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in the feed-forward induction of 11ß-hydroxysteroid dehydrogenase type 1 expression by cortisol in human amnion fibroblasts
1 School of Life Sciences, Fudan University, 220 Handan Road, Shanghai 200433, People’s Republic of China
2 Department of Obstetrics and Gynecology, No. 401 Hospital, Qingdao, China
3 Fetal and Maternal Care Hospital of Changning District, Shanghai, China
4 Department of Obstetrics and Gynecology, University of Cincinnati, Cincinnati, Ohio, USA
(Correspondence should be addressed to K Sun; Email: sunkang2000{at}yahoo.com)
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Abstract |
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but not C/EBPß could bind this promoter region upon cortisol stimulation of amnion fibroblasts. In conclusion, we demonstrated that GR and C/EBP were involved in cortisol-induced 11ß-HSD1 mRNA expression via binding to 11ß-HSD1 promoter in amnion fibroblasts, which may cast a feed-forward production of cortisol in the fetal membranes at the end of gestation. |
Introduction |
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GCs are important hormones in pregnancy. The placenta, fetal membranes, and the fetus are all GC targets during pregnancy. GCs have been shown to stimulate rather than inhibit prostaglandin and corticotrophin-releasing hormone production in human fetal membranes and placenta (Casey et al. 1985, Potestio et al. 1988, Karalis et al. 1996, King et al. 2002, Sun et al. 2003). These paradoxical effects of GCs on human fetal membranes and placenta are believed to be part of the positive feedback loops involved in human parturition (Whittle et al. 2001). It is well known that GCs are indispensable hormones for cell differentiation and organ maturation in the fetus (Gonzales et al. 1986), but excess of GCs impedes fetal growth (Shams et al. 1998, Seckl et al. 2000). In consideration of all these above effects of GCs in pregnancy, understanding the metabolism of GCs by 11ß-HSD could undoubtedly have an important impact on the outcome of pregnancy.
Our previous studies have shown that 11ß-HSD1 and 11ß-HSD2 are differentially distributed in human fetal membranes and placenta, with 11ß-HSD1 mainly distributed in the fetal membranes and 11ß-HSD2 exclusively distributed in the placenta (Sun et al. 1997a,b). 11ß-HSD2 in the placenta provides a barrier for maternal cortisol entering the fetal circulation (Seckl et al. 2000). Although the physiological functions of 11ß-HSD1 in the fetal membranes are less clear, it is believed that 11ß-HSD1 in the fetal membranes may either provide an extra-adrenal source of cortisol for the fetus or simply amplify the local actions of cortisol in the fetal membranes (Murphy 1977, Alfaidy et al. 2003).
Human amnion fibroblasts are enriched not only in 11ß-HSD1 but also in prostaglandin-synthesizing enzymes (Sun et al. 2003). Therefore, it is particularly of interest to examine the regulation of 11ß-HSD1 expression in these cells in terms of prostaglandin synthesis. Our previous study has shown that dexamethasone, the synthetic GC, could concurrently increase the expressions of 11ß-HSD1 and the key enzymes involved in prostaglandin synthesis such as cytosolic phospholipase A2 and cyclooxygenase 2 in human amnion fibroblasts (Sun & Myatt 2003, Sun et al. 2003). However, the feedback effect of cortisol, the endogenous product of 11ß-HSD1-catalyzed reaction, on 11ß-HSD1 expression has not been characterized in human amnion fibroblasts.
Sequence analysis revealed a putative GC-response element (GRE) and several consensus CCAAT boxes in human 11ß-HSD1 promoter (Tannin et al. 1991), which may bind either GR or CCAAT/enhancer-binding proteins (C/EBPs) respectively. However, we do not know whether the putative GRE and CCAAT boxes are truly involved in the regulation of 11ß-HSD1 expression by GCs. C/EBPs have been reported to be closely related to the actions of GCs in a number of tissues (Rudiger et al. 2002, Roth et al. 2004, Yang et al. 2005a,b, Woltje et al. 2006). Although C/EBPs have been shown to be involved in basal expression of human or mouse 11ß-HSD1 gene transcription (Williams et al. 2000, Gout et al. 2006), it is not known whether C/EBPs play any roles in the regulation of 11ß-HSD expression by GCs. To address the above issues, we examined the feedback regulation of 11ß-HSD1 mRNA expression by cortisol in cultured human amnion fibroblasts and explored the underlying molecular mechanisms.
