HIV-1 Capture and Transmission by Dendritic Cells: The Role of Viral Glycolipids and the Cellular Receptor Siglec-1

Dendritic cells (DCs) are essential in order to combat invading viruses and trigger antiviral responses. Paradoxically, in the case of HIV-1, DCs might contribute to viral pathogenesis through trans-infection, a mechanism that promotes viral capture and transmission to target cells, especially after DC maturation. In this review, we highlight recent evidence identifying sialyllactosecontaining gangliosides in the viral membrane and the cellular lectin Siglec-1 as critical determinants for HIV-1 capture and storage by mature DCs and for DC-mediated trans-infection of T cells. In contrast, DC-SIGN, long considered to be the main receptor for DC capture of HIV-1, plays a minor role in mature DC-mediated HIV-1 capture and trans-infection.

NLRC5 shields T lymphocytes from NK-cell-mediated elimination under inflammatory conditions.

NLRC5 is a transcriptional regulator of MHC class I (MHCI), which maintains high MHCI expression particularly in T cells. Recent evidence highlights an important NK-T-cell crosstalk, raising the question on whether NLRC5 specifically modulates this interaction. Here we show that NK cells from Nlrc5-deficient mice exhibit moderate alterations in inhibitory receptor expression and responsiveness. Interestingly, NLRC5 expression in T cells is required to protect them from NK-cell-mediated elimination upon inflammation. Using T-cell-specific Nlrc5-deficient mice, we show that NK cells surprisingly break tolerance even towards 'self' Nlrc5-deficient T cells under inflammatory conditions. Furthermore, during chronic LCMV infection, the total CD8(+) T-cell population is severely decreased in these mice, a phenotype reverted by NK-cell depletion. These findings strongly suggest that endogenous T cells with low MHCI expression become NK-cell targets, having thus important implications for T-cell responses in naturally or therapeutically induced inflammatory conditions.

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.

In vivo co-ordinated interactions between inhibitory systems to control glutamate mediated hippocampal excitability.

We present an overview of the long-term adaptation of hippocampal neurotransmission to cholinergic and GABAergic deafferentation caused by excitotoxic lesion of the medial septum. Two months after septal microinjection of 2.7 nmol a -amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), a 220% increase of GABA A receptor labelling in the hippo- campal CA3 and the hilus was shown, and also changes in hippocampal neurotransmission characterised by in vivo microdialysis and HPLC. Basal amino acid and purine extra- cellular levels were studied in control and lesioned rats. In vivo effects of 100 m M KCl perfusion and adenosine A 1 receptor blockade with 1,3-dipropyl- 8-cyclopentylxanthine (DPCPX) on their release were also investigated. In lesioned animals GABA, glutamate and glutamine basal levels were decreased and taurine, adenosine and uric acid levels increased. A similar response to KCl infusion occurred in both groups except for GABA and glutamate, which release decreased in lesioned rats. Only in lesioned rats, DPCPX increased GABA basal level and KCl-induced glutamate release, and decreased glutamate turnover. Our results evidence that an excitotoxic septal lesion leads to increased hippocampal GABA A receptors and decreased glutamate neurotransmis- sion. In this situation, a co-ordinated response of hippocampal retaliatory systems takes place to control neuron excitability.

