Endothelin 1

Cloning and Expression

A family of structurally and pharmacologically distinct peptides, the endothelins, have been identified and sequenced in humans (Inoue et al., 1989). Three isoforms of human endothelin have been identified: endothelin-1, -2, and -3. Endothelin-1 is a potent, 21-amino acid vasoconstrictor peptide produced by vascular endothelial cells. Inoue et al. (1989) cloned the full length of the human preproendothelin-1 gene and the corresponding cDNA and determined the complete nucleotide sequence. The human preproendothelin-1 mRNA consists of 2,026 nucleotides, excluding the poly(A) tail. Endothelin-1 was originally isolated from the supernatant of porcine aortic endothelial cell cultures and is the most potent vasoconstrictor known. Subsequent cloning and sequence analysis from a human placental cDNA library showed that human endothelin-1 is identical to porcine endothelin. In addition to its vasoconstrictor action, endothelin has effects on the central nervous system and on neuronal excitability.

Gordon et al. (2013) stated that EDN1 is translated as a 212-amino acid preproprotein that undergoes a series of proteolytic processing events: cleavage by a signal peptidase to produce proEDN1; cleavage by furin (136950) at 2 sites in proEDN1 to liberate the 38-amino acid bigEDN1; and cleavage of bigEDN1 by ECE enzymes to produce the mature, bioactive EDN1 peptide consisting of 21 amino acids.

Gene Structure

Inoue et al. (1989) determined that the EDN1 gene is composed of 5 exons distributed over 6,836 bp.

Benatti et al. (1993) demonstrated that at least 2 preproendothelin-1 mRNAs are produced from a single gene by use of different promoters; the 2 molecules share the same coding sequence but differ in the 5-prime untranslated region. Analysis of the tissue distribution of the 2 mRNAs showed a tissue-type specificity for one mRNA in brain and heart tissues.

Biochemical Features

Crystal Structure

Shihoya et al. (2016), reported the crystal structures of human endothelin type B receptor (131244) in the ligand-free form and in complex with the endogenous agonist endothelin-1. The structures and mutation analysis revealed the mechanism for the isopeptide selectivity between endothelin-1 and -3. Transmembrane helices 1, 2, 6, and 7 move and envelop the entire endothelin peptide in a virtually irreversible manner. The agonist-induced conformational changes are propagated to the receptor core and the cytoplasmic G protein-coupling interface, and probably induce conformational flexibility in TM6. A comparison with the M2 muscarinic receptor (CHRM2; 118493) suggested a shared mechanism for signal transduction in class A G protein-coupled receptors.

Mapping

Bloch et al. (1989) localized the ET1 gene to human chromosome 6. By Southern blot analysis of somatic cell hybrid DNAs and by in situ hybridization, Arinami et al. (1991) confirmed the assignment of EDN1 to chromosome 6 and regionalized it to 6p24-p23. By pairwise linkage analysis, Pages et al. (1993) placed EDN1 distal to D6S89 (maximum lod = 78.98 at theta = 0.059) and proximal to F13A1 (maximum lod = 38.65 at theta = 0.113).

Maemura et al. (1996) mapped the Edn1 gene to mouse chromosome 13, where the mouse mutation congenital hydrocephalus (ch) is also mapped.

Gene Function

Using in situ hybridization in studies of postmortem material from the spinal cord and dorsal root ganglia, Giaid et al. (1989) found evidence of expression of endothelin-1 mRNA in distinct neuronal cell types of the dorsal ganglia and spinal cord.

Maemura et al. (1996) found that the highest expression of Edn1 mRNA was detected in the lung in adult mice, whereas in the embryo the gene is predominantly expressed in the epithelium and mesenchyme of the pharyngeal arches and in the endothelium of the large arteries.

To investigate the influence of pregnancy-specific hormonal environment on expression of ET1 and the ET1 receptor (EDNR), Bourgeois et al. (1997) cultured and characterized vascular smooth muscle cells from stem villi vessels. They investigated whether the muscular layer of stem villi vessels could be a site of the ET1 expression described in the placenta, and they examined this expression in placental vascular smooth muscle cells (PVSMCs). Peptide precursors prepro-ET1 and prepro-ET3 mRNAs were identified in stem villi vessels, whereas only prepro-ET1 mRNA was observed in PVSMCs. The authors characterized EDNR expressed by these cells in comparison with the muscular layer of stem villi vessels. Whereas both EDNRA (131243) and EDNRB (131244) are present in stem villi vessels, they found that PVSMCs exclusively express EDNRA. They described an alternatively spliced EDNRA transcript that is generated by exclusion of exon 3 in stem villi vessels and PVSMCs. The authors concluded that alternative splicing mechanisms of EDNRA mRNA could constitute a control of the abundance of active EDNRA in terms of contractility.

