Transcriptional activation of the GLUT2 gene by the IPF-1/STF-1/IDX-1 homeobox factor.

The homeodomain protein PDX-1, referred as IPF-1/STF-1/IDX-1, is a transcriptional factor that plays a critical role in the control of several genes expressed in the pancreatic islet. PDX-1 gene expression has been previously shown to be reduced in cultured beta-cell lines chronically exposed to high glucose concentrations. As the glucose transporter type 2 (GLUT2) gene expression is selectively decreased in the beta-pancreatic cells of experimental models of diabetes, we postulated that the loss of GLUT2 gene expression in the pancreatic islets of diabetic animals may be due to the loss of PDX-1 transacting function on the GLUT2 gene. We, therefore, investigated the potential role of PDX-1 in the transcriptional control of GLUT2. We have identified a repeat of a TAAT motif (5'-TAATA-ATAACA-3') conserved in the sequence of the human and murine GLUT2 promoters. Recombinant PDX-1 binds to this GLUT2TAAT motif in electrophoretic mobility shift experiments. PDX-1 antiserum detects the formation of the complex of PDX-1 with the GLUT2TAAT motif in nuclear extracts from the pancreatic insulin-secreting cell line, beta TC3. The GLUT2TAAT motif was mutated in the murine GLUT2 promoter (-1308/+49 bp) linked to a luciferase reporter gene and transfected into beta TC3 cells. Compared with the transcriptional activity of the wild type promoter, that of the mutated promoter decreases by 41%. Multiple copies of the GLUT2TAAT motif were ligated 5' to a heterologous promoter and transfected into a PDX-1-expressing cell line (beta TC3) and into cell lines lacking the homeobox factor (InR1-G9 and JEG-3). The GLUT2TAAT motif mediates the activation of the heterologous promoter in the PDX-1-expressing cell line but not in InR1-G9 or JEG-3 cell lines. Furthermore, cotransfection in a PDX-1-deficient cell line with the expression vector encoding PDX-1 transactivates specifically the heterologous promoter containing the multimerized GLUT2TAAT motif. These data demonstrate that the murine GLUT2 promoter is controlled by the PDX-1 homeobox factor through the identified GLUT2TAAT motif.

The gene MAPK8IP1, encoding islet-brain-1, is a candidate for type 2 diabetes.

Type 2 diabetes is a polygenic and genetically heterogeneous disease . The age of onset of the disease is usually late and environmental factors may be required to induce the complete diabetic phenotype. Susceptibility genes for diabetes have not yet been identified. Islet-brain-1 (IB1, encoded by MAPK8IP1), a novel DNA-binding transactivator of the glucose transporter GLUT2 (encoded by SLC2A2), is the homologue of the c-Jun amino-terminal kinase-interacting protein-1 (JIP-1; refs 2-5). We evaluated the role of IBi in beta-cells by expression of a MAPK8IP1 antisense RNA in a stable insulinoma beta-cell line. A 38% decrease in IB1 protein content resulted in a 49% and a 41% reduction in SLC2A2 and INS (encoding insulin) mRNA expression, respectively. In addition, we detected MAPK8IP1 transcripts and IBi protein in human pancreatic islets. These data establish MAPK8IP1 as a candidate gene for human diabetes. Sibpair analyses performed on i49 multiplex French families with type 2 diabetes excluded MAPK8IP1 as a major diabetogenic locus. We did, however, identify in one family a missense mutation located in the coding region of MAPK8IP1 (559N) that segregated with diabetes. In vitro, this mutation was associated with an inability of IB1 to prevent apoptosis induced by MAPK/ERK kinase kinase 1 (MEKK1) and a reduced ability to counteract the inhibitory action of the activated c-JUN amino-terminal kinase (JNK) pathway on INS transcriptional activity. Identification of this novel non-maturity onset diabetes of the young (MODY) form of diabetes demonstrates that IB1 is a key regulator of 3-cell function.

Regulation of insulin secretion by phosphatidylinositol-4,5-bisphosphate.

