Galectin-3 Up-regulation In Hypoxic And Nutrient Deprived Microenvironments Promotes Cell Survival.

Galectin-3 (gal-3) is a β-galactoside binding protein related to many tumoral aspects, e.g. angiogenesis, cell growth and motility and resistance to cell death. Evidence has shown its upregulation upon hypoxia, a common feature in solid tumors such as glioblastoma multiformes (GBM). This tumor presents a unique feature described as pseudopalisading cells, which accumulate large amounts of gal-3. Tumor cells far from hypoxic/nutrient deprived areas express little, if any gal-3. Here, we have shown that the hybrid glioma cell line, NG97ht, recapitulates GBM growth forming gal-3 positive pseudopalisades even when cells are grafted subcutaneously in nude mice. In vitro experiments were performed exposing these cells to conditions mimicking tumor areas that display oxygen and nutrient deprivation. Results indicated that gal-3 transcription under hypoxic conditions requires previous protein synthesis and is triggered in a HIF-1α and NF-κB dependent manner. In addition, a significant proportion of cells die only when exposed simultaneously to hypoxia and nutrient deprivation and demonstrate ROS induction. Inhibition of gal-3 expression using siRNA led to protein knockdown followed by a 1.7-2.2 fold increase in cell death. Similar results were also found in a human GBM cell line, T98G. In vivo, U87MG gal-3 knockdown cells inoculated subcutaneously in nude mice demonstrated decreased tumor growth and increased time for tumor engraftment. These results indicate that gal-3 protected cells from cell death under hypoxia and nutrient deprivation in vitro and that gal-3 is a key factor in tumor growth and engraftment in hypoxic and nutrient-deprived microenvironments. Overexpression of gal-3, thus, is part of an adaptive program leading to tumor cell survival under these stressing conditions.

Functional microarray analysis suggests repressed cell-cell signaling and cell survival-related modules inhibit progression of head and neck squamous cell carcinoma

Background: Cancer shows a great diversity in its clinical behavior which cannot be easily predicted using the currently available clinical or pathological markers. The identification of pathways associated with lymph node metastasis (N+) and recurrent head and neck squamous cell carcinoma (HNSCC) may increase our understanding of the complex biology of this disease. Methods: Tumor samples were obtained from untreated HNSCC patients undergoing surgery. Patients were classified according to pathologic lymph node status (positive or negative) or tumor recurrence (recurrent or non-recurrent tumor) after treatment (surgery with neck dissection followed by radiotherapy). Using microarray gene expression, we screened tumor samples according to modules comprised by genes in the same pathway or functional category. Results: The most frequent alterations were the repression of modules in negative lymph node (N0) and in non-recurrent tumors rather than induction of modules in N+ or in recurrent tumors. N0 tumors showed repression of modules that contain cell survival genes and in non-recurrent tumors cell-cell signaling and extracellular region modules were repressed. Conclusions: The repression of modules that contain cell survival genes in N0 tumors reinforces the important role that apoptosis plays in the regulation of metastasis. In addition, because tumor samples used here were not microdissected, tumor gene expression data are represented together with the stroma, which may reveal signaling between the microenvironment and tumor cells. For instance, in non-recurrent tumors, extracellular region module was repressed, indicating that the stroma and tumor cells may have fewer interactions, which disable metastasis development. Finally, the genes highlighted in our analysis can be implicated in more than one pathway or characteristic, suggesting that therapeutic approaches to prevent tumor progression should target more than one gene or pathway, specially apoptosis and interactions between tumor cells and the stroma.

