title: Inflammatory cytokines increase extracellular procathepsin D in permanent and primary endothelial cell cultures.
abstract: The protease cathepsin D (Cath D) and its proteolytically inactive proform, procathepsin D (ProCath D), turned out to be multifunctional within and outside the cell. Elevated levels of ProCath D occur in malignant tumors and in organs under chronic inflammation. One important source for this increase of ProCath D might be endothelial cells. Here we examined the expression of Cath D in the human endothelial cell line EA.hy 926 and in primary endothelial cells isolated from human umbilical cord veins (HUVEC). After serum-free incubation with or without human interferon-gamma (hIFN-gamma) and/or human tumor necrosis factor-alpha (hTNF-alpha) immature and mature Cath D forms were examined in cell extracts and in cell-conditioned medium concentrates by Western blotting. Lysates of EA.hy 926 cells as well as of HUVEC contained active Cath D as two-chain form, but only negligible amounts of ProCath D and Cath D intermediates. Yet both endothelial cell cultures accumulated ProCath D in their conditioned media in the absence of any stimulus. The treatment with hIFN-gamma and/or hTNF-alpha had little effect on intracellular levels of Cath D, whereas the cytokine stimulation increased the extracellular presence of ProCath D in both endothelial cell cultures. The extracellular increase of ProCath D was not related to induction of apoptosis, as validated by cleaved caspase-3 in cell lysates. Acidification of cytokine-treated media converted ProCath D into Cath D, which was associated with cathepsin-like activity using a fluorogenic substrate-linked assay. We conclude, in vitro, endothelial cells are a cytokine-dependent source for extracellular ProCath D.
authors.affiliation: Institute of Anatomy, University of Leipzig, Liebigstrasse 13, D-04103 Leipzig, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Erdmann
authors.forename: Albert
authors.initials: A
authors.lastname: Ricken
authors.forename: Katja
authors.initials: K
authors.lastname: Hummitzsch
authors.forename: Claudia
authors.initials: C
authors.lastname: Merkwitz
authors.forename: Nicole
authors.initials: N
authors.lastname: Schliebe
authors.forename: Frank
authors.initials: F
authors.lastname: Gaunitz
authors.forename: Rainer
authors.initials: R
authors.lastname: Strotmann
authors.forename: Katharina
authors.initials: K
authors.lastname: Spanel-Borowski
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism; D002402:Cathepsin D / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D003470:Culture Media; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects*; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism*; D005110:Extracellular Space; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007371:Interferon-gamma / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D003470:Culture Media; D004792:Enzyme Precursors; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma; D016282:Aspartic Acid Endopeptidases; C058345:procathepsin D; D002402:Cathepsin D