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Endocrinology Vol. 138, No. 3 898-904
Copyright © 1997 by The Endocrine Society

Articles

Retinoic Acids Increase 17ß-Hydroxysteroid Dehydrogenase Type 1 Expression in JEG-3 and T47D Cells, but the Stimulation Is Potentiated by Epidermal Growth Factor, 12-O-Tetradecanoylphorbol-13-Acetate, and Cyclic Adenosine 3',5'-Monophosphate Only in JEG-3 Cells1

Yun-shang Piao, Hellevi Peltoketo, Annukka Jouppila and Reijo Vihko

Biocenter Oulu and Department of Clinical Chemistry, University of Oulu, Oulu, Finland

Address all correspondence and requests for reprints to: Dr. Hellevi Peltoketo, Biocenter Oulu, and Department of Clinical Chemistry, University of Oulu, Kajaanintie 50, FIN-90220 Oulu, Finland. E-mail: hpeltoke{at}whoccr.oulu.fi


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human 17ß-hydroxysteroid dehydrogenase type 1 (17HSD type 1) primarily catalyzes the reduction of low activity estrone to high activity estradiol in ovarian granulosa cells and placental trophoblasts. 17HSD type 1 is also present in certain peripheral tissues, such as breast tissue. In the present study we investigated the effects of retinoic acids (RAs) together with other stimuli known to modulate estradiol production and/or cell growth on expression of 17HSD type 1 in JEG-3 choriocarcinoma cells and estrogen-responsive T47D breast cancer cells. Treatment of cultured JEG-3 and T47D cells with all-trans-RA and 9-cis-RA increased reductive 17HSD activity and 17HSD type 1 messenger RNA expression severalfold in both cell lines. On the other hand, epidermal growth factor (EGF), Ca ionophore, the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA), and cAMP elevated 17HSD type 1 expression only in JEG-3 cells. Correspondingly, the effects of RAs were potentiated by EGF, TPA, and cAMP in JEG-3 cells, whereas no such phenomenon was observed in T47D cells. In JEG-3 cells, simultaneous administration of RAs with TPA and EGF maximally resulted in approximately 40- and 20-fold increases in 17HSD type 1 messenger RNA expression, respectively. The present data indicate that RAs may stimulate estradiol biosynthesis by regulating 17HSD type 1 expression in certain breast cancer and choriocarcinoma cells. The results suggest that interaction of multiple regulatory pathways is involved in maintaining high 17HSD type 1 expression in the placenta. In addition, regulation of 17HSD type 1 expression may be different in trophoblast cells from that in breast epithelial cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PLACENTA plays a vital role in the maintenance and development of pregnancy by strongly influencing the endocrine milieu of the feto-maternal unit. Among the several endocrine alterations is estrogen production, which greatly increases during pregnancy. After the first 7 weeks of gestation, nearly all estrogens produced are synthesized in the placenta from fetal precursors (1). In placental syncytiotrophoblasts, P450aromatase and 17ß-hydroxysteroid dehydrogenase (17HSD) activities convert dehydroepiandrosterone and dehydroepiandrosterone sulfate, derived from fetal sources, to estrone (E1) and estradiol (E2) (2, 3). In addition, 16{alpha}-hydroxydehydroepiandrosterone is abundantly converted into estriol during gestation.

In the human placenta, aromatization of androgens and reduction of low activity E1 into E2 are catalyzed by P450aromatase and 17HSD type 1, as in ovarian granulosa cells (4, 5). In addition to 17HSD type 1, the human placenta expresses 17HSD type 2 (6, 7, 8), which catalyzes oxidation of E2 to E1, the opposite reaction of that catalyzed by the type 1 enzyme. The type 2 enzyme also possesses androgenic 17HSD and 20{alpha}-hydroxysteroid dehydrogenase activities (7). The activity of both of the 17HSD enzymes increases steadily up to the 20th week of pregnancy, but the activity of the type 1 enzyme is constantly considerably greater than that of the type 2 enzyme (6). On the other hand, the human placenta does not express detectable amounts of 17HSD type 3, the enzyme mainly converting androstenedione to testosterone in the testis (9). This suggests that fetal C19 precursors are predominantly aromatized to E1 and then further catalyzed to E2, rather than being converted to T and then aromatized to E2. Together, the data point to the importance of 17HSD type 1 in placental E2 production.

