AMPK boosts ADAM10 shedding activity in human aortic endothelial cells by promoting Rab14-dependent ADAM10 cell surface translocation.

A disintegrin and metalloprotease 10 (ADAM10) regulates the expression of cell surface receptors such as tumor necrosis factor receptor 1, toll-like receptor 4, and the receptor for advanced glycation end products (RAGE) by cleaving their extracellular regions. To function as a sheddase, ADAM10 should translocate from the intracellular compartments to the cell surface, but the translocation mechanism remains unclear. In this study, we explored the possible role of adenosine monophosphate-activated protein kinase (AMPK) in the induction of ADAM10 shedding activity. In cultured human aortic endothelial cells (HAECs), 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), an AMPK activator, boosted ADAM10 cell surface translocation and ectodomain shedding of RAGE. ADAM10 inhibition with GI 254023X and ADAM10 siRNA silencing both prevented AICAR-induced RAGE ectodomain shedding. AICAR increased AMPK phosphorylation as well. Both Compound C-mediated AMPK inhibition and AMPKα1-siRNA-mediated AMPK depletion suppressed AICAR-induced ADAM10 cell surface translocation and RAGE ectodomain shedding. On the other hand, siRNA knockdown of Rab14, a small GTPase that facilitates the intracellular trafficking of transmembrane proteins, prevented AICAR-induced ADAM10 cell surface translocation and RAGE ectodomain shedding. In conclusion, AMPK activation is an obvious inducer of ADAM10 shedding activity. Our findings suggest that AMPK boosts ADAM10 shedding activity in HAECs by promoting Rab14-dependent ADAM10 cell surface translocation.

FGFR2 mutations promote endometrial cancer progression through dual engagement of EGFR and Notch signalling pathways.

BACKGROUND: Mutations in the receptor tyrosine kinase gene fibroblast growth factor receptor 2 (FGFR2) occur at a high frequency in endometrial cancer (EC) and have been linked to advanced and recurrent disease. However, little is known about how these mutations drive carcinogenesis. METHODS: Differential transcriptomic analysis and two-step quantitative real-time PCR (qRT-PCR) assays were applied to identify genes differentially expressed in two cohorts of EC patients carrying mutations in the FGFR2 gene as well as in EC cells harbouring mutations in the FGFR2. Candidate genes and target signalling pathways were investigated by qRT-PCR assays, immunohistochemistry and bioinformatics analysis. The functional roles of differently regulated genes were analysed using in vitro and in vivo experiments, including 3D-orthotypic co-culture systems, cell proliferation and migration protocols, as well as colony and focus formation assays together with murine xenograft tumour models. The molecular mechanisms were examined using CRISPR/Cas9-based loss-of-function and pharmacological approaches as well as luciferase reporter techniques, cell-based ectodomain shedding assays and bioinformatics analysis. RESULTS: We show that common FGFR2 mutations significantly enhance the sensitivity to FGF7-mediated activation of a disintegrin and metalloprotease (ADAM)17 and subsequent transactivation of the epidermal growth factor receptor (EGFR). We further show that FGFR2 mutants trigger the activation of ADAM10-mediated Notch signalling in an ADAM17-dependent manner, highlighting for the first time an intimate cooperation between EGFR and Notch pathways in EC. Differential transcriptomic analysis in EC cells in a cohort of patients carrying mutations in the FGFR2 gene identified a strong association between FGFR2 mutations and increased expression of members of the Notch pathway and ErbB receptor family. Notably, FGFR2 mutants are not constitutively active but require FGF7 stimulation to reprogram Notch and EGFR pathway components, resulting in ADAM17-dependent oncogenic growth. CONCLUSIONS: These findings highlight a pivotal role of ADAM17 in the pathogenesis of EC and provide a compelling rationale for targeting ADAM17 protease activity in FGFR2-driven cancers.

The role of ADAM10 in astrocytes: Implications for Alzheimer's disease.

