Kinase signaling cascades that modulate peroxisome proliferator-activated receptors.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors involved in lipid and glucose homeostasis, inflammation and wound healing. In addition to ligand binding, phosphorylation can also regulate PPARs; the biological effects of phosphorylation depend on the stimulus, the kinase, the PPAR isotype, the residue modified, the cell type and the promoter investigated. The study of this dual regulation mode, which allows PPARs to integrate signals conveyed by lipophilic ligands with those coming from the plasma membrane, may ultimately offer new therapeutic strategies.

A-Kinase Anchoring Protein (AKAP)-Lbc Anchors a PKN-based Signaling Complex Involved in α1-Adrenergic Receptor-induced p38 Activation.

The mitogen-activated protein kinases (MAPKs) pathways are highly organized signaling systems that transduce extracellular signals into a variety of intracellular responses. In this context, it is currently poorly understood how kinases constituting these signaling cascades are assembled and activated in response to receptor stimulation to generate specific cellular responses. Here, we show that AKAP-Lbc, an A-kinase anchoring protein (AKAP) with an intrinsic Rho-specific guanine nucleotide exchange factor activity, is critically involved in the activation of the p38α MAPK downstream of α(1b)-adrenergic receptors (α(1b)-ARs). Our results indicate that AKAP-Lbc can assemble a novel transduction complex containing the RhoA effector PKNα, MLTK, MKK3, and p38α, which integrates signals from α(1b)-ARs to promote RhoA-dependent activation of p38α. In particular, silencing of AKAP-Lbc expression or disrupting the formation of the AKAP-Lbc·p38α signaling complex specifically reduces α(1)-AR-mediated p38α activation without affecting receptor-mediated activation of other MAPK pathways. These findings provide a novel mechanistic hypothesis explaining how assembly of macromolecular complexes can specify MAPK signaling downstream of α(1)-ARs.

Yellow submarine of the Wnt/Frizzled signaling: submerging from the G protein harbor to the targets.

The Wnt/Frizzled signaling pathway plays multiple functions in animal development and, when deregulated, in human disease. The G-protein coupled receptor (GPCR) Frizzled and its cognate heterotrimeric Gi/o proteins initiate the intracellular signaling cascades resulting in cell fate determination and polarization. In this review, we summarize the knowledge on the ligand recognition, biochemistry, modifications and interacting partners of the Frizzled proteins viewed as GPCRs. We also discuss the effectors of the heterotrimeric Go protein in Frizzled signaling. One group of these effectors is represented by small GTPases of the Rab family, which amplify the initial Wnt/Frizzled signal. Another effector is the negative regulator of Wnt signaling Axin, which becomes deactivated in response to Go action. The discovery of the GPCR properties of Frizzled receptors not only provides mechanistic understanding to their signaling pathways, but also paves new avenues for the drug discovery efforts.

Genetic architecture of ambulatory blood pressure in the general population: insights from cardiovascular gene-centric array.

Genetic determinants of blood pressure are poorly defined. We undertook a large-scale, gene-centric analysis to identify loci and pathways associated with ambulatory systolic and diastolic blood pressure. We measured 24-hour ambulatory blood pressure in 2020 individuals from 520 white European nuclear families (the Genetic Regulation of Arterial Pressure of Humans in the Community Study) and genotyped their DNA using the Illumina HumanCVD BeadChip array, which contains ≈50 000 single nucleotide polymorphisms in >2000 cardiovascular candidate loci. We found a strong association between rs13306560 polymorphism in the promoter region of MTHFR and CLCN6 and mean 24-hour diastolic blood pressure; each minor allele copy of rs13306560 was associated with 2.6 mm Hg lower mean 24-hour diastolic blood pressure (P=1.2×10(-8)). rs13306560 was also associated with clinic diastolic blood pressure in a combined analysis of 8129 subjects from the Genetic Regulation of Arterial Pressure of Humans in the Community Study, the CoLaus Study, and the Silesian Cardiovascular Study (P=5.4×10(-6)). Additional analysis of associations between variants in gene ontology-defined pathways and mean 24-hour blood pressure in the Genetic Regulation of Arterial Pressure of Humans in the Community Study showed that cell survival control signaling cascades could play a role in blood pressure regulation. There was also a significant overrepresentation of rare variants (minor allele frequency: <0.05) among polymorphisms showing at least nominal association with mean 24-hour blood pressure indicating that a considerable proportion of its heritability may be explained by uncommon alleles. Through a large-scale gene-centric analysis of ambulatory blood pressure, we identified an association of a novel variant at the MTHFR/CLNC6 locus with diastolic blood pressure and provided new insights into the genetic architecture of blood pressure.

