Clathrin pit-mediated endocytosis of neutrophil elastase and cathepsin G by cancer cells.
Neutrophil elastase (NE) is a neutrophil-derived serine proteinase with broad substrate specificity. We have recently demonstrated that NE is capable of entering tumor cell endosomes and processing novel intracellular substrates. In the current study, we sought to determine the mechanism by which NE enters tumor cells. Our results show that NE enters into early endosomal antigen-1(+) endosomes in a dynamin- and clathrin-dependent but flotillin-1- and caveolin-1-independent fashion. Cathepsin G (but not proteinase-3) also enters tumor endosomes via the same mechanism. We utilized (125)I-labeled NE to demonstrate that NE binds to the surface of cancer cells. Incubation of radiolabeled NE with lung cancer cells displays a dissociation constant (K(d)) of 284 nm. Because NE is known to bind to heparan sulfate- and chondroitin sulfate-containing proteoglycans, we treated cells with glycanases to remove these confounding factors, which did not significantly diminish cell surface binding or endosomal entry. Thus, NE and CG bind to the surface of cancer cells, presumably to a cell surface receptor, and subsequently undergo clathrin pit-mediated endocytosis.
Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, 15261.
Alyssa D
AD
Gregory
Department of Pediatrics, Division of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, 15261.
Pamela
P
Hale
Department of Pediatrics, Division of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, 15261.
David H
DH
Perlmutter
Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, 15261; Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109; Division of Pulmonary and Critical Care, University of Washington, Seattle, Washington 98195. Electronic address: houghton@fhcrc.org.
A McGarry
AM
Houghton
D000818:Animals; D016466:CHO Cells; D056649:Cathepsin G / Q000235:genetics / Q000378:metabolism*; D051242:Caveolin 1 / Q000235:genetics / Q000378:metabolism; D002809:Chondroitin Sulfates / Q000235:genetics / Q000378:metabolism; D002966:Clathrin / Q000235:genetics / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D004705:Endocytosis; D006801:Humans; D019272:Leukocyte Elastase / Q000235:genetics / Q000378:metabolism*; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D021381:Protein Transport / Q000235:genetics
D051242:Caveolin 1; D002966:Clathrin; D009363:Neoplasm Proteins; D002809:Chondroitin Sulfates; C534094:CTSG protein, human; D056649:Cathepsin G; D019272:Leukocyte Elastase