MFG-E8/lactadherin regulates cyclins D1/D3 expression and enhances the tumorigenic potential of mammary epithelial cells.

Milk fat globule-EGF factor 8 (MFG-E8) is a glycoprotein highly expressed in breast cancer that contributes to tumor progression through largely undefined mechanisms. By analyzing publicly available gene expression profiles of breast carcinomas, we found that MFG-E8 is highly expressed in primary and metastatic breast carcinomas, associated with absent estrogen receptor expression. Immunohistochemistry analysis of breast cancer biopsies revealed that MFG-E8 is expressed on the cell membrane as well as in the cytoplasm and nucleus. We also show that increased expression of MFG-E8 in mammary carcinoma cells increases their tumorigenicity in immunodeficient mice, and conversely, its downregulation reduces their in vivo growth. Moreover, expression of MFG-E8 in immortalized mammary epithelial cells promotes their growth and branching in three-dimensional collagen matrices and induces the expression of cyclins D1/D3 and N-cadherin. A mutant protein unable to bind integrins can in part exert these effects, indicating that MFG-E8 function is only partially dependent on integrin activation. We conclude that MFG-E8-dependent signaling stimulates cell proliferation and the acquisition of mesenchymal properties and contributes to mammary carcinoma development.

Ataxia telangiectasia mutated (ATM) inhibition transforms human mammary gland epithelial cells.

Carriers of mutations in the cell cycle checkpoint protein kinase ataxia telangiectasia mutated (ATM), which represent 1-2% of the general population, have an increased risk of breast cancer. However, experimental evidence that ATM deficiency contributes to human breast carcinogenesis is lacking. We report here that in MCF-10A and MCF-12A cells, which are well established normal human mammary gland epithelial cell models, partial or almost complete stable ATM silencing or pharmacological inhibition resulted in cellular transformation, genomic instability, and formation of dysplastic lesions in NOD/SCID mice. These effects did not require the activity of exogenous DNA-damaging agents and were preceded by an unsuspected and striking increase in cell proliferation also observed in primary human mammary gland epithelial cells. Increased proliferation correlated with a dramatic, transient, and proteasome-dependent reduction of p21(WAF1/CIP1) and p27(KIP1) protein levels, whereas little or no effect was observed on p21(WAF1/CIP1) or p27(KIP1) mRNAs. p21(WAF1/CIP1) silencing also increased MCF-10A cell proliferation, thus identifying p21(WAF1/CIP1) down-regulation as a mediator of the proliferative effect of ATM inhibition. Our findings provide the first experimental evidence that ATM is a human breast tumor suppressor. In addition, they mirror the sensitivity of ATM tumor suppressor function and unveil a new mechanism by which ATM might prevent human breast tumorigenesis, namely a direct inhibitory effect on the basal proliferation of normal mammary epithelial cells.

Increased Wnt signaling triggers oncogenic conversion of human breast epithelial cells by a Notch-dependent mechanism.

Wnt and Notch signaling have long been established as strongly oncogenic in the mouse mammary gland. Aberrant expression of several Wnts and other components of this pathway in human breast carcinomas has been reported, but evidence for a causative role in the human disease has been missing. Here we report that increased Wnt signaling, as achieved by ectopic expression of Wnt-1, triggers the DNA damage response (DDR) and an ensuing cascade of events resulting in tumorigenic conversion of primary human mammary epithelial cells. Wnt-1-transformed cells have high telomerase activity and compromised p53 and Rb function, grow as spheres in suspension, and in mice form tumors that closely resemble medullary carcinomas of the breast. Notch signaling is up-regulated through a mechanism involving increased expression of the Notch ligands Dll1, Dll3, and Dll4 and is required for expression of the tumorigenic phenotype. Increased Notch signaling in primary human mammary epithelial cells is sufficient to reproduce some aspects of Wnt-induced transformation. The relevance of these findings for human breast cancer is supported by the fact that expression of Wnt-1 and Wnt-4 and of established Wnt target genes, such as Axin-2 and Lef-1, as well as the Notch ligands, such as Dll3 and Dll4, is up-regulated in human breast carcinomas.

Highly inducible synthesis of heterologous proteins in epithelial cells carrying a glucocorticoid-responsive vector.

