Annexin AI processing is associated with caspase-dependent apoptosis in BZR cells

Annexins are widely distributed and have been described in lung as well as in other cells and tissues. Annexin I (ANX AI) is a member of the calcium-dependent phospholipid binding protein family. Besides its anti-inflammatory function, ANX AI has been involved in several mechanisms such as the Erk repression pathway or apoptosis. To investigate the role of ANX AI on apoptosis in broncho-alveolar cells, we have constructed a plasmid containing the ANX AI full length cDNA. Transfected BZR cells displayed a higher level of both forms of ANX AI (33 and 33 kDa) as well as a decrease in cell viability (two-fold versus cells transfected with an empty vector). In order to analyse the endogenous ANX AI processing time stimulus-induced apoptosis, BZR cells were treated with a commonly used inducer, i.e. C2 ceramides. In these conditions, microscopic analysis revealed chromatin condenstaion in dying cells and the Bcl-2, Bcl-x(L)/ Bax mRNA balance was altered. Caspase-3 is one of the key executioners of apoptosis, being responsible for the cleavage of many proteins such as the nuclear enzyme poly-(ADP-ribose) polymerase (PARP). We demonstrate that Caspase-3 was activated after 4 h treatment in the presence of ceramide leading to the clevage of PARP. Dose-response experiments revealed that cell morohology and viability modifications following ceramide treatment were accompanied by an increase in endogenous ANX AI processing. Interestingly, in both ceramide and transfection experiments, the ANX AI cleaved form was enhanced whereas per-treatment with the caspase-inhibitor Z-VAD-fmk abolished ANX AI cleavage. In conclusion, thsi study demonstrates a complex regulatory role of caspase-dependent apoptosis where ANX AI is processed at the N-terminal region which could give susceptibility to apoptosis upon ceramide treatment. (C) 2003 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.