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Mol. Hum. Reprod. Advance Access originally published online on December 9, 2005
Molecular Human Reproduction 2005 11(12):847-852; doi:10.1093/molehr/gah242
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© The Author 2005. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Transforming growth factor ß1 regulates angiotensin II type I receptor gene expression in the extravillous trophoblast cell line SGHPL-4

C.L. Tower1, S.L. Chappell1, K. Morgan1, N. Kalsheker1, P.N. Baker2 and L.J. Morgan1,3

1Division of Clinical Chemistry, Institute of Genetics, School of Molecular Medical Sciences, University Hospital, Nottingham and 2Maternal and Fetal Health Research Centre, St. Mary’s Hospital, Whitworth Park, Manchester, UK

3 To whom correspondence should be addressed at: Clinical Chemistry, A Floor West Block, University Hospital, Nottingham NG7 2UH, UK. E-mail: linda.morgan{at}nottingham.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The angiotensin II type 1 (AT1) receptor, transforming growth factor ß1 (TGFß1) and Oncostatin M (OSM) control key pathways that may be important during placentation. Although interactions between them exist in other tissues, trophoblast cells have not been investigated. Extravillous trophoblast cells, SGHPL-4, were stimulated with 10 ng/ml TGFß1 ± 100 ng/ml OSM for 24 h. Real-time PCR showed that AT1 expression increased 2.76-fold [95% confidence interval (CI) = 1–6.74, P = 0.05] in response to TGFß1 and 4.21-fold (95% CI = 1.33–11.76, P = 0.03) with TGFß1 + OSM. Luciferase reporter gene constructs containing three haplotypes of the 59 flanking region of the AT1 receptor gene were transfected into SGHPL-4 and HepG2 cells and stimulated with 0.1, 1 and 10 ng/ml TGFß1 and 50 ng/ml OSM. Responses were dose and cell dependent. Luciferase activity increased in HepG2 cells in response to TGFß1 alone or together with OSM (P < 0.001); transcriptional activation differed between AT1 receptor gene haplotypes. In SGHPL-4 cells, luciferase activity was reduced on exposure to low concentrations of TGFß1 or high concentrations of TGFß1 combined with OSM (P = 0.003); the response was unaffected by haplotype. Interaction between AT1 and TGFß1 is a novel observation in trophoblast and suggests new avenues for the study of placentation.

Key words: angiotensin type 1 receptor gene/gene regulation/transforming growth factor ß1/trophoblast


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Transforming growth factor ß1 (TGFß1) is known to regulate many biological processes such as cellular proliferation, differentiation and migration, in addition to production of the extracellular matrix (Piek et al., 1999Go). It exists in three distinct isoforms, TGFß1, –ß2 and –ß3, all of which are present in the human placenta and are likely to play an important role in placental development (Caniggia et al., 1999Go; Simpson et al., 2002Go). Experimental evidence suggests that TGFß1 inhibits trophoblast invasion (Graham and Lala, 1991Go), proliferation (Graham et al., 1992Go) and migration (Irving and Lala, 1995Go) and limits degradation of the extracellular matrix by extravillous trophoblast (Graham, 1997Go). Since TGFß1 is produced predominantly by the maternal decidua, these authors proposed that it provides a mechanism to prevent over-invasion.

All components of the renin angiotensin system have been described within the utero-placental unit (Paul et al., 1993Go). In particular, angiotensin II type I (AT1) receptors are present in the placenta throughout pregnancy (Cooper et al., 1999Go) and have been localized to cytotrophoblast, syncytiotrophoblast, extravillous trophoblast and perivascular cells (Li et al., 1998Go). It is unclear whether the AT1 receptor has a role in early placentation. Work in other cell systems has demonstrated that, in addition to its well-known effects in the cardiovascular system and on salt and water homeostasis, it also mediates processes likely to be important for successful placentation, including cellular proliferation and growth (Dinh et al., 2001Go) and angiogenesis (Fernandez et al., 1985Go; Le Noble et al., 1991Go). Recent work has suggested that angiotensin II, acting through AT1 receptors, reduced the invasion of trophoblast cells (HTR-8/SVneo) into a Matrigel-coated filter, probably because of increased generation of the plasminogen activator inhibitor-1 (PAI-I) (Xia et al., 2002Go).

