Native and oxidized low-density lipoproteins stimulate endothelin-converting enzyme-1 expression in human endothelial cells

This study addressed the question how different lipoproteins modulate the expression of endothelin-converting enzyme-1 (ECE-1) in human endothelial cells. The effect of native and oxidized low-density lipoproteins (nLDL, oxLDL) on expression of ECE-1, prepro-endothelin-1, and endothelin-1 peptide was studied in primary cultures of human endothelial cells. Native and oxidized LDL increased ECE-1 mRNA after 1 h, reaching its maximum at 100 microg/ml (1.9- and 2.5-fold, respectively). Furthermore, ECE-1 protein expression, prepro-endothelin-1 mRNA, and endothelin-1 peptide release were increased in response to nLDL or oxLDL. Induction of ECE-1 by nLDL and of prepro-endothelin-1 by oxLDL was reduced by protein kinase C inhibition. Increased expression of ECE-1 mRNA by oxLDL and of prepro-endothelin-1 by nLDL was blocked by an angiotensin II receptor type 1 antagonist. Our data provide evidence for a new mechanism how increased LDL plasma levels might contribute to enhanced endothelin-1 release in patients with hypercholesterolemia.     

Role of Protein Kinase C in Endothelin Converting Enzyme-1 trafficking and shedding from endothelial cells

This study aimed to determine the consequences of Protein Kinase C (PKC) mediated Endothelin Converting Enzyme-1 (ECE-1) phosphorylation and its relationship to ECE-1 expression and shedding. The proteins on the surface of EA.hy926 cells were labelled with EZ-Link NHS-SS-Biotin both prior to (control) and following stimulation by 2 μM phorbol 12-myristate 13-acetate (PMA) which activates PKC. The biotinylated proteins were isolated using neutravidin beads, resolved by gel electrophoresis and analysed by western blotting using anti-ECE-1 antibodies. Significant increase in ECE-1 expression at the cell surface was observed following stimulation by PMA, compared to unstimulated control cells (170 ± 32.3% of control, n = 5). The ECE-1 activity (expressed as μM substrate cleaved/min) was determined by monitoring the cleavage of a quenched fluorescent substrate. The specificity of cleavage was confirmed using the ECE-1 inhibitor CGS35066. The stimulation of cells by PMA (1 μM, 6 h) significantly increased the ECE-1 activity (0.28 ± 0.02; n = 3) compared to the control (0.07 ± 0.02; n = 3). This increase was prevented by prior incubation with the PKC inhibitor bisindolymaleimide (BIM; 2 μM for 1 h; 0.10 ± 0.01; n = 3). Treatment with PMA also increased the activity of ECE-1 in the media (0.18 ± 0.01; n = 3) compared to control (0.08 ± 0.01; n = 3). In addition, this study confirmed by western immunoblotting that only the extracellular region of ECE-1 is released from the cell surface. These data indicate for the first time that PKC activation induces the trafficking and shedding of ECE to and from the cell surface, respectively.     

Protein Kinase C Regulates the Cell Surface Activity of Endothelin-Converting Enzyme-1

The potent vasoconstrictor endothelin is a 21 amino acid peptide whose principal physiological function is to regulate vascular tone. The generation of endothelin is crucially dependent on the local presence and activity of endothelin converting enzyme-1 (ECE-1) expressed on the surface of vascular endothelial cells. In this study, we have shown in endothelial cells that the enzyme is phosphorylated, and that phosphorylation is increased by phorbol ester stimulation of protein kinase C (PKC). Furthermore, by monitoring specific ECE-1 activity on the surface of live cells, we also show that following PKC activation, enzyme activity is significantly increased at the cell surface, where it is positioned to catalyse the generation of active endothelin. We believe this novel finding is unprecedented for a peptide processing enzyme. Indeed, this new knowledge regarding the control of endothelin production by regulating ECE-1 activity at the cell surface opens up a new area of endothelin biology and will provide novel insights into the physiology and pathophysiology of endothelin and endothelin-associated diseases. In addition, the information generated in these studies may provide valuable new insights into potential extra- and intracellular targets for the pharmacological and perhaps even therapeutic regulation of endothelin production and thus vascular tone.Australian Peptide Conference Issue.     

