Ectodomain Shedding Research Articles

MiR-21-3p regulates AGE/RAGE signalling and improves diabetic atherosclerosis.

To explore the effects of miR-21-3p on diabetic atherosclerosis. Using enzyme-linked immunosorbent assay (ELISA), we also detected the levels of soluble receptor for advanced glycation endproducts RAGE (sRAGE) in the cellular supernatant of vascular endothelial cells after transfecting them with adenovirus vector having miR-21-3p mimic or inhibitor. We found decrease in the expression levels of miR-21-3p in vascular endothelial cells (VECs) induced by high-concentration glucose. We also observed that the introduction of miR-21-3p mimic significantly increased the expression of ADAM10 in the VECs. Similarly, significantly higher levels of sRAGE were found in the cultured supernatant after administration of miR-21-3p mimic in human vein endothelial cells. The production of reactive oxygen species and expression of inflammatory cytokines in VECs induced by LPS and high-concentration glucose were significantly decreased after administration of miR-21-3p. in vivo studies revealed that intravenous injection of miR-21-3p at regular intervals would reduce the area of atherosclerotic lesion and elevate the serum levels of sRAGE in atherosclerotic diabetic mice. miR-21-3p may be beneficial in diabetic atherosclerosis by promoting the cleaved form of sRAGE and inhibition of RAGE/NADPH oxidase signalling depending on the increased expression of ADAM10. SIGNIFICANCE OF THE STUDY: We identified a novel microRNA, miR-21-3p, which is characteristically at elevated levels in serum derived from diabetic patients and responsible for target degradation of ADAM10 mRNA. Further, we show that miR-21-3p aggravates the atherosclerotic lesion via dysfunction of the ectodomain shedding of molecular binding RAGE in the diabetic atherosclerotic mice.

The Role of Collagen XVII in Cancer: Squamous Cell Carcinoma and Beyond.

Alterations in the extracellular matrix (ECM) likely facilitate the first steps of cancer cell metastasis and supports tumor progression. Recent data has demonstrated that alterations in collagen XVII (BP180), a transmembrane protein and structural component of the ECM, can have profound effects on cancer invasiveness. Collagen XVII is a homotrimer of three α1 (XVII) chains. Its intracellular domain contains binding sites for plectin, integrin β4, and BP230, while the extracellular domain facilitates interactions between the cell and the ECM. Collagen XVII and its shed ectodomain have been implicated in cell motility and adhesion and are believed to promote tumor development and invasion. A strong association of collagen XVII ectodomain shedding and tumor invasiveness occurs in squamous cell carcinoma (SCC). Aberrant expression of collagen XVII has been reported in many epithelial cancers, ranging from squamous cell carcinoma to colon, pancreatic, mammary, and ovarian carcinoma. Thus, in this review, we focus on collagen XVII's role in neoplasia and tumorigenesis. Lastly, we discuss the importance of targeting collagen XVII and its ectodomain shedding as a novel strategy to curb tumor growth and reduce metastatic potential.

Substrate-selective protein ectodomain shedding by ADAM17 and iRhom2 depends on their juxtamembrane and transmembrane domains.

The metalloprotease ADAM17 (a disintegrin and metalloprotease 17) regulates EGF-receptor and TNFα signaling, thereby not only protecting the skin and intestinal barrier, but also contributing to autoimmunity. ADAM17 can be rapidly activated by many stimuli through its transmembrane domain (TMD), with the seven membrane-spanning inactive Rhomboids (iRhom) 1 and 2 implicated as candidate regulatory partners. However, several alternative models of ADAM17 regulation exist that do not involve the iRhoms, such as regulation through disulfide bond exchange or through interaction with charged phospholipids. Here, we report that a non-activatable mutant of ADAM17 with the TMD of betacellulin (BTC) can be rescued by restoring residues from the ADAM17 TMD, but only in Adam17-/- cells, which contain iRhoms, not in iRhom1/2-/- cells. We also provide the first evidence that the extracellular juxtamembrane domains (JMDs) of ADAM17 and iRhom2 regulate the stimulation and substrate selectivity of ADAM17. Interestingly, a point mutation in the ADAM17 JMD identified in a patient with Tetralogy of Fallot, a serious heart valve defect, affects the substrate selectivity of ADAM17 toward Heparin-binding epidermal growth factor like growth factor (HB-EGF), a crucial regulator of heart valve development in mice. These findings provide new insights into the regulation of ADAM17 through an essential interaction with the TMD1 and JMD1 of iRhom2.

