A non-canonical role of endothelin converting enzyme 1 (ECE1) in promoting lung cancer development via directly targeting protein kinase B (AKT).

BACKGROUND: Lung cancer is the second most common malignancy in the world, and lung adenocarcinoma (LUAD) in particular is the leading cause of cancer death worldwide. Endothelin converting enzyme 1 (ECE1) is a membrane-bound metalloprotease involved in endothelin-1 (ET-1) processing and regulates vasoconstriction. However, very few studies have reported the involvement of ECE1 in regulating tumor cell proliferation, and the mechanism remains poorly understood. Therefore, we aimed to determine the role of ECE1 in lung cancer development. METHODS: The Cancer Genome Atlas database and Kaplan-Meier plotter were used to assess the association between ECE1 and lung cancer. The expression of ECE1 was detected using immunohistochemistry staining and western blotting. A variety of in vitro assays were performed to evaluate the effects of ECE1 on the colony formation, proliferation, migration and invasion using ECE1 knockdown lung cancer cells. The gene expression profiles regulated by ECE1 were investigated by RNA sequencing. An immunoprecipitation assay and immunofluorescence assay were used to evaluate the mechanism underlying the regulatory effect of ECE1 on protein kinase B (AKT). The effect of ECE1 on tumor development was assessed by xenografted lung cancer cells in either C57BL/6 mice or nude mice. RESULTS: ECE1 was upregulated in LUAD and correlated with the poor prognosis of patients with LUAD. Functional studies showed that knockdown of ECE1 retarded the progression of tumors formed by lung cancer cells at least partly by inhibiting tumor cell proliferation. Moreover, ECE1 accelerated tumor cell proliferation through promoting AKT activation dispensable of its canonical target ET-1. Mechanically, ECE1 interacted with the pleckstrin homology (PH) domain of AKT and facilitated its translocation to the plasma membrane for activation. Furthermore, the inhibition of AKT activity counteracted the lung cancer cell growth inhibition observed both in vitro and in xenografts caused by ECE1 suppression. CONCLUSIONS: The present study reveals a non-canonical function of ECE1 in regulating AKT activation and cell proliferation, which provides the basis for the development of a novel strategy for the intervention of cancer including LUAD by abrogating ECE1-AKT signaling.

ERN1 knockdown modifies the impact of glucose and glutamine deprivations on the expression of EDN1 and its receptors in glioma cells.

Objective. The aim of the present investigation was to study the impact of glucose and gluta-mine deprivations on the expression of genes encoding EDN1 (endothelin-1), its cognate receptors (EDNRA and EDNRB), and ECE1 (endothelin converting enzyme 1) in U87 glioma cells in response to knockdown of ERN1 (endoplasmic reticulum to nucleus signaling 1), a major signaling pathway of endoplasmic reticulum stress, for evaluation of their possible implication in the control of glioma growth through ERN1 and nutrient limitations. Methods. The expression level of EDN1, its receptors and converting enzyme 1 in control U87 glioma cells and cells with knockdown of ERN1 treated by glucose or glutamine deprivation by quantitative polymerase chain reaction was studied. Results. We showed that the expression level of EDN1 and ECE1 genes was significantly up-regulated in control U87 glioma cells exposure under glucose deprivation condition in comparison with the glioma cells, growing in regular glucose containing medium. We also observed up-regulation of ECE1 gene expression in U87 glioma cells exposure under glutamine deprivation as well as down-regulation of the expression of EDN1 and EDNRA mRNA, being more significant for EDN1. Furthermore, the knockdown of ERN1 signaling enzyme function significantly modified the response of most studied gene expressions to glucose and glutamine deprivation conditions. Thus, the ERN1 knockdown led to a strong suppression of EDN1 gene expression under glucose deprivation, but did not change the effect of glutamine deprivation on its expression. At the same time, the knockdown of ERN1 signaling introduced the sensitivity of EDNRB gene to both glucose and glutamine deprivations as well as completely removed the impact of glucose deprivation on the expression of ECE1 gene. Conclusions. The results of this study demonstrated that the expression of endothelin-1, its receptors, and ECE1 genes is preferentially sensitive to glucose and glutamine deprivations in gene specific manner and that knockdown of ERN1 significantly modified the expression of EDN1, EDNRB, and ECE1 genes in U87 glioma cells. It is possible that the observed changes in the expression of studied genes under nutrient deprivation may contribute to the suppressive effect of ERN1 knockdown on glioma cell proliferation and invasiveness.

