Mash-2 likely affects other processes involved in trophoblast differentiation. In mice, targeted mutation of the mash-2 gene resulted in death of embryos at 10 days post coitum due to placental failure (29). The mutant placentas lacked spongiotrophoblasts and had an increased number of giant cells (nonproliferative differentiated cells) (29). It is apparent that Mash-2 is required for generation and maintenance of spongiotrophoblast precursors and could possibly block differentiation of giant cells. Mash-2 expression also was found to be dramatically decreased during differentiation of a rat choriocarcinoma cell line (RCHO-1 cells), into giant cells in culture (27).

Unlike the human, the mouse placenta does not synthesize estrogens or express P450arom (46). However, in studies using transgenic mice, we found that human CYP19I.1-501:hGH fusion genes were highly expressed in a developmental and placenta-specific manner, exclusively within the labyrinthine trophoblast (47). The labyrinthine layer contains syncytial cells that are analogous to the syncytiotrophoblast of human placenta. These findings suggest that the transcription factors that mediate placenta-specific expression of the CYP19 gene are highly conserved among species, although the response elements that bind these factors within the mouse cyp19 gene are not. Moreover, the activating transcription factors are likely restricted to the mouse labyrinthine trophoblast, further suggesting its analogy to the human syncytiotrophoblast. Interestingly, transgene expression is first detected in the mouse placenta at E10.5, a time when Mash-2 expression is greatly diminished (48). The decrease in endogenous Mash-2 expression in association with elevated expression of the CYP19I.1-501:hGH transgene suggests that the human homolog of this transcription factor may normally block CYP19 gene expression in proliferating cytotrophoblasts.

The findings presented herein, therefore, suggest for the first time that Mash-2 may function as a "hypoxia-induced" transcription factor that blocks differentiative function of the syncytiotrophoblast layer. A number of HLH transcription factors that contain a PAS (per-ARNT-Sim) domain have been implicated as mediators of the hypoxic response. These include hypoxia-inducible factor (HIF)-1{alpha}, HIF-1ß (ARNT), and human endothelial PAS protein-1 (EPAS-1). HIF-1{alpha} and EPAS-1 protein levels have been found to be markedly increased during hypoxia and to increase transcription of a number of hypoxia-induced genes (49). Surprisingly, expression levels of both HIF-1{alpha} and ARNT were found to be markedly increased during human trophoblast differentiation in 20% O2, while EPAS-1 expression remained relatively unchanged (41). When the cells were cultured under hypoxic conditions, the levels of HIF-1{alpha} and ARNT decreased slightly, while EPAS-1 levels were relatively unaffected (41). Since HIF-1{alpha} (50) and EPAS-1 (51) are primarily regulated at the level of protein stability, their roles as hypoxia-regulated transcription factors in trophoblast differentiation remain to be defined.

In summary, we have found that hypoxia prevents the induction of CYP19 gene expression that occurs in association with differentiation of human trophoblast cells in culture. Culture of trophoblast cells in a hypoxic environment prevented the decline in Mash-2 expression that normally occurs in association with human cytotrophoblast differentiation. The finding that Mash-2 overexpression inhibited fusion of cytotrophoblasts to form syncytiotrophoblast and the associated induction of endogenous CYP19 gene expression, and markedly reduced expression CYP19 promoter activity in transfected placental cells, suggests that Mash-2 may play an important role as a hypoxia-inducible transcription factor in human trophoblast. We suggest that with increased vascularization of the placenta and increased oxygen availability to the cytotrophoblast cells of the chorionic villi, there is decreased expression of Mash-2. This removes the brakes on the cascade of cellular differentiation and placental hormone production. Studies are in progress to define the mechanisms whereby Mash-2 acts to regulate syncytiotrophoblast differentiation and the induction of CYP19 gene expression.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Primary Culture of Human Trophoblast Cells and Maintenance of Choriocarcinoma Cell Lines
Midtrimester human placental tissues were obtained in accordance with the Donors Anatomical Gift Act of the State of Texas after written consent had been obtained. In all cases, consent forms and protocols were approved by the Human Research Review Committee of the University of Texas Southwestern Medical Center at Dallas. A placental primary culture system developed by Kliman et al. (40) was modified for isolation and culture of cytotrophoblasts from midgestation human placenta (21). The placental tissues were washed with HBSS, pH 7.4 (Life Technologies, Inc., Gaithersburg, MD), and then finely minced and digested with 0.125% trypsin in HBSS at 37 C for 30 min. This procedure was repeated three times. At the end of each digestion step, the supernatant was collected, layered over 10 ml serum, and then briefly centrifuged. The pellet was suspended in DMEM (Life Technologies, Inc.), filtered, and then layered over a Percoll gradient (70%–5%). The gradients were centrifuged at 1,200 x g for 20 min at room temperature, and cells in the middle layer (density 1.045–1.062) were collected, washed, and counted. The cells were resuspended in DMEM supplemented with 10% FBS and 1% antibiotic/antimycotic solution (Life Technologies, Inc.) and plated at a density of 2 x 106 cells per dish in 35-mm culture dishes coated with extracellular matrix (ECM). The cells were then incubated at 37 C in a humidified atmosphere of either 95% air/5% CO2 (20% O2) or placed in a modular incubator chamber (Billups-Rothenberg, Inc., Del Mar, CA) in which they were maintained in an atmosphere containing 2% O2, 93% N2, 5% CO2. Cells were cultured overnight and the medium was then changed to DMEM containing 2% FBS.

