The alpha1-adrenergic receptors in cardiac hypertrophy: Signaling mechanisms and functional implications.

Cardiac hypertrophy is a complex remodeling process of the heart induced by physiological or pathological stimuli resulting in increased cardiomyocyte size and myocardial mass. Whereas cardiac hypertrophy can be an adaptive mechanism to stressful conditions of the heart, prolonged hypertrophy can lead to heart failure which represents the primary cause of human morbidity and mortality. Among G protein-coupled receptors, the α1-adrenergic receptors (α1-ARs) play an important role in the development of cardiac hypertrophy as demonstrated by numerous studies in the past decades, both in primary cardiomyocyte cultures and genetically modified mice. The results of these studies have provided evidence of a large variety of α1-AR-induced signaling events contributing to the defining molecular and cellular features of cardiac hypertrophy. Recently, novel signaling mechanisms have been identified and new hypotheses have emerged concerning the functional role of the α1-adrenergic receptors in the heart. This review will summarize the main signaling pathways activated by the α1-AR in the heart and their functional implications in cardiac hypertrophy.

Mechanisms of Antifungal Drug Resistance.

Antifungal therapy is a central component of patient management for acute and chronic mycoses. Yet, treatment choices are restricted because of the sparse number of antifungal drug classes. Clinical management of fungal diseases is further compromised by the emergence of antifungal drug resistance, which eliminates available drug classes as treatment options. Once considered a rare occurrence, antifungal drug resistance is on the rise in many high-risk medical centers. Most concerning is the evolution of multidrug- resistant organisms refractory to several different classes of antifungal agents, especially among common Candida species. The mechanisms responsible are mostly shared by both resistant strains displaying inherently reduced susceptibility and those acquiring resistance during therapy. The molecular mechanisms include altered drug affinity and target abundance, reduced intracellular drug levels caused by efflux pumps, and formation of biofilms. New insights into genetic factors regulating these mechanisms, as well as cellular factors important for stress adaptation, provide a foundation to better understand the emergence of antifungal drug resistance.

Mechanisms of OGT-mediated HCF-1 protein maturation

Post-translational protein modifications are crucial for many fundamental cellular and extracellular processes and greatly contribute to the complexity of organisms. Human HCF-1 is a transcriptional co-regulator that undergoes complex protein maturation involving reversible and irreversible post-translational modifications. Upon synthesis as a large precursor protein, HCF-1 undergoes extensive reversible glycosylation with β-N-acetylglucosamine giving rise to O-linked-β-N-acetylglucosamine (O-GlcNAc) modified serines and threonines. HCF-1 also undergoes irreversible site-specific proteolysis, which is important for one of HCF-1's major functions - the regulation of the cell-division cycle. HCF-1 O-GlcNAcylation and site-specific proteolysis are both catalyzed by a single enzyme with an unusual dual enzymatic activity, the O-GlcNAc transferase (OGT). HCF-1 is cleaved by OGT at any of six highly conserved 26 amino acid repeated sequences (HCF-1PRO repeats), but the mechanisms and the substrate requirements for OGT-mediated cleavage are not understood. In the present work, I characterized substrate requirements for OGT-mediated cleavage and O-GlcNAcylation of HCF-1. I identified key elements within the HCF-1PRO-repeat sequence that are important for proteolysis. Remarkably, an invariant single amino acid side-chain within the HCF-1PRO-repeat sequence displays particular OGT-binding properties and is essential for proteolysis. Additionally, I characterized substrate requirements for proteolysis outside of the HCF-1PRO repeat and identified a novel, highly O-GlcNAcylated OGT-binding sequence that enhances cleavage of the first HCF-1PRO repeat. These results link OGT association and its O-GlcNAcylation activities to HCF-1PRO-repeat proteolysis.

Fetal programming and epigenetic mechanisms in arterial hypertension.

