Combined CSL and p53 downregulation promotes cancer-associated fibroblast activation.

Stromal fibroblast senescence has been linked to ageing-associated cancer risk. However, density and proliferation of cancer-associated fibroblasts (CAFs) are frequently increased. Loss or downmodulation of the Notch effector CSL (also known as RBP-Jκ) in dermal fibroblasts is sufficient for CAF activation and ensuing keratinocyte-derived tumours. We report that CSL silencing induces senescence of primary fibroblasts from dermis, oral mucosa, breast and lung. CSL functions in these cells as a direct repressor of multiple senescence- and CAF-effector genes. It also physically interacts with p53, repressing its activity. CSL is downmodulated in stromal fibroblasts of premalignant skin actinic keratosis lesions and squamous cell carcinomas, whereas p53 expression and function are downmodulated only in the latter, with paracrine FGF signalling as the probable culprit. Concomitant loss of CSL and p53 overcomes fibroblast senescence, enhances expression of CAF effectors and promotes stromal and cancer cell expansion. The findings support a CAF activation-stromal co-evolution model under convergent CSL-p53 control.

A signaling mechanism coupling netrin-1/deleted in colorectal cancer chemoattraction to SNARE-mediated exocytosis in axonal growth cones

Directed cell migration and axonal guidance are essential steps in neural development. Both processes are controlled by specific guidance cues that activate the signaling cascades that ultimately control cytoskeletal dynamics. Another essential step in migration and axonal guidance is the regulation of plasmalemma turnover and exocytosis in leading edges and growth cones. However, the cross talk mechanisms linking guidance receptors and membrane exocytosis are not understood. Netrin-1 is a chemoattractive cue required for the formation of commissural pathways. Here, we show that the Netrin-1 receptor deleted in colorectal cancer (DCC) forms a protein complex with the t-SNARE (target SNARE) protein Syntaxin-1 (Sytx1). This interaction is Netrin-1 dependent both in vitro and in vivo, and requires specific Sytx1 and DCC domains. Blockade of Sytx1 function by using botulinum toxins abolished Netrin-1-dependent chemoattraction of axons in mouse neuronal cultures. Similar loss-of-function experiments in the chicken spinal cord in vivo using dominant-negative Sytx1 constructs or RNAi led to defects in commissural axon pathfinding reminiscent to those described in Netrin-1 and DCC loss-of-function models. We also show that Netrin-1 elicits exocytosis at growth cones in a Sytx1-dependent manner. Moreover, we demonstrate that the Sytx1/DCC complex associates with the v-SNARE (vesicle SNARE) tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) and that knockdown of TI-VAMP in the commissural pathway in the spinal cord results in aberrant axonal guidance phenotypes. Our data provide evidence of a new signaling mechanism that couples chemotropic Netrin-1/DCC axonal guidance and Sytx1/TI-VAMP SNARE proteins regulating membrane turnover and exocytosis.

A signaling mechanism coupling netrin-1/deleted in colorectal cancer chemoattraction to SNARE-mediated exocytosis in axonal growth cones

Directed cell migration and axonal guidance are essential steps in neural development. Both processes are controlled by specific guidance cues that activate the signaling cascades that ultimately control cytoskeletal dynamics. Another essential step in migration and axonal guidance is the regulation of plasmalemma turnover and exocytosis in leading edges and growth cones. However, the cross talk mechanisms linking guidance receptors and membrane exocytosis are not understood. Netrin-1 is a chemoattractive cue required for the formation of commissural pathways. Here, we show that the Netrin-1 receptor deleted in colorectal cancer (DCC) forms a protein complex with the t-SNARE (target SNARE) protein Syntaxin-1 (Sytx1). This interaction is Netrin-1 dependent both in vitro and in vivo, and requires specific Sytx1 and DCC domains. Blockade of Sytx1 function by using botulinum toxins abolished Netrin-1-dependent chemoattraction of axons in mouse neuronal cultures. Similar loss-of-function experiments in the chicken spinal cord in vivo using dominant-negative Sytx1 constructs or RNAi led to defects in commissural axon pathfinding reminiscent to those described in Netrin-1 and DCC loss-of-function models. We also show that Netrin-1 elicits exocytosis at growth cones in a Sytx1-dependent manner. Moreover, we demonstrate that the Sytx1/DCC complex associates with the v-SNARE (vesicle SNARE) tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) and that knockdown of TI-VAMP in the commissural pathway in the spinal cord results in aberrant axonal guidance phenotypes. Our data provide evidence of a new signaling mechanism that couples chemotropic Netrin-1/DCC axonal guidance and Sytx1/TI-VAMP SNARE proteins regulating membrane turnover and exocytosis.

