Functional significance of a hereditary adenine insertion variant in the 59-UTR of the endothelin-1 gene Katrin Popowski a , Bernhard Sperker a , Heyo K. Kroemer a , Ulrich John b , Michael Laule c , Karl Stangl c and Ingolf Cascorbi a Endothelin-1 (ET-1) is known as a potent vasoconstrictor peptide and stimulator of cell proliferation. The human preproendothelin-1 mRNA contains a frequent adenine insertion polymorphism (allele frequency 0.28) within the 59-untranslated region (59-UTR), 138 bp downstream of the transcription start site, which was assumed to be related to hypertension. This 59-UTR variant could putatively influence the mRNA secondary structure and stability, efficacy of translation initiation, or binding of sequence- specific mRNA-binding proteins. By cloning the entire ET-1 gene 59-UTR in a pGL3 vector and transfection of two cell lines, we studied the effects on luciferase expression in vitro. Luciferase activity was significantly increased in the insertion variant (I) compared to the wild-type (D) variant for both COS1 (2.97 6 0.12 versus 2.17 6 0.10; P 0.002) and HepG2 cells (5.42 6 0.90 versus 3.68 6 0.37; P 0.002). Investigations performed ex vivo using human umbilical vein endothelial cells were performed to examine the influence of genotypes on the formation of mRNA and protein. Preproendothelin-1-mRNA was quantified in relation to GAPDH by a realtime polymerase chain reaction. Homozygous I-carriers showed significant elevated mRNA levels compared to I/D and I/I-carriers (I/I 9.03 6 1.86, I/D 2.07 6 1.15, D/D 2.33 6 0.99; P 0.001). ET-1 protein expression, determined by enzyme-linked immunosorbent assay, was increased among I-carriers (I/I 814 6 144, I/D 528 6 103, D/D 556 6 75 pg/ml; P 0.001). The observed effects may be due to an enhanced mRNA stability because the half-life of mRNA consisting of the I- variant was prolonged (35.4 6 7.9 versus 19.9 6 4.5 min). We were able to show that the 138 I/D polymorphism is of functional importance for ET-1 expression, and this may have consequences for vessel tonus regulation. Pharmacogenetics 13:445–451 & 2003 Lippincott Williams & Wilkins Pharmacogenetics 2003, 13:445–451 Keywords: endothelin, +138 I/D, polymorphism, expression a Peter Holtz Research Center of Pharmacology and Experimental Therapeutics, Department of Pharmacology, b Institut of Epidemiology, Ernst Moritz Arndt University Greifswald and c Department of Medicine I, University Medical Center Charite ´ , Humboldt University, Berlin, Germany. Sponsorship: This work was supported by the German Federal Ministry of Education, Science, Research and Technology (grant 01 GG 9845/5, 01ZZ96030) and an institutional grant of APO GEPHA Dresden and Robert- Bosch-Foundation Stuttgart. Correspondence and requests for reprints to Ingolf Cascorbi, Institut fu ¨r Pharmakologie, Ernst-Moritz-Arndt-Universita ¨ t, Friedrich-Loeffler-Strasse 20/21, D-17487 Greifswald, Germany. Tel: +49 3834 865650; fax: +49 3834 865651; e-mail: cascorbi@uni-greifswald.de Received 18 February 2003 Accepted 10 May 2003 Introduction Endothelin-1 (ET-1), one of the most important vaso- constrictor peptides in the human vascular system [1], acts in a paracrine and autocrine manner on its specific endothelin receptors. Although vasoconstriction by ET- 1 is mediated by ET A receptor on vascular smooth muscle cells, relaxation depends on ET B receptors located on endothelial cells [2]. It is assumed that the mode of action, vasoconstriction or relaxation, predomi- nantly depends on the ET-1 concentration, the condi- tions of vascular beds and the distribution of ET receptors [3–5]. The ubiquitous occurrence of ET-1 (e.g. in cardiac myocytes and macrophages) suggests that the peptide may participate in complex regulatory mechanisms in various organs [6]. Biosynthesis of ET-1 is modulated by growth factors and cytokines [7], vasoactive substances [8], as well as hypoxia and shear stress [9]. The 21-residue human ET-1, similar to many growth factors and peptide hormones, is initially synthesized as a larger inactive precursor preproendothelin-1 (ppET-1) [10]. The 2026-bp ppET mRNA is encoded by the human 6836-bp ET-1 gene, localized on chromosome 6p24–23, which contains five exons and four interven- ing sequences. Exon 1 contains the whole 59-untrans- lated region (268 bp) and the coding sequence for the first 21 amino acid residues of ppET-1 [11]. Systematic sequence analysis of the ET-1 gene disclosed certain single nucleotide polymorphisms [12]. One of these genetic variants, a frequent adenine insertion located 138 bp downstream of the transcription start site in the 59-UTR, showed a significant association with hyper- tension in a small case–control study comprising 100 patients with essential hypertension who were com- pared to matched controls [13]. Moreover, female pa- tients who were homozygous for the insertion showed significantly higher levels of systolic and diastolic blood pressure [14], suggesting that the insertion polymorph- ism may contribute to the inter-individual variability of Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Original article 445 0960-314X & 2003 Lippincott Williams & Wilkins DOI: 10.1097/01.fpc.0000054108.48725.2a