Much of the early research into AD relies on a neuron-centric view of the brain, however, evidence of multiple altered cellular interactions between glial cells and the vasculature early in AD has been demonstrated. As such, alterations in astrocyte function are widely recognized a contributing factor in the pathogenesis of AD. The processes by which astrocytes may be involved in AD make them an interesting target for therapeutic intervention, but in order for this to be most effective, there is a need for the specific mechanisms involving astrocyte dysfunction to be investigated. "α disintegrin and metalloproteinase" 10 (ADAM10) is capable of proteolytic cleavage of the amyloid precursor protein which prevents amyloid-ß generation. As such ADAM10 has been identified as an interesting enzyme in AD pathology. ADAM10 is also known to play a role in a significant number of cellular processes, most notable in notch signaling and in inflammatory processes. There is a growing research base for the involvement of ADAM10 in regulating astrocytic function, primarily from an immune perspective. This review aims to bring together available evidence for ADAM10 activity in astrocytes, and how this relates to AD pathology.

Liraglutide, a glucagon-like peptide-1 receptor agonist, induces ADAM10-dependent ectodomain shedding of RAGE via AMPK activation in human aortic endothelial cells.

AIMS: Glucagon-like peptide-1 alleviates the deleterious effects of advanced glycation end products (AGEs), but the underlying mechanisms are not fully understood. In this study, we investigated the protective mechanism using liraglutide, a glucagon-like peptide-1 receptor agonist, in cultured human aortic endothelial cells (HAECs). MAIN METHODS: Following liraglutide treatment in HAECs, the receptor for AGEs (RAGE) was measured in both cell lysate and culture supernatant, the cytosolic free Ca2+ level was monitored using Fluo-4 AM, the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) was analyzed, and immunofluorescence staining was used to visualize a disintegrin and metalloprotease 10 (ADAM10) on the cell surface. KEY FINDINGS: Liraglutide (100 nM) induced ectodomain shedding of RAGE within 30 min and inhibited the expression of intercellular adhesion molecule-1 (ICAM-1) induced by AGEs of bovine serum albumin (AGE-BSA). Further experiments revealed that liraglutide rapidly increases extracellular Ca2+ influx through L-type calcium channels and activates AMPK, resulting in the translocation of ADAM10 to the cell surface, whereas siRNA-mediated ADAM10 depletion prevents liraglutide-induced ectodomain shedding of RAGE and eliminates liraglutide's inhibitory effect on AGE-BSA-induced ICAM-1 expression. Moreover, compound C-mediated AMPK inhibition and siRNA-mediated AMPK depletion both prevented ADAM10 translocation to the cell surface and ADAM10-mediated ectodomain shedding of RAGE. SIGNIFICANCE: Liraglutide reduces the number of intact RAGE on the cell surface by inducing ADAM10-mediated ectodomain shedding, which decreases the inflammatory effects of AGEs. AMPK activated by extracellular Ca2+ influx is critically involved in the translocation of ADAM10 to the cell surface, where it cleaves RAGE.

Nuclear Paraspeckle Assembly Transcript 1 Enhances Hydrogen Peroxide-Induced Human Vascular Smooth Muscle Cell Injury by Regulating miR-30d-5p/A Disintegrin and Metalloprotease 10.

BACKGROUND: Nuclear paraspeckle assembly transcript 1 (NEAT1) has been reported to be involved in the progression of many cancers; however, the role and mechanisms underlying NEAT1 in abdominal aortic aneurysm (AAA) remain unclear.Methods and Results: The expression of NEAT1, miR-30d-5p and A disintegrin and metalloprotease 10 (ADAM10) was measured by qRT-PCR and western blot. Functional experiments were conducted by using a CCK-8 assay, EDU assay, flow cytometry, western blot, ELISA, and commercial kits. The target relation was confirmed by dual-luciferase reporter assay and the RIP assay. It was then found that NEAT1 was upregulated in peripheral blood of AAA patients ~3.46-fold, smooth muscle cells (SMCs) isolated from AAA tissues ~2.6-fold and in a hydrogen peroxide (H2O2)-induced injury model of human vascular SMC (HVSMCs) ~2.0- and 3.9-fold at 50 µmol/L and 200 µmol/L H2O2treatment, respectively. NEAT1 deletion attenuated H2O2-induced cell proliferation promotion (40.0% vs. 74.3%), apoptosis inhibition (25.0% vs. 13.5%), and reduction of inflammatory response and oxidative stress in HVSMCs. Mechanistically, NEAT1 targeted miR-30d-5p to prevent the degradation of its target, ADAM10, in HVSMCs. Further rescue experiments suggested miR-30d-5p inhibition mitigated the effects of NEAT1 deletion on H2O2-induced HVSMCs. Moreover, ADAM10 overexpression counteracted the inhibitory functions of miR-30d-5p on H2O2-evoked HVSMC injury. CONCLUSIONS: NEAT1 promoted H2O2-induced HVSMC injury by inducing cell apoptosis, inflammation and oxidative stress through miR-30d-5p/ADAM10 axis, indicating the possible involvement of NEAT1 in the pathogenesis of AAA.

