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Recently, we identified and cloned a membrane-based estrogen receptor (ER), ER-α36, which is transcribed from a previously unidentified promoter in the first intron of the original ERα gene. We cloned the 5′ flanking region of ER-α36 and found the cloned DNA sequence possessed strong promoter activity. A single transcription initiation site was mapped and a number of putative regulatory elements for various transcription factors such as the Wilms’ tumor suppressor, WT1 and estrogen receptor (ER) were identified in this region. Transient co-transfection experiments further demonstrated that ER-α suppresses ER-α36 promoter activity in an estrogen-independent manner, which can be released by ER-α36 itself.
Diverse functions of estrogens are mediated by estrogen receptors: ER-α and ER-α. Both ER-α and ER-α function as ligand-dependent transcription factors and consist of an N-terminal activation function 1 (AF1) domain, a DNA-binding domain, and a C-terminal ligand-binding domain that harbors an activation function 2 (AF2) domain [1–4] [YZ1]. The liganded ERs readily form homodimers or heterodimers that interact with the palindromic estrogen response-element (ERE) in the promoter regions of estrogen responsive genes and stimulate gene transcription [1–4]. Alternatively, ER-α may act indirectly by tethering to other transcription factors, such as Sp1 and AP1 to modulate activities of these transcription factors, which in turn regulates downstream gene expression [1–5].
For the past 30 years, accumulating evidence demonstrates a rapid (within seconds or minutes) estrogen action that cannot be explained by the genomic signaling pathway that usually requires hours to reach maximal gene activation [6–11]. This pathway is also called the “non-classical”, “extra-nuclear”, “non-genomic” or “membrane-associated” estrogen signaling pathway [8,9]. However, the identity of the membrane-based estrogen receptor that mediates these rapid estrogen effects has not been well established.
Recently, we identified and cloned a variant of ER-α with a molecular weight of 36 kDa that is transcribed from a previously unidentified promoter located in the first intron of the original ER-α (ER-α66) gene [12,13]. This novel isoform of ER-α, ER-α36 lacks both transcriptional activation domains of ER-α (AF1 and AF2), but retains the DNA-binding domain and partial dimerization and ligand-binding domains. ER-α36 is predominantly expressed on the cell surface and mediates the membrane-initiated estrogen signaling , indicating that ER-α36 is a membrane-based estrogen receptor and a novel player in mitogenic estrogen signaling.
Our recent study also revealed that ERα36 is not only expressed in ER-positive breast cancer but also expressed in ER-negative breast cancer that lacks expression of ER-α66 , further indicating that ER-α36 is subjected to a totally different transcriptional regulation from the original ER-α. As an important mediator of estrogen signaling, ER-α36 must be regulated dynamically and strictly to maintain a normal estrogen signaling. Dysregulation of its expression may be involved in development of a number of human diseases including breast cancer.
In order to elucidate the molecular mechanisms underlying regulation of this biologically important gene, we isolated and cloned the 5′-flanking region of ER-α36, and characterized the general features of this region.
MCF10A cells were obtained from Karmanos Cancer Institute, Detroit, MI. Human embryonic kidney (HEK) 293 cells, human breast cancer cells, MCF7, T47D. MDA-MB-231, MDA-MB-436, HB3396 and human lung cancer cells H226 were obtained from American Type Culture Collection (ATCC). All cells were maintained at 37 °C in a 5% CO2 atmosphere in appropriate tissue culture medium.
Expression vectors, pCR3.1-ER-α66 and pCR3.1-ER-α46 were obtained from Dr. Zafar Nawaz at the University of Miami Sylvester Comprehensive Cancer. The expression vectors pCR3.1-ER-α36 was constructed as described before . Amounts of the expression vectors used in each transfection assay were normalized by the empty expression vector pCR3.1-ER-α36. The luciferase assays were performed using the Dual Luciferase Assay kit from Promega according to the manufacturer’s recommendation.
