Publications by authors named "Nicolas Vitale"

107 Publications

Somatostatin analogue pasireotide (SOM230) inhibits catecholamine secretion in human pheochromocytoma cells.

Cancer Lett 2021 Oct 9. Epub 2021 Oct 9.

Centre National de la Recherche Scientifique, Université de Strasbourg, Institut des Neurosciences Cellulaires et Intégratives, F-67000, Strasbourg, France. Electronic address:

Increasingly common, neuroendocrine tumors (NETs) are regarded nowadays as neoplasms potentially causing debilitating symptoms and life-threatening medical conditions. Pheochromocytoma is a NET that develops from chromaffin cells of the adrenal medulla, and is responsible for an excessive secretion of catecholamines. Consequently, patients have an increased risk for clinical symptoms such as hypertension, elevated stroke risk and various cardiovascular complications. Somatostatin analogues are among the main anti-secretory medical drugs used in current clinical practice in patient with NETs. However, their impact on pheochromocytoma-associated catecholamine hypersecretion remains incompletely explored. This study investigated the potential efficacy of octreotide and pasireotide (SOM230) on human tumor cells directly cultured from freshly resected pheochromocytomas using an implemented catecholamine secretion measurement by carbon fiber amperometry. SOM230 treatment efficiently inhibited nicotine-induced catecholamine secretion both in bovine chromaffin cells and in human tumor cells whereas octreotide had no effect. Moreover, SOM230 specifically decreased the number of exocytic events by impairing the stimulation-evoked calcium influx as well as the nicotinic receptor-activated inward current in human pheochromocytoma cells. Altogether, our findings indicate that SOM230 acts as an inhibitor of catecholamine secretion through a mechanism involving the nicotinic receptor and might be considered as a potential anti-secretory treatment for patients with pheochromocytoma.
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http://dx.doi.org/10.1016/j.canlet.2021.10.009DOI Listing
October 2021

A Lipidomics Approach to Measure Phosphatidic Acid Species in Subcellular Membrane Fractions Obtained from Cultured Cells.

Bio Protoc 2021 Jun 20;11(12):e4066. Epub 2021 Jun 20.

Centre National de la Recherche Scientifique, Université de Strasbourg, Institut des Neurosciences Cellulaires et Intégratives, 67000 Strasbourg, France.

Over the last decade, lipids have emerged as possessing an ever-increasing number of key functions, especially in membrane trafficking. For instance, phosphatidic acid (PA) has been proposed to play a critical role in different steps along the secretory pathway or during phagocytosis. To further investigate in detail the precise nature of PA activities, we need to identify the organelles in which PA is synthesized and the PA subspecies involved in these biological functions. Indeed, PA, like all phospholipids, has a large variety based on its fatty acid composition. The recent development of PA sensors has helped us to follow intracellular PA dynamics but has failed to provide information on individual PA species. Here, we describe a method for the subcellular fractionation of RAW264.7 macrophages that allows us to obtain membrane fractions enriched in specific organelles based on their density. Lipids from these membrane fractions are precipitated and subsequently processed by advanced mass spectrometry-based lipidomics analysis to measure the levels of different PA species based on their fatty acyl chain composition. This approach revealed the presence of up to 50 different species of PA in cellular membranes, opening up the possibility that a single class of phospholipid could play multiple functions in any given organelle. This protocol can be adapted or modified and used for the evaluation of other intracellular membrane compartments or cell types of interest.
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http://dx.doi.org/10.21769/BioProtoc.4066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8260265PMC
June 2021

Protocol for electron microscopy ultrastructural localization of the fusogenic lipid phosphatidic acid on plasma membrane sheets from chromaffin cells.

STAR Protoc 2021 Jun 12;2(2):100464. Epub 2021 Apr 12.

Centre National de la Recherche Scientifique, Université de Strasbourg, Institut des Neurosciences Cellulaires et Intégratives, 67000 Strasbourg, France.

The glycerophospholipid phosphatidic acid (PA) is a key player in regulated exocytosis, but little is known about its localization at the plasma membrane. Here, we provide a protocol for precisely determining the spatial distribution of PA at exocytotic sites by electron microscopy. Using primary bovine chromaffin cells expressing a PA sensor (Spo20p-GFP), we describe the process for cell stimulation and detergent-free preparation of plasma membrane sheets. The protocol can be applied to other cell models and to distinct membrane lipids. For complete details on the use and execution of this protocol, please refer to Tanguy et al. (2020).
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http://dx.doi.org/10.1016/j.xpro.2021.100464DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8065343PMC
June 2021

Ral GTPases promote breast cancer metastasis by controlling biogenesis and organ targeting of exosomes.

Elife 2021 Jan 6;10. Epub 2021 Jan 6.

INSERM UMR_S1109, Tumor Biomechanics, Strasbourg, France.

Cancer extracellular vesicles (EVs) shuttle at distance and fertilize pre-metastatic niches facilitating subsequent seeding by tumor cells. However, the link between EV secretion mechanisms and their capacity to form pre-metastatic niches remains obscure. Using mouse models, we show that GTPases of the Ral family control, through the phospholipase D1, multi-vesicular bodies homeostasis and tune the biogenesis and secretion of pro-metastatic EVs. Importantly, EVs from RalA or RalB depleted cells have limited organotropic capacities and are less efficient in promoting metastasis. RalA and RalB reduce the EV levels of the adhesion molecule MCAM/CD146, which favors EV-mediated metastasis by allowing EVs targeting to the lungs. Finally, RalA, RalB, and MCAM/CD146, are factors of poor prognosis in breast cancer patients. Altogether, our study identifies RalGTPases as central molecules linking the mechanisms of EVs secretion and cargo loading to their capacity to disseminate and induce pre-metastatic niches in a CD146-dependent manner.
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http://dx.doi.org/10.7554/eLife.61539DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822591PMC
January 2021

Phosphatidic acid: Mono- and poly-unsaturated forms regulate distinct stages of neuroendocrine exocytosis.