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Materials and Methods |
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Human fetal membranes were collected from patients undergoing elective cesarean section at term according to the guidelines set forth in a protocol that is in compliance with the Institutional Review Board of Fudan University and University of Cincinnati. Amnion was peeled off chorion and washed thoroughly in cold PBS (pH 7.5) to get rid of the residual blood. Amnion was then digested with 0.125% trypsin (Sigma) and 0.02% DNase (Sigma) twice for 30 min at 37 °C. The digestion media were discarded, and the remaining amnion tissue was washed vigorously with PBS to get rid of residual epithelial cells. The remaining tissue was then subject to digestion with 0.1% collagenase (Roche) at 37 °C for1 h. The digest ion medium was collected and centrifuged at 2300 r.p.m. Cell pellets were resuspended in Dulbecco’s modified Eagle’s medium (DMEM; Sigma) and loaded onto a discontinuous Percoll (Amersham Biosciences) gradient column (5, 20, 40, and 60%). The gradient column was centrifuged at 2500 g. and a single band of cells around 20% Percoll concentration was collected. Cells were plated at a density of 1.5x106 cells per well in a six-well plate in DMEM without phenol red containing 10% fetal calf serum (FCS) and antibiotic–antimycotic solution. The cell identity prepared as described above has been verified previously and more than 90% of the cells are fibroblasts (Sun et al. 2003). The amnion fibroblasts were cultured for 2–3 days at 37 °C and 5% CO2 in air. On the day of treatments, the medium was changed to FCS-free medium and the cells were then treated with cortisol (0.01, 0.1, and 1 µM) for 24 h with and without the GR antagonist RU486 (1 µM), or in the presence of transfection of C/EBP-specific dominant-negative expression CMV500 plasmid (AC/EBP) or empty CMV500 plasmid (courtesy of Dr Vinson C, National Cancer Institute, Center for Cancer Research, NIH). AC/EBP has been shown to have global inhibition on all C/EBPs (Olive et al. 1996). The cells were transfected with 0.5 µg/well CMV500-AC/EBP or empty CMV500 plasmids using lipofectamine 2000 (Invitrogen) in Opti-MEM (Gibco) 12 h before cortisol treatment. In another set of experiments, the cells were also treated with the mRNA transcription inhibitor 5,6-dichlorobenzimidazole riboside (DRB, 75 µM; Sigma) 1 h prior to cortisol treatment and then DRB treatment was continued with cortisol treatment for another 24 h. At the end of the above treatments, cells were collected for total RNA extraction using RNeasy kit (Qiagen). RNA (1.0 µg) was reverse transcribed with oligo(dT)12–18 primers using Superscript II kit (Life Technologies Inc.) for subsequent measurement of 11ß-HSD1 mRNA level with real-time PCR (RT-PCR).
Measurements of 11ß-HSD1 mRNA levels with RT-PCR
To measure 11ß-HSD1 mRNA levels in response to the above treatments, quantitative RT-PCR (qRT-PCR) analysis was performed with the primer sequences as follows: sense 5'-CTCAACCACATCACCAACAC-3' and antisense 5'-TCTGATGGAGGAGAAGAACC-3'. To control sampling errors, qRT-PCR for housekeeping gene ß-actin was routinely performed on each sample. The primer sequences for human ß-actin were as follows: sense 5'-GGGAAATCGTGCGTGA-CATTAAG-3' and antisense 5'-TGTGTTGGCGTACAGG-TCTTTG-3'. Reaction solution of qRT-PCR consisted of 2.0 µl diluted RT-PCR product, 0.2 µM of each paired primers and power SYBR Green PCR Master Mix (Toyobo, Osaka, Japan). The annealing temperature was set at 61 °C, and amplification cycles were set at 45 cycles. The absolute mRNA levels in each sample were calculated according to a standard curve set up using serial dilutions of known amounts of specific templates against corresponding cycle threshold (Ct) values. Then, the ratio of the target gene over ß-actin in each sample was obtained to normalize the expression of the target gene. The specificity of the primers has been verified previously (Sun & Myatt 2003).