The role of PIP(2) in pancreatic beta cell function was examined here using the beta cell line MIN6B1. Blocking PIP(2) with PH-PLC-GFP or PIP5KIgamma RNAi did not impact on glucose-stimulated secretion although susceptibility to apoptosis was increased. Over-expression of PIP5KIgamma improved cell survival and inhibited secretion with accumulation of endocytic vacuoles containing F-actin, PIP(2), transferrin receptor, caveolin 1, Arf6 and the insulin granule membrane protein phogrin but not insulin. Expression of constitutively active Arf6 Q67L also resulted in vacuole formation and inhibition of secretion, which was reversed by PH-PLC-GFP co-expression. PIP(2) co-localized with gelsolin and F-actin, and gelsolin co-expression partially reversed the secretory defect of PIP5KIgamma-over-expressing cells. RhoA/ROCK inhibition increased actin depolymerization and secretion, which was prevented by over-expressing PIP5KIgamma, while blocking PIP(2) reduced constitutively active RhoA V14-induced F-actin polymerization. In conclusion, although PIP(2) plays a pro-survival role in MIN6B1 cells, excessive PIP(2) production because of PIP5KIgamma over-expression inhibits secretion because of both a defective Arf6/PIP5KIgamma-dependent endocytic recycling of secretory membrane and secretory membrane components such as phogrin and the RhoA/ROCK/PIP5KIgamma-dependent perturbation of F-actin cytoskeleton remodelling.

Agonist-induced internalization and recycling of the glucagon-like peptide-1 receptor in transfected fibroblasts and in insulinomas.

Glucagon-like peptide-1 (GLP-1) is the most potent stimulator of glucose-induced insulin secretion and its pancreatic beta-cell receptor is a member of a new subfamily of G-protein-coupled receptors which includes the receptors for vasoactive intestinal polypeptide, secretin and glucagon. Here we studied agonist-induced GLP-1 receptor internalization in receptor-transfected Chinese hamster lung fibroblasts using three different approaches. First, iodinated GLP-1 bound at 4 degrees C to transfected cells was internalized with a t 1/2 of 2-3 min following warming up of the cells to 37 degrees C. Secondly, exposure to GLP-1 induced a shift in the distribution of the receptors from plasma membrane-enriched to endosomes-enriched membrane fractions, as assessed by Western blot detection of the receptors using specific antibodies. Thirdly, continuous exposure of GLP-1 receptor-expressing cells to iodinated GLP-1 led to a linear accumulation of peptide degradation products in the medium following a lag time of 20-30 min, indicating a continuous cycling of the receptor between the plasma membrane and endosomal compartments. Potassium depletion and hypertonicity inhibited transferrin endocytosis, a process known to occur via coated pit formation, as well as GLP-1 receptor endocytosis. In contrast to GLP-1, the antagonist exendin-(9-39) did not lead to receptor endocytosis. Surface re-expression following one round of GLP-1 receptor endocytosis occurred with a half-time of about 15 min. The difference in internalization and surface re-expression rates led to a progressive redistribution of the receptor in intracellular compartments upon continuous exposure to GLP-1. Finally, endogenous GLP-1 receptors expressed by insulinoma cells were also found to be internalized upon agonist binding. Together our data demonstrate that the GLP-1 receptor is internalized upon agonist binding by a route similar to that taken by single transmembrane segment receptors. The characterization of the pathway and kinetics of GLP-1-induced receptor endocytosis will be helpful towards understanding the role of internalization and recycling in the control of signal transduction by this receptor.