The factors and mechanisms maintaining high 17HSD type 1 expression in placental syncytiotrophoblasts are largely unknown. In T47D breast cancer cells, retinoic acids (RAs), derivatives of vitamin A, increase 17HSD type 1 messenger RNA (mRNA) expression (10). The 17HSD type 1 gene, HSD17B1 (previously also called EDH17B2) contains a functional RA receptor-binding site (11). Nutritional signals, such as RAs, have also been suggested to regulate placental trophoblast cell function (12), and RAs have been demonstrated to affect regulation of the biosynthesis of several essential placental products, such as progesterone, hCG subunits, and human placental lactogen (12, 13, 14). In the present study, we investigated the effects of RAs on 17HSD type 1 expression in JEG-3 choriocarcinoma cells. This syncytiotrophoblast-like cell line has retained the capability of producing E2 from C19 precursors (15) and, therefore, has been widely used for investigation of the regulation of P450aromatase and 17HSD type 1 gene expression (11, 16, 17). The effects of RAs on 17HSD type 1 expression were also studied in combination with other factors, such as epidermal growth factor (EGF) and activators of protein kinase C, cAMP-dependent, and Ca2+ pathways, which have been demonstrated to regulate 17HSD type 1 gene expression in placental cells (16, 18, 19, 20). Finally, the influences of these factors, which are all also modulators of estrogen-dependent cell growth, were compared with their effects in the breast cancer cell line T47D.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and reagents
The isotopes [2,4,6,7-3H]E1 (101 Ci/mmol), [2,4,6,7-3H]E2 (98 Ci/mmol), and [{alpha}-32P]deoxy-CTP (3000 Ci/mmol) were purchased from Amersham Life Science (Little Chalfont, UK). Unlabeled E1 and E2 were obtained from Steraloids (Wilton, NH), and RNA molecular markers were purchased from BRL (Gaithersburg, MD). 8-Bromo-cAMP (free acid; 8BrcAMP) and EGF were obtained from Boehringer Mannheim (Mannheim, Germany). 12-O-Tetradecanoylphorbol-13-acetate (TPA) and calcium ionophore A23187 were products of Calbiochem-Novabiochem (La Jolla, CA) and Molecular Probes (Eugene, OR), respectively. All-trans-retinoic acid (at-RA) was obtained from Sigma Chemical Co. (St. Louis, MO), and 9-cis-RA was a gift from F. Hoffman La Roche (Basel, Switzerland). Plates for cell culture were purchased from Nunc Internal Medicine (Roskilde, Denmark). All media, buffers, supplements, and reagents for cell culture were obtained from Sigma Chemical Co. and Life Technologies (Grand Island, NY). Other reagents not mentioned in the text were obtained from Sigma Chemical Co., Boehringer Mannheim, and Merck (Darmstadt, Germany).

Cell culture and treatment of cells
JEG-3 human choriocarcinoma and T47D breast cancer cell lines were obtained from the American Type Culture Collection (Rockville, MD). JEG-3 cells were maintained in Eagle’s MEM supplemented with 10% FCS, 2 mM glutamine, 1 mM sodium pyruvate, and 1 mM nonessential amino acids. T47D cells were cultured in RPMI 1640 medium supplemented with 10% FCS, 1 mM nonessential amino acids, and 0.2 IU insulin/ml. For RNA and immunoblotting analysis, 2.5 x 106 JEG-3 cells and 5.0 x 106 T47D cells were applied to 10-cm plates, and the cells were allowed to attach to the plate for 24 h. The media were then replaced by media containing 5% FCS treated twice with dextran-coated charcoal (DCC-FCS), and the cells were cultured for an additional 4 h before each treatment. The cells were subjected to various stimuli in media containing 5% DCC-FCS for the indicated times before RNA isolation.

