Christophe Girard

Mechanisms underlying excitatory effects of group I metabotropic glutamate receptors via inhibition of 2P domain K+ channels

Group I metabotropic glutamate receptors (mGluRs) are implicated in diverse processes such as learning, memory, epilepsy, pain and neuronal death. By inhibiting background K+ channels, group I mGluRs mediate slow and long‐lasting excitation. The main neuronal representatives of this K+ channel family (K2P or KCNK) are TASK and TREK. Here, we show that in cerebellar granule cells and in heterologous expression systems, activation of group I mGluRs inhibits TASK and TREK channels. D‐myo‐inositol‐1,4,5‐triphosphate and phosphatidyl‐4,5‐inositol‐biphosphate depletion are involved in TASK channel inhibition, whereas diacylglycerols and phosphatidic acids directly inhibit TREK channels. Mechanisms described here with group I mGluRs will also probably stand for many other receptors of hormones and neurotransmitters.

p11, an annexin II subunit, an auxiliary protein associated with the background K+ channel, TASK-1

TASK‐1 belongs to the 2P domain K+ channel family and is the prototype of background K+ channels that set the resting membrane potential and tune action potential duration. Its activity is highly regulated by hormones and neurotransmitters. Although numerous auxiliary proteins have been described to modify biophysical, pharmacological and expression properties of different voltage‐ and Ca2+‐sensitive K+ channels, none of them is known to modulate 2P domain K+ channel activity. We show here that p11 interacts specifically with the TASK‐1 K+ channel. p11 is a subunit of annexin II, a cytoplasmic protein thought to bind and organize specialized membrane cytoskeleton compartments. This association with p11 requires the integrity of the last three C‐terminal amino acids, Ser‐Ser‐Val, in TASK‐1. Using series of C‐terminal TASK‐1 deletion mutants and several TASK‐1–GFP chimeras, we demonstrate that association with p11 is essential for trafficking of TASK‐1 to the plasma membrane. p11 association with the TASK‐1 channel masks an endoplasmic reticulum retention signal identified as Lys‐Arg‐Arg that precedes the Ser‐Ser‐Val sequence.

Emerging roles of secreted phospholipase A2 enzymes: Lessons from transgenic and knockout mice

Among the emerging phospholipase A(2) (PLA(2)) superfamily, the secreted PLA(2) (sPLA(2)) family consists of low-molecular-mass, Ca(2+)-requiring extracellular enzymes with a His-Asp catalytic dyad. To date, more than 10 sPLA(2) enzymes have been identified in mammals. Individual sPLA(2)s exhibit unique tissue and cellular localizations and enzymatic properties, suggesting their distinct pathophysiological roles. Despite numerous enzymatic and cell biological studies on this enzyme family in the past two decades, their precise in vivo functions still remain largely obscure. Recent studies using transgenic and knockout mice for several sPLA(2) enzymes, in combination with lipidomics approaches, have opened new insights into their distinct contributions to various biological events such as food digestion, host defense, inflammation, asthma and atherosclerosis. In this article, we overview the latest understanding of the pathophysiological functions of individual sPLA(2) isoforms fueled by studies employing transgenic and knockout mice for several sPLA(2)s.

Pancreatic two P domain K+ channels TALK-1 and TALK-2 are activated by nitric oxide and reactive oxygen species.

This study firstly shows with in situ hybridization on human pancreas that TALK‐1 and TALK‐2, two members of the 2P domain potassium channel (K2P) family, are highly and specifically expressed in the exocrine pancreas and absent in Langherans islets. On the contrary, expression of TASK‐2 in mouse pancreas is found both in the exocrine pancreas and in the Langherans islets. This study also shows that TALK‐1 and TALK‐2 channels, expressed in Xenopus oocytes, are strongly and specifically activated by nitric oxide (obtained with a mixture of sodium nitroprussate (SNP) and dithiothreitol (DTT)), superoxide anion (obtained with xanthine and xanthine oxidase) and singlet oxygen (obtained upon photoactivation of rose bengal, and with chloramine T). Other nitric oxide and reactive oxygen species (NOS and ROS) donors, as well as reducing conditions were found to be ineffective on TALK‐1, TALK‐2 and TASK‐2 (sin‐1, angelis salt, SNP alone, tBHP, H2O2, and DTT). These results suggest that, in the exocrine pancreas, specific members of the NOS and ROS families could act as endogenous modulators of TALK channels with a role in normal secretion as well as in disease states such as acute pancreatitis and apoptosis.