According to computer analysis, ER-α36 mRNA is initiated from a promoter located in the first intron of ER-α genomic DNA. Using a human placenta genomic DNA library (Clontech Laboratories Inc.) as a template, the 5′-flanking region of ER-α36 was amplified by a genomic PCR kit (Clontech Laboratories Inc.), using a primer set containing XhoI or HindIII restriction sites at the ends, respectively: forward primer 5′-CCCTCGAGGGTACCCGCGCCCGC-3′ and reverse primer 5′-CCAAGCTTAAAAATTCGTGAACACGAAGG-3′. The amplified 751 bp DNA product was cloned into a TA cloning pCR2.1 vector (Invitrogen) and verified by DNA sequencing on both strands. The putative transcription factor binding sites in the 5′ flanking region of ER-α36 gene were analyzed using the AliBaba 2.1 promoter analysis software.
The DNA fragment generated by genomic PCR was cloned upstream of a luciferase reporter gene in a promoter-less pGL2-basic luciferase reporter vector (Promega Co.). The resulted plasmid was named as pGL2-ER36. The 5′-deletion mutants of ER-α36 promoter were produced by PCR method from the pGL2-ER36 plasmid. Primers were designed with HindIII and XhoI sites at the ends of the products (Table 1). The PCR products were purified and cloned into the HindIII and XhoI sites upstream of a luciferase reporter gene in the pGL2-basic vector (Promega Co.). All plasmids were then verified by restriction enzyme digestion and DNA sequencing. The seven deletion mutants were named pGL2-ER36 [1–7].
The transcription initiation site in the 5′ flanking sequence of ER-α36 was identified with the 5′ RACE method. Human placenta “marathon-ready” cDNA from Clontech Inc. was used to perform 5′ RACE according to the manufacturer’s instruction. An ER-α36 gene-specific primer 1 (GSP1) was designed (Table 2) for the touch-down PCR, and paired with an adaptor primer 1 (AP1) provided by the kit. Secondary PCR was performed with the gene specific primer 2 (GSP2) and nested primer AP2 from the kit. The PCR products from the 5′ RACE were cloned into the TA cloning pCR2.1 vector and examined by DNA sequencing.
Total RNA was isolated with Trizol (Invitrogen) from cells according to the manufacture’s instructions. RNA integrity was examined by gel-electrophoresis with 6% formaldehyde gel. 500 ng total RNA was reverse transcribed with Protoscript II RT-PCR kit (New England BioLabs). PCR was performed in a final volume of 50 μl containing 2 μl RT transcript, 0.2 μM of each primer, 25 μl Taq 2X Master Mix. The following primers were used: ER-α66 forward primer: 5′-CACTCAACAGCGTGTCTCCGA-3′; ER-α66 reverse primer: 5′-CCAATCTTTCTCTGCCACCCTG-3′; ER-α36 forward primer 5′-CACTCAACAGCGTGTCTCCGA-3′; ER-α36 reverse primer 5′-CC AATCTTTCTCTGCCACCCTG-3′; β-actin forward primer 5′-TGACGGGGTCACCCACACTGTGCCCATCTA-3′; β-actin reverse primer 5′-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3′. Samples were amplified: ER-α36 35 cycles, ER-α66 35 cycles and β-actin, 25 cycles. PCR products (20 μl) were electrophoresed on 1.2% agarose gel and visualized under ultraviolet light after ethidium bromide staining. Fragment sizes were 286 bp for ER-α36, 200 bp for ER-α66 and 661 bp for β-actin. The band densities of ER-α36, ER-α66 and β-actin were determined with a video-densitometry system (Molecular Imager ChemiDoc XRS System, Bio-Rad). Subsequently, the ER-α36:B-actin ratio and ER-α66:B-actin ratio (β-actin mRNAs were detected in the same RT sample) were calculated with the Quantity One software.
Data were summarized as the mean ± standard error (S.E.) using GraphPad InStat software program. The student t-test was also used, and the significance was accepted for P-values of less than 0.05.
