Sheddase Activity Research Articles

Extracellular sphingomyelinase activity impairs TNF-α-induced endothelial cell death via ADAM17 activation and TNF receptor 1 shedding.

ADAM17, a prominent member of the “Disintegrin and Metalloproteinase” (ADAM) family, is an important regulator of endothelial cell proliferation and cell survival. The protease controls vital cellular functions through cleavage of growth factors, cytokines and their receptors including transforming growth factor-alpha (TGF-α), tumor necrosis factor-alpha (TNF-α) and TNF-α receptor 1 (TNFR1). TNF-α is the major inducer of endothelial cell death in cardiovascular diseases. The latter are also characterized by elevated plasma and tissue levels of extracellular sphingomyelinase (SMase). Whether the SMase affects ADAM activity and thus endothelial cell function has not been addressed to date. Here, we analyzed the effect of SMase on ADAM17-mediated shedding in COS7 cells and in human umbilical vein endothelial cells (HUVECs). Exposure to SMase significantly increased ADAM17-mediated release of alkaline-phosphatase (AP)-tagged TGF-α in COS7 cells and shedding of endogenously expressed TNFR1 in HUVECs. We previously presented evidence that surface exposure of phosphatidylserine (PS) is pivotal for ADAM17 to exert sheddase function. We found that SMase treatment led to PS externalization in both cell types. Transient non-apoptotic PS exposure is often mediated by Ca2+-dependent phospholipid scramblases. Accordingly, the Ca2+-chelator EGTA markedly reduced the breakdown of phospholipid asymmetry and shedding of TGF-α and TNFR1. Moreover, sheddase activity was significantly diminished in the presence of the competing PS-headgroup OPLS. SMase-stimulated TNFR1 shedding strikingly diminished TNF-α-induced signalling cascades and endothelial cell death. Taken together, our data suggest that SMase activity might act as protective factor for endothelial cells in cardiovascular diseases.

Inhibition of Shedding of Low-Density Lipoprotein Receptor-Related Protein 1 Reverses Cartilage Matrix Degradation in Osteoarthritis.

ObjectiveThe aggrecanase ADAMTS‐5 and the collagenase matrix metalloproteinase 13 (MMP‐13) are constitutively secreted by chondrocytes in normal cartilage, but rapidly endocytosed via the cell surface endocytic receptor low‐density lipoprotein receptor–related protein 1 (LRP‐1) and subsequently degraded. This endocytic system is impaired in osteoarthritic (OA) cartilage due to increased ectodomain shedding of LRP‐1. The aim of this study was to identify the LRP‐1 sheddase(s) in human cartilage and to test whether inhibition of LRP‐1 shedding prevents cartilage degradation in OA.MethodsCell‐associated LRP‐1 and soluble LRP‐1 (sLRP‐1) released from human cartilage explants and chondrocytes were measured by Western blot analysis. LRP‐1 sheddases were identified by proteinase inhibitor profiling and gene silencing with small interfering RNAs. Specific monoclonal antibodies were used to selectively inhibit the sheddases. Degradation of aggrecan and collagen in human OA cartilage was measured by Western blot analysis using an antibody against an aggrecan neoepitope and a hydroxyproline assay, respectively.ResultsShedding of LRP‐1 was increased in OA cartilage compared with normal tissue. Shed sLRP‐1 bound to ADAMTS‐5 and MMP‐13 and prevented their endocytosis without interfering with their proteolytic activities. Two membrane‐bound metalloproteinases, ADAM‐17 and MMP‐14, were identified as the LRP‐1 sheddases in cartilage. Inhibition of their activities restored the endocytic capacity of chondrocytes and reduced degradation of aggrecan and collagen in OA cartilage.ConclusionShedding of LRP‐1 is a key link to OA progression. Local inhibition of LRP‐1 sheddase activities of ADAM‐17 and MMP‐14 is a unique way to reverse matrix degradation in OA cartilage and could be effective as a therapeutic approach.

Ectodomain Shedding by ADAM17: Its Role in Neutrophil Recruitment and the Impairment of This Process during Sepsis.

