Neural stem cells adopt tumorigenic properties by constitutively activated NF-kappaB and subsequent VEGF up-regulation

One of the challenges in stem cell research is to avoid transformation during cultivation. We studied high passage subventricular zone derived neural stem cells (NSCs) cultures of adult rats in the absence of growth factors epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). We termed this culture exogenous growth factor independent neural stem cells (GiNSCs). GiNSCs expressed stemness markers, displayed a high constitutive NF-kappaB activity and an increased, aberrant, polyploid DNA content. GiNSCs showed a tumorigenic phenotype and formed colonies in a soft agar assay. Microarray analysis showed the up-regulation of the NF-kappaB target gene vascular endothelial growth factor (VEGF). In contrast, proneuronal genes were down-regulated. Under neuronal differentiation conditions GiNSCs adopted a glioma-like phenotype, with nuclear p53, preserving high amounts of Nestin positive cells and prolonged proliferation. Neutralization of VEGF strongly inhibited proliferation and induced differentiation. In a gain of function approach, the transfection of NSCs with constitutively active upstream kinase IKK-2 led to constitutively activated NF-kappaB, proliferation in absence of growth factors and augmented VEGF secretion. In a rescue experiment a reduction of NF-kappaB activity by overexpression of IkappaB-AA1 was able to shift the morphology toward an elongated cell form, increased cell death, and decreased proliferation. Thus GiNSCs may provide a potent tool in cancer research, as their exogenous cytokine independent proliferation and their constitutively high NF-kappaB expression presumes cancerous properties observed in gliomas. In addition, this study might add a novel mechanism for detecting oncogenic transformation in therapeutic stem cell cultures.

Early weaning accelerates the differentiation of mucous neck cells in rat gastric mucosa: Possible role of TGF alpha/EGFR

The development of the gastric mucosa is controlled by hormones, growth factors and feeding behavior. Early weaning (EW), which means the abrupt interruption of suckling, increases proliferation and differentiation in the rat gastric epithelium. Transforming growth factor alpha(TGF alpha) is secreted in the stomach, binds to the epidermal growth factor receptor( EGFR) and may control cell proliferation, differentiation and migration. Here, we investigated the influence of suckling-weaning transition on the differentiation of mucous neck cells in the stomach and its association to the expression of TGF alpha and EGFR. Fifteen-day-old Wistar rats were divided into two groups: suckling( control), in which pups were kept with the dam, and early weaning( EW), in which rats were separated from their mother and fed with hydrated powdered chow. TGF alpha and EGFR levels were increased at 18 days in EW animals compared to control ones (p<0.05). Histochemical reactions with Periodic Acid-Schiff reagent+Alcian Blue or Bandeiraea simplicifolia II lectin were used to stain the mucous neck cells and showed an increase in this cell population throughout EW, which was more pronounced at 17 days when compared to suckling pups (p<0.05). These morphological results were confirmed by RT-PCR for mucin 6. The levels of mucin 6 mRNA were higher in EW animals from the 16th to the 18th day(1-3 days post-weaning) when compared to the respective control group. Inhibition of EGFR through AG1478 administration to EW animals prevented the expansion of mucous neck cell population induced by EW (p<0.05). Therefore, early weaning up regulated TGF alpha/EGFR expression and induced differentiation of mucous neck cells. Moreover, we showed that EGFR takes part in the maturation of this cell population. We conclude that regular suckling-weaning transition is crucial to guarantee the development of the gastric mucosa. (C) 2009 International Society of Differentiation. Published by Elsevier Ltd. All rights reserved.

The Autoimmune Regulator (AIRE), Which Is Defective in Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy Patients, Is Expressed in Human Epidermal and Follicular Keratinocytes and Associates With the Intermediate Filament Protein Cytokeratin 17

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome, which is caused by mutation of the autoimmune regulator (AIRE) gene, is a highly variable disease characterized by multiple endocrine failure, chronic mucocutaneous candidiasis, and various ectodermal defects. AIRE is a transcriptional regulator classically expressed in medullary thymic epithelial cells, monocytes, macrophages, and dendritic cells. Previous studies have suggested that AIRE can shuttle between the nucleus and cytoplasm of cells, although its cytoplasmic functions are poorly characterized. Through mass spectrometry analysis of proteins co-immunoprecipitating with cytoplasmic AIRE, we identified a novel association of AIRE with the intermediate filament protein cytokeratin 17 (K17) in the THP-1 monocyte cell line. We confirmed AIRE expression in HaCaT epidermal keratinocytes, as well as its interaction with K17. Confocal microscopy of human fetal and adult scalp hair follicles demonstrated a cytoplasmic pattern of AIRE staining that moderately colocalized with K17. The cytoplasmic association of AIRE with the intermediate filament network in human epidermal and follicular keratinocytes may provide a new path to understanding the ectodermal abnormalities associated with the APECED syndrome. (Am J Pathol 2011, 178:983-988; DOI: 10.1016/j.ajpath.2010.12.007)

Evaluation of the Integrity of Horse Hoof Dermal and Epidermal Tissues Collected by Dorsal Transmural Access

