Molecular Human Reproduction, Vol. 8, No. 4, 318-325,
April 2002
© 2002 European Society of Human Reproduction and Embryology
Reproductive endocrinology |
Transforming growth factor-ß1 inhibits steroidogenesis in human trophoblast cells
Department of Biology, York University, 4700 Keele St, Toronto, Ontario, M3J 1P3, Canada
Abstract
Transforming growth factor-ß (TGF-ß) is an important regulator of placental development and function. In this study, we have investigated the effect of TGF-ß1 on steroidogenesis, as well as its sites of action in the steroidogenic pathway by using a choriocarcinoma cell line, JEG-3, and a normal trophoblast cell line (NPC). The effect of TGF-ß1 on progesterone and estradiol production was evaluated in the absence or presence of a membrane-permeable analogue of cholesterol and some intermediate substrates of steroidogenic enzymes. The effect of TGF-ß1 on P450 aromatase (P450arom) mRNA levels was determined by Northern blot analysis. TGF-ß1 significantly decreased progesterone production in both NPC and JEG-3 cells. The inhibitory effect of TGF-ß1 on progesterone production was reversed by addition of 22R-hydroxycholesterol, a membrane-permeable analogue of cholesterol, or pregnenolone. In JEG-3 cells, TGF-ß1 also inhibited estradiol production when androstenedione, but not estrone, was added to the culture. Estradiol production was too low to be detected in NPC cells. Treatment with TGF-ß1 also suppressed aromatase mRNA levels. This study has demonstrated that TGF-ß1 inhibits progesterone and estradiol production by trophoblast cells, and that the sites of TGF-ß1 action on progesterone and estradiol production are likely to be cholesterol transport and P450arom respectively.
estradiol/JEG-3/NPC/progesterone/TGF-ß1
Introduction
Transforming growth factor-ß (TGF-ß) is a 25 kDa dimeric protein that regulates a wide variety of cellular activities in many tissues and organs (Lyons and Moses, 1990
; Massagué, 1990; Massagué and Wotton, 2000). In human placenta, mRNA for three isoforms of TGF-ß (ß1, ß2 and ß3) have been detected (Dungy et al., 1991; Ando et al., 1998; Caniggia et al., 1999). TGF-ß immunoreactivity has also been found in syncytiotrophoblast cells (Vuckovic et al., 1992). In addition, both type I (TßR-I) and type II (TßR-II) TGF-ß receptor mRNA (Ando et al., 1998), as well as TGF-ß binding sites (Mitchell et al., 1992) have been identified in trophoblast cells. These findings suggest that TGF-ß may act as an autocrine/paracrine factor to regulate placental development and function.
Several studies have shown that TGF-ß regulates trophoblast cell proliferation and differentiation. TGF-ß has an antiproliferative effect on first trimester cytotrophoblast cells (Morrish et al., 1991; Graham et al., 1992; Lala and Hamilton, 1996; Li and Zhuang, 1997; Smith et al., 2001), while it increases the formation of multinucleated trophoblast cells (Graham et al., 1992; Lala and Hamilton, 1996). On the other hand, the differentiation of term villous cytotrophoblast cells into the syncytium is inhibited by TGF-ß (Morrish et al., 1991). TGF-ß has also been reported to inhibit the invasiveness of extravillous trophoblast cells (Graham and Lala, 1991; Caniggia et al., 1999; Smith et al., 2001). In addition, TGF-ß1 inhibits HCG and human placental lactogen (hPL) secretion (Morrish et al., 1991; Song et al., 1996).
