Transcriptional and Posttranscriptional Regulation of Fibulin-1 by Estrogens Leads to Differential Induction of Messenger Ribonucleic Acid V
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内分泌学杂志 2005年第2期
Unité Institut National de la Santé et de la Recherche Médicale 540 (A.B., F.M., R.M., C.D., T.M., V.C., P.P.), and Service de Biologie Cellulaire et Hormonale (C.D., T.M., P.P.), Centre Hospitalier Universitaire de Montpellier, H?pital Arnaud de Villeneuve, 34095 Montpellier, France; and Department of Dermatology and Cutaneous Biology (M.L.C.), Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Address all correspondence and requests for reprints to: P. Pujol, Service de Biologie Cellulaire et Hormonale, H?pital Arnaud de Villeneuve, 271, Av G. Giraud, 34095 Montpellier, France. E-mail: p-pujol@chu-montpellier.fr.
Abstract
Fibulin-1 is an extracellular matrix protein overexpressed in epithelial ovarian and breast cancers. In estrogen receptor (ER)-positive ovarian and breast cancer cell lines, fibulin-1 mRNA levels are markedly increased by estrogens. Transfection experiments using fibulin-1 promoter constructs indicate that 17?-estradiol (E2) increases fibulin-1 gene transcription and that ER is more potent than ER? to mediate E2 regulation of the transfected fibulin-1 promoter. Using SL2 cells devoid of specificity protein 1 (Sp1) and site-directed mutagenesis of GC boxes, we evidenced that the E2 regulation occurs through a proximal specificity protein 1 binding site. In addition, we show that fibulin-1C and -1D mRNAs, the two major fibulin-1 splicing variants, are differentially induced by E2. The induction of both mRNAs variants is direct and independent of a newly synthesized protein intermediate. Interestingly, actinomycin D chase experiments demonstrate that E2 treatment selectively shortens the fibulin-1D mRNA half-life. This indicates that estrogens affect differentially the stability of fibulin-1 variants and may explain the lower accumulation of fibulin-1D mRNA on E2 treatment. In conclusion, our data show that estrogens, via ER, are key regulators of fibulin-1 expression at both the transcriptional and posttranscriptional levels. The preferential induction of the fibulin-1C variant, which is overexpressed in ovarian and breast cancer, might play an important role in estrogen-promoted carcinogenesis.
Introduction
IT IS WELL DOCUMENTED that the mitogenic action of estrogens is critical in the etiology and progression of human breast and gynecological cancers (1, 2). The promoting effect of estrogens has been recently highlighted by the results of large prospective studies (3, 4, 5, 6), showing that estradiol intake during menopause increased the risk of breast cancer. In ovarian cancer, although the question is still debated (7), several recent prospective studies (8, 9, 10, 11) indicated an increased risk for women undergoing long-term estrogen replacement therapy.
The effects of estrogens are mediated by two estrogen receptors (ERs), ER (NR3A1) and ER? (NR3A2), which belong to a large family of nuclear receptors (12, 13). ER and ER? classically mediate their action by ligand-dependent binding to the estrogen-responsive element (ERE) of target genes, leading to their transcription regulation (12, 13, 14, 15). Estrogens also transcriptionally regulate target genes via ERs through ERE-independent modes of action. These effects are mediated through promoter elements that bind various transcription factors, including activator protein-1 (16) or specificity protein (Sp1) (17) binding sites.
E2 regulation of proteins has been well documented in breast cancer (18), but little is known about E2 regulation of proteins in ovarian cancers. In previous studies, we have shown that the secretion of the extracellular matrix fibulin-1 protein is induced by estrogens in ER-positive ovarian cancer cell lines (19, 20). We also demonstrated that fibulin-1 is overexpressed in epithelial ovarian cancer (19, 21) as reported by others in breast cancer (22, 23). Fibulin-1 could be involved in cell motility and anchorage-independent growth of tumors cells (24, 25, 26). These cellular functions likely play a crucial role in ovarian cancer tumor progression because the local spread of cancer cells within the peritoneal cavity and in lymphatic vessels contributes to the high mortality and late diagnosis of ovarian cancer (27). Other studies have shown that fibulin-1 could have a role in cellular differentiation. Indeed, fibulin-1 interacts with growth factors such as pro-HB-epidermal growth factor (EGF), NovH, or ?-amyloid (28, 29, 30) and could regulate the cell bioavailability of these factors. Moreover, fibulin-1 binds the papilloma virus E6 protein and could prevent p53 degradation by viral proteins, thus allowing the cell cycle and proliferation regulation and inhibiting E6-mediated transformation (31).
Fibulin-1 is composed of four different splicing isoforms (A, B, C, and D) (32, 33, 34), with variants 1C and 1D being the major forms expressed in ovaries (35). Fibulin-1 variants may have distinct biological properties as suggested by the recent discovery of fibulin-1D variant involvement in the synpolydactyly, a hand malformation syndrome (36). Alteration in fibulin-1D expression, whereas fibulin-1C is normally expressed, seems to reduce apoptosis in interdigital tissues and cause observed malformations. The different binding affinity of fibulin-1 variants with protein partners such as nidogen-1 (37) could be a molecular explanation for their distinct roles.
Very little is known about fibulin-1 regulation and splicing variant expression. We previously demonstrated that fibulin-1 mRNA is induced by estrogens (35) in ER+ ovarian cancer cell lines. The aim of the present study was to further investigate the regulatory mechanisms that govern the expression of fibulin-1. We analyzed fibulin-1 mRNA accumulation in response to various steroid hormones and peptide growth factors in ovarian and breast cancer cells. We showed that E2 regulation involves a direct transcriptional regulation, which requires ER acting through the proximal Sp1 site, and a specific decrease in fibulin-1D mRNA stability. This complex regulation leads to a differential accumulation of variant forms C and D mRNAs, which could be of importance in ovarian and breast tumorigenesis.
Materials and Methods
Cell lines
The ovarian cancer cell lines BG-1, SKOV3, PEO-4, OVCAR-3, and PEO-14 and the breast cancer cell lines MCF-7 were used in this study. BG-1 (38) (obtained from Dr. C. E. Welander, Emory University, Atlanta, GA), MCF-7, and SKOV3 cells (obtained from the American Type Culture Collection, Manassas, VA) were cultured in DMEM/F12 (Invitrogen, Cergy-Pontoise, France) supplemented with 10% fetal calf serum (FCS, Invitrogen). PEO-14, PEO-4 (obtained from Dr. S. P. Langdon, Hospital of Edinburgh, UK), and OVCAR-3 cells (obtained from the American Type Culture Collection) were cultured in RPMI 1640 (Invitrogen) supplemented with 10% FCS. Cells were stripped from endogenous steroids by 7-d weaning in DMEM/F12 or RPMI 1640 without phenol red containing 10% FCS treated with dextran-coated charcoal. Drosophila SL2 cells were grown at 25 C without CO2 in SF900II medium (Life Technologies, Grand Island, NY) supplemented with penicillin and streptomycin sulfate.
Plasmid construction
pSG5-ER and pSG5-ER? correspond to the wild-type human ER and ER? cDNAs cloned into pSG5 plasmid (Stratagene, La Jolla, CA) under control of the Simian virus 40 promoter. A pCMV-?-Gal reporter was used as an internal control and corresponds to the ?-Gal cDNA cloned in the pCMV5 plasmid (Stratagene). PpacSp1 was obtained from R. Tjian (Howard Hughes Medical Institute, University of California, San Diego, CA).
As previously described by Castoldi and Chu (39), all fibulin-1 promoter-reporter constructs were obtained by subcloning various genomic fragments prepared by restriction enzyme digestion into the polylinker of phRG-basic vector (Promega, Charbonnier, France), which contains a firefly luciferase gene. The common 3'-end of the constructs was situated at +101 and their 5' ends corresponded to –38 bp (pHF1), –68 bp (pHF2), –285 bp (pHF3), and –3505 bp (pHF4) upstream from the transcription start site.
The pHF232 and pHF257 plasmids corresponded to the pHF2 construct containing a mutation in the Sp1 binding sites, located respectively at –32 and –57 bp upstream from the transcription start site. Mutations were introduced using the QuikChange XL site-directed mutagenesis kit (Stratagene) according to the manufacturer’s instructions and were both verified by sequencing. The primers used to generate the mutated Sp1 sites at –32 and –57 bp were respectively: 5'-CCGCGCCCTCCTCCCGGTTGGGATAATTGAACGG-3' and 5'-CAGCCCATGGGCCCCTTCTCCGCCGCGCCCTCC-3'.