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Bulliform cells in Loudetiopsis chrysothrix (Nees) Conert and Tristachya leiostachya Nees (Poaceae): structure in relation to function

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

EPIDERMAL GROWTH-FACTOR AND TRANSFORMING GROWTH-FACTOR-ALPHA CHARACTERISTICS OF HUMAN ORAL-CARCINOMA CELL-LINES

This study examined the expression of epidermal growth factor (EGF) cell-surface receptors, the response to exogenous ligand and the autocrine production of transforming growth factor a (TGF-a) in normal and carcinoma-derived human oral keratinocytes. One of eight malignant cell lines overexpressed EGF receptors, while the remainder expressed receptor numbers similar to normal cells. Exogenous EGF stimulated incorporation of tritiated thymidine in a dose-dependent manner. In keratinocytes expressing normal numbers of EGF receptors, the cellular response to exogenous EGF correlated positively with total EGF receptor number. SCC-derived keratinocytes produced more TGF-a than normal cells. There was no statistical correlation between the autocrine production of TGF-a, EGF cell-surface receptor expression and cellular response to exogenous EGF. While the growth-stimulatory effects of exogenous TGF-cl were inhibited by the addition of a neutralising antibody, the presence of this antibody in conditioned medium failed to produce a similar decrease in growth. The results indicate that overexpression of EGF receptors is not an invariable characteristic of human oral squamous carcinoma-derived cell lines. Further, the contribution of TGF-a to the growth of normal and carcinoma-derived human oral keratinocytes in vitro may be less significant than previously documented.

Salivary epithelial cells as model to study immune response against cutaneous pathogens

The human skin not only provides passive protection as a physical barrier against external injury, but also mediates active surveillance via epidermal cell surface receptors that recognize and respond to potential invaders. Primary keratinocytes and immortalized cell lines, the commonly used sources to investigate immune responses of cutaneous epithelium are often difficult to obtain and/or potentially exhibit changes in cellular genetic make-up. Here we investigated the possibility of using salivary epithelial cells (SEC) to evaluate the host response to cutaneous microbes. Elevated secretion of IFN-γ and IL-12 was observed in the SEC stimulated with Staphylococcus aureus, a transient pathogen of the skin, as mono species biofilm as compared to SEC stimulated with a commensal microbe, the Staphylococcus epidermidis. Co-culture of the SEC with both microbes as dual species biofilm elicited maximum cytokine response. Stimulation with S. aureus alone but not with S. epidermidis alone induced maximum toll-like receptor-2 (TLR-2) expression in the SEC. Exposure to dual species biofilm induced a sustained upregulation of TLR-2 in the SEC for up to an hour. The data support novel application of the SEC as efficient biospecimen that may be used to investigate personalized response to cutaneous microflora. © 2013 Wiley Periodicals, Inc.

Imunolocalização do fator de crescimento semelhante à insulina (IGF-I) e do fator de crescimento epidermal (EGF) em ovários caninos e seus efeitos sobre a maturação in vitro de oócitos: avaliação nuclear

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Pyrostegia venusta heptane extract containing saturated aliphatic hydrocarbons induces apoptosis on B16F10-Nex2 melanoma cells and displays antitumor activity in vivo

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Stem cells in embryonic skin development

The skin is a complex stratified organ which acts not only as a permeability barrier and defense against external agents, but also has essential thermoregulatory, sensory and metabolic functions. Due to its high versatility and activity, the skin undergoes continuous self-renewal to repair damaged tissue and replace old cells. Consequently, the skin is a reservoir for adult stem cells of different embryonic origins. Skin stem cell populations reside in the adult hair follicle, sebaceous gland, dermis and epidermis. However, the origin of most of the stem cell populations found in the adult epidermis is still unknown. Far more unknown is the embryonic origin of other stem cells that populate the other layers of this tissue. In this review we attempt to clarify the emergence, structure, markers and embryonic development of diverse populations of stem cells from the epidermis, dermis and related appendages such as the sebaceous gland and hair follicle.

NKp46+ cells express granulysin in multiple cutaneous adverse drug reactions

The spectrum of cutaneous adverse drug reactions (cADRs) ranges from benign presentations to severe life-threatening forms such as toxic epidermal necrolysis (TEN). In TEN, granulysin has been shown to be the key cytotoxic molecule. Still, little is known about the expression of granulysin in other cADRs. As an important source of granulysin, natural killer (NK) cells are of major interest in cADRs. Recently, NKp46 has been identified as the most selective NK-cell marker. However, the role of NKp46(+) cells in cADRs and their contribution to granulysin expression remain to be elucidated.

