Mechanisms of hyperoxia-induced senescence in primary human lung fibroblasts

Tatyana A Klimova

doi:https://doi.org/10.21985/N24B1K

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Mechanisms of hyperoxia-induced senescence in primary human lung fibroblasts

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Senescence, permanent, irreversible replication arrest that occurs in all primary cells studied to date, is considered a cellular model of aging. Recently, senescence has gained attention as a potential tumor-suppressing mechanism. However, despite the obvious importance of senescence, its exact mechanisms remain unclear. One current hypothesis to explain senescence postulates that it is caused by oxidative damage from reactive oxygen species (ROS). ROS are generated normally by the cell's mitochondria, and increase as the cell ages, leading to senescence. Senescence-inducing stressors may elevate ROS to critical levels, also leading to senescence. In support of this theory, previous studies have shown that replicative lifespan was longer in fibroblasts cultured in low oxygen (hypoxia) and decreased under high oxygen concentration (hyperoxia). The canonical interpretation had been that cells generate fewer ROS under hypoxia, and more under hyperoxia, extending or shortening replicative lifespan, respectively. However, other studies have shown that hypoxia paradoxically elevates ROS levels. Our group further determined that, under hypoxia, increased ROS play a signaling role to delay senescence. This thesis further tests the oxidative theory of aging by determining the mechanisms of hyperoxia-induced senescence. The results demonstrate that exposure to 70% O2 leads to stress-induced senescence. Hyperoxia exposure also elevates mitochondrial, but not cytosolic, ROS levels. Importantly, however, overexpression of antioxidant proteins was not sufficient to prevent hyperoxia-induced senescence. Stabilizing mitochondrial iron-sulfur cluster proteins by frataxin overexpression also failed to prevent hyperoxia-induced senescence. Hyperoxia depletes cells of ATP and, therefore, upregulates AMPK, which can lead to senescence. However, overexpression of a kinase-dead mutant of LKB1, which prevented AMPK activation, did not prevent hyperoxia-induced senescence. Knocking down p21 via shRNA, or suppression of the p16/pRb pathway by BMI1 or HPV16-E7 overexpression, was also insufficient to prevent hyperoxia-induced senescence. However, suppressing p53 function resulted in partial rescue from senescence, suggesting that hyperoxia-induced senescence involves p53 but not p21. Suppressing both the p53 and pRb pathways resulted in almost complete protection, indicating that both pathways cooperate in hyperoxia-induced senescence. Collectively, these results indicate that hyperoxia induces senescence through a ROS-independent mechanism that involves activity of both the p53 and pRb pathways.

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