Effect of Global Brain Ischemia on Amyloid Precursor Protein Metabolism and Expression of Amyloid-Degrading Enzymes in Rat Cortex: Role in Pathogenesis of Alzheimer's Disease.

The incidence of Alzheimer's disease (AD) increases significantly following chronic stress and brain ischemia which, over the years, cause accumulation of toxic amyloid species and brain damage. The effects of global 15-min ischemia and 120-min reperfusion on the levels of expression of the amyloid precursor protein (APP) and its processing were investigated in the brain cortex (Cx) of male Wistar rats. Additionally, the levels of expression of the amyloid-degrading enzymes neprilysin (NEP), endothelin-converting enzyme-1 (ECE-1), and insulin-degrading enzyme (IDE), as well as of some markers of oxidative damage were assessed. It was shown that the APP mRNA and protein levels in the rat Cx were significantly increased after the ischemic insult. Protein levels of the soluble APP fragments, especially of sAPPß produced by ß-secretase, (BACE-1) and the levels of BACE-1 mRNA and protein expression itself were also increased after ischemia. The protein levels of APP and BACE-1 in the Cx returned to the control values after 120-min reperfusion. The levels of NEP and ECE-1 mRNA also decreased after ischemia, which correlated with the decreased protein levels of these enzymes. However, we have not observed any changes in the protein levels of insulin-degrading enzyme. Contents of the markers of oxidative damage (di-tyrosine and lysine conjugates with lipid peroxidation products) were also increased after ischemia. The obtained data suggest that ischemia shifts APP processing towards the amyloidogenic ß-secretase pathway and accumulation of the neurotoxic Aß peptide as well as triggers oxidative stress in the cells. These results are discussed in the context of the role of stress and ischemia in initiation and progression of AD.

Intracellular metalloprotease activity controls intraneuronal Aß aggregation and limits secretion of Aß via exosomes.

Accumulating evidence suggests that the abnormal aggregation of amyloid-ß (Αß) peptide in Alzheimer's disease (AD) begins intraneuronally, within vesicles of the endosomal-lysosomal pathway where Aß is both generated and degraded. Metalloproteases, including endothelin-converting enzyme (ECE)-1 and -2, reside within these vesicles and normally limit the accumulation of intraneuronally produced Aß. In this study, we determined whether disruption of Aß catabolism could trigger Aß aggregation within neurons and increase the amount of Aß associated with exosomes, small extracellular vesicles derived from endosomal multivesicular bodies. Using cultured cell lines, primary neurons, and organotypic brain slices from an AD mouse model, we found that pharmacological inhibition of the ECE family of metalloproteases increased intracellular and extracellular Aß levels and promoted the intracellular formation of Aß oligomers, a process that did not require internalization of secreted Aß. In vivo, the accumulation of intraneuronal Aß aggregates was accompanied by increased levels of both extracellular and exosome-associated Aß, including oligomeric species. Neuronal exosomes were found to contain both ECE-1 and -2 activities, suggesting that multivesicular bodies are intracellular sites of Aß degradation by these enzymes. ECE dysfunction could lead to the accumulation of intraneuronal Aß aggregates and their subsequent release into the extracellular space via exosomes.-Pacheco-Quinto, J., Clausen, D., Pérez-González, R., Peng, H., Meszaros, A., Eckman, C. B., Levy, E., Eckman, E. A. Intracellular metalloprotease activity controls intraneuronal Aß aggregation and limits secretion of Aß via exosomes.

Endothelin-converting enzyme-1 regulates glucagon-like peptide-1 receptor signalling and resensitisation.

