Differential β₂-adrenergic receptor expression defines the phenotype of non-tumorigenic and malignant human breast cell lines

Breast cancer is the most frequent malignancy in women. Several reports demonstrated that adrenergic receptors (ARs) are involved in breast cancer. Here we observed that epinephrine (Epi), an endogenous AR agonist, caused opposite effects in non-tumorigenic (MCF-10A and HBL-100) and tumor cells (MCF-7 and MDA-MB-231). Thus, Epi, in non-tumor breast cells, as well as isoproterenol (β-agonist), in all cell lines, maintained a benign phenotype, decreasing cell proliferation and migration, and stimulating cell adhesion. β-AR expression and cAMP levels were higher in MCF-10A than in MCF-7 cells. β₂-AR knock-down caused a significant increase of cell proliferation and migration, and a decrease of cell adhesion both in basal and in Iso-stimulated conditions. Coincidently, β₂-AR over-expression induced a significant decrease of cell proliferation and migration, and an increase of cell adhesion. Therefore, β₂-AR is implied in cell phenotype and its agonists or antagonists could eventually complement cancer therapy.Fil: Gargiulo, Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Copsel, Sabrina Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología; ArgentinaFil: Rivero, Ezequiel Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Galés, Céline. Inserm; FranciaFil: Sénard, Jean Michel. Inserm; FranciaFil: Luthy, Isabel Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Davio, Carlos Alberto. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología; ArgentinaFil: Bruzzone, Ariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

The experimental power of FR900359 to study Gq-regulated biological processes.

Despite the discovery of heterotrimeric αβγ G proteins ∼25 years ago, their selective perturbation by cell-permeable inhibitors remains a fundamental challenge. Here we report that the plant-derived depsipeptide FR900359 (FR) is ideally suited to this task. Using a multifaceted approach we systematically characterize FR as a selective inhibitor of Gq/11/14 over all other mammalian Gα isoforms and elaborate its molecular mechanism of action. We also use FR to investigate whether inhibition of Gq proteins is an effective post-receptor strategy to target oncogenic signalling, using melanoma as a model system. FR suppresses many of the hallmark features that are central to the malignancy of melanoma cells, thereby providing new opportunities for therapeutic intervention. Just as pertussis toxin is used extensively to probe and inhibit the signalling of Gi/o proteins, we anticipate that FR will at least be its equivalent for investigating the biological relevance of Gq

A Cell-Permeable Inhibitor to Trap Gαq Proteins in the Empty Pocket Conformation

In spite of the crucial role of heterotrimeric G proteins as molecular switches transmitting signals from G protein-coupled receptors, their selective manipulation with small molecule, cell-permeable inhibitors still remains an unmet challenge. Here, we report that the small molecule BIM-46187, previously classified as pan-G protein inhibitor, preferentially silences Gαq signaling in a cellular context-dependent manner. Investigations into its mode of action reveal that BIM traps Gαq in the empty pocket conformation by permitting GDP exit but interdicting GTP entry, a molecular mechanism not yet assigned to any other small molecule Gα inhibitor to date. Our data show that Gα proteins may be “frozen” pharmacologically in an intermediate conformation along their activation pathway and propose a pharmacological strategy to specifically silence Gα subclasses with cell-permeable inhibitors

Plasticité des récepteurs couplés aux protéines G et signalisation

Les récepteurs couplés aux protéines G (RCPG) représentent la plus grande famille de récepteurs de surface régulant l’ensemble des fonctions physiologiques et constituent aujourd’hui des cibles médicamenteuses majeures. Jusqu’à récemment, le mode de fonctionnement de ces récepteurs était assimilé à un interrupteur on/off, présentant le ligand physiologique agoniste comme capable d’allumer l’ensemble des voies de signalisation associées à ce récepteur. Le concept d’agonisme biaisé a laissé place très récemment à la notion de sélectivité fonctionnelle des ligands, capables de privilégier l’activation d’une voie de signalisation particulière par rapport à une autre et, potentiellement, d’améliorer l’efficacité des médicaments en diminuant les effets secondaires indésirables. Néanmoins, la publication récente de nouveaux biosenseurs ultrasensibles capables de sonder les premiers événements de la signalisation en aval du récepteur actif, associés à l’analyse d’effecteurs de la signalisation plus en aval viennent remettre en question la définition de l’agonisme biaisé. Cette synthèse fait le point sur l’importance de la sensibilité des essais fonctionnels dans l’identification des agonistes biaisés et sur les stratégies futures de cartographie pharmacologique permettant la sélection de candidats médicaments aux effets thérapeutiques optimisés

[New talk between receptor and trimeric G proteins: "an intertwined body dance"]

