CUL4B Impedes Stress-induced Cellular Senescence By Dampening A P53-reactive Oxygan Species Positive Feedback Loop

p53 functions as an important tumor suppressor and is mutated or lost in more than 50% of human malignancies. p53 knockout mice spontaneously develop multiple types of tumors. p53 protein is very unstable with a short half-life (5-30 min). Diverse stress signals could induce p53 activation, resulting in the increase of p53 protein level and half life. Reactive oxygen species (ROS) are chemically reactive molecules produced by cellular aerobic metabolism, including hydroxyl radical (HO.), superoxide anion radical (O2-) and hydrogen peroxide (H2O2). ROS could function as a second messenger in cell signaling and thus engage in modulation of mutiple cellular physiological processes.Tumor suppressor p53 can modulate the level of intracellular reactive oxygen species (ROS). It can either alleviate oxidative stress under physiological and mildly stressed conditions or exacerbate oxidative stress under highly stressed conditions. The prooxidative and antioxidant activities of p53 are both mediated by p53 target genes. The downstream genes encoding prooxidative or antioxidant proteins can be transcriptionally regulated by p53 and change the level of intracellular ROS. Multiple p53 target genes, such as CDKN1A, PIG3, PUMA, BAX, and GPX1, regulate the level of intracellular ROS in response to stress.CUL4B functions as a scaffold to assemble a variety of ubiquitin ligases that participate in a broad spectrum of biological processes including cell cycle progression, DNA replication, and transcriptional regulation. It is essential for cellular proliferation and is upregulated in many types of cancer.Senescence and apoptosis are two important cell fates after strong stimulus. There are preferences in cell fates for different cell types. For example, cervical cancer HeLa cells and human embryonic kidney 293 cells tend to undergo apoptosis, while human osteosarcoma U2OS cells and normal human fibroblasts (NHF) tend to enter senescence. We determined the function of CUL4B in senescence using U2OS and NHF. Our results showed that the protein level of CUL4B dramatically decreased during stress-induced senescence and the decrease of CUL4B protein accelerated premature cell senescence.p53 modulates intracellular ROS levels and intracellular ROS can also activate p53, which suggests that p53 and ROS may form a feedback loop. Under high or persistent stress, activated p53 exacerbates oxidative stress by upregulation of prooxidative genes such as CDKN1A and PIG3. Increased ROS levels may further enhance p53 activation, thus forming a positive p53-ROS feedback loop that can drive cells into cellular senescence or apoptosis. We found that in our experimental system, cellular senescence was driven by a positive p53-ROS feedback loop, and CUL4B attenuated cellular senescence by repressing the activation of this positive feedback loop.CUL4A and CUL4B share approximately 80% protein sequence homology, and have similar functions in many cellular processes. CUL4A was reported to participate in the proteolysis of p53. We found that CUL4B expression level is negatively correlated with p53 stability under oxidative stress. CUL4B knockdown led to an extended half-life of p53 protein and overexpression of CUL4B accelerated the degradation of p53 protein. We also detected the protein-protein interaction of p53 and CUL4B in NHF cells. Further ubiquitination experiments proved that CUL4B promoted p53 ubiquitination in response to stress.In summary, our study revealed the function of CUL4B in senescence. CUL4B expression significantly decreased after stress stimulus. The downregulation of CUL4B led to an extended half-life of p53 protein by blocking the ubiquitination of p53, and thus induced persistently activation of p53-ROS positive feedback loop, resulting in alleviated cell proliferation and enhanced premature senescence. On the other hand, the upregulation of CUL4B accelerated the degradation of p53, impedes stress-induced cellular senescence by dampening a p53-reactive oxygen species positive feedback loop. Our results suggested CUL4B plays a crucial role in restricting p53-ROS positive feedback loop and premature senescence. Moreover, this study deepened our knowledge of p53 in stress-induced senescence and upstream regulation of p53 in this process.PART ONECUL4B is downregulated during stress-induced senescenceCullin-RING ligases are the largest family of E3 ubiquitin ligases and CUL4B is an important member of cullin-RING ubiquitin ligase complex. CUL4B plays critical roles in epigenetic modification and multiple biological processes. Excessive expression of CUL4B in multiple tumor tissues promotes cell proliferation and knockdown of CUL4B inhibits cell proliferation. What’s more, CUL4B regulates expression of several senescence related genes, such as p16 and CXCR2, through