Neutrophils are specialized at killing bacteria and are recruited from the blood in a rapid and robust manner during infection. A cascade of adhesion events direct their attachment to the vascular endothelium and migration into the underlying tissue. A disintegrin and metalloproteinase 17 (ADAM17) functions in the cell membrane of neutrophils and endothelial cells by cleaving its substrates, typically in a cis manner, at an extracellular site proximal to the cell membrane. This process is referred to as ectodomain shedding and it results in the downregulation of various adhesion molecules and receptors, and the release of immune regulating factors. ADAM17 sheddase activity is induced upon cell activation and rapidly modulates intravascular adhesion events in response to diverse environmental stimuli. During sepsis, an excessive systemic inflammatory response against infection, neutrophil migration becomes severely impaired. This involves ADAM17 as indicated by increased levels of its cleaved substrates in the blood of septic patients, and that ADAM17 inactivation improves neutrophil recruitment and bacterial clearance in animal models of sepsis. Excessive ADAM17 sheddase activity during sepsis thus appears to undermine in a direct and indirect manner the necessary balance between intravascular adhesion and de-adhesion events that regulate neutrophil migration into sites of infection. This review provides an overview of ADAM17 function and regulation and its potential contribution to neutrophil dysfunction during sepsis.

Modification of proteolytic activity matrix analysis (PrAMA) to measure ADAM10 and ADAM17 sheddase activities in cell and tissue lysates.

Increases in expression of ADAM10 and ADAM17 genes and proteins have been evaluated, but not validated as cancer biomarkers. Specific enzyme activities better reflect enzyme cellular functions, and might be better biomarkers than enzyme genes or proteins. However, no high throughput assay is available to test this possibility. Recent studies have developed the high throughput real-time proteolytic activity matrix analysis (PrAMA) that integrates the enzymatic processing of multiple enzyme substrates with mathematical-modeling computation. The original PrAMA measures with significant accuracy the activities of individual metalloproteinases expressed on live cells. To make the biomarker assay usable in clinical practice, we modified PrAMA by testing enzymatic activities in cell and tissue lysates supplemented with broad-spectrum non-MP enzyme inhibitors, and by maximizing the assay specificity using systematic mathematical-modeling analyses. The modified PrAMA accurately measured the absence and decreases of ADAM10 sheddase activity (ADAM10sa) and ADAM17sa in ADAM10-/- and ADAM17-/- mouse embryonic fibroblasts (MEFs), and ADAM10- and ADAM17-siRNA transfected human cancer cells, respectively. It also measured the restoration and inhibition of ADAM10sa in ADAM10-cDNA-transfected ADAM10-/- MEFs and GI254023X-treated human cancer cell and tissue lysates, respectively. Additionally, the modified PrAMA simultaneously quantified with significant accuracy ADAM10sa and ADAM17sa in multiple human tumor specimens, and showed the essential characteristics of a robust high throughput multiplex assay that could be broadly used in biomarker studies. Selectively measuring specific enzyme activities, this new clinically applicable assay is potentially superior to the standard protein- and gene-expression assays that do not distinguish active and inactive enzyme forms.

Targeting ADAM17 Sheddase Activity in Cancer.

A disintegrin and metalloprotease (ADAM)17 is a sheddase, capable of releasing the ectodomains of membrane proteins such as growth factors (e.g. Epidermal Growth Factor Receptor ligands), cytokines and their receptors, adhesion and signaling molecules. These activities regulate several physiological and pathological processes including inflammation, tumor growth and metastatic progression. In this review, we will summarize ADAM17 biology and focus on its role in cancer and the possible usage of ADAM17 inhibitors in cancer therapy. Recent achievements in this area include the development of small molecule metalloprotease inhibitors with enhanced specificity for ADAM17, monoclonal antibodies, and synthetic short RNA molecules for gene silencing. These approaches successfully inhibited cancer cell growth and invasiveness or sensitized them to cytotoxic drugs, ionizing radiations or targeted therapies, in preclinical studies. These findings suggest the repositioning of ADAM17 inhibitors, which have yet proven unsuccessful as anti-inflammatory agents, for the development of new anti-cancer therapies, particularly in EGFR ligand-dependent cancers. Future studies should address ADAM17 inhibitors as short-term treatments in combination with different anti-cancer therapies.

Inhibiting GPIbα Shedding Preserves Post-Transfusion Recovery and Hemostatic Function of Platelets After Prolonged Storage.

