Role of c-Jun concentration in neuronal cell death

A dimer of the basic region leucine zipper proteins c-Jun and c-Fos constitutes the classical activator protein-1 (AP-1) transcription factor. c-Jun is thought to play essential roles in many important cellular pathways, including the control of proliferation and cell death. To investigate the roles of c-Jun and c-Fos concentrations in the regulation of neuronal cell death, we generated conditional alleles by fusing c-Jun and c-Fos to the ligand binding domain of the murine estrogen receptor (ER), with the aim of controlling the biological activities of c-Jun and c-Fos by the synthetic ligand 4-hydroxytamoxifen (4OHT). Transient transfection experiments revealed an increase in AP-1 activity following transfection of an expression vector encoding a c-Jun/estrogen receptor fusion protein (c-JunER) and stimulation with 4OHT. In contrast, a c-Fos/estrogen receptor fusion protein (c-FosER) was only weakly active in HT22 immortalized hippocampal cells and in PC12 pheochromocytoma cells. Highest levels of AP-1 activity were obtained by cotransfection of c-FosER and c-JunER and stimulation with 4OHT. Using retroviral gene transfer, we generated HT22 and PC12 cells expressing either c-JunER or c-FosER. The AP-1 activity was moderately increased in 4OHT-treated HT22 and PC12 cells expressing c-JunER, whereas no biological activity was observed in cells expressing c-FosER. We tested the influence of 4OHT-activated c-JunER or c-FosER upon cell survival and cell death by quantification of mitochondrial reduction capacity of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan dye crystals. We did not observe any 4OHT-dependent decrease in cell survival in cells expressing c-JunER or c-FosER. Likewise, the number of pycnic nuclei did not increase in HT22 or PC12 cells expressing c-JunER or c-FosER. We conclude that an increase in the c-Jun concentration is not sufficient to trigger neuronal cell death. We propose that it is not the concentration of c-Jun that is critical for cell survival but rather the concentration of active, i.e., phosphorylated c-Jun.