Christine Payré

The M-type receptor PLA2R regulates senescence through the p53 pathway

Senescence is a stable proliferative arrest induced by various stresses such as telomere erosion, oncogenic or oxidative stress. Compelling evidence suggests that it acts as a barrier against tumour development. Describing new mechanisms that favour an escape from senescence can thus reveal new insights into tumorigenesis. To identify new genes controlling the senescence programme, we performed a loss‐of‐function genetic screen in primary human fibroblasts. We report that knockdown of the M‐type receptor PLA2R (phospholipase A2 receptor) prevents the onset of replicative senescence and diminishes stress‐induced senescence. Interestingly, expression of PLA2R increases during replicative senescence, and its ectopic expression results in premature senescence. We show that PLA2R regulates senescence in a reactive oxygen species–DNA damage–p53‐dependent manner. Taken together, our study identifies PLA2R as a potential new tumour suppressor gene crucial in the induction of cellular senescence through the activation of the p53 pathway.

Group X phospholipase A2 is released during sperm acrosome reaction and controls fertility outcome in mice

Ejaculated mammalian sperm must undergo a maturation process called capacitation before they are able to fertilize an egg. Several studies have suggested a role for members of the secreted phospholipase A2 (sPLA2) family in capacitation, acrosome reaction (AR), and fertilization, but the molecular nature of these enzymes and their specific roles have remained elusive. Here, we have demonstrated that mouse group X sPLA2 (mGX) is the major enzyme present in the acrosome of spermatozoa and that it is released in an active form during capacitation through spontaneous AR. mGX-deficient male mice produced smaller litters than wild-type male siblings when crossed with mGX-deficient females. Further analysis revealed that spermatozoa from mGX-deficient mice exhibited lower rates of spontaneous AR and that this was associated with decreased in vitro fertilization (IVF) efficiency due to a drop in the fertilization potential of the sperm and an increased rate of aborted embryos. Treatment of sperm with sPLA2 inhibitors and antibodies specific for mGX blocked spontaneous AR of wild-type sperm and reduced IVF success. Addition of lysophosphatidylcholine, a catalytic product of mGX, overcame these deficiencies. Finally, recombinant mGX triggered AR and improved IVF outcome. Taken together, our results highlight a paracrine role for mGX during capacitation in which the enzyme primes sperm for efficient fertilization and boosts premature AR of a likely phospholipid-damaged sperm subpopulation to eliminate suboptimal sperm from the pool available for fertilization.

Epitope Spreading of Autoantibody Response to PLA2R Associates with Poor Prognosis in Membranous Nephropathy

The phospholipase A2 receptor (PLA2R1) is the major autoantigen in idiopathic membranous nephropathy. However, the value of anti-PLA2R1 antibody titers in predicting patient outcomes is unknown. Here, we screened serum samples from 50 patients positive for PLA2R1 for immunoreactivity against a series of PLA2R1 deletion mutants covering the extracellular domains. We identified reactive epitopes in the cysteine-rich (CysR), C-type lectin domain 1 (CTLD1), and C-type lectin domain 7 (CTLD7) domains and confirmed the reactivity with soluble forms of each domain. We then used ELISAs to stratify 69 patients positive for PLA2R1 by serum reactivity to one or more of these domains: CysR (n=23), CysRC1 (n=14), and CysRC1C7 (n=32). Median ELISA titers measured using the full-length PLA2R1 antigens were not statistically different between subgroups. Patients with anti-CysR-restricted activity were younger (P=0.008), had less nephrotic range proteinuria (P=0.02), and exhibited a higher rate of spontaneous remission (P=0.03) and lower rates of renal failure progression (P=0.002) and ESRD (P=0.01) during follow-up. Overall, 31 of 69 patients had poor renal prognosis (urinary protein/creatinine ratio >4 g/g or eGFR<45 ml/min per 1.73 m(2) at end of follow-up). High anti-PLA2R1 activity and epitope spreading beyond the CysR epitope were independent risk factors of poor renal prognosis in multivariable Cox regression analysis. Epitope spreading during follow-up associated with disease worsening (n=3), whereas reverse spreading from a CysRC1C7 profile back to a CysR profile associated with favorable outcome (n=1). We conclude that analysis of the PLA2R1 epitope profile and spreading is a powerful tool for monitoring disease severity and stratifying patients by renal prognosis.