PURPOSE OF REVIEW: To provide an overview of available evidence of the potential role of epigenetics in the pathogenesis of hypertension and vascular dysfunction. RECENT FINDINGS: Arterial hypertension is a highly heritable condition. Surprisingly, however, genetic variants only explain a tiny fraction of the phenotypic variation and the term 'missing heritability' has been coined to describe this phenomenon. Recent evidence suggests that phenotypic alteration that is unrelated to changes in DNA sequence (thereby escaping detection by classic genetic methodology) offers a potential explanation. Here, we present some basic information on epigenetics and review recent work consistent with the hypothesis of epigenetically induced arterial hypertension. SUMMARY: New technologies that enable the rigorous assessment of epigenetic changes and their phenotypic consequences may provide the basis for explaining the missing heritability of arterial hypertension and offer new possibilities for treatment and/or prevention.

Role of Connexins and Pannexins in the Pancreas.

The pancreas produces enzymes with a digestive function and hormones with a metabolic function, which are produced by distinct cell types of acini and islets, respectively. Within these units, secretory cells coordinate their functioning by exchanging information via signals that flow in the intercellular spaces and are generated either at distance (several neural and hormonal inputs) or nearby the pancreatic cells themselves (inputs mediated by membrane ionic-specific channels and by ionic- and metabolite-permeant pannexin channels and connexin "hemichannels"). Pancreatic secretory cells further interact via the extracellular matrix of the pancreas (inputs mediated by integrins) and directly with neighboring cells, by mechanisms that do not require extracellular mediators (inputs mediated by gap and tight junction channels). Here, we review the expression and function of the connexins and pannexins that are expressed by the main secretory cells of the exocrine and endocrine pancreatic cells. Available data show that the patterns of expression of these proteins differ in acini and islets, supporting distinct functions in the physiological secretion of pancreatic enzymes and hormones. Circumstantial evidence further suggests that alterations in the signaling provided by these proteins are involved in pancreatic diseases.

MicroRNAs and the functional β cell mass: For better or worse.

Insulin secretion from pancreatic β cells plays a central role in the control of blood glucose levels. The amount of insulin released by β cells is precisely adjusted to match organism requirements. A number of conditions that arise during life, including pregnancy and obesity, can result in a decreased sensitivity of insulin target tissues and a consequent rise in insulin needs. To preserve glucose homoeostasis, the augmented insulin demand requires a compensatory expansion of the pancreatic β cell mass and an increase in its secretory activity. This compensatory process is accompanied by modifications in β cell gene expression, although the molecular mechanisms underlying the phenomenon are still poorly understood. Emerging evidence indicates that at least part of these compensatory events may be orchestrated by changes in the level of a novel class of gene regulators, the microRNAs. Indeed, several of these small, non-coding RNAs have either positive or negative impacts on β cell proliferation and survival. The studies reviewed here suggest that the balance between the actions of these two groups of microRNAs, which have opposing functional effects, can determine whether β cells expand sufficiently to maintain blood glucose levels in the normal range or fail to meet insulin demand and thus lead, as a consequence, towards diabetes manifestation. A better understanding of the mechanisms governing changes in the microRNA profile will open the way for the development of new strategies to prevent and/or treat both type 2 and gestational diabetes.