Mapping the active site of endothelin-converting enzyme-1 through subsite specificity and mutagenesis studies: a comparison with neprilysin

Endothelin-converting enzyme-1 (ECE-1) is a membrane-bound zinc metallopeptidase that is homologous to neprilysin in amino acid sequence. A major in vivo function of ECE-1 is the generation of endothelin-1, a potent vasoconstrictor, from big endothelin-1. ECE-1 is also potentially involved in the processing or degradation of other peptide hormones. In this study we have used substrates based on the sequence of the COOH-terminal half of big endothelin-1 to examine the subsite specificity of recombinant ECE-1. The big endothelin-1 [16-38] peptides were systematically varied at either position 21 (P(1)) or position 22 (P'(1)) and used in steady-state kinetic analyses of ECE-1. The results indicate that the S(1) pocket of ECE-1 is relatively nonselective, but that the S'(1) subsite of ECE-1 has a preference for large hydrophobic side chains. The peptidyl carboxydipeptidase activity of ECE-1 was also characterized, revealing that substrates with COOH-terminal carboxylates are highly preferred over the cognate amides and esters. A site-directed mutagenesis study was carried out to identify the active-site amino acid residues specifically involved in binding to the COOH-terminal carboxylate of substrates. The data indicate that Arg(133) of ECE-1, which corresponds to Arg(102) of neprilysin that has been identified as an active-site residue of neprilysin involved in binding to the free carboxylate of some substrate peptides, may not play the same role. However, the low activity observed for an ECE-1 Arg(726) mutant is consistent with a role for this arginine residue in the binding of substrates, a role which has been ascribed to arginine residues in both thermolysin (Arg(203)) and neprilysin (Arg(717)).     

Endothelin-converting enzyme-1 expression in acute and chronic liver injury in fibrogenesis

Endothelin-1 (ET-1) induces contraction, proliferation, and collagen synthesis of activated hepatic stellate cells and is a potent mediator of portal hypertension. Endothelin-converting enzyme-1 (ECE-1) generates ET-1 from the inactive precursor big-endothelin-1. The cellular distribution and activity of ECE-1 in the liver is unknown. Hepatic fibrogenesis was induced in rats by CCl₄ administration and secondary biliary cirrhosis after 6 weeks of complete bile duct occlusion (BDO). The tissue ET-1 and ET receptor protein levels were quantified, the ECE-1 isoform mRNAs were measured by RNase protection assay and ECE-1 activity was analyzed. ECE-1a and -b mRNA were upregulated in biliary cirrhosis and in CCl₄-injured livers, whereas ECE-1c mRNA remained unchanged. ECE-1 activity was increased after BDO and peaked at 12?h after acute CCl₄-intoxication. Tissue levels of ET-1, ET_A- and ET_B receptors were elevated 7-, 5-, and 4.6-fold in cirrhotic rats, respectively. ECE-1 activity increased following BDO and acute CCl₄-intoxication. In conclusion, ECE-1a and -b RNAs are upregulated in fibrogenesis, indicating that these isoforms play a central role in ET-1 generation during fibrogenesis and portal hypertension.     

The ultimate tryptophan residue of neprilysin 2 is not involved in protein maturation and enzymatic activity

Modeling the three-dimensional structure of neprilysin 2 (NEP2) using the crystal structure of neprilysin as template revealed that their active sites share many common features, though slight differences therein cannot completely account for their specific pharmacological profiles. Recent evidence also suggest that residues outside the active site can play crucial functions in the maturation and enzymatic activity of these metalloproteases. To further explore the functions of amino acids in the acquisition and maintenance of the NEP2 structure, site-directed mutagenesis of conserved residues involved in the enzymatic activity of ECE-1 was performed. In particular, the ultimate tryptophan residue of ECE-1 was recently shown to be important in its activation. This residue was thus mutated in the secreted isoform of NEP2, as were proline residues located in its vicinity. Expression of these mutants in AtT20 cells and study of their secretion and catalytic activities shows that while the ultimate tryptophan residue of the NEP2 sequence is not essential to its proper and activity, structural changes in its vicinity can have a severe impact on the maturation processes involved in the activation of NEP2.     