The platelet storage lesion accelerates platelet clearance after transfusion, but the underlying molecular mechanism remains elusive. Although inhibiting sheddase activity hampers clearance of platelets with storage lesion, the target platelet protein responsible for ectodomain shedding-induced clearance is not definitively identified. Monoclonal antibody 5G6 was developed recently to bind specifically human platelet receptor glycoprotein (GP)Ibα and inhibit its shedding but not shedding of other receptors. Here, the role of GPIbα shedding in platelet clearance after transfusion was addressed. Both human leukoreduced apheresis-derived platelets and transgenic mouse platelets expressing human GPIbα were stored at room temperature in the presence and absence of 5G6 Fab fragment. At various time points, aliquots of stored platelets were analyzed and compared. 5G6 Fab inhibited GPIbα shedding in both platelets during storage and preserved higher level of GPIbα on the platelet surface. Compared with age-matched control platelets, 5G6 Fab-stored platelets exhibited similar levels of platelet activation, degranulation, and agonist-induced aggregation. 5G6 Fab-stored human GPIbα platelets exhibited significantly higher post-transfusion recovery and in vivo hemostatic function in recipient mice than control platelets. Consistently, 5G6 Fab-stored, 8-day-old human platelets produced similar improvement in post-transfusion recovery in immunodeficient mice and in ex vivo thrombus formation over collagen under shear flow. Specific inhibition of GPIbα shedding in the stored platelets improves post-transfusion platelet recovery and hemostatic function, providing clear evidence for GPIbα shedding as a cause of platelet clearance. These results suggest that specific inhibition of GPIbα shedding may be used to optimize platelet storage conditions.

Serum activity of angiotensin converting enzyme 2 is decreased in patients with acute ischemic stroke.

Levels of angiotensin converting enzyme 2 (ACE2), a cardio and neuro-protective carboxypeptidase, are dynamically altered after stroke in preclinical models. We sought to characterize the previously unexplored changes in serum ACE2 activity of stroke patients and the mechanism of these changes. Serum samples were obtained from patients during acute ischemic stroke (n=39), conditions mimicking stroke (stroke-alert, n=23), or from control participants (n=20). Enzyme activity levels were analyzed by fluorometric assay and correlated with clinical variables by regression analyses. Serum ACE2 activity was significantly lower in acute ischemic stroke as compared to both control and stroke-alert patients, followed by an increase to control levels at three days. Serum ACE2 activity significantly correlated with the presence of ischemic stroke after controlling for other factors (P=0.01). Additional associations with ACE2 activity included a positive correlation with systolic blood pressure at presentation in stroke-alert (R2=0.24, P=0.03), while stroke levels showed no correlation (R2=0.01, P=0.50). ACE2 sheddase activity was unchanged between groups. These dynamic changes in serum ACE2 activity in stroke, which concur with preclinical studies, are not likely to be driven primarily by acute changes in blood pressure or sheddase activity. These findings provide new insight for developing therapies targeting this protective system in ischemic stroke.

Phosphatidylserine exposure is required for ADAM17 sheddase function.

ADAM17, a prominent member of the ‘Disintegrin and Metalloproteinase' (ADAM) family, controls vital cellular functions through cleavage of transmembrane substrates. Here we present evidence that surface exposure of phosphatidylserine (PS) is pivotal for ADAM17 to exert sheddase activity. PS exposure is tightly coupled to substrate shedding provoked by diverse ADAM17 activators. PS dependency is demonstrated in the following: (a) in Raji cells undergoing apoptosis; (b) in mutant PSA-3 cells with manipulatable PS content; and (c) in Scott syndrome lymphocytes genetically defunct in their capacity to externalize PS in response to intracellular Ca2+ elevation. Soluble phosphorylserine but not phosphorylcholine inhibits substrate cleavage. The isolated membrane proximal domain (MPD) of ADAM17 binds to PS but not to phosphatidylcholine liposomes. A cationic PS-binding motif is identified in this domain, replacement of which abrogates liposome-binding and renders the protease incapable of cleaving its substrates in cells. We speculate that surface-exposed PS directs the protease to its targets where it then executes its shedding function.

Role of matrix metalloproteases in the kinetics of leukocyte-endothelial adhesion in post-capillary venules.