Syndecans in Inflammation at a Glance.

Syndecans are transmembrane proteoglycans with heparan and chondroitin sulfate chains attached to their extracellular domain. Like many proteoglycans, they interact with a large number of ligands, such as growth factors, adhesion receptors, soluble small molecules, proteinases, and other extracellular matrix proteins to initiate downstream signaling pathways. Syndecans play a major role in inflammation, mainly by regulating leukocyte extravasation and cytokine function. At the same time, syndecans can undergo cytokine mediated changes in their expression levels during inflammation. The function of syndecans during inflammation appears to depend on the stage of inflammation, sulfation of heparan/chondroitin sulfate chains, the rate of ectodomain shedding and the solubility of the ectodomains. From the current literature, it is clear that syndecans are not only involved in the initial recruitment of pro-inflammatory molecules but also in establishing a balanced progression of inflammation. This review will summarize how cell surface and soluble syndecans regulate multiple aspects of inflammation.

From the Editor's Desk…

From the Editor's Desk…

Role of Endothelial ADAM17 in Early Vascular Changes Associated with Diabetic Retinopathy.

ADAM17, a disintegrin and metalloproteinase 17, is a transmembrane metalloproteinase that regulates bioavailability of multiple membrane-bound proteins via ectodomain shedding. ADAM17 activity was shown to contribute to a number of vascular pathologies, but its role in the context of diabetic retinopathy (DR) is not determined. We found that expression and enzymatic activity of ADAM17 are upregulated in human diabetic postmortem retinas and a mouse model of streptozotocin-induced diabetes. To further investigate the contribution of ADAM17 to vascular alterations associated with DR, we used human retinal endothelial cells (HREC) treated with ADAM17 neutralizing antibodies and exposed to glucidic stress and streptozotocin-induced endothelial ADAM17 knockout mice. Evaluation of vascular permeability, vascular inflammation, and oxidative stress was performed. Loss of ADAM17 in endothelial cells markedly reduced oxidative stress evidenced by decreased levels of superoxide, 3-nitrotyrosine, and 4-hydroxynonenal and decreased leukocyte-endothelium adhesive interactions in vivo and in vitro. Reduced leukostasis was associated with decreased vascular permeability and was accompanied by downregulation of intercellular adhesion molecule-1 expression. Reduction in oxidative stress in HREC was associated with downregulation of NAD(P)H oxidase 4 (Nox4) expression. Our data suggest a role for endothelial ADAM17 in DR pathogenesis and identify ADAM17 as a potential new therapeutic target for DR.

Dapagliflozin Inhibits Cell Adhesion to Collagen I and IV and Increases Ectodomain Proteolytic Cleavage of DDR1 by Increasing ADAM10 Activity.