Collagen type I selectively activates ectodomain shedding of the discoidin domain receptor 1: involvement of Src tyrosine kinase

The discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that is highly expressed in breast carcinoma cells. Upon binding to collagen, DDR1 undergoes autophosphorylation followed by limited proteolysis to generate a tyrosine phosphorylated C-terminal fragment (CTF). Although it was postulated that this fragment is formed as a result of shedding of the N-terminal ectodomain, collagen-dependent release of the DDR1 extracellular domain has not been demonstrated. We now report that, in conjunction with CTF formation, collagen type I stimulates concentration-dependent, saturable shedding of the DDR1 ectodomain from two carcinoma cell lines, and from transfected cells. In contrast, collagen did not promote cleavage of other transmembrane proteins including the amyloid precursor protein (APP), ErbB2, and E-cadherin. Collagen-dependent tyrosine phosphorylation and proteolysis of DDR1 in carcinoma cells were reduced by a pharmacologic Src inhibitor. Moreover, expression of a dominant negative Src mutant protein in human embryonic kidney cells inhibited collagen-dependent phosphorylation and shedding of co-transfected DDR1. The hydroxamate-based metalloproteinase inhibitor TAPI-1 (tumor necrosis factor-alpha protease inhibitor-1), and tissue inhibitor of metalloproteinase (TIMP)-3, also blocked collagen-evoked DDR1 shedding, but did not reduce levels of the phosphorylated CTF. Neither shedding nor CTF formation were affected by the gamma-secretase inhibitor, L-685,458. The results demonstrate that collagen-evoked ectodomain cleavage of DDR1 is mediated in part by Src-dependent activation or recruitment of a matrix- or disintegrin metalloproteinase, and that CTF formation can occur independently of ectodomain shedding. Delayed shedding of the DDR1 ectodomain may represent a mechanism that limits DDR1-dependent cell adhesion and migration on collagen matrices.     

Secretion of endothelin converting enzyme-1a: The hydrophobic signal anchor domain alone is not sufficient to promote membrane localization

Endothelin converting enzyme-1 (ECE-1) is a type II membrane protein that is important for the proteolytic activation of big endothelin-1 to endothelin-1. Although the highly conserved zinc-binding motif is known to be located in the extracellular domain, the role(s) of the N-terminal and membrane-spanning signal anchor domains in the biosynthesis and function of ECE-1 isoforms, ECE-1a, ECE-1b, and ECE-1c, remain undetermined. In this study, we provide evidence that the deletion of the cytoplasmic N-terminal tail (residues 1-55) of ECE-1a results in the cleavage of a potential signal peptide located in the signal anchor domain leading to the partial secretion of the recombinant enzyme into the media. However, the truncation of N-terminal and/or signal anchor domain does not affect the activity of ECE-1a. Therefore, our results demonstrate that the hydrophobic signal anchor domain alone is not sufficient for the membrane anchoring of ECE-1a and that the N-terminal domain of ECE-1a is important for membrane targeting as well as the intracellular localization of the enzyme.     

Tumor necrosis factor-alpha-converting enzyme (TACE/ADAM-17) mediates the ectodomain cleavage of intercellular adhesion molecule-1 (ICAM-1)

Ectodomain shedding has emerged as an important regulatory step in the function of transmembrane proteins. Intercellular adhesion molecule-1 (ICAM-1), an adhesion receptor that mediates inflammatory and immune responses, undergoes shedding in the presence of inflammatory mediators and phorbol 12-myristate 13-acetate (PMA). The shedding of ICAM-1 in ICAM-1-transfected 293 cells upon PMA stimulation and in endothelial cells upon tumor necrosis factor-alpha stimulation was blocked by metalloproteinase inhibitors, whereas serine protease inhibitors were ineffective. p-Aminophenylmercuric acetate, a mercuric compound that is known to activate matrix metalloproteinases, up-regulated ICAM-1 shedding. TIMP-3 (but not TIMP-1 or -2) effectively blocked cleavage. This profile suggests the involvement of the ADAM family of proteases in the cleavage of ICAM-1. The introduction of enzymatically active tumor necrosis factor-alpha-converting enzyme (TACE) into ICAM-1-expressing cells up-regulated cleavage. Small interfering RNA directed against TACE blocked ICAM-1 cleavage. ICAM-1 transfected into TACE-/- fibroblasts did not show increased shedding over constitutive levels in the presence of PMA, whereas cleavage did occur in ICAM-1-transfected TACE+/+ cells. These results indicate that ICAM-1 shedding is mediated by TACE. Blocking the shedding of ICAM-1 altered the cell adhesive function, as ICAM-1-mediated cell adhesion was up-regulated in the presence of TACE small interfering RNA and TIMP-3, but not TIMP-1. However, cleavage was found to occur at multiple sites within the stalk domain of ICAM-1, and numerous point mutations within the region did not affect cleavage, indicating that TACE-mediated cleavage of ICAM-1 may not be sequence-specific.     

