The functional role of TOP2beta PARylation in myeloid cells senescence-associated secretory phenotype (SASP) and atherosclerosis induction following ionizing radiation

Poly (ADP-ribose) polymerase (PARP) and type II topoisomerase β (TOP2β) are crucial for DNA repair and cellular homeostasis. PARP catalyzes poly (ADP-ribose) polymerization on proteins (PARylation), essential for DNA repair and cancer treatment, and its inhibition reduces inflammation and reactive oxygen species (ROS) production. TOP2β maintains DNA stability. Although the inhibition of TOP2β PARylation has been reported to impede TOP2β-DNA binding and spermatid chromatin remodeling, its role in myeloid cell (MC) function remains unclear.

To investigate the impact of PARP and TOP2β on senescence-associated secretory phenotype (SASP) induction and mitochondrial (mt) dysfunction in MCs in the context of low dose of ionizing radiation (IR, 2-5 Gy)-induced atherogenesis.

We utilized MC-specific Top2β homozygous knockout (TOP2β-MKO) mice and wild-type (WT) mice. Bone marrow-derived macrophages (BMDMs) were isolated and analyzed for PARP activation and SASP. A partial left carotid artery ligation (PLCL) model with hypercholesteremia was used to evaluate IR-induced atherosclerosis.

IR-induced PARP activation mediated NAD+ depletion, leading to mt dysfunction and mtROS production. Depleted TOP2β enhanced IR-induced SASP and mtROS, while PARP inhibitors mitigated this mtROS induction, highlighting the role of the PARP-TOP2β pathway in triggering SASP and mt dysfunction after IR in MCs. IR increased TOP2β ubiquitination and degradation, which was inhibited by proteasome inhibitors and Makorin Ring Finger Protein 1 (an E3 ubiquitin ligase) siRNA, with TOP2β PARylation necessary for its degradation in MCs. Our study also found that TOP2β PARylation increased after IR, and this effect was inhibited by the PARP inhibitor olaparib. PARP1 depletion completely inhibited TOP2β degradation, indicating that PARP1 is crucial for this process. To explore this further, we overexpressed a TOP2β D969A PARylation-site mutation, which significantly decreased TOP2β PARylation and inhibited TOP2β degradation. This demonstrates that PARylation is necessary for TOP2β degradation. Furthermore, TOP2β PARylation and PARP activation formed a positive feedback loop after IR exposure in MCs. Previously, we reported that PARP inhibitors can attenuate IR-induced atherosclerosis when applied at the time of IR only. In TOP2β-MKO mice, TOP2β depletion intensified PARP activation and inflammation, accelerating atherosclerosis. Lastly, in cancer patients undergoing radiotherapy (RT), TOP2β expression decreased, and NAD+ was depleted in peripheral blood mononuclear cells, suggesting the role of PARP-TOP2β in accelerating senescence in patients after RT.

TOP2β PARylation plays significant roles in up-regulating SASP and inducing mt dysfunction in MCs exposed to IR. Targeting the PARP-TOP2β pathway may offer therapeutic potential for mitigating mitochondrial dysfunction and inflammation in IR-induced cardiovascular disease.