Construction of 5' deleted 11ß-HSD1 gene promoters into pGL3 enhancer plasmid carrying luciferase reporter gene
Cloned human 11ß-HSD1 gene promoter, 1178 bp upstream to the translation start site, was originally cloned from human DNA sequence of clone RP1-28O10 on chromosome 1q32.2-41 (Genbank no. AL031316
[GenBank]
) in Dr Kaiping Yang’s laboratory (University of Western Ontario, Ontario, Canada). Progressive 5' deletion of the cloned 11ß-HSD1 gene promoter was produced with PCR using a combination of a common antisense primer (located immediately before the translation start site) and a series of 5' end sense primers (progressing towards the transcription start site) as paired primer sets. The sequences and annealing positions of these primers are shown in Fig. 1
. Restriction sites for KpnI and XhoI are designed into the sense and antisense primers respectively, so that the amplified products carried restriction sites on the 5' and 3' ends (Fig. 1). Correspondingly various lengths of 5' deleted 11ß-HSD1 gene promoters were produced as follows: –1085, –1005, –857, –467, –204, –84, and –64 bp upstream to the transcription start site. All the sub-cloned promoters also carried 93 bp upstream to the translation start site (Fig. 1). Specific nucleotide mutation was introduced into the predicted sequence responsible for the effect of cortisol by designing the mutation sites into the sense primers (Fig. 1, underlined nucleotides). After obtaining the PCR products, the PCR products were cut with KpnI and XhoI and ligated into the polycloning sites of pGL3 enhancer vector upstream to the firefly luciferase reporter gene (Promega). All the above sub-cloned sequences and mutation sites were verified by examining the size of PCR products upon gel electrophoresis as well as by sequencing.
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Amnion epithelium-derived WISH cells were obtained from the American Type Culture Collection (Rockville, MD, USA) with the intention of overcoming relatively poor transfection efficiency in primary cells. Passage of WISH cells was carried out with trypsin digestion and the cells were subsequently grown in a 24-well plate in DMEM containing 10% FCS and antibiotic–antimycotic solution and were allowed to grow to about 85–90% confluence at 37 °C and 5% CO2 in air before the start of transfection and treatments. On the day of transfection, the cells were washed with PBS and the medium was changed to antibiotic-free DMEM containing 10% FCS. Then, the cells were co-transfected with 0.5 µg/well of empty pGL3 enhancer plasmid or pGL3 enhancer plasmid carrying 11ß-HSD1 promoter-driven firefly luciferase gene and 0.05 µg/well of phRL vector carrying renilla luciferase gene using lipofectamine 2000 in Opti-MEM. The phRL vector was used for transfection efficiency control. In another set of experiment, the cells were co-transfected with 0.5 µg/well CMV500-AC/EBP plasmid or with 0.5 µg/well empty CMV-500 plasmid along with the constructed pGL3 enhancer plasmid. All the above transfection were carried out for 12 h.
After 12 h of transfection, the culture medium was changed to serum-free medium and the cells were treated with cortisol (final concentration: 0.001, 0.01, 0.1, and 1 µM) in the presence and absence of RU486 (1 µM) for 24 h. The culture media were then discarded and the cells were washed with PBS and lysed with lysis buffer (Promega). The cellular debris was pelleted by centrifugation (12 000 g, 1 min at 4 °C). The supernatant was used for luciferase activity assay using Dual-Luciferase assay system (Promega) measuring firefly and renilla luciferase of the same samples with a luminometer. The ratio of 11ß-HSD1 promoter-driven firefly luciferase activity against renilla luciferase activity was obtained in order to correct differential transfection efficiency in each well and to express the promoter activity.
