Proliferating cell nuclear antigen acts as a cytoplasmic platform controlling human neutrophil survival

Cytosolic proliferating cell nuclear antigen (PCNA) binds to procaspases and protects human neutrophils from apoptosis

Rôles du Proliferating Cell Nuclear Antigen et de p21/waf1, protéines régulatrices du cycle cellulaire dans l'apoptose du polynucléaire neutrophile : Intérêt potentiel dans la modulation de l'inflammation

Notre laboratoire s'intéresse à la régulation de la balance survie/apoptose du polynucléaireneutrophile, phénomène important dans la résolution de l'inflammation. En effet, le polynucléaire neutrophilea un rôle essentiel dans la défense infectieuse mais il possède également un fort potentiel pro-inflammatoire, àcause des médiateurs hautement toxiques qu'il peut libérer lorsqu'il est stimulé. Le "Proliferating Cell NuclearAntigen" (PCNA) est une protéine de 36 kDa conservée au cours de l'évolution. C'est une protéineindispensable à la prolifération cellulaire de par son rôle dans la réplication et la réparation de l'ADN.L’implication de PCNA dans la régulation de l’apoptose a également été mis en évidence mais cette propriétéde PCNA est jusqu’à présent restreinte aux cellules proliférantes. De manière intéressante, la présence dePCNA dans le cytosol des polynucléaire neutrophiles, cellules en fin de différenciation et qui ne prolifèrentpas, a été observée au laboratoire. L’objectif de mon travail a porté sur l’étude des fonctions de PCNA dans labalance survie / apoptose du neutrophile ainsi que son mécanisme d’action.L'analyse in vitro des polynucléaire neutrophiles isolés de prélèvements sanguins de sujets sains apermis d’identifier une nouvelle fonction de PCNA: un rôle anti-apoptotique dans le cytosol du polynucléaireneutrophile. En effet, nous avons montré une corrélation entre la survie du polynucléaire neutrophile etl’expression accrue de PCNA dans son cytosol. Cette observation est vraie pour une apoptose constitutive oupharmacologique (induite par la gliotoxine, inhibiteur de la voie NFκB ou par l'anti-Fas, agissant par la voiedes récepteurs de mort). Nous avons ensuite analysé les mécanismes par lesquels PCNA est anti-apoptotique.Le peptide "p21 carboxy-terminal" provenant de l’inhibiteur de kinases cycline-dépendantes p21, augmentel'apoptose du polynucléaire neutrophile en entrant en compétition avec des partenaires de PCNA. Nous avonségalement identifié de nouveaux partenaires de PCNA dans le cytosol des polynucléaires neutrophiles par desapproches de co-immunoprécipitation, en particulier les pro-caspases-3, -8, -9 et -10. PCNA séquestrerait lespro-caspases et aurait ainsi un rôle dans la survie du polynucléaire neutrophile.Le rôle anti-apoptotique de PCNA a été confirmé dans des cellules myéloïdes (PLB-985) différenciéesen polynucléaire neutrophiles et stablement transfectées avec l’ADN complémentaire de PCNA. Le mutant dePCNA dans le domaine interdomain-connecting loop (ICL) a été également transfecté dans les cellules PLB-985. Après différenciation de ces cellules en polynucléaire neutrophiles, ce mutant, contrairement à la protéinesauvage, ne présentait pas l'activité anti-apoptotique. Ces résultats suggèrent que la fixation de partenairesprotéiques sur le domaine d'interaction ICL pourrait être responsable de l'activité anti-apoptotique de PCNA.Grâce à la même approche, nous avons montré l'effet pro-apoptotique de la protéine p21 dans les cellulesPLB-985 différenciées en polynucléaire neutrophiles.Mon projet a permis l'identification de nouvelles protéines dont l'expression est modulée au cours del'apoptose du polynucléaire neutrophile. Nous avons déterminé les fonctions des protéines PCNA et p21dans le cytosol des polynucléaire neutrophiles, respectivement anti-apoptotique et pro-apoptotique. Cesrésultats permettent donc une meilleure compréhension des mécanismes de l’apoptose despolynucléaires neutrophiles et donc, par voie de conséquence, potentiellement de nouvelles voies derecherche à explorer pour des traitements anti-inflammatoires

Systems-Genetics-Based Inference of a Core Regulatory Network Underlying White Fat Browning

