NADPH oxidase NOX4 mediates stellate cell activation and hepatocyte cell death during liver fibrosis development.

A role for the NADPH oxidases NOX1 and NOX2 in liver fibrosis has been proposed, but the implication of NOX4 is poorly understood yet. The aim of this work was to study the functional role of NOX4 in different cell populations implicated in liver fibrosis: hepatic stellate cells (HSC), myofibroblats (MFBs) and hepatocytes. Two different mice models that develop spontaneous fibrosis (Mdr2−/−/p19ARF−/−, Stat3Δhc/Mdr2−/−) and a model of experimental induced fibrosis (CCl4) were used. In addition, gene expression in biopsies from chronic hepatitis C virus (HCV) patients or non-fibrotic liver samples was analyzed. Results have indicated that NOX4 expression was increased in the livers of all animal models, concomitantly with fibrosis development and TGF-β pathway activation. In vitro TGF-β-treated HSC increased NOX4 expression correlating with transdifferentiation to MFBs. Knockdown experiments revealed that NOX4 downstream TGF-β is necessary for HSC activation as well as for the maintenance of the MFB phenotype. NOX4 was not necessary for TGF-β-induced epithelial-mesenchymal transition (EMT), but was required for TGF-β-induced apoptosis in hepatocytes. Finally, NOX4 expression was elevated in patients with hepatitis C virus (HCV)-derived fibrosis, increasing along the fibrosis degree. In summary, fibrosis progression both in vitro and in vivo (animal models and patients) is accompanied by increased NOX4 expression, which mediates acquisition and maintenance of the MFB phenotype, as well as TGF-β-induced death of hepatocytes.

Nuclear receptor peroxisome proliferator activated receptor (PPAR) beta/delta in skin wound healing and cancer

We review the functions of peroxisome proliferator activated receptor (PPAR) beta/delta in skin wound healing and cancer. In particular, we highlight the roles of PPAR beta/delta in inhibiting keratinocyte apoptosis at wound edges via activation of the PI3K/PKB alpha/Akt1 pathway and its role during re-epithelialization in regulating keratinocyte adhesion and migration. In fibroblasts, PPAR beta/delta controls IL-1 signalling and thereby contributes to the homeostatic control of keratinocyte proliferation. We discuss its therapeutic potential for treating diabetic wounds and inflammatory skin diseases such as psoriasis and acne vulgaris. PPAR beta/delta is classified as a tumour growth modifier; it is activated by chronic low-grade inflammation, which promotes the production of lipids that, in turn, enhance PPAR beta/delta transcription activity. Our earlier,work unveiled a cascade of events triggered by PPAR beta/delta that involve the oncogene Src, which promotes ultraviolet-induced skin cancer in mice via enhanced EGFR/Erk1/2 signalling and the expression of epithelial-to-mesenchymal transition (EMT) markers. Interestingly, PPAR beta/delta expression is correlated with the expression of SRC and EMT markers in human skin squamous cell carcinoma. Furthermore, there is a positive interaction between PPAR beta/delta, SRC, and TGF beta 1 at the transcriptional level in various human epithelial cancers. Taken together, these observations suggest the need for evaluating PPAR beta/delta modulators that attenuate or increase its activity, depending on the therapeutic target.

Integrin-Specific Mechanoresponses to Compression and Extension Probed by Cylindrical Flat-Ended AFM Tips in Lung Cells

Cells from lung and other tissues are subjected to forces of opposing directions that are largely transmitted through integrin-mediated adhesions. How cells respond to force bidirectionality remains ill defined. To address this question, we nanofabricated flat-ended cylindrical Atomic Force Microscopy (AFM) tips with ~1 µm2 cross-section area. Tips were uncoated or coated with either integrin-specific (RGD) or non-specific (RGE/BSA) molecules, brought into contact with lung epithelial cells or fibroblasts for 30 s to form focal adhesion precursors, and used to probe cell resistance to deformation in compression and extension. We found that cell resistance to compression was globally higher than to extension regardless of the tip coating. In contrast, both tip-cell adhesion strength and resistance to compression and extension were the highest when probed at integrin-specific adhesions. These integrin-specific mechanoresponses required an intact actin cytoskeleton, and were dependent on tyrosine phosphatases and Ca2+ signaling. Cell asymmetric mechanoresponse to compression and extension remained after 5 minutes of tip-cell adhesion, revealing that asymmetric resistance to force directionality is an intrinsic property of lung cells, as in most soft tissues. Our findings provide new insights on how lung cells probe the mechanochemical properties of the microenvironment, an important process for migration, repair and tissue homeostasis.