Demonstration of GR expression in WISH cells with PCR and western blotting analysis
Nuclear protein and total RNA were extracted from some of the WISH cells treated with cortisol (1 µM) overnight using an extraction kit from Active Motif (Carlsbad, CA, USA) and an RNeasy kit from Qiagen respectively. The expression of GR protein was examined following a standard western blotting protocol. Briefly, 50 µg protein of each sample were electrophoresed in 12% Tris–glycine gel (Invitrogen) and transferred to the nitrocellulose blot. The blot was blocked with nonfat milk solution and incubated with 1:1000 dilution of rabbit antibody against GR (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight. After washing, the blot was incubated with secondary anti-rabbit IgG antibody conjugated with horseradish peroxidase (Santa Cruz) for 1 h. The enhanced chemiluminescence detection system (Amersham) was used to detect the bands with peroxidase activity. For measurement of GR mRNA level, total RNA was reverse transcribed with oligo(dT)12–18 primers using Superscript II kit (Life Technologies Inc.) and subsequent standard PCR protocol was followed with the primer sequences as follows: sense 5'-GGCAATACCAGGTTTCAGGAACTTACA-3' and antisense 5'-ATTTCACCATCTACTCTCCCAT-CACTG-3'. Reaction solution of PCR consisted of 2.0 µl diluted RT-PCR product, and 0.2 µM of each paired primers. The annealing temperature was set at 60 °C and amplification cycles were set at 45 cycles. The PCR product was electrophoresed in 1.5% agarose gel.
Demonstration of GR and C/EBPßbinding to 11ß-HSD1 promoter with chromatin immunoprecipitation (ChIP) assay in human amnion fibroblasts
Primary human amnion fibroblasts were prepared as described above and plated in 10-cm Petri dishes at a density of 107 cells/dish. The cells were treated with cortisol (1 µM) for 12 h before the start of ChIP assay using a kit from Upstate Biotechnology (Temecula, CA, USA) and a method modified from the manufacturer’s protocol. Upon termination of treatment, the cells were fixed with 1% formaldehyde to cross-link the transcription factors to chromatin DNA. After washing with PBS, the cells were incubated with glycine (125 mM) for 30 min and then scraped off the dish in cold PBS containing protease inhibitor cocktail. The cells were spun down at 720 g at 4 °C and were resuspended with ice-cold lysis buffer supplemented with protease inhibitor cocktail and incubated on ice for 30 min. The cells were then gently broken up on ice using a Dounce homogenizer to aid nuclei release. The nuclei were spun down at 2400 g, 4 °C and were resuspended in digestion buffer supplemented with protease inhibitor cocktail. Enzymatic shearing of the chromatin DNA was then carried out at 37 °C. The shearing time was optimized according to the pilot study to produce an optimized size of input DNA around 500 bp. The shearing was stopped by adding ice-cold EDTA and the sheared DNA was centrifuged at 2500 g at 4 °C. The supernatant containing sheared DNA was collected for subsequent immunoprecipitation. The diluted sheared DNA was incubated by shaking with GR antibody (Santa Cruz) or C/EBP antibody (Santa Cruz) or C/EBPß antibody (Santa Cruz) or normal serum as negative control for 12 h at 4 °C. Protein A agarose/salmon sperm DNA was then added to the above solution according to the animal source the antibody was derived from and incubated for 2 h at 4 °C. The antibody/protein/DNA/agarose complex was then spun down at 2500 g and resuspended in low salt buffer and allowed to pass through a filter column (0.45 µm). The column was washed repeatedly with low and high salt buffers, LiCl and TE buffer in order to minimize the nonspecific background. After washing, the filter column was eluted with elution buffer (1% SDS, 0.1 M NaHCO3) and the flow through was collected for subsequent reverse cross-linking with 5 M NaCl at 65 °C for 4 h. At the end of reverse cross-linking, 0.5 M EDTA, 1 M Tris–HCl (pH 6.5), and proteinase K were added and incubated for 1 h at 45 °C. The sheared DNA recovered from reverse cross-linking was extracted with DNA extraction kit for further PCR analysis.