Following nutrient ingestion, glucagon-like peptide 1 (GLP-1) is secreted from intestinal L-cells and mediates anti-diabetic effects, most notably stimulating glucose-dependent insulin release from pancreatic ß-cells but also inhibiting glucagon release, promoting satiety and weight reduction and potentially enhancing or preserving ß-cell mass. These effects are mediated by the GLP-1 receptor (GLP-1R), which is a therapeutic target in type 2 diabetes. Although agonism at the GLP-1R has been well studied, desensitisation and resensitisation are perhaps less well explored. An understanding of these events is important, particularly in the design and use of novel receptor ligands. Here, using either HEK293 cells expressing the recombinant human GLP-1R or the pancreatic ß-cell line, INS-1E with endogenous expressesion of the GLP-1R, we demonstrate GLP-1R desensitisation and subsequent resensitisation following removal of extracellular GLP-1 7-36 amide. Resensitisation is dependent on receptor internalisation, endosomal acidification and receptor recycling. Resensitisation is also regulated by endothelin-converting enzyme-1 (ECE-1) activity, most likely through proteolysis of GLP-1 in endosomes and the facilitation of GLP-1R dephosphorylation and recycling. Inhibition of ECE-1 activity also increases GLP-1-induced activation of extracellular signal-regulated kinase and generation of cAMP, suggesting processes dependent upon the lifetime of the internalised ligand-receptor complex.

Changes of Endothelin-1 and Nitric Oxide Systems in Brain Tissue During Mild Hypothermia in a Porcine Model of Cardiac Arrest.

BACKGROUND: Our previous study found that mild hypothermia (MH) after resuscitation reduced cerebral microcirculation, but the mechanism was not elucidated. The aim of this study was to clarify changes of endothelin-1 (ET-1) and nitric oxide (NO) systems in brain tissue during hypothermia after resuscitation. METHODS: Twenty-six domestic male Beijing Landrace pigs were used in this study. MH was intravascularly induced 1 h after resuscitation from 8-min ventricular fibrillation. Core temperature was reduced to 33 °C and maintained until 8 h after resuscitation, and then animals were euthanized. ET-1 and NO levels in brain tissue and peripheral plasma were measured. Expression of endothelin-converting enzyme-1 (ECE-1), endothelin A receptor (ET-AR), endothelin-B receptor, and nitric oxide synthase (NOS) in brain tissue was determined by Western blot analysis. RESULTS: Compared with non-hypothermia (NH) treatment, MH after resuscitation significantly increased the level of endothelin-1 and reduced the level of NO in peripheral blood and brain tissue. Cerebral expression of ECE-1 and ET-AR was significantly increased during MH after resuscitation. Moreover, MH significantly decreased inducible NOS expression compared with the NH group. CONCLUSIONS: The ET-1 system is activated, while inducible NOS is inhibited in brain tissue during MH after resuscitation.

Ligand-Specific Factors Influencing GLP-1 Receptor Post-Endocytic Trafficking and Degradation in Pancreatic Beta Cells.

The glucagon-like peptide-1 receptor (GLP-1R) is an important regulator of blood glucose homeostasis. Ligand-specific differences in membrane trafficking of the GLP-1R influence its signalling properties and therapeutic potential in type 2 diabetes. Here, we have evaluated how different factors combine to control the post-endocytic trafficking of GLP-1R to recycling versus degradative pathways. Experiments were performed in primary islet cells, INS-1 832/3 clonal beta cells and HEK293 cells, using biorthogonal labelling of GLP-1R to determine its localisation and degradation after treatment with GLP-1, exendin-4 and several further GLP-1R agonist peptides. We also characterised the effect of a rare GLP1R coding variant, T149M, and the role of endosomal peptidase endothelin-converting enzyme-1 (ECE-1), in GLP1R trafficking. Our data reveal how treatment with GLP-1 versus exendin-4 is associated with preferential GLP-1R targeting towards a recycling pathway. GLP-1, but not exendin-4, is a substrate for ECE-1, and the resultant propensity to intra-endosomal degradation, in conjunction with differences in binding affinity, contributes to alterations in GLP-1R trafficking behaviours and degradation. The T149M GLP-1R variant shows reduced signalling and internalisation responses, which is likely to be due to disruption of the cytoplasmic region that couples to intracellular effectors. These observations provide insights into how ligand- and genotype-specific factors can influence GLP-1R trafficking.