Biotechnology applied to Annona species: a review

Annonaceae is an ancient family of plants including approximately 50 genera growing worldwide in a quite restricted area with specific agroclimatic requirements. Only few species of this family has been cultivated and exploited commercially and most of them belonging to the genus Annona such as A. muricata, A. squamosa, the hybrid A. cherimola x A. squamosa and specially Annona cherimola: the cherimoya, commercially cultivated in Spain, Chile, California, Florida, México, Australia, Ecuador, Peru, Brazil, New Zealand and several countries in South and Central America. The cherimoya shows a high degree of heterozygosis, and to obtain homogeneous and productive orchards it is necessary to avoid the propagation by seeds of this species. Additionally, the traditional methods of vegetative propagation were inefficient and inadequate, due to the low morphogenetic potential of this species, and the low rooting rate. The in vitro tissue culture methods of micropropagation can be applied successfully to cherimoya and other Annona sp to overcome these problems. Most of the protocols of micropropagation and regeneration were developed using the cultivar Fino de Jete, which is the major cultivar in Spain. First it is developed the method to micropropagate the juvenile material of cherimoya (ENCINA et al., 1994), and later it was optimized a protocol to micropropagate adult cherimoya genotypes selected by outstanding agronomical traits (PADILLA and ENCINA, 2004) and further it was improved the process through micrografting (PADILLA and ENCINA, 2011).At the present time we are involved in inducing and obtaining new elite genotypes, as part of a breeding program for the cherimoya and other Annonas, using and optimizing different methodologies in vitro: a) Adventitious organogenesis and regeneration from cellular cultures (ENCINA, 2004), b) Ploidy manipulation of the cherimoya, to obtain haploid, tetraploid and triploid plants (seedless), c) Genetic transformation, for the genes introduction to control the postharvest processes and the genes introduction to provide resistance to pathogen and insects and d) Micropropagation and regeneration of other wild Annona or related Annonaceae species such as: Annona senegalensis, A. scleroderma, A. montana, A. reticulata, A. glabra, A. diversifolia and Rollinia sp.

Fibroblast surface-associated FGF-2 promotes contact-dependent colorectal cancer cell migration and invasion through FGFR-SRC signaling and integrin αvβ5-mediated adhesion.

Carcinoma-associated fibroblasts were reported to promote colorectal cancer (CRC) invasion by secreting motility factors and extracellular matrix processing enzymes. Less is known whether fibroblasts may induce CRC cancer cell motility by contact-dependent mechanisms. To address this question we characterized the interaction between fibroblasts and SW620 and HT29 colorectal cancer cells in 2D and 3D co-culture models in vitro. Here we show that fibroblasts induce contact-dependent cancer cell elongation, motility and invasiveness independently of deposited matrix or secreted factors. These effects depend on fibroblast cell surface-associated fibroblast growth factor (FGF) -2. Inhibition of FGF-2 or FGF receptors (FGFRs) signaling abolishes these effects. FGFRs activate SRC in cancer cells and inhibition or silencing of SRC in cancer cells, but not in fibroblasts, prevents fibroblasts-mediated effects. Using an RGD-based integrin antagonist and function-blocking antibodies we demonstrate that cancer cell adhesion to fibroblasts requires integrin αvβ5. Taken together, these results demonstrate that fibroblasts induce cell-contact-dependent colorectal cancer cell migration and invasion under 2D and 3D conditions in vitro through fibroblast cell surface-associated FGF-2, FGF receptor-mediated SRC activation and αvβ5 integrin-dependent cancer cell adhesion to fibroblasts. The FGF-2-FGFRs-SRC-αvβ5 integrin loop might be explored as candidate therapeutic target to block colorectal cancer invasion.