Dapagliflozin, empagliflozin, tofogliflozin, selective inhibitors of sodium-glucose cotransporter 2 (SGLT2), is used clinically to reduce circulation glucose levels in patients with type 2 diabetes mellitus by blocking the reabsorption of glucose by the kidneys. Dapagliflozin is metabolized and inactivated by UGT1A9. Empagliflozin is metabolized and inactivated by UGT1A9 and by other related isoforms UGT2B7, UGT1A3, and UGT1A8. Tofogliflozin is metabolized and inactivated by five different enzymes CYP2C18, CYP3A4, CYP3A5, CYP4A11, and CYP4F3. Dapagliflozin treatment of HCT116 cells, which express SGLT2 but not UGT1A9, results in the loss of cell adhesion, whereas HepG2 cells, which express both SGLT2 and UGT1A9, are resistant to the adhesion-related effects of dapagliflozin. PANC-1 and H1792 cells, which do not express either SGLT2 or UGT1A9, are also resistant to adhesion related effects of dapagliflozin. On the other hand, either empagliflozin or tofogliflozin treatment of HCT116, HepG2, PANC-1, and H1792 cells are resistant to the adhesion-related effects as observed in dapagliflozin treated HCT116 cells. Knockdown of UGT1A9 by shRNA in HepG2 cells increased dapagliflozin sensitivity, whereas the overexpression of UGT1A9 in HCT116 cells protected against dapagliflozin-dependent loos of cell adhesion. Dapagliflozin treatment had no effect on cellular interactions with fibronectin, vitronectin, or laminin, but it induced a loss of interaction with collagen I and IV. In parallel, dapagliflozin treatment reduced protein levels of the full-length discoidin domain receptor I (DDR1), concomitant with appearance of DDR1 cleavage products and ectodomain shedding of DDR1. In line with these observations, unmetabolized dapagliflozin increased ADAM10 activity. Dapagliflozin treatment also significantly reduced Y792 tyrosine phosphorylation of DDR1 leading to decrement of DDR1 function and detachment of cancer cells. Concomitant with these lines of results, we experienced that CEA in patients with colon cancer, which express SGLT2 but not UGT1A9, and type 2 diabetes mellitus treated by dapagliflozin in addition to chemotherapy was decreased (case 1). CEA in patients with colon cancer, which express SGLT2 but not UGT1A9, and type 2 diabetes mellitus was treated by dapagliflozin alone after radiation therapy was decreased but started to rise after cessation of dapagliflozin (case 2). CA19-9 in two of patients with pancreatic cancer and type 2 diabetes mellitus was resistant to the combination therapy of dapagliflozin and chemotherapy (case 3 and 4 respectively). PIVKAII in patients with liver cancer and type 2 diabetes mellitus, and CYFRA in patients with squamous lung cancer and type 2 diabetes mellitus was also resistant the combination therapy of dapagliflozin and chemotherapy (case 5 and 6 respectively). Taken together, these data suggest a potential role for dapagliflozin anticancer therapy against colon cancer cells that express SGLT2, but not UGT1A9.

Collagen XVII Processing and Blistering Skin Diseases.

Collagen XVII (COL17) is a hemidesmosomal transmembrane protein in the skin, which, in several autoimmune blistering skin diseases, may be targeted by autoantibodies. In addition, loss-of-function mutations in the COL17A1 gene induce a subtype of junctional epidermolysis bullosa. The extracellular domain of COL17 can be physiologically cleaved from the cell surface by ADAM family proteins in a process known as ectodomain shedding. COL17 ectodomain shedding is thought to be associated with the migration and proliferation of keratinocytes. Furthermore, the C-terminal cleavage of COL17 may be associated with basement membrane formation. COL17 can be targeted by various proteases, including MMP9, neutrophil elastase, plasmin and granzyme B, which may be associated with blister formation in pemphigoid diseases. Interestingly, cleavage of COL17 may induce neoepitopes on the proteolysed fragments, and such induction is associated with dynamic structural changes. This review summarizes the current understanding of cleavage of COL17, and how such cleavage relates to blistering skin diseases.

MET ectodomain shedding is associated with poor disease-free survival of patients diagnosed with oral squamous cell carcinoma.

Ectodomain shedding unleashes the aggressive nature of the MET oncogene product. Using specific C- and N-terminal MET antibodies (D1C2 and A2H2-3), MET protein status (i.e., no MET, decoy MET, transmembranous C-terminal MET with or without the ectodomain) was investigated in oral squamous cell carcinoma. For the cancers showing transmembranous C-terminal MET, the impact of ectodomain shedding on prognosis was investigated. To examine ectodomain shedding, reduced lysates of oral squamous cell carcinoma cell lines were immunoblotted using D1C2 and an ELISA was performed on culture media using A2H2-3. In addition, reduced lysates of fresh frozen tissues of 30 oral squamous cell carcinoma were immunoblotted using D1C2 and immunohistochemistry was performed on corresponding formalin-fixed paraffin-embedded tissues using both antibodies on parallel sections. To examine MET protein status, differences between membranous D1C2 and A2H2-3 immunoreactivities were scored using parallel tissue microarray sections representing 156 oral squamous cell carcinoma. The prognostic value of ectodomain shedding was examined using Cox regression analysis for disease-free survival and overall survival. Ectodomain shedding was observed in all cell lines, 43% (n = 13) of fresh frozen and 50% (n = 15) of formalin-fixed paraffin-embedded cancers (27% overlap, n = 8). The tissue microarray showed no MET in 23% (n = 36), decoy MET in 9% (n = 14), and transmembranous C-terminal MET in 68% (n = 106) of examined cancers. Within the latter group, ectodomain shedding occurs in 36% (n = 38) of the cases and is independently associated with poor disease-free survival (HR = 2.41; 95% CI, 1.35-4.30 and P = 0.003)-though not overall survival (HR = 1.64; 95% CI, 0.92-2.94 and P = 0.095)-after correcting for factors known to influence survival. In conclusion, MET ectodomain shedding occurs in transmembranous C-terminal MET positive oral squamous cell carcinoma and is independently associated with disease-free survival. These findings might aid in designing companion diagnostics for targeted therapies directed against MET.

