Serine protease inhibitors suppress cytochrome c-mediatedcaspase-9 activation and apoptosis during hypoxia-reoxygenation.
We have shown that reoxygenation of hypoxic rat kidney proximaltubule cells leads to apoptosis. This is mediated by translocation ofBax from the cytosol to mitochondria, accompanied by release ofmitochondrial cytochrome c (cyt.c). The present studyhas examined the proteolytic mechanisms responsible for apoptosisduring hypoxia-reoxygenation. Caspases were activated duringhypoxia, as shown by cleavage of fluorogenic peptide substrates. By5 h caspase-3-like activity to cleave carbobenzoxy-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin was increased approx. 30-fold. Thiswas accompanied by specific processing of pro-caspase-3, -8 and -9 intoactive forms. Caspase activation during hypoxia was blocked bycarbobenzoxy-Val-Ala-Asp-fluoromethyl ketone and overexpression of Bcl-2. Of particular interest, caspase activation was also suppressed bythe chymotryptic inhibitors N-tosyl-L-phenylalaninechloromethyl ketone (TPCK) and Ala-Pro-Phe chloromethyl ketone (APF),and the general serine protease inhibitor 4-(2-aminoethyl)benzenesulphonyl fluoride. Inhibition of caspase activationby these compounds resulted in arrest of apoptosis. On the other hand,the serine protease inhibitors did not prevent release of mitochondrialcyt.c during hypoxia, suggesting that these compounds blockeda critical step in post-mitochondrial caspase activation. Furtherstudies using an in vitro reconstitution model showedthat cyt. c/dATP stimulated caspase-9 processing and downstreamcaspase activation were significantly suppressed in the presence ofTPCK and APF. Based on these results, we speculate that serineproteases may be involved in post-mitochondrial apoptotic events thatlead to activation of the initiator, caspase-9.
Dong
Z
Z
Department of Pathology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA. dong@uthscsa.edu
Saikumar
P
P
Patel
Y
Y
Weinberg
J M
JM
Venkatachalam
M A
MA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001692:Biological Transport / Q000187:drug effects; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D015687:Cell Hypoxia / Q000502:physiology*; D002460:Cell Line; D048429:Cell Size / Q000187:drug effects; D003574:Cytochrome c Group / Q000037:antagonists & inhibitors* / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D003838:Deoxyadenine Nucleotides / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004792:Enzyme Precursors / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D051381:Rats; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*
D003574:Cytochrome c Group; D003838:Deoxyadenine Nucleotides; D004792:Enzyme Precursors; D019253:Proto-Oncogene Proteins c-bcl-2; D015842:Serine Proteinase Inhibitors; D000255:Adenosine Triphosphate; C505410:Casp3 protein, rat; C505528:Casp8 protein, rat; C506435:Casp9 protein, rat; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; C026600:2'-deoxyadenosine triphosphate; D010100:Oxygen