Plasma ADAM-10 levels and functional outcome of acute primary basal ganglia hemorrhage.

BACKGROUND: The a-secretase A disintegrin and metalloprotease-10 (ADAM-10) may have deleterious effects in acute brain injury. This study was designed to discern if a relationship between plasma ADAM-10 levels and functional outcome exists in patients with intracerebral hemorrhage (ICH). METHODS: A total of 109 patients with basal ganglia hemorrhage and 100 healthy controls were included. Their plasma ADAM-10 levels were gauged. Ninety-day prognosis was assessed and poor outcome was defined as death or major disability (modified Rankin Scale score of 3 or greater). RESULTS: Plasma ADAM-10 levels were substantially elevated in patients, as compared to controls. ADAM-10 levels were independently correlated with hematoma size and National Institutes of Health Stroke Scale (NIHSS) score. Plasma ADAM-10, NIHSS score and hematoma size emerged as the independent predictors for 90-day poor outcome. Under receiver operating characteristic curve, plasma ADAM-10 levels exhibited similar prognostic capability, as compared to hematoma size and NIHSS score; moreover, it significantly improved prognostic abilities of NIHSS and hematoma size. CONCLUSIONS: Rising plasma ADAM-10 levels are independently related to increasing severity and poor long-term functional outcome after hemorrhagic stroke, substantializing serum ADAM-10 as a useful prognostic biomarker of ICH.

Members of the Fibroblast Growth Factor Receptor Superfamily Are Proteolytically Cleaved by Two Differently Activated Metalloproteases.

Fibroblast growth factor receptors (FGFRs) are a family of receptor tyrosine kinases that have been associated not only with various cellular processes, such as embryonic development and adult wound healing but also enhanced tumor survival, angiogenesis, and metastatic spread. Proteolytic cleavage of these single-pass transmembrane receptors has been suggested to regulate biological activities of their ligands during growth and development, yet little is known about the proteases responsible for this process. In this study, we monitored the release of membrane-anchored FGFRs 1, 2, 3, and 4 in cell-based assays. We demonstrate here that metalloprotease-dependent metalloprotease family, ADAM10 and ADAM17. Loss- and gain-of-function studies in murine embryonic fibroblasts showed that constitutive shedding as well as phorbol-ester-induced processing of FGFRs 1, 3, and 4 is mediated by ADAM17. In contrast, treatment with the calcium ionophore ionomycin stimulated ADAM10-mediated FGFR2 shedding. Cell migration assays with keratinocytes in the presence or absence of soluble FGFRs suggest that ectodomain shedding can modulate the function of ligand-induced FGFR signaling during cell movement. Our data identify ADAM10 and ADAM17 as differentially regulated FGFR membrane sheddases and may therefore provide new insight into the regulation of FGFR functions.

Pharmacologic Inhibition of ADAM10 Attenuates Brain Tissue Loss, Axonal Injury and Pro-inflammatory Gene Expression Following Traumatic Brain Injury in Mice.