Maggi et al. (2000) demonstrated that in FNC-B4 cells, which are derived from a human fetal olfactory epithelium, both sex steroids and odorants regulate GnRH secretion. They found biologic activity of EDN1 in this GnRH-secreting neuronal cell. In situ hybridization and immunohistochemistry revealed gene and protein expression of EDN1 and its converting enzyme (ECE1; 600423) in both fetal olfactory mucosa and FNC-B4 cells. Experiments with radiolabeled EDN1 and EDN3 (131242) strongly indicated the presence of 2 classes of binding sites, corresponding to the ETA (16,500 sites/cell) and the ETB receptors (8,700 sites/cell). Functional studies using selective analogs indicated that these 2 classes of receptors subserve distinct functions in human GnRH-secreting cells. The ETA receptor subtype mediated an increase in intracellular calcium and GnRH secretion.

Endothelin-1 inhibits active Na-K transport by as much as 50% in the renal tubule and other tissues (Zeidel et al., 1989). Okafor and Delamere (2001) noted that the presence of low levels of ET1 in aqueous humor combined with the potential for release of ET1 from ciliary processes suggested that the crystalline lens could be exposed to ET1 in vivo. They studied the influence of ET1 on active Na-K transport in the porcine lens. Their results suggested that ET1 inhibited active lens Na-K transport by activating EDNRA and EDNRB. Activation of the ET receptors also caused an increase in cytoplasmic calcium concentration in cultured lens epithelial cells. Both responses to ET1 appear to have a tyrosine kinase step.

Udono et al. (2001) explored the effects of hypoxia on the production and secretion of adrenomedullin (ADM; 103275) and endothelin in human retinal pigment epithelial (RPE) cells. They found that ADM mRNA levels and immunoreactive ADM levels in the medium were increased by hypoxia in all 3 RPE cell lines studied. Immunoreactive ET1 was detected in 2 cultured media. Hypoxia treatment for 28 hours increased immunoreactive ET1 levels approximately 1.3-fold in 1 cultured cell medium but decreased it in 2 cell lines. Treatment with ADM ameliorated the hypoxia-induced decrease in the cell number. Exogenous ET1 had no significant effect on the number of cells under normoxia or hypoxia. Udono et al. (2001) concluded that the ADM induced by hypoxia may have protective roles against hypoxic cell damage in RPE cells.

Napolitano et al. (2000) investigated the interactions between ET1 and the nitric oxide (NO) system in the fetoplacental unit. They examined the mRNA expression of ET1, inducible NO synthase (iNOS; 163730), and endothelial NOS (eNOS; 163729) in human cultured placental trophoblastic cells obtained from preeclamptic (189800) and normotensive pregnancies. ET1 expression was increased in preeclampsia cells, whereas iNOS, which represents the main source of NO synthesis, was decreased; conversely, eNOS expression was increased. ET1 was able to influence its own expression as well as NOS isoform expression in normal and preeclampsia trophoblastic cultured cells. The findings suggested the existence of a functional relationship between ET(s) and NOS isoforms that could constitute the biologic mechanism leading to the reduced placental blood flow and increased resistance to flow in the fetomaternal circulation that are characteristic of the pathophysiology of preeclampsia.

Pache et al. (2002) tested the hypothesis that plasma endothelin-1 would be increased in 4 patients with biopsy-proven giant cell arteritis (187360). All patients showed significantly increased plasma levels of endothelin-1, although the clinical relevance of the increase required further evaluation.

Jamal and Schneider (2002) found that ultraviolet induction of EDN1 through EDNRB downregulated E-cadherin (192090) and associated catenin proteins in human melanocytes and melanoma cells. Downregulation of E-cadherin through this pathway involved the downstream activation of caspase-8 (601763), but not the distal executioner caspases, and it did not lead to apoptosis. EDN1 also induced a transient association between caspase-8 and E-cadherin/beta-catenin (116806) complexes. Jamal and Schneider (2002) concluded that inhibition of E-cadherin through this pathway would tend to promote melanoma invasion.