Alpha2-Adrenoceptor-mediated vascular responses induced by dexmedetomidine

Alpha2-Adrenoceptors are cell-surface G protein coupled receptors that mediate many of the effects of the catecholamines noradrenaline and adrenaline. The three human α2-adrenoceptor subtypes are widely expressed in different tissues and organs, and they mediate many different physiological and pharmacological effects in the central and peripheral nervous system and as postsynaptic receptors in target organs. Previous studies have demonstrated that α2-adrenoceptors mediate both vascular constriction and dilatation in humans. Large inter-individual variation has been observed in the vascular responses to α2-adrenoceptor activation in clinical studies. All three receptor subtypes are potential drug targets. It was therefore considered important to further elucidate the details of adrenergic vascular regulation and its genetic variation, since such knowledge may help to improve the development of future cardiovascular drugs and intensive care therapies. Dexmedetomidine is the most selective and potent α2-adrenoceptor agonist currently available for clinical use. When given systemically, dexmedetomidine induces nearly complete sympatholysis already at low concentrations, and postsynaptic effects, such vasoconstriction, can be observed with increasing concentrations. Thus, local infusions of small doses of dexmedetomidine into dorsal hand veins and the application of pharmacological sympathectomy with brachial plexus block provide a means to assess drug-induced peripheral vascular responses without interference from systemic pharmacological effects and autonomic nervous system regulation. Dexmedetomidine was observed to have biphasic effects on haemodynamics, with an initial decrease in blood pressure at low concentrations followed by substantial increases in blood pressure and coronary vascular resistance at high concentrations. Plasma concentrations of dexmedetomidine that significantly exceeded the recommended therapeutic level did not reduce myocardial blood flow below the level that is observed with the usual therapeutic concentrations and did not induce any evident myocardial ischaemia in healthy subjects. Further, it was demonstrated that dexmedetomidine also had significant vasodilatory effects through activation of endothelial nitric oxide synthesis, and thus when the endothelial component of the blood vessel response to dexmedetomidine was inhibited, peripheral vasoconstriction was augmented. Hand vein constriction responses to α2-adrenoceptor activation by dexmedetomidine were only weakly associated with the constriction responses to α1-adrenoceptor activation, pointing to independent cellular regulation by these two adrenoceptor classes. Substantial inter-individual variation was noted in the venous constriction elicited by activation of α2-adrenoceptors by dexmedetomidine. In two study populations from two different continents, a single nucleotide polymorphism in the PRKCB gene was found to be associated with the dorsal hand vein constriction response to dexmedetomidine, suggesting that protein kinase C beta may have an important role in the vascular α2-adrenoceptor signalling pathways activated by dexmedetomidine.

The Structure and Function of αI Domains in Collagen Receptor and Leukocyte Integrins

Integrins are heterodimeric, signaling transmembrane adhesion receptors that connect the intracellular actin microfilaments to the extracellular matrix composed of collagens and other matrix molecules. Bidirectional signaling is mediated via drastic conformational changes in integrins. These changes also occur in the integrin αI domains, which are responsible for ligand binding by collagen receptor and leukocyte specific integrins. Like intact integrins, soluble αI domains exist in the closed, low affinity form and in the open, high affinity form, and so it is possible to use isolated αI domains to study the factors and mechanisms involved in integrin activation/deactivation. Integrins are found in all mammalian tissues and cells, where they play crucial roles in growth, migration, defense mechanisms and apoptosis. Integrins are involved in many human diseases, such as inflammatory, cardiovascular and metastatic diseases, and so plenty of effort has been invested into developing integrin specific drugs. Humans have 24 different integrins, four of which are collagen receptor (α1β1, α2β1, α10β1, α11β1) and five leukocyte specific integrins (αLβ2, αMβ2, αXβ2, αDβ2, αEβ7). These two integrin groups are quite unselective having both primary and secondary ligands. This work presents the first systematic studies performed on these integrin groups to find out how integrin activation affects ligand binding and selectivity. These kinds of studies are important not only for understanding the partially overlapping functions of integrins, but also for drug development. In general, our results indicated that selectivity in ligand recognition is greatly reduced upon integrin activation. Interestingly, in some cases the ligand binding properties of integrins have been shown to be cell type specific. The reason for this is not known, but our observations suggest that cell types with a higher integrin activation state have lower ligand selectivity, and vice versa. Furthermore, we solved the three-dimensional structure for the activated form of the collagen receptor α1I domain. This structure revealed a novel intermediate conformation not previously seen with any other integrin αI domain. This is the first 3D structure for an activated collagen receptor αI domain without ligand. Based on the differences between the open and closed conformation of the αI domain we set structural criteria for a search for effective collagen receptor drugs. By docking a large number of molecules into the closed conformation of the α2I domain we discovered two polyketides, which best fulfilled the set structural criteria, and by cell adhesion studies we showed them to be specific inhibitors of the collagen receptor integrins.

The hyperglycemia induced by angiotensin II in rats is mediated by AT1 receptors

We have shown that the renin-angiotensin system (RAS) is involved in glucose homeostasis during acute hemorrhage. Since almost all of the physiological actions described for angiotensin II were mediated by AT1 receptors, the present experiments were designed to determine the participation of AT1 receptors in the hyperglycemic action of angiotensin II in freely moving rats. The animals were divided into two experimental groups: 1) animals submitted to intravenous administration of angiotensin II (0.96 nmol/100 g body weight) which caused a rapid increase in plasma glucose reaching the highest values at 5 min after the injection (33% of the initial values, P<0.01), and 2) animals submitted to intravenous administration of DuP-753 (losartan), a non-peptide antagonist of angiotensin II with AT1-receptor type specificity (1.63 µmol/100 g body weight as a bolus, iv, plus a 30-min infusion of 0.018 µmol 100 g body weight-1 min-1 before the injection of angiotensin II), which completely blocked the hyperglycemic response to angiotensin II (P<0.01). This inhibitory effect on glycemia was already demonstrable 5 min (8.9 ± 0.28 mM, angiotensin II, N = 9 vs 6.4 ± 0.22 mM, losartan plus angiotensin II, N = 11) after angiotensin II injection and persisted throughout the 30-min experiment. Controls were treated with the same volume of saline solution (0.15 M NaCl). These data demonstrate that the angiotensin II receptors involved in the direct and indirect hyperglycemic actions of angiotensin II are mainly of the AT1-type.

