IKKα affects the susceptibility of primary human osteoarthritis chondrocytes to oxidative stress-induced DNA damage by tuning autophagy

Abstract

The functional derangement affecting human chondrocytes during osteoarthritis (OA) onset and progression is sustained by the failure of major homeostatic mechanisms. This makes them more susceptible to oxidative stress (OS), which can induce DNA damage responses and exacerbate stress-induced senescence. The knockdown (KD) of IκB kinase α (IKKα), a dispensable protein in healthy articular cartilage physiology, was shown to increase the survival and replication potential of human primary OA chondrocytes. Our recent findings showed that the DNA Mismatch Repair pathway only partially accounts for the reduced susceptibility to OS of IKKαKD cells. Here we therefore investigated other ROS-mediated DNA damage and repair mechanisms. We exposed IKKαWT and IKKαKD chondrocytes to sub-cytotoxic hydrogen peroxide and evaluated the occurrence of double-strand breaks (DSB), 8-oxo-2'-deoxyguanosine (8-oxo-dG) and telomere shortening. ROS exposure was able to significantly increase the number of γH2AX foci (directly related to the number of DSB) in both cell types, but IKKα deficient cells undergoing cell division were able to better recover compared to their IKKα proficient counterpart. 8-oxo-dG signal proved to be the highest DNA damage signal among those investigated, located in the mitochondria and with a slightly higher intensity in IKKα proficient cells immediately after OS exposure. Furthermore, ROS significantly reduced telomere length both in IKKαWT and IKKαKD, with the former showing more pervasive effects, especially in dividing cells. Assessment of the HIF-1α>Beclin-1>LC3B axis after recovery from OS showed that IKKα deficient cells exhibited a more efficient autophagic machinery that allowed them to better cope with oxidative stress, possibly through the turnover of damaged mitochondria. Higher Beclin-1 levels likely helped in rescuing dividing cells (identified by coupled cell cycle analysis) because of Beclin-1's involvement in both autophagy and mitotic spindle organization. Therefore, our data further confirm the higher capacity of IKKαKD chondrocytes to cope with oxidative stress-induced DNA damage.

Keywords: 8-oxo-dG; Autophagy; Chondrocyte aging; DNA damage and repair; Osteoarthritis (OA); Oxidative stress (OS); Telomere; γH2AX.