Recombinant human prolactin promotes human beta cell survival via inhibition of extrinsic and intrinsic apoptosis pathways

Transplantation of pancreatic islets constitutes a promising alternative treatment for type 1 diabetes. However, it is limited by the shortage of organ donors. Previous results from our laboratory have demonstrated beneficial effects of recombinant human prolactin (rhPRL) treatment on beta cell cultures. We therefore investigated the role of rhPRL action in human beta cell survival, focusing on the molecular mechanisms involved in this process. Human pancreatic islets were isolated using an automated method. Islet cultures were pre-treated in the absence or presence of rhPRL and then subjected to serum starvation or cytokine treatment. Beta cells were labelled with Newport green and apoptosis was evaluated using flow cytometry analysis. Levels of BCL2 gene family members were studied by quantitative RT-PCR and western blot. Caspase-8, -9 and -3 activity, as well as nitric oxide production, were evaluated by fluorimetric assays. The proportion of apoptotic beta cells was significantly lowered in the presence of rhPRL under both cell death-induced conditions. We also demonstrated that cytoprotection may involve an increase of BCL2/BAX ratio, as well as inhibition of caspase-8, -9 and -3. Our study provides relevant evidence for a protective effect of lactogens on human beta cell apoptosis. The results also suggest that the improvement of cell survival may involve, at least in part, inhibition of cell death pathways controlled by the BCL2 gene family members. These findings are highly relevant for improvement of the islet isolation procedure and for clinical islet transplantation.

A lipocalin sequence signature modulates cell survival

Lipocalins are beta-barrel proteins, which share three conserved motifs in their amino acid sequence. In this study, we identified by a peptide mapping approach, a seven-amino acid sequence related to one of these motifs (motif 2) that modulates cell survival. A synthetic peptide based on an insect lipocalin displayed cytoprotective activity in serum-deprived endothelial cells and leucocytes. This activity was dependent on nitric oxide synthase. This sequence was found within several lipocalins, including apolipoprotein D, retinol binding protein, lipocalin-type prostaglandin D synthase, and many unknown proteins, suggesting that it is a sequence signature and a lipocalin conserved property. (C) 2010 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Constitutive mutants of the GM-CSF receptor reveal multiple pathways leading to myeloid cell survival, proliferation, and granulocyte-macrophage differentiation

Activation of the granulocyte-macrophage colony-stimulating factor (GM-CSF) family of receptors promotes the survival, proliferation, and differentiation of cells of the myeloid compartment. Several signaling pathways are activated downstream of the receptor, however it is not clear how these induce specific biologic outcomes. We have previously identified 2 classes of constitutively active mutants of the shared signaling subunit, human (h) betac, of the human GM-CSF/interieukin-3 (IL-3)/IL-5 receptors that exhibit different modes of signaling. In a factor-dependent bipotential myeloid cell line, FDB1, an activated mutant containing a substitution in the transmembrane domain (V449E) induces factor-independent proliferation and survival, while mutants in the extracellular domain induce factor-independent granulocyte-macrophage differentiation. Here we have used further mutational analysis to demonstrate that there are nonredundant functions for several regions of the cytoplasmic domain with regard to mediating proliferation, viability, and differentiation, which have not been revealed by previous studies with the wild-type GM-CSF receptor. This unique lack of redundancy has revealed an association of a conserved membrane-proximal region with viability signaling and a critical but distinct role for tyrosine 577 in the activities of each class of mutant.