RNA isolation and Northern blot analyses of 17HSD type 1 mRNA
Total RNAs from cultured cells were isolated using TRIzol reagent from Life Technologies according to the manufacturer’s instructions. Thirty micrograms of total RNA were subjected to 1% (wt/vol) agarose-formaldehyde gel electrophoresis, followed by transfer to Hybond nylon membrane (Amersham International, Aylesbury, UK) by capillarity and cross-linking by UV radiation (21). The membranes were then prehybridized for 4 h at 42 C in 5 x SSPE [1 x SSPE = 0.15 M NaCl, 10 mM sodium phosphate (pH 7.4), and 0.1 mM EDTA] containing 50% (vol/vol) formamide, 0.1% BSA (wt/vol), 0.1% Ficoll (wt/vol), 0.1% (wt/vol) polyvinylpyrrolidone, 0.5% (wt/vol) SDS, and 20 µg salmon sperm DNA/ml. The membranes were further hybridized at 42 C overnight with the 32P-labeled 1.0-kilobase (kb) EcoRI-SacI fragment of human 17HSD type 1 complementary DNA (cDNA) as a probe (22). After hybridization, they were washed twice for 15 min each time in 2 x SSPE containing 0.1% SDS and then once with 1 x SSPE containing 0.1% SDS for 30 min. Finally, the membranes were exposed to Kodak XAR films (Rochester, NY) for 3–4 days. To control the amount of mRNA applied to the gel, membranes were also hybridized with rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe. The autoradiographic signals on the film were measured using a Molecular Dynamics 300 A computing densitometer (Molecular Dynamics, Sunnyvale, CA). Each experiment was repeated, and a representative figure from each analysis is shown.

Western blot analyses of 17HSD type 1 protein
For immunoblotting analyses, each treatment of JEG-3 cells was carried out in quadruplicate. Cultured JEG-3 cells were then treated with trypsin, washed, and sonicated in buffer A containing 20% glycerol (23), after which two samples from each treatment were combined. Cytosolic proteins from JEG-3 cell lysates were separated by SDS-PAGE, followed by electrophoretic transfer of proteins onto a Hybond ECL membrane (Amersham Life Science) (24). The membranes were immunostained using 1:400 diluted rabbit polyclonal antibody raised against human 17HSD type 1 (23) as a primary antibody and horseradish peroxidase-linked F(ab')2 fragments of antirabbit Ig (Amersham Life Science) as secondary antibody. An ECL Western blotting procedure (Amersham Life Science) was finally used to reveal the immunoreactive proteins. The membranes were exposed to Cronex 4 film (DuPont, Bad Homburg, Germany) for periods ranging from 5 sec to 5 min, after which the autoradiographic signals were measured using a Molecular Dynamics 300 A computing densitometer.

Reductive and oxidative 17HSD activity measurements
JEG-3 cells (2.5 x 105/well) and T47D cells (5.0 x 105/well) were applied to six-well plates and allowed to attach to the plates for 24 h. The cells were then cultured in medium with 5% DCC-FCS for 4 h, after which they were exposed to various stimuli for 24 h. Control samples were treated similarly, but without stimuli. For measuring reductive 17HSD activity, the media were removed from the wells and 2 ml of serum-free medium (MEM for JEG-3 cells and RPMI-1640 for T47D cells) containing 500 nM unlabeled E1 and 2.5 x 106 cpm [2,4,6,7-3H]E1 were applied to the plate. Correspondingly, for the measurement of oxidative 17HSD activity, 500 nM unlabeled E2 and 2.5 x 106 cpm [2,4,6,7-3H]E2 were included in the medium. The cells were then incubated for the indicated times at 37 C in cell culture conditions, and the subsequent steps followed the method described by Miettinen et al. (25). The results are given as the mean ± range from two independent experiments, in each of which duplicate samples were analyzed.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of RA on 17HSD activities and 17HSD type 1 mRNA expression in JEG-3 choriocarcinoma and T47D breast cancer cells
To study the role of RA in the regulation of 17HSD type 1 expression in JEG-3 cells, the cells were treated with different doses of at-RA, ranging from 0.1–1000 nM, for 6–48 h, and the influence on 17HSD type 1 gene expression was followed using Northern blot analysis. Increased 1.3-kb 17HSD type 1 mRNA expression could clearly be detected after stimulation for 48 h with 1.0 nM at-RA, and the expression was enhanced dose dependently up to 1.0 µM (Fig. 1Go, left panel). Again, after induction for 6 h with 1.0 µM at-RA, increases in 1.3-kb 17HSD type 1 mRNA were observed, and mRNA expression was continually increased up to 48 h (Fig. 1Go, right panel). In contrast to 1.3-kb mRNA, the expression of 2.3-kb 17HSD type 1 mRNA was not affected by at-RA treatment.



