Pierre Vacher | Université de Bordeaux (original) (raw)

Papers by Pierre Vacher

Cancer Research, 2013

Triple-negative breast cancers (TNBC) lacking estrogen and progesterone receptors and HER2 amplif... more Triple-negative breast cancers (TNBC) lacking estrogen and progesterone receptors and HER2 amplification have a relatively high risk of metastatic dissemination, but the mechanistic basis for this risk is not understood. Here we report that serum levels of CD95L are higher in TNBC patients compared to other breast cancer patients. Metalloprotease-mediated cleavage of CD95L expressed by endothelial cells surrounding tumors generates a gradient that promotes cell motility, due to formation of an unconventional CD95-containing receptosome termed the motility-inducing signaling complex. Formation of this complex was instrumental for Nox3driven ROS generation. Mechanistic investigations revealed a Yes-Orai1-EGFR-PI3K pathway that triggered migration of TNBC cells exposed to CD95L. Our findings establish a prometastatic function for metalloprotease-cleaved CD95L in TNBCs, revisiting its role in carcinogenesis.

Neuroendocrinology, 1996

Arachidonic acid (AA) has been implicated in signaling actions in several cell types including en... more Arachidonic acid (AA) has been implicated in signaling actions in several cell types including endocrine cells. In the present study, we investigated the effect of exogenous AA on GH release from dispersed pituitary cells and tried to elucidate the mechanism involved in this process. We show that AA stimulates GH release in a dose- and extracellular calcium-dependent manner. The effects of AA on cytosolic calcium concentration ([Ca2+]i) were studied using dual-emission microspectrofluorimetry in identified somatotropes. AA (1 microM) induced an increase in intracellular calcium concentration ([Ca2+]i) by stimulating Ca2+ influx through dihydropyridine-sensitive, voltage-dependent calcium channels. In these cells, the effects of AA were only reduced by the inhibition of protein kinase C (PKC) activity, suggesting that the fatty acid may act by both PKC-dependent and PKC-independent pathways. In order to determine whether AA metabolites were involved in the effects attributed to AA, and, if so, which ones, we inhibited the three arachidonate metabolic pathways: cyclo-oxygenase by indomethacin (50 microM), lipoxygenase by nordihydroguaiaretic acid (NGDA, 50 microM), and epoxygenase by 5,8,11, 14-eicosatetraynoic acid (ETYA, 10 microM). NGDA and ETYA reduced the effects of AA on GH release (50 and 74%, respectively) and inhibited the [Ca2+]i response, whereas indomethacin slightly potentiated both AA-induced GH release and [Ca2+]i increase. As these results suggested that lipoxygenase metabolites may be responsible for AA-induced Ca2+ influx and GH release, we tested the effects of 5-, 12- and 15-hydroperoxyeicosatetraenoic acids (5-, 12- and 15-HpETE) on [Ca2+]i and GH release. They all stimulated calcium influx and GH release in a dose-dependent manner, 12-HpETE being more potent than 5- and 15-HpETE. We conclude that lipoxygenase metabolites of arachidonic acid, particularly 12-HpETE, may be involved in the GH secretion mechanism, probably by facilitating Ca2+ influx via L-type Ca2+ channels.

Annals of the New York Academy of Sciences, 1989

Journal of immunology (Baltimore, Md. : 1950), 2015

The anti-CD20 mAb, rituximab, is routinely used to treat B cell malignancies. However, a majority... more The anti-CD20 mAb, rituximab, is routinely used to treat B cell malignancies. However, a majority of patients relapse. An improvement in the complete response was obtained by combining rituximab with chemotherapy, at the cost of increased toxicity. We reported that rituximab induced the colocalization of both the Orai1 Ca(2+) release-activated Ca(2+) channel (CRAC) and the endoplasmic reticulum Ca(2+) sensor stromal interaction molecule 1 with CD20 and CD95 into a cluster, eliciting a polarized store-operated Ca(2+) entry (SOCE). We observed that blocking this Ca(2+) entry with downregulation of Orai1, pharmacological inhibitors, or reducing calcemia with hypocalcemic drugs sensitized human B lymphoma cell lines and primary human lymphoma cells to rituximab-induced apoptosis in vitro, and improved the antitumoral effect of rituximab in xenografted mice. This revealed that Ca(2+) entry exerted a negative feedback loop on rituximab-induced apoptosis, suggesting that associating CRAC c...