Glutathione deficit impairs myelin maturation: relevance for white matter integrity in schizophrenia patients.

Schizophrenia pathophysiology implies both abnormal redox control and dysconnectivity of the prefrontal cortex, partly related to oligodendrocyte and myelin impairments. As oligodendrocytes are highly vulnerable to altered redox state, we investigated the interplay between glutathione and myelin. In control subjects, multimodal brain imaging revealed a positive association between medial prefrontal glutathione levels and both white matter integrity and resting-state functional connectivity along the cingulum bundle. In early psychosis patients, only white matter integrity was correlated with glutathione levels. On the other side, in the prefrontal cortex of peripubertal mice with genetically impaired glutathione synthesis, mature oligodendrocyte numbers, as well as myelin markers, were decreased. At the molecular levels, under glutathione-deficit conditions induced by short hairpin RNA targeting the key glutathione synthesis enzyme, oligodendrocyte progenitors showed a decreased proliferation mediated by an upregulation of Fyn kinase activity, reversed by either the antioxidant N-acetylcysteine or Fyn kinase inhibitors. In addition, oligodendrocyte maturation was impaired. Interestingly, the regulation of Fyn mRNA and protein expression was also impaired in fibroblasts of patients deficient in glutathione synthesis. Thus, glutathione and redox regulation have a critical role in myelination processes and white matter maturation in the prefrontal cortex of rodent and human, a mechanism potentially disrupted in schizophrenia.

Dysregulated ADAM10-Mediated Processing of APP during a Critical Time Window Leads to Synaptic Deficits in Fragile X Syndrome.

The Fragile X mental retardation protein (FMRP) regulates neuronal RNA metabolism, and its absence or mutations leads to the Fragile X syndrome (FXS). The β-amyloid precursor protein (APP) is involved in Alzheimer's disease, plays a role in synapse formation, and is upregulated in intellectual disabilities. Here, we show that during mouse synaptogenesis and in human FXS fibroblasts, a dual dysregulation of APP and the α-secretase ADAM10 leads to the production of an excess of soluble APPα (sAPPα). In FXS, sAPPα signals through the metabotropic receptor that, activating the MAP kinase pathway, leads to synaptic and behavioral deficits. Modulation of ADAM10 activity in FXS reduces sAPPα levels, restoring translational control, synaptic morphology, and behavioral plasticity. Thus, proper control of ADAM10-mediated APP processing during a specific developmental postnatal stage is crucial for healthy spine formation and function(s). Downregulation of ADAM10 activity at synapses may be an effective strategy for ameliorating FXS phenotypes.

Reticular dysgenesis-associated AK2 protects hematopoietic stem and progenitor cell development from oxidative stress.

Adenylate kinases (AKs) are phosphotransferases that regulate the cellular adenine nucleotide composition and play a critical role in the energy homeostasis of all tissues. The AK2 isoenzyme is expressed in the mitochondrial intermembrane space and is mutated in reticular dysgenesis (RD), a rare form of severe combined immunodeficiency (SCID) in humans. RD is characterized by a maturation arrest in the myeloid and lymphoid lineages, leading to early onset, recurrent, and overwhelming infections. To gain insight into the pathophysiology of RD, we studied the effects of AK2 deficiency using the zebrafish model and induced pluripotent stem cells (iPSCs) derived from fibroblasts of an RD patient. In zebrafish, Ak2 deficiency affected hematopoietic stem and progenitor cell (HSPC) development with increased oxidative stress and apoptosis. AK2-deficient iPSCs recapitulated the characteristic myeloid maturation arrest at the promyelocyte stage and demonstrated an increased AMP/ADP ratio, indicative of an energy-depleted adenine nucleotide profile. Antioxidant treatment rescued the hematopoietic phenotypes in vivo in ak2 mutant zebrafish and restored differentiation of AK2-deficient iPSCs into mature granulocytes. Our results link hematopoietic cell fate in AK2 deficiency to cellular energy depletion and increased oxidative stress. This points to the potential use of antioxidants as a supportive therapeutic modality for patients with RD.