There is growing evidence that the AT1 receptor plays a role in the pathogenesis of fetal growth restriction and pre-eclampsia. Lower levels of placental AT1 receptor binding sites have been described in both conditions (Knock et al., 1994Go; Li et al., 1998Go), and polymorphisms within the AT1 receptor gene (AGTR1) have been associated with pre-eclampsia and fetal growth restriction (Morgan et al., 1998Go; Tower et al., in pressGo). Of particular interest, we recently reported a significant distortion of transmission of maternal AGTR1 haplotypes in fetal growth restriction (Tower et al., in press).

Interactions between the renin-angiotensin system and TGFß1 have been well described in renal and cardiac tissues, with most of the studies describing the effect of angiotensin II on TGFß1 expression (Kim et al., 1995Go; Rosenkranz, 2004Go). Reports on the effect of TGFß1 on the AT1 receptor are few and conflicting. TGFß1 was recently shown to reduce angiotensin II binding in rabbit renal cells (Park and Han, 2002Go). In contrast, earlier work in human adrenal cells showed an increase in angiotensin II binding and AT1 receptor mRNA in response to TGFß1 (Lebrathon et al., 1994Go). Thus the effects may be both species and cell specific.

Pregnancy is a state of altered immune competence, and cytokines are thought to be important intercellular signal molecules required during implantation and placental development (Robertson et al., 2003Go). The multifunctional cytokine Oncostatin M (OSM) is produced by T lymphocytes and monocytes and is similar in structure to leukaemia inhibitory factor (LIF) and interleukin-11 (IL-11). It acts through the JAK-signal transducers and activators of transcription (STAT) pathway to activate transcription factors regulating the type II acute phase response genes (Moshage, 1997Go). Healthy pregnant women have raised plasma levels of OSM, consistent with the heightening of some elements of the immune response during pregnancy (Ogata et al., 2000Go). It is expressed in chorionic and decidual tissue throughout pregnancy and stimulates production of human chorionic gonadotrophin by chorionic tissue (Ogata et al., 2000Go). Its precise role is yet to be defined, although experiments in genetically manipulated mice have shown that similar molecules such as LIF and IL-11 play key roles in implantation (Robb et al., 2002Go). Interestingly, a synergistic effect between OSM and TGFß1 on gene expression in other tissues has been described (Boutten et al., 1998Go).

Given the likely roles of the AT1 receptor, TGFß1 and OSM during placentation, this study was conducted to investigate the interaction between them in the extravillous trophoblast cell line SGHPL-4. The response of the AT1 receptor gene to stimulation with OSM and TGFß1 was assessed by real-time PCR measurement of AGTR1 mRNA. To explore the molecular mechanism of transcriptional regulation of AGTR1 by OSM and TGFß1, luciferase reporter gene constructs containing 990 base pairs from the 5' end and flanking region of the gene were transfected into SGHPL-4 cells and the hepatoma cell line HepG2. This region includes three DNA sequences with homology to binding sites for activated Smad factors, the effectors of TGFß1. It also includes a binding site for STAT3, a transcription factor activated by OSM. Furthermore, three haplotypes common in western European populations are defined by four single nucleotide polymorphisms in the 5' flanking region. Constructs representing each of these haplotypes were therefore examined by luciferase reporter gene assays to assess haplotype-specific differences in transcriptional regulation.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Cell culture
The extravillous trophoblast cell line, SGHPL-4, was kindly donated by Professor Guy Whitley of St. George’s Hospital Medical School, London. The hepatoma cell line (HepG2) was obtained from the European Collection of Cell Cultures (Salisbury, UK). HepG2 cells were utilized for comparison as they were known to express the AT1 receptor, and a transfection protocol was in regular use in our laboratory. Reagents were obtained from GIBCO Invitrogen Life Technologies (Paisley, UK) unless stated otherwise.