Powering Amyloid Beta Degrading Enzymes: A Possible Therapy for Alzheimer's Disease.

The accumulation of amyloid beta (Aß) in the brain is believed to play a central role in the development and progression of Alzheimer's disease. Revisions to the amyloid cascade hypothesis now acknowledge the dynamic equilibrium in which Aß exists and the importance of enzymes involved in the production and breakdown of Aß in maintaining healthy Aß levels. However, while a wealth of pharmacological and immunological therapies are being generated to inhibit the Aß-producing enzymes, ß-site APP cleavage enzyme 1 and γ-secretase, the therapeutic potential of stimulating Aß-degrading enzymes such as neprilysin, endothelin-converting enzyme-1 and insulin-degrading enzyme remains relatively unexplored. Recent evidence indicates that increasing Aß degradation as opposed to inhibiting synthesis is a more effective strategy to prevent Aß build-up. Therefore Aß degrading enzymes have become valuable targets of therapy. In this review, we discuss the pathway of Aß synthesis and clearance along with the opportunities they present for therapeutic intervention, the benefits of increasing the expression/activity of Aß-degrading enzymes, and the untapped therapeutic potential of enzyme activation.

Ligand-Specific Signaling Profiles and Resensitization Mechanisms of the Neuromedin U2 Receptor.

The structurally related, but distinct neuropeptides, neuromedin U (NmU) and neuromedin S (NmS) are ligands of two G protein-coupled NmU receptors (NMU1 and NMU2). Hypothalamic NMU2 regulates feeding behavior and energy expenditure and has therapeutic potential as an anti-obesity target, making an understanding of its signaling and regulation of particular interest. NMU2 binds both NmU and NmS with high affinity, resulting in receptor-ligand co-internalization. We have investigated whether receptor trafficking events post-internalization are biased by the ligand bound and can therefore influence signaling function. Using recombinant cell lines expressing human NMU2, we demonstrate that acute Ca2+ signaling responses to NmU or NmS are indistinguishable and that restoration of responsiveness (resensitization) requires receptor internalization and endosomal acidification. The rate of NMU2 resensitization is faster following NmU compared with NmS exposure, but is similar if endothelin-converting enzyme-1 activity is inhibited or knocked down. Although acute activation of extracellular signal-regulated kinase (ERK) is also similar, activation by NMU2 is longer lasting if NmS is the ligand. Furthermore, when cells are briefly challenged before removal of free, but not receptor-bound ligand, activation of ERK and p38 mitogen-activated protein kinase by NmS is more sustained. However, only NmU responses are potentiated and extended by endothelin-converting enzyme-1 inhibition. These data indicate that differential intracellular ligand processing produces different signaling and receptor resensitization profiles and add to the findings of other studies demonstrating that intracellular ligand processing can shape receptor behavior and signal transduction.

Upregulation of miR-199a-5p Protects Spinal Cord Against Ischemia/Reperfusion-Induced Injury via Downregulation of ECE1 in Rat.