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.

Twist1 Is a TNF-Inducible Inhibitor of Clock Mediated Activation of Period Genes.

BACKGROUND: Activation of the immune system affects the circadian clock. Tumor necrosis factor (TNF) and Interleukin (IL)-1β inhibit the expression of clock genes including Period (Per) genes and the PAR-bZip clock-controlled gene D-site albumin promoter-binding protein (Dbp). These effects are due to cytokine-induced interference of E-box mediated transcription of clock genes. In the present study we have assessed the two E-box binding transcriptional regulators Twist1 and Twist2 for their role in cytokine induced inhibition of clock genes. METHODS: The expression of the clock genes Per1, Per2, Per3 and of Dbp was assessed in NIH-3T3 mouse fibroblasts and the mouse hippocampal neuronal cell line HT22. Cells were treated for 4h with TNF and IL-1β. The functional role of Twist1 and Twist2 was assessed by siRNAs against the Twist genes and by overexpression of TWIST proteins. In luciferase (luc) assays NIH-3T3 cells were transfected with reporter gene constructs, which contain a 3xPer1 E-box or a Dbp E-box. Quantitative chromatin immunoprecipitation (ChIP) was performed using antibodies to TWIST1 and CLOCK, and the E-box consensus sequences of Dbp (CATGTG) and Per1 E-box (CACGTG). RESULTS: We report here that siRNA against Twist1 protects NIH-3T3 cells and HT22 cells from down-regulation of Period and Dbp by TNF and IL-1β. Overexpression of Twist1, but not of Twist2, mimics the effect of the cytokines. TNF down-regulates the activation of Per1-3xE-box-luc, the effect being prevented by siRNA against Twist1. Overexpression of Twist1, but not of Twist2, inhibits Per1-3xE-box-luc or Dbp-E-Box-luc activity. ChIP experiments show TWIST1 induction by TNF to compete with CLOCK binding to the E-box of Period genes and Dbp. CONCLUSION: Twist1 plays a pivotal role in the TNF mediated suppression of E-box dependent transactivation of Period genes and Dbp. Thereby Twist1 may provide a link between the immune system and the circadian timing system.

Short stature and primary ovarian insufficiency possibly due to chromosomal position effect in a balanced X;1 translocation.

BACKGROUND: Primary ovarian insufficiency (POI) is defined as a primary ovarian defect characterized by absent menarche (primary amenorrhea), a decrease in the initial primordial follicle number, high follicle-stimulating hormone (FSH) levels and hypoestrogenism. Although the etiology of a majority of POI cases is not yet identified, several data suggest that POI has a strong genetic component. Conventional cytogenetic and molecular analyses have identified regions of the X chromosome that are associated with ovarian function, as well as POI candidate genes, such as FMR1 and DIAPH2. Here we describe a 10.5-year-old girl presenting with high FSH and luteinizing hormone (LH) levels, pathologic GH stimulation arginine and clonidine tests, short stature, pterygium, ovarian dysgenesis, hirsutism and POI. RESULTS: Cytogenetic analysis demonstrated a balanced reciprocal translocation between the q arms of chromosomes X and 1, with breakpoints falling in Xq21 and 1q41 bands. Molecular studies did not unravel any chromosome microdeletion/microduplication, and no XIST-mediated inactivation was found on the derivative chromosome 1. Interestingly, through immunofluorescence assays, we found that part of the Xq21q22 trait, translocated to chromosome 1q41, was late replicating and therefore possibly inactivated in 30 % metaphases both in lymphocytes and skin fibroblasts, in addition to a skewed 100 % inactivation of the normal X chromosome. These findings suggest that a dysregulation of gene expression might occur in this region. Two genes mapping to the Xq translocated region, namely DIAPH2 and FMR1, were found overexpressed if compared with controls. CONCLUSIONS: We report a case in which gonadal dysgenesis and POI are associated with over-expression of DIAPH2 gene and of FMR1 gene in wild type form. We hypothesize that this over-expression is possibly due to a phenomenon known as "chromosomal position effect", which accounts for gene expression variations depending on their localization within the nucleus. For the same effect a double mosaic inactivation of genes mapping to the Xq21-q22 region, demonstrated by immunofluorescence assays, may be the cause of a functional Xq partial monosomy leading to most Turner traits of the proband's phenotype.