Meprinα transactivates the epidermal growth factor receptor (EGFR) via ligand shedding, thereby enhancing colorectal cancer cell proliferation and migration

Meprinα, an astacin-type metalloprotease is overexpressed in colorectal cancer cells and is secreted in a non-polarized fashion, leading to the accumulation of meprinα in the tumor stroma. The transition from normal colonocytes to colorectal cancer correlates with increased meprinα activity at primary tumor sites. A role for meprinα in invasion and metastatic dissemination is supported by its pro-angiogenic and pro-migratory activity. In the present study, we provide evidence for a meprinα-mediated transactivation of the EGFR signaling pathway and suggest that this mechanism is involved in colorectal cancer progression. Using alkaline phosphatase-tagged EGFR ligands and an ELISA assay, we demonstrate that meprinα is capable of shedding epidermal growth factor (EGF) and transforming growth factor-α (TGFα) from the plasma membrane. Shedding was abrogated using actinonin, an inhibitor for meprinα. The physiological effects of meprinα-mediated shedding of EGF and TGFα were investigated with human colorectal adenocarcinoma cells (Caco-2). Proteolytically active meprinα leads to an increase in EGFR and ERK1/2 phosphorylation and subsequently enhances cell proliferation and migration. In conclusion, the implication of meprinα in the EGFR/MAPK signaling pathway indicates a role of meprinα in colorectal cancer progression.

Epidermal caspase-3 cleavage associated with interferon-gamma-expressing lymphocytes in acute atopic dermatitis lesions

Keratinocyte apoptosis mediated by Fas/Fas ligand molecular interactions and subsequent caspase activation is believed to play an important role in the pathogenesis of atopic dermatitis (AD), in particular for the formation of spongiosis. To estimate epidermal caspase activation in normal and AD skin under in vivo conditions, we analysed caspase-3 cleavage by immunohistology. In normal skin as well as non-lesional AD skin, we detected caspase-3 cleavage in single cells of the basal layer. In contrast, in acute lesional AD skin, we not only obtained evidence for increased expression of cleaved caspase-3 in keratinocytes of the basal layer but also observed caspase-3 cleavage in one or more layers of the spinous cell layer, in particular in spongiotic areas. Short-term topical treatment of the skin lesions with tacrolimus or pimecrolimus abolished the expression of cleaved caspase-3 in the spinous layer. Moreover, epidermal caspase-3 cleavage correlated with the numbers of dermal interferon-gamma (IFN-gamma)-expressing CD4+ and CD8+ lymphocytes in skin lesions of AD patients, supporting the view that IFN-gamma is important for the activation of proapoptotic pathways in keratinocytes. This is also confirmed by the observation of increased Fas expression on keratinocytes in acute AD lesions that was markedly reduced following topical calcineurin inhibitor treatment. These data suggest that caspase-3 cleavage in the spinous layer of the epidermis is a pathologic event contributing to spongiosis formation in AD, whereas cleavage of caspase-3 in basal cells might represent a physiologic mechanism within the process of epidermal renewal.

Expression of HB-EGF by retinal pigment epithelial cells in vitreoretinal proliferative disease

The heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been implicated in wound-healing processes of various tissues. However, it is not known whether HB-EGF may represent a factor implicated in overstimulated wound-healing processes of the retina during proliferative retinopathies. Therefore, we investigated whether human retinal pigment epithelial (RPE) cells, which are crucially involved in proliferative retinopathies, express and respond to HB-EGF. RPE cells express mRNAs for various members of the EGF-related growth factor family, among them for HB-EGF, as well as for the EGF receptors ErbB1, -2, -3, and -4. The gene expression of HB-EGF is stimulated in the presence of transforming and basic fibroblast growth factors and by oxidative stress and is suppressed during chemical hypoxia. Exogenous HB-EGF stimulates proliferation and migration of RPE cells and the gene and protein expression of the vascular endothelial growth factor (VEGF). HB-EGF activates at least three signal transduction pathways in RPE cells including the extracellular signal-regulated kinases (involved in the proliferation-stimulating action of HB-EGF), p38 (mediates the effects on chemotaxis and secretion of VEGF), and the phosphatidylinositol-3 kinase (necessary for the stimulation of chemotaxis). In epiretinal membranes of patients with proliferative retinopathies, HB-EGF immunoreactivity was partially colocalized with the RPE cell marker, cytokeratins; this observation suggests that RPE cell-derived HB-EGF may represent one factor that drives the uncontrolled wound-healing process of the retina. The stimulating effect on the secretion of VEGF may suggest that HB-EGF is also implicated in the pathological angiogenesis of the retina.

Tie2 receptor expression and phosphorylation in cultured cells and mouse tissues

Accumulating experimental evidence indicates that endothelial cell growth and blood vessel morphogenesis are processes that are governed by the activity of specifically expressed receptor tyrosine kinases (RTKs). We have used two new rat monoclonal antibodies (mAbs) to study the expression and phosphorylation of one such receptor, mouse Tie2 (tyrosine kinase that contains immunoglobulin-like loops and epidermal-growth-factor-similar domains 2]), in transfected cells, endothelioma cell lines and mouse tissues. The Tie2 receptor was found to be constitutively autophosphorylated when over-expressed in COS7 cells. In contrast, the endogenous Tie2 protein was not phosphorylated in endothelioma cell lines. However, in these cell lines, Tie2 could be induced to become tyrosine phosphorylated, and this activation was found to be independent of Tie1. Studying Tie2 receptor activity during angiogenesis in mouse development, the receptor was only weakly phosphorylated in the early postnatal mouse brain whereas a stronger activation could be detected in mouse embryos at day 10.5 post coitum.