SGHPL-4 cells were cultured in nutrient mixture F-10 Ham with L-glutamine (1 mM) containing 10% fetal bovine serum (FBS), 2% penicillin/streptomycin (10 000 U/ml penicillin, 10 000 µg/ml streptomycin) and 1% amphotericin B (250 µg/ml) using previously described methods (Crocker et al., 2001Go). HepG2 cells were cultured as previously described (Morgan et al., 2001Go).

mRNA expression studies by real-time PCR
Cells were washed in 1x phosphate-buffered saline (PBS) before stimulation. Each stimulant was added to 6 ml of complete media to give final concentrations of 10 ng/ml TGFß1 and 100 ng/ml OSM (both R&D Systems, Abingdon, UK). Cells were stimulated for 24 h since effects have been previously described at this time-point (Graham, 1997Go; Boutten et al., 1998Go; Tse et al., 2002Go); a flask containing 6 ml complete media only was included with each experiment to allow comparison to basal levels. A minimum of three independent experiments were conducted. After 24 h, cells were washed and trypsinized.

RNA was extracted from the resulting cell pellet with the Absolutely RNATM RT–PCR Miniprep kit (Stratagene, La Jolla, CA, USA) using the manufacturer’s protocol with modifications for small samples. The concentration and purity of the resulting RNA were estimated by measuring absorbance at 260 nm and 280 nm taking the average of two or three readings. RNA was stored at –80°C.

First strand cDNA synthesis was achieved by the addition of 200 ng of random primers (Promega, Southampton, UK) to 1 µg of RNA per sample, made up to a volume of 26.5 µl with water. Samples were held at 70°C for 10 min to allow primer annealing, then cooled on ice and spun. Reverse transcription was performed by the addition of 50 mM Tris–HCl, pH 8.3, 50 mM KCl, 4 mM MgCl2, 10 mM DTT, 1 mM dNTPs, 20 U ribonuclease inhibitor and 200 U RevertAidTM H M-MuLV Reverse Transcriptase (MBI Fermentas, Sunderland, UK) to a volume of 40 µl per sample. This was prepared in a master mix to limit variability. Samples were incubated at 25°C for 10 min, 42°C for 1 h then 70°C for 10 min.

Real-time PCR was used to investigate AT1 receptor expression in response to TGFß1 and OSM. Normalization was calculated relative to the expression of thioredoxin reductase (TXN) since constant low–medium levels of expression of this gene have been described in a range of adult and fetal tissues (Warrington et al., 2000Go). AT1 was amplified using forward primer 5'-TGTCAGCATTGATCGATACC and reverse primer 5'-TGACTTTGGCTACAAGCATT. TXN was amplified using forward primer 5'-TTGGAGCATCCTATGTCG and reverse primer 5'-CTAACCATAACAGTGACGCC. The molecular beacon sequence (probe) for AT1 was 5'HEX-CGACCGAAATGAAGTCCCGCCTTCGACTCGGTCG-DABCYL and for TXN 5'FAM-CAGCTGCGTGCGCTGGATTTCTTGCTGGCAGCTG-DABCYL (MWG Biotech, Milton Keynes, UK). Real-time PCR was conducted on the MX4000TM Real Time Multiplex PCR instrument (Stratagene) and optimal conditions were determined experimentally.

Each PCR was conducted in duplicate using 1 g cDNA template. A standard curve of known serial dilutions (1:2, 1:4, 1:8, 1:16, 1:32, 1:64) of SGHPL-4 cDNA was included with each set of reactions. AGTR1 and TXN were amplified using the following conditions (Stratagene Brilliant® Quantitative PCR Core Reagent kit): 1x Core PCR buffer, 6 mM MgCl2, 200 nM probe, 0.8 mM dNTP, 30 nM ROX reference dye, 1 U SureStart® Taq, with 300 nM each primer for AT1 or 100 nM TXN forward primer and 300 nM TXN reverse primer, made up to 50 µl with water. Samples were incubated at 95°C for 5 min followed by 35 cycles of 95°C for 30 s, 53°C for 1 min and 72°C for 1 min. Experimental samples were compared to the standard curve to estimate the amount of starting template for each gene.