A glucocorticoid-responsive vector is described which allows for the highly inducible expression of complementary DNAs (cDNAs) in stably transfected mammalian cell lines. This vector, pLK-neo, composed of a variant mouse mammary tumor virus long terminal repeat promoter, containing a hormone regulatory element, a Geneticin resistance-encoding gene in a simian virus 40 transcription unit, and a polylinker insertion site for heterologous cDNAs, was used to express the polymeric immunoglobulin (poly-Ig) receptor and the thymocyte marker, Thy-1, in Madin-Darby canine kidney (MDCK) cells and in murine fibroblast L cells. A high level of poly-Ig receptor or Thy-1 mRNA accumulation was observed in MDCK cells in response to dexamethasone with a parallel ten- to 200-fold increase in protein synthesis depending on the recombinant protein and the transfected cell clone.

Antagonistic roles of Notch and p63 in controlling mammary epithelial cell fates.

The breast epithelium has two major compartments, luminal and basal cells, that are established and maintained by poorly understood mechanisms. The p53 homolog, p63, is required for the formation of mammary buds, but its function in the breast after birth is unknown. We show that in primary human breast epithelial cells, maintenance of basal cell characteristics depends on continued expression of the p63 isoform, DeltaNp63, which is expressed in the basal compartment. Forced expression of DeltaNp63 in purified luminal cells confers a basal phenotype. Notch signaling downmodulates DeltaNp63 expression and mimics DeltaNp63 depletion, whereas forced expression of DeltaNp63 partially counteracts the effects of Notch. Consistent with Notch activation specifying luminal cell fate in the mammary gland, Notch signaling activity is specifically detected in mice at sites of pubertal ductal morphogenesis where luminal cell fate is determined. Basal cells in which Notch signaling is active show decreased p63 expression. Both constitutive expression of DeltaNp63 and ablation of Notch signaling are incompatible with luminal cell fate. Thus, the balance between basal and luminal cell compartments of the breast is regulated by antagonistic functions of DeltaNp63 and Notch.Cell Death and Differentiation advance online publication, 9 April 2010; doi:10.1038/cdd.2010.37.

Proteomic analysis of cytokine induced proteins in human intestinal epithelial cells: implications for inflammatory bowel diseases.

A role for cytokine regulated proteins in epithelial cells has been suggested in the pathogenesis of inflammatory bowel diseases (IBD). The aim of this study was to identify such cytokine regulated targets using a proteomic functional approach. Protein patterns from (35)S-radiolabeled homogenates of cultured colon epithelial cells were compared before and after exposure to interferon-gamma, interleukin-1beta and interleukin-6. Proteins were separated by two-dimensional polyacrylamide gel electrophoresis. Both autoradiographies and silver stained gels were analyzed. Proteins showing differential expression were identified by tryptic in-gel digestion and mass spectrometry. Metabolism related proteins were also investigated by Western blot analysis. Tryptophanyl-tRNA synthetase, indoleamine-2,3-dioxygenase, heterogeneous nuclear ribonucleoprotein JKTBP, interferon-induced 35kDa protein, proteasome subunit LMP2 and arginosuccinate synthetase were identified as cytokine modulated proteins in vitro. Using purified epithelial cells from patients, overexpression of indoleamine-2,3-dioxygenase, an enzyme involved in tryptophan metabolism, was confirmed in Crohn's disease as well as in ulcerative colitis, as compared to normal mucosa. No such difference was found in diverticulitis. Potentially, this observation opens new avenues in the treatment of IBD.

Facilitated glucose transporters in epithelial cells.

The molecular cloning of facilitated sugar transporters has led to the identification of a family of transport molecules having similar functions, but possessing specific kinetic and regulatory properties. These transporter isoforms are characterized by different primary structures, specific tissue localization, and polarized expression within the same epithelial cells. The use of Xenopus oocytes for the functional expression of different members of this transporter family has been of considerable value in defining the kinetic properties and sugar specificities of the different isoforms. The expression of chimeric or variously mutated transporters should, in the near future, permit the determination of the structural basis for their kinetic properties and sugar specificities. cDNA probes and antipeptide antibodies specific for each isoform are now being used to determine their specific regulation during development and in different states of altered glucose homeostasis. The variety of molecular forms implicated in the apparently simple task of sugar uptake or transepithelial transport has been surprising. With the available molecular tools now in hand, it will be possible to study these mechanisms in much greater detail.