Group X Phospholipase A2 Stimulates the Proliferation of Colon Cancer Cells by Producing Various Lipid Mediators

Among mammalian secreted phospholipases A2 (sPLA2s), the group X enzyme has the most potent hydrolyzing capacity toward phosphatidylcholine, the major phospholipid of cell membrane and lipoproteins. This enzyme has recently been implicated in chronic inflammatory diseases such as atherosclerosis and asthma and may also play a role in colon tumorigenesis. We show here that group X sPLA2 [mouse (m)GX] is one of the most highly expressed PLA2 in the mouse colon and that recombinant mouse and human enzymes stimulate proliferation and mitogen-activated protein kinase activation of various colon cell lines, including Colon-26 cancer cells. Among various recombinant sPLA2s, mGX is the most potent enzyme to stimulate cell proliferation. Based on the use of sPLA2 inhibitors, catalytic site mutants, and small interfering RNA silencing of cytosolic PLA2α and M-type sPLA2 receptor, we demonstrate that mGX promotes cell proliferation independently of the receptor and via its intrinsic catalytic activity and production of free arachidonic acid and lysophospholipids, which are mitogenic by themselves. mGX can also elicit the production of large amounts of prostaglandin E2 and other eicosanoids from Colon-26 cells, but these lipid mediators do not play a role in mGX-induced cell proliferation because inhibitors of cyclooxygenases and lipoxygenases do not prevent sPLA2 mitogenic effects. Together, our results indicate that group X sPLA2 may play an important role in colon tumorigenesis by promoting cancer cell proliferation and releasing various lipid mediators involved in other key events in cancer progression.

Group X Secreted Phospholipase A2 Limits the Development of Atherosclerosis in LDL Receptor–Null Mice

Objective—Several secreted phospholipases A2 (sPLA2s), including group IIA, III, V, and X, have been linked to the development of atherosclerosis, which led to the clinical testing of A-002 (varespladib), a broad sPLA2 inhibitor for the treatment of coronary artery disease. Group X sPLA2 (PLA2G10) has the most potent hydrolyzing activity toward phosphatidylcholine and is believed to play a proatherogenic role. Methods and Results—Here, we show that Ldlr–/– mice reconstituted with bone marrow from mouse group X–deficient mice (Pla2g10–/–) unexpectedly display a doubling of plaque size compared with Pla2g10+/+ chimeric mice. Macrophages of Pla2g10–/– mice are more susceptible to apoptosis in vitro, which is associated with a 4-fold increase of plaque necrotic core in vivo. In addition, chimeric Pla2g10–/– mice show exaggerated T lymphocyte (Th)1 immune response, associated with enhanced T-cell infiltration in atherosclerotic plaques. Interestingly, overexpression of human PLA2G10 in murine bone marrow cells leads to significant reduction of Th1 response and to 50% reduction of lesion size. Conclusion—PLA2G10 expression in bone marrow cells controls a proatherogenic Th1 response and limits the development of atherosclerosis. The results may provide an explanation for the recently reported inefficacy of A-002 (varespladib) to treat patients with coronary artery disease. Indeed, A-002 is a nonselective sPLA2 inhibitor that inhibits both proatherogenic (groups IIA and V) and antiatherogenic (group X) sPLA2s. Our results suggest that selective targeting of individual sPLA2 enzymes may be a better strategy to treat cardiovascular diseases.

Prediction of membranous nephropathy recurrence after transplantation by monitoring of anti-PLA2R1 (M-type phospholipase A2 receptor) autoantibodies: a case series of 15 patients