Wnt secretion and gradient formation.

Concentration gradients formed by the lipid-modified morphogens of the Wnt family are known for their pivotal roles during embryogenesis and adult tissue homeostasis. Wnt morphogens are also implicated in a variety of human diseases, especially cancer. Therefore, the signaling cascades triggered by Wnts have received considerable attention during recent decades. However, how Wnts are secreted and how concentration gradients are formed remains poorly understood. The use of model organisms such as Drosophila melanogaster has provided important advances in this area. For instance, we have previously shown that the lipid raft-associated reggie/flotillin proteins influence Wnt secretion and spreading in Drosophila. Our work supports the notion that producing cells secrete Wnt molecules in at least two pools: a poorly diffusible one and a reggie/flotillin-dependent highly diffusible pool which allows morphogen spreading over long distances away from its source of production. Here we revise the current views of Wnt secretion and spreading, and propose two models for the role of the reggie/flotillin proteins in these processes: (i) reggies/flotillins regulate the basolateral endocytosis of the poorly diffusible, membrane-bound Wnt pool, which is then sorted and secreted to apical compartments for long-range diffusion, and (ii) lipid rafts organized by reggies/flotillins serve as "dating points" where extracellular Wnt transiently interacts with lipoprotein receptors to allow its capture and further spreading via lipoprotein particles. We further discuss these processes in the context of human breast cancer. A better understanding of these phenomena may be relevant for identification of novel drug targets and therapeutic strategies.