The consensus molecular subtypes of colorectal cancer.

Colorectal cancer (CRC) is a frequently lethal disease with heterogeneous outcomes and drug responses. To resolve inconsistencies among the reported gene expression-based CRC classifications and facilitate clinical translation, we formed an international consortium dedicated to large-scale data sharing and analytics across expert groups. We show marked interconnectivity between six independent classification systems coalescing into four consensus molecular subtypes (CMSs) with distinguishing features: CMS1 (microsatellite instability immune, 14%), hypermutated, microsatellite unstable and strong immune activation; CMS2 (canonical, 37%), epithelial, marked WNT and MYC signaling activation; CMS3 (metabolic, 13%), epithelial and evident metabolic dysregulation; and CMS4 (mesenchymal, 23%), prominent transforming growth factor-β activation, stromal invasion and angiogenesis. Samples with mixed features (13%) possibly represent a transition phenotype or intratumoral heterogeneity. We consider the CMS groups the most robust classification system currently available for CRC-with clear biological interpretability-and the basis for future clinical stratification and subtype-based targeted interventions.

Food intake and weight of lactating rats maintained on different protein-calorie diets, and pup growth

Studies on rats maintained on low-protein-calorie diets during the lactation period show that food intake decreases. This process results in weight loss and a delay in litter development. The purpose of the present study was to determine the alterations in food intake, maternal weight and litter growth during lactation when dams were exposed to diets with different levels of protein and carbohydrate. Female Wistar rats receiving one of 4 different diets, A (N = 14), B (N = 14), C (N = 9) and D (N = 9), were used. Diet A contained 16% protein and 66% carbohydrate; diet B, 6% protein and 77% carbohydrate; diet C, 6% protein and 66% carbohydrate; diet D, 16% protein and 56% carbohydrate. Thus, C and D diets were hypocaloric, while A and B were isocaloric. The intake of a low-protein diet in groups B and C affected the weight of dams and litters during the last two weeks of lactation, while the low-calorie diets limited the growth of D litters at 21 days compared with A litters, but had no effect on the weight of D dams. Group B showed an increase in intake during the first five days of lactation, resulting in a behavioral calorie compensation due to the increase in carbohydrate content, but the intake decreased during the last part of lactation. Food intake regulation predominantly involves the recruitment of a variety of peripheral satiety systems that attempt to decrease the central feeding command system.

Pattern of Wnt ligand expression during chick eye development

The dorsoventral axis of the eye is determined prior to optic cup invagination. A variety of signaling pathways have been implicated in the maintenance of the optic dorsoventral axis, including, but not limited to, bone morphogenetic protein 4, Sonic Hedgehog and retinoic acid. Here, we investigated the possible contribution of Wnt ligands to the establishment or maintenance of the optic axis by analyzing their expression pattern during early chick optic development. We performed in situ hybridization of Wnt-1, Wnt-3a, Wnt-4, and Wnt-5a during the optic vesicle, early optic cup and established optic cup stages and focused our analysis on the optic region. Our data showed that Wnt-5a, but none of the others, is expressed in the dorsal region of the eye starting from the Hamburger and Hamilton stage 14 (HH14). These results are supported by cryosections of the labeled optic region, which further reveal that Wnt-5a is expressed only in the dorsal retinal pigmented epithelium. Thus, we propose that Wnt-5a is a marker for dorsal retinal pigmented epithelium in chick embryos from HH14 to HH19.

