NF‐κB/IKK activation by small extracellular vesicles within the SASP

Extracellular Vesicles

Cellular senescence plays an important role in different biological and pathological conditions. Senescent cells communicate with their microenvironment through a plethora of soluble factors, metalloproteases and extracellular vesicles (EV). Although much is known about the role that soluble factors play in senescence, the downstream signalling pathways activated by EV in senescence is unknown. To address this, we performed a small molecule inhibitor screen and have identified the IκB kinases IKKε, IKKα and IKKβ as essential for senescence mediated by EV (evSASP). By using pharmacological inhibitors of IKKε, IKKα and IKKβ, in addition to CRISPR/Cas9 targeting their respective genes, we find these pathways are important in mediating senescence. In addition, we find that senescence activation is dependent on canonical NF-κB transcription factors where siRNA targeting p65 prevent senescence. Importantly, these IKK pathways are also relevant to ageing as knockout of IKKA, IKKB and IKKE avoid the activation of senescence. Altogether, these findings open a new potential line of investigation in the field of senescence by targeting the negative effects of the evSASP independent of particular EV contents.

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Cigarette smoke (CS) represents one of the most relevant environmental risk factors for several chronic pathologies. Tissue damage caused by CS exposure is mediated, at least in part, by oxidative stress induced by its toxic and pro-oxidant components. Evidence demonstrates that extracellular vesicles (EVs) released by various cell types exposed to CS extract (CSE) are characterized by altered biochemical cargo and gained pathological properties. In the present study, we evaluated the content of oxidized proteins and phospholipid fatty acid profiles of EVs released by human bronchial epithelial BEAS-2B cells treated with CSE. This specific molecular characterization has hitherto not been performed. After confirmation that CSE reduces viability of BEAS-2B cells and elevates intracellular ROS levels, in a dose-dependent manner, we demonstrated that 24 h exposure at 1% CSE, a concentration that only slight modifies cell viability but increases ROS levels, was able to increase carbonylated protein levels in cells and released EVs. The release of oxidatively modified proteins via EVs might represent a mechanism used by cells to remove toxic proteins in order to avoid their intracellular overloading. Moreover, 1% CSE induced only few changes in the fatty acid asset in BEAS-2B cell membrane phospholipids, whereas several rearrangements were observed in EVs released by CSE-treated cells. The impact of changes in acyl chain composition of CSE-EVs accounted for the increased saturation levels of phospholipids, a membrane parameter that might influence EV stability, uptake and, at least in part, EV-mediated biological effects. The present in vitro study adds new information concerning the biochemical composition of CSE-related EVs, useful to predict their biological effects on target cells. Furthermore, the information regarding the presence of oxidized proteins and the specific membrane features of CSE-related EVs can be useful to define the utilization of circulating EVs as marker for diagnosing of CS-induced lung damage and/or CS-related diseases.

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