The endothelial glycocalyx serves as a barrier to leukocyte (WBC)-endothelium (EC) adhesion. Shedding of glycans, by matrix metalloproteases (MMPs) exposes EC integrin receptors to facilitate firm adhesion. However, the effect of shedding on the strength of the adhesive bond remains to be determined. Examine the effect of MMP inhibition on the kinetics of WBC-EC adhesion under normal and inflammatory conditions to delineate differences in the duration and number of adhesive bonds. WBC adhesion in post-capillary venules was observed in rat mesentery. Adhesion duration and off-rates (KOFF) were correlated with shear stress during adhesion in response to 1 µM fMLP or 0.5 µM doxycycline (doxy, to inhibit MMP activation). Doxy increased mean adhesion time significantly from 2.5 (control) to 5.6 s, whereas fMLP increased it 8-fold to 20 s, which was not affected by pre-treatment with doxy. Estimates of the number of adhesive bonds (simplified Bell-model) revealed a significantly greater increase with fMLP compared to doxy alone, with no effect on fMLP by pretreatment with doxy. With doxy alone, KOFF was significantly 4-fold greater compared to fMLP, suggesting a much weaker bond. Although the increased number of bonds by MMP inhibition with doxy alone and fMLP were similar, the bonds due to doxy appeared weaker as evidenced by their shorter duration, and lesser reduction in KOFF relative to control. Thus doxy limits the availability of integrin binding sites during fMLP stimulated adhesion, but has a pro-adhesive effect due to increased ligands for WBC binding that arises from inhibition of normal sheddase activity on the EC.

The relationship of miR-122-5p with trastuzumab therapy in breast cancer.

e11593 Background: Recently, microRNA-122’s functions in breast cancer (BC) remain unknown. HER2 receptors are known cleaved by an ectodomain sheddase, ADAM10, to liberate HER2 extracellular domain (ECD) in most metastatic cases. So, trastuzumab cannot inactivate HER2 by binding ECD portion of it. Our aim is to suppress HER2 sheddase activity with miR-122-5p that selectively inhibits ADAM10 expression and increase activity of trastuzumab. Also, we aimed to analyze advancing of apoptosis by miR-122-5p together with Trastuzumab in SKBR3 (HER2+) cell line. Methods: MiR-122-5p and ADAM10 expressions were measured in breast cancer cell lines (CRL-2329,MDA-MB-231,CRL-1500,MCF-7,SK-BR-3) and in CRL-4010 (control) by Real-time PCR. MiR-122-5p and ADAM10 expressions were analyzed in 71 breast cancer patients’ tumor and normal tissues. Also, SKBR3 cells (HER2+) were transfected with miR-122-5p mimic, inhibitor and miRNA negative control and trastuzumab was administered to miRNA transfected and non-transfected SKBR3...

A pathogenic trace of Tannerella forsythia - shedding of soluble fully active tumor necrosis factor α from the macrophage surface by karilysin.

Tannerella forsythia is implicated as a pathogen causing chronic and aggressive periodontitis. However, its virulence factors, including numerous putative proteases, are mostly uncharacterized. Karilysin is a newly described matrix metalloprotease-like enzyme of T. forsythia. Since pathogen-derived proteases may affect the host defense system via modulation of the cytokine network, the aim of this study was to determine the influence of karilysin on tumor necrosis factor-α (TNF-α). The results showed that karilysin cleaved the membrane form of TNF-α on the surface of macrophages, and that this led to an increased concentration of soluble TNF-α in the conditioned medium. Importantly, despite partial degradation of soluble TNF-α by karilysin, the released cytokine retained its biological activity, inducing apoptosis and stimulating autocrine pathway of pro-inflammatory gene expression. Notably, the observed effect required proteolytic activity by karilysin, since a catalytically inactive mutant of the enzyme did not affect TNF-α secretion. The shedding was independent of the activity of ADAM17, a major endogenous TNF-α converting enzyme. Karilysin-dependent TNF-α release from the cell surface is likely to occur in vivo because human plasma, the main constituent of gingival crevicular fluid, only slightly affected the sheddase activity of karilysin. Taken together, these results indicate that karilysin modulates the host immune response through regulation of TNF-α secretion, and should therefore be considered as a new virulence factor of T. forsythia.

