ADAM8 expression in breast cancer derived brain metastases: Functional implications on MMP-9 expression and transendothelial migration in breast cancer cells.

Metastatic breast cancer affects long-term survival and is a major cause of cancer death for women worldwide. The Metalloprotease-Disintegrin ADAM8 promotes breast cancer development and brain metastasis in a mouse breast cancer model. Here, abundant ADAM8 expression was detected in primary human breast tumors and associated brain metastases. To investigate the function of ADAM8 in metastasis, MB-231 breast cancer cells with ADAM8 knockdown (MB-231_shA8) and scramble control cells (MB-231_shCtrl) were analyzed for their capability to develop metastases. In vitro, formation of metastatic complexes in hanging drops is dependent on ADAM8 and blocked by ADAM8 inhibition. MB-231_shA8 in contrast to MB-231_shCtrl cells were impaired in transmigration through an endothelial and a reconstituted blood-brain barrier. Out of 23 MMP and 22 ADAM genes, only the MMP-9 gene was affected by ADAM8 knockdown in MB-231_shA8 cells. Following re-expression of wild-type ADAM8 in contrast to ADAM8 lacking the cytoplasmic domain in MB-231_shA8 cells caused increased levels of activated pERK1/2 and pCREB (S133) that were associated with elevated MMP-9 transcription. Application of ADAM8 and MMP-9 antibodies reduced transmigration of MB-231 cells suggesting that ADAM8 affects transmigration of breast cancer cells by MMP-9 regulation. ADAM8-dependent transmigration was confirmed in Hs578t cells overexpressing ADAM8. Moreover, transmigration of MB-231 and Hs578t cells was significantly reduced for cells treated with an antibody directed against P-selectin glycoprotein ligand (PSGL-1), a substrate of ADAM8. From these data we conclude that ADAM8 promotes early metastatic processes such as transendothelial migration by upregulation of MMP-9 and shedding of PSGL-1 from breast cancer cells.

The metalloproteinase ADAM8 promotes leukocyte recruitment in vitro and in acute lung inflammation.

Alveolar leukocyte recruitment is a hallmark of acute lung inflammation and involves transmigration of leukocytes through endothelial and epithelial layers. The disintegrin and metalloproteinase (ADAM) 8 is expressed on human isolated leukocytic cells and can be further upregulated on cultured endothelial and epithelial cells by proinflammatory cytokines. By shRNA-mediated knockdown we show that leukocytic ADAM8 is required on monocytic THP-1 cells for chemokine-induced chemotaxis as well as transendothelial and transepithelial migration. Furthermore, ADAM8 promotes αL-integrin upregulation and THP-1 cell adhesion to endothelial cells. On endothelial cells ADAM8 enhances transendothelial migration and increases cytokine-induced permeability. On epithelial cells the protease facilitates migration in a wound closure assay but does not affect transepithelial leukocyte migration. Blood leukocytes and bone marrow-derived macrophages (BMDM) from ADAM8-deficient mice show suppressed chemotactic response. Intranasal application of LPS to mice is accompanied with ADAM8 upregulation in the lung. In this model of acute lung inflammation ADAM8-deficient mice are protected against leukocyte infiltration. Finally, transfer experiments of BMDM in mice indicate that ADAM8 exerts a promigratory function predominantly on leukocytes. Our study provides in vitro and in vivo evidence that ADAM8 on leukocytes holds a proinflammatory function in acute lung inflammation by promoting alveolar leukocyte recruitment.

Stimulated release and functional activity of surface expressed metalloproteinase ADAM17 in exosomes.