MOLECULAR REORGANIZATION FOLLOWING SENSORY STIMULATION IN THE ADULT MOUSE BARREL CORTEX

Sensory information is an important factor in shaping neuronal circuits during development and adulthood. In the barrel cortex of adult rodents, cells from layer IV are able to adapt their functional state to an increased flow of sensory information from the mystacial whisker follicles. Previous studies in our group have shown that whisker stimulation induces the formation of inhibitory synapses in the corresponding barrel (Knott et al., 2002) and decreases neuronal responses toward the deflection of the stimulated whisker (Quairiaux et al., 2007). Together these observations have turned the barrel cortex into a model to study homeostatic plasticity. At the cellular level, neuronal activity triggers intracellular signaling cascades leading to a transcriptional response. To further characterize the molecular pathways involved in the synaptic changes after whisker stimulation in the adult mouse, a previous doctoral student in our group performed a microarray analysis on laser-dissected barrels in sections through layer IV. This study identified the regulation (up and down) of a series of genes in the stimulated barrels (thesis of Johnston-Wenger, 2010). We here focused on ten genes that presented the highest fold change according to the microarray analysis. Out of these genes, 7 are known as neuronal activity-dependent genes (Tnncl, Nptx2, Sorcs3, Ptgs2, Nr4a2, Npas4 and Adcyapl) whereas three have so far not been related to neuronal plasticity (Scn7a, Pcdhl5 and Cede3). The study aimed at confirming the results of the microarray analysis and localizing molecular modifications in the stimulated barrel column at the cellular level. In situ hybridization for Pcdhl5 after different periods of whisker stimulation (3, 6, 9, 15, 24 hrs) allowed us to confirm that the 1.25 fold change used for the microarray analysis is an appropriate threshold for considering a regulation significant after sensory-stimulation. Moreover, we confirmed with in situ hybridization a significant upregulation of the genes of interest in the stimulated barrels. In situ hybridization and immunohistochemistry allowed us to observe the distribution of the genes of interest and the corresponding protein products at the cellular level. Three observations were made: 1) alterations of the expression was restricted to the stimulated barrels for all genes tested; 2) within a barrel column not all cells responded to whisker stimulation with an altered gene expression; 3) in the stimulated barrels, two different patterns of mRNA and protein expression can be distinguished. We hypothesize that this segregation of the activity-induced gene expression reflects the segregation of the two principal thalamocortical pathways conveying the sensory information to the barrel cortex. Moreover, only neurons reaching the critical threshold will modify their gene expression program resulting in structural as well as physiological modifications that prevent the subsequent propagation of the excess of excitation to the postsynaptic targets. The activity-induced gene expression is therefore adapted in a cell-type-specific manner to induce a homeostatic response to the entire neuronal network involved in the integration of the sensory information. This to our knowledge the first study showing the distinct, but complementary contribution of the two thalamocortical pathways in experience-dependent plasticity in the adult mouse barrel cortex. -- L'information sensorielle nous permet de continuellement façonner nos circuits neuronaux autant durant le développement qu'à l'âge adulte. Chez le rongeur l'information sensorielle perçue par les vibrisses est intégrée au niveau du cortex somatosensoriel