Luciferase reporter gene assays
Cloning of the AGTR1 promoter and 5' flanking region
The AT1 receptor gene (AGTR1) is numbered throughout relative to GenBank AF245699 [GenBank] .1. Reporter gene constructs containing 990 base pairs of AGTR1 (g.4823–5813), including 34 base pairs of the 5' end of exon 1 and 956 base pairs of the 5' flanking region and the luciferase reporter gene were generated using the Gateway cloning system (Invitrogen, Paisley, UK). A diagram of the region is given in Figure 1. This region contains polymorphisms defining the three common haplotypes in UK white Caucasians (Plummer et al., 2004Go). Separate constructs were generated for each haplotype. Haplotype A: g.4955T, g.5052T, g.5245C, g.5612A; haplotype B: g.4955T, g.5052T, g.5245T, g.5612A and haplotype C: g.4955A, g.5052G, g.5245T, g.5612G. This region of AGTR1 was amplified using forward primer 5'attB1-TGCAATTGGCATATCCATCA and reverse primer 5'attB2-GGTCCAGACGTCCTGTCACT using genomic DNA template from homozygous individuals. The primers with Gateway recombination sites attB1 and attB2 were obtained from Invitrogen. These PCR products were used to generate Gateway entry clones using the Gateway BP reaction, according to the manufacturer’s instructions. The attB1 and attB2 sites enable a site-specific recombination of the PCR product, in the correct orientation, into the donor plasmid. This DNA was then transformed into library efficiency DH5{alpha} Escherichia coli cells (Invitrogen), as previously described (Morgan et al., 2001Go). The transformation mix was spread onto prewarmed CG (Circle Grow rich bacterial growth media, Bio 101 Inc, Anachem, Luton, UK)—agar plates made with 4 g/100 ml CG, 1.5 g/100 ml agar and 50 µg/ml kanamycin and grown overnight at 37°C. Single colonies of each transformant were inoculated into 5 ml CG containing 50 µg/ml kanamycin and grown overnight in a shaking incubator at 37°C. Plasmid DNA was extracted from 1.5 ml of culture using the QIAprep® spin mini prep kit (Qiagen, Crawley, UK) following the manufacturer’s protocol. Presence of the insert was confirmed by PCR using primers given above (without the att sites) and direct sequencing of plasmid DNA using the sense primer 5'-AGCAGCAACGCCCCTCACTA.


Figure 1
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  		Figure 1. Diagram of the AGTR1 construct (not to scale). Numbering is relative to reference sequence AF245699 [GenBank] .1, with exon 1 beginning at g.5779. The black lines represent the location of the polymorphisms g.4955T>A, g.5052T>G, g.5245C>T and g.5612A>G. The hatchings represent transcription factor binding sites for Fast-1 Smad at g.4905–4918 and g.5240–5254, Smad3 at g.5805–5813 and signal transducers and activators of transcription (STAT) at g.5193–5211.

 

The AGTR1 5' flanking region was then introduced into the luciferase expression clone using the Gateway LR reaction, following the manufacturer’s instructions (Gateway cloning system). In common with the BP reaction, this reaction uses att recombination sites. The destination vector was generated by adapting the pGL3-enhancer vector (Promega) to contain the Gateway cloning sites upstream of the luciferase reporter gene. The pGL3-enhancer vector contains the SV40 enhancer downstream of the luc+ reporter gene. The resulting DNA was then transformed into library efficiency DH5{alpha} E. coli cells (Invitrogen), with substitution of 50 µg ampicillin for kanamycin. Single colonies of each transformant were inoculated into 5 ml CG containing 50 µg/ml ampicillin and grown overnight in a shaking incubator at 37°C. Plasmid DNA was obtained as above and the presence of the insert was confirmed initially by digesting the plasmid clone with restriction enzymes BamHI and NcoI. Plasmids were then sequenced throughout the full length using sense primer 5'-CTAGCAAAATAGGCTGTCCC and antisense primer 5'-CTTTATGTTTTTGGCGTCTTCC (Promega). This confirmed both the presence of the insert in the pGL3-enhancer vector in the correct orientation and the presence of the desired haplotypes. Sequencing revealed two PCR-generated single base changes in two of the constructs that did not occur in the genomic sample. These were G>A at position 648 in haplotype A (g.5471 G>A AF 245699.1) and A>T at position 5 in haplotype C (g.4827 A>T AF245699 [GenBank] .1).