Glia, 2002

Prolactin (PRL) has several physiological effects on peripheral tissues and the brain. This hormo... more Prolactin (PRL) has several physiological effects on peripheral tissues and the brain. This hormone acts via its membrane receptor (PRL-R) to induce cell differentiation or proliferation. Using reverse transcription-polymerase chain reaction (RT-PCR) combined with Southern blot analysis, we detected PRL-R transcripts in a human glioma cell line (U87-MG) and in primary cultured human glioblastoma cells. These transcripts were deleted or not in their extracellular domains. We examined the effects of PRL on intracellular free Ca 2ϩ concentration ([Ca 2ϩ ] i ) in these cells in order to improve our understanding of the PRL transduction mechanism, which is still poorly documented. [Ca 2ϩ ] i was measured by microspectrofluorimetry using indo-1 as the Ca 2ϩ fluorescent probe. Spatiotemporal aspects of PRL-induced Ca 2ϩ signals were investigated using high-speed fluo-3 confocal imaging. We found that physiological concentrations (0.4-4 nM) of PRL-stimulated Ca 2ϩ entry and intracellular Ca 2ϩ mobilization via a tyrosine kinase-dependent mechanism. The two types of Ca 2ϩ responses observed were distinguishable by their kinetics: one showing a slow (type I) and the other a fast (type II) increase in [Ca 2ϩ ] i . The amplitude of PRL-induced Ca 2ϩ increases may be sufficient to provoke several physiological responses, such as stimulating proliferation. Furthermore, PRL induced a dose-dependent increase in [ 3 H]thymidine incorporation levels and in cellular growth and survival, detected by the MTT method. These data indicate that PRL induced mitogenesis of human glioma cells. GLIA 38:200-214, 2002.

Endocrinology, 2007

Prolonged exposure of ␤-cells to high glucose (glucotoxicity) diminishes insulin secretion in res... more Prolonged exposure of ␤-cells to high glucose (glucotoxicity) diminishes insulin secretion in response to glucose and has been linked to altered generation of metabolism-secretion coupling factors. We have investigated whether glucotoxicity may also alter calcium handling and late steps in secretion such as exocytosis. Clonal INS-1E ␤-cells cultured at high glucose (20 or 30 mM vs. 5.5 mM) for 72 h exhibited elevated basal intracellular calcium ([Ca 2؉ ] i ), which was K ATP -channel dependent and due to long-term activation of protein kinase A. An increased amplitude and shortened duration of depolarization-evoked rises in [Ca 2؉ ] i were apparent. These changes were probably linked to the observed increased filling of intracellular stores and to short-term activation of protein kinase A. Insulin secretion was reduced not only by acute stimulation with either glucose or KCl but more importantly by direct calcium stimulation of permeabilized cells. These findings indicate a defect in the final steps of exocytosis. To con-firm this, we measured expression levels of some 30 proteins implicated in trafficking/exocytosis of post-Golgi vesicles. Several proteins required for calcium-induced exocytosis of secretory granules were down-regulated, such as the soluble N-ethylmaleimide-sensitive factor-sensitive factor attachment receptor (SNARE) proteins VAMP-2 [vesicle (v)-SNARE, vesicle-associated membrane protein 2] and syntaxin 1 as well as complexin. VAMP-2 was also reduced in human islets. In contrast, cell immunostaining and expression levels of several fluorescent proteins suggested that other post-trans-Golgi trafficking steps and compartments are preserved and that cells were not degranulated. Thus, these studies indicate that, in addition to known metabolic changes, glucotoxicity impedes generation of signals for secretion and diminishes the efficiency of late steps in exocytosis. (Endocrinology