Expression of human endothelin-converting enzyme isoforms: role of angiotensin II

A key step in endothelin-1 (ET-1) synthesis is the proteolytic cleavage of big ET-1 by the endothelin-converting enzyme-1 (ECE-1). Four alternatively spliced isoforms, ECE-1a to ECE-1d, have been discovered; however, regulation of the expression of specific ECE-1 isoforms is not well understood. Therefore, we stimulated primary human umbilical vein endothelial cells (HUVECs) with angiotensin II (Ang II). Furthermore, expression of ECE-1 isoforms was determined in internal mammary arteries of patients undergoing coronary artery bypass grafting surgery. Patients had received one of 4 therapies: angiotensin-converting enzyme inhibitors (ACE-I), Ang II type 1 receptor blockers (ARB), HMG-CoA reductase inhibitors (statins), and a control group that had received neither ACE-I, ARB (that is, treatment not interfering in the renin-angiotensin system), nor statins. Under control conditions, ECE-1a is the dominant isoform in HUVECs (4.5+/-2.8 amol/microg RNA), followed by ECE-1c (2.7+/-1.0 amol/microg), ECE-1d (0.49+/-0.17 amol/microg), and ECE-1b (0.17+/-0.04 amol/microg). Stimulation with Ang II did not change the ECE-1 expression pattern or the ET-1 release. We found that ECE-1 mRNA expression was higher in patients treated with statins than in patients treated with ARB therapy (5.8+/-0.76 RU versus 3.0+/-0.4 RU), mainly attributed to ECE-1a. In addition, ECE-1a mRNA expression was higher in patients receiving ACE-I therapy than in patients receiving ARB therapy (1.68+/-0.27 RU versus 0.83+/-0.07 RU). We conclude that ECE-1a is the major ECE-1 isoform in primary human endothelial cells. Its expression in internal mammary arteries can be regulated by statin therapy and differs between patients with ACE-I and ARB therapy.     

Hormonal regulation and cell-specific expression of endothelin-converting enzyme 1 isoforms in bovine ovarian endothelial and steroidogenic cells

Endothelin-converting enzyme 1 (ECE-1) is a key enzyme in the biosynthesis of endothelin 1 (ET-1), a potent regulator of ovarian function. Different ECE-1 isoforms are localized in distinct intracellular compartments. Thus, the spatial and temporal pattern of ECE-1 expression determines the site of big ET-1 activation and the bioavailability of ET-1. This study was undertaken to investigate the hormonal regulation and cell-specific expression of ECE-1 isoforms in endothelial and steroidogenic cells of bovine follicles and corpora lutea (CL). Using enriched follicular and luteal cell subpopulations and in situ hybridization techniques, we showed that the ECE-1 gene is expressed by both endothelial and steroidogenic cells; however, the intracellular ECE-1a isoform was present only in ET-1-expressing endothelial cells. Steroidogenic cells in follicles or in CL, deficient in ET-1, expressed only the plasma membrane ECE-1b isoform. The intensity of antisense ECE-1 labeling in the granulosa cell layer increased with follicular size; insulin-like growth factor I and insulin upregulated ECE-1 expression when cultured with granulosa cells, suggesting that these growth factors may increase ECE-1 in growing follicles. In contrast, ET-1 and LH downregulated ECE-1 in steroidogenic cells. This effect could account for low ECE (and ET-1) levels, which characterize the early luteal phase. These findings suggest that ECE-1 is regulated during different stages of the cycle in a physiologically relevant manner. The hormonal regulation and intracellular localization of bovine ECE-1 isoforms revealed in this study may provide new insights into ET-1 biosynthesis and mode of action in different cellular microenvironments within the ovary.     