Transfection and stimulation of SGHPL-4 and HepG2 cell lines
Transfection of the luciferase reporter vector containing the putative AGTR1 promoter region into SGHPL-4 and HepG2 was conducted using Promega’s TfxTM-20 reagents using previously described methods (Morgan et al., 2001Go). Cells were also transfected with the pRL-SV40 control reporter (pRL), containing Renilla luciferase (Promega), to control for transfection efficiency. Each experiment included the three AGTR1 haplotypes, the pGL3-control vector (containing both the SV40 promoter and enhancer) as a positive control and the pGL3-enhancer vector alone to provide a baseline for comparison of the experimental construct. In a separate set of three experiments, cells transfected with the pGL3-enhancer alone were also stimulated with TGFß1 and OSM. After transfection, the old medium was aspirated from the wells, cells washed in 1x PBS and stimulated for 24 h with 1 ml warmed serum-free media containing the following stimulants: basal (no additive), TGFß1 0.1 ng/ml, TGFß1 1 ng/ml, TGFß1 10 ng/ml, TGFß1 10 ng/ml + OSM 50 ng/ml and OSM 50 ng/ml. Within a single experiment, each condition was conducted in triplicate. Cells were harvested and the Luciferase assay (Promega) performed as described in the manufacturer’s protocol with minor modifications (Morgan et al., 2001Go). Sequential readings of firefly and renilla luciferase reporter activities were taken using the Turner Designs Model TD-20/20 Luminometer (Turner Biosystems, Sunnyvale, CA, USA). Light output was recorded in relative luminescence units (RLU) and the ratio of firefly : renilla luciferase activity used to represent normalized luciferase activity. An average of the triplicate wells from a single experiment was used in the analysis and three independent experiments of each condition were conducted.

Statistical analysis
Statistical tests were performed using SPSS for Windows version 11.0.1. For the RNA expression experiments, the ratio of stimulated expression to basal expression was calculated to give fold over basal. There was no response of TXN expression to any stimulant, hence correction to this gene was used to normalize for inconsistencies between experiments. For transfection analysis, the ratio of firefly to Renilla luciferase was used. The promoter activity of each construct was normalized to the activity of the pGL3-enhancer. Therefore, the reporter gene activity conferred by the presence of the construct was expressed as fold change over pGL3-enhancer. To investigate the effect of TGFß1 and OSM stimulation, the ratio of stimulated to unstimulated (basal) activity was used. To normalize the distribution of data, all ratios were log transformed. Parametric tests were performed using Student’s t-test and univariate analysis of variance in a general linear model, with haplotype and stimulation conditions as independent variables.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
AT1 expression in response to TGFß1 and OSM
In SGHPL-4 cells there was a 2.76-fold [95% confidence interval (CI) = 1–6.74, P = 0.05] increase in expression of AT1 mRNA measured by real-time PCR in response to stimulation with 10 ng/ml TGFß1 and a 4.21-fold (95% CI = 1.33–11.76, P = 0.03) increase in AT1 response to 10 ng/ml TGFß1 and 100 ng/ml OSM together (Figure 2).


Figure 2
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  		Figure 2. Angiotensin II type 1 (AT1) mRNA expression in SGHPL-4 cells in response to stimulation with transforming growth factor ß1 (TGFß1) and Oncostatin M (OSM). Cells were stimulated with 10 ng/ml TGFß1 ± 100 ng/ml OSM. Results are expressed as the mean fold change over basal (unstimulated) expression and corrected to the expression of the housekeeping gene thioredoxin reductase. Error bars represent ±SE of the mean.