Ischemia-reperfusion (I/R)-induced spinal cord injury can cause apoptotic damage and subsequently act as a blood-spinal cord barrier damage. MicroRNAs (miRNAs) contributed to the process of I/R injury by regulating their target mRNAs. miR-199a-5p is involved in brain and heart I/R injury; however, its function in the spinal cord is not yet completely clarified. In this study, we investigated the role of miR-199a-5p on spinal cord I/R via the endothelin-converting enzyme 1, especially the apoptosis pathway. In the current study, the rat spinal cord I/R injury model was established, and the Basso Beattie Bresnahan scoring, Evans blue staining, HE staining, and TUNEL assay were used to assess the I/R-induced spinal cord injury. The differentially expressed miRNAs were screened using microarray. miR-199a-5p was selected by unsupervised hierarchical clustering analysis. The dual-luciferase reporter assay was used for detecting the regulatory effects of miR-199a-5p on ECE1. In addition, neuron expression was detected by immunostaining assay, while the expressions of p-ERK, ERK, p-JNK, JNK, caspase-9, Bcl-2, and ECE1 were evaluated by Western blot. The results indicated the successful establishment of the I/R-induced spinal cord injury model; the I/R induced the damage to the lower limb motor. Furthermore, 18 differentially expressed miRNAs were detected in the I/R group compared to the sham group, and miR-199a-5p protected the rat spinal cord injury after I/R. Moreover, miR-199a-5p negatively regulated ECE1, and silencing the ECE1 gene also protected the rat spinal cord injury after I/R. miR-199a-5p or silencing of ECE1 also regulated the expressions of caspase-9, Bcl-2, p-JNK, p-ERK, and ECE1 in rat spinal cord injury after I/R. Therefore, we demonstrated that miR-199a-5p might protect the spinal cord against I/R-induced injury by negatively regulating the ECE1, which could aid in developing new therapeutic strategies for I/R-induced spinal cord injury.

Nkx2.5 regulates endothelin converting enzyme-1 during pharyngeal arch patterning.

In gnathostomes, dorsoventral (D-V) patterning of neural crest cells (NCC) within the pharyngeal arches is crucial for the development of hinged jaws. One of the key signals that mediate this process is Endothelin-1 (EDN1). Loss of EDN1 binding to the Endothelin-A receptor (EDNRA) results in loss of EDNRA signaling and subsequent facial birth defects in humans, mice and zebrafish. A rate-limiting step in this crucial signaling pathway is the conversion of immature EDN1 into a mature active form by Endothelin converting enzyme-1 (ECE1). However, surprisingly little is known about how Ece1 transcription is induced or regulated. We show here that Nkx2.5 is required for proper craniofacial development in zebrafish and acts in part by upregulating ece1 expression. Disruption of nkx2.5 in zebrafish embryos results in defects in both ventral and dorsal pharyngeal arch-derived elements, with changes in ventral arch gene expression consistent with a disruption in Ednra signaling. ece1 mRNA rescues the nkx2.5 morphant phenotype, indicating that Nkx2.5 functions through modulating Ece1 expression or function. These studies illustrate a new function for Nkx2.5 in embryonic development and provide new avenues with which to pursue potential mechanisms underlying human facial disorders.

Endothelin-converting enzymes degrade α-synuclein and are reduced in dementia with Lewy bodies.

We have examined the roles of the endothelin-converting enzyme-1 and -2 (ECE-1 and ECE-2) in the homeostasis of α-synuclein (α-syn) and pathogenesis of Lewy body disease. The ECEs are named for their ability to convert inactive big endothelin to the vasoactive peptide endothelin-1 (EDN1). We have found that ECE-1 and ECE-2 cleave and degrade α-syn in vitro and siRNA-mediated knockdown of ECE-1 and ECE-2 in SH-SY5Y neuroblastoma cells significantly increased α-syn both intracellularly (within the cell lysate) (p < 0.05 for both ECE-1 and -2) and extracellularly (in the surrounding medium) (p < 0.05 for ECE-1 and p = 0.07 for ECE-2). Double immunofluorescent labelling showed co-localization of ECE-1 and ECE-2 with α-syn within the endolysosomal system (confirmed by a proximity ligation assay). To assess the possible relevance of these findings to human Lewy body disease, we measured ECE-1 and ECE-2 levels by sandwich ELISA in post-mortem samples of cingulate cortex (a region with a predilection for Lewy body pathology) in dementia with Lewy bodies (DLB) and age-matched controls. ECE-1 (p < 0.001) and ECE-2 (p < 0.01) levels were significantly reduced in DLB and both enzymes correlated inversely with the severity of Lewy body pathology as indicated by the level of α-syn phosphorylated at Ser129 (r = -0.54, p < 0.01 for ECE-1 and r = -0.49, p < 0.05 for ECE-2). Our novel findings suggest a role for ECEs in the metabolism of α-syn that could contribute to the development and progression of DLB.