The ECM-coated dishes were prepared from confluent monolayers of Madin-Darby canine kidney cells (ATCC CRL 6253) (2–5 x 106 cells per dish) that were treated with 0.5% deoxycholate for 5 min. The ECM dishes were washed three times with HBSS and stored at 37 C until used. BeWo cells were purchased from ATCC and grown in F-12K medium (Life Technologies, Inc.) containing 15% FBS.

Tritiated Water Assay of Aromatase Activity in Placental Cells
Aromatase activity was assayed in placental cells in monolayer culture using a tritiated water assay as described previously (52). The [1ß-3H]androstenedione (NEN Life Science Products, Boston, MA) was added to the culture medium for 1 h. Culture medium was then removed and placed in ice-cold 30% (wt/vol) trichloroacetic acid. The incorporation of tritium from [1ß-3H]androstenedione into water was assayed in aqueous scintillation fluid after extraction with 4 volumes of chloroform and 1 volume of dextran-charcoal suspension. Aromatase activity is expressed as picomoles of [1ß-3H]androstenedione metabolized to estrogen/min/mg protein. The adherent cells were analyzed for protein by the method of Lowry et al. (53).

Northern Analysis of Placental Cell mRNAs
Total RNA was isolated from placental cells by the method of Chirgwin et al. (54). Briefly, cells in monolayer culture were washed with PBS, lysed in 4 M guanidinium isothiocyanate, followed by centrifugation for 16 h at 42,000 rpm through a cesium chloride cushion (5.7 M). The RNA samples were then ethanol precipitated, resuspended in water, and quantitated at 260 nm. Cytotrophoblast and syncytiotrophoblast polyadenylated RNA was isolated using Poly(A) Quick mRNA Isolation Kit (Stratagene, La Jolla, CA) following the manufacturer’s protocol. Total RNA (20 µg) or poly (A)+ RNA (3 µg) was size-fractionated on a 7.4% formaldehyde/0.9% agarose gel, transferred to Zeta-Probe Blotting Membrane (Bio-Rad Laboratories, Inc. Hercules, CA), and then hybridized overnight at 65 C to a 32P-labeled human aromatase cDNA probe (55) made by Prime-It RmT Random Primer Labeling Kit (Stratagene). A 1.5-kb rat Mash-2 cDNA insert (kindly provided by Dr. Jane Johnson, University of Texas Southwestern) and cDNA inserts (1.1 kb) for human Id1 and Id2 (kindly provided by Dr. Judith Campisi, University of California, Berkeley) were used as probes for detecting mRNAs encoding the human homolog of Mash-2, Id1, and Id2 expression, respectively. The membrane was then washed in 0.1% SDS-0.1x NaCl-sodium citrate (SSC) solution, and the relative levels of mRNA were assessed by autoradiography. Northern blots were reprobed with ß-actin cDNA (ATCC, Manassas, VA) or 28S rRNA to assess loading and transfer of RNA. Autoradiograms were scanned by densitometry, and relative levels of specific mRNA were normalized either to ß-actin mRNA or 28S rRNA to correct for RNA loading and transfer.

Morphological Analysis
Cells were cultured on glass coverslips in DMEM containing 2% FBS in atmospheres of 20% or 2% O2, as described above. After culture, the cells were rinsed with PBS and fixed in 75% ethanol. Hematoxylin and eosin Y were used to stain nuclei and cytoplasm, respectively. Morphology was analyzed by light microscopy.

BrdU Incorporation
Cells were cultured on glass coverslips in DMEM containing 2% FBS in atmospheres of 20% or 2% O2. After 48 h, the culture medium was aspirated, and fresh medium containing 1 µM BrdU (Sigma, St. Louis, MO) was added (34). After 24 h of further incubation, the cells were rinsed with PBS and fixed in absolute methanol at 4 C. Cellular DNA was denatured by incubating the coverslips in 2 M HCl for 60 min at 37 C. The acid was neutralized by immersing the coverslips in 0.1 M borate buffer, pH 8.5. The cells were then incubated with anti-BrdU-fluorescein (Roche Molecular Biochemicals, Indianapolis, IN). Cells were counterstained with hematoxylin. The coverslips were then dehydrated and mounted on slides.