primaire (appelé en anglais « barrel cortex ») dont les cellules de la couche IV sont capables d'adapter leur état fonctionnel en réponse à une augmentation d'activité neuronale. Ce modèle expérimental a permis à notre groupe de recherche d'observer des changements rapides du circuit neuronal en fonction de l'activité sensorielle. En effet, la stimulation continue d'une vibrisse d'une souris adulte pendant 24 heures induit non seulement un remaniement synaptique (Knott et al., 2002), mais également des changements physiologiques au niveau des neurones du tonneau correspondant (Quairiaux et al., 2007). Ces observations nous permettent d'affirmer que le « barrel cortex » est un modèle approprié pour y étudier la plasticité synaptique. Au niveau cellulaire, l'activité neuronale déclenche des cascades de signalisation intracellulaire résultant en une réponse transcriptionnelle. Afin de caractériser les voies moléculaires impliquées dans la plasticité synaptique, une puce à ARN nous a permis de comparer l'expression de gènes entre un tonneau correspondant à une vibrisse stimulée et un tonneau d'une vibrisse non-stimulée (Nathalie). Cette analyse a révélé un certain nombre de gènes régulés de manière positive ou négative par l'augmentation de l'activité neuronale. Nous nous sommes concentrés sur 10 gènes dont l'expression est fortement régulée. L'expression de sept d'entre eux a déjà été démontrée comme dépendante de l'activité neuronale (Tnncl, Nptx2, Sorcs3, Ptgs2, Nr4a2, Npas4 otAdcyapl) alors que l'expression des trois autres (Scn7a, Pcdhl5 et Cedei) n'a pour le moment pas encore été liée à la plasticité neuronale. Le but de cette thèse est de confirmer les résultats de la puce à ARN et de déterminer dans quel type cellulaire ces gènes sont exprimés. L'hybridation in situ pour le gène Pcdhl5, après différentes périodes de stimulation des vibrisses (3, 6, 9, 15 et 24 heures), nous a permis de confirmer que le seuil de 1.25x utilisé dans l'analyse de la puce à ARN est approprié pour considérer qu'un gène est régulé de manière significative par la stimulation sensorielle. Nous avons également pu confirmer à l'aide de cette technique que la stimulation sensorielle augmente significativement l'expression de ces dix gènes. L'expression de ces gènes au niveau cellulaire a été observée à l'aide des techniques d'hybridation in situ et d'immunohistochimie. Trois observations ont été faites : 1) la régulation de ces gènes est restreinte aux tonneaux correspondants aux vibrisses stimulées ; 2) au niveau d'une colonne corticale correspondant aux vibrisses stimulées, seules certaines cellules présentent une altération de leur expression génique ; 3) au niveau des tonneaux stimulés, deux profils d'expression d'ARNm et de protéines sont observés. Notre hypothèse est que cette distribution pourrait correspondre à la terminaison ségrégée des deux voies thalamocortical qui amènent l'information sensorielle dans le cortex cérébral. De plus, seul les neurones atteignant le seuil critique d'activation modifient leur expression génique en réponse à la stimulation sensorielle. Ces changements d'expression géniques vont permettre à la cellule de modifier ses propriétés structurales et physiologiques de manière a prevenir la propagation d'un excès d'activité neuronale au niveau de ses cibles postsynaptics. L'activité neuronale agit donc spécifiquement sur certains types cellulaires de maniere a induire une réponse homéostatique au niveau du réseau neuronal impliqué dans l'integration de l'information sensorielle. Nos travaux démontrent pour une première fois que les deux voies sensorielles contribuent d'une manière distincte et complémentaire à la plasticité corticale induite par un changement de l'activité sensorielle chez la souris adulte.