PCR primers for amplifying DNA fragments immunoprecipitated by GR, C/EBP and C/EBPß antibodies were designed according to the results of 11ß-HSD1 promoter analysis study to ensure the primers spanned the predicted promoter region responsible for the regulation by cortisol. The positions and sequences of these primers are illustrated in Fig. 8. Real-time PCR was performed on the DNA fragments. Some of the PCRs were stopped at 23 cycles and PCR products were analyzed with 2% agarose gel electrophoresis. Some of the PCRs were continued to obtain Ct values. The relative level of DNA fragments was calculated according to a standard curve set up using serial dilutions of the PCR product.
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In order to further confirm the putative GRE- and C/EBP-binding sites in 11ß-HSD1 promoter, EMSA was performed using biotin end-labeled double-stranded oligonucleotides designed according to the 11ß-HSD1 promoter-driven reporter gene study. The sense oligonucleotide sequence for putative GRE is as follows: 5'-GAATCCAGTCCTGTA-CAGTCATGAGCTT-3', which corresponds to the promoter region from –207 to –178 bp spanning the predicted GRE (underlined). The sense oligonucleotide sequence for putative C/EBP-binding sites is as follows: 5'-CCACCCAAAGCCAATCGCTGCTCTGA-3', which corresponds to the promoter region from –86 to –61 bp spanning the predicted C/EBP-binding site (underlined). To verify the specific binding to the predicted GR and C/EBP sites by the transcription factors, oligonucleotide sequences carrying the same mutations (bold letters) in the predicted GRE and CCAAT as in the reporter gene study were also utilized in the binding reaction. Nuclear protein was extracted from cultured human amnion fibroblasts stimulated by cortisol (1 µM) for 12 h using an extraction kit from Active Motif. The binding reaction of nuclear protein (3 µg) and biotin-labeled oligonucleotides with or without mutations (15 fmol) was carried out overnight at 4 °C in the presence of binding buffer (10 mM Tris, 50 mM KCl, pH 7.5), poly (dI.dC; 50 ng/µl) with or without 200-fold molar excess of unlabeled specific competitor sequences or GR antibody (1 µg, Santa Cruz) or C/EBP antibody (1 µg, Santa Cruz) or C/EBPß antibody (1 µg, Santa Cruz). The binding solution was then electrophoresed in a 6% DNA retardation gel (Invitrogen) in 0.5xTBE at 100 V and transferred to a nylon membrane using a semi-dry transfer unit at 380 mA for 30 min. The transferred DNA was cross-linked to the membrane under u.v.-light. The biotin-labeled DNA on the membrane was detected using a LightShift Chemiluminescent EMSA kit (Pierce Biotechnology, Rockford, IL, USA) according to a protocol provided by the manufacturer. Briefly, after blocking, the membrane was incubated with streptavidin–horseradish peroxidase conjugate/blocking buffer. The membrane was then washed and incubated in substrate equilibration buffer. After discarding the substrate equilibration buffer, the membrane was incubated with substrate (luminol/enhancer/stable peroxide solution) for streptavidin–horseradish peroxidase for 1 min and finally the membrane was exposed to X-ray film.
Statistical analyses
All data are reported as mean±S.E.M. Paired Student’s t-tests or two-way ANOVA followed by the Student–Newman–Keuls test were used where applicable to assess significant differences between groups. Significance was set at P<0.05. The values for n refer to the number of experiments performed with primary cells prepared from different patients or from different passages of WISH cells.
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Results |
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Treatment of human amnion fibroblasts with cortisol for 24 h caused an average of 3.6- and 7.9-fold increases in 11ß-HSD1 mRNA expression respectively at 0.1 and 1 µM, but not at 0.01 µM cortisol (Fig. 2). Treatment of the cells with the mRNA transcription inhibitor DRB (75 µM) completely blocked the induction of 11ß-HSD1 mRNA expression by cortisol (0.1 µM; Fig. 3, top panel), suggesting that this induction was dependent on the ongoing transcription. The induction of 11ß-HSD1 mRNA expression by cortisol (0.1 µM) could also be completely blocked by the GR antagonist RU486 (1 µM, Fig. 3, middle panel) and partially blocked by global inhibition of C/EBPs with transfection of AC/EBP into the cells (Fig. 3, bottom panel).
