O220	Cathepsin D induces endothelial cell apoptosis via protein degradation of thioredoxin.
J.Haendeler, J.Hoffmann, V.Tischler, A.M.Zeiher, S.Dimmeler
University of Frankfurt/Internal Medicine III/Molecular Cardiology, Frankfurt/Main, DE.

Thioredoxin 1 (Trx) is a known redox regulator, which plays a crucial role for the apoptosis inhibition in endothelial cells. Oxidative stress is known to induce apoptosis in endothelial cells. Therefore, we investigated the effects of oxidative stress on the known redox regulator Trx. Incubation of endothelial cells with 100 µM and 200 µM H2O2 induced apoptosis in endothelial cells already after 12 h (346 +/- 97 % increase with 100 µM H2O2 and 562 +/- 99 % increase with 200 µM H2O2, p<0.05). Next, we determined the protein levels of Trx after treatment with H2O2. Incubation with H2O2 significantly decreased Trx protein levels as early as 6 hours, whereas incubation with H2O2 for 3 and 6 hours did notalter Trx mRNA as detected by real time PCR. To investigate the underlying mechanism for Trx protein degradation, we coincubated endothelial cells with H2O2 and the protease inhibitors, pepstatin A, leupeptin, ZVAD-CHO, MG132 and E64c for 6 hours. The only protease inhibitor, which completely inhibited H2O2-induced reduction of Trx protein, was pepstatin A. Pepstatin A is known to inhibit cathepsin D and pepsin. Therefore, we used an in vitro assay to determine whether cathepsin D degrades Trx protein. Incubation of unstimulated cell lysates with recombinant cathepsin D significantly reduced Trx protein levels, which was completely blocked by pepstatin A preincubation. Next, we determined the functional relevance of Trx protein degradation on H2O2-induced endothelial cell apoptosis. Coincubation with pepstatin A significantly inhibited H2O2 induced apoptosis (100 µM H2O2: 5.2 +/- 1.3 % apoptotic cells; 100 µM H2O2 + 10 µg/ml pepstatin A: 2.2 +/- 1.2 % apoptotic cells, p<0.02). To further underscore a direct role of cathepsin D for Trx protein degradation and apoptosis signaling, we cloned cathepsin D and overexpressed cathepsin D into endothelial cells. Overexpression of cathepsin D reduced Trx protein levels (54 +/- 18 % reduction of Trx protein) and induced apoptosis in endothelial cells (354 +/- 43 % increase in apoptosis induction, p<0.01). Conclusion: Cathepsin D plays an important role in H2O2-induced apoptosis signalling in endothelial cells. Protein degradation of Trx,which is an essential reactive oxygen species scavenging protein, is a prerequisite for cathepsin D-dependent apoptosis induction in endothelial cells.

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