Progesterone and estradiol produced by the placenta are known to play important roles during pregnancy. Steroid biosynthesis begins with the transport of the substrate cholesterol to the first enzyme in the steroidogenic pathway, cytochrome P450 cholesterol side-chain cleavage enzyme complex (P450scc), located in the inner membrane of mitochondria. This is a major step for the physiological regulation of steroidogenesis (Strauss et al., 1999). P450scc catalyzes the conversion of cholesterol to pregnenolone, which is then converted to progesterone via the action of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) (Albrecht and Pepe, 1990, 1998). The conversion of androgens to estrogens is catalysed by P450 aromatase (P450arom). In vivo, dehydroepiandrosterone produced by the fetal adrenal gland is used by the trophoblast cells as the precursor for estradiol (Albrecht and Pepe, 1998). In primary cultured trophoblast cells and in choriocarcinoma cell lines, estradiol can be produced when an androgen substrate, such as androstenedione, is supplied to the culture medium (Ritvos, 1988). Androstenedione is converted to estrone by aromatase and estrone can be converted to estradiol by type 1 17ß-HSD (Albrecht and Pepe, 1998). Several hormones, such as GnRH (Siler-Khodr et al., 1986, 1987), HCG (Albrecht and Pepe, 1990; Chaudhary et al., 1992) and activin-A (Petraglia et al., 1989, 1994; Song et al., 1996; Ni et al., 2000) are known to stimulate progesterone and estradiol production. In contrast, inhibitory regulators of steroidogenesis in the placenta are not well understood. Using the tritiated water assay, it has been reported that TGF-ß1 suppresses basal and activin-induced aromatase activity in cultured human placental cells (Song et al., 1996), suggesting that TGF-ß inhibits estrogen production.
JEG-3 cells, a choriocarcinoma cell line, produce many peptide and steroid hormones (including HCG, progesterone, and estradiol) found in normal trophoblast cells and have been widely used as a placental trophoblast model to study hormone production (Ringler and Strauss, 1990). This cell line was used in the present study to investigate the effect of TGF-ß on progesterone and estradiol production, as well as the potential sites of TGF-ß action. The effect of TGF-ß on progesterone production was also examined in a normal placental cytotrophoblast (NPC). The NPC cell line was established from isolated cytotrophoblast cells of first trimester placenta (Li et al., 1996; Li and Zhuang, 1997). These cells produce progesterone, GnRH and a small amount of HCG (Li et al., 1996). The proliferation of this cell line is regulated by several growth factors, including TGF-ß (Li and Zhuang, 1997), and recently, we have detected mRNA for several Smads, the downstream signalling molecules of TGF-ß, in the NPC cells (Wu et al., 2001).
Materials and methods
Cell culture
The JEG-3 cell line was obtained from American Type Cell Culture (Rockville, MD, USA). The cells were cultured in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, and 100 µg/ml streptomycin (all reagents were purchased from Invitrogen Canada Inc., Burlington, ON, Canada). NPC cells were maintained in serum-free Ham's F-12 and Dulbecco's modified Eagle's medium (FD medium, F-12:DMEM = 1:1) (Invitrogen), supplemented with 10 ng/ml epidermal growth factor, 10 µg/ml insulin, 0.1% bovine serum albumin, 2 mmol/l glutamine, 10–7 mol/l dexamethasone, 1.75 mol/l HEPES (Sigma–Aldrich Canada Ltd, Oakville, ON, Canada), 100 IU/ml penicillin, and 100 µg/ml streptomycin, as previously described (Li et al., 1996; Li and Zhuang, 1997; Wu et al., 2001).
Steroid production
Cells were seeded in 24-well plates at a density of 5x104 cells/well. One day after plating, cells were treated with control medium (MEM) or TGF-ß1 (Research Diagnostics Inc., Flanders, NJ, USA) at various concentrations in serum-free MEM for 48 h (n = 6 wells). To determine which steps in the steroidogenic pathway are affected by TGF-ß1, cells were treated with TGF-ß1 (10 ng/ml) in the presence or absence of 22R-hydroxycholesterol (5 µg/ml) or 20 µmol/l pregnenolone (Sigma–Aldrich). For experiments involving estradiol production, 200 nmol/l androstenedione or estrone (Sigma–Aldrich) was added to the culture medium as a precursor for estradiol. To investigate the interaction between activin and TGF-ß1, cells were treated with control medium, TGF-ß1, follistatin-288 (kindly provided by Dr A.F.Parlow, National Hormone and Pituitary Program, Rockville, MD, USA), or the combination of TGF-ß1 and follistatin-288. At the end of each experiment, culture medium was collected and cells were trypsinized. Cell numbers were determined using a haemocytometer. Concentrations of progesterone and estradiol in the media were measured using specific radioimmunoassay as previously described (Ni et al., 2000). The assays have detectable limits of 150 pg/ml for progesterone and 40 pg/ml for estradiol, respectively. Both inter- and intra-assay coefficients of variation for these assays were <10%.