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In order to overcome the relative difficulties in transfecting primary amnion fibroblasts, a human amnion epithelium-derived immortalized cell line, WISH cells, was used for promoter analysis study. Analysis with PCR and western blot revealed GR mRNA and protein expression in WISH cells (Fig. 4, top panel). All the plasmid constructs containing different lengths of 11ß-HSD1 promoter exhibited basal promoter activity when transfected into WISH cells, while the empty pGL3 enhancer plasmid bearing no promoter exhibited mostly background activity (Fig. 4, open columns). The basal promoter activity was significantly increased when the 5' end of –1085 bp was deleted to –1005 bp and was further dramatically increased when deleted to –857 bp (Fig. 4 open columns). There was no further change with deletion from –857 to –467 bp (Fig. 4, open columns). However, the basal activity was dramatically decreased with deletion from –467 to –204 bp (Fig. 4, open columns). Further modest decrease was observed with deletion from –204 to –84 bp, but there was no further change with deletion from –84 to–64 bp (Fig. 4, open columns). These observations suggest that repressors for promoter activity might exist in the promoter region upstream to –857 bp, while enhancers for promoter activity might exist in the promoter region between –857 and –204 bp.
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). Moreover, similar fold of cortisol-induced increases was observed among the promoters of –204, –1005, and –1085 bp, which was significantly greater than the fold of increases induced by cortisol in promoters of –467 and –857 bp (P<0.05). These observations suggest that the high basal activity of –467 and –857 bp might impede further increases upon cortisol stimulation. In consideration of the dramatic decrease in cortisol-induced promoter activity with deletion from –204 to –84 bp (Fig. 4) and the similar fold of cortisol-induced increases among the promoters of –204, –1005, and –1085 bp, we inferred that the promoter region downstream to –204 bp and upstream to –84 bp might bear a crucial cortisol-response element, although the involvement of other promoter regions both upstream to –204 bp and downstream to –84 bp in cortisol’s induction could not be ruled out. Such an element was shown to be present in the promoter region between –84 and –64 bp, though appeared to be less important, as cortisol caused a modest increase of promoter activity of the –84 bp (Fig. 4). Effect of GR antagonist RU486 and co-transfection of AC/EBP plasmid on cortisol-induced 11ß-HSD1 promoter activity in WISH cells
Sequence analysis of the promoter region downstream to –204 bp and upstream to –64 bp with transcription element search system (TESS) revealed a putative GRE and several CCAAT boxes in this region. Based on this analysis, we further studied the effect of cortisol in this region in relation to the roles of GR- and CCAAT-binding proteins, C/EBPs. Results showed that cortisol induced the promoter activity of –204 bp in a dose-dependent manner (Fig. 5, top panel). Co-treatment of the cells with cortisol (0.1 µM) and the GR antagonist RU486 (1 µM) completely blocked the induction of the promoter activity of –204 bp by cortisol (Fig. 5, bottom panel), suggesting that the induction was fully dependent on GR activation. Despite this, the induction of the promoter activity of –204 bp by cortisol (0.1 µM) could also be partially blocked by global inhibition of C/EBPs with co-transfection of AC/EBP plasmid into the cells (Fig. 6, top panel), suggesting that in addition to the dominant role of GR, C/EBPs might also be involved in the induction of promoter activity by cortisol. However, the involvement of C/EBPs was more likely to be secondary to GR activation as GR antagonist completely blocked the induction by cortisol. Nevertheless, inhibition of C/EBPs with AC/EBP plasmid transfection completely inhibited cortisol (0.1 µM)-induced promoter activity of –84 bp (Fig. 6, bottom panel), suggesting that C/ EBPs, possibly secondary to GR activation, were the transcription factor responsible for cortisol’s induction in the region downstream to –84 bp and upstream to –64 bp.