The α-secretase A disintegrin and metalloprotease 10 (ADAM10) regulates various physiological and pathophysiological processes. Despite its broad functional implications during development, plasticity, and disease, no pharmacological approaches to inhibit ADAM10 in acute brain injury have been reported. Here, we examined the effects of the ADAM10 inhibitor GI254023X on the neurological and histopathological outcome after experimental traumatic brain injury (TBI). C57BL/6N mice were subjected to the controlled cortical impact (CCI) model of TBI or sham procedure and received GI254023X or vehicle during the acute phase of injury (n = 40, 100 mg/kg, 25% DMSO, 0.1 M Na2CO3, intraperitoneal, 30 min and 24 h after TBI). GI254023X treatment did not improve neurological deficits from 1 to 7 days post-injury (dpi) but animals treated with GI254023X exhibited smaller brain lesions compared to vehicle treatment. Determination of brain mRNA expression by quantitative PCR showed that TBI-induced up-regulation of Adam10 and Adam17 was not influenced by GI254023X but the up-regulation of the matrix metalloproteinase genes Mmp2 and Mmp9 was attenuated. GI254023X treatment further increased the T cell marker Cd247 but did not affect blood brain barrier integrity, as assessed by Occludin mRNA expression and IgG brain extravasation. However, in agreement with neuroprotective effects of ADAM10 inhibition, GI254023X treatment attenuated axonal injury, as indicated by decreased generation of spectrin breakdown products (SBDPs) and decreased immunostaining using anti-non-phosphorylated neurofilament (SMI-32). Interestingly, reduced axonal injury in GI254023X-treated animals coincided with subtle mRNA dysregulation in the glutamate receptor subunit genes Gria1 and Grin2b. Quantitative PCR also revealed that GI254023X mitigated up-regulation of the pro-inflammatory markers Il6, Tnfa, and Lcn2 but not the up-regulation of the pan-microglia marker Aif1, the M2 microglia marker Arg1 and the reactive astrocyte marker Gfap. Taken together, the ADAM10 inhibitor GI254023X attenuates brain tissue loss, axonal injury and pro-inflammatory gene expression in the CCI model of TBI. These results suggest that ADAM10 may represent a therapeutic target in the acute phase of TBI.

Human umbilical cord mesenchymal stem cells-derived exosomal microRNA-451a represses epithelial-mesenchymal transition of hepatocellular carcinoma cells by inhibiting ADAM10.

Exosomes derived from human umbilical cord mesenchymal stem cells (hucMSCs) expressing microRNAs (miRNAs) have been highlighted in human cancers. However, the detailed molecular mechanism of hucMSCs-derived exosomal miR-451a on hepatocellular carcinoma (HCC) remains further investigation. Our study aims to explore the impact of exosomal miR-451a on the progression of HCC. Expression of miR-451a and a disintegrin and metalloprotease 10 (ADAM10) in HCC tissues and adjacent normal tissues were determined. The exosomes were extracted from hucMSCs and co-cultured with Hep3B and SMMC-7721 cell lines. After the treatment of relative exosomes or exosome inhibitor GW4869 in Hep3B and SMMC-7721 cells, the paclitaxel resistance and malignant phenotypes of HCC cells were measured. Moreover, the effect of hucMSCs-derived exosomes on the expression of miR-451a and ADAM10 in HCC cells was assessed. The targeting relationship between miR-451a and ADAM10 was verified to detect the impact of ADAM10-wild type and ADAM10-mutant type (MUT) on HCC cell processes. Low expression of miR-451a and high expression of ADAM10 indicated a poor prognosis of HCC patients. MiR-451a was up-regulated while ADAM10 was down-regulated in HCC cells after co-culture with HucMSC-derived exosomes. The exosomes elevated miR-451a and inhibited ADAM10 to suppress the paclitaxel resistance, cell cycle transition, proliferation, migration and invasion, and promote apoptosis of HCC cells. ADAM10 was verified to be a target gene of miR-451a. ADAM10-MUT promoted HCC process independent of miR-451a mimic. HucMSC-derived exosomal miR-451a could restrict the epithelial-mesenchymal transition of HCC cells by targeting ADAM10, which might provide new targets for HCC treatment.

ß2-AR activation promotes cleavage and nuclear translocation of Her2 and metastatic potential of cancer cells.