 

Effect of AGTR1 haplotype on reporter gene activity
The presence of the AGTR1 construct within the pGL3-enhancer vector increased luciferase activity 39- to 117-fold (P < 0.001, t-test; Figure 3). This is of comparable magnitude to the pGL3-control vector containing the SV40 promoter. The AGTR1 construct had approximately double the activity in the SGHPL-4 cells compared to HepG2 under basal conditions (P < 0.01, t-test). There were no differences in luciferase activity between the three AGTR1 haplotypes (A, B and C) under basal conditions in either cell line [P > 0.40, analysis of variance (ANOVA)].


Figure 3
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  		Figure 3. Luciferase reporter gene activity of the AGTR1 haplotypes A, B and C. Activity is given as the mean fold response relative to the PGL3-enhancer construct. The pGL3-control (pGL3C) construct is a positive control included for comparison. Each experimental construct contained the following AGTR1 haplotypes: A, g.4955T, g.5052T, g.5245C, g.5612A; B, g.4955T, g.5052T, g.5245T, g.5612A and C, g.4955A, g.5052G, g.5245T, g.5612G. Error bars represent ±SE of the mean.

 

Response of the AGTR1 construct to TGFß1 and OSM stimulation
Stimulation of the pGL3-enhancer showed no significant response with the exception of OSM stimulation of HepG2 cells (1.3-fold, P = 0.002). Therefore, when considering stimulation of the AGTR1 construct in HepG2 with OSM, this is calculated as fold response relative to pGL3-enhancer stimulated with OSM.

In both cell lines a dose-dependent response to TGFß1 stimulation was observed. In HepG2 cells, increased luciferase activity was seen with increasing concentrations of TGFß1 (P < 0.001, trend analysis; Figure 4). In SGHPL-4 cells, the dose response was characterized by reduced luciferase activity on exposure to 0.1 ng/ml TGFß1, with significant increases in activity in response to increased concentrations of TGFß1 (P = 0.008, trend analysis; Figure 5). There were no differences in the responses between haplotypes in SGHPL-4 (P = 0.23). In HepG2 cells, haplotype C (g.4955A, g.5052G, g.5245T, g.5612G) showed a significantly lower response to stimulation with TGFß1 (P = 0.003; Figure 4).


Figure 4
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  		Figure 4. Luciferase reporter gene activity of the AGTR1 haplotypes in response to transforming growth factor ß1 (TGFß1) and Oncostatin M (OSM) stimulation in HepG2 cells. Bars represent fold response relative to basal (unstimulated expression). Error bars represent ±SE of the mean. AGTR1 haplotypes: A, g.4955T, g.5052T, g.5245C, g.5612A; B, g.4955T, g.5052T, g.5245T, g.5612A and C, g.4955A, g.5052G, g.5245T, g.5612G. *, P = 0.01 and **, P < 0.001 (trend analysis) compared with basal activity. OSM = OSM and TGF = TGFß1.

 

Figure 5
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  		Figure 5. Luciferase reporter gene activity of AGTR1 haplotypes in response to transforming growth factor ß1 (TGFß1) and Oncostatin M (OSM) stimulation in SGHPL-4 cells. Bars represent fold response relative to basal (unstimulated expression). Error bars represent ±SE of the mean. AGTR1 haplotypes: A, g.4955T, g.5052T, g.5245C, g.5612A; B, g.4955T, g.5052T, g.5245T, g.5612A and C, g.4955A, g.5052G, g.5245T, g.5612G. *, P < 0.01 and **, P < 0.005 (trend analysis) compared with basal activity. OSM = OSM and TGF = TGFß1.

 