Transient transfection and reporter assays
Approximately 5.105 MCF-7 cells were plated in 12-well plates 48 h before transfection. Cells were cotransfected with 2 μg of each firefly luciferase reporter construct, 0.5 μg of pSG5-hER or -?, and 0.5 μg of the internal reference pCMV-?-Gal reporter plasmid. Transfections were performed overnight with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocols. Cells were then treated with 10–8 M 17?-estradiol (E2) or ethanol vehicle. Twenty-four hours after E2 treatment, the cells were lysed directly in the plates with 200 μl of cell culture lysis reagent (Promega). Luciferase activity was measured on 100 μl of lysate aliquots for 10 sec after injection of luciferase detection solution [20 mM tricine (pH 7.8), 1.07 mM (MgCO3)4 Mg(OH)2 5H2O, 2.67 mM MgSO4, 1 mM EDTA, 0.53 mM ATP, 0.47 mM luciferin, and 0.27 mM coenzyme A] in an LKB luminometer (BioMérieux, Marcy l’Etoile, France). The induction of luciferase activity is indicated in arbitrary units, corrected by ?-Gal activity and the values obtained for pGL2 basic transfected cells. Absorbance of the ?-Gal assay was read in a microtitertek at 420 nm. All transfections were performed in triplicate and repeated at least three times. Drosophila SL2 cells (5 x 106) were plated in 24-well plates, and transfection was carried out using Lipofectamine 2000 (Invitrogen). Each well was transfected with 0.25 μg reporter plasmid, 0.5 μg ?-Gal plasmid, and pPac-Sp1 (0–125 ng) or pSG5-HEGO expression vectors (0–125 ng) adjusted, when necessary, by the addition of empty vectors to equalize the total DNA transfected. Twenty-four hours after transfection, the cells were harvested and assayed for luciferase activity as described above.
Northern blot
For RNA extraction, cells were harvested at 80% confluence. Total RNA was extracted using the RN easy midikit (Qiagen, Courtaboeuf, France). Twenty micrograms total RNA were electrophoresed in 1% agarose gel containing formaldehyde and transferred to Hybond-N+ membrane (Amersham Corp., Les Ulis, France). The fibulin-1 probes used for Northern blot analysis of C and D variants were PCR-generated fragments of 442 and 541 bp, respectively, as previously described (35). The 18S and fibulin-1 probes were 32P-labeled by the Radprime DNA labeling system (Invitrogen). Hybridization was performed overnight using Ultrahyb hybridization buffer (Ambion, Austin, TX), and washes were performed as previously described (40). When required, the membranes were stripped of probe by boiling in 0.5% sodium dodecyl sulfate solution and rehybridized. Signals were quantified using PC BAS 1000 reader (Fujix).
Reverse transcription and real-time quantitative PCR
Classically, 1 μg total RNA was reverse transcribed as previously described (35). Real-time PCR quantification was performed using a SYBR Green approach (Light Cycler; Roche Diagnostics, Indianapolis, IN). PCR was carried out in a final volume of 20 μl using 1 μl of each primer (10 μM), 4 μl of the supplied enzyme mix, 9 μl H2O, and finally 5 μl of the template diluted at 1:10. After a 10-min preincubation at 95 C, runs corresponded to 45 cycles of 15 sec at 95 C (denaturation), 6 sec at 65 C (annealing), and 12 sec at 72 C (amplification). Primers for fibulin-1C and fibulin-1D (35) or hypoxanthine-guanine phosphoribosyl transferase (HPRT) (41) were previously described. PCR products were subjected to melting curves analysis using the light cycler system to exclude the amplification of unspecific products. For each sample, fibulin-1 mRNA levels were corrected for HPRT mRNA levels (reference gene).
Statistical analysis
The results were presented as mean ± SD. Statistical analysis was performed by Mann-Whitney U test. A value of P < 0.05 was considered to be statistically significant.
Results
Regulation of fibulin-1 mRNA in ovarian and breast cancer cells
Previous studies by our laboratory have shown that E2 induced fibulin-1 expression in ovarian cancer cells at both the mRNA and protein levels (19, 20, 35). As shown in Fig. 1A, E2 increased by 3-fold the level of fibulin-1C mRNA, the major form of fibulin-1 variant expressed in ovarian cancer, in the ER + BG-1, PEO4, and SKOV3 ovarian cancer cell lines. The pure antiestrogen ICI 182, 780 did not modify the basal expression of fibulin-1C mRNA but completely abolished its induction by E2 in BG-1 cells. By contrast, no estrogen induction of fibulin-1C was observed in the PEO-14 and OVCAR-3 ER-negative ovarian cancer cell lines (Fig. 1B). Because of the low level of expression of fibulin-1 in MCF-7 breast cancer cells, the fibulin-1 mRNA could not be detected by Northern blot experiments. We therefore set up the quantification of fibulin-1C mRNA using real-time quantitative RT-PCR (Fig. 1C). This experiments indicated that the same regulation of fibulin-1C mRNA by E2 (3-fold) was obtained in BG-1 and MCF-7 cells despite a 300-fold lower expression in MCF-7 cells.
FIG. 1. Regulation of fibulin-1C expression by E2 in ovarian and breast cancer cells. A, The BG-1 ER+ ovarian cancer cells were treated for 48 h with control vehicle ethanol (C), E2 (10–8 M), ICI 182, 780 (I) (10–7 M), or a combination (E2+I). PEO-4 and SKOV3 were treated with control vehicle ethanol, E2, and ICI 182, 780 in the same conditions used for BG-1 cells. mRNA accumulation was quantified by Northern blot. The results are expressed as n-fold induction after normalization by 18S of fibulin-1C (Fib-1C) mRNA content in vehicle treated cells. B, Same experiment as in A with the ER-ovarian cancer cells PEO-14 and OVCAR-3. C, BG-1 and MCF-7 cells were treated for 48 h with control vehicle ethanol (C) or E2 (10–8 M). mRNA was quantified by real-time quantitative RT-PCR. The results are expressed in arbitrary units and correspond at the fibulin-1C expression after normalization by HPRT. Values are the means ± SD of three independent experiments.
We then studied whether other nuclear receptor ligands could regulate the expression of fibulin-1C mRNA in ovarian cancer cells. As shown in Fig. 2A, treatment of BG-1 cells with retinoic acid or nonmetabolizable androgens or progestins (R1881 and R5020, respectively) had no effects on fibulin-1C mRNA expression. By contrast, glucocorticoids such as dexamethasone slightly decreased fibulin-1C mRNA levels, as previously reported in bone marrow (42).
FIG. 2. Regulation of fibulin-1C (Fib-1C) expression by other effectors in BG-1. A, BG-1 cells were treated with control vehicle ethanol (C), E2 (10–8 M), R1881 (10–9 M), R5020 (10–6 M), dexamethasone (DXM) (10–6 M), retinoic acid (RA) (5.10–6 M), or RU486 (10–6 M) for 48 h. Total RNA was processed by Northern blot, and the hybridization was performed with fibulin-1C or 18S probes. The quantification results of Northern blot experiments after normalization by 18S RNA levels are indicated below. The results are expressed as n-fold induction after normalization by 18S of fibulin-1C mRNA content in vehicle-treated cells. *, P < 0.05 vs. vehicle-treated cells (C). B, BG-1 cells were treated with control vehicle ethanol (C), E2 (10–8 M), EGF (100 ng/ml), or IGF-I (5 nM) alone or combined with E2 (10–8 M) for 48 h or with CT (1 μg/ml) and cAMP (AMPc) (1 mM) for 24 h. The previous treatments were carried out after 7 d starvation in DMEM/F12 without phenol red with dextran-coated charcoal-treated FCS as in all the other experiments. The culture medium was then replaced by DMEM/F12 without FCS for 24 h before the addition of growth factors, CT, and cAMP in DMEM/F12 alone. The results are expressed as n-fold induction after normalization by 18S of fibulin-1C mRNA content in vehicle-treated cells. *, P < 0.05 vs. vehicle-treated cells (C).