Fluorescent ligands for studying neuropeptide receptors by confocal microscopy

This paper reviews the use of confocal microscopy as it pertains to the identification of G-protein coupled receptors and the study of their dynamic properties in cell cultures and in mammalian brain following their tagging with specific fluorescent ligands. Principles that should guide the choice of suitable ligands and fluorophores are discussed. Examples are provided from the work carried out in the authors' laboratory using custom synthetized fluoresceinylated or BODIPY-tagged bioactive peptides. The results show that confocal microscopic detection of specifically bound fluorescent ligands permits high resolution appraisal of neuropeptide receptor distribution both in cell culture and in brain sections. Within the framework of time course experiments, it also allows for a dynamic assessment of the internalization and subsequent intracellular trafficking of bound fluorescent molecules. Thus, it was found that neurotensin, somatostatin and mu- and delta-selective opioid peptides are internalized in a receptor-dependent fashion and according to receptor-specific patterns into their target cells. In the case of neurotensin, this internalization process was found to be clathrin-mediated, to proceed through classical endosomal pathways and, in neurons, to result in a mobilization of newly formed endosomes from neural processes to nerve cell bodies and from the periphery of cell bodies towards the perinuclear zone. These mechanisms are likely to play an important role for ligand inactivation, receptor regulation and perhaps also transmembrane signaling.

A receptor for infectious and cellular prion protein

Prions are an unconventional form of infectious agents composed only of protein and involved in transmissible spongiform encephalopathies in humans and animals. The infectious particle is composed by PrPsc which is an isoform of a normal cellular glycosyl-phosphatidylinositol (GPI) anchored protein, PrPc, of unknown function. The two proteins differ only in conformation, PrPc is composed of 40% a helix while PrPsc has 60% ß-sheet and 20% a helix structure. The infection mechanism is trigged by interaction of PrPsc with cellular prion protein causing conversion of the latter's conformation. Therefore, the infection spreads because new PrPsc molecules are generated exponentially from the normal PrPc. The accumulation of insoluble PrPsc is probably one of the events that lead to neuronal death. Conflicting data in the literature showed that PrPc internalization is mediated either by clathrin-coated pits or by caveolae-like membranous domains. However, both pathways seem to require a third protein (a receptor or a prion-binding protein) either to make the connection between the GPI-anchored molecule to clathrin or to convert PrPc into PrPsc. We have recently characterized a 66-kDa membrane receptor which binds PrPc in vitro and in vivo and mediates the neurotoxicity of a human prion peptide. Therefore, the receptor should have a role in the pathogenesis of prion-related diseases and in the normal cellular process. Further work is necessary to clarify the events triggered by the association of PrPc/PrPsc with the receptor.

Angiotensin II-mediated vascular smooth muscle cell growth signaling

The mechanism by which Ang II stimulates the growth of vascular smooth muscle cells was investigated by measuring the phosphorylation of mitogen-activated protein kinases ERK 1 and ERK 2. Ca2+ ionophore was found to have effects practically analogous to Ang II. We found that the signaling pathway involves the activation of epidermal growth factor receptor (EGFR) kinase, activation of the adaptor proteins Shc and Grb2, and the small G-protein Ras. Although the mechanism of AT1- (or Ca2+)-induced activation of EGFR is not yet clear, we have found that calcium-dependent protein kinase CAKß/PYK2 and c-Src are involved in this process. These studies indicate a transactivation mechanism that utilizes EGFR as a bridge between a Gq-coupled receptor and activation of phosphotyrosine generation.