Genetic variability of vascular endothelial growth factor and prognosis of head and neck cancer in a Brazilian population

Vascular endothelial growth factor (VEGF) is one of the most potent endothelial cell mitogens and plays a critical role in angiogenesis. Polymorphisms in this gene have been evaluated in patients with several types of cancer. The objectives of this study were to determine if there was an association of the -1154G/A polymorphism of the VEGF gene with head and neck cancer and the interaction of this polymorphism with lifestyle and demographic factors. Additionally, the distribution of the VEGF genotype was investigated with respect to the clinicopathological features of head and neck cancer patients. The study included 100 patients with histopathological diagnosis of head and neck squamous cell carcinoma. Patients with treated tumors were excluded. A total of 176 individuals 40 years or older were included in the control group and individuals with a family history of neoplasias were excluded. Analysis was performed after extraction of genomic DNA using the real-time PCR technique. No statistically significant differences between allelic and genotype frequencies of -1154G/A VEGF polymorphism were identified between healthy individuals and patients. The real-time PCR analyses showed a G allele frequency of 0.72 and 0.74 for patients and the control group, respectively. The A allele showed a frequency of 0.28 for head and neck cancer patients and 0.26 for the control group. However, analysis of the clinicopathological features showed a decreased frequency of the A allele polymorphism in patients with advanced (T3 and T4) tumors (OR = 0.36; 95%CI = 0.14-0.93; P = 0.0345). The -1154A allele of the VEGF gene may decrease the risk of tumor growth and be a possible biomarker for head and neck cancer. This polymorphism is associated with increased VEGF production and may have a prognostic importance.

Étude du dysfonctionnement du compartiment des cellules B chez des patients à différents stades d’infection par le virus d’immunodéficience humaine (VIH)

Les anomalies phénotypiques et fonctionnelles des lymphocytes B (LB) sont typiques d'une infection au VIH et se traduisent principalement par une activation polyclonale, une perte de la mémoire immunitaire ainsi qu'une réponse humorale déficiente et des phénomènes auto-immunitaires souvent précurseurs de lymphomes B. Ces anomalies se retrouvent principalement chez les patients lors de la phase chronique de la maladie et semblent être reliées en partie au niveau de la charge virale ainsi qu'à un compartiment de lymphocytes T CD4+ altéré. Cependant, quoique controversé, des éléments d’activation polyclonale ont également été observés chez les non-progresseurs à long terme (LTNPs) qui présentent une charge virale faible et un compartiment T CD4+ semblable aux individus séronégatifs. Ainsi, les objectifs principaux de cette étude sont 1) d’établir une chronologie des anomalies du compartiment des cellules B chez des individus infectés par le VIH qui ont une progression différente de la maladie (PHI normaux, rapides, sains et LTNP). 2) corréler les niveaux sériques du stimulateur de lymphocytes B (BLyS), un facteur de croissance des cellules B, avec les phénotypes observés chez ces mêmes patients. L’hyperglobulinémie, les niveaux sériques de BLyS et d’auto-anticorps ont été mesuré longitudinalement chez une cohorte d’individus en primo-infection (PHI) avec des progressions différentes de la maladie (rapides et normaux), LTNP et sujets sains. Nos résultats démontrent que l’activation polyclonale des LB survient indépendamment de la vitesse de progression et persiste chez les LTNP ou malgré une thérapie antirétrovirale efficace chez les progresseurs rapides. Des niveaux élevés de BLyS dans le sérum des progresseurs rapides corrèlent avec des fréquences altérées de monocytes et cellules dendritiques, suggérant un rôle de celles-ci dans l’atteinte du compartiment des cellules B.

Le rôle de la signalisation Wnt dans le phénotype anormal des ostéoblastes ostéoarthrosiques