Total RNA extraction, probe preparation, and Northern blot analysis
JEG-3 cells were seeded at a density of 106 cells/60 mm dish. Treatments were carried out with medium alone (control) or with 10 ng/ml TGF-ß1 in the presence of 200 nmol/l androstenedione. The cells were harvested at 6, 12 and 24 h after TGF-ß1 treatment and total RNA was extracted using the Trizol reagent (Invitrogen) following the manufacturer's suggested protocol. Total RNA (30 µg) samples were separated by electrophoresis and transferred onto Hybond-N-membranes (Amersham Pharmacia Biotech, Inc.).
To obtain a specific probe for P450arom, RT–PCR was first carried out. Samples of 2 µg of human placental polyA+ RNA (Clontech Laboratories Inc., Palo Alto, CA, USA) were reverse-transcribed using the First Strand cDNA Synthesis Kit and oligo-dT12–18 primers (Amersham) according to the manufacturer's method. Specific primers were designed based on the published human P450arom (Chen et al., 1988) sequence and used in PCR to amplify partial cDNA for P450arom. The primers used were: Arom1: 5'-GCTGCAGTGCATCGGTATGCA-3'; and Arom2: 5'-ACTCGAGTCTGTGCATCCTTC-3'. The expected PCR product was excised from the gel and the DNA fragment was recovered using the Geneclean II kit (Bio 101, Vista, CA, USA). The DNA fragment was then ligated into the PCR2.1 plasmid vector using the Topo TA cloning kit (Invitrogen). Several clones were sequenced using an ABI 373A sequencer at the York University Core Facility for Molecular Biology. After confirming its identity, the insert DNA was labelled with 32P-dATP (Amersham) using a NEBlot kit (New England BioLabs Inc., Berverly, MA, USA). Hybridizations were carried out in ULTRAhyb solution (Ambion, Austin, TX, USA) containing the 32P-labelled probe at 42°C for 16–18 h. Membranes were washed under high stringency conditions. Human 18S ribosomal RNA (kindly provided by Dr D.Hood, Department of Kinesiology, York University) was used as an internal control to normalize the loading of RNA samples in these analyses.
Statistical analysis
Steroid hormone levels and cell numbers were expressed as the mean ± SEM of six replicate culture wells from a single experiment. Each experiment was repeated three times. Messenger RNA levels were expressed as the mean ± SEM of four experiments. RNA levels were normalized against 18S rRNA and expressed as a percentage of the control of each experiment to allow pooling of data from different experiments. For multiple group comparisons, one-way analysis of variance was used followed by Scheffé's multiple test. For comparison between two groups, Student's t-test was performed. P < 0.05 was considered to be statistically significant.
Results
TGF-ß1 inhibits progesterone production
NPC cells were treated with various doses of TGF-ß1 for 48 h and progesterone levels in the cultured medium were determined. With increasing concentrations of TGF-ß1, there were decreases in progesterone levels in the medium (Figure 1A). TGF-ß1 treatment also caused decreases in cell numbers in a dose-dependent manner (Figure 1B). When progesterone levels in the culture medium were normalized by cell numbers, a significant reduction was observed in the 10 ng/ml TGF-ß1-treated group (Figure 1C). Using a [3H]thymidine assay, we found that TGF-ß1 significantly inhibited NPC cell proliferation (data not shown).
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A similar treatment was performed in JEG-3 cells. Again, TGF-ß1 caused an inhibition of progesterone production in a dose-dependent manner. TGF-ß1, at 1 and 10 ng/ml, significantly decreased progesterone concentration in the culture medium (Figure 2A
, C), but did not affect cell numbers (Figure 2B).