Proteolytic Processing of Neuregulin 2

Neuregulin 2 (NRG2) belongs to the EGF family of growth factors. Most of this family members require proteolytic cleavage to liberate their ectodomains capable of binding and activating their cognate ErbB receptors. To date, most of the studies investigating proteolytic processing of neuregulins focused on NRG1, which was shown to undergo ectodomain shedding by several ADAM proteases and BACE1 and the remaining fragment was further cleaved by γ-secretase. Recently, NRG2 attracted more attention due to its role in the neurogenesis and modulation of behaviors associated with psychiatric disorders. In this study, we used genetic engineering methods to identify proteases involved in proteolytic processing of murine NRG2. Using non-neuronal cell lines as well as cultures of primary hippocampal neurons, we demonstrated that the major proteases responsible for releasing NRG2 ectodomain are ADAM10 and BACE2. Co-expression of NRG2 and BACE2 in neurons of certain brain structures including medulla oblongata and cerebellar deep nuclei was confirmed via immunohistochemical staining. The cleavage of NRG2 by ADAM10 or BACE2 generates a C-terminal fragment that serves as a substrate for γ-secretase. We also showed that murine NRG2 is subject to post-translational modifications, substantial glycosylation of its extracellular part, and phosphorylation of the cytoplasmic tail.

DPF is a cell-density sensing factor, with cell-autonomous and non-autonomous functions during Dictyostelium growth and development

BackgroundCellular functions can be regulated by cell-cell interactions that are influenced by extra-cellular, density-dependent signaling factors. Dictyostelium grow as individual cells in nutrient-rich sources, but, as nutrients become depleted, they initiate a multi-cell developmental program that is dependent upon a cell-density threshold. We hypothesized that novel secreted proteins may serve as density-sensing factors to promote multi-cell developmental fate decisions at a specific cell-density threshold, and use Dictyostelium in the identification of such a factor.ResultsWe show that multi-cell developmental aggregation in Dictyostelium is lost upon minimal (2-fold) reduction in local cell density. Remarkably, developmental aggregation response at non-permissive cell densities is rescued by addition of conditioned media from high-density, developmentally competent cells. Using rescued aggregation of low-density cells as an assay, we purified a single, 150-kDa extra-cellular protein with density aggregation activity. MS/MS peptide sequence analysis identified the gene sequence, and cells that overexpress the full-length protein accumulate higher levels of a development promoting factor (DPF) activity than parental cells, allowing cells to aggregate at lower cell densities; cells deficient for this DPF gene lack density-dependent developmental aggregation activity and require higher cell density for cell aggregation compared to WT. Density aggregation activity co-purifies with tagged versions of DPF and tag-affinity-purified DPF possesses density aggregation activity. In mixed development with WT, cells that overexpress DPF preferentially localize at centers for multi-cell aggregation and define cell-fate choice during cytodifferentiation. Finally, we show that DPF is synthesized as a larger precursor, single-pass transmembrane protein, with the p150 fragment released by proteolytic cleavage and ectodomain shedding. The TM/cytoplasmic domain of DPF possesses cell-autonomous activity for cell-substratum adhesion and for cellular growth.ConclusionsWe have purified a novel secreted protein, DPF, that acts as a density-sensing factor for development and functions to define local collective thresholds for Dictyostelium development and to facilitate cell-cell communication and multi-cell formation. Regions of high DPF expression are enriched at centers for cell-cell signal-response, multi-cell formation, and cell-fate determination. Additionally, DPF has separate cell-autonomous functions for regulation of cellular adhesion and growth.

