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.
Institute of Pharmacology and Toxicology, Medical Faculty, RWTH Aachen University, Aachen, Germany.
Esther
E
Groth
Institute of Pharmacology and Toxicology, Medical Faculty, RWTH Aachen University, Aachen, Germany.
Jessica
J
Pruessmeyer
Institute of Pharmacology and Toxicology, Medical Faculty, RWTH Aachen University, Aachen, Germany.
Aaron
A
Babendreyer
Institute of Pharmacology and Toxicology, Medical Faculty, RWTH Aachen University, Aachen, Germany.
Julian
J
Schumacher
Institute of Pharmacology and Toxicology, Medical Faculty, RWTH Aachen University, Aachen, Germany.
Tobias
T
Pasqualon
Institute of Pharmacology and Toxicology, Medical Faculty, RWTH Aachen University, Aachen, Germany.
Daniela
D
Dreymueller
Institute for Biochemistry, Christian-Albrechts-University, Kiel, Germany.
Shigeki
S
Higashiyama
Laboratory of Tumor and Development Biology, (GIGA-Cancer), University of Liege and CHU Liege, Liege, Belgium.
Inken
I
Lorenzen
Department of Cell Growth and Tumor Regulation, Proteo-Medicine Research Center, Ehime University, Toon, Shitsukawa, Ehime, Japan.
Joachim
J
Grötzinger
Laboratory of Tumor and Development Biology, (GIGA-Cancer), University of Liege and CHU Liege, Liege, Belgium.
Didier
D
Cataldo
Institute of Pharmacology and Toxicology, Medical Faculty, RWTH Aachen University, Aachen, Germany. Electronic address: aludwig@ukaachen.de.
Andreas
A
Ludwig
D000072283:A549 Cells; D000072197:ADAM10 Protein / Q000378:metabolism; D000072198:ADAM17 Protein / Q000235:genetics / Q000378:metabolism*; D066258:Amphiregulin / Q000378:metabolism; D053829:Amyloid Precursor Protein Secretases / Q000378:metabolism; D020013:Calcium Signaling; D002352:Carrier Proteins / Q000378:metabolism; D042783:Endothelial Cells / Q000201:enzymology; D004789:Enzyme Activation; D055354:Exosomes / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008070:Lipopolysaccharides / Q000494:pharmacology; D021962:Membrane Microdomains / Q000201:enzymology; D008565:Membrane Proteins / Q000378:metabolism; D009000:Monocytes / Q000201:enzymology; D021381:Protein Transport; D034622:RNA Interference; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D016211:Transforming Growth Factor alpha / Q000378:metabolism
D066258:Amphiregulin; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008070:Lipopolysaccharides; D008565:Membrane Proteins; C580845:RHBDF2 protein, human; D016211:Transforming Growth Factor alpha; D053829:Amyloid Precursor Protein Secretases; D000072197:ADAM10 Protein; C491236:ADAM10 protein, human; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human; D013755:Tetradecanoylphorbol Acetate
ADAM10; ADAM17; Metalloproteinase; exosomes; inflammation; shedding