Adv Biol Regul 2021 01 28;79:100772. Epub 2020 Nov 28.

Centre National de la Recherche Scientifique, Université de Strasbourg, Institut des Neurosciences Cellulaires et Intégratives, F-67000 Strasbourg, France. Electronic address:

Lipids have emerged as important actors in an ever-growing number of key functions in cell biology over the last few years. Among them, glycerophospholipids are major constituents of cellular membranes. Because of their amphiphilic nature, phospholipids form lipid bilayers that are particularly useful to isolate cellular content from the extracellular medium, but also to define intracellular compartments. Interestingly, phospholipids come in different flavors based on their fatty acyl chain composition. Indeed, lipidomic analyses have revealed the presence in cellular membranes of up to 50 different species of an individual class of phospholipid, opening the possibility of multiple functions for a single class of phospholipid. In this review we will focus on phosphatidic acid (PA), the simplest phospholipid, that plays both structural and signaling functions. Among the numerous roles that have been attributed to PA, a key regulatory role in secretion has been proposed in different cell models. We review here the evidences that support the idea that mono- and poly-unsaturated PA control distinct steps in hormone secretion from neuroendocrine cells.
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http://dx.doi.org/10.1016/j.jbior.2020.100772DOI Listing
January 2021

Transmission Electron Microscopy and Tomography on Plasma Membrane Sheets to Study Secretory Docking.

Methods Mol Biol 2021 ;2233:301-309

Centre National de la Recherche Scientifique, Université de Strasbourg, Institut des Neurosciences Cellulaires et Intégratives, Strasbourg, France.

To study the formation and the architecture of exocytotic site, we generated plasma membrane (PM) sheets on electron microscopy grids to visualize the membrane organization and quantitatively analyze distributions of specific proteins and lipids. This technique allows observing the cytoplasmic face of the plasma membrane by transmission electron microscope. The principle of this approach relies on application of mechanical forces to break open cells. The exposed inner membrane surface can then be visualized with different electron-dense colorations, and specific proteins or lipids can be detected with gold-conjugated probes. Moreover, the membrane sheets are sufficiently resistant to support automated acquisition of multiple-tilt projections, and thus electron tomography allows to obtain three-dimensional (3D) ultrastructural images of secretory granule docked to the plasma membrane.
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http://dx.doi.org/10.1007/978-1-0716-1044-2_20DOI Listing
March 2021

Bovine Chromaffin Cells: Culture and Fluorescence Assay for Secretion.

Methods Mol Biol 2021 ;2233:169-179

Centre National de la Recherche Scientifique, Université de Strasbourg, Institut des Neurosciences Cellulaires et Intégratives, Strasbourg, France.

Over the last four decades, chromaffin cells originating from the adrenal medulla have been probably one of the most popular cell models to study neurosecretion at the molecular level. Accordingly, numerous seminal discoveries in the field, including the characterization of role of the cytoskeleton, fusogenic lipids, and soluble N-ethylmaleimide-sensitivefactor attachment protein receptor (SNARE) proteins, have been made using this model. In this chapter, we describe a standard method currently used to isolate and culture bovine chromaffin cells, and we illustrate a catecholamine secretion assay based on the successive transformation of adrenaline into adrenochrome and adrenolutine for fluorescence measurements. We also provide some guidelines for efficient cell recovery and for the use of this assay in the laboratory.
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http://dx.doi.org/10.1007/978-1-0716-1044-2_11DOI Listing
March 2021

Measurements of Compensatory Endocytosis by Antibody Internalization and Quantification of Endocytic Vesicle Distribution in Adrenal Chromaffin Cells.

Methods Mol Biol 2021 ;2233:43-51

Centre National de la Recherche Scientifique, Université de Strasbourg, Institut des Neurosciences Cellulaires et Intégratives, Strasbourg, France.

Plasma membrane proteins are amenable to endocytosis assays since they are easily labeled by reagents applied in the extracellular medium. This has been widely exploited to study constitutive endocytosis or ligand-induced receptor endocytosis. Compensatory endocytosis is the mechanism by which components of secretory vesicles are retrieved after vesicle fusion with the plasma membrane in response to cell stimulation and a rise in intracellular calcium. Luminal membrane proteins from secretory vesicles are therefore transiently exposed at the plasma membrane. Here, we described an antibody-based method to monitor compensatory endocytosis in chromaffin cells and present an image-based analysis to quantify endocytic vesicles distribution.
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http://dx.doi.org/10.1007/978-1-0716-1044-2_3DOI Listing
March 2021

RALB GTPase: a critical regulator of DR5 expression and TRAIL sensitivity in KRAS mutant colorectal cancer.

Cell Death Dis 2020 10 29;11(10):930. Epub 2020 Oct 29.

Drug Resistance Group, Patrick G. Johnston Centre for Cancer Research, School of Medicine, Dentistry and Biomedical Science, Queen's University Belfast, 97 Lisburn Road, Belfast, BT9 7AE, UK.