Mechanisms Underlying Endothelin-1 Level Elevations Caused by Excessive Fluoride Exposure.

OBJECTIVE: To explore the mechanisms underlying endothelin-1 (ET-1) elevations induced by excessive fluoride exposure. METHODS: We measured serum and bone fluoride ion content and plasma ET-1 levels and compared these parameters among different groups in an animal model. We also observed morphological changes in the aorta and endothelium of rabbits. In cell experiments, human umbilical vein endothelial cells (HUVECs) were treated with varying concentrations of NaF for 24h, with or without 10 µM U0126 pretreatment for 1 h. ET-1 levels in culture fluid and intracellular reactive oxygen species (ROS) levels, as well as ET1 gene, endothelin-converting enzyme-1 (ECE-1), extracellular signal-regulating kinase 1/2 (ERK1/2), pERK1/2 expression levels and RAS activation were measured and compared among the groups. RESULTS: Plasma ET-1 levels of rabbits increased significantly in fluorinated groups compared with those in the control group. The rabbit thoracic aortas became slightly hardened in fluorinated groups compared with those in the control group, and some vacuoles were present in the endothelial cell cytoplasm of the rabbits in fluorinated groups. In our cell experiments, ET1 gene and ECE-1 expression levels in HUVECs and ET-1 expression levels in the cell culture supernatants increased significantly in some experimental groups compared with those in the control group. These trends paralleled the changes in intracellular ROS levels, RAS activation, and the pERK1/2-to-ERK1/2 ratio. After U0126 was added, ECE-1 expression and ET-1 levels decreased significantly. CONCLUSION: Excessive fluoride exposure leads to characteristic endothelial damage (vacuoles), thoracic aorta hardening, and plasma ET-1 level elevations in rabbits. In addition, the ROS-RAS-MEK1/2-pERK1/2/ERK1/2 pathway plays a crucial-and at least partial-role in ET-1 over-expression, which is promoted by excessive fluoride exposure.

Angiogenesis in the skin of SHARPIN-deficient mice with chronic proliferative dermatitis.

Angiogenesis is a common feature of pathological processes including wound healing, tumor formation, and chronic inflammation. Chronic inflammation can also be associated with dilation or proliferation of lymph vessels. We examined blood vessels and lymphatics and the expression of pro- and anti-angiogenic genes in the skin of SHARPIN-deficient mice which spontaneously develop a chronic proliferative dermatitis (cpdm). The number of blood vessels in the dermis of cpdm mice increased with age as the inflammation progressed. Lymphatics identified by labeling for LYVE1 and podoplanin were moderately dilated, but they were not increased in number. The expression of proangiogenic Vegfa, Flt1 and anti-angiogenic Sema3a mRNA was increased. VEGFA was primarily localized in keratinocytes of cpdm skin. There was also increased expression of Ece1 and Pdpn mRNA. Podoplanin was restricted to lymphatic endothelial cells in normal skin, but fibroblasts in cpdm skin also reacted with anti-podoplanin antibodies indicating that they were activated. The expression of other angiogenic and lymphangiogenic factors was not altered or decreased. These results indicate that cpdm mice may be a useful model to study the pathogenesis of angiogenesis in chronic inflammation.

Endothelin as a local regulating factor in the bovine oviduct.

Endothelin (EDN) is a possible regulating factor of oviductal motility, which is important for the transport of gametes and embryo. To clarify the factors that control the secretion of EDN in the bovine oviduct, the expression of EDNs, EDN-converting enzymes (ECEs) and EDN receptors (EDNRs) were investigated. All isoforms of EDN (EDN1-3), ECE (ECE1 and ECE2) and EDNR (EDNRA and EDNRB) were immunolocalised in the epithelial cells of the ampulla and the isthmus. EDNRs were also immunolocalised in smooth-muscle cells. The mRNA expression of EDN2 and ECE2 was higher in cultured ampullary oviductal epithelial cells than in isthmic cells. The expression of EDN1, EDN2 and ECE2 in the ampullary tissue was highest on the day of ovulation. Oestradiol-17ß increased EDN2 and ECE1 expression, while progesterone increased only ECE1 expression in cultured ampullary epithelial cells. These results indicate that EDNs are produced by epithelial cells and their target site is smooth-muscle and epithelial cells, and suggest that ovarian steroids are regulators of endothelin synthesis in ampullary oviductal epithelial cells.