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  		Figure 1. Dose and time responses of 17HSD type 1 mRNA expression to at-RA in JEG-3 cells. The cells were maintained and stimulated as described in Materials and Methods. For dose-response experiments, cells were treated with 0.1, 1.0, 10, 100, and 1000 nM at-RA for 48 h (left panel). To test the effect of the duration of RA treatment, cells were maintained in the presence of 1.0 µM at-RA for 6, 12, 24, and 48 h (right panel). Thirty micrograms of total RNA were loaded into each lane. In the upper panels, the position of the 1.3-kb mRNA of 17HSD type 1 is indicated; in the lower panels, hybridization with a reference probe, GAPDH cDNA (exposure time, 20 h), is demonstrated.

 
at-RA induction of 17HSD type 1 mRNA expression was reflected by 17HSD activities in both JEG-3 and T47D cells. In both cell types, reduction from E1 to E2 was the predominant 17HSD activity (Fig. 2Go, A and B), and it could be induced by at-RA (Fig. 2Go, C and D). Treatment of the cells with at-RA for 24 h before activity measurements led to 2.6- and 8.8-fold average increases in the reduction of E1 to E2 in JEG-3 and T47D cells, respectively. at-RA also enhanced oxidative 17HSD activity in both JEG-3 and T47D cells, but to a lesser extent, i.e. approximately 1.5- and 2.5-fold, respectively (Fig. 2Go, E and F).



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  		Figure 2. 17HSD activities in cultured JEG-3 and T47D cells and the effect of at-RA. The cells were maintained as described in Materials and Methods, after which they were incubated for the indicated times or for 1 h in serum free-medium containing substrates E1 or E2 for the measurement of 17HSD activities. A and B, Reductive and oxidative estrogenic 17HSD activities in JEG-3 and T47D cells with no stimuli. C and D, Reductive 17HSD activity in JEG-3 and T47D cells after 1.0 µM at-RA administration for 24 h. E and F, Oxidative 17HSD activity in JEG-3 and T47D cells after induction with 1.0 µM at-RA for 24 h. Note that the scale is different in blots C and D from that in blots E and F.

 
Interaction of RAs with cAMP, TPA, and EGF on reductive 17HSD activity and 17HSD type 1 mRNA expression in JEG-3 and T47D cells
In addition to at-RA, 8BrcAMP, TPA, and EGF increased reductive 17HSD activity in JEG-3 choriocarcinoma cells (Fig. 3Go, upper panel). In contrast, in T47D breast cancer cells, 8BrcAMP, TPA, and EGF had no effect on the activity (Fig. 3Go, lower panel). In line with these results, TPA and EGF potentiated the influence of at-RA in JEG-3 cells, whereas in T47D cells they had no significant effect on at-RA induction. mRNA analysis confirmed the results; differences between the cell lines were even more clearly detectable in the Northern blot analysis (Fig. 4Go) than in the activity assays (Fig. 3Go). Scanning of the Northern blot films revealed that in JEG-3 cells, simultaneous administration of RAs with TPA and EGF resulted maximally in 41- and 23-fold increases in 17HSD type 1 expression, respectively. Interestingly, 8BrcAMP appeared to increase the effect of RA on 17HSD type 1 expression in JEG-3 cells, but decrease the effect in T47D cells (Figs. 3Go and 5Go). Similar to at-RA treatment alone, the double treatments affected only the 1.3-kb 17HSD type 1 mRNA, and not the 2.3-kb mRNA. Administration of 9-cis-RA led to identical responses in 17HSD type 1 mRNA expression as treatment with at-RA in both JEG-3 and T47D cells (Fig. 4Go).



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  		Figure 3. Interaction of at-RA with cAMP, TPA, and EGF on reductive 17HSD activity in cultured JEG-3 and T47D cells. JEG-3 cells (upper panel) and T47D cells (lower panel) were incubated with 1.0 µM at-RA (bar 2), 1.5 mM 8BrcAMP (bar 3), 20 nM TPA (bar 4), 50 ng EGF/ml (bar 5), 1.0 µM at-RA plus 1.5 mM 8BrcAMP (bar 6), 1.0 µM at-RA plus 20 nM TPA (bar 7), and 1.0 µM at-RA plus 50 ng EGF/ml (bar 8) for 24 h. After treatment, reductive 17HSD activity was measured, as described in Materials and Methods and Fig. 2Go, together with the activity of nontreated cells (bar 1).