RAS mutant (MT) metastatic colorectal cancer (mCRC) is resistant to MEK1/2 inhibition and remains a difficult-to-treat group. Therefore, there is an unmet need for novel treatment options for RASMT mCRC. RALA and RALB GTPases function downstream of RAS and have been found to be key regulators of several cell functions implicated in KRAS-driven tumorigenesis. However, their role as regulators of the apoptotic machinery remains to be elucidated. Here, we found that inhibition of RALB expression, but not RALA, resulted in Caspase-8-dependent cell death in KRASMT CRC cells, which was not further increased following MEK1/2 inhibition. Proteomic analysis and mechanistic studies revealed that RALB depletion induced a marked upregulation of the pro-apoptotic cell surface TRAIL Death Receptor 5 (DR5) (also known as TRAIL-R2), primarily through modulating DR5 protein lysosomal degradation. Moreover, DR5 knockdown or knockout attenuated siRALB-induced apoptosis, confirming the role of the extrinsic apoptotic pathway as a regulator of siRALB-induced cell death. Importantly, TRAIL treatment resulted in the association of RALB with the death-inducing signalling complex (DISC) and targeting RALB using pharmacologic inhibition or RNAi approaches triggered a potent increase in TRAIL-induced cell death in KRASMT CRC cells. Significantly, high RALB mRNA levels were found in the poor prognostic Colorectal Cancer Intrinsic Subtypes (CRIS)-B CRC subgroup. Collectively, this study provides to our knowledge the first evidence for a role for RALB in apoptotic priming and suggests that RALB inhibition may be a promising strategy to improve response to TRAIL treatment in poor prognostic RASMT CRIS-B CRC.
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http://dx.doi.org/10.1038/s41419-020-03131-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7596570PMC
October 2020

Annexin A2 Egress during Calcium-Regulated Exocytosis in Neuroendocrine Cells.

Cells 2020 09 9;9(9). Epub 2020 Sep 9.

Centre National de la Recherche Scientifique, Université de Strasbourg, Institut des Neurosciences Cellulaires et Intégratives, F-67000 Strasbourg, France .

Annexin A2 (AnxA2) is a calcium- and lipid-binding protein involved in neuroendocrine secretion where it participates in the formation and/or stabilization of lipid micro-domains required for structural and spatial organization of the exocytotic machinery. We have recently described that phosphorylation of AnxA2 on Tyr is critical for exocytosis. Considering that Tyr phosphorylation is known to promote AnxA2 externalization to the outer face of the plasma membrane in different cell types, we examined whether this phenomenon occurred in neurosecretory chromaffin cells. Using immunolabeling and biochemical approaches, we observed that nicotine stimulation triggered the egress of AnxA2 to the external leaflets of the plasma membrane in the vicinity of exocytotic sites. AnxA2 was found co-localized with tissue plasminogen activator, previously described on the surface of chromaffin cells following secretory granule release. We propose that AnxA2 might be a cell surface tissue plasminogen activator receptor for chromaffin cells, thus playing a role in autocrine or paracrine regulation of exocytosis.
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http://dx.doi.org/10.3390/cells9092059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564067PMC
September 2020

Mono- and Poly-unsaturated Phosphatidic Acid Regulate Distinct Steps of Regulated Exocytosis in Neuroendocrine Cells.

Cell Rep 2020 08;32(7):108026

Centre National de la Recherche Scientifique, Université de Strasbourg, Institut des Neurosciences Cellulaires et Intégratives, UPR-3212 67000 Strasbourg, France. Electronic address:

Specific forms of fatty acids are well known to have beneficial health effects, but their precise mechanism of action remains elusive. Phosphatidic acid (PA) produced by phospholipase D1 (PLD1) regulates the sequential stages underlying secretory granule exocytosis in neuroendocrine chromaffin cells, as revealed by pharmacological approaches and genetic mouse models. Lipidomic analysis shows that secretory granule and plasma membranes display distinct and specific composition in PA. Secretagogue-evoked stimulation triggers the selective production of several PA species at the plasma membrane near the sites of active exocytosis. Rescue experiments in cells depleted of PLD1 activity reveal that mono-unsaturated PA restores the number of exocytotic events, possibly by contributing to granule docking, whereas poly-unsaturated PA regulates fusion pore stability and expansion. Altogether, this work provides insight into the roles that subspecies of the same phospholipid may play based on their fatty acyl chain composition.
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http://dx.doi.org/10.1016/j.celrep.2020.108026DOI Listing
August 2020

The atypical Rho GTPase RhoU interacts with intersectin-2 to regulate endosomal recycling pathways.

J Cell Sci 2020 08 27;133(16). Epub 2020 Aug 27.

Centre National de la Recherche Scientifique, Université de Strasbourg, Institut des Neurosciences Cellulaires et Intégratives, F-67000 Strasbourg, France

Rho GTPases play a key role in various membrane trafficking processes. RhoU is an atypical small Rho GTPase related to Rac/Cdc42, which possesses unique N- and C-terminal domains that regulate its function and its subcellular localization. RhoU localizes at the plasma membrane, on endosomes and in cell adhesion structures where it governs cell signaling, differentiation and migration. However, despite its endomembrane localization, RhoU function in vesicular trafficking has been unexplored. Here, we identified intersectins (ITSNs) as new binding partners for RhoU and showed that the second PxxP motif at the N terminus of RhoU mediated interactions with the SH3 domains of ITSNs. To evaluate the function of RhoU and ITSNs in vesicular trafficking, we used fluorescent transferrin as a cargo for uptake experiments. We showed that silencing of either RhoU or ITSN2, but not ITSN1, increased transferrin accumulation in early endosomes, resulting from a defect in fast vesicle recycling. Concomitantly, RhoU and ITSN2 colocalized to a subset of Rab4-positive vesicles, suggesting that a RhoU-ITSN2 interaction may occur on fast recycling endosomes to regulate the fate of vesicular cargos.
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http://dx.doi.org/10.1242/jcs.234104DOI Listing
August 2020

Hormones Secretion and Rho GTPases in Neuroendocrine Tumors.