OSM alone (50ng/ml) increased luciferase activity in HepG2 (P = 0.01; Figure 4) but not in SGHPL-4 (P = 0.18; Figure 5). In HepG2 cells, there was an increase in luciferase activity when stimulated with 50 ng/ml OSM and 10 ng/ml TGFß1 together compared with basal conditions (P < 0.001; Figure 4), but this did not differ significantly from the response to 10 ng/ml TGFß1 alone (P = 0.76). A fall in luciferase activity relative to basal conditions following stimulation with TGFß1 and OSM was seen in SGHPL-4 cells (P = 0.003; Figure 5), but again this was not significantly different from the response to TGFß1 alone (P = 0.18). Haplotype did not significantly affect response to stimulation with OSM, with or without TGFß1, in SGHPL-4 (P = 0.23) or HepG2 (P = 0.16).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
This is the first study to investigate the interaction between TGFß1 and AT1 receptors in cells relevant to human placentation. These experiments have demonstrated that AGTR1 mRNA expression in the extravillous trophoblast cell line SGHPL-4 is up-regulated by TGFß1 acting alone (2.76-fold increase) or in combination with OSM (4.21-fold increase). Up-regulation by OSM acting alone was not statistically significant, nor did the addition of OSM to TGFß1 result in a significant increase in AGTR1 mRNA expression, indicating that OSM plays a minor role if any in the regulation of this gene in these cells under these conditions. Subsequent experiments using luciferase reporter gene constructs containing 990 base pairs from the 5' end and flanking region of AGTR1 indicated that this region is a powerful activator of transcription in SGHPL-4 cells, resulting in a more than 100-fold increase in reporter gene activity under basal conditions. Furthermore, transcriptional activation in SGHPL-4 cells was two-fold higher than that observed in HepG2 cells, suggesting the operation of cell-specific transcription factors. Stimulation of transfected cells confirmed that this region is responsive to TGFß1. In HepG2 cells, there was a dose-related up-regulation of luciferase reporter gene expression in response to increasing concentrations of TGFß1. In contrast, in SGHPL-4 cells there was an unexpected down-regulation of reporter gene expression at a low concentration (0.1 ng/ml) of TGFß1, suggesting the presence of cell-specific repressor activity. Nevertheless, increasing concentrations of TGFß1 resulted in relative increases in reporter gene expression in SGHPL-4 cells, achieving levels approximating basal expression at a TGFß1 concentration of 10 ng/ml. The response to OSM also showed cell-specific differences, with up-regulation of luciferase activity observable in HepG2 cells, and down-regulation recorded in SGHPL-4 cells in response to OSM combined with TGFß1.

TGFß1 is likely to be an important growth factor for successful placentation. It interacts with three receptor subtypes, I, II and III, to produce activation and phosphorylation of the Smad proteins 2 and 3. These then oligomerize with Smad4 to produce the Smad complex. The Smad complex translocates to the nucleus and associates with a DNA binding protein known as forkhead activin signal transducer-1 (Fast-1). The complex then binds to specific DNA sequences leading to transcriptional activation of the target gene (Massagué, 1998Go; Goumans et al., 2003Go). Normal extravillous trophoblast expresses all three TGFß1 receptors as well as members of the Smad family signalling molecules (Xu et al., 2001Go). The AGTR1 construct used in these experiments contains three regions with high homology to sequences involved in TGFß1 signal transduction (MatInspector software, http://www.genomatix.de) (Figure 1). Two consensus binding site sequences for Fast-1 Smad interacting protein are located at g.4905–4918 and g.5240–5254 and a consensus sequence for Smad3 binding is located at g.5805–5813 (Figure 1). Some or all of these sites may be involved in the AGTR1 response to TGFß1 observed in reporter gene experiments. Four further consensus sequences for Fast-1 Smad binding sites and one potential Smad4 binding site are present within 680 bases upstream of the constructs examined in this study, and these may also be active in the intact gene. This may explain the discordance between AGTR1 mRNA up-regulation observed in SGHPL-4 cells in response to 10 ng/ml TGFß1 and the lack of luciferase reporter gene response in the same cells under similar conditions.

An alternative explanation for this paradox is that TGFß1 is influencing the other stages in the control of mRNA levels. Several levels of control are exerted to produce steady state mRNA levels, including the rate of transcription initiation, transcription elongation, pre-mRNA splicing, mRNA stability, mRNA transport and the rate of translation. The two main points of control are generally believed to be the rate of transcription and mRNA stability. Reporter gene assays only assess functional activity of the insert, as an indication of the rate of transcription initiation. Hence, control of mRNA stability may be contributing to the rise in AT1 mRNA levels in response to TGFß1. Indeed, experimental evidence exists suggesting that rodent AT1 receptors are regulated by post-transcriptional mechanisms such as control of translation and RNA stability (Lassègue et al., 1995Go; Krishnamurthi et al., 1998Go; Nickenig et al., 1998Go).