L’ostéoarthrose (OA) est une pathologie de forte incidence affectant les articulations. Elle est caractérisée principalement par une dégradation du cartilage articulaire, un déséquilibre au niveau du remodelage osseux et une sclérose de l’os sous-chondral. L’étiologie de cette pathologie reste encore méconnue, cependant il semble de plus en plus que tous les tissus composant l’articulation soient affectés dans cette pathologie. L’importance du rôle de l’os dans le développement de l’OA est incontestable et représente donc une cible thérapeuthique intéressante. Des études effectuées par tomodensitométrie ont démontré une structure et une organisation anormales du tissu osseux des patients OA. Parallèlement, les cultures primaires d’ostéoblastes (Ob) humains OA issus de l’os sous-chondral démontrent un phénotype altéré et une faible minéralisation in vitro. La signalisation Wnt, essentielle dans l’embryogenèse, a montré avoir un rôle clé dans la régulation de l’ostéogenèse en régulant notamment la différenciation terminale des Ob. Le facteur de croissance transformant-β1 (TGF-β1), un facteur agissant notamment sur la prolifération et sur le début de la différenciation des Ob, est surexprimé par les Ob OA et pourrait moduler cette signalisation. Aussi, deux populations de patients OA peuvent être différenciées in vitro par la production de prostaglandines E2 (PGE2) par leurs Ob et les PGE2, dans une étude sur le cancer colorectal, ont montré moduler la signalisation Wnt. Notre hypothèse de travail est que l’activation de la voie de signalisation Wnt/β-caténine est diminuée dans les Ob OA. Cette diminution est responsable de la sous-minéralisation et de l’altération du phénotype des Ob humains OA. Par ailleurs, DKK2, dont l’expression est contrôlée par TGF-β1, est responsable de la diminution de l’activité Wnt/β-caténine et les PGE2 peuvent en partie corriger cette situation. L’objectif général de cette étude est d’une part, de démontrer le rôle de TGF-β1, DKK2 et de PGE2 sur l’altération de la signalisation Wnt/β-caténine et d’autre part, de démontrer le lien entre TGF-β1 et DKK2 et l’effet de ces derniers sur le phénotype des Ob. Dans cette étude on a montré que la signalisation canonique Wnt est altérée dans les Ob OA et que cela était responsable de l’altération du phénotype des Ob OA. On a montré, parmi les acteurs de la signalisation Wnt, que l’expression de l’antagoniste Dickkopf-1 (DKK1) était relativement similaire entre les Ob OA et normaux contrairement à celle de l’antagoniste DKK2 qui était augmentée et à celle de l’agoniste Wnt7B qui était diminuée dans les Ob OA. On a également montré que les PGE2 pouvaient potentialiser l’activité de la signalisation Wnt dans les Ob OA. L’inhibition de DKK2 a permis d’augmenter l’activité de la signalisation Wnt et de corriger le phénotype anormal ainsi que d’augmenter la minéralisation des Ob OA. L’inhibition de TGF-β1, un facteur aussi surexprimé dans les Ob OA, a également permis la correction du phénotype et l’augmentation de la minéralisation dans les Ob OA. L’inhibition de TGF-β1 a aussi menée à l’inhibition de DKK2. Le contraire ne fût pas observé démontrant ainsi la régulation de DKK2 par TGF-β1. En conclusion, la signalisation canonique Wnt est diminuée dans les Ob OA et cela est dû au niveau élevé de DKK2 dans ces Ob. TGF-β1 régule positivement DKK2 et donc la surexpression de TGF-β1 entraîne celle de DKK2 ce qui a pour conséquences d’altérer le phénotype des Ob. Les PGE2 ont aussi montré pouvoir potentialiser l’activité de la signalisation Wnt et auraient donc un rôle positif. Ensemble, ces données suggèrent que ces altérations au niveau des Ob OA pourraient être responsables de la structure osseuse anormale observée chez les patients OA.

Association entre les dérégulations des cellules dendritiques et les altérations des lymphocytes B présentes chez les patients infectés par le VIH

