Multiple intracellular pathways interfere with the activation of a CPP32-like protease induced by serum deprivation of AKR-2B cells.
As previously described, confluent AKR-2B fibroblasts rapidly disintegrate upon removal of serum. Platelet-derived growth factor isoforms AB or BB (PDGF-AB, -BB) added immediately after serum deprivation caused complete survival of the cells without initiating proliferation (Simm et al., 1994, J. Cell. Physiol. 160, 295). Here the role of cAMP as a protective agent was investigated by using forskolin or 8-Br-cAMP. Both reagents afforded high cellular protection. The phorbolester TPA, an activator of protein kinase C isoforms, also exerted a high protection against cell death (ED50 = 7 nM). Unexpectedly colchicine (ED50 = 1.5 microM) an inhibitor of tubulin polymerization also protected cells from death. The protective effects of PDGF-BB and TPA were dependent on protein synthesis as indicated by their complete suppression by cycloheximide (CHx). Surprisingly, forskolin and 8-Br-cAMP remained effective even in the presence of CHx. Detailed studies of several signalling pathways were performed. These investigations showed no interference between PDGF-BB and cAMP-dependent pathways at the early stage of signal transduction. As previously described, the ICE-like protease inhibitor tyr-val-ala-asp-chloromethylketone (YVAD-cmk) protected cells from death (Simm et al., 1997, J. Cell Sci. 110, 819-828). As shown here, a substantial protection was also achieved by the addition of two other caspase inhibitors: asp-glu-val-asp-aldehyde (DEVD-cho; ED50 = 100 microM) and benzoylcarbonyl-asp-glu-val-asp-chloromethylketone (Z-DEVD-cmk; ED50 = 100 microM). The activity of caspases was studied using either tyr-val-ala-asp-aminomethylcoumarine (YVAD-amc) or asp-glu-val-asp-aminomethylcoumarine (DEVD-amc) as substrates. There was no activation of a YVADase, whereas as pronounced increase in DEVDase activity was found with a maximum 3 h after serum removal. Cross competition experiments in vitro showed that the latter activity is inhibited also by low concentrations of YVAD-cmk (300-600 nM), suggesting that both inhibitors inactivated the same target protease. Remarkably all tested protective reagents lead to an inhibition of the DEVDase activity in intact cells. Since these reagents act via distinct intracellular pathways, the existence of a regulatory element upstream of the DEVDase is proposed which integrates signals from a variety of pathways.
Schäfer
R
R
Theodor-Boveri-Institut, Department of Physiological Chemistry II, Würzburg, Germany.
Karbach
D
D
Hoppe
J
J
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D000925:Anticoagulants / Q000494:pharmacology; D000077214:Becaplermin; D002273:Carcinogens / Q000494:pharmacology; D053148:Caspase 3; D020169:Caspases; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D003078:Colchicine / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D003374:Coumarins / Q000494:pharmacology; D016895:Culture Media, Serum-Free / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004792:Enzyme Precursors / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D005456:Fluorescent Dyes / Q000494:pharmacology; D051379:Mice; D008806:Mice, Inbred AKR; D009842:Oligopeptides / Q000494:pharmacology; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D020574:Proto-Oncogene Proteins c-sis; D015398:Signal Transduction / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000590:Amino Acid Chloromethyl Ketones; D000925:Anticoagulants; D002273:Carcinogens; D003374:Coumarins; D016895:Culture Media, Serum-Free; D015853:Cysteine Proteinase Inhibitors; D004792:Enzyme Precursors; D005456:Fluorescent Dyes; C098738:N-acetyl-tyrosyl-valyl-alanyl-aspartyl chloromethyl ketone; D009842:Oligopeptides; D010982:Platelet-Derived Growth Factor; D011500:Protein Synthesis Inhibitors; D020574:Proto-Oncogene Proteins c-sis; C102580:acetyl-aspartyl-glutamyl-valyl-aspartyl-amino-4-methylcoumarin; C108637:aspartyl-glutamyl-valyl-aspartal; D000077214:Becaplermin; D005576:Colforsin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000242:Cyclic AMP; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; D013755:Tetradecanoylphorbol Acetate; D003078:Colchicine