Tumor necrosis factor-α-induced a disintegrin and metalloprotease 10 increases apoptosis resistance in prostate cancer cells

In developed countries, prostate cancer (PCa) is the second most frequently diagnosed type of cancer and the third most common cause of cancer-related mortality in males. Compared with western countries, the morbidity rate of PCa in China is markedly lower, however, it is rising annually. The etiology of PCa is unclear, therefore, to investigate how a disintegrin and metalloprotease 10 (ADAM10) functions in PCa, ADAM10 mRNA and protein levels induced by tumor necrosis factor (TNF)-α were identified using polymerase chain reaction and flow cytometry, respectively. To investigate the mechanism of ADAM10 activity in PCa, specific inhibitors were used, and DNA transfection and RNA interference technology were employed to identify the interaction between ADAM10 and the Fas ligand (L). The results indicated that TNF-α induced ADAM10 expression in a time- and dose-dependent manner through the p38 mitogen-activated protein kinase/necrosis factor-κB signaling pathway. ADAM10 hydrolyzed FasL and contributed to apoptosis resistance of the tumor cells. These observations indicate a promising therapeutic modality for the treatment of apoptosis-resistant PCa, by targeting ADAM10 sheddase activity.

Stress immunity in lymphomas: mesenchymal cells as a target of therapy

The role of lymphocytes in eliminating lymphoma cells is based on the interaction between activating receptors on lymphocytes and target surface ligands on lymphoma cells. Stress-related immunity can be triggered both in Hodgkin's (HL) and non-Hodgkin lymphomas (NHL), through the activation of the NKG2D receptor on CD8+ T and gammadelta T lymphocytes, by NKG2D-ligands (NKG2D-L), as the MHC class-I related molecules MIC-A/B and the UL16-binding proteins 1-4 (ULBPs), expressed on lymphoma cells. Furthermore, NKG2D-L can be shed and interact with NKG2D on effector lymphocytes affecting the recognition of lymphoma cells. Proteolytic cleavage of MIC-A is known to depend on the thiol isomerase ERp5 and the disintegrins and metalloproteinases ADAM10 and ADAM17, which also cleave ULPBs. Mesenchymal stromal cells (MSC) are relevant in regulating effector T lymphocytes-mediated lymphoma surveillance. Indeed, MSC can be seen as targets of potential new therapeutic schemes acting on lymphoma microenvironment, to redirect the stress immune response and avoid escape strategies, by inducing stress molecules, inhibiting sheddase activity, shifting cytokine production to Th1 pattern and blocking Treg differentiation.

Targeting the sheddase activity of ADAM17 by an anti-ADAM17 antibody D1(A12) inhibits head and neck squamous cell carcinoma cell proliferation and motility via blockage of bradykinin induced HERs transactivation.

A disintegrin and metalloproteinase 17 (ADAM17) regulates key cellular processes including proliferation and migration through the shedding of a diverse array of substrates such as epidermal growth factor receptor (EGFR) ligands. ADAM17 is implicated in the pathogenesis of many diseases including rheumatoid arthritis and cancers such as head and neck squamous cell carcinoma (HNSCC). As a central mediator of cellular events, overexpressed EGFR is a validated molecular target in HNSCC. However, EGFR inhibition constantly leads to tumour resistance. One possible mechanism of resistance is the activation of alternative EGFR family receptors and downstream pathways via the release of their ligands. Here, we report that treating human HNSCC cells in vitro with a human anti-ADAM17 inhibitory antibody, D1(A12), suppresses proliferation and motility in the absence or presence of the EGFR tyrosine kinase inhibitor (TKI) gefitinib. Treatment with D1(A12) decreases both the endogenous and the bradykinin (BK)-stimulated shedding of HER ligands, accompanied by a reduction in the phosphorylation of HER receptors and downstream signalling pathways including STAT3, AKT and ERK. Knockdown of ADAM17, but not ADAM10, also suppresses HNSCC cell proliferation and migration. Furthermore, we show that heregulin (HRG) and heparin-binding epidermal growth factor like growth factor (HB-EGF) predominantly participate in proliferation and migration, respectively. Taken together, these results demonstrate that D1(A12)-mediated inhibition of cell proliferation, motility, phosphorylation of HER receptors and downstream signalling is achieved via reduced shedding of ADAM17 ligands. These findings underscore the importance of ADAM17 and suggest that D1(A12) might be an effective targeted agent for treating EGFR TKI-resistant HNSCC.

