No abstract

Key Points• ADAM10 but not ADAM17 on leukocytes is essential for chemokine-induced signaling, adhesion, cytoskeletal rearrangement, and migration.• Leukocyte-expressed ADAM10 promotes leukocyte recruitment and edema formation in a murine model of acute pulmonary inflammation.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 r GTPase-family activation, F-actin polymerization, adhesion to fibronectin, and upregulation of a 5 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 2/2 or Vav-Adam17 2/2 mice). In lipopolysaccharide-induced acute pulmonary inflammation, alveolar recruitment of neutrophils and monocytes was transiently increased in Vav-Adam17 2/2 but steadily reduced in Vav-Adam10 2/2 mice. This deficit in alveolar leukocyte recruitment was also observed in LysM-Adam10 2/2 mice lacking ADAM10in myeloid cells and correlated with protection against edema formation. Thus, with regard to leukocyte migration, leukocyteexpressed ADAM10 but not ADAM17 displays proinflammatory activities and may therefore serve as a target to limit inflammatory cell recruitment. (Blood. 2014;123(26):4077-4088)

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.

The work described in this paper demonstrates that very small protein and DNA structures can be applied to various substrates without denaturation using aerosol printing technology. This technology allows high-resolution deposition of various nanoscaled metal and biological suspensions. Before printing, metal and biological suspensions were formulated and then nebulized to form an aerosol which is aerodynamically focused on the printing module of the system in order to achieve precise structuring of the nanoscale material on a substrate. In this way, it is possible to focus the aerosol stream at a distance of about 5 mm from the printhead to the surface. This technology is useful for printing fluorescence-marked proteins and printing enzymes without affecting their biological activity. Furthermore, higher molecular weight DNA can be printed without shearing. The advantages, such as printing on complex, non-planar 3D structured surfaces, and disadvantages of the aerosol printing technology are also discussed and are compared with other printing technologies. In addition, miniaturized sensor structures with line thicknesses in the range of a few micrometers are fabricated by applying a silver sensor structure to glass. After sintering using an integrated laser or in an oven process, electrical conductivity is achieved within the sensor structure. Finally, we printed BSA in small micrometre-sized areas within the sensor structure using the same deposition system. The aerosol printing technology combined with material development offers great advantages for future-oriented applications involving biological surface functionalization on small areas. This is important for innovative biomedical micro-device development and for production solutions which bridge the disciplines of biology and electronics.

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.