Role of ACTH receptor in adrenocortical tumor formation

Adrenocorticotrophin (ACTH) is the major regulatory hormone of steroid synthesis and secretion by adrenocortical cells. The actions of ACTH are mediated by its specific membrane receptor (ACTH-R). The human ACTH-R gene was recently cloned, allowing systematic determination of its sequence, expression and function in adrenal tumorigenesis. The presence of oncogenic mutations of the ACTH-R gene in adrenocortical tumors has been reported. Direct sequencing of the entire coding region of the ACTH-R gene of sporadic adrenocortical adenomas and carcinomas did not reveal constitutive activating mutations, indicating that this mechanism is not frequent in human adrenocortical tumorigenesis. Recent studies demonstrated allelic loss of the ACTH-R gene in a subset of sporadic adrenocortical tumors using a PstI polymorphism located in the promoter region of the ACTH-R gene. Loss of heterozygosity of the ACTH-R was analyzed in 20 informative patients with a variety of benign and malignant adrenocortical tumors. Three of them showed loss of heterozygosity of the ACTH-R gene. In addition, Northern blot experiments demonstrated reduced expression of ACTH-R mRNA in these three tumors with loss of heterozygosity, suggesting the functional significance of this finding at the transcriptional level. Deletion of the ACTH-R gene seems to be involved in a subset of human adrenocortical tumors, contributing to cellular dedifferentiation.

Strain-dependent effects of diazepam and the 5-HT2B/2C receptor antagonist SB 206553 in spontaneously hypertensive and Lewis rats tested in the elevated plus-maze

The 5-HT2B/2C receptor antagonist SB 206553 exerts anxiolytic effects in rat models of anxiety. However, these effects have been reported for standard rat strains, thus raising the issue of SB 206553 effects in rat strains displaying different levels of anxiety. Herein, the effects of SB 206553 in a 5-min elevated plus-maze test of anxiety were compared to those of the reference anxiolytic, diazepam, in two rat strains respectively displaying high (Lewis rats) and low (spontaneously hypertensive rats, SHR) anxiety. Diazepam (0.37, 0.75, or 1.5 mg/kg; 30 min before testing) increased in a dose-dependent manner the behavioral measures in SHR, but not in Lewis rats. On the other hand, SB 206553 (1.25, 2.5, or 5 mg/kg; 30 min before testing) failed to alter the anxiety parameters in both strains, whereas it increased closed arm entries in Lewis rats, suggesting that it elicited hyperactivity in the latter strain. Accordingly, the hypolocomotor effect of the nonselective 5-HT2B/2C receptor agonist m-chlorophenylpiperazine (1.5 mg/kg ip 20 min before a 15-min exposure to an activity cage) was prevented by the 1.25 and 2.5 mg/kg doses of SB 206553 in Lewis rats and SHR, respectively. Compared with SHR, Lewis rats may display a lower response to benzodiazepine-mediated effects and a more efficient control of locomotor activity by 5-HT2B/2C receptors.

Protective effect of N-acetylcysteine against oxygen radical-mediated coronary artery injury

The present study investigated the protective effect of N-acetylcysteine (NAC) against oxygen radical-mediated coronary artery injury. Vascular contraction and relaxation were determined in canine coronary arteries immersed in Kreb's solution (95% O2-5% CO2), incubated or not with NAC (10 mM), and exposed to free radicals (FR) generated by xanthine oxidase (100 mU/ml) plus xanthine (0.1 mM). Rings not exposed to FR or NAC were used as controls. The arteries were contracted with 2.5 µM prostaglandin F2alpha. Subsequently, concentration-response curves for acetylcholine, calcium ionophore and sodium fluoride were obtained in the presence of 20 µM indomethacin. Concentration-response curves for bradykinin, calcium ionophore, sodium nitroprusside, and pinacidil were obtained in the presence of indomethacin plus Nomega-nitro-L-arginine (0.2 mM). The oxidative stress reduced the vascular contraction of arteries not exposed to NAC (3.93 ± 3.42 g), compared to control (8.56 ± 3.16 g) and to NAC group (9.07 ± 4.0 g). Additionally, in arteries not exposed to NAC the endothelium-dependent nitric oxide (NO)-dependent relaxation promoted by acetylcholine (1 nM to 10 µM) was also reduced (maximal relaxation of 52.1 ± 43.2%), compared to control (100%) and NAC group (97.0 ± 4.3%), as well as the NO/cyclooxygenase-independent receptor-dependent relaxation provoked by bradykinin (1 nM to 10 µM; maximal relaxation of 20.0 ± 21.2%), compared to control (100%) and NAC group (70.8 ± 20.0%). The endothelium-independent relaxation elicited by sodium nitroprusside (1 nM to 1 µM) and pinacidil (1 nM to 10 µM) was not affected. In conclusion, the vascular dysfunction caused by the oxidative stress, expressed as reduction of the endothelium-dependent relaxation and of the vascular smooth muscle contraction, was prevented by NAC.