Hepatocyte Growth Factor Activator Inhibitor Type 1 Is a Suppressor of Intestinal Tumorigenesis

Hepatocyte growth factor activator inhibitor type 1 (HAI-1/SPINT1) is a membrane-bound serine protease inhibitor expressed on the surface of epithelial cells. Although HAI-1/SPINT1 is abundantly expressed in the intestinal epithelium, its role in intestinal tumorigenesis is not known. In this study, we investigated the role of Hai-1/Spint1 in intestinal tumorigenesis using mouse models. The membranous Hai-1/Spint1 immunoreactivity was decreased in murine Apc(Min/+) tumors and also in carcinogen (azoxymethane treatment followed by dextran sodium sulfate administration)-induced colon tumors compared with the adjacent non-neoplastic epithelium. The decreased immunoreactivity appeared to be due to sheddase activity of membrane-type 1 matrix metalloprotease. Then, we examined the effect of intestine-specific deletion of Spint1 gene on Apc(Min/+) mice. The loss of Hai-1/Spint1 significantly accelerated tumor formation in Apc(Min/+) mice and shortened their survival periods. Activation of HGF was enhanced in Hai-1/Spint1-deficient Apc(Min/+) intestine. Gene expression profiling revealed upregulation of the Wnt/β-catenin signaling circuit, claudin-2 expression, and angiogenesis not only in tumor tissue but also in the background mucosa without macroscopic tumors in Hai-1/Spint1-deficient Apc(Min/+) intestine. Intestinal deletion of Spint1 also enhanced the susceptibility to carcinogen-induced colon tumorigenicity of wild-type Apc mice. Our findings suggest that HAI-1/SPINT1 has a crucial role in suppressing intestinal tumorigenesis, which implies a novel link between epithelial cell surface serine protease inhibitors and protection from carcinogenic stimuli.

Pancreatic ACE2 shedding is associated with impaired glycemia in high fat diet‐fed mice.

Angiotensin converting enzyme 2 (ACE2) activity decreases with the progression of type 2 diabetes and restoration of ACE2 appears to be beneficial in this disease. We hypothesized that shedding of pancreatic ACE2 might be associated with impaired glycemia in high‐fat diet (HFD)‐fed mice. Pancreatic ACE2 and ADAM17 (a sheddase) activities (FU/min/μg protein) were assessed in 6‐month old C57BL/6J mice (N=5/group) fed with HFD or regular diet (RD) for 16 weeks. HFD‐fed mice showed lower pancreatic ACE2 activity (144 ±17 vs. 218 ±20; P<0.05) associated with higher ADAM17 activity (60.5 ±0.4 vs. 53.1 ±1.8; P<0.05) compared to RD mice. To restore ACE2 activity, we treated HFD‐fed mice with a lentivirus coding for mACE2 or GFP (pancreas injection, 10^6 TU/ml in 100 μl saline) at the 13th week of HFD‐feeding. Three weeks later, ACE2‐treated mice not only exhibited higher pancreatic ACE2 (225.4 ±18.3) and reduced ADAM17 (55.5 ±1.4) activity but also reduced Ang‐II levels (2.1 ±0.3 vs. 3.4 ±0.2 ng/g of pancreas) and improved glucose tolerance (AUC= 46153 ±1646 vs. 56045 ±2552 mg/dl*min.) versus GFP‐injected mice (P<0.05). Altogether, our data suggest that pancreatic ACE2 shedding might be a possible mechanism contributing to impaired glycemia in HFD‐fed mice. Hence, restoration of ACE2 could be a promising therapeutic target for managing HFD‐induced type 2 diabetes.

Adipose tissue artery‐derived TNF impairs dilation of skeletal muscle resistance arteries in obesity

Despite its critical importance, factors responsible for altering the vasomotor behaviour of resistance arteries in obesity is poorly understood. We tested the hypothesis that soluble inflammatory mediators are released from arteries of adipose tissue (ATA), which act remotely on skeletal muscle resistance arteries (SMRA) to interfere with their dilator function. ATA and SMRA were dissected from the heart and gracilis muscle of lean or obese mice (60% of fat feeding, 3‐month), and changes in diameter was measured in pressurized (80 mmHg) arteries with video‐microscopy. The endothelium‐dependent dilation to acetylcholine (ACh) was significantly reduced in SMRA of obese mice. When SMRA from lean mice were serially connected to ATA from obese mice with a glass capillary, we found that dilation of SMRA to ACh was greatly reduced. When the ATA from obese mice were pre‐incubated with TIMP‐3, an inhibitor of ADAM‐17, ACh‐induced dilation of SMRA was significantly enhanced. Similarly, preservation of dilation was observed when ATA and SMRA were pre‐incubated with a TNF blocking antibody. Taken together, we identified ATA as the direct source of TNF, which via ADAM‐17‐dependent sheddase activity manner is released from ATA to interfere with dilation of skeletal muscle resistance arteries in obesity.