Recruitment of brite/beige cells, known as browning of white adipose tissue (WAT), is an efficient way to turn an energy-storing organ into an energy-dissipating one and may therefore be of therapeutic value in combating obesity. However, a comprehensive understanding of the regulatory mechanisms mediating WAT browning is still lacking. Here, we exploit the large natural variation in WAT browning propensity between inbred mouse strains to gain an inclusive view of the core regulatory network coordinating this cellular process. Combining comparative transcriptomics, perturbation-based validations, and gene network analyses, we present a comprehensive gene regulatory network of inguinal WAT browning, revealing up to four distinct regulatory modules with key roles for uncovered transcriptional factors, while also providing deep insights into the genetic architecture of brite adipogenesis. The presented findings therefore greatly increase our understanding of the molecular drivers mediating the intriguing cellular heterogeneity and plasticity of adipose tissue

Cytoplasmic proliferating cell nuclear antigen connects glycolysis and cell survival in acute myeloid leukemia

International audienceCytosolic 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

Cytosolic PCNA interacts with p47phox and controls NADPH oxidase NOX2 activation in neutrophils.

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

Skewed peripheral B- and T-cell compartments in patients with ANCA-associated vasculitis

International audienceObjectives: To characterize lymphocytes dysregulation in patients with granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA).Methods: Using flow cytometry, we analysed B- and T-cell subsets in peripheral blood from 37 untreated patients with active disease (29 GPA and 8 MPA) and 22 healthy controls (HCs).Results: GPA patients had increased Th2 (1.8 vs 1.0%, P = 0.02), Th9 (1.1 vs 0.2%, P = 0.0007) and Th17 (1.4 vs 0.9%, P = 0.03) cells compared with HC. Patients with MPO-ANCAs had significantly more CD21- B cells than HC or PR3-ANCA patients (6.9 vs 3.3% and 4.4%, P = 0.01). CD69 expressing B cells were significantly higher in GPA and MPA (3.0 and 5.9 vs 1.4%, P = 0.02 and P = 0.03, respectively) compared with HC, whereas B-cell activating factor-receptor expression was decreased in GPA and MPA (median fluorescence intensity ratio 11.8 and 13.7 vs 45.1 in HC, P < 0.0001 and P = 0.003, respectively). Finally, IL-6-producing B cells were increased in GPA vs HC (25.8 vs 14.9%, P < 0.0001) and decreased in MPA vs HC (4.6 vs 14.9%, P = 0.005), whereas TNF-α-producing B cells were lower in both GPA and MPA patients compared with controls (15 and 8.4 vs 30%, P = 0.01 and P = 0.006, respectively).Conclusion: Skewed T-cell polarization towards Th2, Th9 and Th17 responses characterizes GPA, whereas B-cell populations are dysregulated in both GPA and MPA with an activated phenotype and a decreased B-cell activating factor-receptor expression. Finally, inflammatory B cells producing IL-6 are dramatically increased in GPA, providing an additional mechanism by which rituximab could be effective

Neutrophil-expressed p21/waf1 Favors Inflammation Resolution in Pseudomonas aeruginosa Infection

Neutrophil-associated inflammation during Pseudomonas aeruginosa lung infection is a determinant of morbidity in cystic fibrosis (CF). Neutrophil apoptosis is a key factor in inflammation resolution and is controlled by cytosolic proliferating cell nuclear antigen (PCNA). p21/Waf1, a cyclin-dependent kinase inhibitor, is a partner of PCNA and its mRNA is upregulated in human neutrophils during lipopolysaccharide challenge. We here show that after 7 days of persistent infection with Pseudomonas aeruginosa, neutrophilic inflammation was more prominent in p21-/- compared to wild type mice. Notably, no intrinsic defect in the phagocytosis of apoptotic cells by macrophages was found in p21-/- compared to wild type mice. Inflammatory cells analysis in the peritoneal lavages after zymosan-induced peritonitis showed a significant increased number of neutrophils at 48h in p21-/- compared with wild type mice. In vitro analysis was consistent with a delayed neutrophil apoptosis in p21-/- compared with wild type mice. Ectopic expression of p21/waf1 in neutrophil-differentiated PLB985 cells potentiated apoptosis and reversed the prosurvival effect of PCNA. In human neutrophils, p21 mRNA was induced by TNF-\u3b1, G-CSF and LPS. Neutrophils isolated from CF patients showed enhanced survival, which was reduced after treatment with a carboxy-peptide derived from the sequence of p21/waf1. Notably, p21/waf1 was detected by immunohistochemistry in neutrophils within lung from CF patients. Our data reveals a novel role for p21/waf1 in the resolution of inflammation via its ability to control neutrophil apoptosis. This mechanism may be relevant in the neutrophil dominated inflammation observed in CF and other chronic inflammatory lung conditions