Membrane-bound Gaussia luciferase as a tool to track shedding of membrane proteins from the surface of extracellular vesicles

Extracellular vesicles (EVs) released by cells play a role in intercellular communication. Reporter and targeting proteins can be modified and exposed on the surface of EVs to investigate their half-life and biodistribution. A characterization of membrane-bound Gaussia luciferase (mbGluc) revealed that its signal was detected also in a form smaller than common EVs (<70 nm). We demonstrated that mbGluc initially exposed on the surface of EVs, likely undergoes proteolytic cleavage and processed fragments of the protein are released into the extracellular space in active form. Based on this observation, we developed a new assay to quantitatively track shedding of membrane proteins from the surface of EVs. We used this assay to show that ectodomain shedding in EVs is continuous and is mediated by specific proteases, e.g. metalloproteinases. Here, we present a novel tool to study membrane protein cleavage and release using both in vitro and in vivo models.

Trypanosoma cruzi: cell surface dynamics in trypomastigotes of different strains.

Capping and shedding of ectodomains in Trypanosoma cruzi may be triggered by different ligands. Here, we analysed the mobility and shedding of cell surface components of living trypomastigotes of the Y strain and the CL Brener clone in the presence of poly-L-lysine, cationized ferritin (CF) and Concanavalin A (Con A). Poly-L-lysine and CF caused intense shedding in Y strain parasites. Shedding was less intense in CL Brener trypomastigotes, and approximately 10% of these parasites did not show any decrease in poly L-lysine or CF labelling. Binding of Con A induced low-intensity shedding in Y strain and redistribution of Con A-binding sites in CL Brener parasites. Trypomastigotes of the Y strain showed intense labelling with anti-〈-galactosyl antibodies, resulting in the lysis of approximately 30% of their population, in contrast with what was observed in CL Brener parasites. Incubation with Con A and CF protected trypomastigotes of the Y strain from lysis by anti-αGal. The last treatment did not interfere with the survival of the CL Brener parasites. This study corroborates with the idea that a ligand can differentially modulate the cell surface of T. cruzi, depending on the strain used, resulting in variable immune system responses and recognition by host cells.

Proteases as Secreted Exoproteins in Mycoplasmas from Ruminant Lungs and Their Impact on Surface-Exposed Proteins.

Many mycoplasma species are isolated from the ruminant lungs as either saprophytes or true pathogens. These wall-less bacteria possess a minimal genome and reduced metabolic capabilities. Accordingly, they rely heavily on their hosts for the supply of essential metabolites and, notably, peptides. Seven of 13 ruminant lung-associated Mycoplasma (sub)species were shown to possess caseinolytic activity when grown in rich media and assessed with a quantitative fluorescence test. For some species, this activity was detected in spent medium, an indication that proteases were secreted outside the mycoplasma cells. To identify these proteases, we incubated concentrated washed cell pellets in a defined medium and analyzed the supernatants by tandem mass spectrometry. Secreted-protease activity was detected mostly in the species belonging to the Mycoplasma mycoides cluster (MMC) and, to a lesser extent, in Mycoplasma bovirhinis Analyzing a Mycoplasma mycoides subsp. capri strain, chosen as a model, we identified 35 expressed proteases among 55 predicted coding genes, of which 5 were preferentially found in the supernatant. Serine protease S41, acquired by horizontal gene transfer, was responsible for the caseinolytic activity, as demonstrated by zymography and mutant analysis. In an M. capricolum mutant, inactivation of the S41 protease resulted in marked modification of the expression or secretion of 17 predicted surface-exposed proteins. This is an indication that the S41 protease could have a role in posttranslational cleavage of surface-exposed proteins and ectodomain shedding, whose physiological impacts still need to be explored.IMPORTANCE Few studies pertaining to proteases in ruminant mycoplasmas have been reported. Here, we focus on proteases that are secreted outside the mycoplasma cell using a mass spectrometry approach. The most striking result is the identification, within the Mycoplasma mycoides cluster, of a serine protease that is exclusively detected outside the mycoplasma cells and is responsible for casein digestion. This protease may also be involved in the posttranslational processing of surface proteins, as suggested by analysis of mutants showing a marked reduction in the secretion of extracellular proteins. By analogy, this finding may help increase understanding of the mechanisms underlying this ectodomain shedding in other mycoplasma species. The gene encoding this protease is likely to have been acquired via horizontal gene transfer from Gram-positive bacteria and sortase-associated surface proteases. Whether this protease and the associated ectodomain shedding are related to virulence has yet to be ascertained.