International audienc

Le réveil des cardiomyocytes adultes résidents

Les pathologies cardiaques, et en particulier l’infarctus du myocarde, conduisent inéluctablement à la mort des cardiomyocytes adultes favorisant le développement de l’insuffisance cardiaque. Les efforts en médecine cardiaque régénératrice se sont concentrés jusqu’à présent sur l’utilisation des cellules souches, laissant de côté les cardiomyocytes préexistants, considérés comme étant dans un état postmitotique. Néanmoins, des données récentes obtenues chez le poisson zèbre et les mammifères relancent le débat sur la capacité proliférative de ces cellules. Dans cette revue, nous proposons un état des lieux des connaissances de la capacité proliférative et régénératrice des cardiomyocytes résidents, et discutons certains aspects mécanistiques. Dans le futur, l’identification précise des mécanismes moléculaires permettant à ces cellules de reprendre leur prolifération devrait permettre d’ouvrir de nouvelles perspectives thérapeutiques en régénération cardiaque

Probing heterotrimeric G protein activation: applications to biased ligands.

International audienceCell surface G protein-coupled receptors (GPCRs) drive numerous signaling pathways involved in the regulation of a broad range of physiologic processes. Today, they represent the largest target for modern drugs development with potential application in all clinical fields. Recently, the concept of "ligand-directed trafficking" has led to a conceptual revolution in pharmacological theory, thus opening new avenues for drug discovery. Accordingly, GPCRs do not function as simple on-off switch but rather as filters capable of selecting the activation of specific signals and thus generating texture responses to ligands, a phenomenon often referred to as ligand-biased signaling. Also, one challenging task today remains optimization of pharmacological assays with increased sensitivity so to better appreciate the inherent texture of ligands. However, considering that a single receptor has pleiotropic signaling properties and that each signal can crosstalk at different levels, biased activity remains thus difficult to evaluate. One strategy to overcome these limitations would be examining the initial steps following receptor activation. Even, if some G protein independent functions have been recently described, heterotrimeric G protein activation remains a general hallmark for all GPCRs families and the first cellular event subsequent to agonist binding to the receptor. Herein, we review the different methodologies classically used or recently developed to monitor G protein activation and discussed them in the context of G protein biased-ligands

Biophysical properties of cardiomyocyte surface explored by multiparametric AFM

International audiencePeakForce Quantitative Nanomechanical Mapping (PeakForce QNM) multiparametric AFM mode was adapted to qualitative and quantitative study of the lateral membrane of cardiomyocytes (CMs), extending this powerful mode to the study of soft cells. On living CM, PeakForce QNM depicted the crests and hollows periodic alternation of cell surface architecture previously described using AFM Force Volume (FV) mode. PeakForce QNM analysis provided better resolution in terms of pixel number compared to FV mode and reduced acquisition time, thus limiting the consequences of spontaneous living adult CM dedifferentiation once isolated from the cardiac tissue. PeakForce QNM mode on fixed CMs clearly visualized subsarcolem-mal mitochondria (SSM) and their loss following formamide treatment, concomitant with the interfibrillar mitochondria climbing up and forming heaps at the cell surface. Interestingly, formamide-promoted SSM loss allowed visualization of the sarcomeric apparatus ultrastructure below the plasma membrane. High PeakForce QNM resolution led to better contrasted mechanical maps than FV mode and provided correlation between adhesion, dissipation, mechanical and topographical maps. Modified hydrophobic AFM tip enhanced contrast on adhesion and dissipation maps and suggested that CM surface crests and hollows exhibit distinct chemical properties. Finally, two-dimensional Fast Fourier Transform to objectively quantify AFM maps allowed characterization of periodicity of both sarcomeric Z-line and M-band. Overall, this study validated PeakForce QNM as a valuable and innovative mode for the exploration of living and fixed CMs. In the future, it could be applied to depict cell membrane architectural, mechanical and chemical defects as well as sarcomeric abnormalities associated with cardiac diseases

Atomic Force Microscopy: an innovative technology to explore cardiomyocyte cell surface in cardiac physio/pathophysiology

International audienceAtomic Force Microscopy (AFM) has emerged these recent years as a multifunctional toolbox for studying biological samples in physiological conditions. Although its use has spread among biologists community, cardiology remains a scientific field where not been extensively used yet. Heart diseases are nowadays a major human threat, and cause the death of millions of people each year. A convergent point to all heart diseases seems to be related to the defect of the cardiomyocyte, the contractile unit of the he reason, many scientists got interested in this cell type. However, very few studies use a technology such as AFM and its derivatives (force spectroscopy, multiparametric imaging) to explore this cell. The aim of this review is thus to give a comprehensive an interest of the biophysical approach made possible by AFM studies. We will show how AFM has been and can be used to study fix living cardiomyocytes, and, how combined with other types of microscopy, it can help getting a better understanding o pathologies or drugs. This review is the first dedicated to the use of AFM technics in cardiology, and gives new insights in fundamental questions surrounding cardiomyocytes, that can be answered using such a technology