ER-α36 is a membrane-based estrogen receptor that mediates non-genomic estrogen signaling and stimulates cell growth . ER-α36 is expressed in specimens from ER-negative breast cancer patients that lack expression of ER-α66 , suggesting that its expression is regulated independently and differently from the ER-α66 gene. However, the molecular mechanisms by which ER-α36 gene transcription is regulated are unknown. We decided to clone the promoter region of ER-α36 and to study its transcriptional regulation. To this aim, we designed primers corresponding to the 5′ flanking sequence upstream of the ER-α36 cDNA according to the published genomic sequence of the ER-α66 gene (ESR1, Gene bank AY425004). A genomic PCR assay was performed using human genomic DNA from placenta. An expected 751 bp PCR product was cloned into a TA cloning vector (pCR2.1), and completely sequenced in both directions. Sequence analysis revealed that the 5′ flanking region contains a high G + C content, a non-canonical TATA box, but lacks the CCAAT box. A number of SP1 sites and AP1 sites were identified in the 5′ flanking region of ER-α36 (Fig. 1). The region also contains putative binding sites for the following transcription factors: AhR (aryl hydrocarbon receptor), Erg-1 (ETS-related gene 1), NF-κB (nuclear factor kappa B), WT1 (the Wilms’ tumor suppressor), PU.1, GATA-1, Elk-1 and GR (glucocorticoid receptor). This region lacks consensus palindromic ERE site (5′-GGTCAnnnTGACC-3′); however, it contains an imperfect ERE half site (5′-AGTCA-3′, located at −369 to −365, relative to the transcription initiation site) that is overlapped with an AP-1 binding site (Fig. 1).
To map the transcription start site of ER-α36, we used human placenta “marathon-ready” cDNA as templates to perform 5′ RACE assay and cloned PCR products into the TA cloning pCR2.1 vector. The DNA clones were sequenced from both directions. We found all DNA clones contained sequences matched exactly to the sequences of the ER-α66 genomic DNA. Two clones contained identical sequences corresponding to the 5′ end of the ER-α36 cDNA. After comparing these two DNA sequences with the 5′ flanking sequence of ER-α36 gene, we mapped the first nucleotide of the transcription initiation at the nucleotide A (+1) 238 bp upstream of the translation initiation site (Fig. 1).
To determine that the 5′ flanking region of ER-α36 gene contains promoter activity, we placed the 751 bp DNA fragment in the front of the firefly luciferase gene of the pGL2-basic vector to generate the plasmid pGL2-ER36. The plasmid pGL2-ER36 was transiently transfected into HEK293 cells and the promoter activity was assessed by measuring luciferase activity in the transfected HEK293 cells. As shown in Fig. 2, pGL2-ER36 exhibited about 14-fold higher promoter activity compared to the empty vector that produced barely detectable luciferase activity. The basal activity of the ER-α36 promoter is about half of that of the SV-40 promoter/enhancer activity in HEK293 cells (Fig. 2).
To further characterize the 5′ flanking region of the ER-α36 gene, we constructed a set of seven reporter vectors, containing successive 5′ deletion of the ER-α36 promoter (Fig. 3). These deletion mutants were then transfected into three cell lines: HEK293, ER-negative breast cancer MDA-MB-231 cells and ER-positive breast cancer MCF7 cells, and the luciferase activities of these constructs were then measured. In HEK293 cells, a significant increase of promoter activity was observed with the removal of 5′ sequence up to nucleotide −584 bp (relative to the transcription initiation site), indicating the presence of negative regulatory element(s) in the region from −736 bp to −584 bp in HEK 293 cells. Further truncation to −513 bp returned the promoter activity to the full-length promoter activity, suggesting the presence of positive regulatory element(s) in the region −584 bp to −513 bp. In ER-positive breast cancer MCF7 cells, however, a decrease of promoter activity was observed in the deletion of the DNA sequence −736 bp to −675 bp, indicating the presence of enhancer element(s) in this region. The promoter activity was without significant changes in the remaining deletion constructs. In ER-negative breast cancer MDA-MB-231 cells, there are no significant changes of the promoter activities of all deletion mutants. In three cell lines, the promoter activity was dramatically reduced after the putative TATA box was deleted, indicating its essential role in the optimal promoter activity of ER-α36 gene. Taken together, these results demonstrated that both negative and positive regulatory regions controlled the basal promoter activity of ER-α36 gene depending on cell context.