As BG-1 cells were described to be responsive to peptide growth factors such as IGF-I and EGF (43), we investigated whether these mitogenic molecules could also regulate fibulin-1 expression or interfere with its E2-regulation. EGF and IGF-I significantly inhibited fibulin-1C mRNA expression (Fig. 2B) and, when administered in combination with E2, partially blocked estrogen induction (especially IGF-I). These results suggest that these growth factor pathways may be involved in the regulation of fibulin-1 expression and could cross-talk with estrogen signaling. In addition, cAMP and cholera toxin (CT), which both mimic constitutive activation of the G protein-coupled receptor signaling pathway, also negatively affected fibulin-1C mRNA expression (Fig. 2B).
Transcriptional regulation of fibulin-1 gene expression by E2
To further investigate the transcriptional regulation of fibulin-1 gene by E2, we performed transient transfection experiments using four different promoter constructs. The common 3'-ends of these constructs corresponded to +101 bp downstream from the transcription start site and their 5'-ends corresponded to –38, –68, –285, and –3505 bp for pHF1, pHF2, pHF3, and pHF4 constructs, respectively (Fig. 3). These experiments were carried out in MCF-7 cells because of their facility of transfection, compared with BG-1 cells.
FIG. 3. Study of estradiol effects on the fibulin-1 promoter. A, Nucleotide sequence of the human fibulin-1 promoter. The transcription start is denoted as +1 and shown by an arrow. Putative regulatory elements (underlined) and the TATA-like sequenced (boxed) are also indicated. B, Schematic representation of the four different fibulin-1 promoter constructs used in this study. They contain up to upstream the luciferase reporter gene (+1 represents the transcription start site). The position of Sp1 sites and half-EREs is also shown. Upon transfection of the four constructs into MCF-7 cells, luciferase activity was measured after treatment with E2 (10–8 M) or ethanol vehicle, compared with cotransfected CMV-?-Gal. Values are the means ± SD of three independent experiments. *, P < 0.05 vs. control of pHF1. C, Luciferase activity was measured after transient transfection of pHF4 and pSG5-ER, pSG5-ER?, or the empty pSG5 plasmid in MCF-7 cells. Cells were treated with ethanol vehicle (C), E2 (10–8 M), 4-hydroxy tamoxifen (TAM) (10–7 M), or ICI 182, 780 (ICI) (10–7 M) for 24 h. Values are the means ± SD of three independent experiments. *, P < 0.05 vs. control of pSG5. #, P value not significant.
According to Castoldi and Chu (39), a basal luciferase activity was obtained with pHF1, and it significantly increased with pHF2 and pHF3 reporter constructs. By contrast, the distal region of the promoter (–3505 to –285 bp) had no additional effect on the transcriptional activity of the fibulin-1 promoter (compare pHF4 and pHF3 constructs in Fig. 3B). Interestingly, on E2-stimulation, a significant increase in luciferase activity was obtained with the pHF2, -3, and -4 reporter constructs but not with the pHF1 reporter plasmid (Fig. 3B). This suggested that an important regulatory element was located between –38 and –68 bp.
To determine the respective roles of the two ERs in the regulation of fibulin-1 expression, the pHF4 construct was cotransfected in MCF-7 breast cancer cells with a human (h)ER or hER? expression vector. In the presence of E2, we observed a significant increase in luciferase activity in hER cotransfected cells, compared with cells transfected with the empty pSG5 plasmid (Fig. 3C). By contrast, hER? was not able to change the magnitude of E2 induction. This was in agreement with our previous results obtained on the endogenous fibulin-1 gene after infection of MDA-MB-231 ER-breast cancer cells with a recombinant adenovirus containing either ER or ER? cDNA (35). The partial and pure antiestrogens treatment with 4-hydroxy-tamoxifen or the ICI 182, 780 compound had no significant effect on basal transcriptional activity of the pHF4 reporter construct (Fig. 3C).
Role of Sp1 in E2 regulation of fibulin-1 gene expression
The fibulin-1 proximal promoter region contains two binding sites for the Sp1 transcription factor, located at –32 and –57 bp upstream from the transcription start site (Fig. 3, A and B) (39). The Sp1 transcription factor has been frequently involved in mediating transcriptional responses to E2 through recruitment of ER via protein-protein interactions (44). Based on our deletion analysis (Fig. 3), the Sp1 binding site located at the –57 bp position could be critical for fibulin-1 regulation by E2.
The importance of the Sp1 transcription factor in the regulation of fibulin-1 promoter was first assessed in Drosophila SL2 cells that do not express Sp1 proteins (45). In these cells, cotransfection of the pHF4 construct with increasing concentrations of Sp1 expression plasmid strongly increased luciferase activity, thus demonstrating the response of the fibulin-1 promoter to Sp1 (Fig. 4A). Interestingly, in SL2 cells, the same pHF4 construct did not confer E2 responsiveness, whereas a marked induction was noticed for an ERE-containing reporter construct (Fig. 4B). These results indicate that the Sp1 transcription factor is necessary not only for the basal expression of fibulin-1 gene but also for its regulation by estrogens.
FIG. 4. Sp1 is required for fibulin-1 induction by estrogens. A, SL2 cells were transiently transfected with the pHF4 construct together with increasing concentrations (0, 5, 25, or 125 ng) of Sp1 expression vector. Luciferase activity was measured 48 h later and corrected for the corresponding ?-Gal activity. Values are the means ± SD of two independent transfections performed in triplicate. *, P < 0.05 vs. control (0 ng Sp1). B, The pHF4 construct or a reporter vector containing an ERE in front of the ?-globin (Glob) promoter and the luciferase gene were transiently transfected into SL2 cells together with the expression vector for ER. Cells were then treated or not with E2 (10 nM), and luciferase activity was measured 24 h later as described in A. *, P < 0.05 vs. vehicle-treated cells (C). C, SL2 cells were transiently transfected with the pHF2 constructs containing the wild-type sequence or mutated on the Sp1 sites at position –57 or –32 in combination with either the expression vector for Sp1 (50 ng) or the corresponding empty vector. Luciferase activity was quantified as in A. *, P < 0.05 vs. corresponding control. D, MCF-7 cells were transiently transfected with the pHF1 or pHF2 constructs containing the wild-type sequence or mutated on the Sp1 sites at position –57 or –32 together with the expression vector for ER. Cells were then treated or not with E2 (10 nM), and luciferase activity was measured 24 h later as described before. Values are the means ± SD of two independent transfections performed in triplicate. *, P < 0.05 vs. corresponding control.
To further demonstrate the role of Sp1 transcription factor in the E2-regulation of fibulin-1, we generated a pHF2 construct with a mutation in the Sp1 site at position –57 (named pHF257), which appeared to be the critical element for E2 responsiveness according to data shown in Fig. 3B. As a control, we also mutated the second putative Sp1 binding site (named pHF232) present in the pHF2 construct. As shown in Fig. 4C, the effect of Sp1 in SL2 cells was completely abolished on pHF257, compared with pHF2 or pHF232, thus confirming the role of the distal GC box. As expected, when transfected in MCF-7 cells, the pHF257 mutant no longer conferred E2-regulation, compared with the wild-type counterpart or to the pHF232 mutant (Fig. 4D).
Altogether, these data demonstrated that the transcriptional regulation of fibulin-1 gene by E2 required the presence of the Sp1 transcription factor acting through its response element located at –57 bp from the start site.
E2 differentially regulates fibulin-1C and -1D mRNA expression and stability
Fibulin-1 mRNA is expressed as several variants differing in their 3' untranslated region. We have previously shown that the C and D isoforms were the two major variants present in ovarian tissues, fibulin-1C being predominant in ovarian cancer (35). By Northern blot, we compared the E2-regulation of fibulin-1C and -1D mRNA in BG-1 cells. Interestingly, estrogen induction was higher for fibulin-1 mRNA variant C than variant D (Fig. 5, A and B). Using real-time quantitative PCR, we also found the same magnitude of differential regulation by E2 of the two fibulin-1 mRNA variants in both BG-1 and MCF-7 cells (Fig. 5C).
FIG. 5. E2 regulation of fibulin-1C (Fib-1C) and -1D (Fib-1D) mRNA in BG-1 and MCF-7 cells. A, BG-1 cells were treated with ethanol vehicle (C) or E2 (10–8 M) for 48 h. Northern blots were performed with 20 μg total RNA, and hybridization was carried out with fibulin-1C or -1D or 18S probes. B, Quantification of Northern blot experiments shown in A after normalization according to 18S RNA levels. The results are expressed as n-fold induction of E2-treated vs. vehicle-treated cells and are means of three independent experiments. C, MCF-7 cells were treated with ethanol (C) or E2 (10–8 M) for 48 h, and fibulin-1C and -D mRNAs were quantified by real-time quantitative RT-PCR. The results are expressed in arbitrary units after normalization by HPRT. Values are the means ± SD of three independent experiments. For data shown in B and C, statistical analysis indicated that the fold induction of fibulin-1C or -1D mRNAs on E2 stimulation was significant (P < 0.05).