 


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  		Figure 4. Interaction of RAs with TPA and EGF on 17HSD type 1 mRNA expression in JEG-3 and T47D cells. at-RA or 9-cis-RA (1.0 µM; lanes 2–3), 20 nM TPA (lane 4), 50 ng EGF/ml (lane 5), 1.0 µM at-RA with 20 nM TPA or 50 ng EGF/ml (lanes 6–7), 1.0 µM 9-cis-RA with 20 nM TPA or 50 ng EGF/ml (lanes 8–9) were administered to JEG-3 (A) and T47D (B) cells for 24 h. The treated and nontreated (lane 1) cells were collected for isolation of total RNA, 30 µg of which were loaded into each lane. The positions of 1.3-kb mRNA of 17HSD type 1 are indicated by the arrowheads on the left, and hybridization obtained with a GAPDH probe was used as an RNA loading control (exposure time, 20 h). The numbers represent the degree of intensity (fold) of the 17HSD type 1 mRNA signals detected by a densitometer.

 


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  		Figure 5. Interaction of at-RA with cAMP on 17HSD type 1 mRNA expression in JEG-3 and T47D cells. at-RA (1.0 µM), 1.5 mM 8BrcAMP, and 1.0 µM at-RA plus 1.5 mM 8BrcAMP were administered to T47D (left panel) and JEG-3 (right panel) cells for 24 h. The treated and nontreated cells were collected for isolation of total RNA, 30 µg of which were loaded into each lane. Positions of 2.3- and 1.3-kb mRNAs of 17HSD type 1 are indicated by the arrowheads on the left, and hybridization obtained with a GAPDH probe was used as a RNA loading control. The numbers represent the degree of intensity (fold) of the 1.3-kb 17HSD type 1 mRNA signals detected by a densitometer.

 
According to the scanning results, the stimuli administered increased 1.3-kb 17HSD type 1 mRNA expression to a greater extent than 17HSD activity. When the strength of the various signals varied, Northern blot analysis was a semiquantitative, rather than a quantitative, method; thus, the results from the activity and mRNA measurements were not completely comparable. The amounts of 17HSD type 1 protein from selected samples were, however, also measured to clarify the difference. Immunoblotting analyses of type 1 enzyme were thus performed using JEG-3 cells treated with at-RA, TPA, or both of them, and the luminometric signals were quantified by densitometer. Scanning demonstrated that treatment of JEG-3 cells with at-RA and TPA separately resulted in approximately 4-fold increases in 17HSD type 1 protein, whereas their combination generated 13- to 19-fold induction (Fig. 6Go).



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  		Figure 6. Effects of at-RA and TPA on 17HSD type 1 protein concentration in JEG-3 cells. Two milligrams of cytosolic proteins isolated from JEG-3 cells after mock stimulation (lanes 1 and 2) and stimulation with 1.0 µM at-RA (lanes 3 and 4), 20 nM TPA (lane 5), and 1.0 µM at-RA plus 20 nM TPA (lanes 6 and 7) were subjected to SDS-PAGE for immunoblotting analysis. The figure depicts the film after exposure for 1 min with the immunoblotting membrane, and the numbers under the panel represent the degree of intensity (fold) of the corresponding signals compared with the mean of the values for the mock-treated samples.

 
Interaction of calcium ionophore with RA, cAMP, TPA, and EGF on 17HSD type 1 mRNA expression in JEG-3 cells
To investigate whether a Ca2+-dependent pathway is involved in placental 17HSD type 1 gene expression, JEG-3 cells were exposed to the Ca ionophore A23187, alone and together with at-RA, 8BrcAMP, TPA, and EGF. A23187 increased 1.3-kb 17HSD type 1 mRNA expression in a time-dependent fashion, and maximal induction with 0.5 µM A23187 was achieved in 6–12 h. By 24 h of treatment, the effect of the Ca ionophore on the expression of 17HSD type 1 mRNA had disappeared (data not shown). Stimulation by the Ca ionophore could be enhanced by all of the other factors tested (Fig. 7Go). The strongest synergistic effect was achieved by the combination of calcium ionophore and at-RA, whereas administration of A23187 with 8BrcAMP, TPA, and EGF also resulted in strong increases in 1.3-kb 17HSD type 1 mRNA expression.



