Transforming growth factor-beta inhibits aromatase gene transcription in human trophoblast cells via the Smad2 signaling pathway

JEG-3 cells were purchased from American Type Culture Collection (Rockville, MD). The cells were cultured in minimal essential medium (MEM, Canadian Life Technologies, Inc.) containing 10% fetal bovine serum (FBS, Sigma-Aldrich Canada Ltd, Oakville, ON) and antibiotics (100 IU/m penicillin, and 100 μg/ml streptomycin, purchased from Invitrogen Canada Inc. Burlington, ON).

Expression constructs for constitutively active and dominant negative ALK5, and dominant negative TβRII were kindly provided by Dr. L. Attisano (Univ of Toronto). Luciferase reporter constructs were generated using pGL3 basic luciferase reporter vector (Promega, Madison, WI). To obtain DNA fragments containing different lengths of the exon I.1 5' flanking sequence (+120 to -2538, +120 to -1333, +120 to -714, +120 to -422, and +120 to -117), PCR was performed using genomic DNA extracted from JEG-3 cells as the template. All PCRs were carried out for 30 cycles using a common antisense primer, 5'-AGATTAAGAGATACATACGCG-3', and a specific sense primers (5'-CCCGGTACCCCAGATGATCTTTCCCAGGAA-3' for Arom -2538 construct; 5'-CTGTGGAACCATGAGCCAATT-3' for Arom-1333; 5'-GCATTGGAGATACGGAAGTAA-3' for Arom -714; 5'-TGTAGAACAATGTGGTGTGTG-3' for Arom-422; and 5'-AGACCTTGCTGAGATTAGATC-3' for Arom-117). The resulting DNA fragments were first cloned into pCRII vector using a TOPO cloning kit (Invitrogen). The inserts were then cut out from pCRII vector using KpnI and XhoI (for Arom -1333, Arom -714 bp, Arom -422 bp and Arom -117 bp) and KpnI and NheI (for Arom -2541) respectively, and then subcloned into the pGL3-basic vector. All promoter constructs were fully sequenced to verify their identities.

JEG-3 cells were seeded into 6-well plates at a density of 2 × 10⁵ cells/well and incubated in MEM containing 10%FBS for 24 hours. Transient transfection was carried out using a 25 kDa polyethylenimine (PEI, Sigma-Aldrich) as previously described [24]. After transfection for 4 h, the medium was replaced with MEM supplied with 10% FBS and cells were allowed to be recovered for different lengths of time. After treatment with TGF-β1, cells were then lysed in 250 μl of lysis buffer (20 mM Tris PH 7.4/0.1% TritonX-100). The supernatant was collected for both luciferase and β-gal assays. To determine the luciferase activity, 100 μl of luciferase substrate (Promega) was added to 30 μl supernatant and the total light emission during the initial 10 seconds of the reaction was measured using a luminometer. For β-gal assay, 30 μl of supernatant was added into 270 μl of reaction mixture (1 ml contains 244 μl of 4 mg/ml of 2-Nitrophenyl-β-D-Galactopyranoside (ONPG), 2.22 μl of 0.5 M MgSO₄, 3.89 μl of β-mercaptoethanol, 186.2 μl of 0.4 M sodium phosphate buffer, and 563.9 μl of water, and all chemicals were purchased from Sigma-Aldrich), mixed well and incubated at 37°C for 30 min or until a yellow color change was detectable. The reaction was then terminated by the addition of 500 μl of 1 M Na₂CO₃ and absorbance at 410 nm was measured using a spectrophotomer. The luciferase assay data were normalized by β-gal activity and expressed as relative luciferase activity.

Cells were plated into 6-well plates at the density of 2 × 10⁵cells/well and allowed to grow overnight. To determine the effect of TGF-β1 on aromatase gene transcription, cells were either treated with different concentrations of TGF-β1 for 2 hours or 1 ng/ml of TGF-β1 for different time periods (2 h, 6 h, 12 h and 24 h).

Smad2 expression was knockdown using its siRNA. The published Smad2 siRNA sequence (UCUUUGUGCAGAGCCCCAAtt [25]) was cloned into the pSuper vector (Oligoengine, Seattle, WA), and JEG-3 cells were transiently transfected with either the control pSuper vector or the siRNA for Smad2 for 4 h. After recovering for 12, 24, 48 and 72 h, protein extract was prepared from cells and Western blotting was performed using an antibody against human Smad2 (Cell Signalling). To determine the effect of Smad2 silencing on TGF-β action, cells were first transfected with the control or siRNA plasmid for 4 h and recovered for 24 h before being treated with TGF-β1 (1 ng/ml) for 6 h.

The whole cell extraction was carried out as described previously with some modification [26]. Briefly, JEG-3 cells were plated using a density of 6 × 10⁵ cells/dish in 60-mm dishes and incubated overnight. Following treatment with 1 ng/ml TGF-β1 for 30 min, the cells were lysed in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, and 1% SDS) containing 1 mM dithiothreitol, 1 mM Na₃VO₄, 5 mM NaF, 100 mM EDTA, 10 mg/ml aprotinin, and 100 mM phenylmethysulfonyl fluoride. Nuclear proteins and cytoplasm proteins were extracted using Nuclear Extraction Kit (Panomics) following the manufacturer's protocols and stored at -20°C until Western blot analysis. Proteins (40 μg) were separated by SDS-polyacrylamide gel electrophoresis in 12% gels. The proteins were transferred electrophoretically onto polyvinylidene difluoride membranes (Immobilon-P, Millipore Corp., Bedford, MA). After blocking with 5% milk in TBS-T, the membranes were incubated with rabbit anti-human phospho-Smad2 (1:1000 dilution, Cell Signaling) or phosphor-Smad3 (1:1000 dilution, Cell Signaling), or mouse anti-human actin (1:1000 dilution, Sigma) antibodies at 4°C overnight. The membranes were subsequently probed with horseradish peroxidase-conjugated anti-rabbit or anti-mouse (1:5000 dilution, Amersham) at room temperature for 1 h. Signals were detected using a ECL-Plus kit (Amersham) according to the instructions of the manufacturer.