Mechanisms of B cell survival induced by BAFF, APRIL and their receptors

RESUME : BAFF est un membre de 1a famille du TNF qui contrôle l'homéostasie des lymphocytes B. BAFF lie les récepteurs TACI, BCMA et BAFF-R sur les cellules B, tandis qu'APRIL, son proche homologue, lie seulement TACI et BCMA. BAFF et APRIL sont des protéines transmembranaires pouvant -être relâchées sous forme de cytokines trimériques solubles suite à un clivage protéolytique. Le BAFF soluble peut s'assembler en 60-mère. Les rôles physiologiques des BAFF membranaires et solubles sont inconnnus. Nous avons étudié la capacité de diverses formes de BAFF et APRIL à activer différents récepteurs. BAFF-R répond à toutes les formes dé BAFF, tandis que TACI nécessite du BAFF ou de l'APRIL membranaire ou oligomérisé pour être activé et pour transmettre des signaux de survie dans les lymphocytes B primaires. TACI ne répond pas aux ligands trimériques bien qu'il puisse les lier. TACI est essentiel pour la réponse humorale aux antigènes présentant des épitoges répétitifs, une réponse qui est indépendante des lymphocytes T (réponse TI-2). Des souris exprimant moins de BAFF ont un pourcentage modérément réduit de lymphocytes B et leur réponse TI-2 est atténuée. Par contre, des souris qui n'expriment que du BAFF membranaire ont encore moins de cellules B mais répondent efficacement aux antigènes TI-2. Ces résultats suggèrent que le BAFF soluble est impliqué dans le maintien de la population des lymphocytes B, alors que le BAFF membranaire peut activer TACI lors d'are réponse TI-2. Le BAFF 60-mère est un autre activateur potentiel de TACI in vivo. Le BAFF 60-mère existe dans des surnageants de cellules productrices de BAFF mais n'est pas détecté dans le plasma de souris saines, même lorsqu'elles présentent des niveaux élevés de BAFF. BAFF 60-mère est néanmoins présent dans le plasma de souris transgéniques pour BAFF et de souris déficientes en TACI. Comme ces deux lignées présentent des signes d'autoimmunité, ces résultats suggèrent que la présence de BAFF 60-mère pourrait être liée à des conditions pathologiques. Summary : The TNF family ligand BAFF is essential for B cell homeostasis. BAFF binds to the receptors TACI, BCMA and BAFF-R on B cells, whereas its close homolog APRIL binds to TACI and BCMA only. BAFF and APRIL are transmembrane proteins, which can be proteolytically processed to release trimeric soluble cytokines. Soluble BAFF 3-mer can further assemble in a 60-mer. The physiological roles of membrane-bound and soluble BAFF are unknown. We studied the ability of various forms of BAFF and APRIL to signal through different receptors. BAFF-R responded to all forms of BAFF, but TACI required membrane-bound, cross-licked or oligomeric BAFF or APRIL in order to transmit productive signals in primary B cells. TACI was unresponsive to trimeric ligands, although it could bind them. TACI is essential for T-cell independent antibody responses to antigens with repetitive epitopes (TI-2 responses). Mice expressing lower than normal levels of BAFF displayed a moderate B cell reduction and impaired TI-2 responses, whereas mice expressing membrane-bound BAFF displayed severe B cell reduction, but unimpaired TI-2 responses. These results suggest that processed BAFF is involved in the maintenance of the B cell pool and that membrane-bound BAFF can activate TACI during T-cell independent humoral responses. BAFF 60-mer is another potential activator of TACI in vivo. BAFF 60-mer was detected in the supernatant of BAFF-producing cells, but not in the plasma of healthy mice with either norma1 or elevated BAFF levels. It was however present in sera of BAFF transgenic mice and TACI-/- mice, both of which suffer from autoimmunity, suggesting that GAFF 60-mer may be linked to pathogenic conditions.

BAFF and the regulation of B cell survival.

The TNF family member BAFF is a fundamental survival factor for B cells. BAFF binds to three receptors, only one of which, BAFF-R, does not cross-react with the BAFF-related ligand APRIL. The survival function of BAFF on B cells is mediated mainly by BAFF-R and is particularly effective in transitional B cells. BAFF depletion leads to a considerable decrease in mature B cells, without apparent effect on B cell genesis. Consistently, BAFF overexpression results in an expanded B cell compartment and autoimmunity in mice. Elevated amounts of BAFF can be found in the serum of patients suffering from autoimmune diseases. The BAFF system is a promising target for the treatment of autoimmune diseases.

Neural crest cell survival is dependent on Rho kinase and is required for development of the mid face in mouse embryos.

