Protective effects of neurotrophic factors on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis of murine adrenal chromaffin cell line tsAM5D.

We previously established the murine adrenal chromaffin cell line tsAM5D, which was immortalized with the temperature-sensitive simian virus 40 large T-antigen. tsAM5D cells have the capacity to differentiate into neuron-like cells in response to neurotrophic factors when the culture temperature is shifted from 33 to 39 degrees C. In this model system, the temperature shift in the absence of neurotrophic factors led to cell death. Hoechst staining analysis revealed that typical apoptotic nuclei appeared in a time-dependent manner after the temperature shift. Upon shifting to 39 degrees C, the degradation of T-antigen was accompanied by the transcriptional activation of p53 protein. Among the p53 target genes, death receptor 5 (DR5), which is the receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), showed the highest level of induction. Interestingly, TRAIL-neutralizing antibody protected tsAM5D cells from the temperature shift-induced apoptotic cell death by blocking the activation of caspase-8 and -3, indicating the involvement of TRAIL-mediated death signaling in the temperature shift-induced apoptosis. Glial cell line-derived neurotrophic factor (GDNF) inhibited the TRAIL-mediated activation of caspase-8 in tsAM5D cells exposed to 39 degrees C and cooperated with basic fibroblast growth factor and ciliary neurotrophic factor. Interestingly, the temperature shift induced oligomerization of DR5, which is the earliest process necessary for transduction of the death signal. This oligomerization was inhibited by treatment with GDNF plus ciliary neurotrophic factor but not by that with GDNF alone or GDNF plus basic fibroblast growth factor. These results are discussed with respect to the intracellular mechanism underlying the protective function of neurotrophic factors against TRAIL-mediated death signaling.