Soluble and Catalytically Active Endothelin Converting Enzyme-1 is Present in Cerebrospinal Fluid of Subarachnoid Hemorrhage Patients

Endothelin converting Enzyme-1 (ECE-1) is essential for the production of Endothelin-1 (ET-1), which is associated with vasospasm following subarachnoid hemorrhage (SAH). We have previously demonstrated the presence of a catalytically active soluble form of ECE-1 in the media of endothelial cells. We aimed to determine if this form of ECE-1 exists in vivo, in cerebrospinal fluid (CSF) of SAH patients. We examined CSF taken from SAH subjects for the presence of soluble ECE-1 using a bradykinin based quenched fluorescent substrate assay. We obtained further confirmation by characterizing the CSF mediated cleavage products of BigET-1 and BigET_18–34 (6 μg/ml) using mass spectrometry. The specificity of cleavage was confirmed using the ECE-1 inhibitor {"type":"entrez-protein","attrs":{"text":"CGS35066","term_id":"877962710","term_text":"CGS35066"}}CGS35066 5nmol/L. SAH CSF samples had mean ECE-1 activity of 0.127 ± 0.037 μmols of substrate cleaved/μl of CSF/24 h. The C-terminal peptides generated upon the cleavage of BigET-1 and BigET_18–34 were detected 48 h after incubation of these substrates with CSF. Cleavage of these substrates was inhibited by {"type":"entrez-protein","attrs":{"text":"CGS35066","term_id":"877962710","term_text":"CGS35066"}}CGS35066. Results of Western blots also produced strong evidence for the presence of truncated soluble ECE-1 in CSF. These results strongly suggest the presence of a truncated but catalytically active form of ECE-1 in the CSF of SAH subjects. Further studies are necessary to determine the biological significance of soluble ECE-1 in CSF of SAH subjects, including an association with vasospasm after SAH.Endothelin-1 (ET-1)¹ is the most potent vasoconstrictor in the central nervous system. Elevated levels of ET-1 in cerebrospinal fluid (CSF) have been implicated in the pathogenesis of cerebral vasospasm following subarachnoid hemorrhage (SAH) (1). However, it is not known whether the production of ET-1 within the CSF space contributes to the pathogenesis of vasospasm. ET-1 is produced upon the cleavage of its precursor BigET-1 by the highly specific metalloprotease Endothelin Converting Enzyme-1 (ECE-1). The rate-limiting step of ET-1 production is the expression and localization of ECE-1, whose catalytic activity is confined to the extracellular C-terminal domain. Previously, we have demonstrated that the catalytically active C terminus can be shed from the cell surface (2). This results in the release of a truncated but catalytically active form of ECE-1 into the extracellular milieu.Although the presence of a soluble form of ECE-1 has been demonstrated in vitro, it is yet to be shown in any human biological fluid. In this study, we have used a combination of mass spectrometry, Western blotting as well as quenched fluorescent substrate (QFS) based enzyme assays to demonstrate for the first time the presence of catalytically active, soluble form of ECE-1 in CSF of SAH subjects.     

Hydrogen peroxide regulation of bovine endothelin-converting enzyme-1

Vascular injury leads to the production of reactive oxygen species (ROS), but the mechanisms by which ROS contribute to vascular pathology are not completely understood. We hypothesized that ROS increase endothelin converting enzyme (ECE-1) expression. We found that glucose oxidase (GO) increases ECE-1 mRNA, protein, and activity in bovine aortic endothelial cells. Catalase abolishes this effect. Glucose oxidase treatment of endothelial cells transactivates the ECE-1 promoter. The ECE-1 promoter element that mediates this response to GO is located between -444 and -216 bp. This region contains a STAT response element, and GO activates STAT-3 binding to this STAT response element. Our data suggest that STAT3 mediates hydrogen peroxide induction of ECE-1 expression.     

