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This study investigates the mechanism of cell death induced by cadmium (Cd) in Chinese hamster ovary (CHO) cells. Cells exposed to 4 microM Cd for 24 h did not show signs of apoptosis, such as DNA fragmentation and caspase-3 activation. The pro-apoptotic (Bax) or anti-apoptotic (Bcl-2 and Bcl-xL) protein levels in the Bcl-2 family were not altered. However, an increase in propidium iodide uptake and depletion of ATP, characteristics of necrotic cell death, were observed. Cd treatment increased the intracellular calcium (Ca2+) level. Removal of the Ca2+ by a chelator, BAPTA-AM, efficiently inhibited Cd-induced necrosis. The increased Ca2+ subsequently mediated calpain activation and intracellular ROS production. Calpains then triggered mitochondrial depolarization resulting in cell necrosis. Cyclosporin A, an inhibitor of mitochondrial permeability transition, recovered the membrane potential and reduced the necrotic effect. The generated ROS reduced basal NF-kappaB activity and led cells to necrosis. An increase of NF-kappaB activity by its activator, PMA, attenuated Cd-induced necrosis. Calpains and ROS act cooperatively in this process. The calpain inhibitor and the ROS scavenger synergistically inhibited Cd-induced necrosis. Results in this study suggest that Cd stimulates Ca2+-dependent necrosis in CHO cells through two separate pathways. It reduces mitochondrial membrane potential by activating calpain and inhibits NF-kappaB activity by increasing the ROS level.

Mesenchymal stem cells (MSCs) have been used in clinical studies to treat neurological diseases and damage. However, implanted MSCs do not achieve their regenerative effects by differentiating into and replacing neural cells. Instead, MSC secretome components mediate the regenerative effects of MSCs. MSC‐derived extracellular vesicles (EVs)/exosomes carry cargo responsible for rescuing brain damage. We previously showed that EP4 antagonist‐induced MSC EVs/exosomes have enhanced regenerative potential to rescue hippocampal damage, compared with EVs/exosomes from untreated MSCs. Here we show that EP4 antagonist‐induced MSC EVs/exosomes promote neurosphere formation in vitro and increase neurogenesis and neuritogenesis in damaged hippocampi; basal MSC EVs/exosomes do not contribute to these regenerative effects. 2′,3′‐Cyclic nucleotide 3′‐phosphodiesterase (CNP) levels in EP4 antagonist‐induced MSC EVs/exosomes are 20‐fold higher than CNP levels in basal MSC EVs/exosomes. Decreasing elevated exosomal CNP levels in EP4 antagonist‐induced MSC EVs/exosomes reduced the efficacy of these EVs/exosomes in promoting β3‐tubulin polymerization and in converting toxic 2′,3′‐cAMP into neuroprotective adenosine. CNP‐depleted EP4 antagonist‐induced MSC EVs/exosomes lost the ability to promote neurogenesis and neuritogenesis in damaged hippocampi. Systemic administration of EV/exosomes from EP4‐antagonist derived MSC EVs/exosomes repaired cognition, learning, and memory deficiencies in mice caused by hippocampal damage. In contrast, CNP‐depleted EP4 antagonist‐induced MSC EVs/exosomes failed to repair this damage. Exosomal CNP contributes to the ability of EP4 antagonist‐elicited MSC EVs/exosomes to promote neurogenesis and neuritogenesis in damaged hippocampi and recovery of cognition, memory, and learning. This experimental approach should be generally applicable to identifying the role of EV/exosomal components in eliciting a variety of biological responses.

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