Analysis of GPCR properties of Frizzled receptors and establishment of the "in vitro" platform for screening of Frizzled agonists/antagonists as potential therapeutic agents

Morphogens of the Wnt protein family are the secreted lipoglycoprotein ligands which initiate several pathways heavily involved in the coordination of various developmental stages of organisms in the majority of animal species. Deregulation of these pathways in the adult leads to formation and sustaining of multiple types of cancer. The latter notion is reinforced by the fact that the very discovery of the first Wnt ligand was due to its role as the causative factor of carcinogenic transformation (Nusse and Varmus, 1982). Nowadays our knowledge on Wnt signaling has "moved with the times" and these pathways were identified to be often crucial for tumor formation, its interactions with the microenvironment, and promotion of the metastases (Huang and Du, 2008; Zerlin et al., 2008; Jessen, 2009). Thus the relevance of the pathway as the target for drug development has further increased in the light of modern paradigms of the complex cancer treatments which target also spreading and growth- promoting factors of tumors by specific and highly efficient substances (Pavet et al., 2010). Presently the field of the Wnt-targeting drug research is almost solely dominated by assays based on transcriptional activation induced by the signaling. This approach resulted in development of a number of promising substances (Lee et al., 2011). Despite its effectiveness, the method nevertheless suffers from several drawbacks. Among the major ones is the fact that this approach is prone to identify compounds targeting rather downstream effectors of the pathway, which are indiscriminately used by all the subtypes of the Wnt signaling. Additionally, proteins which are involved in several signaling cascades and not just the Wnt pathway turn out as targets of the new compounds. These issues increase risks of side effects due to off-target interactions and blockade of the pathway in healthy cells. In the present work we put forward a novel biochemical approach for drug development on the Wnt pathway. It targets Frizzleds (Fzs) - a family of 7-transmbembrane proteins which serve as receptors for Wnt ligands. They offer unique properties for the development of highly specific and effective drugs as they control all branches of the Wnt signaling. Recent advances in the understanding of the roles of heterotrimeric G proteins downstream from Fzs (Katanaev et al., 2005; Liu et al., 2005; Jernigan et al., 2010) suggest application of enzymatic properties of these effectors to monitor the receptor-mediated events. We have applied this knowledge in practice and established a specific and efficient method based on utilization of a novel high-throughput format of the GTP-binding assay to follow the activation of Fzs. This type of assay is a robust and well-established technology for the research and screenings on the GPCRs (Harrison and Traynor, 2003). The conventional method of detection involves the radioactively labeled non-hydrolysable GTP analog [35S]GTPyS. Its application in the large-scale screenings is however problematic which promoted development of the novel non-radioactive GTP analog GTP-Eu. The new molecule employs phenomenon of the time-resolved fluorescence to provide sensitivity comparable to the conventional radioactive substance. Initially GTP-Eu was tested only in one of many possible types of GTP-binding assays (Frang et al., 2003). In the present work we expand these limits by demonstrating the general comparability of the novel label with the radioactive method in various types of assays. We provide a biochemical characterization of GTP-Eu interactions with heterotrimeric and small GTPases and a comparative analysis of the behavior of the new label in the assays involving heterotrimeric G protein effectors. These developments in the GTP-binding assay were then applied to monitor G protein activation by the Fz receptors. The data obtained in mammalian cultured cell lines provides for the first time an unambiguous biochemical proof for direct coupling of Fzs with G proteins. The specificity of this interaction has been confirmed by the experiments with the antagonists of Fz and by the pertussis toxin-mediated deactivation. Additionally we have identified the specificity of Wnt3a towards several members of the Fz family and analyzed the properties of human Fz-1 which was found to be the receptor coupled to the Gi/o family of G proteins. Another process playing significant role in the functioning of every GPCR is endocytosis. This phenomenon can also be employed for drug screenings on GPCRs (Bickle, 2010). In the present work we have demonstrated that Drosophila Fz receptors are involved in an unusual for many GPCRs manifestation of the receptor-mediated internalization. Through combination of biochemical approaches and studies on Drosophila as the model organism we have shown that direct interactions of the Fzs and the α-subunit of the heterotrimeric G protein Go with the small GTPase Rab5 regulate internalization of the receptor in early endosomes. We provide data uncovering the decisive role of this self-promoted endocytosis in formation of a proper signaling output in the canonical as well as planar cell polarity (PCP) pathways regulated by Fz. The results of this work thus establish a platform for the high-throughput screening to identify substances active in the cancer-related Wnt pathways. This methodology has been adjusted and applied to provide the important insights in Fz functioning and will be instrumental for further investigations on the Wnt-mediated pathways.