Activity of daptomycin- and vancomycin-loaded poly-epsilon-caprolactone microparticles against mature staphylococcal biofilms.

The aim of the present study was to develop novel daptomycin-loaded poly-epsilon-caprolactone (PCL) microparticles with enhanced antibiofilm activity against mature biofilms of clinically relevant bacteria, methicillin-resistant Staphylococcus aureus (MRSA) and polysaccharide intercellular adhesin-positive Staphylococcus epidermidis. Daptomycin was encapsulated into PCL microparticles by a double emulsion-solvent evaporation method. For comparison purposes, formulations containing vancomycin were also prepared. Particle morphology, size distribution, encapsulation efficiency, surface charge, thermal behavior, and in vitro release were assessed. All formulations exhibited a spherical morphology, micrometer size, and negative surface charge. From a very early time stage, the released concentrations of daptomycin and vancomycin were higher than the minimal inhibitory concentration and continued so up to 72 hours. Daptomycin presented a sustained release profile with increasing concentrations of the drug being released up to 72 hours, whereas the release of vancomycin stabilized at 24 hours. The antibacterial activity of the microparticles was assessed by isothermal microcalorimetry against planktonic and sessile MRSA and S. epidermidis. Regarding planktonic bacteria, daptomycin-loaded PCL microparticles presented the highest antibacterial activity against both strains. Isothermal microcalorimetry also revealed that lower concentrations of daptomycin-loaded microparticles were required to completely inhibit the recovery of mature MRSA and S. epidermidis biofilms. Further characterization of the effect of daptomycin-loaded PCL microparticles on mature biofilms was performed by fluorescence in situ hybridization. Fluorescence in situ hybridization showed an important reduction in MRSA biofilm, whereas S. epidermidis biofilms, although inhibited, were not eradicated. In addition, an important attachment of the microparticles to MRSA and S. epidermidis biofilms was observed. Finally, all formulations proved to be biocompatible with both ISO compliant L929 fibroblasts and human MG63 osteoblast-like cells.

Nitric oxide-induced p53 accumulation and regulation of inducible nitric oxide synthase expression by wild-type p53.

The tumor suppressor gene product p53 plays an important role in the cellular response to DNA damage from exogenous chemical and physical mutagens. Therefore, we hypothesized that p53 performs a similar role in response to putative endogenous mutagens, such as nitric oxide (NO). We report here that exposure of human cells to NO generated from an NO donor or from overexpression of inducible nitric oxide synthase (NOS2) results in p53 protein accumulation. In addition, expression of wild-type (WT) p53 in a variety of human tumor cell lines, as well as murine fibroblasts, results in down-regulation of NOS2 expression through inhibition of the NOS2 promoter. These data are consistent with the hypothesis of a negative feedback loop in which endogenous NO-induced DNA damage results in WT p53 accumulation and provides a novel mechanism by which p53 safeguards against DNA damage through p53-mediated transrepression of NOS2 gene expression, thus reducing the potential for NO-induced DNA damage.

Growth factor- and cytokine-driven pathways governing liver stemness and differentiation.