Cancers (Basel) 2020 Jul 10;12(7). Epub 2020 Jul 10.

Institut des Neurosciences Cellulaires et Intégratives, Centre National de la Recherche Scientifique, Université de Strasbourg, F-67000 Strasbourg, France.

Neuroendocrine tumors (NETs) belong to a heterogeneous group of neoplasms arising from hormone secreting cells. These tumors are often associated with a dysfunction of their secretory activity. Neuroendocrine secretion occurs through calcium-regulated exocytosis, a process that is tightly controlled by Rho GTPases family members. In this review, we compiled the numerous mutations and modification of expression levels of Rho GTPases or their regulators (Rho guanine nucleotide-exchange factors and Rho GTPase-activating proteins) that have been identified in NETs. We discussed how they might regulate neuroendocrine secretion.
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http://dx.doi.org/10.3390/cancers12071859DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7408961PMC
July 2020

High prevalence for obesity in severe COVID-19: Possible links and perspectives towards patient stratification.

Biochimie 2020 Dec 8;179:257-265. Epub 2020 Jul 8.

Centre National de la Recherche Scientifique, Université de Strasbourg, Institut des Neurosciences Cellulaires et Intégratives, F-67000, Strasbourg, France; Member of the scientific council of the Groupe d'Etude et de Recherche en Lipidomique (GERLI), France. Electronic address:

It is becoming obvious that in addition to aging and various hearth pathologies, excess of body weight, especially obesity is a major risk factor for severity of COVID-19 infection. Intriguingly the receptor for SARS-CoV-2 is ACE2, a member of the angiotensin receptor family that has a relatively large tissue distribution. This observation likely explains the multitude of symptoms that have been described from human patients. The adipose tissue also expresses ACE2, suggesting that adipocytes are potentially infected by SARS-CoV-2. Here we discuss some of the potential contribution of the adipose tissue to the severity of the infection and propose some aspects of obese patients metabolic phenotyping to help stratification of individuals with high risk of severe disease.
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http://dx.doi.org/10.1016/j.biochi.2020.07.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7340594PMC
December 2020

Fibrillarin Ribonuclease Activity is Dependent on the GAR Domain and Modulated by Phospholipids.

Cells 2020 05 6;9(5). Epub 2020 May 6.

Biochemistry and Molecular Plant Biology Department, Centro de Investigación Científica de Yucatán, A.C. Calle 43 No. 130, Colonia Chuburná de Hidalgo, Mérida C.P. 97200, Yucatán, Mexico.

Fibrillarin is a highly conserved nucleolar methyltransferase responsible for ribosomal RNA methylation across evolution from Archaea to humans. It has been reported that fibrillarin is involved in the methylation of histone H2A in nucleoli and other processes, including viral progression, cellular stress, nuclear shape, and cell cycle progression. We show that fibrillarin has an additional activity as a ribonuclease. The activity is affected by phosphoinositides and phosphatidic acid and insensitive to ribonuclease inhibitors. Furthermore, the presence of phosphatidic acid releases the fibrillarin-U3 snoRNA complex. We show that the ribonuclease activity localizes to the GAR (glycine/arginine-rich) domain conserved in a small group of RNA interacting proteins. The introduction of the GAR domain occurred in evolution in the transition from archaea to eukaryotic cells. The interaction of this domain with phospholipids may allow a phase separation of this protein in nucleoli.
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http://dx.doi.org/10.3390/cells9051143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290794PMC
May 2020

The receptor tyrosine kinase EPHB6 regulates catecholamine exocytosis in adrenal gland chromaffin cells.

J Biol Chem 2020 05 22;295(22):7653-7668. Epub 2020 Apr 22.

Research Centre, Centre hospitalier de l'Université de Montréal (CRCHUM), Montreal, Quebec, Canada

The erythropoietin-producing human hepatocellular receptor EPH receptor B6 (EPHB6) is a receptor tyrosine kinase that has been shown previously to control catecholamine synthesis in the adrenal gland chromaffin cells (AGCCs) in a testosterone-dependent fashion. EPHB6 also has a role in regulating blood pressure, but several facets of this regulation remain unclear. Using amperometry recordings, we now found that catecholamine secretion by AGCCs is compromised in the absence of EPHB6. AGCCs from male knockout (KO) mice displayed reduced cortical F-actin disassembly, accompanied by decreased catecholamine secretion through exocytosis. This phenotype was not observed in AGCCs from female KO mice, suggesting that testosterone, but not estrogen, contributes to this phenotype. Of note, reverse signaling from EPHB6 to ephrin B1 (EFNB1) and a 7-amino acid-long segment in the EFNB1 intracellular tail were essential for the regulation of catecholamine secretion. Further downstream, the Ras homolog family member A (RHOA) and FYN proto-oncogene Src family tyrosine kinase (FYN)-proto-oncogene c-ABL-microtubule-associated monooxygenase calponin and LIM domain containing 1 (MICAL-1) pathways mediated the signaling from EFNB1 to the defective F-actin disassembly. We discuss the implications of EPHB6's effect on catecholamine exocytosis and secretion for blood pressure regulation.
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http://dx.doi.org/10.1074/jbc.RA120.013251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7261780PMC
May 2020

Chromogranin A preferential interaction with Golgi phosphatidic acid induces membrane deformation and contributes to secretory granule biogenesis.

