Cytosolic proliferating cell nuclear antigen (PCNA), a scaffolding protein involved in DNA replication, has been described as a key element in survival of mature neutrophil granulocytes, which are non-proliferating cells. Herein, we demonstrated an active export of PCNA involved in cell survival and chemotherapy resistance. Notably, daunorubicin-resistant HL-60 cells (HL-60R) have a prominent cytosolic PCNA localization due to increased nuclear export compared to daunorubicin-sensitive HL-60 cells (HL-60S). By interacting with nicotinamide phosphoribosyltransferase (NAMPT), a protein involved in NAD biosynthesis, PCNA coordinates glycolysis and survival, especially in HL-60R cells. These cells showed a dramatic increase in intracellular NAD+ concentration as well as glycolysis including increased expression and activity of hexokinase 1 and increased lactate production. Furthermore, this functional activity of cytoplasmic PCNA was also demonstrated in patients with acute myeloid leukemia (AML). Our data uncover a novel pathway of nuclear export of PCNA that drives cell survival by increasing metabolism flux.

Neutrophils produce high levels of reactive oxygen species (ROS) by NADPH oxidase that are crucial for host defense but can lead to tissue injury when produced in excess. We previously described that proliferating cell nuclear antigen (PCNA), a nuclear scaffolding protein pivotal in DNA synthesis, controls neutrophil survival through its cytosolic association with procaspases. We herein showed that PCNA associated with p47phox, a key subunit of NADPH oxidase, and that this association regulated ROS production. Surface plasmon resonance and crystallography techniques demonstrated that the interdomain-connecting loop of PCNA interacted directly with the phox homology (PX) domain of the p47phox. PCNA inhibition by competing peptides or by T2AA, a small-molecule PCNA inhibitor, decreased NADPH oxidase activation in vitro. Furthermore, T2AA provided a therapeutic benefit in mice during trinitro-benzene-sulfonic acid (TNBS)–induced colitis by decreasing oxidative stress, accelerating mucosal repair, and promoting the resolution of inflammation. Our data suggest that targeting PCNA in inflammatory neutrophils holds promise as a multifaceted antiinflammatory strategy.

New therapeutic approaches that can accelerate neutrophil apoptosis under inflammatory conditions to enhance the resolution of inflammation are now under study. Neutrophils are deprived of proliferative capacity and have a tightly controlled lifespan to avoid their persistence at the site of injury. We have recently described that the proliferating cell nuclear antigen (PCNA), a nuclear factor involved in DNA replication and repair of proliferating cells is a key regulator of neutrophil survival. The nuclear-to-cytoplasmic relocalization occurred during granulocytic differentiation and is dependent on a nuclear export sequence thus strongly suggesting that PCNA has physiologic cytoplasmic functions. In this review, we will try to put into perspective the physiologic relevance of PCNA in neutrophils. We will discuss key issues such as molecular structure, post-translational modifications, based on our knowledge of nuclear PCNA, assuming that similar principles governing its function are conserved between nuclear and cytosolic PCNA. The example of cystic fibrosis that features one of the most intense neutrophil-dominated pulmonary inflammation will be discussed. We believe that through an intimate comprehension of the cytosolic PCNA scaffold based on nuclear PCNA knowledge, novel pathways regulating neutrophil survival can be unraveled and innovative agents can be developed to dampen inflammation where it proves detrimental.

We have shown previously that PCNA, a nuclear factor involved in DNA replication and repair in proliferating cells, is localized exclusively in the cytoplasm of neutrophils, where it regulates their survival. Nuclear PCNA functions are tightly linked to its ring-shaped structure, which allows PCNA to bind to numerous partner proteins to orchestrate DNA-related processes. We have shown that only monomeric PCNA can expose its NES to be relocalized from nucleus to cytosol during granulocyte differentiation. This study tested the hypothesis that monomeric PCNA could have a biological role in neutrophils. With the use of a combination of cross-linking and gel-filtration experiments, trimeric and monomeric PCNAs were detected in neutrophil cytosol. The promyelocytic cell line PLB985 was next stably transfected to express the monomeric PCNAY114A mutant to examine its function compared with the WT trimeric PCNA. Monomeric PCNAY114A mutant potentiated DMF-induced differentiation, as evidenced by an increased percentage of CD11b- and gp91phox-positive PLB985PCNAY114A cells and by an increased, opsonized zymosan-triggered NADPH oxidase activity compared with PLB985PCNA or PLB985 cells overexpressing WT PCNA or the empty plasmid, respectively. Regarding antiapoptotic activity, DMF-differentiated PLB985 cells overexpressing WT or the monomeric PCNAY114A mutant displayed a similar antiapoptotic activity following treatment with gliotoxin or TRAIL compared with PLB985. The molecular basis through which cytoplasmic PCNA exerts its antiapoptotic activity in mature neutrophils may, at least in part, be independent of the trimeric conformation.

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