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The biosynthesis of progesterone involves several steps, including cholesterol transport into the inner mitochondrial membrane, conversion of cholesterol to pregnenolone by P450scc and conversion of pregnenolone to progesterone by 3ß-HSD. To determine at which step TGF-ß1 exerts its inhibitory effect on progesterone production, we first tested the effect of TGF-ß1 in the presence and absence of 22R-hydroxycholesterol, a membrane-permeable analogue of cholesterol, in JEG-3 cells. As shown in Figure 3
, TGF-ß1 caused a significant decrease in progesterone levels in the absence of 22R-hydroxycholesterol. However, when 22R-hydroxycholesterol was added, TGF-ß1 no longer inhibited progesterone production. This result suggests that P450scc and 3ß-HSD are not affected by TGF-ß1. We then confirmed this notion by testing the effect of TGF-ß1 in the presence of pregnenolone. Indeed, the inhibitory effect of TGF-ß1 on progesterone production was also reversed by pregnenolone (Figure 4).
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TGF-ß1 inhibits estradiol production and aromatase mRNA expression
Since the NPC cells produce very little estradiol from androstenedione, this part of the study was only conducted in JEG-3 cells. JEG-3 cells were incubated with or without TGF-ß1 in the presence of androstenedione. As shown in Figure 5
, treatment with TGF-ß1 resulted in dose-dependent decreases in estradiol concentrations; a significant inhibition of estradiol production was observed after 1 and 10 ng/ml TGF-ß1 treatment (Figure 5A, C). Again, cell numbers were not affected by the TGF-ß1 treatment (Figure 5B).
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The conversion of androstenedione to estradiol involves two enzymes, P450arom and 17ß-HSD1; therefore, we compared the effect of TGF-ß1 on estradiol production from two precursors: androstenedione and estrone. In the presence of androstenedione, TGF-ß1 (10 ng/ml) again inhibited estradiol production; however, TGF-ß1 had no effect on estradiol levels when estrone was used as the precursor (Figure 6
). These data suggest that TGF-ß1 inhibits aromatase, but not 17ß-HSD, activity. To investigate the regulation of aromatase by TGF-ß, Northern blot analysis was carried out to determine if TGF-ß1 affects P450arom mRNA levels. A transcript of 3.4 kb was detected in JEG-3 cells by the P450arom probe (Figure 7A). Treatment with TGF-ß1 did not affect aromatase mRNA levels at 6 and 12 h after treatment. However, at 24 h after treatment, the P450arom mRNA level was significantly lower in TGF-ß1-treated samples when compared to the control group (Figure 7B).
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TGF-ß and follistatin had additive inhibitory effects on steroid production
Previously, we have shown that activin-A stimulated progesterone and estradiol production by JEG-3 cells (Ni et al., 2000
). To determine if the inhibitory effect of TGF-ß1 on steroid production was mediated via effects on activin, we used an activin binding protein, follistatin-288, to block activin action and determined how this would affect the action of TGF-ß1 on progesterone and estradiol production. As shown in Figure 8, follistatin-288 slightly, while TGF-ß1 significantly, decreased progesterone and estradiol levels. When TGF-ß1 and follistatin-288 were added together, the decreases in progesterone and estradiol levels were greater than those observed with either treatment alone.
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Discussion
TGF-ß has been suggested to be an autocrine/paracrine regulator of human placental development. In this study, we further examined the role of TGF-ß1 by testing the effects of TGF-ß1 on progesterone and estradiol production. We demonstrated that TGF-ß1 inhibits progesterone production, probably via inhibition of cholesterol transport, and estradiol production by suppressing P450arom. In both NPC and JEG-3 cells, we found that TGF-ß1 inhibited progesterone production in a dose-dependent manner. TGF-ß1 also significantly inhibited estradiol production in JEG-3 cells. It has been reported that TGF-ß1 inhibited aromatase activity, as measured by the tritiated water assay, in cultured term placental trophoblast cells (Song et al., 1996). Although no estradiol level was measured in that study, a decrease in aromatase activity would be expected to result in a decrease in estrogen production. Thus, it is likely that TGF-ß1 can regulate progesterone and estradiol production in normal human trophoblast cells.