Prolonged hypersecretion of catecholamine induced by chronic stress may correlate with malignant progression of cancer. ß2-adrenergic receptor (ß2-AR) overexpressed in certain cancer cells may translate the signals from neuroendocrine system to malignant signals by interacting with oncoproteins, such as Her2. In the present study, we demonstrate that catecholamine stimulation activates the expression and proteolytic activity of ADAM10 by modulating the expression of miR-199a-5p and SIRT1 and also confirm that catecholamine induction triggers the activities of γ-secretase, leading to shedding of Her2 extracellular domain (ECD) by ADAM10 and subsequent intramembranous cleavage of Her2 intracellular domain (ICD) by presenilin-dependent γ-secretase, nuclear translocation of Her2 ICD, and enhanced transcription of tumor metastasis-associated gene COX-2. Chronic stimulation of catecholamine strongly promotes the invasive activities of cancer cells in vitro and spontaneous tumor lung metastasis in mice. Furthermore, nuclear localization of Her2 was significantly correlated with overexpression of ß2-AR in human breast cancer tissues, indicating that catecholamine-induced ß2-AR activation plays decisive roles in tumor metastasis. Our data also reveal that an unknown mechanism by which the regulated intramembrane proteolysis (RIP) initiated by ß2-AR-mediated signaling controls a novel Her2-mediated signaling transduction.

Endothelial deletion of ADAM10, a key regulator of Notch signaling, causes impaired decidualization and reduced fertility in female mice.

During the initiation of pregnancy, the vasculature of the implantation site expands rapidly, yet little is known about this process or its role in fertility. Here, we report that endothelial-specific deletion of a disintegrin and metalloprotease 10 (ADAM10), an essential regulator of Notch signaling, results in severe subfertility in mice. We found that implantation sites develop until 5.5 days post conception (dpc) but are resorbed by 6.5 dpc in A10ΔEC mice. Analysis of the mutant implantation sites showed impaired decidualization and abnormal vascular patterning compared to controls. Moreover, RNA-seq analysis revealed changes in endothelial cell marker expression consistent with defective ADAM10/Notch signaling in samples from A10ΔEC mice, suggesting that this signaling pathways is essential for the physiological function of endometrial endothelial cells during early pregnancy. Our findings raise the possibility that impaired endothelial cell function could be a cause for repeated pregnancy loss (RPL) and infertility in humans.

Epigallocatechin-3-gallate downregulates lipopolysaccharide signaling in human aortic endothelial cells by inducing ectodomain shedding of TLR4.

Epigallocatechin-3-gallate (EGCG), the most abundant polyphenol in green tea leaves, has anti-inflammatory effects. In this study, we investigated the mechanism by which EGCG attenuates the effects of lipopolysaccharide (LPS), an agonist of toll-like receptor 4 (TLR4), in cultured human aortic endothelial cells (HAECs). The increase in the expression of intercellular adhesion molecule-1 (ICAM-1) induced by LPS (100 ng/ml) was effectively attenuated by pretreatment with EGCG (50 µM). Importantly, EGCG treatment resulted in a rapid reduction of cellular TLR4, which was accompanied by an increase in the N-terminal fragment of TLR4 in the culture supernatant, indicating that EGCG induces ectodomain shedding of TLR4. EGCG increased cytosolic Ca2+ by inducing the release of intracellular stored Ca2+ and the influx of extracellular Ca2+; accordingly, EGCG-induced ectodomain shedding of TLR4 was nullified by pretreatment with BAPTA-AM (10 µM), an intracellular Ca2+ chelator. EGCG induced translocation of a disintegrin and metalloprotease 10 (ADAM10) to the cell surface, which was also blocked by BAPTA-AM. Treatment with ADAM10 inhibitor (GI254023X, 2 µM) and siRNA-mediated depletion of ADAM10 prevented EGCG-induced ectodomain shedding of TLR4 and abolished the inhibitory effect of EGCG on LPS-induced ICAM-1 expression. Collectively, these findings suggest that EGCG decreases cell surface TLR4 in HAECs by inducing ADAM10-mediated ectodomain shedding, and thereby attenuates the effects of LPS. This is a new mechanism of the suppressive effect of EGCG on LPS signaling.

