The fate of pro-TNF-alpha following inhibition of metalloprotease-dependent processing to soluble TNF-alpha in human monocytes.
Human monocytes rapidly produce TNF-alpha following activation by bacterial LPS. The message for TNF-alpha encodes a 26-kDa protein that is proteolytically processed to the secreted 17-kDa form. Sequencing of the N terminus of the protein secreted by monocytes shows processing of the 26-kDa pro-TNF-alpha to a mature form at the projected metalloprotease cleavage site to generate 17-kDa TNF-alpha with the N terminus VRSSSR-. The addition of hydroxamic acid-based metalloprotease inhibitors to the cell culture is capable of blocking >95% of the production of soluble TNF-alpha and leads to a transient, but reproducible, increase in cell surface TNF-alpha as measured by FACS analysis. The cell surface TNF-alpha was demonstrated to increase the cell's ability to kill L929 tumor targets and induce PG production from human gingival fibroblasts. The buildup of cell surface TNF-alpha is unable to account for the TNF-alpha that is not secreted when inhibitor is present. Pulse-chase analysis of the cells demonstrates rapid degradation of the pro-TNF-alpha that remains unprocessed in the monocytes. Through inhibition of processing and secretion by brefeldin A, processing was shown to occur at a postendoplasmic reticulum site and is closely associated with movement to the cell surface.
Solomon
K A
KA
Department of Inflammatory Diseases Research, DuPont Merck Pharmaceutical Co., Wilmington, DE 19880, USA.
Covington
M B
MB
DeCicco
C P
CP
Newton
R C
RC
D001692:Biological Transport / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008070:Lipopolysaccharides / Q000276:immunology / Q000494:pharmacology; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D009000:Monocytes / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D011498:Protein Precursors / Q000276:immunology / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology / Q000378:metabolism*
D004791:Enzyme Inhibitors; D008070:Lipopolysaccharides; D011498:Protein Precursors; D014409:Tumor Necrosis Factor-alpha; D008666:Metalloendopeptidases