We have shown that reoxygenation of hypoxic rat kidney proximal tubule cells leads to apoptosis. This is mediated by translocation of Bax from the cytosol to mitochondria, accompanied by release of mitochondrial cytochrome c (cyt.c). The present study has examined the proteolytic mechanisms responsible for apoptosis during hypoxia-reoxygenation. Caspases were activated during hypoxia, as shown by cleavage of fluorogenic peptide substrates. By 5 h caspase-3-like activity to cleave carbobenzoxy-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin was increased approx. 30-fold. This was accompanied by specific processing of pro-caspase-3, -8 and -9 into active forms. Caspase activation during hypoxia was blocked by carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone and overexpression of Bcl-2. Of particular interest, caspase activation was also suppressed by the chymotryptic inhibitors N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and Ala-Pro-Phe chloromethyl ketone (APF), and the general serine protease inhibitor 4-(2-aminoethyl)benzenesulphonyl fluoride. Inhibition of caspase activation by these compounds resulted in arrest of apoptosis. On the other hand, the serine protease inhibitors did not prevent release of mitochondrial cyt.c during hypoxia, suggesting that these compounds blocked a critical step in post-mitochondrial caspase activation. Further studies using an in vitro reconstitution model showed that cyt.c/dATP stimulated caspase-9 processing and downstream caspase activation were significantly suppressed in the presence of TPCK and APF. Based on these results, we speculate that serine proteases may be involved in post-mitochondrial apoptotic events that lead to activation of the initiator, caspase-9.
Serine protease inhibitors suppress cytochrome c-mediated caspase-9 activation and apoptosis during hypoxia–reoxygenation
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Zheng DONG, Pothana SAIKUMAR, Yogendra PATEL, Joel M. WEINBERG, Manjeri A. VENKATACHALAM; Serine protease inhibitors suppress cytochrome c-mediated caspase-9 activation and apoptosis during hypoxia–reoxygenation. Biochem J 1 May 2000; 347 (3): 669–677. doi: https://doi.org/10.1042/bj3470669
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