As shown in Fig. 6, cycloheximide had no significant effect on E2 induction of either fibulin-1C or fibulin-1D, suggesting that regulation of both variants was not dependent upon de novo synthesis of a protein intermediate. We then measured fibulin-1C and -1D mRNA half-lives by actinomycin D chase experiments to compare their respective stabilities in the presence of E2 in BG-1 cells. The apparent half-life of both fibulin-1C and 1-D mRNA was approximately 8 h in the absence of E2 (Fig. 7, A and B). E2 treatment did not affect the fibulin-1C mRNA half-life (Fig. 7A), whereas it decreased that of fibulin-1D mRNA (5.5 h, Fig. 7B). This differential stability of the two mRNAs isoforms on E2 treatment could therefore explain the lower E2 induction of fibulin-1D mRNA steady-state levels, compared with fibulin-1C (Figs. 5 and 6).
FIG. 6. Direct E2 stimulation of fibulin-1C (Fib-1C) and -1D (Fib-1D) mRNA in BG-1 cells. A, BG-1 cells were treated for 1 h with cycloheximide (30 μg/ml) (CHX) before adding E2 (10–8 M) with the antibiotic still present. Cells were scrapped 15 h after E2 treatment. Northern blot was performed with 20 μg total RNA, and hybridization was conducted with fibulin-1C, fibulin-1D, and 18S probes. B, Quantification of Northern blot experiments after normalization by 18S RNA levels. The results are means of three independent experiments. Statistical analysis comparing the E2 induction of fibulin-1C or -1D mRNAs with or without cycloheximide treatment did not show any significant difference.
FIG. 7. Stability of fibulin-1C (Fib-1C) and -1D (Fib-1D) mRNAs in response to E2. A, BG-1 cells were treated or not with E2 (10–8 M) for 48 h and then incubated for different times (0, 5, and 10 h) with actinomycin D (3 μg/ml) before RNA extraction. Northern blot was performed with total RNA, and hybridization was carried out with fibulin-1C (A) or fibulin-1D (B). The same membrane was rehybridized with the 18S probes. B, Quantification of Northern blot experiments shown in A after normalization by 18S RNA levels. The results are expressed as 100% of fibulin-1C or fibulin-1D/18S RNA levels without actinomycin D treatment (0 h). The results are means of three independent experiments. *, P < 0.05 vs. corresponding vehicle-treated cells (C).
Discussion
In this study, we characterized a complex regulation of fibulin-1 expression by E2 in ovarian and breast cancer cells leading to a differential accumulation of fibulin-1C and D mRNA isoforms. Up-regulation of fibulin-1 expression under estrogen stimulation takes place at the transcriptional level, and this induction involves ER action through a Sp1 binding site. We also demonstrate the existence of a negative control at the posttranscriptional level because E2 treatment appears to decrease specifically the stability of fibulin-1D mRNA.
Mechanism of transcriptional regulation by E2
The recent cloning of the fibulin-1 promoter (39) showed that the Sp1 site at –57 bp upstream from the transcription start site was responsible for part of the basal promoter activity via Sp1 and Sp3 transcription factors. Our results demonstrate that the two half-ERE consensus sequences present on the pHF4 construct do not appear to be necessary for efficient E2 regulation. By contrast, several lines of evidence indicate that Sp1 transcription factor binding on the promoter was required for E2 regulation of fibulin-1 gene transcription. First, the deletion analysis mapped the critical regulatory element to sequences a region between –38 and –68 bp, which contains one of the two GC boxes. Second, no regulation was obtained in Drosophila cells devoid of Sp1 protein. In addition, treatment of MCF-7 cells with mithramycin A, which inhibits the interaction of Sp1 with its DNA target sequence (46), significantly reduced E2 induction of luciferase activity expressed from the pHF4 reporter vector (data not shown). Finally, the mutagenesis of the distal Sp1 binding site located at –57 bp completely abolished E2-induction of the fibulin-1 promoter.
Other studies have highlighted the role of the ER-Sp1 interaction in E2 activation of gene transcription (17, 47). In line with data showing that Sp1-mediated E2 regulation was supported more efficiently by ER than ER? (48), our data showed that the transfected reporter constructs was regulated via ER but not ER?. We obtained the same results on the endogenous fibulin-1 gene after adenoviral infection of ER-breast cancer cells (35).
Posttranscriptional regulation by E2
Our data demonstrate that posttranscriptional events are likely to be responsible for the differential regulation of the fibulin-1C and -1D variants. The two isoforms derive via alternative 3' splice sites from the same pre-mRNA transcribed from a single promoter. The spliced region encoding fibulin-1C contains the proximal exon 17, whereas fibulin-1D mRNA encompasses the more distal exons 18, 19, and 20.
There are many steps in the pathway of RNA metabolism at which steroid hormones might act to alter the levels of a given mRNA. Estrogen regulation of fibulin-1 isoforms could take place at the level of mRNA splicing (49). Other steroids such as progestins have been shown to regulate alternative splicing of reporter genes through mechanisms that could involve nuclear receptor coregulators (50). Further work will be necessary to precise whether such mechanisms could be involved in the hormonal control of the fibulin-1 gene.
The results presented herein strongly suggest that the differential induction of fibulin-1C and -1D mRNAs by estrogens in breast and ovarian cancer cells is associated with a specific regulation of mRNA stability. Indeed, the results obtained in the present study demonstrate that the fibulin-1D mRNA half-life is decreased in the presence of E2, whereas hormone treatment did not modify the apparent stability of fibulin-1C mRNA. Little is known about the hormonal control of mRNA half-life, but several reports have described the role of adenylate/uridylate-rich elements (AREs) located in the 3'-untranslated regions (UTRs). These AREs represent a combination of functionally and structurally distinct sequence motifs or domains, such as AUUUA motifs, UUAUUUA(U/A)(U/A) nonamers, U stretches, and/or a U-rich domain (51). A number of proteins that bind these AREs and increase mRNA stability are induced by estrogens, such as vigilin or E-RmRNASF, which stabilizes, respectively, the vitellogenin (52) and apolipoprotein II mRNAs (53). Fewer estrogen-induced proteins associated with ARE-containing mRNAs are involved in mRNA destabilization. The Xenopus polysomal ribonuclease 1 is implicated in the destabilization of albumin mRNA (54), and AUF1/hnRNPD (55) is involved in mRNA destabilization of c-myc mRNA (56, 57). It has been also demonstrated that TIS11 (also named TPA-inducible sequence 11 or TTP) (58), TIS11b, and TIS11d bind to the AREs of TNF, granulocyte macrophage colony-stimulating factor, and IL-3 mRNAs and induce their destabilization (59, 60, 61). Interestingly, TIS11b is induced by numerous mitogenic factors in different cells (62) and estrogens in rat uterine cells (63).
The fibulin-1D 3'UTR has a higher uridin-rich content than fibulin-1C and we could hypothesized that the 3'-UTR of fibulin-1D mRNA could be more sensitive to degradation by one of mechanisms describe above or by a still-unknown ribonuclease. Further work will be required to characterize the sequences and factors involved in the specific E2-induced degradation of fibulin-1D mRNA.
Expression of fibulin-1 isoforms and physiopathological relevance
The differential expression of fibulin-1C and -1D variants under estrogen stimulation provide, to our knowledge, a first example of variant-specific regulation by estrogens due to a specific destabilization of splicing variant.
Interestingly, an increased fibulin-1C to fibulin-1D ratio was previously noted in ovarian cancers, compared with normal ovaries (35). This overexpression of variant C in cancer may thus be related to estrogen stimulation. Fibulin-1 is overexpressed during breast carcinogenesis, although no information concerning the splicing variants involved is available (22, 23). By contrast, fibulin-1 expression appeared weak or absent in cancer tissues such as neurofibrosarcoma, fibrosarcoma, osteocarcinoma, rhabdomyosarcoma, Wilms, and bladder tumors, which do not express ERs (25). Despite the fact that no strong (35) or even negative (23) correlations between fibulin-1 and ER or -? expression levels were found, it is likely that estrogens play an important role in fibulin-1 expression in vivo. This regulation could alter fibulin-1 expression both quantitatively and qualitatively by modulating the ratio between the two main isoforms. Further investigations are underway to understand the relevance of the regulation of fibulin-1 variants in ovarian physiology, particularly during hormone fluctuation periods such as ovulation, and cancer.