Endothelin-1 is a pain mediator that is involved in the pathogenesis of pain states ranging from trauma to cancer. It is a potent vasoactive peptide and appears to be implicated in the pathogenesis of pain associated with ischemic states (such as coronary artery disease or sickle cell anemia), and inflammation (such as arthritis) in addition to cancer. Endothelin-1 is synthesized by keratinocytes in normal skin and is locally released after cutaneous injury. While it is able to trigger pain through its actions on endothelin-A receptors (EDNRA; 131243) of local nociceptors, it can coincidentally produce analgesia through endothelin-B receptors (EDNRB; 131244). Khodorova et al. (2003) mapped an endogenous analgesic circuit, in which endothelin-B receptor activation induces the release of beta-endorphin from keratinocytes and the activation of G protein-coupled inwardly rectifying potassium channels (GIRKs, also called Kir-3) linked to opioid receptors on nociceptors. These results indicated the existence of an intrinsic feedback mechanism to control peripheral pain in skin, and established keratinocytes as an endothelin-B receptor-operated opioid pool.

Osteoblastic bone metastases are common in prostate and breast cancer patients. Yin et al. (2003) sought to determine mechanisms by which tumor cells stimulate new bone formation. They identified 3 breast cancer cell lines that cause osteoblastic metastases in a mouse model and secrete endothelin-1. Tumor-produced endothelin-1 stimulated new bone formation in vitro and osteoblastic metastases in vivo via the endothelin-A receptor. Treatment with an orally active endothelin-A receptor antagonist dramatically decreased bone metastases and tumor burden in mice inoculated with cancer cells of a particular line. Yin et al. (2003) concluded that tumor-producing endothelin-1 may have a major role in the establishment of osteoblastic bone metastases and that endothelin-A receptor blockade (Remuzzi et al., 2002) is a promising form of therapy.

Chauhan et al. (2004) described a model of chronic ET1 administration to the rat optic nerve and evaluated its effect on retinal ganglion cell and axon survival. ET1 led to a mean reduction in optic nerve blood flow of 68%. This resulted in a time-dependent loss of retinal ganglion cells and their axons without apparent change in the optic disc topography.

Campia et al. (2004) examined forearm blood flow responses to intraarterial injection of an endothelin-A receptor blocker in 37 normotensive and 27 hypertensive patients. In hypertensive patients, the vasodilator effect of the blocker was significantly higher in blacks than in whites (p = 0.01), whereas blood flow was not significantly affected in black or white healthy controls. Campia et al. (2004) concluded that hypertensive blacks have enhanced EDNRA-dependent vasoconstrictor tone, which they suggested might be related to increased production of ET1.

Placental growth factor (PGF; 601121) upregulates ET1 expression via HIF1-alpha (HIF1A; 603348). Using primary human endothelial cells and cell lines, Li et al. (2015) found that PGF also upregulated ET1 via a pathway involving PAX5 (167414) and microRNA-648 (MIR648; 616205). They showed that MIR648 directly targeted the 3-prime UTR of ET1 and destabilized the transcript, thereby reducing ET1 translation. Overexpression and knockdown studies revealed that PGF reduced MIR648 content indirectly by downregulating PAX5, a positive regulator of MIR648 expression.

Molecular Genetics

High Density Lipoprotein Cholesterol

Pare et al. (2007) used a candidate gene approach to study the genetics of coronary artery disease (CAD) in the Saguenay-Lac-Saint-Jean (SLSJ) region of northeastern Quebec. The SLSJ region is inhabited by an archetypal 'founder effect' population of approximately 280,000 individuals, which was subjected to a first bottleneck with the establishment of New France by French settlers in the 17th-18th century and then to a second bottleneck with the founding of the SLSJ region in the 19th century. Consequently, only approximately 600 ancestors contributed up to 70% of the genetic pool (Heyer and Tremblay, 1995). It was anticipated that the population would show decrease in allelic and genetic heterogeneity, 2 phenomena that hinder dissection of the genetic architecture of complex traits. The project involved the analysis of 884 individuals from 142 families (with average sibships of 5.7) as well as 558 cases and control subjects from the SLSJ region, with the use of 1,536 SNPs in 103 candidate genes. Suggestive linkage for high density lipoprotein (HDL) cholesterol was observed on chromosome 1p36.22. Furthermore, several associations that remained significant after Bonferroni correction for multiple testing were observed with lipoprotein-related traits as well as plasma concentrations of adiponectin (605441). Of note, HDL cholesterol levels were associated with a lys198-to-asn (K198N) substitution in the EDN1 gene (rs5370; 131240.0001) in a sex-specific manner, as well as with an SNP located 7.7 kb upstream of lecithin:cholesterol acyltransferase (LCAT; 606967). Whereas the other observed associations had been previously described, these 2 were not. Using an independent validation sample of 806 individuals, Pare et al. (2007) confirmed the EDN1 association (p less than 0.005), whereas the LCAT association was nonsignificant (p = 0.12).