Arginine vasopressin attenuates the effects of TNF-α in aortic endothelial cells by inducing ectodomain shedding of TNF receptor 1.

In septic shock, arginine vasopressin (AVP) is commonly used as a vasopressor to restore blood pressure. Exogenous AVP may have anti-inflammatory effects as well. We investigated whether AVP modulates the effects of tumor necrosis factor-α (TNF-α) in human aortic endothelial cells (HAECs). TNF-α stimulated intercellular adhesion molecule-1 expression, while AVP pretreatment attenuated this effect of TNF-α. Upon treatment with AVP, extracellular Ca2+ entered the cells rapidly through L-type calcium channels, which in turn induced cell surface translocation of a disintegrin and metalloprotease 10 (ADAM10) and ectodomain shedding of tumor necrosis factor receptor 1 (TNFR1). On the other hand, siRNA depletion of ADAM10 suppressed AVP-induced ectodomain shedding of TNFR1 and eliminated the inhibitory effect of AVP against TNF-α. Depletion of oxytocin receptor also abolished AVP-induced extracellular Ca2+ influx, AVP-induced ectodomain shedding of TNFR1 and the inhibitory effect of AVP against TNF-α. These findings suggest that AVP decreases the responsiveness of HAECs to TNF-α by inducing ADAM10-dependent ectodomain shedding of TNFR1. Extracellular Ca2+ influx through L-type calcium channels was essential for ADAM10 activation. This effect of AVP was mediated through the oxytocin receptor.

Impairment of amyloid precursor protein alpha-processing in cerebral microvessels of type 1 diabetic mice.

The mechanisms underlying dysfunction of cerebral microvasculature induced by type 1 diabetes (T1D) are not fully understood. We hypothesized that in cerebral microvascular endothelium, α-processing of amyloid precursor protein (APP) is impaired by T1D. In cerebral microvessels derived from streptozotocin (STZ)-induced T1D mice protein levels of APP and its α-processing enzyme, a disintegrin and metalloprotease 10 (ADAM10) were significantly decreased, along with down-regulation of adenylate cyclase 3 (AC3) and enhanced production of thromboxane A2 (TXA2). In vitro studies in human brain microvascular endothelial cells (BMECs) revealed that knockdown of AC3 significantly suppressed ADAM10 protein levels, and that activation of TXA2 receptor decreased APP expression. Furthermore, levels of soluble APPα (sAPPα, a product of α-processing of APP) were significantly reduced in hippocampus of T1D mice. In contrast, amyloidogenic processing of APP was not affected by T1D in both cerebral microvessels and hippocampus. Most notably, studies in endothelial specific APP knockout mice established that genetic inactivation of APP in endothelium was sufficient to significantly reduce sAPPα levels in the hippocampus. In aggregate, our findings suggest that T1D impairs non-amyloidogenic processing of APP in cerebral microvessels. This may exert detrimental effect on local concentration of neuroprotective molecule, sAPPα, in the hippocampus.

ADAM10 controls the differentiation of the coronary arterial endothelium.

The coronary vasculature is crucial for normal heart function, yet much remains to be learned about its development, especially the maturation of coronary arterial endothelium. Here, we show that endothelial inactivation of ADAM10, a key regulator of Notch signaling, leads to defects in coronary arterial differentiation, as evidenced by dysregulated genes related to Notch signaling and arterial identity. Moreover, transcriptome analysis indicated reduced EGFR signaling in A10ΔEC coronary endothelium. Further analysis revealed that A10ΔEC mice have enlarged dysfunctional hearts with abnormal myocardial compaction, and increased expression of venous and immature endothelium markers. These findings provide the first evidence for a potential role for endothelial ADAM10 in cardioprotective homeostatic EGFR signaling and implicate ADAM10/Notch signaling in coronary arterial cell specification, which is vital for normal heart development and function. The ADAM10/Notch signaling pathway thus emerges as a potential therapeutic target for improving the regenerative capacity and maturation of the coronary vasculature.

Impact of ADAM10 gene polymorphisms on hepatocellular carcinoma development and clinical characteristics.