Comprehensive analysis of the endothelin system in the kidneys of mice, rats, and humans.

The intrarenal endothelin (ET) system is an established moderator of kidney physiology and mechanistic contributor to the pathophysiology and progression of chronic kidney disease in humans and rodents. The aim of the present study was to characterize ET system by combining single cell RNA sequencing (scRNA-seq) data with immunolocalization in human and rodent kidneys of both sexes. Using publicly available scRNA-seq data, we assessed sex and kidney disease status (human), age and sex (rats), and diurnal expression (mice) on the kidney ET system expression. In normal human biopsies of both sexes and in rodent kidney samples, the endothelin-converting enzyme-1 (ECE1) and ET-1 were prominent in the glomeruli and endothelium. These data agreed with the scRNA-seq data from these three species, with ECE1/Ece1 mRNA enriched in the endothelium. However, the EDN1/Edn1 gene (encodes ET-1) was rarely detected, even though it was immunolocalized within the kidneys, and plasma and urinary ET-1 excretion are easily measured. Within each species, there were some sex-specific differences. For example, in kidney biopsies from living donors, men had a greater glomerular endothelial cell endothelin receptor B (Ednrb) compared with women. In mice, females had greater kidney endothelial cell Ednrb than male mice. As commercially available antibodies did not work in all species, and RNA expression did not always correlate with protein levels, multiple approaches should be considered to maintain required rigor and reproducibility of the pre- and clinical studies evaluating the intrarenal ET system.

Cancer Stem Cell and Aggressiveness Traits Are Promoted by Stable Endothelin-Converting Enzyme-1c in Glioblastoma Cells.

Glioblastoma (GBM) is the most common and aggressive type of brain tumor due to its elevated recurrence following treatments. This is mainly mediated by a subpopulation of cells with stemness traits termed glioblastoma stem-like cells (GSCs), which are extremely resistant to anti-neoplastic drugs. Thus, an advancement in the understanding of the molecular processes underlying GSC occurrence should contribute significantly towards progress in reducing aggressiveness. High levels of endothelin-converting enzyme-1 (ECE1), key for endothelin-1 (ET-1) peptide activation, have been linked to the malignant progression of GBM. There are four known isoforms of ECE1 that activate ET-1, which only differ in their cytoplasmic N-terminal sequences. Isoform ECE1c is phosphorylated at Ser-18 and Ser-20 by protein kinase CK2, which increases its stability and hence promotes aggressiveness traits in colon cancer cells. In order to study whether ECE1c exerts a malignant effect in GBM, we designed an ECE1c mutant by switching a putative ubiquitination lysine proximal to the phospho-serines Lys-6-to-Arg (i.e., K6R). This ECE1cK6R mutant was stably expressed in U87MG, T98G, and U251 GBM cells, and their behavior was compared to either mock or wild-type ECE1c-expressing clone cells. ECE1cK6R behaved as a highly stable protein in all cell lines, and its expression promoted self-renewal and the enrichment of a stem-like population characterized by enhanced neurospheroid formation, as well as increased expression of stem-like surface markers. These ECE1cK6R-derived GSC-like cells also displayed enhanced resistance to the GBM-related chemotherapy drugs temozolomide and gemcitabine and increased expression of the ABCG2 efflux pump. In addition, ECE1cK6R cells displayed enhanced metastasis-associated traits, such as the modulation of adhesion and the enhancement of cell migration and invasion. In conclusion, the acquisition of a GSC-like phenotype, together with heightened chemoresistance and invasiveness traits, allows us to suggest phospho-ECE1c as a novel marker for poor prognosis as well as a potential therapeutic target for GBM.