Acknowledgments
The authors thank Professor J. P. Daurès for statistical analysis.
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Address all correspondence and requests for reprints to: P. Pujol, Service de Biologie Cellulaire et Hormonale, H?pital Arnaud de Villeneuve, 271, Av G. Giraud, 34095 Montpellier, France. E-mail: p-pujol@chu-montpellier.fr.
Abstract
Fibulin-1 is an extracellular matrix protein overexpressed in epithelial ovarian and breast cancers. In estrogen receptor (ER)-positive ovarian and breast cancer cell lines, fibulin-1 mRNA levels are markedly increased by estrogens. Transfection experiments using fibulin-1 promoter constructs indicate that 17?-estradiol (E2) increases fibulin-1 gene transcription and that ER is more potent than ER? to mediate E2 regulation of the transfected fibulin-1 promoter. Using SL2 cells devoid of specificity protein 1 (Sp1) and site-directed mutagenesis of GC boxes, we evidenced that the E2 regulation occurs through a proximal specificity protein 1 binding site. In addition, we show that fibulin-1C and -1D mRNAs, the two major fibulin-1 splicing variants, are differentially induced by E2. The induction of both mRNAs variants is direct and independent of a newly synthesized protein intermediate. Interestingly, actinomycin D chase experiments demonstrate that E2 treatment selectively shortens the fibulin-1D mRNA half-life. This indicates that estrogens affect differentially the stability of fibulin-1 variants and may explain the lower accumulation of fibulin-1D mRNA on E2 treatment. In conclusion, our data show that estrogens, via ER, are key regulators of fibulin-1 expression at both the transcriptional and posttranscriptional levels. The preferential induction of the fibulin-1C variant, which is overexpressed in ovarian and breast cancer, might play an important role in estrogen-promoted carcinogenesis.
Introduction
IT IS WELL DOCUMENTED that the mitogenic action of estrogens is critical in the etiology and progression of human breast and gynecological cancers (1, 2). The promoting effect of estrogens has been recently highlighted by the results of large prospective studies (3, 4, 5, 6), showing that estradiol intake during menopause increased the risk of breast cancer. In ovarian cancer, although the question is still debated (7), several recent prospective studies (8, 9, 10, 11) indicated an increased risk for women undergoing long-term estrogen replacement therapy.
The effects of estrogens are mediated by two estrogen receptors (ERs), ER (NR3A1) and ER? (NR3A2), which belong to a large family of nuclear receptors (12, 13). ER and ER? classically mediate their action by ligand-dependent binding to the estrogen-responsive element (ERE) of target genes, leading to their transcription regulation (12, 13, 14, 15). Estrogens also transcriptionally regulate target genes via ERs through ERE-independent modes of action. These effects are mediated through promoter elements that bind various transcription factors, including activator protein-1 (16) or specificity protein (Sp1) (17) binding sites.
E2 regulation of proteins has been well documented in breast cancer (18), but little is known about E2 regulation of proteins in ovarian cancers. In previous studies, we have shown that the secretion of the extracellular matrix fibulin-1 protein is induced by estrogens in ER-positive ovarian cancer cell lines (19, 20). We also demonstrated that fibulin-1 is overexpressed in epithelial ovarian cancer (19, 21) as reported by others in breast cancer (22, 23). Fibulin-1 could be involved in cell motility and anchorage-independent growth of tumors cells (24, 25, 26). These cellular functions likely play a crucial role in ovarian cancer tumor progression because the local spread of cancer cells within the peritoneal cavity and in lymphatic vessels contributes to the high mortality and late diagnosis of ovarian cancer (27). Other studies have shown that fibulin-1 could have a role in cellular differentiation. Indeed, fibulin-1 interacts with growth factors such as pro-HB-epidermal growth factor (EGF), NovH, or ?-amyloid (28, 29, 30) and could regulate the cell bioavailability of these factors. Moreover, fibulin-1 binds the papilloma virus E6 protein and could prevent p53 degradation by viral proteins, thus allowing the cell cycle and proliferation regulation and inhibiting E6-mediated transformation (31).
Fibulin-1 is composed of four different splicing isoforms (A, B, C, and D) (32, 33, 34), with variants 1C and 1D being the major forms expressed in ovaries (35). Fibulin-1 variants may have distinct biological properties as suggested by the recent discovery of fibulin-1D variant involvement in the synpolydactyly, a hand malformation syndrome (36). Alteration in fibulin-1D expression, whereas fibulin-1C is normally expressed, seems to reduce apoptosis in interdigital tissues and cause observed malformations. The different binding affinity of fibulin-1 variants with protein partners such as nidogen-1 (37) could be a molecular explanation for their distinct roles.
Very little is known about fibulin-1 regulation and splicing variant expression. We previously demonstrated that fibulin-1 mRNA is induced by estrogens (35) in ER+ ovarian cancer cell lines. The aim of the present study was to further investigate the regulatory mechanisms that govern the expression of fibulin-1. We analyzed fibulin-1 mRNA accumulation in response to various steroid hormones and peptide growth factors in ovarian and breast cancer cells. We showed that E2 regulation involves a direct transcriptional regulation, which requires ER acting through the proximal Sp1 site, and a specific decrease in fibulin-1D mRNA stability. This complex regulation leads to a differential accumulation of variant forms C and D mRNAs, which could be of importance in ovarian and breast tumorigenesis.
Materials and Methods
Cell lines
The ovarian cancer cell lines BG-1, SKOV3, PEO-4, OVCAR-3, and PEO-14 and the breast cancer cell lines MCF-7 were used in this study. BG-1 (38) (obtained from Dr. C. E. Welander, Emory University, Atlanta, GA), MCF-7, and SKOV3 cells (obtained from the American Type Culture Collection, Manassas, VA) were cultured in DMEM/F12 (Invitrogen, Cergy-Pontoise, France) supplemented with 10% fetal calf serum (FCS, Invitrogen). PEO-14, PEO-4 (obtained from Dr. S. P. Langdon, Hospital of Edinburgh, UK), and OVCAR-3 cells (obtained from the American Type Culture Collection) were cultured in RPMI 1640 (Invitrogen) supplemented with 10% FCS. Cells were stripped from endogenous steroids by 7-d weaning in DMEM/F12 or RPMI 1640 without phenol red containing 10% FCS treated with dextran-coated charcoal. Drosophila SL2 cells were grown at 25 C without CO2 in SF900II medium (Life Technologies, Grand Island, NY) supplemented with penicillin and streptomycin sulfate.
Plasmid construction
pSG5-ER and pSG5-ER? correspond to the wild-type human ER and ER? cDNAs cloned into pSG5 plasmid (Stratagene, La Jolla, CA) under control of the Simian virus 40 promoter. A pCMV-?-Gal reporter was used as an internal control and corresponds to the ?-Gal cDNA cloned in the pCMV5 plasmid (Stratagene). PpacSp1 was obtained from R. Tjian (Howard Hughes Medical Institute, University of California, San Diego, CA).
As previously described by Castoldi and Chu (39), all fibulin-1 promoter-reporter constructs were obtained by subcloning various genomic fragments prepared by restriction enzyme digestion into the polylinker of phRG-basic vector (Promega, Charbonnier, France), which contains a firefly luciferase gene. The common 3'-end of the constructs was situated at +101 and their 5' ends corresponded to –38 bp (pHF1), –68 bp (pHF2), –285 bp (pHF3), and –3505 bp (pHF4) upstream from the transcription start site.
The pHF232 and pHF257 plasmids corresponded to the pHF2 construct containing a mutation in the Sp1 binding sites, located respectively at –32 and –57 bp upstream from the transcription start site. Mutations were introduced using the QuikChange XL site-directed mutagenesis kit (Stratagene) according to the manufacturer’s instructions and were both verified by sequencing. The primers used to generate the mutated Sp1 sites at –32 and –57 bp were respectively: 5'-CCGCGCCCTCCTCCCGGTTGGGATAATTGAACGG-3' and 5'-CAGCCCATGGGCCCCTTCTCCGCCGCGCCCTCC-3'.