A disintegrin and metalloprotease (ADAM) family proteins are type-I transmembrane glycoproteins with multiple functions in cell adhesion, migration, proteolysis and signaling. ADAM10 is a member of the ADAM family reportedly involved in cancer progression and has been shown to be overexpressed in hepatocellular carcinoma (HCC) tissues and significantly associated with tumor progression and shortened survival. This study investigated ADAM10's single nucleotide polymorphisms (SNPs) and their association to HCC development and regulation. Real-time polymerase chain reaction was used to analyze five SNPs of ADAM10 in 333 patients with HCC and 1196 controls without cancer. The results indicated that of the 333 patients with HCC, those who carried ADAM10 rs514049 (AC + CC) variants had a higher risk of developing lymph node metastasis (odds ratio [OR] = 5.087, p = 0.027), and those who carried ADAM10 rs653765 (GA + AA) variants had a higher risk of developing distant metastasis (OR = 3.346, p = 0.020) and higher levels of α-fetoprotein. In conclusion, our study demonstrated that the SNPs of ADAM10 are involved in HCC progression. ADAM10 SNPs may be used as therapeutic targets to evaluate poor prognoses for HCC.

A disintegrin and metalloprotease 10-containing exosomes derived from nasal polyps promote angiogenesis and vascular permeability.

Abnormal angiogenesis and vascular permeability is important for the formation of nasal polyps (NPs). Increasing evidence has indicated that exosomes serve a vital role in modulating angiogenesis and vascular permeability. A disintegrin and metalloprotease 10 (ADAM10), an important type of proteinase that is overexpressed in various diseases, can influence angiogenesis and vascular permeability and has been observed in healthy nasal exosomes. To the best of our knowledge, the expression levels and the function of ADAM10 in NLF­derived exosomes from NPs has not been demonstrated previously. In order to determine the influence of exosomes derived from nasal lavage fluid (NLF) on angiogenesis and vascular permeability, 25 nasal polyp patients and 15 healthy volunteers were enrolled in the present study. NLF was collected from all of the subjects. Exosomes were isolated from NLF, visualized under transmission electron microscope and identified using western blot analysis. The effect of exosomes on human umbilical vein endothelial cells (HUVECs) was measured by tube formation and permeability assays in vitro. The expression of exosomal ADAM10 was also analyzed by western blotting. NLF­derived exosomes from NPs influenced proliferation, tube formation and the permeability of HUVECs. ADAM10 was highly expressed in NLF­derived exosomes from NPs when compared with healthy volunteers. Thus, NLF­derived exosomes from NPs promoted angiogenesis and vascular permeability, which may be associated with abundant ADAM10 in NP exosomes.

Glomerular endothelial cell maturation depends on ADAM10, a key regulator of Notch signaling.

The principal function of glomeruli is to filter blood through a highly specialized filtration barrier consisting of a fenestrated endothelium, the glomerular basement membrane and podocyte foot processes. Previous studies have uncovered a crucial role of endothelial a disintegrin and metalloprotease 10 (ADAM10) and Notch signaling in the development of glomeruli, yet the resulting defects have not been further characterized nor understood in the context of kidney development. Here, we used several different experimental approaches to analyze the kidneys and glomeruli from mice lacking ADAM10 in endothelial cells (A10ΔEC mice). Scanning electron microscopy of glomerular casts demonstrated enlarged vascular diameter and increased intussusceptive events in A10ΔEC glomeruli compared to controls. Consistent with these findings, genes known to regulate vessel caliber (Apln, AplnR and Vegfr3) are significantly upregulated in A10ΔEC glomeruli. Moreover, transmission electron microscopy revealed the persistence of diaphragms in the fenestrae of A10ΔEC glomerular endothelial cells, which was corroborated by the elevated expression of the protein PLVAP/PV-1, an integral component of fenestral diaphragms. Analysis of gross renal vasculature by light sheet microscopy showed no major alteration of the branching pattern, indicating a localized importance of ADAM10 in the glomerular endothelium. Since intussusceptions and fenestrae with diaphragms are normally found in developing, but not mature glomeruli, our results provide the first evidence for a crucial role of endothelial ADAM10, a key regulator of Notch signaling, in promoting the development and maturation of the glomerular vasculature.