Neural crest cells (NCC) give rise to much of the tissue that forms the vertebrate head and face, including cartilage and bone, cranial ganglia and teeth. In this study we show that conditional expression of a dominant-negative (DN) form of Rho kinase (Rock) in mouse NCC results in severe hypoplasia of the frontonasal processes and first pharyngeal arch, ultimately resulting in reduction of the maxilla and nasal bones and severe craniofacial clefting affecting the nose, palate and lip. These defects resemble frontonasal dysplasia in humans. Disruption of the actin cytoskeleton, which leads to abnormalities in cell-matrix attachment, is seen in the RockDN;Wnt1-cre mutant embryos. This leads to elevated cell death, resulting in NCC deficiency and hypoplastic NCC-derived craniofacial structures. Rock is thus essential for survival of NCC that form the craniofacial region. We propose that reduced NCC numbers in the frontonasal processes and first pharyngeal arch, resulting from exacerbated cell death, may be the common mechanism underlying frontonasal dysplasia.

Extracellular matrix molecules enhance the neurotrophic effect of schwann cell-like differentiated adipose-derived stem cells and increase cell survival under stress conditions.

Since the first reports of induction of adipose-derived stem cells (ASC) into neuronal and glial cell phenotypes, expectations have increased regarding their use in tissue engineering applications for nerve repair. Cell adhesion to extracellular matrix (ECM) is a basic feature of survival, differentiation, and migration of Schwann cells (SC) during nerve regeneration, and fibronectin and laminin are two key molecules of this process. Interaction between ECM and SC-like differentiated ASC (dASC) could potentially improve the neurotrophic potential of the stem cells. We have investigated the effect of ECM molecules on SC-like dASC in terms of proliferation, adhesion, and cell viability. Fibronectin and laminin did not affect the proliferation of dASC when compared with cell adherent tissue culture plastic, but significantly improved viability and cell attachment when dASC were exposed to apoptotic conditions. To assess the influence of the ECM molecules on dASC neurotrophic activity, dASC were seeded onto ECM-coated culture inserts suspended above dorsal root ganglia (DRG) sensory neurons. Neurite outgrowth of DRG neurons was enhanced when dASC were seeded on fibronectin and laminin when compared with controls. When DRG neurons and dASC were in direct contact on the various surfaces there was significantly enhanced neurite outgrowth and coculture with laminin-conditioned dASC produced the longest neurites. Compared with primary SCs, dASC grown on laminin produced similar levels of neurite outgrowth in the culture insert experiments but neurite length was shorter in the direct contact groups. Anti β1 integrin blocking antibody could inhibit baseline and dASC evoked neurite elongation but had no effect on outgrowth mediated by laminin-conditioned dASC. ECM molecules had no effect on the levels of nerve growth factor and brain-derived neurotrophic factor secretion from dASC. The results of the study suggest that ECM molecules can significantly improve the potential of dASC for nerve regeneration.

Mir-190b negatively contributes to the Trypanosoma cruzi- infected cell survival by repressing PTEN protein expression

Chagas disease, which is caused by the intracellular protozoanTrypanosoma cruzi, is a serious health problem in Latin America. The heart is one of the major organs affected by this parasitic infection. The pathogenesis of tissue remodelling, particularly regarding cardiomyocyte behaviour after parasite infection, and the molecular mechanisms that occur immediately following parasite entry into host cells are not yet completely understood. Previous studies have reported that the establishment of parasitism is connected to the activation of the phosphatidylinositol-3 kinase (PI3K), which controls important steps in cellular metabolism by regulating the production of the second messenger phosphatidylinositol-3,4,5-trisphosphate. Particularly, the tumour suppressor PTEN is a negative regulator of PI3K signalling. However, mechanistic details of the modulatory activity of PTEN on Chagas disease have not been elucidated. To address this question, H9c2 cells were infected with T. cruzi Berenice 62 strain and the expression of a specific set of microRNAs (miRNAs) were investigated. Our cellular model demonstrated that miRNA-190b is correlated to the decrease of cellular viability rates by negatively modulating PTEN protein expression in T. cruzi-infected cells.

mTORC2 regulates PGE2-mediated endothelial cell survival and migration.