BACKGROUND The predictive value of anti-M-type phospholipase A2 receptor (PLA2R1) autoantibodies for membranous nephropathy (MN) recurrence after renal transplantation remains controversial. METHODS Our aim was to monitor anti-PLA2R1 IgG4 activity using a sensitive enzyme-linked immunosorbent assay in 15 kidney transplant recipients with MN, and to test the correlation between antibody titres and MN recurrence. RESULTS Five patients never exhibited anti-PLA2R1 antibodies, and one of them relapsed. Ten patients (67%) had IgG4 anti-PLA2R1 antibodies at the time of transplantation and during follow-up. The presence of IgG4 anti-PLA2R1 antibodies at the time of kidney transplantation does not imply MN recurrence (P = 0.600, n = 15). However, a positive IgG4 anti-PLA2R1 activity during follow-up (>Month 6) was a significant risk factor for MN relapse (P = 0.0048, n = 10). Indeed, four patients had persistent IgG4 anti-PLA2R1 activity after transplantation and relapsed. Among them, one was successfully treated with rituximab. Another had persistently high IgG4 anti-PLA2R1 activity and exhibited a histological relapse but no proteinuria while on treatment with renin-angiotensin system inhibitors. In contrast, the six other patients who did not relapse exhibited a decrease of their IgG4 anti-PLA2R1 activity following transplant immunosuppression, including two with proteinuria due to biopsy-proven differential diagnoses. A weak transplant immunosuppressive regimen was also a risk factor of MN recurrence (P = 0.0048, n = 10). Indeed, the six patients who received both an induction therapy and a combined treatment with calcineurin inhibitors/mycophenolate exhibited a decrease of IgG4 anti-PLA2R1 activity and did not relapse, while the four patients who did not receive this strong immunosuppressive treatment association had persistently high IgG4 anti-PLA2R1 activity and relapsed. CONCLUSION The monitoring of IgG4 anti-PLA2R1 titres during follow-up helps to predict MN recurrence, and a strong immunosuppressive treatment of anti-PLA2R1 positive patients may prevent recurrence.

Group X secreted phospholipase A 2 induces lipid droplet formation and prolongs breast cancer cell survival

BackgroundAlterations in lipid metabolism are inherent to the metabolic transformations that support tumorigenesis. The relationship between the synthesis, storage and use of lipids and their importance in cancer is poorly understood. The human group X secreted phospholipase A2 (hGX sPLA2) releases fatty acids (FAs) from cell membranes and lipoproteins, but its involvement in the regulation of cellular FA metabolism and cancer is not known.ResultsHere we demonstrate that hGX sPLA2 induces lipid droplet (LD) formation in invasive breast cancer cells, stimulates their proliferation and prevents their death on serum deprivation. The effects of hGX sPLA2 are shown to be dependent on its enzymatic activity, are mimicked by oleic acid and include activation of protein kinase B/Akt, a cell survival signaling kinase. The hGX sPLA2-stimulated LD biogenesis is accompanied by AMP-activated protein kinase (AMPK) activation, up-regulation of FA oxidation enzymes and the LD-coating protein perilipin 2, and suppression of lipogenic gene expression. Prolonged activation of AMPK inhibited hGX sPLA2-induced LD formation, while etomoxir, an inhibitor of FA oxidation, abrogated both LD formation and cell survival. The hGX sPLA2-induced changes in lipid metabolism provide a minimal immediate proliferative advantage during growth under optimal conditions, but they confer to the breast cancer cells a sustained ability to resist apoptosis during nutrient and growth factor limitation.ConclusionOur results identify hGX sPLA2 as a novel modulator of lipid metabolism that promotes breast cancer cell growth and survival by stimulating LD formation and FA oxidation.

Group X Secreted Phospholipase A2 Proenzyme Is Matured by a Furin-like Proprotein Convertase and Releases Arachidonic Acid inside of Human HEK293 Cells

Background: Group X secreted phospholipase A2 is an enzyme produced as a proenzyme that plays an important role in arachidonic acid release. Results: Group X phospholipase A2 is matured intracellularly by a furin-like proprotein convertase and releases arachidonic acid during secretion. Conclusion: Group X phospholipase A2 can release arachidonic acid intracellularly. Significance: Group X phospholipase A2 may produce lipid mediators during secretion. Among mammalian secreted phospholipases A2 (sPLA2s), group X sPLA2 has the most potent hydrolyzing activity toward phosphatidylcholine and is involved in arachidonic acid (AA) release. Group X sPLA2 is produced as a proenzyme and contains a short propeptide of 11 amino acids ending with a dibasic motif, suggesting cleavage by proprotein convertases. Although the removal of this propeptide is clearly required for enzymatic activity, the cellular location and the protease(s) involved in proenzyme conversion are unknown. Here we have analyzed the maturation of group X sPLA2 in HEK293 cells, which have been extensively used to analyze sPLA2-induced AA release. Using recombinant mouse (PromGX) and human (ProhGX) proenzymes; HEK293 cells transfected with cDNAs coding for full-length ProhGX, PromGX, and propeptide mutants; and various permeable and non-permeable sPLA2 inhibitors and protease inhibitors, we demonstrate that group X sPLA2 is mainly converted intracellularly and releases AA before externalization from the cell. Most strikingly, the exogenous proenzyme does not elicit AA release, whereas the transfected proenzyme does elicit AA release in a way insensitive to non-permeable sPLA2 inhibitors. In transfected cells, a permeable proprotein convertase inhibitor, but not a non-permeable one, prevents group X sPLA2 maturation and partially blocks AA release. Mutations at the dibasic motif of the propeptide indicate that the last basic residue is required and sufficient for efficient maturation and AA release. All together, these results argue for the intracellular maturation of group X proenzyme in HEK293 cells by a furin-like proprotein convertase, leading to intracellular release of AA during secretion.