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Based upon the above promoter studies, we introduced nucleotide mutations (underlined and bold letters) into the putative GRE (CTGTACAG) at –197 to –190 bp of the –204 bp promoter and a C/EBP-binding site (CCAAT box, CCAATC) at –76 to –71 bp of the –84 bp promoter (Fig. 1). Although single nucleotide mutation of the putative GRE at position –192 bp (from C to T) did not affect the induction by cortisol (data not shown), two nucleotide mutations at positions –194 and –193 bp (from TA to CG) partially inhibited the induction of the promoter activity of –204 bp by cortisol (Fig. 7, top panel), while two nucleotide mutations of the CCAAT box at positions –75 and –73 bp (from C and A to T and G respectively) almost completely blocked the induction of the promoter activity of –84 bp by cortisol (Fig. 7, bottom panel). These observations further confirmed the involvement of the predicted GRE and CCAAT in cortisol-induced 11ß-HSD1 promoter activity on the one hand and also suggested that in addition to GRE, other elements such as CCAAT were also involved in cortisol’s induction on the other hand.
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ChIP assay showed that treatment of human amnion fibroblasts with cortisol (1 µM) for 12 h caused association of GR and C/EBP to 11ß-HSD1 promoter as reflected by the abundance of PCR products amplified using primers spanning the predicted promoter sequences (Fig. 8, middle panel). Analysis with qRT-PCR also revealed that the amount of DNA precipitated by GR or C/EBP antibody was more in cortisol stimulation group than that of without cortisol stimulation group (Fig. 8, bottom panel). However, there were no obvious differences between cortisol and without cortisol-treated groups using DNA precipitated by C/EBPß antibody (data not shown), suggesting that cortisol caused no obvious binding of C/EBPß to 11ß-HSD1 promoter.
EMSA revealed that nuclear protein extracted from amnion fibroblasts stimulated by cortisol for 12 h could bind the oligonucleotides at positions of –207 to –178 bp spanning the predicted GRE producing distinguishable shifted band upon gel electrophoresis (Fig. 9, upper). Incubation with GR antibody but not C/EBP antibody supershifted the band (Fig. 9, upper left). Although there was some binding of the mutated oligonucleotides bearing the putative GRE with the nuclear protein, incubation of GR antibody caused no obvious supershift of the binding (Fig. 9, upper right). Likewise, nuclear protein derived from amnion fibroblasts simulated by cortisol could also bind the oligonucleotides spanning the predicted CCAAT box at positions of –86 to –61 bp and produced distinguishable shifted band upon gel electrophoresis (Fig. 9, bottom). Incubation of the CCAAT oligonucleotides with C/EBP antibody but not C/EBPß antibody supershifted the band (Fig. 9, bottom left), while incubation of the mutated oligonucleotides bearing CCAAT with C/EBP antibody produced no obvious supershifted band (Fig. 9, bottom right). These observations were consistent with the results of ChIP assay, suggesting C/EBP rather than C/EBPß was involved in the induction of 11ß-HSD1 promoter activity by cortisol. In addition, there were no obvious shifts observed in incubation without nuclear protein (far left lane in each gel picture of Fig. 9) and all the shifts and supershifts produced by nuclear protein and antibodies were abolished when excessive nonlabeled-specific oligonucleotides were included in the incubation (far right lane in each gel picture of Fig. 9), suggesting that the binding of nuclear protein to the oligonucleotides was specific.
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Discussion |
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Tannin et al. (1991) isolated a human cDNA clone encoding 11ß-HSD1 from a human testis cDNA library. Their primer extension analysis using liver RNA indicated that transcription starts 93 bp upstream from the translation start site. Their data also showed that there was no TATA box in the 5' untranslated region, but there were a consensus CCAAT box at –76 bp upstream from the start of transcription and a palindromic sequence resembling part of a GRE at –188 bp upstream from the start of transcription. In this study, we found that cortisol induced similar extent of increases in the promoter activities of –204, –1005, and –1085 bp, and the induction was greatly decreased when deletion progressed from –204 to –84 bp and disappeared when deletion progressed to –64 bp. These observations suggested that a crucial GRE might be present in the promoter region between –204 and –84 bp, although other promoter regions could also participate, such as the one in between –84 and –64 bp as demonstrated in this study. Coincidently, the promoter region between –204 and –84 bp contains the putative GRE and the promoter region between –84 and –64 bp contains CCAAT box respectively, as speculated by Tannin et al. (1991).