Breast stem cells and hormonal mechanisms in carcinogenesis

A woman's risk of breast cancer is strongly affected by her reproductive history. The hormonal milieu is also a key determinant of the course of the disease. Combining mouse genetics with tissue recombination techniques, we have established that the female reproductive hormones, estrogens, progesterone, and prolactin, act sequentially on the mammary epithelium to trigger distinct developmental steps. The hormones impinge directly on a subset of luminal mammary epithelial cells that express the respective hormone receptors and act as sensor cells translating and amplifying systemic signals into local stimuli. Local signaling is stage and age specific. During puberty, estrogens promote proliferation using the EGF family member, amphiregulin, as essential paracrine mediator. In adulthood, progesterone, rather than estrogen, is the major inducer of stem cell activation and cell proliferation of the mammary epithelium. Hormonal signaling modulates crucial developmental pathways that impinge on mammary stem cell populations, while Notch signaling, by inhibiting p63, is central to mammary cell fate determination. Cell proliferation occurs in two waves. The first results from direct stimulation of the small fraction of hormone receptor positive cells. It is followed by a second wave of progesterone-induced proliferation involving mostly hormone receptor negative cells, in which RANKL is a key mediator. A model in which repeated activation of paracrine signaling by progesterone with resulting stem cell activation promotes breast carcinogenesis is proposed.

Liposome-mediated transfection of fetal lung epithelial cells: DNA degradation and enhanced superoxide toxicity.

Cationic liposomes, 1:1 (mol/mol) 1,2-dioleoyldimethylammonium chloride-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, were used to transfect primary cultures of distal rat fetal lung epithelial cells with pCMV4-based plasmids. A DNA-to-lipid ratio of 1:10 to 1:15 (wt/wt) optimized DNA uptake over a 24-h exposure. At a fixed DNA-to-lipid ratio of 1:15, chloramphenicol acetyltransferase (CAT) reporter gene expression declined at lipid concentrations > 2.5 nmol/cm2 cell surface area, whereas DNA uptake remained concentration dependent. CAT expression peaked 48 h after removal of the liposome-DNA complex, declining thereafter. Reporter gene expression was increased, and supercoiled cDNA degradation was reduced by the addition of 0.2 mM nicotinamide and 10 microM chloroquine. Rat fetal lung epithelial cells transfected with two different expression cassettes had an increased susceptibility to superoxide-mediated cytotoxicity. This could be attributed to a nonspecific delivery of exogenous DNA or some other copurified factor. The DNA-dependent increase in superoxide-mediated cytotoxicity, but not basal levels of cytotoxicity, was inhibited by the addition of 0.2 mM nicotinamide and 10 microM chloroquine.

Selective absence of CD8+ TCRalpha beta+ intestinal epithelial cells in transgenic mice expressing beta2-microglobulin-associated ligands exclusively on thymic cortical epithelium.

Whereas interactions between the TCRalpha beta and self MHC:peptide complexes are clearly required for positive selection of mature CD4(+) and CD8(+) T cells during intrathymic development, the role of self or foreign ligands in maintaining the peripheral T cell repertoire is still controversial. In this report we have utilized keratin 14-beta2-microglobulin (K14-beta2m)-transgenic mice expressing beta2m-associated ligands exclusively on thymic cortical epithelial cells to address the possible influence of TCR:ligand interactions in peripheral CD8(+) T cell homeostasis. Our data indicate that CD8(+) T cells in peripheral lymphoid tissues are present in normal numbers in the absence of self MHC class I:peptide ligands. Surprisingly, however, steady state homeostasis of CD8(+) T cells in the intestinal epithelium is severely affected by the absence of beta2m-associated ligands. Indeed TCRalpha beta(+) IEL subsets expressing CD8alpha beta or CD8alpha alpha are both dramatically reduced in K14-beta2m mice, suggesting that the development, survival or expansion of CD8(+) IEL depends upon interaction of the TCR with MHC class I:peptide or other beta2m-associated ligands elsewhere than on thymic cortical epithelium. Collectively, our data reveal an unexpected difference in the regulation of CD8(+) T cell homeostasis by beta2m-associated ligands in the intestine as compared to peripheral lymphoid organs.