Group V Phospholipase A2 in Bone Marrow-derived Myeloid Cells and Bronchial Epithelial Cells Promotes Bacterial Clearance after Escherichia coli Pneumonia

Background: GV sPLA2 hydrolyzes bacterial phospholipids. Myeloid and non-myeloid cells in lung express GV sPLA2. Result: Deletion of GV sPLA2 impairs leukocyte accumulation and bacterial clearance after lung infection with E. coli. Conclusion: GV sPLA2 regulates the innate immune response to E. coli pneumonia. Significance: Inhibiting GV sPLA2 to treat inflammatory diseases could impair the immune response to bacterial infection. Group V-secreted phospholipase A2 (GV sPLA2) hydrolyzes bacterial phospholipids and initiates eicosanoid biosynthesis. Here, we elucidate the role of GV sPLA2 in the pathophysiology of Escherichia coli pneumonia. Inflammatory cells and bronchial epithelial cells both express GV sPLA2 after pulmonary E. coli infection. GV−/− mice accumulate fewer polymorphonuclear leukocytes in alveoli, have higher levels of E. coli in bronchoalveolar lavage fluid and lung, and develop respiratory acidosis, more severe hypothermia, and higher IL-6, IL-10, and TNF-α levels than GV+/+ mice after pulmonary E. coli infection. Eicosanoid levels in bronchoalveolar lavage are similar in GV+/+ and GV−/− mice after lung E. coli infection. In contrast, GV+/+ mice have higher levels of prostaglandin D2 (PGD2), PGF2α, and 15-keto-PGE2 in lung and express higher levels of ICAM-1 and PECAM-1 on pulmonary endothelial cells than GV−/− mice after lung infection with E. coli. Selective deletion of GV sPLA2 in non-myeloid cells impairs leukocyte accumulation after pulmonary E. coli infection, and lack of GV sPLA2 in either bone marrow-derived myeloid cells or non-myeloid cells attenuates E. coli clearance from the alveolar space and the lung parenchyma. These observations show that GV sPLA2 in bone marrow-derived myeloid cells as well as non-myeloid cells, which are likely bronchial epithelial cells, participate in the regulation of the innate immune response to pulmonary infection with E. coli.

Human group X secreted phospholipase A2 induces dendritic cell maturation through lipoprotein-dependent and -independent mechanisms

OBJECTIVE Increased secreted phospholipase A(2) (sPLA(2)) activity has been documented in several inflammatory disorders. Among sPLA(2)s, the human group X (hGX)-sPLA(2) has the highest catalytic activity towards phosphatidylcholine (PC), the major phospholipid of cell membranes and blood lipoproteins. hGX-sPLA(2) has been detected in human atherosclerotic lesions, indicating that sPLA(2)s are an important link between lipids and inflammation, both involved in atherosclerosis. The presence of dendritic cells (DC), the most potent antigen presenting cells, in atherosclerotic lesions has raised the question about their role in disease progression. METHODS AND RESULTS In this study, we show that hGX-sPLA(2)-treated LDL induces human monocyte-derived DC maturation, resulting in a characteristic mature DC phenotype and enhanced DC ability to activate IFNγ secretion from T cells. hGX-sPLA(2) phospholipolysis of LDL produces high levels of lipid mediators, such as lysophosphatidylcholine (LPC) and free fatty acids (FFAs), which also modulate DC maturation. The major molecular species of LPC containing a palmitic or stearic acid esterified in the sn-1 position induce DC maturation, whereas the FFAs can positively or negatively modulate DC maturation depending on their nature. hGX-sPLA(2) added alone can also activate DC in vitro through the hydrolysis of the DC membrane phospholipids leading, however, to a different cytokine profile secretion pattern than the one observed with hGX-sPLA(2)-phospholipolysed LDL. CONCLUSION hGX-sPLA(2) secreted in inflamed tissues can contribute to local DC maturation, resulting in pro-Th1 cells, through the production of various lipid mediators from hydrolysis of either LDL and/or cell plasma membrane.