Interference between DNA binding activities of AP-1 and GR transcription factors in rat thymocytes undergoing dexamethasone-induced apoptosis.

Abstract

The early molecular events of glucocorticoid-induced apoptosis have been 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 evaluated by binding studies using the tritiated ligand, we found that nuclear accumulation of radioactive complexes occurred in the first hour of incubation, and was followed by a progressive decline. This trend was confirmed by immunoblotting of nuclear proteins using a monoclonal anti-glucocorticoid receptor antibody. When the kinetics of binding activity to AP-1 and GRE sequences were studied, using nuclear extracts prepared from Dex-treated thymocytes in gel shift assays, we found peaks at 1 and 2 h after Dex treatment, and a return to basal levels in the following hours. Binding specificity was proved by competition studies using non-radioactive sequences, including mutated AP-1. Unexpectedly, however, protein binding to GRE was better competed for by AP-1 sequence than by GRE itself. Data obtained using the super gel shift assay suggested that AP-1/Jun can be responsible for the high affinity for the GRE sequence. Thus, we report here for the first time that an interference between AP-1 and GR in the binding to DNA consensus sequences-previously described in other biological systems-also occurs during apoptosis induced by glucocorticoids in lymphoid cells.