Decreased acute vascular remodeling, anaphylaxis and delayed type hypersensitivity in PPARβ/δ-deficient mice

Endothelial cells form a semi-permeable barrier that participates in the exchange of plasma fluids, proteins and cells, and helps to maintain the physiological functions of organs as well as circulatory homeostasis. Vascular permeability and vasodilatation are increased during acute and chronic inflammation, cancer and wound healing. This is mediated by exposure to certain vascular permeability increasing factors, such as vascular endothelial growth factor (VEGF). The peroxisome proliferator-activated receptors (PPAR) belong to the nuclear hormone receptor (NHRs) family of ligand-activated transcription factors. Three isotypes, PPARa, PPARp/5 and PPARy have been identified. They are all expressed in endothelial cells (ECs). Recent data have demonstrated their involvement in important mechanisms for vasculogenesis and angiogenesis, such as cell proliferation/differentiation, directional sensing/migration, and survival. PPARs were reported to modulate the expression of pro-angiogenic soluble factors, such as VEGF-A and may also participate in the regulation of expression of VEGF receptors. The aim of the present work was to elucidate the role of PPARp/δ in endothelial cell functions important for angiogenesis as well as in vascular permeability and vasodilatation. Using organ culture models of mouse aorta expiants, cultures of human umbilical vein endothelial cells (HUVECs) and genetically modified mouse models, we studied the consequences of loss and gain of PPARp/5 activity on endothelial cell functions. In the first part of this study, we show that the activation of PPARp/δ promotes EC outgrowth in murine aorta expiants. In vivo we observed that dermal vessel acute permeability in response to VEGF-A stimulation is strongly impaired in PPARfi/δ -I- animals. Additionally, observation of the dermal vessel morphology showed a clear enlargement of the wild-type dermal vessels upon VEGF-A injection, whereas vessels of PPARp/5 -/- animals showed almost no enlargement. The impaired response to VEGF stimulation in the knock-out animals was not due to structural or morphological abnormalities. Based on this data, we suggest that PPARp/5 may act on intracellular signaling cascades in ECs, downstream of the VEGF-A receptor. In the second part of this study, we address the relevance of PPARβ/δ vascular functions in pathophysiological inflammatory conditions, such as delayed- type hypersensitivity (DTH) reaction and anaphylaxis in mice. The DTH reaction is a cell-mediated immune reaction to protein, bacterial and viral antigens, whereas anaphylaxis is the most severe form of allergic reaction. In these in vivo models, we demonstrated that the absence of PPARβ/δ in ECs prevents the formation of severe edema in the DTH reaction, and that Ρ PARβ/δ accelerates recovery following systemic anaphylaxis, at least partially through the control of vascular permeability. Our data not only describe a novel function of PPARβ/δ in vessel permeability and vasodilatation, but also open new routes of research for the development of vessel permeability/vasodilatation regulating agents. - Les cellules endothéliales qui bordent la face interne des vaisseaux sanguins forment l'endothélium, une barrière semi-perméable qui régule les échanges de fluides, de protéines et de cellules immunes entre la circulation et les organes. L'endothélium participe également au maintien de la fonction des organes et de l'homéostasie circulatoire. La perméabilité vasculaire augmente dans des situations inflammatoires aigties ou chroniques, dans les tumeurs, et pendant la réparation de blessures. Cette augmentation de perméabilité est due à la production de facteurs sécrétés, tels que le Vascular Endothelial Growth Factor (VEGF-A), la thrombine ou I'histamine. Lès récepteurs nucléaires Peroxisome Proliferator-Activated Receptors (PPAR) sont des facteurs de transcription mis en activité par des ligands. Trois isotypes de PPARs, PPARa, ΡΡΑΡβ/δ and PPARy ont été caractérisés. Ils sont exprimés dans les cellules endothéliales, et des travaux récents ont montré qu'ils régulent des comportements cellulaires importants pour la vasculogenèse et l'angiogenèse, tels que la prolifération, la différenciation, la migration, et la survie des cellules. Ils régulent également la production de VEGF-A par divers types cellulaires. Le but de ce travail était d'élucider le rôle de PPARβ/δ dans la régulation de la perméabilité vasculaire, plus particulièrement dans les cellules endothéliales. Grâce à des cultures d'expiants d'aortes de souris, à la culture d'une lignée endothéliale humaine (HUVECs) et de souris génétiquement modifiées, nous avons étudié le rôle de PPARβ/δ dans les cellules endothéliales, dans des situations gain et perte de fonction du récepteur. Dans la première partie de ce travail, nous avons montré les propriétés pro-angiogéniques de PPARβ/δ dans des explants d'aortes. In vivo, nous avons observé l'absence d'hyperperméabilité aiguë induite par le VEGF-A, la thrombine et I'histamine chez les souris PPARβ/δ -/-. De plus, l'analyse morphologique des vaisseaux dans le derme des souris après stimulation par VEGF- A a confirmé l'absence de réponse à la stimulation. Ces analyses morphologiques nous ont également permis de montrer que l'absence de réponse aiguë n'était pas due à un défaut de structure des vaisseaux dermiques chez les souris PPARp/δ -/-. Sur la base de ces résultats, nous proposons que PPARp/δ régule des voies de signalisation intracellulaires dans les cellules endothéliales, voie de signalisation impliquées dans la régulation de la perméabilité vasculaire: Dans la seconde partie du travail, nous avons étudié l'importance de la régulation de la perméabilité vasculaire par PPARβ/δ dans des situations pathophysiologiques impliquant une hyperperméabilité aiguë des vaisseaux : une réaction d'hypersensibilité cutanée retardée d'une part (delayed-type hypersensitivity, DTH), et un choc anaphylactique d'autre part. Dans ces deux modèles induits expérimentalement chez la souris, l'absence de PPARβ/δ prévient en partie la formation de l'oedème inflammatoire local (DTH), et accélère la récupération (anaphylaxie), au moins partiellement en réglant la perméabilité vasculaire. Ces résultats ouvrent un nouveau champs d'étude quant au rôle de PPARβ/δ dans les vaisseaux et à d'éventuelles applications thérapeutiques dans des pathologies inflammatoires.

Molecular insights for optimizing T cell receptor specificity against cancer.

