Examining the role of caspase-2 in skeletal muscle cell differentiation

Abstract

Prior research has indicated a crucial role for apoptotic signaling in skeletal muscle cell differentiation. Although a number of caspases (-3, -8, -9) have been implicated in this process, few prior investigations have identified a role for the most enigmatic member of the caspase family, caspase-2. Due to its unique nuclear localization as well as its purported roles in cell cycle regulation and DNA damage response; caspase-2 is a likely candidate for regulating differentiation. In order to examine the role of caspase-2 in myocyte differentiation, we assessed enzyme activity throughout the time course of C2C12 differentiation. Additionally, we stably transfected C2C12 cells with caspase-2 shRNA to assess the impact of a caspase-2 knockdown on myocyte differentiation. Finally, we identified the subcellular localization of caspase-2 and p21 throughout the early stages of differentiation. Enzyme activity of caspase-2 transiently increased more than two-fold within 24 hours of differentiation induction, with levels returning to normal by day 7, indicating that the enzyme likely plays a role in the differentiation process. Furthermore, knockdown of caspase-2 dramatically impaired myotube formation and induction of cell cycle inhibitor p21 and myogenic regulatory factor myogenin. Caspase-3 activity was also ablated in the caspase-2 knockdown C2C12 cells. Finally, subcellular fractionation of C2C12 cells at early time points in differentiation revealed a nuclear retention of both caspase-2 and p21 throughout the process. Given the nuclear localization of caspase-2 and p21 as well as the impairment in p21 induction in caspase-2 KD cells, we propose that the role of caspase-2 in myocyte differentiation is to regulate p21 induction at the onset of differentiation. Collectively, the results of this study highlight a novel function for caspase-2 in regulating myocyte differentiation.