β-actin is required for mitochondria clustering and ROS generation in TNF-induced, caspase-independent cell death

Abstract

Tumor necrosis factor (TNF)-α induces caspase-independent cell death in the fibrosarcoma cell line L929. This cell death has a necrotic phenotype and is dependent on production of reactive oxygen species (ROS) in the mitochondria. To identify genes involved in this TNF-induced, ROS-dependent cell death pathway, we utilized retrovirus insertion-mediated random mutagenesis to generate TNF-resistant L929 cell lines and we subsequently identified genes whose mutations are responsible for the TNF-resistant phenotype. In one such resistant line, β-actin was disrupted by viral insertion, and subsequent reconstitution of β-actin expression levels in the mutant line Actin^mut restored its sensitivity to TNF. Resistance to TNF in Actin^mut cells is signal specific since the sensitivity to other death stimuli is either unchanged or even increased. Comparable NF-κB activation and p38 phosphorylation in TNF-treated wild-type and Actin^mut cells also indicates that reduced expression of actin only selectively blocked some of the TNF-induced cellular changes. Actin cleavage involved in apoptosis does not occur in TNF-treated L929 cell death, as in HeLa cells. Consistent over-expression of a caspase-cleaved product, a 15 kDa actin fragment, had no effect on TNF-induced necrosis of L929 cell. By contrast, TNF-induced mitochondria clustering and ROS production were dramatically reduced in Actin^mut cells, indicating that actin-deficiency-mediated TNF resistance is most likely due to impaired mitochondrial responses to TNF stimulation. Our findings suggest that a full complement of actin is required for transduction of a cell death signal to mitochondria in TNF-treated L929 cells.