Since there is one imperfect ERE half site (−369 bp to −365 bp) in the ER-α36 promoter, we decided to test whether ER-α66 regulates ER-α36 promoter activity using HEK293 cells that express no detectable level of endogenous ER-α. The results from transient co-transfection indicated that co-transfection of ER-α66 expression vector with pGL-ER36 reporter plasmid resulted in about 2-fold repression of ER-α36 promoter activity (Fig. 4) and this suppression was not changed in the presence of 17α-estrodial (data not shown). However, the combination of ER-α66 and ER-α36 or ER-α46, another ER-α variant that lacks the AF-1 domain  released the suppression activity of ER-α66 while co-transfection with ER-α36 or ER-α46 expression vector alone has no effect on ER-α36 promoter activity (Fig. 4). These results suggested that ER-α66 suppresses ER-α36 promoter activity in an estrogen-independent manner presumably through the AF1 domain, which can be released by co-expression of either ER-α46 or ER-α36 itself both of which lack the AF1 domain.
We also examined expression patterns of ER-α66 and 36 in established breast cancer cell lines with RT-PCR analysis. ER-α36 was not expressed in normal mammary epithelial cells MCF10A, weakly expressed in ER-positive breast cancer cells such as MCF7 and T47D both of which express high levels of ER-α66. ER-α36 was highly expressed in ER-negative breast cancer cells such as MDA-MB-231 cells that lack ER-α66 expression (Fig. 5). However, the ER-negative breast cancer MDA-MB-436 cells express low levels of ER-α36. ER-α36 was also highly expressed in the ER-positive breast cancer cells H3396 that have high levels of ER-α46 expression (Fig. 5, data not shown). ER-α36 was also detected in lung cancer H226 cells that lack ER-α66 expression (Fig. 5). The different expression patterns of ER-α66 and 36 in these cancer cells further indicated that ER-α36 is transcriptionally regulated differently from ER-α66.
In this study, we have isolated and cloned a DNA fragment containing the promoter region of ER-α36 gene that is located in the first intron of the human ER-α66 gene. Functional analysis demonstrated that this DNA sequence exhibited strong promoter activity, indicating that this region contains ER-α36 promoter. We also further determined a single transcription start site at the nucleotide A 238 bp upstream of the translation initiation site. Sequence analysis revealed that the ER-α36 promoter region contains a high G + C content, a non-canonical TATA box and multiple Sp1 binding sites. Deletion of these Sp1 sites and the non-canonical TATA box resulted in significantly decreased promoter activity, suggesting these DNA sequences are critical for the optimum ER-α36 promoter activity.
To further analyze ER-α36 promoter, we constructed a series of 5′ truncations of the ER-α36 promoter region and tested promoter activities of these truncated promoters with transient transfection in three cell lines: HEK293 cells, ER-positive breast cancer MCF7 cells and ER-negative breast cancer MDA-MB-231 cells. HEK293 cells express undetectable levels of both ER-α66 and 36; MCF7 cells weakly express ER-α36 but highly express ER-α66 while ER-negative breast cancer MDA-MB-231 cells only express ER-α36. These cell lines presumably provide different genetic background to test ER-α36 promoter activity. In HEK293 cells, the highest transcriptional activity was observed for the promoter deletion 2 (removal of DNA sequence from −675 bp to −584 bp), suggesting existence of negative element(s) in this region. We observed a Wilms’ tumor suppressor WT1 binding site within this region. In preliminary study, we found that WT1 expression inhibited ER-α36 promoter activity through this binding site (unpublished data), suggesting that deletion of the WT1 binding site may release WT1 suppression activity in HEK293 cells that highly express the transcription suppressor WT1. It is worth noting that loss of WT1 expression was implicated in development of breast cancer . Aberrant methylation of WT1 promoter was also found in breast cancer [16,17]. Our laboratory previously demonstrated that over-expression of recombinant WT1 in MDA-MB-231 cells inhibits malignant growth of breast cancer cells . Thus, the finding here that deletion of the WT1 binding site increased ER-α36 promoter activity suggested that WT1 is involved in negative regulation of ER-α36 expression and aberrant WT1 expression may lead to the increased ER-α36 expression observed in breast cancer. We also noticed that several deletion mutants of the ER-α36 flanking region exhibited higher promoter activity in HEK293 cells compared to MCF7 and MDA-MB-231 cells although HEK293 cells express undetectable levels of ER-α36 expression , suggesting that other mechanisms such as DNA methylation may occur in the G + C rich promoter region of ER-α36 and contribute to the regulation of ER-α36 expression.
