15 research outputs found
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
RĂŽles du Proliferating Cell Nuclear Antigen et de p21/waf1, protĂ©ines rĂ©gulatrices du cycle cellulaire dans l'apoptose du polynuclĂ©aire neutrophile : IntĂ©rĂȘt potentiel dans la modulation de l'inflammation
Notre laboratoire s'intĂ©resse Ă la rĂ©gulation de la balance survie/apoptose du polynuclĂ©aireneutrophile, phĂ©nomĂšne important dans la rĂ©solution de l'inflammation. En effet, le polynuclĂ©aire neutrophilea un rĂŽle essentiel dans la dĂ©fense infectieuse mais il possĂšde Ă©galement un fort potentiel pro-inflammatoire, Ă cause des mĂ©diateurs hautement toxiques qu'il peut libĂ©rer lorsqu'il est stimulĂ©. Le "Proliferating Cell NuclearAntigen" (PCNA) est une protĂ©ine de 36 kDa conservĂ©e au cours de l'Ă©volution. C'est une protĂ©ineindispensable Ă la prolifĂ©ration cellulaire de par son rĂŽle dans la rĂ©plication et la rĂ©paration de l'ADN.Lâimplication de PCNA dans la rĂ©gulation de lâapoptose a Ă©galement Ă©tĂ© mis en Ă©vidence mais cette propriĂ©tĂ©de PCNA est jusquâĂ prĂ©sent restreinte aux cellules prolifĂ©rantes. De maniĂšre intĂ©ressante, la prĂ©sence dePCNA dans le cytosol des polynuclĂ©aire neutrophiles, cellules en fin de diffĂ©renciation et qui ne prolifĂšrentpas, a Ă©tĂ© observĂ©e au laboratoire. Lâobjectif de mon travail a portĂ© sur lâĂ©tude des fonctions de PCNA dans labalance survie / apoptose du neutrophile ainsi que son mĂ©canisme dâaction.L'analyse in vitro des polynuclĂ©aire neutrophiles isolĂ©s de prĂ©lĂšvements sanguins de sujets sains apermis dâidentifier une nouvelle fonction de PCNA: un rĂŽle anti-apoptotique dans le cytosol du polynuclĂ©aireneutrophile. En effet, nous avons montrĂ© une corrĂ©lation entre la survie du polynuclĂ©aire neutrophile etlâexpression accrue de PCNA dans son cytosol. Cette observation est vraie pour une apoptose constitutive oupharmacologique (induite par la gliotoxine, inhibiteur de la voie NFÎșB ou par l'anti-Fas, agissant par la voiedes rĂ©cepteurs de mort). Nous avons ensuite analysĂ© les mĂ©canismes par lesquels PCNA est anti-apoptotique.Le peptide "p21 carboxy-terminal" provenant de lâinhibiteur de kinases cycline-dĂ©pendantes p21, augmentel'apoptose du polynuclĂ©aire neutrophile en entrant en compĂ©tition avec des partenaires de PCNA. Nous avonsĂ©galement identifiĂ© de nouveaux partenaires de PCNA dans le cytosol des polynuclĂ©aires neutrophiles par desapproches de co-immunoprĂ©cipitation, en particulier les pro-caspases-3, -8, -9 et -10. PCNA sĂ©questrerait lespro-caspases et aurait ainsi un rĂŽle dans la survie du polynuclĂ©aire neutrophile.Le rĂŽle anti-apoptotique de PCNA a Ă©tĂ© confirmĂ© dans des cellules myĂ©loĂŻdes (PLB-985) diffĂ©renciĂ©esen polynuclĂ©aire neutrophiles et stablement transfectĂ©es avec lâADN complĂ©mentaire de PCNA. Le mutant dePCNA dans le domaine interdomain-connecting loop (ICL) a Ă©tĂ© Ă©galement transfectĂ© dans les cellules PLB-985. AprĂšs diffĂ©renciation de ces cellules en polynuclĂ©aire neutrophiles, ce mutant, contrairement Ă la protĂ©inesauvage, ne prĂ©sentait pas l'activitĂ© anti-apoptotique. Ces rĂ©sultats suggĂšrent que la fixation de partenairesprotĂ©iques sur le domaine d'interaction ICL pourrait ĂȘtre responsable de l'activitĂ© anti-apoptotique de PCNA.GrĂące Ă la mĂȘme approche, nous avons montrĂ© l'effet pro-apoptotique de la protĂ©ine p21 dans les cellulesPLB-985 diffĂ©renciĂ©es en polynuclĂ©aire neutrophiles.Mon projet a permis l'identification de nouvelles protĂ©ines dont l'expression est modulĂ©e au cours del'apoptose du polynuclĂ©aire neutrophile. Nous avons dĂ©terminĂ© les fonctions des protĂ©ines PCNA et p21dans le cytosol des polynuclĂ©aire neutrophiles, respectivement anti-apoptotique et pro-apoptotique. CesrĂ©sultats permettent donc une meilleure comprĂ©hension des mĂ©canismes de lâapoptose despolynuclĂ©aires neutrophiles et donc, par voie de consĂ©quence, potentiellement de nouvelles voies derecherche Ă 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