Wiltshire et al. (2008) analyzed the K198N polymorphism of the EDN1 gene in 1,109 individuals from the general population of Western Australia and 556 patients with coronary artery disease, and found no association with hypertension, systolic blood pressure, lipid levels, insulin resistance, or metabolic syndrome in either population.

Auriculocondylar Syndrome 3

In patients from 2 unrelated consanguineous families with auriculocondylar syndrome-3 (ARCND3; 615706), Gordon et al. (2013) identified homozygosity for missense mutations in the EDN1 gene (131240.0002 and 131240.0003) that segregated with disease in each family.

Isolated Question Mark Ears

In affected individuals from 2 unrelated families with isolated question mark ears (QME; 612798), Gordon et al. (2013) identified heterozygosity for a missense (V64D; 131240.0004) and a nonsense (Y83X; 131240.0005) mutation in the EDN1 gene, respectively. The mutations segregated with disease in each family. Gordon et al. (2013) suggested a model in which heterozygous-null EDN1 alleles result in isolated question mark ears, whereas hypomorphic alleles result in an auriculocondylar syndrome phenotype in homozygotes and no phenotype in heterozygotes.

Exclusion Studies

Berge and Berg (1992) found no relationship between a TaqI DNA polymorphism at the EDN1 locus and the level of normal blood pressure or variability in blood pressure.

Pezzetti et al. (2000) examined the endothelin gene and 3 other genes in the endothelin pathway (ECE1, EDNRA, EDNRB) as possible candidates for orofacial cleft (OFC; 119530). Linkage results indicated that none of these genes is involved in the pathogenesis of OFC.

Animal Model

Kurihara et al. (1994) found that mice homozygous for a knockout of the endothelin-1 gene died of respiratory failure at birth and had morphologic abnormalities of the pharyngeal arch-derived craniofacial tissues and organs. Heterozygous mice produced lower levels of endothelin-1 than wildtype mice and developed elevated blood pressure. The phenotype of the homozygous ET1 deficient mice was quite similar to first pharyngeal arch syndromes, such as Pierre Robin syndrome (261800) and Treacher Collins syndrome (154500).

To clarify the physiologic and pathophysiologic role of ET1, Kurihara et al. (1994) disrupted the mouse Edn1 locus by gene targeting and demonstrated that ET1 is essential to the normal development of pharyngeal arch-derived tissues and organs. In a later study, Kurihara et al. (1995) focused on the phenotypic manifestations in the cardiovascular system of homozygous deficient mice. They found cardiovascular malformations, including interrupted aortic arch (2.3%), tubular hypoplasia of the aortic arch (4.6%), aberrant right subclavian artery (12.9%), and ventricular septal defect with abnormalities of the outflow tract (48.4%). The frequency and extent of these abnormalities were increased by treatment with neutralizing monoclonal antibodies or a selective antagonist to EDNRA. At an earlier embryonic stage, formation of pharyngeal arch arteries and endocardial cushion is disturbed in homozygotes. In situ hybridization by Kurihara et al. (1995) confirmed ET1 expression in the endothelium of the arch arteries and cardiac outflow tract and the endocardial cushion, as well as in the epithelium of the pharyngeal arches. Thus, they concluded that ET1 is involved in the normal development of the heart and great vessels, and circulating ET1 and/or other ET isoforms may cause a functional redundancy, at least partly, through EDNRA.

