The human endothelin-converting enzyme-1 (ECE-1) gene is expressed in two different mRNAs, termed α and β(1) (or b and a) (2), which differ only in the 5′-terminal part. The 3′-terminal part, including the exons coding for the catalytic domain, are identical between these two isoforms. The human α-isoform comprises the first two exons and was described in 1994 by Schmidt et al. (3). The human ECE-1β mRNA, first cloned by Shimada et al. (4), is encoded downstream of the 11-kb large intron 2. ECE-1α and -1β show different tissue-expression levels. The α-isoform is expressed more abundantly in tissues such as pancreas, testes, and prostate gland, whereas the β-form is the predominant one in spleen, placenta, and lung (2).
To prove the hypothesis that expression of the ECE-1β isoforms is regulated by an alternative promoter and not by differential splicing, we sequenced and cloned the genomic region upstream of exon 3. Using a luciferase reporter gene assay, we demonstrated that intronic DNA upstream of the first β-specific exon exerts strong promoter activity in BAECs and HUVECs.
MATERIALS AND METHODS
For details see ref. 9.
Genome library screening and sequencing
A λ phage clone was isolated from a human genomic library(Clontech, Palo Alto, CA, U.S.A.) using an ECE-1-specific probe complementary to exon 3. The isolated phage was sequenced by the Cycle Sequencing method (USB, Cleveland, OH, U.S.A.) using several anti-sense primers derived from the 5′-untranslated region upstream of exon 3.
Subcloning into reporter plasmids
A PCR with Pfu polymerase (Stratagene, Heidelberg, Germany) and primers based on the yielded sequencing data was performed on phage DNA as template. The resulting PCR product (1206 bp) was subcloned into a pGL3-Basic luciferase reporter vector (Promega, Madison, WI, U.S.A.).
Construction of deletion mutants
PCR reactions were performed with an anti-sense primer in the common 3′-terminal region of the putative β-promoter region, directly 5′-terminal of theβ-specific translation initiation codon, and several different sense primers located at positions −267, −498, −736, −973 (bp relative to the putative translation initiation codon of the ECE-1β isoform). PCR products were subcloned into the pGL3-Basic vector.
Cell culture and transfection experiments
ECE-1β promoter luciferase constructs and the control plasmids pGL3-Promoter (SV40) and pGL3-Basic (Promega) were transfected into bovine aortic endothelial cells (BAECs) and CHO-K1 cells using calcium phosphate, and into human umbilical vein endothelial cells (HUVECs) and ECV304 cells using Lipofectine (Life Technologies, Birmingham, AL, U.S.A.). Co-transfection with pSV-β-galactosidase Control vector (Promega) was carried out to standardize for transfection efficiency. Cells were harvested and luciferase and galactosidase activities were measured using a Lumat LB 9501 (Laboratorium, Prof. Dr. Berhold, Bad Wildbad, Germany). Construct-specific transfection represents the mean value of six single transfections.
The β-promoter sequence was analyzed for potential cis-acting elements with the FACTOR algorithm of the HUSAR software (German Cancer Research Center, Heidelberg, Germany).
Transfection of the promoter construct spanning up to position −1,206 bp relative to the first putative translation initiation codon of the ECE-1β isoform resulted in a relative luciferase activity (RLA) of 72.7 in BAECs, 81.6 in HUVECs, 6.3 in EVC304, and 2.0 in CHO-K1 cells (Figs. 1 and 2). Transfection of serial deletion mutants into BAECs showed increasing RLA, depending on fragment length. Fragments −267 and −498 altered luciferase activity by 0.8 and 2.7, respectively. Extension of the construct to position −736 resulted in a RLA of 12.4. Potential cis-acting elements identified in this region include three sites for binding of ETS transcription factors. Further extension to −973 resulted in an RLA of 31. This region contains, e.g., consensus sequences for NFκB, AP-2, ETS proteins, and shear stress-responsive elements (SSREs). In the final extension region between −973 and −1,206, additional AP-2 and ETS sites are localized. Transfection into BAECs of a luciferase reporter vector containing the SV40 promoter increased the RLA 53-fold (Fig. 2). Differences between deletion mutants and differences between cell-specific transfections were significant (p < 0.05), except for BAEC vs. HUVEC.
The previously described β-mRNA of ECE-1 can principally be generated in two ways. First, the α-specific exons 1 and 2 could be spliced out of a common primary transcript. This mechanism is consistent with a single promoter located upstream of exon 1. Second, the β-isoform is regulated by a second promoter which is located in the 11-kb intron upstream of exon 3.
The use of alternative promoters has been shown for several genes, such as human angiotensin I-converting enzyme (5) and the neutral endopeptidase (NEP) (6), whereas genes of some contractile proteins(e.g., myosin) and the troponin-T gene employ differential splicing (7).
Our transfection experiments show that intronic DNA upstream of the first β-specific exon dramatically increases the transcription rate of a downstream reporter gene. This, in conjunction with the work of Valdenaire et al. (2), who mapped multiple transcription start points upstream of exon 3, proves the existence of an alternative β-promoter located in intron 2. Comparison of the relative luciferase activities of the viral SV40 promoter and the ECE-1β promoter in BAECs underlines the activity of the β-promoter in macrovascular endothelial cells. Significant promoter activity in HUVECs was expected for two reasons. First, in HUVECs, ECE-1β mRNA is expressed more abundantly compared to the α-isoform(2). Second, a homologous cellular system was studied with respect to species. The marginal promoter activity observed in CHO cells is consistent with the fact that CHO cells do not express endogenous ECE activity (3,8).
We found reduced β-promoter activity in ECV304 cells, whereas it was shown previously that the ECE-1α promoter is about five times more active then the β-promoter in these cells (9). These results are in accordance with RT-PCR and Northern blot data. In ECV304 cells the α-mRNA level predominates the β-isoform level (2,9). Therefore, we conclude that ECE-1 isoform mRNA levels are largely controlled at the transcriptional level. Our data suggest that the β-promoter acts in a cell-specific manner, a conclusion further confirmed by the observation that human umbilical vein vascular smooth-muscle cells do not express ECE-1β mRNA constitutively (9).
Interestingly there are at least 12 potential binding sites for transcription factors of the ETS family (e.g., Ets-1, Ets-2, PEA3) within the β-promoter. Ets-1 has been shown to play a role in the transcriptional activation of matrix metalloprotease genes(e.g., collagenases) and is expressed in human endothelial cells during embryonic angiogenesis and tumor vascularization (10,11). Recently, upregulation of ECE-1 in the neointima induced by balloon angioplasty of the rat carotid artery has been reported (12). A possible role for Ets-1 in the upregulation of ECE-1 in the neointima remains to be defined.
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8. Shiraki T, Sawamura T, Ikura T, et al. Genetic transfer of ECE activity to CHO-K1 cells. FEBS Lett
9.Orzechowski HD, Richter CM, Funke-Kaiser H, et al. Evidence of alternative promoters directing isoform-specific expression of human endothelin-converting enzyme-1 mRNA in cultured endothelial cells. J Mol Med
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11. Wernert N, Raes MB, Lassalle P, et al. c-ets 1 proto-oncogene is a transcription factor expressed in endothelial cells during tumor vascularization and other forms of angiogenesis in humans. Am J Pathol
12. Minammino T, Kurihara HK, Takahashi M, et al. Endothelinconverting enzyme expression in the rat vascular injury model and human atherosclerosis. Circulation
Keywords:© Lippincott-Raven Publishers
Big endothelins; Endothelin-converting enzymes; ECE-1 isoforms; Luciferase reporter vector