Endocrinology, 1994

The mechanism of transduction of the PRL signal in target cells is poorly understood. We examined... more The mechanism of transduction of the PRL signal in target cells is poorly understood. We examined the effects of PRL on the intracellular free Ca2+ concentration in Chinese hamster ovary cells overexpressing functional PRL receptors. [Ca2+]i was determined by dual emission microspectrofluorimetry using indo-1 as the Ca2+ fluorescent probe. We demonstrate that at physiological concentrations (0.5-5 nM), PRL stimulates Ca2+ entry (type I) and/or induces a mobilization of calcium ions stored in intracellular compartments (type II). Two types of Ca2+ mobilization, distinguishable by their onset kinetics, were observed, a slow mobilization (type IIa; transition time to peak, approximately 10 sec) and a fast mobilization (type IIb; transition time to peak, < 2 sec). PRL responses were delayed (15-120 sec) compared to the well known activation by phosphatidylinositol 4,5 bisphosphate hydrolysis-coupled receptors. This suggests that inositol trisphosphate is not involved in PRL response or that phosphatidylinositol 4,5 bisphosphate hydrolysis is not directly coupled to the PRL receptor. The amplitude of the PRL-induced Ca2+ increases (300-1400 nM) would be sufficient to provoke several physiological responses, such as stimulation of secretion, cell proliferation, or gene activation. However, the relation between the increase in Ca2+ and activation of milk protein genes remains to be established.

Diabetes & Metabolism, 2010

Diabetes & Metabolism, 2011

ABSTRACT Introduction L’augmentation de la [Ca2+]i dans les cellules β constitue l’action clé du ... more ABSTRACT Introduction L’augmentation de la [Ca2+]i dans les cellules β constitue l’action clé du glucose permettant la sécrétion d’insuline et la régulation de l’expression génique. Le glucose induit également la synthèse d’AMPc par les adénylates cyclases, un second messager jusqu’à présent considéré uniquement comme un amplificateur des effets du glucose. Nous avons récemment publié dans Diabetologia qu’une isoforme spécifique d’adénylate cyclase activée par le Ca2+, l’ADCY8, est responsable des effets du GLP-1 et est impliquée dans la glucotoxicité. Dans la présente étude, nous montrons l’importance de l’ADCY8 cette fois dans la réponse au glucose lui-même. Matériels et méthodes La [Ca2+]i a été mesurée avec la sonde INDO-1 dans les cellules INS-1 et primaires de souris. L’activité électrique a été déterminée par enregistrements extracellulaires sur microelectrode arrays et la sécrétion d’insuline par ELISA. Résultats Les réponses calciques, électriques et sécrétoires à 15 mM de glucose sont fortement diminuées par des inhibiteurs des voies dépendantes de l’AMPc (SQ22,536, Rp-cAMPS, H-89). L’ADCY8 joue un rôle central puisque la réponse calcique au glucose est augmentée lorsque l’ADCY8 est surexprimée, fortement réduite par un ARNsh spécifique et totalement restaurée suite à la réIntroduction d’AMPc perméant dans les cellules knockdown pour l’ADCY8. Le mécanisme mis en jeu ici semble spécifique du glucose puisque les réponses calciques et sécrétoires au KCl ne sont altérées ni par le Rp-cAMPS, ni par le knockdown de l’ADCY8. Enfin des mesures réalisées chez des souris knockout pour l’ADCY8 montrent une intolérance au glucose en prise orale sans altération de sensibilité à l’insuline et des réponses calciques au glucose fortement réduites dans les îlots. Conclusion Ces résultats démontrent que l’ADCY8 est indispensable à la réponse des cellules β au glucose. Étant donné son rôle dans la glucotoxicité, ceci suggère fortement l’implication de l’ADCY8 dans le diabète de type 2.