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  		Figure 7. Interaction of calcium ionophore with at-RA, cAMP, TPA, and EGF on 17HSD type 1 mRNA expression in JEG-3 cells. Northern blot analysis of 30 µg total RNA isolated from JEG-3 cells after mock stimulation (lane 1) and stimulation with 0.5 µM A23187 (lane 2), 1.0 µM at-RA (lane 3), 1.5 mM 8BrcAMP (lane 4), 20 nM TPA (lane 5), 50 ng EGF/ml (lane 6), 0.5 µM A23187 plus 1.0 µM at-RA (lane 7), 0.5 µM A23187 plus 1.5 mM 8BrcAMP (lane 8), 0.5 µM A23187 plus 20 nM TPA (lane 9), and 0.5 µM A23187 plus 50 ng EGF/ml (lane 10) for 12 h. 17HSD type 1 mRNA and GAPDH mRNA are indicated by arrows. The numbers represent the fold intensity of the corresponding 17HSD type 1 mRNA signals.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Estrogens have a pivotal role in gestation, from implantation to the start of delivery (26). Accumulating data strongly suggest that increased 17HSD activities are related to the pattern of E2 and E1 formation during pregnancy (6), which is, in turn, associated with human placental growth before 16 weeks (27). Expression of human 17HSD type 1 is not limited to the cells secreting E2 into the circulation, such as ovarian granulosa cells and placental trophoblasts during pregnancy; the enzyme is also expressed in certain target tissues of estrogen action. Immunostaining of the 17HSD type 1 enzyme has been observed in benign and malignant breast (28) and endometrial (3, 29) lesions. Thus, intracellular E2 concentrations, and consequently E2 responses, of target cells can also be modulated by local 17HSD type 1 (30) and its regulation.

Two transcription start points in the HSD17B1 gene result in two 17HSD type 1 mRNAs, 1.3 and 2.3 kb in size (31). In agreement with Reed et al. (10), our present results show that in the cell lines originating from breast epithelial cells as well as from placental trophoblasts, RAs enhance 1.3-kb 17HSD type 1 mRNA expression and 17HSD activity. This is in line with results published by Miettinen et al. (25) showing that 17HSD type 1 protein concentrations correlate with the amount of 1.3-kb 17HSD type 1 mRNA rather than with the amount of the 2.3-kb mRNA, and that in both JEG-3 and T47D cells, the type 1 enzyme is the predominant 17HSD type. The expression and most likely the translational efficacy of the 17HSD type 1 mRNAs differ from each other. The 1.3-kb mRNA is mainly expressed in granulosa cells and cells of trophoblast origin as well as in some breast epithelial cell lines, i.e. in cells expressing 17HSD type 1 protein (4, 8, 20, 25), whereas the longer transcript is widely expressed in various tissues and cell lines, including cells without detectable 17HSD type 1 protein (25). Thus the role of the 2.3 kb mRNA is not fully understood. Increases in 17HSD type 1 expression were reflected in raised 1.3-kb mRNA and protein concentrations and elevated 17HSD activity, although the effects of different stimuli were stronger on the mRNA and protein concentrations than on the enzyme activity. Previously, it was also observed that an addition to the 17HSD type 1 protein concentration is not reflected in a great increase in enzyme activity in vivo (20, 25, 30), as a result of factors, such as substrate and cofactor availability, that can limit the reaction rate in cultured cells. An increase in the 17HSD type 1 concentration further suggests that activity measurements in the cultured cells depict the trend of changes in enzyme expression and thus confirm the results of mRNA analysis, but may not represent the exact intensities of the changes. On the other hand, the differences between the levels of the enzyme activity, mRNA concentration, and protein concentration could to some extent be a result of the semiquantitative nature of Northern and immunoblotting analysis. Reed et al. (10) have also suggested that the 2.3-kb mRNA is responsible to some extent for the constitutive cellular activity of 17HSD type 1, which may explain the difference between the increase in 17HSD activity and level of expression of the 1.3-kb mRNA.

According to our results, the influence of at-RA and 9-cis-RA on 17HSD type 1 expression is similar, which supports the observation that the HSD17B1 gene contains a DR5-RA response element (11). DR5-RA response element is a typical binding site for a retinoid X receptor (RXR)/RA receptor (RAR) dimer, which is activated by at-RA and 9-cis-RA (32). RARs and their isoforms are widely distributed in different tissues; for example, cultured human trophoblasts have been demonstrated to express at least RAR{alpha}, RARß, RAR{gamma}, and RXR{alpha}. During differentiation from cytotrophoblasts to syncytiotrophoblasts, the concentrations of RAR{alpha}, RARß, and RXR{alpha} mRNA further increase (33). Breast cancer tumors and several breast cancer cell lines also contain RARs (34, 35, 36). Thus, the HSD17B1 gene could be one of the target genes of RAs in breast epithelial and placental trophoblast cells. The CYP19 gene, the gene encoding P450aromatase, is also regulated by RAs in JEG-3 cells (37) (our unpublished observation), which further implies that RAs participate in the regulation of estrogen biosynthesis in the placenta. The involvement of RAs in modulation of the production of placental steroid hormone (estrogens and progestins) as well as hCG and human placental lactogen peptide hormone (12, 13, 14) points to the role of RAs as regulators of placental endocrine functions.