Cytotoxic CD8 T cells mediate immunity to pathogens and they are able to eliminate malignant cells. Immunity to viruses and bacteria primarily involves CD8 T cells bearing high affinity T cell receptors (TCRs), which are specific to pathogen-derived (non-self) antigens. Given the thorough elimination of high affinity self/tumor-antigen reactive T cells by central and peripheral tolerance mechanisms, anti-cancer immunity mostly depends on TCRs with intermediate-to-low affinity for self-antigens. Because of this, a promising novel therapeutic approach to increase the efficacy of tumor-reactive T cells is to engineer their TCRs, with the aim to enhance their binding kinetics to pMHC complexes, or to directly manipulate the TCR-signaling cascades. Such manipulations require a detailed knowledge on how pMHC-TCR and co-receptors binding kinetics impact the T cell response. In this review, we present the current knowledge in this field. We discuss future challenges in identifying and targeting the molecular mechanisms to enhance the function of natural or TCR-affinity optimized T cells, and we provide perspectives for the development of protective anti-tumor T cell responses.

The inflammasomes: guardians of the body.

The innate immune system relies on its capacity to rapidly detect invading pathogenic microbes as foreign and to eliminate them. The discovery of Toll-like receptors (TLRs) provided a class of membrane receptors that sense extracellular microbes and trigger antipathogen signaling cascades. More recently, intracellular microbial sensors have been identified, including NOD-like receptors (NLRs). Some of the NLRs also sense nonmicrobial danger signals and form large cytoplasmic complexes called inflammasomes that link the sensing of microbial products and metabolic stress to the proteolytic activation of the proinflammatory cytokines IL-1beta and IL-18. The NALP3 inflammasome has been associated with several autoinflammatory conditions including gout. Likewise, the NALP3 inflammasome is a crucial element in the adjuvant effect of aluminum and can direct a humoral adaptive immune response. In this review, we discuss the role of NLRs, and in particular the inflammasomes, in the recognition of microbial and danger components and the role they play in health and disease.

High-grade meningiomas: new avenues for drug treatment?

PURPOSE OF REVIEW: For standard first-line treatment of high-grade meningiomas, surgical resection and radiotherapy are regarded as standard of care. In the recurrent setting after exhaustion of all local treatment options, no effective therapies are known and several drugs have failed to show efficacy, but novel compounds may offer hope for better disease control. RECENT FINDINGS: Upregulation of proangiogenic molecules and dysregulation of some signaling pathways such as the platelet-derived growth factor and mammalian target of rapamycin are recurrently found in high-grade meningiomas. Furthermore, in-vitro studies and single patient experience indicate that trabectedin may be an effective therapy in this tumor type. Unfortunately, so far there is a lack of conclusive clinical trials to draw definite conclusions of efficacy of these approaches. SUMMARY: There remains a significant unmet need for defining the role of medical therapy in recurrent high-grade meningioma, and more basic research and multicentric well designed trials are needed in this rare and devastating tumor type. Potentially promising novel therapeutics include antiangiogenic drugs, molecular inhibitors of signaling cascades, immunotherapeutics or trabectedin. However, more basic research is required to identify more promising drug targets. VIDEO ABSTRACT AVAILABLE: See the Video Supplementary Digital Content 1 (http://links.lww.com/CONR/A22).

Chemokine receptor trio: CXCR3, CXCR4 and CXCR7 crosstalk via CXCL11 and CXCL12.