Transient transfection and reporter assays
Approximately 5.105 MCF-7 cells were plated in 12-well plates 48 h before transfection. Cells were cotransfected with 2 μg of each firefly luciferase reporter construct, 0.5 μg of pSG5-hER or -?, and 0.5 μg of the internal reference pCMV-?-Gal reporter plasmid. Transfections were performed overnight with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocols. Cells were then treated with 10–8 M 17?-estradiol (E2) or ethanol vehicle. Twenty-four hours after E2 treatment, the cells were lysed directly in the plates with 200 μl of cell culture lysis reagent (Promega). Luciferase activity was measured on 100 μl of lysate aliquots for 10 sec after injection of luciferase detection solution [20 mM tricine (pH 7.8), 1.07 mM (MgCO3)4 Mg(OH)2 5H2O, 2.67 mM MgSO4, 1 mM EDTA, 0.53 mM ATP, 0.47 mM luciferin, and 0.27 mM coenzyme A] in an LKB luminometer (BioMérieux, Marcy l’Etoile, France). The induction of luciferase activity is indicated in arbitrary units, corrected by ?-Gal activity and the values obtained for pGL2 basic transfected cells. Absorbance of the ?-Gal assay was read in a microtitertek at 420 nm. All transfections were performed in triplicate and repeated at least three times. Drosophila SL2 cells (5 x 106) were plated in 24-well plates, and transfection was carried out using Lipofectamine 2000 (Invitrogen). Each well was transfected with 0.25 μg reporter plasmid, 0.5 μg ?-Gal plasmid, and pPac-Sp1 (0–125 ng) or pSG5-HEGO expression vectors (0–125 ng) adjusted, when necessary, by the addition of empty vectors to equalize the total DNA transfected. Twenty-four hours after transfection, the cells were harvested and assayed for luciferase activity as described above.
Northern blot
For RNA extraction, cells were harvested at 80% confluence. Total RNA was extracted using the RN easy midikit (Qiagen, Courtaboeuf, France). Twenty micrograms total RNA were electrophoresed in 1% agarose gel containing formaldehyde and transferred to Hybond-N+ membrane (Amersham Corp., Les Ulis, France). The fibulin-1 probes used for Northern blot analysis of C and D variants were PCR-generated fragments of 442 and 541 bp, respectively, as previously described (35). The 18S and fibulin-1 probes were 32P-labeled by the Radprime DNA labeling system (Invitrogen). Hybridization was performed overnight using Ultrahyb hybridization buffer (Ambion, Austin, TX), and washes were performed as previously described (40). When required, the membranes were stripped of probe by boiling in 0.5% sodium dodecyl sulfate solution and rehybridized. Signals were quantified using PC BAS 1000 reader (Fujix).
Reverse transcription and real-time quantitative PCR
Classically, 1 μg total RNA was reverse transcribed as previously described (35). Real-time PCR quantification was performed using a SYBR Green approach (Light Cycler; Roche Diagnostics, Indianapolis, IN). PCR was carried out in a final volume of 20 μl using 1 μl of each primer (10 μM), 4 μl of the supplied enzyme mix, 9 μl H2O, and finally 5 μl of the template diluted at 1:10. After a 10-min preincubation at 95 C, runs corresponded to 45 cycles of 15 sec at 95 C (denaturation), 6 sec at 65 C (annealing), and 12 sec at 72 C (amplification). Primers for fibulin-1C and fibulin-1D (35) or hypoxanthine-guanine phosphoribosyl transferase (HPRT) (41) were previously described. PCR products were subjected to melting curves analysis using the light cycler system to exclude the amplification of unspecific products. For each sample, fibulin-1 mRNA levels were corrected for HPRT mRNA levels (reference gene).
Statistical analysis
The results were presented as mean ± SD. Statistical analysis was performed by Mann-Whitney U test. A value of P < 0.05 was considered to be statistically significant.
Results
Regulation of fibulin-1 mRNA in ovarian and breast cancer cells
Previous studies by our laboratory have shown that E2 induced fibulin-1 expression in ovarian cancer cells at both the mRNA and protein levels (19, 20, 35). As shown in Fig. 1A, E2 increased by 3-fold the level of fibulin-1C mRNA, the major form of fibulin-1 variant expressed in ovarian cancer, in the ER + BG-1, PEO4, and SKOV3 ovarian cancer cell lines. The pure antiestrogen ICI 182, 780 did not modify the basal expression of fibulin-1C mRNA but completely abolished its induction by E2 in BG-1 cells. By contrast, no estrogen induction of fibulin-1C was observed in the PEO-14 and OVCAR-3 ER-negative ovarian cancer cell lines (Fig. 1B). Because of the low level of expression of fibulin-1 in MCF-7 breast cancer cells, the fibulin-1 mRNA could not be detected by Northern blot experiments. We therefore set up the quantification of fibulin-1C mRNA using real-time quantitative RT-PCR (Fig. 1C). This experiments indicated that the same regulation of fibulin-1C mRNA by E2 (3-fold) was obtained in BG-1 and MCF-7 cells despite a 300-fold lower expression in MCF-7 cells.
FIG. 1. Regulation of fibulin-1C expression by E2 in ovarian and breast cancer cells. A, The BG-1 ER+ ovarian cancer cells were treated for 48 h with control vehicle ethanol (C), E2 (10–8 M), ICI 182, 780 (I) (10–7 M), or a combination (E2+I). PEO-4 and SKOV3 were treated with control vehicle ethanol, E2, and ICI 182, 780 in the same conditions used for BG-1 cells. mRNA accumulation was quantified by Northern blot. The results are expressed as n-fold induction after normalization by 18S of fibulin-1C (Fib-1C) mRNA content in vehicle treated cells. B, Same experiment as in A with the ER-ovarian cancer cells PEO-14 and OVCAR-3. C, BG-1 and MCF-7 cells were treated for 48 h with control vehicle ethanol (C) or E2 (10–8 M). mRNA was quantified by real-time quantitative RT-PCR. The results are expressed in arbitrary units and correspond at the fibulin-1C expression after normalization by HPRT. Values are the means ± SD of three independent experiments.
We then studied whether other nuclear receptor ligands could regulate the expression of fibulin-1C mRNA in ovarian cancer cells. As shown in Fig. 2A, treatment of BG-1 cells with retinoic acid or nonmetabolizable androgens or progestins (R1881 and R5020, respectively) had no effects on fibulin-1C mRNA expression. By contrast, glucocorticoids such as dexamethasone slightly decreased fibulin-1C mRNA levels, as previously reported in bone marrow (42).
FIG. 2. Regulation of fibulin-1C (Fib-1C) expression by other effectors in BG-1. A, BG-1 cells were treated with control vehicle ethanol (C), E2 (10–8 M), R1881 (10–9 M), R5020 (10–6 M), dexamethasone (DXM) (10–6 M), retinoic acid (RA) (5.10–6 M), or RU486 (10–6 M) for 48 h. Total RNA was processed by Northern blot, and the hybridization was performed with fibulin-1C or 18S probes. The quantification results of Northern blot experiments after normalization by 18S RNA levels are indicated below. The results are expressed as n-fold induction after normalization by 18S of fibulin-1C mRNA content in vehicle-treated cells. *, P < 0.05 vs. vehicle-treated cells (C). B, BG-1 cells were treated with control vehicle ethanol (C), E2 (10–8 M), EGF (100 ng/ml), or IGF-I (5 nM) alone or combined with E2 (10–8 M) for 48 h or with CT (1 μg/ml) and cAMP (AMPc) (1 mM) for 24 h. The previous treatments were carried out after 7 d starvation in DMEM/F12 without phenol red with dextran-coated charcoal-treated FCS as in all the other experiments. The culture medium was then replaced by DMEM/F12 without FCS for 24 h before the addition of growth factors, CT, and cAMP in DMEM/F12 alone. The results are expressed as n-fold induction after normalization by 18S of fibulin-1C mRNA content in vehicle-treated cells. *, P < 0.05 vs. vehicle-treated cells (C).
As BG-1 cells were described to be responsive to peptide growth factors such as IGF-I and EGF (43), we investigated whether these mitogenic molecules could also regulate fibulin-1 expression or interfere with its E2-regulation. EGF and IGF-I significantly inhibited fibulin-1C mRNA expression (Fig. 2B) and, when administered in combination with E2, partially blocked estrogen induction (especially IGF-I). These results suggest that these growth factor pathways may be involved in the regulation of fibulin-1 expression and could cross-talk with estrogen signaling. In addition, cAMP and cholera toxin (CT), which both mimic constitutive activation of the G protein-coupled receptor signaling pathway, also negatively affected fibulin-1C mRNA expression (Fig. 2B).