Cell Calcium, 2004

Agents mobilising Ca 2+ from the endoplasmic reticulum are known to activate apoptosis. Whatever ... more Agents mobilising Ca 2+ from the endoplasmic reticulum are known to activate apoptosis. Whatever means are used, the release of Ca 2+ is often followed by a store-dependent entry of Ca 2+ . Whether apoptosis is triggered by the depletion of the stores or by the subsequent store-dependent entry of Ca 2+ is still a matter of controversy. Here we studied apoptosis in CHO cells transfected with the rat neurotensin (NT) receptor, in which the store-dependent entry of Ca 2+ is abolished by repressing the transient receptor potential channel 2 (TRPC2) by an antisense oligonucleotide strategy (TRPC2 − cells) [Cell Calcium 30 ]. When stimulated with thapsigargin (TG), apoptosis occurred in both TRPC2 + and TRPC2 − cells but 12 h earlier in TRPC2 + cells, suggesting that store-dependent entry of Ca 2+ can accelerate the process. The expression and localisation of caspase-12, an enzyme that has been involved in the apoptosis triggered by a stress on the endoplasmic reticulum, was not different in TRPC2 + and TRPC2 − cells. On the contrary, the expression of GADD153 (Growth Arrest and DNA Damage inducible gene 153) triggered by TG treatment depended on external Ca 2+ and occurred earlier in TRPC2 + than in TRPC2 − cells. In these cells, we also noted the presence of K + channels activated by Ca 2+ (K Ca channels). Stimulation of TRPC2 + cells with TG or with NT triggered a long sustained K + current, parallel to [Ca 2+ ] i transients, and resulting in a sustained hyperpolarisation of the cell membrane. K + current and hyperpolarisation were transient and not sustained in TRPC2 − cells. Inhibition of K Ca channels with charybdotoxin dramatically reduced the K + current and also significantly brought down the level of apoptosis, suggesting that a prolonged efflux of K + could be involved in the apoptosis process. We conclude that in CHO cells, store-dependent entry of Ca 2+ can accelerate apoptosis by accelerating the expression of GADD153 and by inducing a prolonged efflux of K + out of the cell.

Biophysical Journal, 2008

We investigate the mode of action of Cateslytin, an antimicrobial peptide, on zwitterionic biomem... more We investigate the mode of action of Cateslytin, an antimicrobial peptide, on zwitterionic biomembranes by performing numerical simulations and electrophysiological measurements on membrane vesicles. Using this natural b-sheet antimicrobial peptide secreted during stress as a model we show that a single peptide is able to form a stable membrane pore of 1 nm diameter of 0.25 nS conductance found both from calculation and electrical measurements. The resulting structure does not resemble the barrel-stave or carpet models earlier predicted, but is very close to that found in the simulation of a-helical peptides. Based on the simulation of a mutated peptide and the effects of small external electric fields, we conclude that electrostatic forces play a crucial role in the process of pore formation.

PLoS Biology, 2011

Patients affected by chronic inflammatory disorders display high amounts of soluble CD95L. This h... more Patients affected by chronic inflammatory disorders display high amounts of soluble CD95L. This homotrimeric ligand arises from the cleavage by metalloproteases of its membrane-bound counterpart, a strong apoptotic inducer. In contrast, the naturally processed CD95L is viewed as an apoptotic antagonist competing with its membrane counterpart for binding to CD95. Recent reports pinpointed that activation of CD95 may attract myeloid and tumoral cells, which display resistance to the CD95-mediated apoptotic signal. However, all these studies were performed using chimeric CD95Ls (oligomerized forms), which behave as the membrane-bound ligand and not as the naturally processed CD95L. Herein, we examine the biological effects of the metalloprotease-cleaved CD95L on CD95-sensitive activated T-lymphocytes. We demonstrate that cleaved CD95L (cl-CD95L), found increased in sera of systemic lupus erythematosus (SLE) patients as compared to that of healthy individuals, promotes the formation of migrating pseudopods at the leading edge of which the death receptor CD95 is capped (confocal microscopy). Using different migration assays (wound healing/Boyden Chamber/endothelial transmigration), we uncover that cl-CD95L promotes cell migration through a c-yes/Ca 2+ /PI3K-driven signaling pathway, which relies on the formation of a CD95-containing complex designated the MISC for Motility-Inducing Signaling Complex. These findings revisit the role of the metalloprotease-cleaved CD95L and emphasize that the increase in cl-CD95L observed in patients affected by chronic inflammatory disorders may fuel the local or systemic tissue damage by promoting tissuefiltration of immune cells.