La dérégulation du compartiment B est une conséquence importante de l’infection par le virus de l’immunodéficience humaine (VIH), qui peut mener à des manifestations autoimmunes et ultimement à des lymphomes B. Parmi les premières anomalies détectées, on dénote l’activation polyclonale, reflétée par la présence d’hyperglobulinémie (hyper-Ig) et des titres élevés d’autoanticorps chez les patients. On observe également une altération des dynamiques des populations, notamment une expansion de la population des cellules matures activées. De plus, les patients évoluent vers l’incapacité de générer une réponse humorale efficace, et sont sujets à une perte de la mémoire immunologique en phase chronique, caractérisée par une diminution de la population des cellules mémoires et par l’épuisement cellulaire. Toutefois, on connaît très peu les mécanismes impliqués dans de telles altérations. Les cellules dendritiques (DC) sont parmi les premières populations cellulaires à rencontrer et à propager le VIH lors d’une infection, et s’en trouvent affectées directement et indirectement, par le virus et ses composantes. On retrouve en effet une diminution des fréquences de DC dans le sang, les muqueuses et les organes lymphoïdes de patients infectés par le VIH, ainsi qu’un blocage au niveau de la maturation cellulaire. Toutefois, un débat perdure quant à l’apparition de ces altérations durant la phase aigüe de l’infection, et à la restauration des fréquences et des fonctions des DC chez les patients sous traitement. Cette controverse est due à la rareté des études longitudinales incluant des suivis qui s’échelonnent de la phase aigüe à la phase chronique de l’infection. Les DC jouent un rôle important dans le développement, la survie et l’activation des lymphocytes B, de façon T-dépendante et T-indépendante, notamment via des facteurs de croissance tel que BLyS (B lymphocyte stimulator). Par conséquent, nous formulons l’hypothèse que dans le cadre d’une infection VIH, les altérations observées au niveau des cellules B sont modulées par les DC. L’objectif majeur de cette étude est donc d’évaluer l’implication potentielle des DC dans les altérations des cellules B au cours de l’infection par le VIH. Pour ce faire, nous avons d’abord caractérisé de façon longitudinale le statut des populations de DC du sang périphérique de patients infectés au VIH et présentant différents types de progression de la maladie. Cela nous a permis d’évaluer la présence d’une corrélation entre les dynamiques de DC et le type de progression. Par la suite, nous avons évalué la capacité des DC à exprimer BLyS, puis mesuré sa concentration ainsi que celles d’autres facteurs de croissance des cellules B dans le plasma des patients. Enfin, nous avons caractérisé le statut des lymphocytes B, en fonction du stade de l’infection et du taux de progression clinique des patients. Cette étude démontre une diminution de la fréquence des populations de DC myéloïdes (mDC) dans le sang de patients infectés par le VIH sujets à une progression clinique. Cette diminution est observée dès le stade aigu de l’infection et au-delà du traitement antirétroviral (ART). Des concentrations élevées de MCP-1 (monocyte chemotactic protein), MIP (macrophage inflammatory protein) -3α et MIP-3β suggèrent la possibilité d’un drainage vers des sites périphériques. Nous observons également des niveaux supérieurs à la normale de précurseurs CD11c+CD14+CD16- en phase chronique, possiblement liés à une tendence de régénération des DC. Les patients en phase chronique présentent de hautes concentrations plasmatiques de BLyS, reflétée par un haut taux d’expression de cette cytokine par les mDC et leurs précurseurs. Parallèlement, nous observons une expansion des cellules B matures activées ainsi que des taux élevés d’IgG et IgA dans le sang de ces patients. De plus, nous constatons l’expansion d’une population de cellules B qui présente à la fois des caractéristiques de cellules B immatures transitionnelles (TI, transitional immature), et de cellules B recirculantes activées de la zone marginale (MZ, marginal zone), considérées ici comme des «précurseurs/activées de la MZ». Cette étude démontre aussi, chez les progresseurs lents, une meilleure préservation du compartiment des DC du sang périphérique, accompagnée d’une augmentation de précurseurs des DC de phénotype CD11c+CD14+CD16+, ainsi que des concentrations plasmatiques et niveaux d’expression normaux de BLyS. Conséquemment, nous n’avons pas observé d’augmentation des cellules B matures activées et des cellules B précurseurs/activées de la MZ. Toutefois, la fréquence des cellules B matures de la MZ est diminuée, reflétant possiblement leur recrutement vers des sites périphériques et leur contribution à un mécanisme actif de contrôle de la progression de la maladie. L’ensemble de ce travail suggère que dans le cadre d’une infection au VIH, les altérations observées au niveau des DC modulent les anomalies des cellules B. Par conséquent, le maintien de l’équilibre des fonctions DC, notamment les fonctions noninflammatoires, pourrait avoir un impact important dans la prévention de la progression de maladies associées aux altérations du compartiment des cellules B.

Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) mediates post-endocytic trafficking of protease-activated receptor 2 and calcitonin receptor-like receptor

The E3 ligase c-Cbl ubiquitinates protease-activated receptor 2 (PAR(2)), which is required for post-endocytic sorting of PAR(2) to lysosomes, where degradation arrests signaling. The mechanisms of post-endocytic sorting of ubiquitinated receptors are incompletely understood. Here, we investigated the role of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), in post-endocytic sorting and signaling of PAR(2). In HEK-PAR(2) cells, PAR(2) activating peptide (PAR(2)-AP) induced PAR(2) trafficking from the cell surface to early endosomes containing endogenous HRS, and then to lysosomes. HRS overexpression or knockdown with small interfering RNA caused formation of enlarged HRS-positive endosomes, where activated PAR(2) and c-Cbl accumulated, and PAR(2) failed to traffic to lysosomes. Overexpression of HRS prevented PAR(2)-AP-induced degradation of PAR(2), as determined by Western blotting. Overexpression of HRS mutant lacking an ubiquitin-binding motif similarly caused retention of PAR(2) in enlarged endosomes. Moreover, HRS overexpression or knockdown caused retention of ubiquitin-resistant PAR(2)Delta14K/R in enlarged HRS-containing endosomes, preventing recycling and resensitization of PAR(2)Delta14K/R. HRS overexpression or knockdown similarly prevented lysosomal trafficking and recycling of calcitonin receptor-like receptor, a non-ubiquitinated receptor that traffics to lysosomes after sustained activation and recycles after transient activation. Thus, HRS plays a critically important role in the post-endocytic sorting of single receptors, PAR(2) and CLR, to both degradative and recycling pathways. This sorting role for HRS is independent of its ubiquitin-interacting motif, and it can regulate trafficking of both ubiquitinated and non-ubiquitinated PAR(2) and non-ubiquitinated CLR. The ultimate sorting decision to degradative or recycling pathways appears to occur downstream from HRS.

Conditional transgenic expression of fibroblast growth factor 9 in the adult mouse heart reduces heart failure mortality after myocardial infarction

BACKGROUND: Fibroblast growth factor 9 (FGF9) is secreted from bone marrow cells, which have been shown to improve systolic function after myocardial infarction (MI) in a clinical trial. FGF9 promotes cardiac vascularization during embryonic development but is only weakly expressed in the adult heart. METHODS AND RESULTS: We used a tetracycline-responsive binary transgene system based on the α-myosin heavy chain promoter to test whether conditional expression of FGF9 in the adult myocardium supports adaptation after MI. In sham-operated mice, transgenic FGF9 stimulated left ventricular hypertrophy with microvessel expansion and preserved systolic and diastolic function. After coronary artery ligation, transgenic FGF9 enhanced hypertrophy of the noninfarcted left ventricular myocardium with increased microvessel density, reduced interstitial fibrosis, attenuated fetal gene expression, and improved systolic function. Heart failure mortality after MI was markedly reduced by transgenic FGF9, whereas rupture rates were not affected. Adenoviral FGF9 gene transfer after MI similarly promoted left ventricular hypertrophy with improved systolic function and reduced heart failure mortality. Mechanistically, FGF9 stimulated proliferation and network formation of endothelial cells but induced no direct hypertrophic effects in neonatal or adult rat cardiomyocytes in vitro. FGF9-stimulated endothelial cell supernatants, however, induced cardiomyocyte hypertrophy via paracrine release of bone morphogenetic protein 6. In accord with this observation, expression of bone morphogenetic protein 6 and phosphorylation of its downstream targets SMAD1/5 were increased in the myocardium of FGF9 transgenic mice. CONCLUSIONS: Conditional expression of FGF9 promotes myocardial vascularization and hypertrophy with enhanced systolic function and reduced heart failure mortality after MI. These observations suggest a previously unrecognized therapeutic potential for FGF9 after MI.