Transcriptional regulation of fibulin-1 gene expression by E2
To further investigate the transcriptional regulation of fibulin-1 gene by E2, we performed transient transfection experiments using four different promoter constructs. The common 3'-ends of these constructs corresponded to +101 bp downstream from the transcription start site and their 5'-ends corresponded to –38, –68, –285, and –3505 bp for pHF1, pHF2, pHF3, and pHF4 constructs, respectively (Fig. 3). These experiments were carried out in MCF-7 cells because of their facility of transfection, compared with BG-1 cells.
FIG. 3. Study of estradiol effects on the fibulin-1 promoter. A, Nucleotide sequence of the human fibulin-1 promoter. The transcription start is denoted as +1 and shown by an arrow. Putative regulatory elements (underlined) and the TATA-like sequenced (boxed) are also indicated. B, Schematic representation of the four different fibulin-1 promoter constructs used in this study. They contain up to upstream the luciferase reporter gene (+1 represents the transcription start site). The position of Sp1 sites and half-EREs is also shown. Upon transfection of the four constructs into MCF-7 cells, luciferase activity was measured after treatment with E2 (10–8 M) or ethanol vehicle, compared with cotransfected CMV-?-Gal. Values are the means ± SD of three independent experiments. *, P < 0.05 vs. control of pHF1. C, Luciferase activity was measured after transient transfection of pHF4 and pSG5-ER, pSG5-ER?, or the empty pSG5 plasmid in MCF-7 cells. Cells were treated with ethanol vehicle (C), E2 (10–8 M), 4-hydroxy tamoxifen (TAM) (10–7 M), or ICI 182, 780 (ICI) (10–7 M) for 24 h. Values are the means ± SD of three independent experiments. *, P < 0.05 vs. control of pSG5. #, P value not significant.
According to Castoldi and Chu (39), a basal luciferase activity was obtained with pHF1, and it significantly increased with pHF2 and pHF3 reporter constructs. By contrast, the distal region of the promoter (–3505 to –285 bp) had no additional effect on the transcriptional activity of the fibulin-1 promoter (compare pHF4 and pHF3 constructs in Fig. 3B). Interestingly, on E2-stimulation, a significant increase in luciferase activity was obtained with the pHF2, -3, and -4 reporter constructs but not with the pHF1 reporter plasmid (Fig. 3B). This suggested that an important regulatory element was located between –38 and –68 bp.
To determine the respective roles of the two ERs in the regulation of fibulin-1 expression, the pHF4 construct was cotransfected in MCF-7 breast cancer cells with a human (h)ER or hER? expression vector. In the presence of E2, we observed a significant increase in luciferase activity in hER cotransfected cells, compared with cells transfected with the empty pSG5 plasmid (Fig. 3C). By contrast, hER? was not able to change the magnitude of E2 induction. This was in agreement with our previous results obtained on the endogenous fibulin-1 gene after infection of MDA-MB-231 ER-breast cancer cells with a recombinant adenovirus containing either ER or ER? cDNA (35). The partial and pure antiestrogens treatment with 4-hydroxy-tamoxifen or the ICI 182, 780 compound had no significant effect on basal transcriptional activity of the pHF4 reporter construct (Fig. 3C).
Role of Sp1 in E2 regulation of fibulin-1 gene expression
The fibulin-1 proximal promoter region contains two binding sites for the Sp1 transcription factor, located at –32 and –57 bp upstream from the transcription start site (Fig. 3, A and B) (39). The Sp1 transcription factor has been frequently involved in mediating transcriptional responses to E2 through recruitment of ER via protein-protein interactions (44). Based on our deletion analysis (Fig. 3), the Sp1 binding site located at the –57 bp position could be critical for fibulin-1 regulation by E2.
The importance of the Sp1 transcription factor in the regulation of fibulin-1 promoter was first assessed in Drosophila SL2 cells that do not express Sp1 proteins (45). In these cells, cotransfection of the pHF4 construct with increasing concentrations of Sp1 expression plasmid strongly increased luciferase activity, thus demonstrating the response of the fibulin-1 promoter to Sp1 (Fig. 4A). Interestingly, in SL2 cells, the same pHF4 construct did not confer E2 responsiveness, whereas a marked induction was noticed for an ERE-containing reporter construct (Fig. 4B). These results indicate that the Sp1 transcription factor is necessary not only for the basal expression of fibulin-1 gene but also for its regulation by estrogens.
FIG. 4. Sp1 is required for fibulin-1 induction by estrogens. A, SL2 cells were transiently transfected with the pHF4 construct together with increasing concentrations (0, 5, 25, or 125 ng) of Sp1 expression vector. Luciferase activity was measured 48 h later and corrected for the corresponding ?-Gal activity. Values are the means ± SD of two independent transfections performed in triplicate. *, P < 0.05 vs. control (0 ng Sp1). B, The pHF4 construct or a reporter vector containing an ERE in front of the ?-globin (Glob) promoter and the luciferase gene were transiently transfected into SL2 cells together with the expression vector for ER. Cells were then treated or not with E2 (10 nM), and luciferase activity was measured 24 h later as described in A. *, P < 0.05 vs. vehicle-treated cells (C). C, SL2 cells were transiently transfected with the pHF2 constructs containing the wild-type sequence or mutated on the Sp1 sites at position –57 or –32 in combination with either the expression vector for Sp1 (50 ng) or the corresponding empty vector. Luciferase activity was quantified as in A. *, P < 0.05 vs. corresponding control. D, MCF-7 cells were transiently transfected with the pHF1 or pHF2 constructs containing the wild-type sequence or mutated on the Sp1 sites at position –57 or –32 together with the expression vector for ER. Cells were then treated or not with E2 (10 nM), and luciferase activity was measured 24 h later as described before. Values are the means ± SD of two independent transfections performed in triplicate. *, P < 0.05 vs. corresponding control.
To further demonstrate the role of Sp1 transcription factor in the E2-regulation of fibulin-1, we generated a pHF2 construct with a mutation in the Sp1 site at position –57 (named pHF257), which appeared to be the critical element for E2 responsiveness according to data shown in Fig. 3B. As a control, we also mutated the second putative Sp1 binding site (named pHF232) present in the pHF2 construct. As shown in Fig. 4C, the effect of Sp1 in SL2 cells was completely abolished on pHF257, compared with pHF2 or pHF232, thus confirming the role of the distal GC box. As expected, when transfected in MCF-7 cells, the pHF257 mutant no longer conferred E2-regulation, compared with the wild-type counterpart or to the pHF232 mutant (Fig. 4D).
Altogether, these data demonstrated that the transcriptional regulation of fibulin-1 gene by E2 required the presence of the Sp1 transcription factor acting through its response element located at –57 bp from the start site.
E2 differentially regulates fibulin-1C and -1D mRNA expression and stability
Fibulin-1 mRNA is expressed as several variants differing in their 3' untranslated region. We have previously shown that the C and D isoforms were the two major variants present in ovarian tissues, fibulin-1C being predominant in ovarian cancer (35). By Northern blot, we compared the E2-regulation of fibulin-1C and -1D mRNA in BG-1 cells. Interestingly, estrogen induction was higher for fibulin-1 mRNA variant C than variant D (Fig. 5, A and B). Using real-time quantitative PCR, we also found the same magnitude of differential regulation by E2 of the two fibulin-1 mRNA variants in both BG-1 and MCF-7 cells (Fig. 5C).
FIG. 5. E2 regulation of fibulin-1C (Fib-1C) and -1D (Fib-1D) mRNA in BG-1 and MCF-7 cells. A, BG-1 cells were treated with ethanol vehicle (C) or E2 (10–8 M) for 48 h. Northern blots were performed with 20 μg total RNA, and hybridization was carried out with fibulin-1C or -1D or 18S probes. B, Quantification of Northern blot experiments shown in A after normalization according to 18S RNA levels. The results are expressed as n-fold induction of E2-treated vs. vehicle-treated cells and are means of three independent experiments. C, MCF-7 cells were treated with ethanol (C) or E2 (10–8 M) for 48 h, and fibulin-1C and -D mRNAs were quantified by real-time quantitative RT-PCR. The results are expressed in arbitrary units after normalization by HPRT. Values are the means ± SD of three independent experiments. For data shown in B and C, statistical analysis indicated that the fold induction of fibulin-1C or -1D mRNAs on E2 stimulation was significant (P < 0.05).