By mediating proteolytic shedding on the cell surface the disintegrin and metalloproteinases ADAM10 and ADAM17 function as critical regulators of growth factors, cytokines and adhesion molecules. We here report that stimulation of lung epithelial A549 tumor cells with phorbol-12-myristate-13-acetate (PMA) leads to the downregulation of the surface expressed mature form of ADAM17 without affecting ADAM10 expression. This reduction could not be sufficiently explained by metalloproteinase-mediated degradation, dynamin-mediated internalization or microdomain redistribution of ADAM17. Instead, surface downregulation of ADAM17 was correlated with the presence of its mature form in exosomes. Exosomal ADAM17 release was also observed in monocytic and primary endothelial cells where it could be induced by stimulation with lipopolysaccharide. Antibody-mediated surface labelling of ADAM17 revealed that at least part of exosomal ADAM17 was oriented with the metalloproteinase domain outside and had been expressed on the cell surface. Suppression of iRHOM2-mediated ADAM17 maturation prevented surface expression and exosomal release of ADAM17. Further, deletion of the protease's C-terminus or cell treatment with a calcium chelator diminished exosomal release as well as surface downregulation of ADAM17, underlining that both processes are closely associated. Co-incubation of ADAM17 containing exosomes with cells expressing the ADAM17 substrates TGFα or amphiregulin lead to increased shedding of both substrates. This was prevented when exosomes were prepared from cells with shRNA-mediated ADAM17 knockdown. These data indicate that cell stimulation can downregulate expression of mature ADAM17 from the cell surface and induce release of exosomal ADAM17, which can then distribute and contribute to substrate shedding on more distant cells.

Cell surface syndecan-1 contributes to binding and function of macrophage migration inhibitory factor (MIF) on epithelial tumor cells.

Surface expressed proteoglycans mediate the binding of cytokines and chemokines to the cell surface and promote migration of various tumor cell types including epithelial tumor cells. We here demonstrate that binding of the chemokine-like inflammatory cytokine macrophage migration inhibitory factor (MIF) to epithelial lung and breast tumor cell lines A549 and MDA-MB231 is sensitive to enzymatic digestion of heparan sulphate chains and competitive inhibition with heparin. Moreover, MIF interaction with heparin was confirmed by chromatography and a structural comparison indicated a possible heparin binding site. These results suggested that proteoglycans carrying heparan sulphate chains are involved in MIF binding. Using shRNA-mediated gene silencing, we identified syndecan-1 as the predominant proteoglycan required for the interaction with MIF. MIF binding was decreased by induction of proteolytic shedding of syndecan-1, which could be prevented by inhibition of the metalloproteinases involved in this process. Finally, MIF induced the chemotactic migration of A549 cells, wound closure and invasion into matrigel without affecting cell proliferation. These MIF-induced responses were abrogated by heparin or by silencing of syndecan-1. Thus, our study indicates that syndecan-1 on epithelial tumor cells promotes MIF binding and MIF-mediated cell migration. This may represent a relevant mechanism through which MIF enhances tumor cell motility and metastasis.

A cytoplasmic C-terminal fragment of Syndecan-1 is generated by sequential proteolysis and antagonizes Syndecan-1 dependent lung tumor cell migration.

Syndecan-1 is a surface expressed heparan sulphate proteoglycan, which is upregulated by several tumor types and involved in tumor cell migration and metastasis. Syndecan-1 is shed from the cell surface and the remaining transmembrane fragment undergoes intramembrane proteolysis by γ-secretase. We here show that this generates a cytoplasmic C-terminal fragment (cCTF). In epithelial lung tumor A549 cells the endogenously produced cCTF accumulated when its proteasomal degradation was blocked with bortezomib and this accumulation was prevented by γ-secretase inhibition. Overexpression of the cCTF suppressed migration and invasion of A549 cells. This inhibitory effect was only seen when endogenous Syndecan-1 was present, but not in Syndecan-1 deficient cells. Further, overexpression of Syndecan-1 cCTF increased the basal activation of Src kinase, focal adhesion kinase (FAK) and Rho GTPase. This was associated with increased adhesion to fibronectin and collagen G and an increased recruitment of paxillin to focal adhesions. Moreover, lung tumor formation of A549 cells in mice was reduced by overexpression of Syndecan-1 cCTF. Finally, delivery of a synthetic peptide corresponding to the Syndecan-1 cCTF suppressed A549 cell migration and increased basal phosphorylation of Src and FAK. Our data indicate that the Syndecan-1 cCTF antagonizes Syndecan-1 dependent tumor cell migration in vitro and in vivo by dysregulating proadhesive signaling pathways and suggest that the cCTF can be used as an inhibitory peptide.

