title: Cathepsin D and H2O2 stimulate degradation of thioredoxin-1: implication for endothelial cell apoptosis.
abstract: Cathepsin D (CatD) is a lysosomal aspartic proteinase and plays an important role in the degradation of proteins and in apoptotic processes induced by oxidative stress, cytokines, and aging. All of these stimuli are potent inducers of endothelial cell apoptosis. Therefore, we investigated the role of CatD in endothelial cell apoptosis and determined the underlying mechanisms. Incubation with 100-500 microm H2O2 for 12 h induced apoptosis in endothelial cells. To determine a role for CatD, we co-incubated endothelial cells with the CatD inhibitor pepstatin A. Pepstatin A as well as genetic knock down of CatD abolished H2O2-induced apoptosis. In contrast, overexpression of CatD wild type but not a catalytically inactive mutant of CatD (CatDD295N) induced apoptosis under basal conditions. To gain insights into the underlying mechanisms, we investigated the effect of CatD on reactive oxygen species (ROS) formation. Indeed, knocking down CatD expression reduced H2O2-induced ROS formation and apoptosis. The major redox regulator in endothelial cells is thioredoxin-1 (Trx), which plays a crucial role in apoptosis inhibition. Thus, we hypothesized that CatD may alter Trx protein levels and thereby promote formation of ROS and apoptosis. Incubation with 100 microm H2O2 for 6 h decreased Trx protein levels, whereas Trx mRNA was not altered. H2O2-induced Trx degradation was inhibited by pepstatin A and genetic knock down of CatD but not by other protease inhibitors. Incubation of unstimulated cell lysates with recombinant CatD significantly reduced Trx protein levels in vitro, which was completely blocked by pepstatin A pre-incubation. Overexpression of CatD reduced Trx protein in cells. Moreover, H2O2 incubation led to a translocation of Trx to the lysosomes prior to the induction of apoptosis. Taken together, CatD induces apoptosis via degradation of Trx protein, which is an essential anti-apoptotic and reactive oxygen species scavenging protein in endothelial cells.
authors.affiliation: Molecular Cardiology, Department of Internal Medicine III, and Department of Physiology, University of Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany. j.haendeler@em.uni-frankfurt.de
authors.forename: Judith
authors.initials: J
authors.lastname: Haendeler
authors.forename: Rüdiger
authors.initials: R
authors.lastname: Popp
authors.forename: Christine
authors.initials: C
authors.lastname: Goy
authors.forename: Verena
authors.initials: V
authors.lastname: Tischler
authors.forename: Andreas M
authors.initials: AM
authors.lastname: Zeiher
authors.forename: Stefanie
authors.initials: S
authors.lastname: Dimmeler
mesh_terms: D000375:Aging; D017209:Apoptosis; D002384:Catalysis; D002402:Cathepsin D / Q000737:chemistry*; D016923:Cell Death; D002469:Cell Separation; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000166:cytology*; D004730:Endothelium, Vascular / Q000166:cytology; D005434:Flow Cytometry; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D015151:Immunoblotting; D008247:Lysosomes / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009154:Mutation; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010436:Pepstatins / Q000737:chemistry / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D021381:Protein Transport; D012313:RNA / Q000378:metabolism; D017382:Reactive Oxygen Species; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013879:Thioredoxins / Q000737:chemistry*; D013997:Time Factors; D014162:Transfection; D014471:Umbilical Veins / Q000166:cytology
chemical_list: D016207:Cytokines; D010436:Pepstatins; D017382:Reactive Oxygen Species; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins; D012313:RNA; D006861:Hydrogen Peroxide; D002402:Cathepsin D; C031375:pepstatin