Diosgenin, an Activator of 1,25D3-MARRS Receptor/ERp57, Attenuates the Effects of TNF-α by Causing ADAM10-Dependent Ectodomain Shedding of TNF Receptor 1.

BACKGROUND/AIMS: We investigated how diosgenin, a steroidal sapogenin, has anti-tumor necrosis factor-α (TNF-α) effects in human aortic endothelial cells (HAECs). METHODS: Tumor necrosis factor receptor 1 (TNFR1) was assessed by Western blot analysis. Intracellular Ca2+ was measured using Fluo-4 AM. Immunofluorescence staining was performed for a disintegrin and metalloprotease 10 (ADAM10). RESULTS: Diosgenin (1 ∼ 100 nM) induced ectodomain shedding of TNFR1 within 30 min and attenuated TNF-α-induced intercellular adhesion molecule-1 (ICAM-1) expression. Upon treatment with diosgenin, extracellular Ca2+ entered into the cells via L-type calcium channels, whereas diosgenin-induced ectodomain shedding of TNFR1 was almost completely inhibited by BAPTA-AM (intracellular Ca2+ chelator), verapamil (L-type calcium channel antagonist) and the absence of extracellular Ca2+. Diosgenin caused translocation of ADAM10 to the cell surface, which was mediated by extracellular Ca2+ influx. Depletion of ADAM10 prevented diosgenin-induced ectodomain shedding of TNFR1 and abolished the inhibitory effect of diosgenin on TNF-α-induced ICAM-1 expression. Diosgenin did not induce extracellular Ca2+ influx and ectodomain shedding of TNFR1 in cells depleted of 1,25D3-membrane associated rapid response steroid-binding receptor (1,25D3-MARRS receptor/ERp57). CONCLUSION: Diosgenin elicits L-type calcium channel-mediated extracellular Ca2+ influx, and thereby induces ADAM10-mediated ectodomain shedding of TNFR1. This effect of diosgenin was exerted through 1,25D3-MARRS receptor/ERp57.

ADAM10 modulates calcitriol-regulated RAGE in cardiomyocytes.

BACKGROUND: Receptor for advanced glycation end products (RAGE) signalling plays a critical role in the pathogenesis of cardiovascular disease. Calcitriol modulates cardiac RAGE expression. This study explored the mechanisms underlying the effect of calcitriol on RAGE and soluble RAGE (sRAGE) expression in cardiomyocytes. MATERIALS AND METHODS: Western blot, ELISA, fluorometric assay and PCR analyses were used to evaluate the RAGE, sRAGE, endogenous secretory RAGE (esRAGE), Jun N-terminal kinase (JNK), and a disintegrin and metalloprotease 10 (ADAM10) expression and enzyme activity in HL-1 atrial myocytes without and with calcitriol (10 and 100 nM), nuclear factor-κB (NF-κB) inhibitor (50 µg/mL), or ADAM10 inhibitor (5 µM) incubation for 48 h. RESULTS: Calcitriol (10 nM) significantly reduced RAGE protein expression and increased sRAGE concentrations in HL-1 cardiomyocytes compared with control cells. These changes were associated with increased protein expression and enzyme activity of ADAM10 and higher mRNA expression of esRAGE. In the presence of ADAM10 inhibitor, however, the suppressive effect of calcitriol on RAGE was diminished. Methylglyoxal (500 µM for 10 min)-mediated JNK phosphorylation was attenuated in the presence of calcitriol (10 nM). Moreover, control and NF-κB inhibitor-treated HL-1 cells had similar RAGE and sRAGE expression, suggesting that calcitriol-mediated RAGE modulation was independent of NF-κB signalling. CONCLUSIONS: We showed that RAGE downregulation and increased sRAGE production by calcitriol were mediated through ADAM10 activation in cardiomyocytes. The results suggest that calcitriol has therapeutic potential in treating RAGE-mediated cardiovascular complications.