Liver is unique in its capacity to regenerate in response to injury or tissue loss. Hepatocytes and other liver cells are able to proliferate and repopulate the liver. However, when this response is impaired, the contribution of hepatic progenitors becomes very relevant. Here, we present an update of recent studies on growth factors and cytokine-driven intracellular pathways that govern liver stem/progenitor cell expansion and differentiation, and the relevance of these signals in liver development, regeneration and carcinogenesis. Tyrosine kinase receptor signaling, in particular, c-Met, epidermal growth factor receptors or fibroblast growth factor receptors, contribute to proliferation, survival and differentiation of liver stem/progenitor cells. Different evidence suggests a dual role for the transforming growth factor (TGF)-β signaling pathway in liver stemness and differentiation. On the one hand, TGF-β mediates progression of differentiation from a progenitor stage, but on the other hand, it contributes to the expansion of liver stem cells. Hedgehog family ligands are necessary to promote hepatoblast proliferation but need to be shut off to permit subsequent hepatoblast differentiation. In the same line, the Wnt family and β-catenin/T-cell factor pathway is clearly involved in the maintenance of liver stemness phenotype, and its repression is necessary for liver differentiation during development. Collectively, data indicate that liver stem/progenitor cells follow their own rules and regulations. The same signals that are essential for their activation, expansion and differentiation are good candidates to contribute, under adequate conditions, to the paradigm of transformation from a pro-regenerative to a pro-tumorigenic role. From a clinical perspective, this is a fundamental issue for liver stem/progenitor cell-based therapies.

Individual differences in gene expression : insights from transcriptional control of the CSL gene and from human population studies

CSL is a key transcription factor, mostly acting as a repressor, which has been shown to have a highly context-dependent function. While known as the main effector of Notch signaling, it can also exert Notch-independent functions. The downstream effects of the Notch/CSL signaling pathway and its involvement in several biological processes have been intensively studied. We recently showed that CSL is important to maintain skin homeostasis, as its specific deletion in mouse dermal fibroblasts -or downmodulation in human stromal fibroblasts- creates an inducing environment for squamous cell carcinoma (SCC) development, possibly due to the conversion of stromal fibroblasts into cancer associated fibroblasts (CAFs). Despite the wide interest in CSL as a transcriptional regulator, the mechanism of its own regulation has so far been neglected. We show here that CSL expression levels differ between individuals, and correlate among others with genes involved in DNA damage response. Starting from this finding we show that in dermal fibroblasts CSL is under transcriptional control of stress inducers such as UVA irradiation and Reactive Oxygen Species (ROS) induction, and that a main player in CSL transcriptional regulation is the transcription factor p53. In a separate line of work, we focused on individual variability, studying the differences in gene expression between human populations in various cancer types, particularly focusing on the Caucasian and African populations. It is indeed widely known that these populations have different incidences and mortalities for various cancers, and response to cancer treatment may also vary between them. We show here several genes that are differentially expressed and could be of interest in the study of population differences in cancer. -- CSL est un facteur de transcription agissant essentiellement comme répresseur, et qui a une fonction hautement dépendant du contexte. C'est l'effecteur principal de la voie de signalisation de Notch, mais il peut également exercer ses fonctions dans une façon Notch- indépendante. Nous avons récemment montré que CSL est important pour maintenir l'homéostasie de la peau. Sa suppression spécifique dans les fibroblastes dermiques de la souris ou dans les fibroblastes stromales humaines crée un environnement favorable pour le développement du carcinome épidermoïde (SCC), probablement en raison de la conversion des fibroblastes en fibroblastes associé au cancer (CAF). Malgré le grand intérêt de CSL comme régulateur transcriptionnel, le mécanisme de sa propre régulation a été jusqu'ici négligée. Nous montrons ici que dans les fibroblastes dermiques CSL est sous le contrôle transcriptionnel de facteurs de stress tels que l'irradiation UVA et l'induction des ROS dont p53 est l'acteur principal de cette régulation. Nous montrons aussi que les niveaux d'expression de CSL varient selon les individus, en corrélation avec d'autres gènes impliqués dans la réponse aux dommages de l'ADN. Dans une autre axe de recherche, concernant la variabilité individuelle, nous avons étudié les différences dans l'expression des gènes dans différents types de cancer entre les populations humaines, en se concentrant particulièrement sur les populations africaines et caucasiennes. Il est en effet bien connu que ces populations montrent des variations dans l'incidence des cancers, la mortalité, ainsi que pour les réponses au traitement. Nous montrons ici plusieurs gènes qui sont exprimés différemment et pourraient être digne d'intérêt dans l'étude du cancer au sein de différentes populations.