Targeting Endothelin in Alzheimer's Disease: A Promising Therapeutic Approach.

Endothelin is a chemical mediator that helps in maintaining balance within the blood-brain barrier by regulating the levels of toxicants and molecules which pass through the brain, suggesting that a rise in its production determines Alzheimer's disease. The inequity in the amyloid ß occurs due to a problem in its clearance from the brain initiating the production of reactive oxygen species and superoxide that activates a cascade wherein the release of inflammatory mediators and various enzymes like endothelin-converting enzymes take place. Furthermore, the cascade increases the levels of endothelin in the brain from endothelial cells. Endothelin levels are upregulated, which can be regulated by modulating the action of endothelin-converting enzymes and endothelin receptors. Hence, endothelin paves a pathway in the treatment of Alzheimer's disease. In this article, we have covered various mechanisms and preclinical studies that support and direct endothelin involvement in the progression of Alzheimer's disease by using various search tools such as PubMed, Science Direct, and Medline. Conclusive outcome data were extracted that all together defy contrivance pathways, potential drugs, endothelin receptors, and endothelin enzymes in our article giving profound importance to target endothelin for prevention and treatment of Alzheimer's disease.

Identification of Alzheimer's disease-associated rare coding variants in the ECE2 gene.

Accumulation of amyloid ß protein (Aß) due to increased generation and/or impaired degradation plays an important role in Alzheimer's disease (AD) pathogenesis. In this report, we describe the identification of rare coding mutations in the endothelin-converting enzyme 2 (ECE2) gene in 1 late-onset AD family, and additional case-control cohort analysis indicates ECE2 variants associated with the risk of developing AD. The 2 mutations (R186C and F751S) located in the peptidase domain in the ECE2 protein were found to severely impair the enzymatic activity of ECE2 in Aß degradation. We further evaluated the effect of the R186C mutation in mutant APP-knockin mice. Overexpression of wild-type ECE2 in the hippocampus reduced amyloid load and plaque formation, and improved learning and memory deficits in the AD model mice. However, the effect was abolished by the R186C mutation in ECE2. Taken together, the results demonstrated that ECE2 peptidase mutations contribute to AD pathogenesis by impairing Aß degradation, and overexpression of ECE2 alleviates AD phenotypes. This study indicates that ECE2 is a risk gene for AD development and pharmacological activation of ECE2 could be a promising strategy for AD treatment.

Endothelin-converting enzyme-1c promotes stem cell traits and aggressiveness in colorectal cancer cells.

Endothelin-1 is a mitogenic peptide that activates several proliferation, survival, and invasiveness pathways. The effects of endothelin-1 rely on its activation by endothelin-converting enzyme-1 (ECE1), which is expressed as four isoforms with different cytoplasmic N termini. Recently, isoform ECE1c has been suggested to have a role in cancer aggressiveness. The N terminus of ECE1c is phosphorylated by protein kinase CK2 (also known as casein kinase 2), and this enhances its stability and promotes invasiveness in colorectal cancer cells. However, it is not known how phosphorylation improves stability and why this is correlated with increased aggressiveness. We hypothesized that CK2 phosphorylation protects ECE1c from N-terminal ubiquitination and, consequently, from proteasomal degradation. Here, we show that lysine 6 is the bona fide residue involved in ubiquitination of ECE1c and its mutation to arginine (ECE1cK6R ) significantly impairs proteasomal degradation, thereby augmenting ECE1c stability, even in the presence of the CK2 inhibitor silmitasertib. Furthermore, colorectal cancer cells overexpressing ECE1cK6R displayed enhanced cancer stem cell (CSC) traits, including increased stemness gene expression, chemoresistance, self-renewal, and colony formation and spheroid formation in vitro, as well as enhanced tumor growth and metastasis in vivo. These findings suggest that CK2-dependent phosphorylation enhances ECE1c stability, promoting an increase in CSC-like traits. Therefore, phospho-ECE1c may be a biomarker of poor prognosis and a potential therapeutic target for colorectal cancer.