Our previous (20) and present results demonstrate that EGF increases 17HSD type 1 gene expression in JEG-3 cells. EGF is an inducer of placental trophoblast differentiation (38), and its receptors (EGFR) are abundantly expressed in trophoblasts (39, 40), suggesting that EGF, via autocrine or paracrine effects, may also participate in placental estrogen production. Simultaneous administration of EGF and RAs led to strongly increased 17HSD type 1 expression in JEG-3 cells. Depending on the cell type, RAs have been found to either increase (41) or decrease (42) EGFR concentrations. Enhanced EGFR expression, however, does not explain the synergism of RAs and EGF in the present study, as RAs did not affect EGFR expression under the conditions used (data not shown). In addition to EGF, TPA, cAMP, and Ca ionophore increased 17HSD type 1 expression and potentiated the effect of RAs only in JEG-3 choriocarcinoma cells, not in T47D breast cancer cells. EGF receptor is expressed in both of the cell lines (43, 44), and protein kinase A (PKA) and protein kinase C (PKC) pathways function in them (19, 45). This suggests that the dissimilar responses of JEG-3 and T47D cells were due to a different pattern of transcription factors and/or their activators in these cell lines, rather than to the absence of the receptors.

Binding of a ligand to the EGF receptor can lead to activation of inositol phospholipid-specific phospholipase C and, consequently, mobilization of intracellular Ca2+ stores and generation of diacylglycerol (46). An increase in the intracellular Ca2+ concentration acts synergistically with phorbol esters, analogs of diacylglycerol, in activating some PKC isoforms, resulting in a cellular response (46). The calcium ionophore, A23187, which induces an increase in Ca2+ influx, can also potentiate the effects of forskolin, an activator of adenylate cyclase. In line with this, Ca ionophore enhanced the effects of 8BrcAMP and TPA on 17HSD type 1 expression in JEG-3 cells. In addition, the Ca ionophore further potentiated the effect of EGF, which was administered to JEG-3 cells at a dose maximally stimulating type 1 enzyme expression. Previously, it was demonstrated that tyrosine kinase activities in the EGF receptor and PKA pathways are involved in 17HSD type 1 regulation by EGF (20). PKA and PKC have been suggested to modulate RA action via phosphorylation of RARs (47, 48), which may be one mechanism involved in the cooperative regulation of 17HSD type 1 by RAs and other factors in JEG-3 cells. In T47D cells, cAMP had the opposite effect on RA induction, demonstrating that a divergent mechanism operates in T47D cells. In several cases, RAs have been demonstrated to counteract TPA action (32). In contrast, the present results from JEG-3 cells show an example of a case in which administration of RAs and TPA led to a synergistic response.

In conclusion, the present results show that RAs together with EGF and protein kinase A and C pathway activators, significantly increase 17HSD type 1 expression in trophoblast-like cells. Thus, RAs together with EGF and other available factors that activate PKA and PKC pathways could be involved in regulating 17HSD type 1 concentrations and, consequently, E2 biosynthesis in placental syncytiotrophoblasts. Furthermore, the interaction of several regulatory pathways is needed to maintain a high 17HSD type 1 concentration in the placenta. Finally, the dissimilar effects of EGF, cAMP, and TPA on 17HSD type 1 expression and on RA induction between JEG-3 and T47D cells demonstrate that the regulatory mechanism controlling 17HSD type 1 in breast cancer cells differs from that in choriocarcinoma cells.


   Acknowledgments
 
We thank Ms. Helmi Konola for her skillful technical assistance.


   Footnotes
 
1 This work was supported by the Research Council for Health (Project 1051135) and the Subcommittee for Development Studies (Project 7267) of the Academy of Finland. The Department of Clinical Chemistry is a WHO Collaborating Center for Research in Human Reproduction supported by the Ministries of Education, Social Affairs and Health, and Foreign Affairs, Finland. Back

Received July 17, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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