The regulation of placental progesterone and estradiol production is a critical event during pregnancy, as the coordinated action of progesterone and estradiol is known to be essential for the normal progression of gestation (Albrecht and Pepe, 1998). The production of progesterone and estradiol is tightly controlled by many placental hormones. GnRH (Siler-Khodr et al., 1986, 1987), HCG (Chaudhary et al., 1992) and activin-A (Petraglia et al., 1989, 1994; Song et al., 1996; Ni et al., 2000) have been shown to stimulate progesterone or estradiol production. Prolactin (Barnea et al., 1989a), growth hormone (Barnea et al., 1989b), insulin-like growth factor (IGF)-I (Nestler, 1987) and IGF-II (Nestler, 1990) have dual effects on steroid hormone production: they stimulate progesterone but inhibit estradiol production. On the other hand, BMP-7 suppresses progesterone production (Martinovic et al., 1996). The present demonstration that TGF-ß1 inhibits progesterone and estradiol production further indicates that steroid levels are maintained by both stimulatory and inhibitory factors. It remains to be elucidated how different regulators interact to control progesterone and estradiol production during pregnancy.
In the present study, we have shown that the inhibitory action of TGF-ß1 on progesterone production is exerted at a step prior to the conversion of cholesterol to pregnenolone. 22R-hydroxycholesterol, a cholesterol analogue that readily passes through the mitochondrial membrane, completely reversed the inhibitory effect of TGF-ß1 on progesterone production. This result indicates that the inhibition occurs before or at the step of cholesterol transport into the inner mitochondrial membrane. This, together with the finding that TGF-ß1 failed to inhibit the conversion of pregnenolone into progesterone, demonstrates that TGF-ß1 does not regulate P450scc or 3ß-HSD. The transport of cholesterol from the outer to inner mitochondrial membrane is known to be a rate-limiting step in steroidogenesis and is under the control of hormones (Struss et al., 1999). In the ovary and adrenal context, this transport process is facilitated by the steroidogenic acute regulatory protein (StAR) (Strauss et al., 1999). Interestingly, TGF-ß1 has been shown to decrease adrenocorticotrophic hormone-induced cortisol production via the inhibition of StAR expression in bovine adrenocortical cells (Brand et al., 1998a) and in a human adrenocortical cell line, H295R (Brand et al., 1998b; 2000). Similarly, TGF-ß has been found to inhibit forskolin-induced StAR expression in a human thecal like tumour cell line (Attia et al., 2000). However, StAR is not expressed in the human placenta (Albrecht and Pepe, 1998; Strauss et al., 1999). A protein named MLN64, which shares some homology with StAR, has been suggested to perform a function similar to that of StAR in human placenta (Watari et al., 1997; Strauss et al., 1999). Whether or not TGF-ß inhibits MLN64 expression remains to be determined in the future.
The present study indicates that aromatase is a target enzyme for TGF-ß in human placenta. TGF-ß1 inhibited the conversion of androstenedione, but not estrone, to estradiol, suggesting that aromatase, but not 17ß-HSD, is regulated by TGF-ß1. This notion is further supported by the finding that treatment with TGF-ß1 caused a significant decrease in aromatase mRNA levels. Similar inhibitory effects on aromatase expression and/or activity of TGF-ß1 have also been observed in human fetal hepatocytes (Rainey et al., 1992), human adipose stromal cells (Simpson et al., 1989) and in skin fibroblasts (Emoto et al., 1991). However, in human osteoblast-like cells and THP-1 cells, TGF-ß1 has been found to increase aromatase gene transcription (Shozu et al., 2000). It is unclear how TGF-ß1 exerts opposite effects on the same enzyme in different tissues. However, since the expression of P450arom is under the control of tissue-specific promoters (Chou et al., 1996; Toda et al., 1996; Simpson et al., 1997; Shozu et al., 1998), it is possible that the TGF-ß1 signalling pathway may interact with different transcription factors to differentially regulate aromatase gene transcription in different tissues.