Interestingly, TGFß1 has been shown to have a dose-dependent effect on angiogenesis, and Smad3 is an important mediator (Goumans et al., 2003Go; Nakagawa et al., 2004Go). TGFß1 has a modulating effect on other angiogenic growth factors including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Low concentrations (0.1–1 ng/ml) of TGFß enhance VEGF- and bFGF-induced migration and invasion whereas high concentrations (10 ng/ml) are inhibitory (Pepper, 1997Go). TGFß1 is generally acknowledged to have an anti-migratory, anti-proliferative and anti-invasive effect on trophoblast (Lala and Chakraborty, 2003Go). There are few reports on the effects of the AT1 receptor in trophoblast. Angiotensin II, acting through the AT1 receptor, was shown to increase levels of PAI-1 and reduce trophoblast invasion (Xia et al., 2002Go). These experiments used a relatively high concentration of angiotensin II (100 nM) and require confirmation in primary cells. However, these findings suggest that TGFß1 and the AT1 receptor may interact to limit trophoblast invasion within the placenta.

There is also growing evidence suggesting that the AT1 receptor may play a role in the pathogenesis of pre-eclampsia and fetal growth restriction. Pre-eclampsia has been associated with maternal autoantibodies to the AT1 receptor (Wallukat et al., 1999Go). These autoantibodies may bind to trophoblast AT1 receptors and result in shallow trophoblast invasion (Xia et al., 2003Go). In addition, a recent study of genetically manipulated mice has suggested that the AT1a receptor plays a key role in the development of pregnancy-induced hypertension and fetal growth restriction (Saito et al., 2004Go). Cross-mating human angiotensinogen transgenic female mice with human renin male transgenic mice generated a phenotype of late gestation hypertension and fetal growth restriction. When the AT1a receptor was knocked out in these mice, the hypertension and growth restriction were much improved. Although fundamental differences exist between mouse and human placentae these findings raise the possibility of an important role for the AT1 receptor.

We have recently reported distortion of transmission of maternal AGTR1 haplotypes represented by the constructs used in this study in fetal growth restriction (Tower et al., in press). The consensus sequence for the Fast-1 Smad interacting protein at g.5240–5254 is introduced by the presence of the T allele at the polymorphic site g.5245C>T (Figure 1). The g.5245T variant was present in both haplotypes B and C. It is therefore of interest to note that in HepG2 cells transcriptional activation in response to TGFß1 was less in constructs containing haplotypes B and C than those containing haplotype A; in the case of haplotype C, this achieved statistical significance. The relevance of these findings to AT1 expression in liver cells in vivo remains to be determined. Transcriptional activation by TGFß1 in SGHPL-4 cells was not affected by the haplotype of the reporter gene construct. It is not clear whether this is a further example of cell-specific differences or a limitation of the experimental model. Real-time PCR showed no statistically significant difference between AGTR1 mRNA expression in response to TGFß1 alone compared with TGFß1 and OSM together (P = 0.28). This suggests that the combined effect may largely be due to TGFß1. Paradoxically, the luciferase reporter gene assays in SGHPL-4 cells suggested that a combination of TGFß1 and OSM caused down-regulation of transcription. It is worth noting that the 5' flanking region of AGTR1 contains a region with high homology to a STAT binding site at nucleotides g.5193–5211 (Figure 1), and a further STAT binding site upstream of the constructs is examined in this study, which may mediate different OSM effects on AT1 expression.

In conclusion, this study has presented some novel findings suggesting a cell-specific interaction between AT1 receptors and TGFß1 in extravillous trophoblast that may be important in successful placentation and therefore, relevant for disorders such as fetal growth restriction and pre-eclampsia. Further detailed mapping of TGFß1 responsive elements in the 5' region of the AGTR1 gene is underway in our laboratory.


   Acknowledgements
 
Dr Clare Tower was funded by a Wellcome Clinical Research Training Fellowship.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on August 10, 2005; revised on October 21, 2005; accepted on October 26, 2005


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