Although chemokines are well established to function in immunity and endothelial cell activation and proliferation, a rapidly growing literature suggests that CXC Chemokine receptors CXCR3, CXCR4 and CXCR7 are critical in the development and progression of solid tumors. The effect of these chemokine receptors in tumorigenesis is mediated via interactions with shared ligands I-TAC (CXCL11) and SDF-1 (CXCL12). Over the last decade, CXCR4 has been extensively reported to be overexpressed in most human solid tumors and has earned considerable attention toward elucidating its role in cancer metastasis. To enrich the existing armamentarium of anti-cancerous agents, many inhibitors of CXCL12-CXCR4 axis have emerged as additional or alternative agents for neo-adjuvant treatments and even many of them are in preclinical and clinical stages of their development. However, the discovery of CXCR7 as another receptor for CXCL12 with rather high binding affinity and recent reports about its involvement in cancer progression, has questioned the potential of "selective blockade" of CXCR4 as cancer chemotherapeutics. Interestingly, CXCR7 can also bind another chemokine CXCL11, which is an established ligand for CXCR3. Recent reports have documented that CXCR3 and their ligands are overexpressed in different solid tumors and regulate tumor growth and metastasis. Therefore, it is important to consider the interactions and crosstalk between these three chemokine receptors and their ligand mediated signaling cascades for the development of effective anti-cancer therapies. Emerging evidence also indicates that these receptors are differentially expressed in tumor endothelial cells as well as in cancer stem cells, suggesting their direct role in regulating tumor angiogenesis and metastasis. In this review, we will focus on the signals mediated by this receptor trio via their shared ligands and their role in tumor growth and progression.

Platforms for high-throughput screening of Wnt/Frizzled antagonists.

Signaling cascades initiated by Wnt lipoglycoproteins and their receptors of the Frizzled family regulate many aspects of animal development and physiology. Improper activation of this signaling promotes carcinogenic transformation and metastasis. Development of agents blocking the Wnt-Frizzled signaling is of prime interest for drug discovery. Despite certain progress no such agents are as yet brought to the market or even to clinical trials. One reason for these delays might be the use of suboptimal readout assays. In this article we overview existing and developing assay platforms to screen for Wnt-Frizzled antagonists. Among those, G protein-activating assays built on the emerging GPCR properties of Frizzleds are highlighted.

Calcineurin blockade prevents cardiac mitogen-activated protein kinase activation and hypertrophy in renovascular hypertension.

Chronic stimulation of the renin-angiotensin system induces an elevation of blood pressure and the development of cardiac hypertrophy via the actions of its effector, angiotensin II. In cardiomyocytes, mitogen-activated protein kinases as well as protein kinase C isoforms have been shown to be important in the transduction of trophic signals. The Ca(2+)/calmodulin-dependent phosphatase calcineurin has also been suggested to play a role in cardiac growth. In the present report, we investigate possible cross-talks between calcineurin, protein kinase C, and mitogen-activated protein kinase pathways in controlling angiotensin II-induced hypertrophy. Angiotensin II-stimulated cardiomyocytes and mice with angiotensin II-dependent renovascular hypertension were treated with the calcineurin inhibitor cyclosporin A. Calcineurin, protein kinase C, and mitogen-activated protein kinase activations were determined. We show that cyclosporin A blocks angiotensin II-induced mitogen-activated protein kinase activation in cultured primary cardiomyocytes and in the heart of hypertensive mice. Cyclosporin A also inhibits specific protein kinase C isoforms. In vivo, cyclosporin A prevents the development of cardiac hypertrophy, and this effect appears to be independent of hemodynamic changes. These data suggest cross-talks between the calcineurin pathway, the protein kinase C, and the mitogen-activated protein kinase signaling cascades in transducing angiotensin II-mediated stimuli in cardiomyocytes and could provide the basis for an integrated model of cardiac hypertrophy.

Heat shock response in photosynthetic organisms: membrane and lipid connections.

The ability of photosynthetic organisms to adapt to increases in environmental temperatures is becoming more important with climate change. Heat stress is known to induce heat-shock proteins (HSPs) many of which act as chaperones. Traditionally, it has been thought that protein denaturation acts as a trigger for HSP induction. However, increasing evidence has shown that many stress events cause HSP induction without commensurate protein denaturation. This has led to the membrane sensor hypothesis where the membrane's physical and structural properties play an initiating role in the heat shock response. In this review, we discuss heat-induced modulation of the membrane's physical state and changes to these properties which can be brought about by interaction with HSPs. Heat stress also leads to changes in lipid-based signaling cascades and alterations in calcium transport and availability. Such observations emphasize the importance of membranes and their lipids in the heat shock response and provide a new perspective for guiding further studies into the mechanisms that mediate cellular and organismal responses to heat stress.