The regulation of the mucosal immune response by secretory IgA

L'immunoglobuline A sécrétoire (SlgA) est l'anticorps qui est dominant dans la réponse humorale des muqueuses. IgA est produit localement sous la forme de dimère ou polymère. Ces derniers sont ensuite relâchés dans les sécrétions muqueuses sous forme d'un complexe avec la pièce sécrétoire (SC). SlgA est capable d'identifier des antigènes microbiens dans l'environnement des muqueuses et de prévenir leur diffusion en bloquant l'adhésion ou la surface des cellules épithéliales. Au-delà de sa fonction classique d'exclusion immune, SlgA peut aussi adhérer aux cellules M présente au niveau des follicules associés à l'épithélium dans des structures organisées appelées plaques de Peyer (PPs) dans le système gastrointestinal. L'interaction sélective avec les SlgA amène cette dernière à être transportée à travers les cellules M Ce procès facilite l'association de l'anticorps avec les cellules dendritiques (DCs) CDllc+CDllb+, localisées dans la région sous-épithéliale des PPs. L'entrée de SlgA ou de microorganismes couverts par la SlgA via ce chemin est cruciale pour la modulation de la réponse immunitaire locale. Dans la première partie du travail, nous avons établi les conditions pour l'analyse de l'interaction entre SlgA et une lignes de DCs dérivée in vitro. Nous avons aussi montré que l'internalisation de la SlgA par les DCs est affecté après traitement avec des inhibiteurs de l'endocytose ou par l'utilisation de compétiteurs moléculaires. En utilisant le microscope confocal, on a observé qu'après internalisation la SlgA suit la voie endocytique, vers le compartiment lysozomal. Dans la deuxième partie du travail, une partie des résultats a été confirmée pour les DCs CDllc+CDllb+ des muqueuses. En outre, l'importance des sucres présents sur la SlgA a été mis en évidence par la réduction de l'interaction avec les DCs des muqueuses après déglycosylation. On a montré pour la première fois à notre connaissance que Dectin-1 et SIGNR3 sont des récepteurs potentiels pour la SlgA sur les DCs des muqueuses de la souris. De plus, les DCs de la souris prélevées des PPs, des ganglions lymphatiques mésentériques (MLNs) et de la rate ont été infectés avec Shigella flexneri (Sf) seule ou en complexe avec SlgA. Les DCs infectées ont montré une augmentation de l'expression des marqueurs co-stimulateurs, une forte production des cytokines pro¬inflammatoires et une induction de la prolifération des cellules T. Après l'association des Sf avec SlgA, les profils pro-inflammatoires des DCs ont diminués. En particulier, SlgA a un effet sur les cytokines sécrétées et sur la prolifération des cellules T. Ces données démontrent le rôle de la SlgA dans la réponse immunitaire par les DCs des muqueuses.

Combined deletion of cathepsin protease family members reveals compensatory mechanisms in cancer.

Proteases are important for regulating multiple tumorigenic processes, including angiogenesis, tumor growth, and invasion. Elevated protease expression is associated with poor patient prognosis across numerous tumor types. Several multigene protease families have been implicated in cancer, including cysteine cathepsins. However, whether individual family members have unique roles or are functionally redundant remains poorly understood. Here we demonstrate stage-dependent effects of simultaneously deleting cathepsin B (CtsB) and CtsS in a murine pancreatic neuroendocrine tumor model. Early in tumorigenesis, the double knockout results in an additive reduction in angiogenic switching, whereas at late stages, several tumorigenic phenotypes are unexpectedly restored to wild-type levels. We identified CtsZ, which is predominantly supplied by tumor-associated macrophages, as the compensatory protease that regulates the acquired tumor-promoting functions of lesions deficient in both CtsB and CtsS. Thus, deletion of multiple cathepsins can lead to stage-dependent, compensatory mechanisms in the tumor microenvironment, which has potential implications for the clinical consideration of selective versus pan-family cathepsin inhibitors in cancer.

Delivery of antigen to nasal-associated lymphoid tissue microfold cells through secretory IgA targeting local dendritic cells confers protective immunity.

BACKGROUND: Transmission of mucosal pathogens relies on their ability to bind to the surfaces of epithelial cells, to cross this thin barrier, and to gain access to target cells and tissues, leading to systemic infection. This implies that pathogen-specific immunity at mucosal sites is critical for the control of infectious agents using these routes to enter the body. Although mucosal delivery would ensure the best onset of protective immunity, most of the candidate vaccines are administered through the parenteral route. OBJECTIVE: The present study evaluates the feasibility of delivering the chemically bound p24gag (referred to as p24 in the text) HIV antigen through secretory IgA (SIgA) in nasal mucosae in mice. RESULTS: We show that SIgA interacts specifically with mucosal microfold cells present in the nasal-associated lymphoid tissue. p24-SIgA complexes are quickly taken up in the nasal cavity and selectively engulfed by mucosal dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin-positive dendritic cells. Nasal immunization with p24-SIgA elicits both a strong humoral and cellular immune response against p24 at the systemic and mucosal levels. This ensures effective protection against intranasal challenge with recombinant vaccinia virus encoding p24. CONCLUSION: This study represents the first example that underscores the remarkable potential of SIgA to serve as a carrier for a protein antigen in a mucosal vaccine approach targeting the nasal environment.