A transmembrane C-terminal fragment of syndecan-1 is generated by the metalloproteinase ADAM17 and promotes lung epithelial tumor cell migration and lung metastasis formation.

Syndecan-1 is a heparan sulfate proteoglycan expressed by endothelial and epithelial cells and involved in wound healing and tumor growth. Surface-expressed syndecan-1 undergoes proteolytic shedding leading to the release of the soluble N-terminal ectodomain from a transmembrane C-terminal fragment (tCTF). We show that the disintegrin and metalloproteinase (ADAM) 17 generates a syndecan-1 tCTF, which can then undergo further intra-membrane proteolysis by γ-secretase. Scratch-induced wound closure of cultured lung epithelial A549 tumor cells associates with increased syndecan-1 cleavage as evidenced by the release of shed syndecan-1 ectodomain and enhanced generation of the tCTF. Both wound closure and the associated syndecan-1 shedding can be suppressed by inhibition of ADAM family proteases. Cell proliferation, migration and invasion into matrigel as well as several signaling pathways implicated in these responses are suppressed by silencing of syndecan-1. These defects of syndecan-1 deficient cells can be overcome by overexpression of syndecan-1 tCTF or a corresponding tCTF of syndecan-4 but not by overexpression of a tCTF lacking the transmembrane domain. Finally, lung metastasis formation of A549 cells in SCID mice was found to be dependent on syndecan-1, and the presence of syndecan-1 tCTF was sufficient for this activity. Thus, the syndecan-1 tCTF by itself is capable of mediating critical syndecan-1-dependent functions in cell proliferation, migration, invasion and metastasis formation and therefore can replace full length syndecan-1 in the situation of increased syndecan-1 shedding during cell migration and tumor formation.

Leukocytes require ADAM10 but not ADAM17 for their migration and inflammatory recruitment into the alveolar space.

Inflammation is a key process in various diseases, characterized by leukocyte recruitment to the inflammatory site. This study investigates the role of a disintegrin and a metalloproteinase (ADAM) 10 and ADAM17 for leukocyte migration in vitro and in a murine model of acute pulmonary inflammation. Inhibition experiments or RNA knockdown indicated that monocytic THP-1 cells and primary human neutrophils require ADAM10 but not ADAM17 for efficient chemokine-induced cell migration. Signaling and adhesion events that are linked to cell migration such as p38 and ρ GTPase-family activation, F-actin polymerization, adhesion to fibronectin, and up-regulation of α5 integrin were also dependent on ADAM10 but not ADAM17. This was confirmed with leukocytes isolated from mice lacking either ADAM10 or ADAM17 in all hematopoietic cells (vav 1 guanine nucleotide exchange factor [Vav]-Adam10(-/-) or Vav-Adam17(-/-) mice). In lipopolysaccharide-induced acute pulmonary inflammation, alveolar recruitment of neutrophils and monocytes was transiently increased in Vav-Adam17(-/-) but steadily reduced in Vav-Adam10(-/-) mice. This deficit in alveolar leukocyte recruitment was also observed in LysM-Adam10(-/-) mice lacking ADAM10 in myeloid cells and correlated with protection against edema formation. Thus, with regard to leukocyte migration, leukocyte-expressed ADAM10 but not ADAM17 displays proinflammatory activities and may therefore serve as a target to limit inflammatory cell recruitment.

Growth arrest-specific protein 1 is a novel endogenous inhibitor of glomerular cell activation and proliferation.