As shown in Fig. 6, cycloheximide had no significant effect on E2 induction of either fibulin-1C or fibulin-1D, suggesting that regulation of both variants was not dependent upon de novo synthesis of a protein intermediate. We then measured fibulin-1C and -1D mRNA half-lives by actinomycin D chase experiments to compare their respective stabilities in the presence of E2 in BG-1 cells. The apparent half-life of both fibulin-1C and 1-D mRNA was approximately 8 h in the absence of E2 (Fig. 7, A and B). E2 treatment did not affect the fibulin-1C mRNA half-life (Fig. 7A), whereas it decreased that of fibulin-1D mRNA (5.5 h, Fig. 7B). This differential stability of the two mRNAs isoforms on E2 treatment could therefore explain the lower E2 induction of fibulin-1D mRNA steady-state levels, compared with fibulin-1C (Figs. 5 and 6).
FIG. 6. Direct E2 stimulation of fibulin-1C (Fib-1C) and -1D (Fib-1D) mRNA in BG-1 cells. A, BG-1 cells were treated for 1 h with cycloheximide (30 μg/ml) (CHX) before adding E2 (10–8 M) with the antibiotic still present. Cells were scrapped 15 h after E2 treatment. Northern blot was performed with 20 μg total RNA, and hybridization was conducted with fibulin-1C, fibulin-1D, and 18S probes. B, Quantification of Northern blot experiments after normalization by 18S RNA levels. The results are means of three independent experiments. Statistical analysis comparing the E2 induction of fibulin-1C or -1D mRNAs with or without cycloheximide treatment did not show any significant difference.
FIG. 7. Stability of fibulin-1C (Fib-1C) and -1D (Fib-1D) mRNAs in response to E2. A, BG-1 cells were treated or not with E2 (10–8 M) for 48 h and then incubated for different times (0, 5, and 10 h) with actinomycin D (3 μg/ml) before RNA extraction. Northern blot was performed with total RNA, and hybridization was carried out with fibulin-1C (A) or fibulin-1D (B). The same membrane was rehybridized with the 18S probes. B, Quantification of Northern blot experiments shown in A after normalization by 18S RNA levels. The results are expressed as 100% of fibulin-1C or fibulin-1D/18S RNA levels without actinomycin D treatment (0 h). The results are means of three independent experiments. *, P < 0.05 vs. corresponding vehicle-treated cells (C).
Discussion
In this study, we characterized a complex regulation of fibulin-1 expression by E2 in ovarian and breast cancer cells leading to a differential accumulation of fibulin-1C and D mRNA isoforms. Up-regulation of fibulin-1 expression under estrogen stimulation takes place at the transcriptional level, and this induction involves ER action through a Sp1 binding site. We also demonstrate the existence of a negative control at the posttranscriptional level because E2 treatment appears to decrease specifically the stability of fibulin-1D mRNA.
Mechanism of transcriptional regulation by E2
The recent cloning of the fibulin-1 promoter (39) showed that the Sp1 site at –57 bp upstream from the transcription start site was responsible for part of the basal promoter activity via Sp1 and Sp3 transcription factors. Our results demonstrate that the two half-ERE consensus sequences present on the pHF4 construct do not appear to be necessary for efficient E2 regulation. By contrast, several lines of evidence indicate that Sp1 transcription factor binding on the promoter was required for E2 regulation of fibulin-1 gene transcription. First, the deletion analysis mapped the critical regulatory element to sequences a region between –38 and –68 bp, which contains one of the two GC boxes. Second, no regulation was obtained in Drosophila cells devoid of Sp1 protein. In addition, treatment of MCF-7 cells with mithramycin A, which inhibits the interaction of Sp1 with its DNA target sequence (46), significantly reduced E2 induction of luciferase activity expressed from the pHF4 reporter vector (data not shown). Finally, the mutagenesis of the distal Sp1 binding site located at –57 bp completely abolished E2-induction of the fibulin-1 promoter.
Other studies have highlighted the role of the ER-Sp1 interaction in E2 activation of gene transcription (17, 47). In line with data showing that Sp1-mediated E2 regulation was supported more efficiently by ER than ER? (48), our data showed that the transfected reporter constructs was regulated via ER but not ER?. We obtained the same results on the endogenous fibulin-1 gene after adenoviral infection of ER-breast cancer cells (35).
Posttranscriptional regulation by E2
Our data demonstrate that posttranscriptional events are likely to be responsible for the differential regulation of the fibulin-1C and -1D variants. The two isoforms derive via alternative 3' splice sites from the same pre-mRNA transcribed from a single promoter. The spliced region encoding fibulin-1C contains the proximal exon 17, whereas fibulin-1D mRNA encompasses the more distal exons 18, 19, and 20.
There are many steps in the pathway of RNA metabolism at which steroid hormones might act to alter the levels of a given mRNA. Estrogen regulation of fibulin-1 isoforms could take place at the level of mRNA splicing (49). Other steroids such as progestins have been shown to regulate alternative splicing of reporter genes through mechanisms that could involve nuclear receptor coregulators (50). Further work will be necessary to precise whether such mechanisms could be involved in the hormonal control of the fibulin-1 gene.
The results presented herein strongly suggest that the differential induction of fibulin-1C and -1D mRNAs by estrogens in breast and ovarian cancer cells is associated with a specific regulation of mRNA stability. Indeed, the results obtained in the present study demonstrate that the fibulin-1D mRNA half-life is decreased in the presence of E2, whereas hormone treatment did not modify the apparent stability of fibulin-1C mRNA. Little is known about the hormonal control of mRNA half-life, but several reports have described the role of adenylate/uridylate-rich elements (AREs) located in the 3'-untranslated regions (UTRs). These AREs represent a combination of functionally and structurally distinct sequence motifs or domains, such as AUUUA motifs, UUAUUUA(U/A)(U/A) nonamers, U stretches, and/or a U-rich domain (51). A number of proteins that bind these AREs and increase mRNA stability are induced by estrogens, such as vigilin or E-RmRNASF, which stabilizes, respectively, the vitellogenin (52) and apolipoprotein II mRNAs (53). Fewer estrogen-induced proteins associated with ARE-containing mRNAs are involved in mRNA destabilization. The Xenopus polysomal ribonuclease 1 is implicated in the destabilization of albumin mRNA (54), and AUF1/hnRNPD (55) is involved in mRNA destabilization of c-myc mRNA (56, 57). It has been also demonstrated that TIS11 (also named TPA-inducible sequence 11 or TTP) (58), TIS11b, and TIS11d bind to the AREs of TNF, granulocyte macrophage colony-stimulating factor, and IL-3 mRNAs and induce their destabilization (59, 60, 61). Interestingly, TIS11b is induced by numerous mitogenic factors in different cells (62) and estrogens in rat uterine cells (63).
The fibulin-1D 3'UTR has a higher uridin-rich content than fibulin-1C and we could hypothesized that the 3'-UTR of fibulin-1D mRNA could be more sensitive to degradation by one of mechanisms describe above or by a still-unknown ribonuclease. Further work will be required to characterize the sequences and factors involved in the specific E2-induced degradation of fibulin-1D mRNA.
Expression of fibulin-1 isoforms and physiopathological relevance
The differential expression of fibulin-1C and -1D variants under estrogen stimulation provide, to our knowledge, a first example of variant-specific regulation by estrogens due to a specific destabilization of splicing variant.
Interestingly, an increased fibulin-1C to fibulin-1D ratio was previously noted in ovarian cancers, compared with normal ovaries (35). This overexpression of variant C in cancer may thus be related to estrogen stimulation. Fibulin-1 is overexpressed during breast carcinogenesis, although no information concerning the splicing variants involved is available (22, 23). By contrast, fibulin-1 expression appeared weak or absent in cancer tissues such as neurofibrosarcoma, fibrosarcoma, osteocarcinoma, rhabdomyosarcoma, Wilms, and bladder tumors, which do not express ERs (25). Despite the fact that no strong (35) or even negative (23) correlations between fibulin-1 and ER or -? expression levels were found, it is likely that estrogens play an important role in fibulin-1 expression in vivo. This regulation could alter fibulin-1 expression both quantitatively and qualitatively by modulating the ratio between the two main isoforms. Further investigations are underway to understand the relevance of the regulation of fibulin-1 variants in ovarian physiology, particularly during hormone fluctuation periods such as ovulation, and cancer.
Acknowledgments
The authors thank Professor J. P. Daurès for statistical analysis.
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