Impact of T cell receptor affinity on the molecular mechanisms modulating CD8 T cell signalling and function against NY-ESO-1 tumour antigen

It is well established that cytotoxic T lymphocytes play a pivotal role in the protection against intracellular pathogens and tumour cells. Such protective immune responses rely on the specific T cell receptor (TCR)-mediated recognition by CD8 T cells of small antigenic peptides presented in the context of class-I Major Histocompatibility Complex molecules (pMHCs) on the surface of infected or malignant cells. The strength (affinity/avidity) of this interaction is a major correlate of protection. Although tumour-reactive CD8 T cells can be observed in cancer patients, anti-tumour immune responses are often ineffective in controlling or eradicating the disease due to the relative low TCR affinity of these cells. To overcome this limitation, tumour-specific CD8 T cells can be genetically modified to express TCRs of improved binding strength against a defined tumour antigen before adoptive cell transfer into cancer patients. We previously generated a panel of TCRs specific for the cancer-testis antigen NY-ESO-l,57.165 with progressively increased affinities for the pMHC complex, thus providing us with a unique tool to investigate the causal link between the surface expression of such TCRs and T cell activation and function. We recently demonstrated that anti-tumour CD8 T cell reactivity could only be improved within physiological affinity limits, beyond which drastic functional declines were observed, suggesting the presence of multiple regulatory mechanisms limiting T cell activation and function in a TCR affinity-dependent manner. The overarching goal of this thesis was (i) to assess the precise impact of TCR affinity on T cell activation and signalling at the molecular level and (ii) to gain further insights on the mechanisms that regulate and delimitate maximal/optimized CD8 T cell activation and signalling. Specifically, by combining several technical approaches we characterized the activation status of proximal (i.e. CD3Ç, Lek, and ZAP-70) and distal (i.e. ERK1/2) signalling molecules along the TCR affinity gradient. Moreover, we assessed the extent of TCR downmodulation, a critical step for initial T cell activation. CD8 T cells engineered with the optimal TCR affinity variants showed increased activation levels of both proximal and distal signalling molecules when compared to the wild-type T cells. Our analyses also highlighted the "paradoxical" status of tumour-reactive CD8 T cells bearing very high TCR affinities, which retained strong proximal signalling capacity and TCR downmodulation, but were unable to propagate signalling distally (i.e. pERKl/2), resulting in impaired cell-mediated functions. Importantly, these very high affinity T cells displayed maximal levels of SHP-1 and SHP-2 phosphatases, two negative regulatory molecules, and this correlated with a partial pERKl/2 signalling recovery upon pharmacological SHP-l/SHP-2 inhibition. These findings revealed the putative presence of inhibitory regulators of the TCR signalling cascade acting very rapidly following tumour-specific stimulation. Moreover, the very high affinity T cells were only able to transiently express enhanced proximal signalling molecules, suggesting the presence of an additional level of regulation that operates through the activation of negative feedback loops over time, limiting the duration of the TCR-mediated signalling. Overall, the determination of TCR-pMHC binding parameters eliciting optimal CD8 T cell activation, signalling, and effector function while guaranteeing high antigen specificity, together with the identification of critical regulatory mechanisms acting proximally in the TCR signalling cascade, will directly contribute to optimize and support the development of future TCR-based adoptive T cell strategies for the treatment of malignant diseases. -- Les lymphocytes T CD8 cytotoxiques jouent un rôle prédominant dans la protection contre les pathogènes intracellulaires et les cellules tumorales. Ces réponses immunitaires dépendent de la spécificité avec