FASEB J 2020 05 29;34(5):6769-6790. Epub 2020 Mar 29.

Laboratoire de Différenciation et Communication Neuronale et Neuroendocrine, Institut de Recherche et d'Innovation Biomédicale de Normandie, Normandie University, UNIROUEN, INSERM, U1239, Rouen, France.

Chromogranin A (CgA) is a key luminal actor of secretory granule biogenesis at the trans-Golgi network (TGN) level but the molecular mechanisms involved remain obscure. Here, we investigated the possibility that CgA acts synergistically with specific membrane lipids to trigger secretory granule formation. We show that CgA preferentially interacts with the anionic glycerophospholipid phosphatidic acid (PA). In accordance, bioinformatic analysis predicted a PA-binding domain (PABD) in CgA sequence that effectively bound PA (36:1) or PA (40:6) in membrane models. We identified PA (36:1) and PA (40:6) as predominant species in Golgi and granule membranes of secretory cells, and we found that CgA interaction with these PA species promotes artificial membrane deformation and remodeling. Furthermore, we demonstrated that disruption of either CgA PABD or phospholipase D (PLD) activity significantly alters secretory granule formation in secretory cells. Our findings show for the first time the ability of CgA to interact with PLD-generated PA, which allows membrane remodeling and curvature, key processes necessary to initiate secretory granule budding.
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http://dx.doi.org/10.1096/fj.202000074RDOI Listing
May 2020

Phosphatidic acid metabolism regulates neuroendocrine secretion but is not under the direct control of lipins.

IUBMB Life 2020 04 22;72(4):533-543. Epub 2020 Jan 22.

Centre National de la Recherche Scientifique, Université de Strasbourg, Institut des Neurosciences Cellulaires et Intégratives, Strasbourg, France.

Phosphatidic acid (PA) produced by phospholipase D1 has been shown to contribute to secretory vesicle exocytosis in a large number of cell models. Among various hypotheses, PA may contribute to recruit and/or activate at the exocytotic site a set of proteins from the molecular machinery dedicated to secretion, but also directly influence membrane curvature thereby favoring membrane rearrangements required for membrane fusion. The release of informative molecules by regulated exocytosis is a tightly controlled process. It is thus expected that PA produced to trigger membrane fusion should be rapidly metabolized and converted in a lipid that does not present similar characteristics. PA-phosphatases of the lipin family are possible candidates as they convert PA into diacylglycerol. We show here that lipin 1 and lipin 2 are expressed in neuroendocrine cells where they are cytosolic, but also partially associated with the endoplasmic reticulum. Silencing of lipin 1 or 2 did not affect significantly either basal or evoked secretion from PC12 cells, suggesting that it is unlikely that conversion of PA into a secondary lipid by lipins might represent a regulatory step in exocytosis in neurosecretory cells. However, in agreement with a model in which PA-metabolism could contribute to prevent entering into exocytosis of additional secretory vesicles, ectopic expression of lipin1B-GFP in bovine chromaffin cells reduced the number of exocytotic events as revealed by carbon fiber amperometry recording. Furthermore, individual spike parameters reflecting fusion pore dynamics were also modified by lipin1B-GFP, suggesting that a tight control of PA levels represents an important regulatory step of the number and kinetic of exocytotic events.
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http://dx.doi.org/10.1002/iub.2229DOI Listing
April 2020

αII-spectrin controls calcium-regulated exocytosis in neuroendocrine chromaffin cells through neuronal Wiskott-Aldrich Syndrome protein interaction.

IUBMB Life 2020 04 20;72(4):544-552. Epub 2019 Dec 20.

Centre National de la Recherche Scientifique, Institut des Neurosciences Cellulaires et Intégratives, Université de Strasbourg, Strasbourg, France.

Besides a fundamental structural role at the plasma membrane, spectrin- and actin-based skeletons have been proposed to participate in various processes including vesicular trafficking. Neuroendocrine cells release hormones and neuropeptides through calcium-regulated exocytosis, a process that is coordinated by a fine remodeling of the actin cytoskeleton. We describe here that calcium-regulated exocytosis is impaired in chromaffin and PC12 cells with reduced αII-spectrin expression levels. Using yeast two-hybrid screening, we show that neuronal Wiskott-Aldrich Syndrome protein (N-WASP) is a partner of the αII-spectrin SH3 domain and demonstrate that secretagogue-evoked N-WASP recruitment at cell periphery is blocked in the absence of αII-spectrin. Additionally, experiments performed with ectopically expressed αII-spectrin mutant unable to bind N-WASP indicated that the interaction between SH3 domain and N-WASP is pivotal for neuroendocrine secretion. Our results extend the list of spectrin interactors and strengthen the idea that αII-spectrin is an important scaffold protein that gathers crucial actin-related players of the exocytic machinery.
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http://dx.doi.org/10.1002/iub.2217DOI Listing
April 2020

Pegvisomant in acromegaly: a multicenter real-life study in Argentina.

Arch Endocrinol Metab 2019 Aug 22;63(4):320-327. Epub 2019 Aug 22.

Departamento de Endocrinología Hospital Carlos G. Durand, CABA, Argentina.

Objective: To describe the long term safety and efficacy of pegvisomant (PEGV), and the predictors of treatment response in patients with acromegaly in the real life setting.

Subjects And Methods: We retrospectively reviewed the clinical, hormonal and radiological data of acromegalic patients treated with PEGV in 17 Argentine centers.