Fibroblast growth factor 2 maintains the neurogenic capacity of embryonic neural progenitor cells in vitro but changes their neuronal subtype specification

Many in vitro systems used to examine multipotential neural progenitor cells (NPCs) rely on mitogens including fibroblast growth factor 2 (FGF2) for their continued expansion. However, FGF2 has also been shown to alter the expression of transcription factors (TFs) that determine cell fate. Here, we report that NPCs from the embryonic telencephalon grown without FGF2 retain many of their in vivo characteristics, making them a good model for investigating molecular mechanisms involved in cell fate specification and differentiation. However, exposure of cortical NPCs to FGF2 results in a profound change in the types of neurons generated, switching them from a glutamatergic to a GABAergic phenotype. This change closely correlates with the dramatic upregulation of TFs more characteristic of ventral telencephalic NPCs. In addition, exposure of cortical NPCs to FGF2 maintains their neurogenic potential in vitro, and NPCs spontaneously undergo differentiation following FGF2 withdrawal. These results highlight the importance of TFs in determining the types of neurons generated by NPCs in vitro. In addition, they show that FGF2, as well as acting as a mitogen, changes the developmental capabilities of NPCs. These findings have implications for the cell fate specification of in vitro-expanded NPCs and their ability to generate specific cell types for therapeutic applications. Disclosure of potential conflicts of interest is found at the end of this article.

RE1 silencing transcription factor/neuron-restrictive silencing factor regulates expansion of adult mouse subventricular zone-derived neural stem/progenitor cells in vitro

Adult neural stem cell (aNSC) activity is tuned by external stimuli through the recruitment of transcription factors. This study examines the RE1 silencing transcription factor (REST) in neural stem/progenitor cells isolated from the subventricular zone of adult mouse brain and provides the first extensive characterization of REST-mediated control of the cellular and molecular properties. This study shows that REST knockdown affects the capacity of progenitor cells to generate neurospheres, reduces cell proliferation, and triggers cell differentiation despite the presence of growth factors. Genome- and transcriptome-wide analyses show that REST binding sites are significantly enriched in genes associated with synaptic transmission and nervous system development and function. Seeking candidate regulators of aNSC function, this study identifies a member of the bone morphogenetic protein (BMP) family, BMP6, the mRNA and protein of which increased after REST knockdown. The results of this study extend previous findings, demonstrating a reciprocal control of REST expression by BMPs. Administration of exogenous BMP6 inhibits aNSC proliferation and induces the expression of the astrocytic marker glial fibrillary acidic protein, highlighting its antimitogenic and prodifferentiative effects. This study suggests that BMP6 produced in a REST-regulated manner together with other signals can contribute to regulation of NSC maintenance and fate. © 2015 Wiley Periodicals, Inc.

MKP-1 mediates glucocorticoid-induced ERK1/2 dephosphorylation and reduction in pancreatic beta-cell proliferation in islets from early lactating mothers

Maternal pancreatic islets undergo a robust increase of mass and proliferation during pregnancy, which allows a compensation of gestational insulin resistance. Studies have described that this adaptation switches to a low proliferative status after the delivery. The mechanisms underlying this reversal are unknown, but the action of glucocorticoids (GCs) is believed to play an important role because GCs counteract the pregnancy-like effects of PRL on isolated pancreatic islets maintained in cell culture. Here, we demonstrate that ERK1/2 phosphorylation (phospho-ERK1/2) is increased in maternal rat islets isolated on the 19th day of pregnancy. Phospho-ERK1/2 status on the 3rd day after delivery (L3) rapidly turns to values lower than that found in virgin control rats (CTL). MKP-1, a protein phosphatase able to dephosphorylate ERK1/2, is increased in islets from L3 rats. Chromatin immunoprecipitation assay revealed that binding of glucocorticoid receptor (GR) to MKP-1 promoter is also increased in islets from L3 rats. In addition, dexamethasone (DEX) reduced phospho-ERK1/2 and increased MKP-1 expression in RINm5F and MIN-6 cells. Inhibition of transduction with cycloheximide and inhibition of phosphatases with orthovanadate efficiently blocked DEX-induced downregulation of phospho-ERK1/2. In addition, specific knockdown of MKP-1 with siRNA suppressed the downregulation of phosphoERK1/2 and the reduction of proliferation induced by DEX. Altogether, our results indicate that downregulation of phospho-ERK1/2 is associated with reduction in proliferation found in islets of early lactating mothers. This mechanism is probably mediated by GC-induced MKP-1 expression.