Fibrinogen-like protein 2 controls sepsis catabasis by interacting with resolvin Dp5.

The mechanisms that drive programmed resolution of inflammation remain elusive. Here, we report the temporal regulation of soluble (s) and transmembrane (m) fibrinogen-like protein 2 (Fgl2) during inflammation and show that both sFgl2 and mFgl2 correlate with the outcome. The expression and ectodomain shedding of Fgl2 are respectively promoted by miR-466l and metalloproteinases (ADAM10 and ADAM17) during inflammation resolution. Deficiency of Fgl2 enhances polymorphonuclear neutrophil (PMN) infiltration but impairs macrophage (MΦ) maturation and phagocytosis and inhibits the production of n-3 docosapentaenoic acid-derived resolvin 5 (RvDp5). In contrast, administration of sFgl2 blunts PMN infiltration as well as promotes PMN apoptosis and RvDp5 biosynthesis. By activating ALX/FPR2, RvDp5 enhances sFgl2 secretion via ADAM17 and synergistically accelerates resolution of inflammation. These results uncover a previously unknown endogenous programmed mechanism by which Fgl2 regulates resolution of inflammation and shed new light on clinical sepsis treatments.

Serine Phosphorylation of L-Selectin Regulates ERM Binding, Clustering, and Monocyte Protrusion in Transendothelial Migration.

The migration of circulating leukocytes toward damaged tissue is absolutely fundamental to the inflammatory response, and transendothelial migration (TEM) describes the first cellular barrier that is breached in this process. Human CD14+ inflammatory monocytes express L-selectin, bestowing a non-canonical role in invasion during TEM. In vivo evidence supports a role for L-selectin in regulating TEM and chemotaxis, but the intracellular mechanism is poorly understood. The ezrin-radixin-moesin (ERM) proteins anchor transmembrane proteins to the cortical actin-based cytoskeleton and additionally act as signaling adaptors. During TEM, the L-selectin tail within transmigrating pseudopods interacts first with ezrin to transduce signals for protrusion, followed by moesin to drive ectodomain shedding of L-selectin to limit protrusion. Collectively, interaction of L-selectin with ezrin and moesin fine-tunes monocyte protrusive behavior in TEM. Using FLIM/FRET approaches, we show that ERM binding is absolutely required for outside-in L-selectin clustering. The cytoplasmic tail of human L-selectin contains two serine (S) residues at positions 364 and 367, and here we show that they play divergent roles in regulating ERM binding. Phospho-S364 blocks direct interaction with ERM, whereas molecular modeling suggests phospho-S367 likely drives desorption of the L-selectin tail from the inner leaflet of the plasma membrane to potentiate ERM binding. Serine-to-alanine mutagenesis of S367, but not S364, significantly reduced monocyte protrusive behavior in TEM under flow conditions. Our data propose a model whereby L-selectin tail desorption from the inner leaflet of the plasma membrane and ERM binding are two separable steps that collectively regulate protrusive behavior in TEM.

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.