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 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.

PLA2R1 Mediates Tumor Suppression by Activating JAK2

Little is known about the physiological role of the phospholipase A2 receptor (PLA2R1). PLA2R1 has been described as regulating the replicative senescence, a telomerase-dependent proliferation arrest. The downstream PLA2R1 signaling and its role in cancer are currently unknown. Senescence induction in response to activated oncogenes is a failsafe program of tumor suppression that must be bypassed for tumorigenesis. We now present evidence that PLA2R1 functions in vitro as a tumor suppressor, the depletion of which is sufficient to escape oncogene-induced senescence (OIS), thereby facilitating oncogenic cell transformation. Furthermore, mice that are genetically deficient in PLA2R1 display increased sensitivity to RAS-induced tumorigenesis by facilitating OIS escape, highlighting its physiological role as a tumor suppressor. Unexpectedly, PLA2R1 activated JAK2 and its effector signaling, with PLA2R1-mediated inhibition of cell transformation largely reverted in JAK2-depleted cells. This finding was unexpected as the JAK2 pathway has been associated mainly with protumoral functions and several inhibitors are currently in clinical trials. Taken together, our findings uncover an unanticipated tumor suppressive role for PLA2R1 that is mediated by targeting downstream JAK2 effector signaling.

PLA2R1 kills cancer cells by inducing mitochondrial stress

Little is known about the biological functions of the phospholipase A2 receptor (PLA2R1) except that it has the ability to bind a few secreted phospholipases A2 (sPLA2s). We have previously shown that PLA2R1 regulates senescence in normal human cells. In this study, we investigated the ability of PLA2R1 to control cancer cell growth. Analysis of expression in cancer cells indicates a marked PLA2R1 decrease in breast cancer cell lines compared to normal or nontransformed human mammary epithelial cells. Accordingly, PLA2R1 ectopic expression in PLA2R1-negative breast cancer cell lines led to apoptosis, whereas a prosenescence response was predominantly triggered in normal cells. PLA2R1 structure-function studies and the use of chemical inhibitors of sPLA2-related signaling pathways suggest that the effect of PLA2R1 is sPLA2-independent. Functional experiments demonstrate that PLA2R1 regulation of cell death is driven by a reactive oxygen species (ROS)-dependent mechanism. While screening for ROS-producing complexes involved in PLA2R1 biological responses, we identified a critical role for the mitochondrial electron transport chain in PLA2R1-induced ROS production and cell death. Taken together, this set of data provides evidence for an important role of PLA2R1 in controlling cancer cell death by influencing mitochondrial biology.

New physiopathological roles for the PLA2R1 receptor in cancer and membranous nephropathy

PLA2R1 is a large transmembrane receptor of 180-kDa that belongs to the superfamily of C-type lectins. It was discovered because of its high affinity for secreted phospholipases A2 (sPLA2), enzymes that play a key role in lipid mediator synthesis. Early PLA2R1 physiological roles include the clearance of sPLA2 from the extracellular medium and/or promotion of their actions. Over the last four years, two independent studies suggested that PLA2R1 plays a role in cancer as a tumor gene suppressor and is the major target antigen of auto-immune antibodies involved in idiopathic membranous nephropathy, a severe human kidney disease. These novel findings shed light on PLA2R1 and pave the way for its use as a reliable biomarker and an attractive therapeutic target in these diseases.