To study the effect of TGF-β1 on aromatase mRNA stability, JEG-3 cells were seeded at a density of 1 × 10⁶ cells/60 mm dish. 24 h after plating, cells were treated with 2 μg/ml actinomycin D (Sigma), either alone or in the presence of 1 ng/ml of TGF-β1, in serum free medium. The cells were then harvested at time 0 (pretreatment) and 6, 12, 24, 36 and 48 h after treatment with TGF-β1. Total RNA was then extracted using Trizol reagent (Invitrogen) following the manufacturer's recommended protocol and reversed transcribed into cDNA as previously described [5]. Aromatase mRNA levels were determined using RT-PCR. GAPDH was used as internal control to normalize the aromatase mRNA level. The GAPDH sense primer was 5'-AAGGTCATCCCTGAGCTGAAC, and antisense primer was 5'-CGCCTGCTTCACCACCTTCTA. Primers used in aromatase PCR were sense: 5'-GCTGCAGTGCATCGGTATGCA-3', and antisense: 5'-ACTCGAGTCTGTGCATCCTTC-3'. Validation assays were performed to determine the proper cycle number and the numbers chosen for aromatase (32 cycles) and GAPDH (22 cycles) are in the linear range of amplification.

Examination of the major promoter region that directs placenta-specific expression of the aromatase gene reveals the presence of four Smad binding elements (SBE) [27], CAGAC, located at +93 to +97, -114 to -118, -118 to -122, and -1186 to -1182 (Fig. 1A). This suggests that the TGF-β family may regulate aromatase transcription via the Smad pathway. Since TGF-β1 decreased aromatase mRNA [5], we therefore tested its effect on aromatase gene transcription. Five promoter constructs containing different lengths of the 5' flanking sequence of the exon I.1, designated as Arom -2538, Arom -1333, Arom -714, Arom -422, and Arom -117, were made and used to test the effect of TGF-β1. JEG-3 cells were transiently transfected with a luciferase construct and then treated with or without TGF-β1. TGF-β1 inhibited promoter activities in Arom -2538, Arom -1333, Arom -714, and Arom -422 constructs. Deletion between -422 and -117 abolished the basal promoter activity as well as the inhibitory effect of TGF-β1 (Fig. 1A).

The dose-dependent and time-course effects of TGF-β1 were subsequently tested using promoter construct Arom -714. A significant decrease in aromatase promoter activity was observed at 2 to 12 h after TGF-β1 treatment (Fig. 1B). At 6 h after treatment, all doses of TGF-β1 (0.1 to 10 ng/ml) significantly inhibited luciferase activity; with a maximal effective dose observed at 1 ng/ml (Fig. 1C).

To confirm that TGF-β1 acts through its specific receptors and the Smad pathway to regulate aromatase gene transcription, several experiments were performed. First, cells were transfected with dominant negative mutant of TβRII, or its vector control (pCMV5) and then treated with or without 1 ng/ml TGF-β1. As shown in Fig. 2A, TGF-β1 treatment decreased luciferase activity in the pCMV5-transfected cells, but not in the dominant negative TβRII-transfected cells, indicating that dominant negative TβRII reversed the inhibitory effect of TGF-β1 on aromatase gene transcription. Second, the involvement of ALK5 in TGF-β1-regulated aromatase transcription was examined using both constitutive active and dominant negative mutants. Transfection of the constitutively active ALK5 inhibited the aromatase promoter activity to a similar extend as TGF-β1 treatment (Fig. 2B). On the other hand, overexpression of dominant negative ALK5 completely blocked the effect of TGF-β1 on aromatase promoter activity (Fig. 2B).

We subsequently investigated if the Smad pathway is involved in the TGF-β1-regulated aromatase transcription. First, protein samples were extracted from cells at 30 min after TGF-β1 treatment and immunoblotting was performed. TGF-β1 strongly induced Smad2 phosphorylation (Fig. 3A). Consistent with our previous report that Smad3 expression level is very low in JEG3 cells [28], pSmad3 was only weakly detected in the TGF-β1-treated cells (Fig. 3A). To further confirm the activation of Smad2, protein samples were prepared from cytoplasmic and nuclear fractions of the control and TGF-β1-treated cells. Treatment with TGF-β1 resulted in strong accumulation of phosphorylated Smad2 in the nucleus (Fig. 3B). To directly examine the role of endogenous Smad2, we generated a siRNA construct to silence Smad2 expression. Compared to the vector control, transfection with siSmad2 construct resulted in a decrease in Smad2 protein levels at 24, 48 and 72 h after transfection (Fig. 3C). When cells were transfected with siSmad2 construct before TGF-β1 treatment, we found that knockdown of Smad2 completely reversed the effect of TGF-β1 on aromatase transcription (Fig. 3D).

Since the decrease in aromatase mRNA levels could also be due to a decrease in its stability, we tested if TGF-β1 regulated aromatase mRNA degradation. Cells were treated with a transcription inhibitor actinomycin D and the decay of aromatase mRNA was measured from 6 to 48 h. At all time points tested, aromatase mRNA levels were significantly lower in TGF-β1-treated cells than in the control cells (Fig. 4). The half-life of aromatase transcript was approximately 36 h and 20 h, respectively, in the control and TGF-β1-treated cells (Fig. 4B), suggesting that TGF-β1 decreased the half-life of aromatase mRNA by 16 h.