Abstract
Proteolytic activities decline with age, resulting in the accumulation of aggregated proteins in aged organisms. To investigate how disturbance of proteostasis causes cellular senescence in proliferating cells, we developed a stress-induced premature senescence (SIPS) model, in which normal human fibroblast MRC-5 cells were treated with either the proteasome inhibitor MG132 or V-ATPase inhibitor bafilomycin A1 (BAFA1). After 5 days of drug treatment, cells showed morphological and functional changes associated with aging along with DNA damage response. Time-course studies revealed significant increase in intracellular and mitochondrial reactive oxygen species (ROS) during and after drug treatment. We also found temporal downregulation of mitochondrial membrane potential during drug treatment, followed by an increase in mitochondrial mass, especially after drug treatment. Notable upregulation of PGC-1α and TFAM proteins confirmed enhanced mitochondrial biogenesis. Furthermore, the protein levels of SOD2 and GPx4, mitochondrial antioxidant enzymes, in the mitochondrial fraction were specifically reduced on day 1 of the treatment. Co-treatment with rapamycin along with MG132 or BAFA1 partially attenuated induction of SIPS by suppressing generation of excess ROS and mitochondrial biogenesis. In conclusion, the present study revealed that disturbance of proteostasis by the inhibitors changes the distribution of nuclear-encoded mitochondrial antioxidant enzymes at an early period of the treatment, which induces mitochondrial ROS and temporal mitochondrial dysfunction. ROS in turn activates stress responses pathways, followed by PGC-1α-mediated mitochondrial biogenesis. Hence, the excessive ROS continuously generated by increased mitochondria can cause deleterious damage to nuclear DNA, cell cycle arrest, and eventual cellular senescence.
Footnotes
In revised version of the manuscript, antibody list was inserted between text and figures.