During embryogenesis, establishment of the circulatory system requires the organized development of the heart and vessels. Six pairs of branchial arch arteries appear in a rostral to caudal direction, and form the precursors of the great vessels and large arteries of the head and neck. The sequential remodeling of the arch arteries together with the regression of the right dorsal aorta results in a highly asymmetric arterial system in the mature organism. An intimate involvement of cardiac neural crest cells in arch artery remodeling is suggested by the phenotype resulting from the ablation of cardiac neural crest in chick embryos, where various types of great vessel abnormalities and septation defects of the outflow tract develop (Kirby and Waldo, 1995). Yanagisawa et al. (1998) and Clouthier et al. (1998) found that mice deficient in Ece1 (600423) or Ednra (131243) develop defects in a subset of cephalic and cardiac neural crest derivatives. The most common great vessel malformations in Ece1 -/- and Ednra -/- mice were found to be interruption of the aortic arch between the left common carotid artery and left subclavian artery (type B interruption of the aortic arch). The second most common defect was absence of the right subclavian artery. Among outflow tract abnormalities, perimembranous interventricular septal defect was observed in almost all embryos with disruption of either gene. In further studies, Yanagisawa et al. (1998) demonstrated that the defects in the mice with gene disruptions were highly similar to those seen in neural crest-ablated chick embryos and in human congenital cardiac defects. The authors demonstrated that signaling mediated by the endothelin-1/endothelin receptor-A pathway plays an essential role in the complex process of aortic arch patterning by affecting the postmigratory cardiac neural crest cell development.

Endothelin-1, a potent vasoconstrictor peptide expressed by endothelium, is also produced in the heart in response to a variety of stresses. It induces hypertrophy in cultured cardiac myocytes but only at concentrations far greater than those found in plasma. Shohet et al. (2004) tested whether endothelin-1 generated by cardiomyocytes in vivo is a local signal for cardiac hypertrophy. To avoid the perinatal lethality seen in systemic Et1 null mice, they used the Cre/loxP system to generate mice with cardiac myocyte-specific disruption of the Et1 gene. They used the alpha-myosin heavy chain (160710) promoter to drive expression of Cre and obtained 75% reduction in Et1 mRNA in cardiac myocytes isolated from these mice at baseline and after stimulation, in vivo, for 24 hours with triiodothyronine (T3). Necropsy measurements of cardiac mass indexed for body weight showed a 57% reduction in cardiac hypertrophy in response to 16 days of exogenous T3 in mice homozygous for the disrupted Et1 allele compared to sibs with an intact Et1 gene. Moreover, in vivo MRI showed only a 3% increase in left ventricular mass indexed for body weight in mice with the disrupted allele after 3 weeks of T3 treatment versus a 47% increase in mice with an intact Et1 gene. Shohet et al. (2004) concluded that ET1, produced locally by cardiac myocytes, and acting in a paracrine/autocrine manner, is an important signal for myocardial hypertrophy that facilitates the response to thyroid hormone.

In a study of regulators of nerve growth factor (NGFB; 162030), Ieda et al. (2004) found that EDN1 specifically upregulated NGFB expression in primary cultured cardiomyocytes. EDN1-induced NGF augmentation was mediated by EDNRA, Gi-beta-gamma (see 139310), PKC (see 176960), the Src family (see 190090), EGFR (131550), MAPK3 (601795), MAPK14 (600289), AP1 (165160), and CEBPD (116898). Either conditioned medium or coculture with EDN1-stimulated cardiomyocytes caused NGF-mediated PC12 cell differentiation. Edn1-deficient mice exhibited reduced NGF expression and norepinephrine concentration in the heart, reduced cardiac sympathetic innervation, excess apoptosis of sympathetic stellate ganglia, and loss of neurons at the late embryonic stage. Cardiac-specific overexpression of NGF in Edn1-deficient mice overcame the reduced sympathetic expression and loss of stellate ganglia neurons. Ieda et al. (2004) concluded that EDN1 plays a critical role in sympathetic innervation of the heart.

Ahn et al. (2004) generated mice with collecting duct-specific knockout of Et1 that had no collecting duct Et1 mRNA and reduced urinary Et1 excretion. On a normal sodium diet, the mice were hypertensive, while body weight, sodium excretion, urinary aldosterone excretion, and plasma renin activity were unchanged. On a high sodium diet, they had increased hypertension, reduced urinary sodium excretion, and excessive weight gain, but showed no difference in aldosterone excretion and plasma renin activity compared to controls. Ahn et al. (2004) concluded that collecting duct-derived ET1 is an important physiologic regulator of renal sodium excretion and systemic blood pressure.

In adult transgenic mice with conditional cardiac-restricted ET1 overexpression, Yang et al. (2004) observed nuclear factor kappa-B (see 164011) translocation, cytokine expression, inflammation and hypertrophy, resulting in dilated cardiomyopathy, congestive heart failure, and death as early as 5 weeks after transgene induction. Significant prolongation of survival was observed with a combined EDNRA/EDNRB antagonist but not with an EDNRA-selective antagonist, consistent with an important role for EDNRB in this model.