GCs act through binding to their cytosolic GR, serving as a transcription factor. Once activated by GCs, GRs forms homodimers and translocate into the nucleus, where they ultimately bind to specific GRE on gene promoters to regulate transcription (Hayashi et al. 2004, Smoak & Cidlowski 2004). However, this mechanism dose not explains all the actions of GCs. Several studies demonstrated that interaction of GR with other transcription factors could provide an alternative pathway for GC actions (Hayashi et al. 2004, Smoak & Cidlowski 2004). GCs have been found to induce C/EBP or C/EBPß expression in a number of tissues or cells (Zilberfarb et al. 2001, Hernandez et al. 2003, Yang et al. 2005a,b). Furthermore, interaction of C/EBP and GR has been found to regulate cell proliferation (Rudiger et al. 2002, Roth et al. 2004). Williams et al. (2000) found that C/EBP was an important regulator of mouse basal 11ß-HSD1 promoter activity in hepatoma cells. In a recent study, C/EBPß was also found to regulate human 11ß-HSD1 promoter activity in mouse adipose cells (Gout et al. 2006). All these findings pointed to a role of C/EBPs in the regulation of 11ß-HSD1 expression at least at the basal status. In this study, we found that the induction of 11ß-HSD1 mRNA expression and promoter activity by cortisol involves not only GR but also C/EBP.
The observation that the induction of 11ß-HSD1 expression could be completely blocked by a GR antagonist suggests a predominant role of GR in this induction. Studies of GRE mutation, ChIP assay, and EMSA not only confirmed the putative GRE in 11ß-HSD1 promoter but also provided evidence for a direct interaction of GR with GRE in 11ß-HSD1 promoter. However, in addition to the direct interaction of GR with GRE, this study also provided evidence for an indirect role of GR in the induction of 11ß-HSD1 promoter activity by cortisol, as the induction of both 11ß-HSD1 mRNA expression and promoter activity by cortisol could also be partially blocked by C/EBP inhibition. A number of studies (Zilberfarb et al. 2001, Hernandez et al. 2003, Yang et al. 2005a,b) have demonstrated that GCs induced C/EBP expression in various tissues. These observations were in favor of the participation of C/EBPs in the induction of 11ß-HSD1 expression by cortisol. Furthermore, the ChIP assay and EMSA in this study revealed that C/EBP rather than C/EBPß was involved in the induction by cortisol. This observation was supported by the study of Williams et al. (2000) showing that C/EBP was a powerful transcription factor for the induction of basal 11ß-HSD1 mRNA expression in the mouse, while C/EBPß functioned mainly as a relative inhibitor of basal 11ß-HSD1 expression.
However, it should also be kept in mind that there are several CCAAT-like sequences both upstream and downstream to –84 bp as revealed by TESS analysis. Using footprint analysis, Williams et al. (2000) also found multiple C/EBP-binding sites scattered along the –812 bp of mouse 11ß-HSD1 promoter. Thus, in addition to the CCAAT box downstream to –84 bp as demonstrated in this study, other CCAAT-like sequences upstream to –84 bp may also participate in the induction of 11ß-HSD1 expression by cortisol.
In conclusion, we demonstrated in this study that the feed-forward regulation of 11ß-HSD1 gene expression by cortisol involves both direct binding of GR to GRE in the promoter and indirect activation of C/EBP binding to the CCAAT box in the promoter in human amnion cells. This feed-forward regulation of 11ß-HSD1 by cortisol may be required for the full local actions of cortisol in the fetal membranes as well as for the fetal maturation at the end of gestation.
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Acknowledgements |
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References |
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Received in final form 9 August 2007
Accepted 23 August 2007
Made available online as an Accepted Preprint 23 August 2007
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