Regulation of ROS-independent ERK signaling rescues replicative cellular senescence in ex vivo expanded human c-kit-positive cardiac progenitor cells

Backgrounds: Although the rescue of cellular senescence during ex vivo expansion of human-derived cardiac progenitor cells (hCPC) is critical for the application of autologous stem cell therapy in cardiovascular disease, the underlying molecular pathways during replicative senescence in hCPC have not been fully defined. Thus, we examined whether the regulation of mitogen-activated protein kinases activation could facilitate the recovery of human c-kit-positive hCPCs (hCPC(c-kit+)) and whether senescence is reactive oxygen species (ROS)-dependent or -independent.

Methods and results: To investigate the molecular pathways of replicative cellular senescence, we first evaluated cellular senescence in ex vivo-expanded hCPC(c-kit+) by using senescence-associated β-galactosidase (SA-β-gal) activity with enlarged cytoplasm and observed increased expression of cell senescence-related pivotal molecules, including TP53, cleavage Mdm2 (cMdm2), and Mdm2. Unexpectedly, we found that the extracellular signal-regulated kinase (ERK) was markedly activated in aged hCPC(c-kit+), with reduced proliferative activity. SA-β-gal activity and cytoplasm size in senescent hCPC(c-kit+) were significantly reduced, with reduced TP53 and cMdm2 expression after treatment with a specific ERK inhibitor (U0126). We examined whether the signaling in ERK inhibitory rescue of hCPC(c-kit+) senescence is ROS-dependent. Interestingly, the increased ROS level was not changed after treatment with a specific ERK inhibitor. Similarly, the increased expression levels of endogenous antioxidant enzymes, e.g., peroxiredoxin (Prdx)-1 and 2, in senescent hCPC(c-kit+) were not changed after treatment with a specific ERK inhibitor.

Conclusions: From the above results, we conclude that the specific inhibition of ERK during cellular senescence might rescue bioactivities of senescent hCPC(c-kit+) in a ROS-independent manner.