Plasmid Constructs
A cDNA containing the protein coding sequence of rat Mash-2 (783 bp) (provided by Dr. Jane Johnson, University of Texas Southwestern) was subcloned into the multiple cloning site of the expression plasmid pCMV5 (provided by Dr. David W. Russell, University of Texas Southwestern). pCMV1-Id1 and pCMV1-Id2 were provided by Dr. Judith Campisi (University of California, Berkley). Fusion genes comprised of 501, 246, 201, 125, and 42 bp of 5'-flanking sequence and 103 bp of untranslated exon I.1 of the human CYP19 gene fused to the human GH structural gene (hGH) as reporter were constructed as described previously (21).

Cell Transfection Assays
Some of the cotransfection assays were carried out using BeWo choriocarcinoma cells, since human trophoblast cells in primary culture cannot be transfected with plasmid DNA using standard techniques (21). The day before transfection, 4 x 105 cells were plated onto 60-mm culture dishes and maintained in 4 ml of growth medium overnight. All cells were transfected with the same amount of plasmid DNA (2 µg reporter plasmid, 2 µg expression plasmid or corresponding empty expression vector, and 0.5 µg ß-gal plasmid). The plasmid DNAs, to be added to each culture dish, were combined in 150 µl of F-12K medium that was devoid of FBS or antibiotics. Thirty microliters of SuperFect transfection reagent (QIAGEN, Valencia, CA) were added to each tube containing plasmid DNA, and the samples were incubated for 5–10 min to allow complex formation. One milliliter of cell growth medium containing serum and antibiotics was added to each reaction tube containing the plasmid DNA and transfection reagent; the total content of each tube was then added to each dish of cultured cells. Cells were then incubated for 3 h at 37 C in an atmosphere of 95% air/5% CO2. The media were then removed by gentle aspiration, and the cells were washed once with 4 ml of PBS. F-12K medium containing 15% FBS (2 ml) was added to each dish. Media were then collected every 24-h for RIA of hGH using a kit (Nichols Institute Diagnostics, San Juan Capistrano, CA). ß-Galactosidase assays were performed using Galato-light kit (Tropix, Inc., Bedford, MA) for normalization of transfection efficiency.

Preparation of Recombinant Adenovirus and Infection of Placental Cells
To generate a Mash-2 recombinant adenovirus, we used a protocol involving in vivo recombination in bacteria (56). In short, the coding sequence of the rat Mash-2 cDNA was subcloned into the pShuttle vector containing a subcloned CMV promoter to generate pShuttle-CMV/Mash-2. The recombinant adenoviral CMV/Mash-2 was generated by cotransformation of pShuttle-CMV/Mash-2 and pAdEasy-1 into electrocompetent BJ5183 bacteria. 293 cells, a permissive human embryonic kidney cell line that expresses adenoviral E1A, were transfected with PacI-digested recombinant adenoviral CMV/Mash-2 DNA for recombinant adenoviral packaging and propagation. Viral plaques were isolated and propagated to produce a lysate containing infectious recombinant virus. Viral DNA was analyzed to confirm the presence of CMV/Mash-2 by restriction endonuclease digestion, PCR, and DNA sequencing (ABI 377 automated sequencer). The recombinant virus was then titered in 293 cells three times to determine the number of infectious particles (plaque-forming units). CMV-ß-gal adenovirus was kindly provided by Dr. Joseph Alcorn (University of Texas Medical School, Houston). Recombinant adenoviruses containing fusion genes comprised of 42, 125, 201, 246, and 501 bp of sequence flanking the 5'-end of exon I.1 of the human CYP19 gene and 103 bp of exon I.1 fused upstream of the hGH structural gene, as reporter, were constructed as described previously (21).

Freshly isolated cytotrophoblasts plated at a density of 2 x 106 cells per 35-mm dish in DMEM containing 10% FBS were infected with recombinant adenoviral particles at a m.o.i. of 0.5–20.0. After an overnight incubation, the media were removed and replaced with fresh DMEM containing 2% FBS. The cells were incubated for up to 72 h; media were collected and replaced daily and analyzed for hGH by RIA.


    ACKNOWLEDGMENTS
 
The authors gratefully acknowledge the technical expertise of Vickey Chau, Margaret Smith, and Barbara Murry, and thank Drs. Margaret Hinshelwood and Linda Margraf for their helpful discussions.


    FOOTNOTES
 
Address requests for reprints to: Carole R. Mendelson, Ph.D., Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9038. E-mail: cmende{at}biochem.swmed.edu

This research was supported by NIH Grant R01 DK-31206.

Received for publication January 3, 2000. Revision received June 9, 2000. Accepted for publication July 7, 2000.


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