Regulation of the epithelial Na+ channel ENaC by Tsg101 in renal epithelial cells

Kidneys are the main regulator of salt homeostasis and blood pressure. In the distal region of the tubule active Na-transport is finely tuned. This transport is regulated by various hormonal pathways including aldosterone that regulates the reabsorption at the level of the ASDN, comprising the late DCT, the CNT and the CCD. In the ASDN, the amiloride-sensitive epithelial Na-channel (ENaC) plays a major role in Na-homeostasis, as evidenced by gain-of function mutations in the genes encoding ENaC, causing Liddle's syndrome, a severe form of salt-sensitive hypertension. In this disease, regulation of ENaC is compromised due to mutations that delete or mutate a PY-motif in ENaC. Such mutations interfere with Nedd4-2- dependent ubiquitylation of ENaC, leading to reduced endocytosis of the channel, and consequently to increased channel activity at the cell surface. After endocytosis ENaC is targeted to the lysosome and rapidly degraded. Similarly to other ubiquitylated and endocytosed plasma membrane proteins (such as the EGFR), it is likely that the multi-protein complex system ESCRT is involved. To investigate the involvement of this system we tested the role of one of the ESCRT proteins, Tsg101. Here we show that Tsg101 interacts endogenously and in transfected HEK-293 cells with all three ENaC sub-units. Furthermore, mutations of cytoplasmic lysines of ENaC subunits lead to the disruption of this interaction, indicating a potential involvement of ubiquitin in Tsg101 / ENaC interaction. Tsg101 knockdown in renal epithelial cells increases the total and cell surface pool of ENaC, thus implying TsglOl and consequently the ESCRT system in ENaC degradation by the endosomal/lysosomal system. - Les reins sont les principaux organes responsables de la régulation de la pression artérielle ainsi que de la balance saline du corps. Dans la région distale du tubule, le transport actif de sodium est finement régulé. Ce transport est contrôlé par plusieurs hormones comme l'aldostérone, qui régule la réabsorption au niveau de l'ASDN, segment comprenant la fin du DCT, le CNT et le CCD. Dans l'ASDN, le canal à sodium épithélial sensible à l'amiloride (ENaC) joue un rôle majeur dans l'homéostasie sodique, comme cela fut démontré par les mutations « gain de fonction » dans les gênes encodant ENaC, causant ainsi le syndrome de Liddle, une forme sévère d'hypertension sensible au sel. Dans cette maladie, la régulation d'ENaC est compromise du fait des mutations qui supprime ou mute le domaine PY présent sur les sous-unités d'ENaC. Ces mutations préviennent l'ubiquitylation d'ENaC par Nedd4-2, conduisant ainsi à une baisse de l'endocytose du canal et par conséquent une activité accrue d'ENaC à la surface membranaire. Après endocytose, ENaC est envoyé vers le lysosome et rapidement dégradé. Comme d'autres protéines membranaires ubiquitylées et endocytées (comme l'EGFR), il est probable que le complexe multi-protéique ESCRT est impliqué dans le transport d'ENaC au lysosome. Pour étudier l'implication du système d'ESCRT dans la régulation d'ENaC nous avons testé le rôle d'une protéine de ces complexes, TsglOl. Notre étude nous a permis de démontrer que TsglOl se lie aux trois sous-unités ENaC aussi bien en co-transfection dans des cellules HEK-293 que de manière endogène. De plus, nous avons pu démontrer l'importance de l'ubiquitine dans cette interaction par la mutation de toutes les lysines placées du côté cytoplasmique des sous-unités d'ENaC, empêchant ainsi l'ubiquitylation de ces sous-unités. Enfin, le « knockdown » de TsglOl dans des cellules épithéliales de rein induit une augmentation de l'expression d'ENaC aussi bien dans le «pool» total qu'à la surface membranaire, indiquant ainsi un rôle pour TsglOl et par conséquent du système d'ESCRT dans la dégradation d'ENaC par la voie endosome / lysosome. - Le corps humain est composé d'organes chacun spécialisé dans une fonction précise. Chaque organe est composé de cellules, qui assurent la fonction de l'organe en question. Ces cellules se caractérisent par : - une membrane qui leur permet d'isoler leur compartiment interne (milieu intracellulaire ou cytoplasme) du liquide externe (milieu extracellulaire), - un noyau, où l'ADN est situé, - des protéines, sortent d'unités fonctionnelles ayant une fonction bien définie dans la cellule. La séparation entre l'extérieure et l'intérieure de la cellule est essentielle pour le maintien des composants de ces milieux ainsi que pour la bonne fonction de l'organisme et des cellules. Parmi ces composants, le sodium joue un rôle essentiel car il conditionne le maintien de volume sanguin en participant au maintien du volume extracellulaire. Une augmentation du sodium dans l'organisme provoque donc une augmentation du volume sanguin et ainsi provoque une hypertension. De ce fait, le contrôle de la quantité de sodium présente dans l'organisme est essentiel pour le bon fonctionnement de l'organisme. Le sodium est apporté par l'alimentation, et c'est au niveau du rein que va s'effectuer le contrôle de la quantité de sodium qui va être retenue dans l'organisme pour le maintien d'une concentration normale de sodium dans le milieu extracellulaire. Le rein va se charger de réabsorber toutes sortes de solutés nécessaires pour l'organisme avant d'évacuer les déchets ou le surplus de ces solutés en produisant l'urine. Le rein va se charger de réabsorber le sodium grâce à différentes protéines, parmi elle, nous nous sommes intéressés à une protéine appelée ENaC. Cette protéine joue un rôle important dans la réabsorption du sodium, et lorsqu'elle fonctionne mal, comme il a pu être observé dans certaines maladies génétiques, il en résulte des problèmes d'hypo- ou d'hypertension. Les problèmes résultant du mauvais fonctionnement de cette protéine obligent donc la cellule à réguler efficacement ENaC par différents mécanismes, notamment en diminuant son expression et en dégradant le « surplus ». Dans cette travail de thèse, nous nous sommes intéressés au mécanisme impliqué dans la dégradation d'ENaC et plus précisément à un ensemble de protéines, appelé ESCRT, qui va se charger « d'escorter » une protéine vers un sous compartiment à l'intérieur de la cellule ou elle sera dégradée.