Results: Seventy-five patients (age range 22-77, 51 females) with acromegaly have been treated with PEGV for up to 118 months (median 27 months). Before PEGV, 97.3% of patients had been treated with medical therapy, surgery and/or radiotherapy, two patients had no previous treatment. At that time, all patients had an IGF-1 above the upper normal limit (ULN) (mean 2.4 x ULN ± 0.98, range 1.25-7). At diagnosis of acromegaly 84% presented macroadenomas, prior to PEGV only 23,5% of patients remained with tumor remnant > 1 cm, the remaining showed normal or less than 1 cm images. Disease control (IGF-1 ≤ 1.2 x ULN) was achieved in 62.9% of patients with a mean dose of 11.8 mg/day. Thirty-four patients (45%) received PEGV monotherapy, while 41 (55%) received combined therapy with either somatostatin analogues and/or cabergoline. Adverse events related to PEGV were: local injection site reaction in 5.3%, elevated liver enzymes in 9.3%, and tumor size growth in 9.8%. Pre-PEGV IGF-I level was the only predictor of treatment response: 2.1 x ULN vs 2.8 x ULN in controlled and uncontrolled patients respectively (p < 0.001).

Conclusion: this long term experience indicates PEGV treatment was highly effective and safe in our series of Argentine patients with acromegaly refractory to standard therapies. Arch Endocrinol Metab. 2019;63(4):320-7.
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http://dx.doi.org/10.20945/2359-3997000000160DOI Listing
August 2019

Regulation of Phospholipase D by Arf6 during FcγR-Mediated Phagocytosis.

J Immunol 2019 05 3;202(10):2971-2981. Epub 2019 Apr 3.

Centre National de la Recherche Scientifique, Université de Strasbourg, Institut des Neurosciences Cellulaires et Intégratives, F-67000 Strasbourg, France

Phagocytosis is an essential element of the immune response, assuring the elimination of pathogens, cellular debris, and apoptotic and tumoral cells. Activation of phagocytosis by the FcγR stimulates phospholipase D (PLD) activity and triggers the production of phosphatidic acid (PA) at the plasma membrane of macrophages, but the regulatory mechanisms involved are still not clearly understood. In this study, we examined the role of the small GTPase Arf6 in the activation of the PLD isoforms during FcγR-mediated phagocytosis. In RAW 264.7 macrophage cells, expressed Arf6-GFP partially colocalized with PLD1-hemagglutinin on intracellular membrane-bound vesicles and with PLD2-hemagglutinin at the plasma membrane. Both PLD isoforms were found to interact with Arf6 during FcγR-mediated phagocytosis as seen by immunoprecipitation experiments. In macrophages stimulated for phagocytosis, Arf6 was observed to be associated with nascent phagosomes. RNA interference knockdown of Arf6 reduced the amount of active Arf6 associated with phagosomes, revealed by the MT2-GFP probe that specifically binds to Arf6-GTP. Arf6 silencing concomitantly decreased PLD activity as well as the levels of PA found on phagosomes and phagocytic sites as shown with the PA probe Spo20p-GFP. Altogether, our results indicate that Arf6 is involved in the regulation of PLD activity and PA synthesis required for efficient phagocytosis.
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http://dx.doi.org/10.4049/jimmunol.1801019DOI Listing
May 2019

EPHB6 controls catecholamine biosynthesis by up-regulating tyrosine hydroxylase transcription in adrenal gland chromaffin cells.

J Biol Chem 2019 04 1;294(17):6871-6887. Epub 2019 Mar 1.

From the Research Centre and

EPHB6 is a member of the erythropoietin-producing hepatocellular kinase (EPH) family and a receptor tyrosine kinase with a dead kinase domain. It is involved in blood pressure regulation and adrenal gland catecholamine (CAT) secretion, but several facets of EPHB6-mediated CAT regulation are unclear. In this study, using biochemical, quantitative RT-PCR, immunoblotting, and gene microarray assays, we found that EPHB6 up-regulates CAT biosynthesis in adrenal gland chromaffin cells (AGCCs). We observed that epinephrine content is reduced in the AGCCs from male Ephb6-KO mice, caused by decreased expression of tyrosine hydroxylase, the rate-limiting enzyme in CAT biosynthesis. We demonstrate that the signaling pathway from EPHB6 to tyrosine hydroxylase expression in AGCCs involves Rac family small GTPase 1 (RAC1), MAP kinase kinase 7 (MKK7), c-Jun N-terminal kinase (JNK), proto-oncogene c-Jun, activator protein 1 (AP1), and early growth response 1 (EGR1). On the other hand, signaling via extracellular signal-regulated kinase (ERK1/2), p38 mitogen-activated protein kinase, and ELK1, ETS transcription factor (ELK1) was not affected by EPHB6 deletion. We further report that EPHB6's effect on AGCCs was via reverse signaling through ephrin B1 and that EPHB6 acted in concert with the nongenomic effect of testosterone to control CAT biosynthesis. Our findings elucidate the mechanisms by which EPHB6 modulates CAT biosynthesis and identify potential therapeutic targets for diseases, such as hypertension, caused by dysfunctional CAT biosynthesis.
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http://dx.doi.org/10.1074/jbc.RA118.005767DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6497964PMC
April 2019

Phosphatidic Acid: From Pleiotropic Functions to Neuronal Pathology.

Front Cell Neurosci 2019 23;13. Epub 2019 Jan 23.

Institut des Neurosciences Cellulaires et Intégratives (INCI), UPR-3212 Centre National de la Recherche Scientifique & Université de Strasbourg, Strasbourg, France.