Copper Modulates Zinc Metalloproteinase-Dependent Ectodomain Shedding of Key Signaling and Adhesion Proteins and Promotes the Invasion of Prostate Cancer Epithelial Cells

A disintegrin and metalloproteinases (ADAMs) and matrix metalloproteinases (MMPs) are zinc metalloproteinases (ZMPs) that catalyze the "ectodomain shedding" of a range of cell surface proteins including signaling and adhesion molecules. These "sheddases" are associated with the invasion and metastasis of a range of cancers. Increased serum and tumor tissue levels of copper are also observed in several cancers, although little is known about how the metal might promote disease progression at the molecular level. In the current study, we investigated whether copper might regulate the ectodomain shedding of two key cell surface proteins implicated in the invasion and metastasis of prostate cancer, the Notch ligand Jagged1 and the adhesion molecule E-cadherin, and whether the metal was able to influence the invasion of the prostate cancer epithelial cell line PC3. Physiological copper concentrations stimulated the ZMP-mediated proteolysis of Jagged1 and E-cadherin in cell culture models, whereas other divalent metals had no effect. Copper-mediated Jagged1 proteolysis was also observed following the pretreatment of cells with cycloheximide and in a cell-free membrane system, indicating a posttranslational mechanism of sheddase activation. Finally, the concentrations of copper that stimulated ZMP-mediated protein shedding also enhanced PC3 invasion; an effect that could be negated using a sheddase inhibitor or copper chelators. Collectively, these data implicate copper as an important factor in promoting prostate cancer cell invasion and indicate that the selective posttranslational activation of ZMP-mediated protein shedding might play a role in this process.

ALCAM/CD166 adhesive function is regulated by the tetraspanin CD9.

ALCAM/CD166 is a member of the immunoglobulin superfamily of cell adhesion molecules (Ig-CAMs) which mediates intercellular adhesion through either homophilic (ALCAM-ALCAM) or heterophilic (ALCAM-CD6) interactions. ALCAM-mediated adhesion is crucial in different physiological and pathological phenomena, with particular relevance in leukocyte extravasation, stabilization of the immunological synapse, T cell activation and proliferation and tumor growth and metastasis. Although the functional implications of ALCAM in these processes is well established, the mechanisms regulating its adhesive capacity remain obscure. Using confocal microscopy colocalization, and biochemical and functional analyses, we found that ALCAM directly associates with the tetraspanin CD9 on the leukocyte surface in protein complexes that also include the metalloproteinase ADAM17/TACE. The functional relevance of these interactions is evidenced by the CD9-induced upregulation of both homophilic and heterophilic ALCAM interactions, as reflected by increased ALCAM-mediated cell adhesion and T cell migration, activation and proliferation. The enhancement of ALCAM function induced by CD9 is mediated by a dual mechanism involving (1) augmented clustering of ALCAM molecules, and (2) upregulation of ALCAM surface expression due to inhibition of ADAM17 sheddase activity.

Cellular Prion Protein Regulates Its Own α-Cleavage through ADAM8 in Skeletal Muscle

The ubiquitously expressed cellular prion protein (PrP(C)) is subjected to the physiological α-cleavage at a region critical for both PrP toxicity and the conversion of PrP(C) to its pathogenic prion form (PrP(Sc)), generating the C1 and N1 fragments. The C1 fragment can activate caspase 3 while the N1 fragment is neuroprotective. Recent articles indicate that ADAM10, ADAM17, and ADAM9 may not play a prominent role in the α-cleavage of PrP(C) as previously thought, raising questions on the identity of the responsible protease(s). Here we show that, ADAM8 can directly cleave PrP to generate C1 in vitro and PrP C1/full-length ratio is greatly decreased in the skeletal muscles of ADAM8 knock-out mice; in addition, the PrP C1/full-length ratio is linearly correlated with ADAM8 protein level in myoblast cell line C2C12 and in skeletal muscle tissues of transgenic mice. These results indicate that ADAM8 is the primary protease responsible for the α-cleavage of PrP(C) in muscle cells. Moreover, we found that overexpression of PrP(C) led to up-regulation of ADAM8, suggesting that PrP(C) may regulate its own α-cleavage through modulating ADAM8 activity.