Alternagin-C, a disintegrin-like protein from the venom of Bothrops alternatus, modulates alpha2ß1 integrin-mediated cell adhesion, migration and proliferation

The alpha2ß1 integrin is a major collagen receptor that plays an essential role in the adhesion of normal and tumor cells to the extracellular matrix. Alternagin-C (ALT-C), a disintegrin-like protein purified from the venom of the Brazilian snake Bothrops alternatus, competitively interacts with the alpha2ß1 integrin, thereby inhibiting collagen binding. When immobilized in plate wells, ALT-C supports the adhesion of fibroblasts as well as of human vein endothelial cells (HUVEC) and does not detach cells previously bound to collagen I. ALT-C is a strong inducer of HUVEC proliferation in vitro. Gene expression analysis was done using an Affimetrix HU-95A probe array with probe sets of ~10,000 human genes. In human fibroblasts growing on collagen-coated plates, ALT-C up-regulates the expression of several growth factors including vascular endothelial growth factor, as well as some cell cycle control genes. Up-regulation of the vascular endothelial growth factor gene and other growth factors could explain the positive effect on HUVEC proliferation. ALT-C also strongly activates protein kinase B phosphorylation, a signaling event involved in endothelial cell survival and angiogenesis. In human neutrophils, ALT-C has a potent chemotactic effect modulated by the intracellular signaling cascade characteristic of integrin-activated pathways. Thus, ALT-C acts as a survival factor, promoting adhesion, migration and endothelial cell proliferation after binding to alpha2ß1 integrin on the cell surface. The biological activities of ALT-C may be helpful as a therapeutic strategy in tissue regeneration as well as in the design of new therapeutic agents targeting alpha2ß1 integrin.

To divide or differentiate? : nestin-mediated regulation of Cdk5 in cell fate decisions

The molecular functions of the non-cell cycle-related Cyclin-dependent kinase 5 (Cdk5) have been of primary interest within the neuroscience field, but novel undertakings are constantly emerging for the kinase in tissue homeostasis, as well as in diseases such as diabetes and cancer. Although Cdk5 activation is predominantly regulated by specific non-cyclin activator protein binding, additional mechanisms have proved to orchestrate Cdk5 signaling in cells. For example, the interaction between the intermediate filament protein nestin and Cdk5 has been proposed to determine cellular fate during neuronal apoptosis through nestin-dependent adjustment of the sensitive balance and turnover of Cdk5 activators. While nestin constitutes a crucial regulatory scaffold for appropriate Cdk5 activation in apoptosis, Cdk5 itself phosphorylates nestin with the consequence of filament reorganization in both neuronal progenitors and differentiating muscle cells. Interestingly, the two proteins are often found coexpressed in various tissues and cell types, proposing that nestin-mediated scaffolding of Cdk5 and its activators may be applicable to other tissue systems as well. In the literature, the molecular functions of nestin have remained in the shade, as it is mostly exploited as a marker protein for progenitor cells. In light of these studies, the aim of this thesis was to assess the importance of the nestin scaffold in regulation of Cdk5 actions in cell fate decisions. This thesis can be subdivided into two major projects: one that studied the nature of the Cdk5-nestin interplay in muscle, and one that assessed their role in prostate cancer. During differentiation of a myoblast cell line, the filament formation properties of nestin was found to be crucial in directing Cdk5 activity, with direct consequences on the process of differentiation. Also the genetic knockout of nestin was found to influence Cdk5 activity, although differentiation per se was not affected. Instead, the genetic ablation of nestin had broad consequences on muscle homeostasis and regeneration. While the nestin-mediated regulation of Cdk5 in muscle was found to act in multiple ways, the connection remained more elusive in cancer models. Cdk5 was, however, established as a significant determinant of prostate cancer proliferation; a behavior uncharacteristic for this differentiation-associated kinase. Through complex and simultaneous regulation of two major prostate cancer pathways, Cdk5 was placed upstream of both Akt kinase and the androgen receptor. Its action on proliferation was nonetheless mainly exerted through the Akt signaling pathway in various cancer models. In summary, this thesis contributed to the knowledge of Cdk5 regulation and functions in two atypical settings; proliferation (in a cancer framework) and muscle differentiation, which is a poorly understood model system in the Cdk5 field. This balance between proliferation and differentiation implemented by Cdk5 is ultimately regulated (where present) by the dynamics of the cytoskeletal nestin scaffold.