Among the cellular lipids, phosphatidic acid (PA) is a peculiar one as it is at the same time a key building block of phospholipid synthesis and a major lipid second messenger conveying signaling information. The latter is thought to largely occur through the ability of PA to recruit and/or activate specific proteins in restricted compartments and within those only at defined submembrane areas. Furthermore, with its cone-shaped geometry PA locally changes membrane topology and may thus be a key player in membrane trafficking events, especially in membrane fusion and fission steps, where lipid remodeling is believed to be crucial. These pleiotropic cellular functions of PA, including phospholipid synthesis and homeostasis together with important signaling activity, imply that perturbations of PA metabolism could lead to serious pathological conditions. In this mini-review article, after outlining the main cellular functions of PA, we highlight the different neurological diseases that could, at least in part, be attributed to an alteration in PA synthesis and/or catabolism.
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http://dx.doi.org/10.3389/fncel.2019.00002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6351798PMC
January 2019

Phosphorylation cycling of Annexin A2 Tyr23 is critical for calcium-regulated exocytosis in neuroendocrine cells.

Biochim Biophys Acta Mol Cell Res 2019 07 2;1866(7):1207-1217. Epub 2019 Jan 2.

INCI, UPR3212 CNRS, Université de Strasbourg, France; Plateforme Imagerie In Vitro, Neuropôle de Strasbourg, 5 rue Blaise Pascal, F-6708 Strasbourg, France. Electronic address:

Annexin A2 (AnxA2) is a calcium and lipid binding protein involved in neuroendocrine secretion. We have previously demonstrated that AnxA2 participates in the formation and/or stabilization of lipid microdomains required for structural and spatial organization of the exocytotic machinery in chromaffin cells. However, the regulation of AnxA2 is not fully understood. Numerous phosphorylation sites have been identified in the amino-terminal domain of AnxA2. Phosphorylation of Ser25 and Tyr23 are well established and confirmed to be functionally significant. In particular, phosphorylation of Tyr23 by the tyrosine kinase pp60Src reduces the binding of AnxA2 to both actin filaments and the plasma membrane, two major actors of exocytosis, thus, we examined whether AnxA2 was phosphorylated on Tyr23 during exocytosis. Using immunolabelling and a biochemical approach, we found that nicotine stimulation triggered the phosphorylation of Anx A2 on Tyr23. The expression of two AnxA2 mutants carrying phosphorylation deficient (Y23A) or phosphomimetic (Y23E) mutations reduced the number exocytotic sites. Furthermore, expression of AnxA2-Y23A inhibited the formation of lipid microdomains, whereas the expression of AnxA2-Y23E altered actin filaments associated with docked granules. These results suggest that phosphorylation/dephosphorylation switch at Tyr23 in AnxA2 is critical for calcium-regulated exocytosis in neuroendocrine cells. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
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http://dx.doi.org/10.1016/j.bbamcr.2018.12.013DOI Listing
July 2019

Role of Phospholipase D-Derived Phosphatidic Acid in Regulated Exocytosis and Neurological Disease.

Handb Exp Pharmacol 2020 ;259:115-130

Institut des Neurosciences Cellulaires et Intégratives, CNRS UPR 3212 and Université de Strasbourg, Strasbourg, France.

Lipids play a vital role in numerous cellular functions starting from a structural role as major constituents of membranes to acting as signaling intracellular or extracellular entities. Accordingly, it has been known for decades that lipids, especially those coming from diet, are important to maintain normal physiological functions and good health. On the other side, the exact molecular nature of these beneficial or deleterious lipids, as well as their precise mode of action, is only starting to be unraveled. This recent improvement in our knowledge is largely resulting from novel pharmacological, molecular, cellular, and genetic tools to study lipids in vitro and in vivo. Among these important lipids, phosphatidic acid plays a unique and central role in a great variety of cellular functions. This review will focus on the proposed functions of phosphatidic acid generated by phospholipase D in the last steps of regulated exocytosis with a specific emphasis on hormonal and neurotransmitter release and its potential impact on different neurological diseases.
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http://dx.doi.org/10.1007/164_2018_180DOI Listing
August 2020

Effects of phospholipase D during cultured osteoblast mineralization and bone formation.

J Cell Biochem 2019 04 15;120(4):5923-5935. Epub 2018 Oct 15.

Univ Lyon, Université Claude Bernard Lyon 1, CNRS UMR 5246, ICBMS, Lyon, France.

Mammalian phospholipase D (PLD) mostly hydrolyzes phosphatidylcholine producing phosphatidic acid. PLD activity was previously detected in different osteoblastic cell models, and was increased by several growth factors involved in bone homeostasis. To confirm possible actions of PLD isoforms during mineralization process, we analyzed their effects in osteoblastic cell models and during bone formation. PLD1 expression, along with PLD activity, increased during differentiation of primary osteoblasts and Saos-2 cells, and peaked at the onset of mineralization. Subsequently, both PLD1 expression and PLD activity decreased, suggesting that PLD1 function is regulated during osteoblast maturation. In contrast, PLD2 expression was not significantly affected during differentiation of osteoblasts. Overexpression of PLD1 in Saos-2 cells improved their mineralization potential. PLD inhibitor Halopemide or PLD1-selective inhibitor, led to a decrease in mineralization in both cell types. On the contrary, the selective inhibitor of PLD2, did not affect the mineralization process. Moreover, primary osteoblasts isolated from PLD1 knockout (KO) mice were significantly less efficient in mineralization as compared with those isolated from wild type (WT) or PLD2 KO mice. In contrast, bone formation, as monitored by high-resolution microcomputed tomography analysis, was not impaired in PLD1 KO nor in PLD2 KO mice, indicating that the lack of PLD1 or that of PLD2 did not affect the bone structure in adult mice. Taken together, our findings indicate that PLD activity, especially which of PLD1 isoform, may enhance the mineralization process in osteoblastic cells. Nonetheless, the lack of PLD1 or PLD2 do not seem to significantly affect bone formation in adult mice.
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http://dx.doi.org/10.1002/jcb.27881DOI Listing
April 2019

Of local translation control and lipid signaling in neurons.