In MCF7 cells, the full-length promoter exhibited highest promoter activity; deletion of the DNA sequence from −736 bp to −675 bp that harbors several Sp1 binding sites dramatically reduced the promoter activity, suggesting these Sp1 sites act as positively regulatory elements. In MDA-MB-231 cells, most deletion mutants display similar promoter activity.
It is well established that ER-α binds to the palindromic estrogen response-element (ERE) in the target genes and regulates gene transcription . It was also reported that ER-α regulates promoters containing ERE half sites . Other studies suggested that ER-α binds to a single ERE half-site closely located with Sp1 binding sites in the presence of Sp1 binding in the promoters of certain estrogen regulated genes such hsp27 , TGF-alpha  and PR . ER-α36 promoter contains an imperfect ERE half site in adjacent to an Sp1 binding site proximal to the TATA box (Fig. 1). In this study, we found that expression of the original 66-kDa ER-α(ER-α66) suppressed ER-α36 promoter activity in an estrogen-independent manner, presumably through binding to the ERE half site. Flouriot et al.  cloned a 46-kDa isoform of ER-α and demonstrated that the 46-kDa isoform lacking the first 173 amino acids (A/B or AF-1 domain) is derived from alternative splicing of the ER-α gene by skipping exon 1. This isoform of ER-α was named as ER-α46 and the original one as ER-α66 . ER-α46 forms homodimers and heterodimers with ER-α66 . Furthermore, the ER-α46/66 heterodimers form preferentially over the ER-α66 homodimers and ER-α46 acts competitively to inhibit transcription activity mediated by the AF-1 domain of ER-α66 but without effect on an AF-2-dependent transactivation . Here, we found that co-expression ER-α46 or ER-36 itself released suppression activity mediated by ER-α66. The molecular mechanism underlying this release of suppression is currently unknown. The competitive binding to the ERE half site by these ER-α isoforms, or heterodimer formation between ER-α66 and ER-α36 or ER-α46 to block the AF1 function of ER-α66, may explain this phenomenon. It was reported that BRCA1 mediates the ligand-independent transcriptional repression activity of the ER-α66 through its AF-1 domain . Thus, it may be possible that BRCA1 is involved in transcriptional suppression activity mediated by ER-α66 and BRCA1 mutations may lead to upregulation of ER-α36 expression and its activity in BRCA1-related breast cancer.
Recently, we found that singling pathways mediated by EGFR, HER2 and GPR30 regulates ER-α36 expression at both transcriptional and post-transcriptional levels (Kang and Wang, unpublished observations), suggesting as an important player in estrogen signaling, ER-α36 is regulated strictly at both mRNA and protein levels. This may also provide an explanation for the observed inconsistence of ER-α36 expression in MDA-MB-436 cells; low mRNA levels but high protein levels observed before . Further study of the molecular mechanisms by which ER-α36 is regulated at the transcription and post-transcription levels will provide novel and important insights for the biological function of ER-α36 in many physiological and pathological processes.
Grant support: NIH grant DK070016 (Z.Y. Wang) and the Nebraska Tobacco Settlement Biomedical Research Program Award (LB-595) to (Z.Y. Wang).