Growth arrest-specific protein-1 (GAS1) is a GPI-anchored protein which is highly expressed in embryonic mouse fibroblasts and inhibits their proliferation. Glomerular mesangial cells release soluble GAS1 protein into the supernatant in vitro. Growth arrest led to GAS1 overexpression and increased release. Secretion involved disintegrin and metalloproteinase 10 and 17 as signified by inhibition experiments. Recombinant soluble GAS1 protein inhibited the proliferation of mesangial cells. Conversely, the induction of mesangial cell proliferation by PDGF-BB or -DD led to downregulation of GAS1 mRNA. Specific ligands of the PDGF α-receptor, PDGF-AA and -CC, had no effect. The GAS1 protein was localized in podocytes in kidneys from healthy rats. During the time course of mesangioproliferative glomerulonephritis in anti-Thy1.1-treated rats, glomerular GAS1 expression decreased prior to the onset of mesangial cell proliferation and increased at later stages during glomerular recovery. Finally, a plasmid expressing soluble GAS1 fused to an Fc fragment was systemically overexpressed in rats with mesangioproliferative glomerulonephritis. This ameliorated renal damage was indicated by decreased albuminuria and serum creatinine. Gas1/Fc-transfected rats also exhibited a reduction of the glomerular mesangial cell activation and proliferation. Thus, GAS1 is a novel endogenous inhibitor of glomerular mesangial cell proliferation and may be a novel therapeutic target in mesangioproliferative glomerular diseases.

Lung endothelial ADAM17 regulates the acute inflammatory response to lipopolysaccharide.

Acute lung injury (ALI) is associated with increased vascular permeability, leukocyte recruitment, and pro-inflammatory mediator release. We investigated the role of the metalloproteinase ADAM17 in endotoxin-induced ALI with focus on endothelial ADAM17. In vitro, endotoxin-mediated induction of endothelial permeability and IL-8-induced transmigration of neutrophils through human microvascular endothelial cells required ADAM17 as shown by inhibition with GW280264X or shRNA-mediated knockdown. In vivo, ALI was induced by intranasal endotoxin-challenge combined with GW280264X treatment or endothelial adam17-knockout. Endotoxin-triggered upregulation of ADAM17 mRNA in the lung was abrogated in knockout mice and associated with reduced ectodomain shedding of the junctional adhesion molecule JAM-A and the transmembrane chemokine CX3CL1. Induced vascular permeability, oedema formation, release of TNF-α and IL-6 and pulmonary leukocyte recruitment were all markedly reduced by GW280264X or endothelial adam17-knockout. Intranasal application of TNF-α could not restore leukocyte recruitment and oedema formation in endothelial adam17-knockout animals. Thus, activation of endothelial ADAM17 promotes acute pulmonary inflammation in response to endotoxin by multiple endothelial shedding events most likely independently of endothelial TNF-α release leading to enhanced vascular permeability and leukocyte recruitment.

The role of ADAM-mediated shedding in vascular biology.

Within the vasculature the disintegrins and metalloproteinases (ADAMs) 8, 9, 10, 12, 15, 17, 19, 28 and 33 are expressed on endothelial cells, smooth muscle cells and on leukocytes. As surface-expressed proteases they mediate cleavage of vascular surface molecules at an extracellular site close to the membrane. This process is termed shedding and leads to the release of a soluble substrate ectodomain thereby critically modulating the biological function of the substrate. In the vasculature several surface molecules undergo ADAM-mediated shedding including tumour necrosis factor (TNF) α, interleukin (IL) 6 receptor α, L-selectin, vascular endothelial (VE)-cadherin, the transmembrane CX3C-chemokine ligand (CX3CL) 1, Notch, transforming growth factor (TGF) and heparin-binding epidermal growth factor (HB-EGF). These substrates play distinct roles in vascular biology by promoting inflammation, permeability changes, leukocyte recruitment, resolution of inflammation, regeneration and/or neovascularisation. Especially ADAM17 and ADAM10 are capable of cleaving many substrates with diverse function within the vasculature, whereas other ADAMs have a more restricted substrate range. Therefore, targeting ADAM17 or ADAM10 by pharmacologic inhibition or gene knockout not only attenuates the inflammatory response in animal models but also affects tissue regeneration and neovascularisation. Recent discoveries indicate that other ADAMs (e.g. ADAM8 and 9) also play important roles in vascular biology but appear to have more selective effects on vascular responses (e.g. on neovascularisation only). Although, targeting of ADAM17 and ADAM10 in inflammatory diseases is still a promising approach, temporal and spatial as well as substrate-specific inhibition approaches are required to minimise undesired side effects on vascular cells.