epigenetic modulation. Thus, we suspected that CUL4B might play a role in cellular senescence. To determine the function of CUL4B in cellular senescence, we first detected the expression of CUL4B in different types of senescence. We found that CUL4B expression was downregulated in stress-induced senescence but not changed in replicative senescence caused by telomere shortage. Moreover, we measured CUL4B expression in mouse ear fibroblasts (MSF) and found that CUL4B was also downregulated in senescent MSF in cell culture. These results suggested CUL4B expression was downregulated in stress-induced senescence but not in replicative senescence. It is worth noted that the decrease of protein level of CUL4B was not accompanied by the change of mRNA level of CUL4B during stress-induced senescence. Therefore, the decrease of CUL4B protein could be mediated by proteolysis or microRNA.PART TWOCUL4B impedes stress-induced cellular senescenceThe expressions of many genes are altered during cellular senescence. The change of expression of a gene could be either the cause or the result of senescence. CUL4B overexpression and knockdown cell lines (NHF and U2OS) were established and used to study the function of CUL4B in stress-induced senescence. Our results showed that after stimulus, SA-β-gal staining rate significantly increased and BrdU corporation significantly decreased in CUL4B knockdown cells; SA-β-gal staining rate significantly decreased and BrdU corporation significantly increased in CUL4B overexpression cells. These results indicated that CUL4B knockdown dramatically increased cellular senescence and CUL4B overexpression significantly inhibited it Consistent with results in part one, CUL4B knockdown did not affect replicative senescence. Moreover, fibroblasts from Cul4b KO mice were exhibited accelerated senescence when compared with those from WT mice. In summary, the change of CUL4B expression in premature cells contributes to cellular senescence.PART THREECUL4B negatively regulates p53-ROS positive feedback loop.Tumor suppressor p53 functions as a transcription factor. p53 proteins are distributed in cytoplasm under physical condition and are transported to the nucleus after activation by stress-induced phosphorylation. The activated p53 binds to the promoter of target genes to regulate their expression. p53 can transactive both prooxidative and antioxidant genes, depending on stress levels and cell types. Under physiological and mildly stressed conditions, p53 can alleviate oxidative stress, but under highly stressed conditions, it will exacerbate oxidative stress. The ROS levels were significantly increased by continuous nutlin3 (an MDM2 antagonist) treatment in p53 wild type cells, but not in p53 null cells. ROS could also activate p53, forming a p53-ROS positive feedback loop. Stress-induced activation of p53 and elevation of ROS level were further enhanced by CUL4B knockdown, but inhibited by CUL4B overexpression. Under strong stress, the p53-ROS positive feedback loop is permanently activated and causes senescence or apoptosis. To investigate the mechanism of CUL4B in senescence, H2O2 and Nutlin-3 were used to treat CUL4B knockdown cells and CUL4B overexpression cells and the ROS levels and p53 activation were measured after 48 hrs. The ROS levels and p53 activation significantly increased in CUL4B knockdown cells, while in CUL4B overexpression cells ROS levels and p53 activation was inhibited. These results indicated CUL4B negatively regulated the p53-ROS positive feedback loop. By NAC pretreatment experiments, we proved that increased senescence or hyper-activated p53 induced by deletion of CUL4B depends on ROS. Double knockdown of CUL4B and p53 blocked senescence, which suggested senescence induced by CUL4B knockdown depended on p53. In summary, CUL4B knockdown induced persistent activation of p53-ROS positive feedback loop and thus promoted cellular senescence; CUL4B overexpression blocked activation of p53-ROS positive feedback loop and thus attenuated stress-induced senescence.PART FOURCUL4B promoted proteolysis of p53 by mediating its ubiquitination.CUL4A and CUL4B share approximately 80% protein sequence homology and have similar functions in multiple cellular processes. CUL4A was reported to mediate p53 ubiquitination proteolysis, so we speculated CUL4B might also mediate p53 ubiquitination. Indeed, we found that CUL4B could promote p53 ubiquitination and proteolysis in response to stress. Under oxidative stress, CUL4B knockdown led to an extended half-life of p53 protein and overexpression of CUL4B accelerated the degradation of p53 protein. Moreover, we also detected the protein-protein interaction of p53 and CUL4B in NHF cells. These results explain how CUL4B inhibits stress-induced cellular senescence. CUL4B functions to destabilize p53, thus compromising a p53-ROS positive feedback loop and attenuating cellular senescence.