Endothelin inhibition delays onset of hyperglycemia and associated vascular injury in type I diabetes: evidence for endothelin release by pancreatic islet beta-cells

This study investigated the role of endothelin-1 for hyperglycemia, vascular, and pancreatic injury in early type I diabetes in non-obese-diabetic (NOD) mice. Endothelium dependent relaxation to acetylcholine and vascular gene expression of endothelin converting enzyme (ECE) isoforms 1 and 2 were studied as indicators of vascular injury. Endothelial NO bioactivity in the aorta was reduced in diabetic NOD mice while vascular expression of ECE-1 and ECE-2 mRNA was increased compared with controls (all p<0.05). Vascular histology was normal in all animals. Unexpectedly, treatment of prediabetic NOD mice for 6 weeks with the orally active ET(A) receptor antagonist BSF461314 prevented onset of diabetes without affecting insulitis severity. ET(A) receptor blockade also restored abnormal endothelial NO bioactivity and reduced ECE-1 and ECE-2 gene expression in NOD mice to levels comparable with healthy controls (p<0.05). Moreover, secretion of endothelin-1 in a time-dependent fashion was observed by pancreatic islet beta-cells cultured in vitro. These data suggest a critical role for ET(A) receptor signaling in the development of autoimmune forms of diabetes and the early vascular injury associated with it.     

Plasma membrane microdomains regulate TACE-dependent TNFR1 shedding in human endothelial cells

Upon stimulation by histamine, human vascular endothelial cells (EC) shed a soluble form of tumour necrosis factor receptor 1 (sTNFR1) that binds up free TNF, dampening the inflammatory response. Shedding occurs through proteolytic cleavage of plasma membrane-expressed TNFR1 catalysed by TNF-α converting enzyme (TACE). Surface expressed TNFR1 on EC is largely sequestered into specific plasma membrane microdomains, the lipid rafts/caveolae. The purpose of this study was to determine the role of these domains in TACE-mediated TNFR1 shedding in response to histamine. Human umbilical vein endothelial cells derived EA.hy926 cells respond to histamine via H1 receptors to shed TNFR1. Both depletion of cholesterol by methyl-β-cyclodextrin and small interfering RNA knockdown of the scaffolding protein caveolin-1 (cav-1), treatments that disrupt caveolae, reduce histamine-induced shedding of membrane-bound TNFR1. Moreover, immunoblotting of discontinuous sucrose gradient fractions show that TACE, such as TNFR1, is present within low-density membrane fractions, concentrated within caveolae, in unstimulated EA.hy926 endothelial cells and co-immunoprecipitates with cav-1. Silencing of cav-1 reduces the levels of both TACE and TNFR1 protein and displaces TACE, from low-density membrane fractions where TNFR1 remains. In summary, we show that endothelial lipid rafts/caveolae co-localize TACE to surface expressed TNFR1, promoting efficient shedding of sTNFR1 in response to histamine.     

Dissociated presenilin-1 and TACE processing of ErbB4 in lung alveolar type II cell differentiation

Neuregulin (NRG) stimulation of ErbB4 signaling is important for type II cell surfactant synthesis. ErbB4 may mediate gene expression via a non-canonical pathway involving enzymatic cleavage releasing its intracellular domain (4ICD) for nuclear trafficking and gene regulation. The accepted model for release of 4ICD is consecutive cleavage by Tumor necrosis factor alpha Converting Enzyme (TACE) and γ-secretase enzymes. Here, we show that 4ICD mediates surfactant synthesis and its release by γ-secretase is not dependent on previous TACE cleavage. We used siRNA to silence Presenilin-1 (PSEN-1) expression in a mouse lung type II epithelial cell line (MLE12 cells), and both siRNA knockdown and chemical inhibition of TACE. Knockdown of PSEN-1 significantly decreased baseline and NRG-stimulated surfactant phospholipid synthesis, expression of the surfactant proteins SP-B and SP-C, as well as 4ICD levels, with no change in ErbB4 ectodomain shedding. Neither siRNA knockdown nor chemical inhibition of TACE inhibited 4ICD release or surfactant synthesis. PSEN-1 cleavage of ErbB4 for non-canonical signaling through 4ICD release does not require prior cleavage by TACE.