Requirements for leukocyte transmigration via the transmembrane chemokine CX3CL1.

The surface-expressed transmembrane CX3C chemokine ligand 1 (CX3CL1/fractalkine) induces firm adhesion of leukocytes expressing its receptor CX3CR1. After shedding by the disintegrins and metalloproteinases (ADAM) 10 and 17, CX3CL1 also acts as soluble leukocyte chemoattractant. Here, we demonstrate that transmembrane CX3CL1 expressed on both endothelial and epithelial cells induces leukocyte transmigration. To investigate the underlying mechanism, we generated CX3CR1 variants lacking the intracellular aspartate-arginine-tyrosine (DRY) motif or the intracellular C-terminus which led to a defect in intracellular calcium response and impaired ligand uptake, respectively. While both variants effectively mediated firm cell adhesion, they failed to induce transmigration and rather mediated retention of leukocytes on the CX3CL1-expressing cell layer. Targeting of ADAM10 led to increased adhesion but reduced transmigration in response to transmembrane CX3CL1, while transmigration towards soluble CX3CL1 was not affected. Thus, transmembrane CX3CL1 mediates leukocyte transmigration via the DRY motif and C-terminus of CX3CR1 and the activity of ADAM10.

A disintegrin and metalloproteinase 17 (ADAM17) mediates inflammation-induced shedding of syndecan-1 and -4 by lung epithelial cells.

Syndecans are cell surface proteoglycans that bind and modulate various proinflammatory mediators and can be proteolytically shed from the cell surface. Within the lung, syndecan-1 and -4 are expressed as transmembrane proteins on epithelial cells and released in the bronchoalveolar fluid during inflammation. We here characterize the mechanism leading to the generation of soluble syndecan-1 and -4 in cultured epithelial cells and murine lung tissue. We show that the bladder carcinoma epithelial cell line ECV304, the lung epithelial cell line A459 and primary alveolar epithelial cells express and constitutively release syndecan-1 and -4. This release involves the activity of the disintegrin-like metalloproteinase ADAM17 as demonstrated by use of specific inhibitors and lentivirally transduced shRNA. Stimulation of epithelial cells with PMA, thrombin, or proinflammatory cytokines (TNFalpha/IFNgamma) led to the down-regulation of surface-expressed syndecan-1 and -4, which was associated with a significant increase of soluble syndecans and cell-associated cleavage fragments. The enhanced syndecan release was not related to gene induction of syndecans or ADAM17, but rather due to increased ADAM17 activity. Soluble syndecan-1 and -4 were also released into the bronchoalveolar fluid of mice. Treatment with TNFalpha/IFNgamma increased ADAM17 activity and syndecan release in murine lungs. Both constitutive and induced syndecan shedding was prevented by the ADAM17 inhibitor. ADAM17 may therefore be an important regulator of syndecan functions on inflamed lung epithelium.

Regulated release and functional modulation of junctional adhesion molecule A by disintegrin metalloproteinases.

