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 5'-untranslated region (5'-UTR), 138 bp downstream of the transcription start site, which was assumed to be related to
hypertension. This 5'-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 5'-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 +/- 0.12 versus 2.17 +/- 0.10; P = 0.002) and HepG2 cells (5.42 +/- 0.90 versus 3.68 +/- 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 +/- 1.86, I/D 2.07 +/- 1.15,
D/D 2.33 +/- 0.99; P = 0.001). ET-1
protein expression, determined by
enzyme-linked
immunosorbent assay, was increased among I-carriers (I/I 814 +/- 144, I/D 528 +/- 103,
D/D 556 +/- 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 +/- 7.9 versus 19.9 +/- 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.