laquelle les récepteurs T (TCR) des lymphocytes CD8 reconnaissent les peptides antigéniques présentés par les molécules du complexe Majeur de Histocompatibilité de classe I (pCMH) à la surface des cellules infectées ou malignes. La force (ou affinité/avidité) de l'interaction du TCR-pCMH est un corrélat majeur de protection. Les réponses immunitaires sont cependant souvent inefficaces et ne permettent pas de contrôler ou d'éliminer les cellules tumorales chez les patients atteint du cancer, et ce à cause de la relative faible reconnaissance des TCRs exprimés par les lymphocytes T CD8 envers les antigènes tumoraux. Afin de surmonter cette limitation, les cellules T anti-tumorales peuvent être génétiquement modifiées en les dotant de TCRs préalablement optimisés afin d'augmenter leur reconnaissance ou affinité contre les antigènes tumoraux, avant leur ré¬infusion dans le patient. Nous avons récemment généré des cellules T CD8 exprimant un panel de TCRs spécifiques pour l'antigène tumoral NY-ESO-l157.16J avec des affinités croissantes, permettant ainsi d'investiguer la causalité directe entre l'affinité du TCR-pCMH et la fonction des cellules T CD8. Nous avons démontré que la réactivité anti-tumorale pouvait être améliorée en augmentant l'affinité du TCR dans une intervalle physiologique, mais au delà duquel nous observons un important déclin fonctionnel. Ces résultats suggèrent la présence de mécanismes de régulation limitant l'activation des cellules T de manière dépendante de l'affinité du TCR. Le but de cette thèse a été (i) de définir l'impact précis de l'affinité du TCR sur l'activation et la signalisation des cellules T CD8 au niveau moléculaire et (ii) d'acquérir de nouvelles connaissances sur les mécanismes qui régulent et délimitent l'activation et la signalisation maximale des cellules T CD8 optimisées. Spécifiquement, en combinant plusieurs approches technologiques, nous avons caractérisé l'état d'activation de différentes protéines de la voie de signalisation proximale (CD3Ç, Lek et ZAP-70) et distale (ERK1/2) le long du gradient d'affinité du TCR, ainsi que l'internalisation du TCR, une étape clef dans l'activation initiale des cellules T. Les lymphocytes T CD8 exprimant des TCRs d'affinité optimale ont montré des niveaux d'activation augmentés des molécules proximales et distales par rapport aux cellules de type sauvage (wild-type). Nos analyses ont également mis en évidence un paradoxe chez les cellules T CD8 équipées avec des TCRs de très haute affinité. En effet, ces cellules anti-tumorales sont capables d'activer leurs circuits biochimiques au niveau proximal et d'internaliser efficacement leur TCR, mais ne parviennent pas à propager les signaux biochimiques dépendants du TCR jusqu'au niveau distal (via phospho-ERKl/2), avec pour conséquence une limitation de leur capacité fonctionnelle. Finalement, nous avons démontré que SHP-1 et SHP-2, deux phosphatases avec des propriétés régulatrices négatives, étaient majoritairement exprimées dans les cellules T CD8 de très hautes affinités. Une récupération partielle des niveaux d'activation de ERK1/2 a pu être observée après l'inhibition pharmacologique de ces phosphatases. Ces découvertes révèlent la présence de régulateurs moléculaires qui inhibent le complexe de signalisation du TCR très rapidement après la stimulation anti-tumorale. De plus, les cellules T de très hautes affinités ne sont capables d'activer les molécules de la cascade de signalisation proximale que de manière transitoire, suggérant ainsi un second niveau de régulation via l'activation de mécanismes de rétroaction prenant place progressivement au cours du temps et limitant la durée de la signalisation dépendante du TCR. En résumé, la détermination des paramètres impliqués dans l'interaction du TCR-pCMH permettant l'activation de voies de signalisation et des fonctions effectrices optimales ainsi que l'identification des mécanismes de régulation au niveau proximal de la cascade de signalisation du TCR contribuent directement à l'optimisation et au développement de stratégies anti-tumorales basées sur l'ingénierie des TCRs pour le traitement des maladies malignes.