Prostaglandin E(2) (PGE(2)) promotes angiogenesis by in part inducing endothelial cell survival and migration. The present study examined the role of mTOR and its two complexes, mTORC1 and mTORC2, in PGE(2)-mediated endothelial cell responses. We used small interfering RNA (siRNA) to raptor or rictor to block mTORC1 or mTORC2, respectively. We observed that down-regulation of mTORC2 but not mTORC1 reduced baseline and PGE(2)-induced endothelial cell survival and migration. At the molecular level, we found that knockdown of mTORC2 inhibited PGE(2)-mediated Rac and Akt activation two important signaling intermediaries in endothelial cell migration and survival, respectively. In addition, inhibition of mTORC2 by prolonged exposure of endothelial cells to rapamycin also prevented PGE(2)-mediated endothelial cell survival and migration confirming the results obtained with the siRNA approach. Taken together these results show that mTORC2 but not mTORC1 is an important signaling intermediary in PGE(2)-mediated endothelial cell responses.

Chicken BAFF--a highly conserved cytokine that mediates B cell survival.

Members of the tumor necrosis factor (TNF) family play key roles in the regulation of inflammation, immune responses and tissue homeostasis. Here we describe the identification of the chicken homologue of mammalian B cell activating factor of the TNF family (BAFF/BLyS). By searching a chicken EST database we identified two overlapping cDNA clones that code for the entire open reading frame of chicken BAFF (chBAFF), which contains a predicted transmembrane domain and a putative furin protease cleavage site like its mammalian counterparts. The amino acid identity between soluble chicken and human BAFF is 76%, considerably higher than for most other known cytokines. The chBAFF gene is most strongly expressed in the bursa of Fabricius. Soluble recombinant chBAFF produced by human 293T cells interacted with the mammalian cell-surface receptors TACI, BCMA and BAFF-R. It bound to chicken B cells, but not to other lymphocytes, and it promoted the survival of splenic chicken B cells in culture. Furthermore, bacterially expressed chBAFF induced the selective expansion of B cells in the spleen and cecal tonsils when administered to young chicks. Our results suggest that like its mammalian counterpart, chBAFF plays an important role in survival and/or proliferation of chicken B cells.

BAFF AND APRIL: a tutorial on B cell survival.

BAFF, a member of the TNF family, is a fundamental survival factor for transitional and mature B cells. BAFF overexpression leads to an expanded B cell compartment and autoimmunity in mice, and elevated amounts of BAFF can be found in the serum of autoimmune patients. APRIL is a related factor that shares receptors with BAFF yet appears to play a different biological role. The BAFF system provides not only potential insight into the development of autoreactive B cells but a relatively simple paradigm to begin considering the balancing act between survival, growth, and death that affects all cells.

Specific Silencing of the REST Target Genes in Insulin-Secreting Cells Uncovers Their Participation in Beta Cell Survival.

The absence of the transcriptional repressor RE-1 Silencing Transcription Factor (REST) in insulin-secreting beta cells is a major cue for the specific expression of a large number of genes. These REST target genes were largely ascribed to a function of neurotransmission in a neuronal context, whereas their role in pancreatic beta cells has been poorly explored. To identify their functional significance, we have generated transgenic mice expressing REST in beta cells (RIP-REST mice), and previously discovered that REST target genes are essential to insulin exocytosis. Herein we characterized a novel line of RIP-REST mice featuring diabetes. In diabetic RIP-REST mice, high levels of REST were associated with postnatal beta cell apoptosis, which resulted in gradual beta cell loss and sustained hyperglycemia in adults. Moreover, adenoviral REST transduction in INS-1E cells led to increased cell death under control conditions, and sensitized cells to death induced by cytokines. Screening for REST target genes identified several anti-apoptotic genes bearing the binding motif RE-1 that were downregulated upon REST expression in INS-1E cells, including Gjd2, Mapk8ip1, Irs2, Ptprn, and Cdk5r2. Decreased levels of Cdk5r2 in beta cells of RIP-REST mice further confirmed that it is controlled by REST, in vivo. Using siRNA-mediated knock-down in INS-1E cells, we showed that Cdk5r2 protects beta cells against cytokines and palmitate-induced apoptosis. Together, these data document that a set of REST target genes, including Cdk5r2, is important for beta cell survival.

A mouse model for improving cell survival of bisected cattle embryos

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