Junctional adhesion molecule A (JAM-A) is a transmembrane adhesive glycoprotein that participates in the organization of endothelial tight junctions and contributes to leukocyte transendothelial migration. We demonstrate here that cultured endothelial cells not only express a cellular 43-kDa variant of JAM-A but also release considerable amounts of a 33-kDa soluble JAM-A variant. This release is enhanced by treatment with proinflammatory cytokines and is associated with the down-regulation of surface JAM-A. Inhibition experiments, loss/gain-of-function experiments, and cleavage experiments with recombinant proteases indicated that cleavage of JAM-A is mediated predominantly by the disintegrin and metalloproteinase (ADAM) 17 and, to a lesser extent, by ADAM10. Cytokine treatment of mice increased JAM-A serum level and in excised murine aortas increased ADAM10/17 activity correlated with enhanced JAM-A release. Functionally, soluble JAM-A blocked migration of cultured endothelial cells, reduced transendothelial migration of isolated neutrophils in vitro, and decreased neutrophil infiltration in a murine air pouch model by LFA-1- and JAM-A-dependent mechanisms. Therefore, shedding of JAM-A by inflamed vascular endothelium via ADAM17 and ADAM10 may not only generate a biomarker for vascular inflammation but could also be instrumental in controlling JAM-A functions in the molecular zipper guiding transendothelial diapedesis of leukocytes.

The good, the bad and the ugly substrates for ADAM10 and ADAM17 in brain pathology, inflammation and cancer.

Various surface molecules undergo regulated cleavage by the disintegrin and metalloproteinases ADAM10 and ADAM17. The list of substrates includes molecules involved in brain pathology, inflammation and cancer. In the brain both proteases mediate neuroprotective cleavage events such as inactivation of amyloid precursor protein. In inflammatory settings signaling of cytokines including TNFalpha and IL-6 is triggered by proteolytic release of soluble agonists and leukocyte recruitment is controlled by the cleavage of adhesion molecules. Moreover, in tumors, ADAM10- and ADAM17-mediated shedding events trigger proliferative signaling via activation of growth factors including ErbB family members. Concepts of either increasing ADAM10- or ADAM17-activity to limit neurodegeneration or suppressing their activity to block inflammation or tumor growth have to be carefully scrutinized for their potential side effects in various tissues and pathologies.

ADAM10 regulates endothelial permeability and T-Cell transmigration by proteolysis of vascular endothelial cadherin.

Vascular endothelial (VE)-cadherin is the major adhesion molecule of endothelial adherens junctions. It plays an essential role in controlling endothelial permeability, vascular integrity, leukocyte transmigration, and angiogenesis. Elevated levels of soluble VE-cadherin are associated with diseases like coronary atherosclerosis. Previous data showed that the extracellular domain of VE-cadherin is released by an unknown metalloprotease activity during apoptosis. In this study, we used gain-of-function analyses, inhibitor studies, and RNA interference experiments to analyze the proteolytic release of VE-cadherin in human umbilical vein endothelial cells (HUVECs). We found that VE-cadherin is specifically cleaved by the disintegrin and metalloprotease ADAM10 in its ectodomain, releasing a soluble fragment and generating a carboxyl-terminal membrane-bound stub, which is a substrate for a subsequent gamma-secretase cleavage. This ADAM10-mediated proteolysis could be induced by Ca(2+) influx and staurosporine treatment, indicating that ADAM10-mediated VE-cadherin cleavage contributes to the dissolution of adherens junctions during endothelial cell activation and apoptosis, respectively. In contrast, protein kinase C activation or inhibition did not modulate VE-cadherin processing. Increased ADAM10 expression was functionally associated with an increase in endothelial permeability. Remarkably, our data indicate that ADAM10 activity also contributes to the thrombin-induced decrease of endothelial cell-cell adhesion. Moreover, knockdown of ADAM10 in HUVECs as well as in T cells by small interfering RNA impaired T-cell transmigration. Taken together, our data identify ADAM10 as a novel regulator of vascular permeability and demonstrate a hitherto unknown function of ADAM10 in the regulation of VE-cadherin-dependent endothelial cell functions and leukocyte transendothelial migration.