ADAM15 mediates upregulation of Claudin-1 expression in breast cancer cells

A Disintegrin and Metalloproteinase-15 (ADAM15) is a transmembrane protein involved in protein ectodomain shedding, cell adhesion and signalling. We previously cloned and characterised alternatively spliced variants of ADAM15 that differ in their intracellular domains and demonstrated correlation of the expression of specific variants with breast cancer prognosis. In this study we have created isogenic cell panels (MDA-MB-231 and MCF-7) expressing five ADAM15 variants including wild-type and catalytically inactive forms. The expression of ADAM15 isoforms in MDA-MB-231 cells led to cell clustering to varying degree, without changes in EMT markers vimentin, slug and E-cadherin. Analysis of tight junction molecules revealed ADAM15 isoform specific, catalytic function dependent upregulation of Claudin-1. The expression of ADAM15A, and to a lesser degree of C and E isoforms led to an increase in Claudin-1 expression in MDA-MB-231 cells, while ADAM15B had no effect. In MCF-7 cells, ADAM15E was the principal variant inducing Claudin-1 expression. Sh-RNA mediated down-regulation of ADAM15 in ADAM15 over-expressing cells reduced Claudin-1 levels. Additionally, downregulation of endogenous ADAM15 expression in T47D cells by shRNA reduced endogenous Claudin-1 expression confirming a role for ADAM15 in regulating Claudin-1 expression. The PI3K/Akt/mTOR pathway was involved in regulating Claudin-1 expression downstream of ADAM15. Immunofluorescence analysis of MDA-MB-231 ADAM15A expressing cells showed Claudin-1 at cell-cell junctions, in the cytoplasm and nuclei. ADAM15 co-localised with Claudin-1 and ZO1 at cell-cell junctions. Immunoprecipitation analysis demonstrated complex formation between ADAM15 and ZO1/ZO2. These findings highlight the importance of ADAM15 Intra Cellular Domain-mediated interactions in regulating substrate selection and breast cancer cell phenotype.

Autoactivation and calpain-1-mediated shedding of hepsin in human hepatoma cells.

Hepsin is a transmembrane serine protease implicated in many biological processes, including hepatocyte growth, urinary protein secretion, auditory nerve development, and cancer metastasis. Zymogen activation is critical for hepsin function. To date, how hepsin is activated and regulated in cells remains an enigma. In this study, we conducted site-directed mutagenesis, cell expression, plasma membrane protein labeling, trypsin digestion, Western blotting, and flow cytometry experiments in human hepatoma HepG2 cells, where hepsin was originally discovered, and SMMC-7721 cells. Our results show that hepsin is activated by autocatalysis on the cell surface but not intracellularly. Moreover, we show that hepsin undergoes ectodomain shedding. In the conditioned medium from HepG2 and SMMC-7721 cells, we detected a soluble fragment comprising nearly the entire extracellular region of hepsin. By testing protease inhibitors, gene knockdown, and site-directed mutagenesis, we identified calpain-1 as a primary protease that acted extracellularly to cleave Tyr52 in the juxtamembrane space of hepsin. These results provide new insights into the biochemical and cellular mechanisms that regulate hepsin expression and activity.

Meprin β induces activities of A disintegrin and metalloproteinases 9, 10, and 17 by specific prodomain cleavage.

Meprin β is a membrane-bound metalloprotease involved in extracellular matrix assembly and inflammatory processes in health and disease. A disintegrin and metalloproteinase (ADAM)10 and ADAM17 are physiologic relevant sheddases of inactive promeprin β, which influences its substrate repertoire and subsequent biologic functions. Proteomic analysis also revealed several ADAMs as putative meprin β substrates. Here, we demonstrate specific N-terminal processing of ADAM9, 10, and 17 by meprin β and identify cleavage sites within their prodomains. Because ADAM prodomains can act as specific inhibitors, we postulate a role for meprin β in the regulation of ADAM activities. Indeed, prodomain cleavage by meprin β caused increased ADAM protease activities, as observed by peptide-based cleavage assays and demonstrated by increased ectodomain shedding activity. Direct interaction of meprin β and ADAM proteases could be shown by immunofluorescence microscopy and immunoprecipitation experiments. As demonstrated by a bacterial activator of meprin β and additional measurement of TNF-α shedding on bone marrow-derived macrophages, meprin β/ADAM protease interactions likely influence inflammatory conditions. Thus, we identified a novel proteolytic pathway of meprin β with ADAM proteases to control protease activities at the cell surface as part of the protease web.-Wichert, R., Scharfenberg, F., Colmorgen, C., Koudelka, T., Schwarz, J., Wetzel, S., Potempa, B., Potempa, J., Bartsch, J. W., Sagi, I., Tholey, A., Saftig, P., Rose-John, S., Becker-Pauly, C. Meprin β induces activities of A disintegrin and metalloproteinases 9, 10, and 17 by specific prodomain cleavage.