STAT3 links IL-22 signaling in intestinal epithelial cells to mucosal wound healing.

Signal transducer and activator of transcription (STAT) 3 is a pleiotropic transcription factor with important functions in cytokine signaling in a variety of tissues. However, the role of STAT3 in the intestinal epithelium is not well understood. We demonstrate that development of colonic inflammation is associated with the induction of STAT3 activity in intestinal epithelial cells (IECs). Studies in genetically engineered mice showed that epithelial STAT3 activation in dextran sodium sulfate colitis is dependent on interleukin (IL)-22 rather than IL-6. IL-22 was secreted by colonic CD11c(+) cells in response to Toll-like receptor stimulation. Conditional knockout mice with an IEC-specific deletion of STAT3 activity were highly susceptible to experimental colitis, indicating that epithelial STAT3 regulates gut homeostasis. STAT3(IEC-KO) mice, upon induction of colitis, showed a striking defect of epithelial restitution. Gene chip analysis indicated that STAT3 regulates the cellular stress response, apoptosis, and pathways associated with wound healing in IECs. Consistently, both IL-22 and epithelial STAT3 were found to be important in wound-healing experiments in vivo. In summary, our data suggest that intestinal epithelial STAT3 activation regulates immune homeostasis in the gut by promoting IL-22-dependent mucosal wound healing.

Reconstituted human polyclonal plasma-derived secretory-like IgM and IgA maintain the barrier function of epithelial cells infected with an enteropathogen.

Intravenous administration of polyclonal and monoclonal antibodies has proven to be a clinically valid approach in the treatment, or at least relief, of many acute and chronic pathologies, such as infection, immunodeficiency, and a broad range of autoimmune conditions. Plasma-derived IgG or recombinant IgG are most frequently used for intravenous or subcutaneous administration, whereas a few IgM-based products are available as well. We have established recently that secretory-like IgA and IgM can be produced upon association of plasma-derived polymeric IgA and IgM with a recombinant secretory component. As a next step toward potential future mucosal administration, we sought to unravel the mechanisms by which these secretory Igs protect epithelial cells located at the interface between the environment and the inside of the body. By using polarized epithelial Caco-2 cell monolayers and Shigella flexneri as a model enteropathogen, we found that polyspecific plasma-derived SIgA and SIgM fulfill many protective functions, including dose-dependent recognition of the antigen via formation of aggregated immune complexes, reduction of bacterial infectivity, maintenance of epithelial cell integrity, and inhibition of proinflammatory cytokine/chemokine production by epithelial cells. In this in vitro model devoid of other cellular or molecular interfering partners, IgM and secretory IgM showed stronger bacterial neutralization than secretory IgA. Together, these data suggest that mucosally delivered antibody preparations may be most effective when combining both secretory-like IgA and IgM, which, together, play a crucial role in preserving several levels of epithelial cell integrity.