TGF-ß is known to regulate proliferation of many types of cells. Similarly, in NPC cells, we found that TGF-ß1 potently inhibited cell proliferation. This result is consistent with previous studies in several trophoblast cell lines derived from normal placenta, such as HTR-8/SVneo (Graham et al., 1993) and ED27 (Smith et al., 2001). However, consistent with previous studies (Graham et al., 1994), we also found that TGF-ß1 did not affect JEG-3 cell growth, confirming that the tumour cells have lost their sensitivity to the growth-inhibitory effect of TGF-ß1. Interestingly, although JEG-3 cell proliferation is no longer sensitive to TGF-ß1, the progesterone production is inhibited by TGF-ß1 to an extent similar to that found in NPC cells. These results suggest that the signalling pathways mediating TGF-ß1-regulated cell proliferation and hormone production are somewhat different. Recently, we have found that Smad mRNA are expressed in JEG-3 and NPC cells (Ni et al., 2000; Wu et al., 2001). We also observed that Smad3 expression is much lower in JEG-3 than in NPC cells (Wu et al., 2001). Nevertheless, the JEG-3 cells appear to have a functional Smad2/3 signalling pathway as TGF-ß can induce the transcriptional activities of several reporter constructs, pAR3-Lux, p3TP-Lux, and pSmad6-Lux (Wu et al., 2001). The induction of these promoters by TGF-ß is known to involve Smads, including Smad3 (Dennler et al., 1998; Nagarajan et al., 1999; Yeo et al., 1999; von Gersdorff et al., 2000). Therefore, it is unlikely that the resistance of JEG-3 cells to the anti-proliferative action of TGF-ß1 is caused by an abnormality in the Smad signalling pathway. In an adrenal cell line, Smad3 has been found to mediate the inhibitory effects of TGF-ß1 on steroidogenic acute regulatory protein mRNA expression (Brand et al., 1998b). Whether or not Smads mediate the action of TGF-ß1 on steroid production in trophoblast cells remains to be investigated.
Activin and TGF-ß belong to the same family of growth factors and have been proposed to use a similar Smad signalling pathway for signal transduction (Massagué and Wotton, 2000). However, they have distinct functions in human placenta. Previous studies have shown that activin and TGF-ß have opposing effects on HCG secretion in a primary culture of trophoblast cells (Song et al., 1996) and on cytotrophoblast outgrowth in first trimester villous explants (Caniggia et al., 1997). We also found that TGF-ß1 inhibits, while activin stimulates, progesterone and estradiol production in JEG-3 cells (Ni et al., 2000; present study). In this study, we tested the possibility that TGF-ß1 inhibits steroid production by inhibiting activin action and/or production from JEG-3 cells. Follistatin, an activin binding protein known to neutralize the effect of activin on estradiol and progesterone production in JEG-3 cells (Ni et al., 2000), was added together with TGF-ß1 to the culture. If TGF-ß1 acts upstream of activin, one would predict that TGF-ß would not have an effect when the activity of activin is already inhibited. However, this is not the case here. We found that follistatin not only did not block TGF-ß1-inhibited steroid production, but also enhanced the inhibitory effect of TGF-ß1. These results indicate that the inhibitory effect of TGF-ß1 on steroid production is not mediated by effects on activin. This finding is in agreement with a study (Song et al., 1996) which found that the effects of TGF-ß1 and activin on HCG secretion and aromatase activity were independent of each other. Cellular and molecular mechanisms whereby activin-A and TGF-ß1 exert opposing actions on steroid production in human placenta are currently under investigation.
In summary, the present study demonstrates, for the first time, that TGF-ß1 has a significant inhibitory effect on progesterone and estradiol production by human trophoblast cells. These inhibitory effects were diminished if the steps of cholesterol transport and aromatization were bypassed, suggesting that the action of TGF-ß1 on steroid production is exerted at the level of cholesterol transport and aromatase activity.
Acknowledgements
This study was supported by a grant from the Canadian Institute of Health Research (MT-14797) to C.P. We thank Dr A.F.Parlow and NHPP for follistatin, Dr D.Hood for the 18S rRNA probe and Drs Y.Wang and L.Zhuang for the NPC cells.
Notes
1 To whom correspondence should be addressed. E-mail: cpeng{at}yorku.ca 
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Submitted on September 18, 2001; accepted on December 7, 2001.
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