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 disinteg rate upon removal of serum. Platelet-derived growth factor isoforms AB or BE (PDGF AB, -BB) added immediately after serum deprivation caused complete survival of the cells without initiating proliferation (Simm ef 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. Bot h reagents afforded high cellular protection. The phorbolester TPA, an activator of protein kinase C isoforms, also exerted a high protectio n against cell. death (ED50 = 7 nM). Unexpectedly colchicine (ED50 = 1 .5 mu M) an inhibitor of tubulin polymerization also protected cells f rom death. The protective effects of PDGF-BB and TPA were dependent on protein synthesis as indicated by their complete suppression by cyclo heximide (CHx). Surprisingly, forskolin and 8-Br-cAMP remained effecti ve even in the presence of CHx. Detailed studies of several signalling pathways were performed, These investigations showed no interference between PBGF-BB and cAMP-dependent pathways at the early stage of sign al transduction, As previously described, the ICE-like protease inhibi tor 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 othe r caspase inhibitors: asp-glu-val-asp-aldehyde (DEVD-cho; ED50 = 100 m u M) and benzoylcarbonyl-asp-glu-val-asp-chloromethylketone (Z-D3EVD-c mk; ED50 = 100 mu M). The activity of caspases was studied using eithe r tyr-val-ala-asp-aminomethylcoumarine (YVAD-amc) or asp-glu-val-asp-a minomethylcoumarine (DEVD-amc) as substrates. There was no activation of a YVADase, whereas as pronounced increase in DEVDase activity was f ound with a maximum 3 h after serum removal. Cross competition experim ents in vitro showed that the latter activity is inhibited also by low concentrations of YVAD-cmk (300-600 nM), suggesting that both inhibit ors inactivated the same target protease. Remarkably all tested protec tive reagents lead to an inhibition of the DEVDase activity in intact cells. Since these reagents act via distinct intracellular pathways, t he existence of a regulatory element upstream of the DEVDase is propos ed which integrates signals from a variety of pathways. (C) 1998 Acade mic Press.