Mechanisms of protein homeostasis in health, aging and disease.

When emerging from the ribosomes, new polypeptides need to fold properly, eventually translocate, and then assemble into stable, yet functionally flexible complexes. During their lifetime, native proteins are often exposed to stresses that can partially unfold and convert them into stably misfolded and aggregated species, which can in turn cause cellular damage and propagate to other cells. In animal cells, especially in aged neurons, toxic aggregates may accumulate, induce cell death and lead to tissue degeneration via different mechanisms, such as apoptosis as in Parkinson's and Alzheimer's diseases and aging in general. The main cellular mechanisms effectively controlling protein homeostasis in youth and healthy adulthood are: (1) the molecular chaperones, acting as aggregate unfolding and refolding enzymes, (2) the chaperone-gated proteases, acting as aggregate unfolding and degrading enzymes, (3) the aggresomes, acting as aggregate compacting machineries, and (4) the autophagosomes, acting as aggregate degrading organelles. For unclear reasons, these cellular defences become gradually incapacitated with age, leading to the onset of degenerative diseases. Understanding these mechanisms and the reasons for their incapacitation in late adulthood is key to the design of new therapies against the progression of aging, degenerative diseases and cancers.

Regulating Mechanisms of Gonadal and Pituitary Tumorigenesis in Mice Producing Human Chorionic Gonadotropin

Human chorionic gonadotropin (hCG) and luteinizing hormone (LH) are structurally and functionally similar glycoprotein hormones acting through the same luteinizing hormone chorionic gonadotropin receptor (LHCGR). The functions of LH in reproduction and hCG in pregnancy are well known. Recently, the expression of LHCGR has been found in many nongonadal tissues and cancers, and this has raised the question of whether LH/hCG could affect the function or tumorigenesis of these nongonadal tissues. We have also previously generated an hCG expressing mouse model presenting nongonadal phenotypes. Using this model it is possible to improve our understanding of nongonadal action of highly elevated LH/hCG. In the current study, we analyzed the effect of moderately and highly elevated hCG levels on male reproductive development and function. The main finding was the appearance of fetal Leydig cell (FLC) adenomas in prepubertal males. However, the development and differentiation of FLCs were not significantly affected. We also show that the function of hCG is different in FLCs and in adult Leydig cells (ALC), because in the latter cells hCG was not able to induce tumorigenesis. In FLCs, LHCGR is not desensitized or downregulated upon ligand binding. In this study, we found that the testicular expression of two G protein-coupled receptor kinases responsible for receptor desensitization or downregulation is increased in adult testis. Results suggest that the lack of LHCGR desensitization or downregulation in FLCs protect testosterone (Te) synthesis, but also predispose FLCs for LH/hCG induced adenomas. However, all the hCG induced nongonadal changes observed in male mice were possible to explain by the elevated Te level found in these males. Our findings indicate that the direct nongonadal effects of elevated LH/hCG in males are not pathophysiologically significant. In female mice, we showed that an elevated hCG level was able to induce gonadal tumorigenesis. hCG also induced the formation of pituitary adenomas (PA), but the mechanism was indirect. Furthermore, we found two new potential risk factors and a novel hormonally induced mechanism for PAs. Increased progesterone (P) levels in the presence of physiological estradiol (E2) levels induced the formation of PAs in female mice. E2 and P induced the expression and nuclear localization of a known cell-cycle regulator, cyclin D1. A calorie restricted diet was also able to prevent the formation of PAs, suggesting that obesity is able to promote the formation of PAs. Hormone replacement therapy after gonadectomy and hormone antagonist therapy showed that the nongonadal phenotypes observed in hCG expressing female mice were due to ovarian hyperstimulation. A slight adrenal phenotype was evident even after gonadectomy in hCG expressing females, but E2 and P replacement was able to induce a similar phenotype in WT females without elevated LH/hCG action. In conclusion, we showed that the direct effects of elevated hCG/LH action are limited only to the gonads of both sexes. The nongonadal phenotypes observed in hCG expressing mice were due to the indirect, gonadal hormone mediated effects of elevated hCG. Therefore, the gonads are the only physiologically significant direct targets of LHCGR signalling.

Bio-inspired Mechanisms for Artificial Self-organised Systems

Self-organization is a growing interdisciplinary field of research about a phenomenon that can be observed in the Universe, in Nature and in social contexts. Research on self-organization tries to describe and explain forms, complex patterns and behaviours that arise from a collection of entities without an external organizer. As researchers in artificial systems, our aim is not to mimic self-organizing phenomena arising in Nature, but to understand and to control underlying mechanisms allowing desired emergence of forms, complex patterns and behaviours. Rather than attempting to eliminate such self-organization in artificial systems, we think that this might be deliberately harnessed in order to reach desirable global properties. In this paper we analyze three forms of self-organization: stigmergy, reinforcement mechanisms and cooperation. The amplification phenomena founded in stigmergic process or in reinforcement process are different forms of positive feedbacks that play a major role in building group activity or social organization. Cooperation is a functional form for self-organization because of its ability to guide local behaviours in order to obtain a relevant collective one. For each forms of self-organisation, we present a case study to show how we transposed it to some artificial systems and then analyse the strengths and weaknesses of such an approach