Adv Biol Regul 2019 01 15;71:194-205. Epub 2018 Sep 15.

Université de Strasbourg, 67084, Strasbourg, France; Institut des Neurosciences Cellulaires et Intégratives, UPR3212 CNRS, 67084, Strasbourg, France.

Fine-tuned regulation of new proteins synthesis is key to the fast adaptation of cells to their changing environment and their response to external cues. Protein synthesis regulation is particularly refined and important in the case of highly polarized cells like neurons where translation occurs in the subcellular dendritic compartment to produce long-lasting changes that enable the formation, strengthening and weakening of inter-neuronal connection, constituting synaptic plasticity. The changes in local synaptic proteome of neurons underlie several aspects of synaptic plasticity and new protein synthesis is necessary for long-term memory formation. Details of how neuronal translation is locally controlled only start to be unraveled. A generally accepted view is that mRNAs are transported in a repressed state and are translated locally upon externally cued triggering signaling cascades that derepress or activate translation machinery at specific sites. Some important yet poorly considered intermediates in these cascades of events are signaling lipids such as diacylglycerol and its balancing partner phosphatidic acid. A link between these signaling lipids and the most common inherited cause of intellectual disability, Fragile X syndrome, is emphasizing the important role of these secondary messages in synaptically controlled translation.
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http://dx.doi.org/10.1016/j.jbior.2018.09.005DOI Listing
January 2019

Phospholipase D: A new mediator during high phosphate-induced vascular calcification associated with chronic kidney disease.

J Cell Physiol 2019 04 12;234(4):4825-4839. Epub 2018 Sep 12.

University of Lyon, Université Claude Bernard Lyon 1 (UCBL), CNRS UMR 5246, Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Lyon, France.

Vascular calcification (VC) is the pathological accumulation of calcium phosphate crystals in one of the layers of blood vessels, leading to loss of elasticity and causing severe calcification in vessels. Medial calcification is mostly seen in patients with chronic kidney disease (CKD) and diabetes. Identification of key enzymes and their actions during calcification will contribute to understand the onset of pathological calcification. Phospholipase D (PLD1, PLD2) is active at the earlier steps of mineralization in osteoblasts and chondrocytes. In this study, we aimed to determine their effects during high-phosphate treatment in mouse vascular smooth muscle cell line MOVAS, in the ex vivo model of the rat aorta, and in the in vivo model of adenine-induced CKD. We observed an early increase in PLD1 gene and protein expression along with the increase in the PLD activity in vascular muscle cell line, during calcification induced by ascorbic acid and β-glycerophosphate. Inhibition of PLD1 by the selective inhibitor VU0155069, or the pan-PLD inhibitor, halopemide, prevented calcification. The mechanism of PLD activation is likely to be protein kinase C (PKC)-independent since bisindolylmaleimide X hydrochloride, a pan-PKC inhibitor, did not affect the PLD activity. In agreement, we found an increase in Pld1 gene expression and PLD activity in aortic explant cultures treated with high phosphate, whereas PLD inhibition by halopemide decreased calcification. Finally, an increase in both Pld1 and Pld2 expression occurred simultaneously with the appearance of VC in a rat model of CKD. Thus, PLD, especially PLD1, promotes VC in the context of CKD and could be an important target for preventing onset or progression of VC.
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http://dx.doi.org/10.1002/jcp.27281DOI Listing
April 2019

The caveolae-associated coiled-coil protein, NECC2, regulates insulin signalling in Adipocytes.

J Cell Mol Med 2018 11 30;22(11):5648-5661. Epub 2018 Aug 30.

Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Córdoba, Spain.

Adipocyte dysfunction in obesity is commonly associated with impaired insulin signalling in adipocytes and insulin resistance. Insulin signalling has been associated with caveolae, which are coated by large complexes of caveolin and cavin proteins, along with proteins with membrane-binding and remodelling properties. Here, we analysed the regulation and function of a component of caveolae involved in growth factor signalling in neuroendocrine cells, neuroendocrine long coiled-coil protein-2 (NECC2), in adipocytes. Studies in 3T3-L1 cells showed that NECC2 expression increased during adipogenesis. Furthermore, NECC2 co-immunoprecipitated with caveolin-1 (CAV1) and exhibited a distribution pattern similar to that of the components of adipocyte caveolae, CAV1, Cavin1, the insulin receptor and cortical actin. Interestingly, NECC2 overexpression enhanced insulin-activated Akt phosphorylation, whereas NECC2 downregulation impaired insulin-induced phosphorylation of Akt and ERK2. Finally, an up-regulation of NECC2 in subcutaneous and omental adipose tissue was found in association with human obesity and insulin resistance. This effect was also observed in 3T3-L1 adipocytes exposed to hyperglycaemia/hyperinsulinemia. Overall, the present study identifies NECC2 as a component of adipocyte caveolae that is regulated in response to obesity and associated metabolic complications, and supports the contribution of this protein as a molecular scaffold modulating insulin signal transduction at these membrane microdomains.
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http://dx.doi.org/10.1111/jcmm.13840DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6201366PMC
November 2018
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