INTERFERENCE BETWEEN DNA-BINDING ACTIVITIES OF AP-1 AND GR TRANSCRIPTION FACTORS IN RAT THYMOCYTES UNDERGOING DEXAMETHASONE-INDUCED APOPTOSIS

The early molecular events of glucocorticoid-induced apoptosis have be en investigated by studying glucocorticoid receptor levels, as well as binding activities to GRE and AP-1 sequences, using nuclear extracts from dexamethasone (Dex)-treated rat thymocytes. When the time-course of glucocorticoid-receptor complexes in nuclei of thymocytes was evalu ated by binding studies using the tritiated ligand, we found that nucl ear accumulation of radioactive complexes occurred in the first hour o f incubation, and was followed by a progressive decline. This trend wa s confirmed by immunoblotting of nuclear proteins using a monoclonal a nti-glucocorticoid receptor antibody. When the kinetics of binding act ivity to AP-1 and GRE sequences were studied, using nuclear extracts p repared from Dex-treated thymocytes in gel shift assays, we found peak s at 1 and 2 h after Dex treatment, and a return to basal levels in th e following hours. Binding specificity was proved by competition studi es using non-radioactive sequences, including mutated AP-1. Unexpected ly, however, protein binding to GRE was better competed for by AP-1 se quence than by GRE itself. Data obtained using the super gel shift ass ay suggested that AP-1/Jun can be responsible for the high affinity fo r the GRE sequence. Thus, we report here for the first time that an in terference between AP-1 and GR in the binding to DNA consensus sequenc es - previously described in other biological systems - also occurs du ring apoptosis induced by glucocorticoids in lymphoid cells.