Role of Notch signaling in epidermis and appendages

Résumé Dans la peau, il a été montré que Notch1 induit l'arrêt de la prolifération et la différentiation des keratinocytes. L'inactivation de Notch1 cause une hyperplasie de l'épiderme et la formation de carcinomes basaux cellulaires. Notre groupe a principalement identifié deux voies de signalisations, la voie Shh et la voie Wnt, qui sont dérégulées en conséquence de l'inactivation de Notch1 dans la peau. Nous avons démontré l'habilité de Notch1 à réprimer la voie Wnt induite par ß-catenin dans les keratinocytes primaires ainsi que dans d'autres types de cellules épithéliales humaines. De plus, nous avons pu déterminer que Notch1 régule cette voie, probablement en favorisant la phosphorylation de ß-catenin par le complexe axin/APC/GSK-3ß. La protéine faisant partie de la voie Wnt, ou la protéine affectant la voie Wnt, qui est régulée par Notch1 est sujette à de plus amples investigations. Un autre but de cette étude a été l'identification de potentiels gènes cibles de Notch1 autres que ceux faisant partie des voies de signalisation Shh et Wnt précédemment évoquées. Ce projet fut abordé par l'analyse de puces à ADN (ISREC et Affymetrix) qui ont été utilisées pour des expériences de gain et de perte de fonction de Notch1 dans des keratinocytes prúmaires. En plus de l'hyperplasie épidermale, les souris Notch1 déficiente ont une perte importante de poils. Nous avons montré que Notch1 est nécessaire pour le développement et l'homéostasie des follicules pileux. En effet, l'inactivation du gène Notch1 mediée par l'activation des kératines 5 ou 14 dans l'épiderme, cause des défauts du cycle ainsi que de la structure des poils. De plus, d'autres appendices de la peau, comme les glandes sudoripares et de Meibomius, ont une structure anormale et sont non fonctionnelles dans les souris Notch1 déficiente. Finalement, nous avons observé que la déficience de Notch1 dans l'épithélium cornéen mène à la formation d'une plaque épidermale opaque sur la cornée. Basé sur l'hypothèse que le défaut des glandes de Meibomius des souris Notch1 déficientes cause des lésions de la surface oculaire, nous avons montré que Notch1 est essentiel pour la cicatrisation de la cornée. Lorsque Notch1 est absent, les cellules souches de l'épithélium cornéen ne sont plus capables de se différentier en cellules cornéennes, mais réparent la blessure en se différentiant en épiderme. Ce résultat indique que Notch1 est essentiel pour la différentiation de cellules souches de la cornée qui sont spécifiquement impliquées dans la réparation de la cornée. De plus, nous avons montré que l'expression de CRBP1 dans l'épithélium cornéen est diminuée en l'absence de Notch1, ceci étant possiblement à l'origine de la formation de la plaque épidermale. Abstract: In the skin, Notch1 has been shown to trigger cell growth arrest and differentiation of keratinocytes. Notch1 inactivation results in epidermal hyperplasia and subsequent formation of basal cell carcinoma-like (BCC-like) tumors. So far our group has identified two main pathways, the Shh and the Wnt pathway, that are deregulated as a consequence of Notch1 inactivation in the skin. We showed the ability of Notch1 to represses ß-catenin-mediated Wnt signaling in primary keratinocytes as well as in other types of human epithelial cells. In addition we were able to determine that Notch1 regulates this pathway possibly by enhancing ß-catenin phosphorylation by the axin/APC/GSK-3ß complex. The exact target protein of the Wnt pathway or target protein that affects the Wnt pathway, and that is regulated by Notch1, is subject of current investigation. Another aim of this study was the identification of possible Notch1 target genes in addition to those of the Shh and Wnt signaling pathways. This was addressed by gene chip analysis using ISREC as well as Affymetrix microarrays for gain and loss of function of Notch1 in mouse primary keratinocytes. In addition to epidermal hyperplasia, Notch1 deficient mice show an important hair loss. We showed that Notch1 is required for postnatal development and homeostasis of hair follicles. Indeed, keratin5 or keratinl4-driven Cre recombinase-mediated inactivation of the Notch1 gene in the epidermis causes perturbations of the hair cycle and structural defects of the hair follicle. Moreover, other skin appendages, like the sweat and Meibomian glands show abnormal morphology and are not functional in the Notch 1 deficient mice. Finally, we observed that Notch1 deficiency in the corneal epithelium leads to the formation of an epidermal corneal plaque. Based on the hypothesis that the Meiboinian gland defect in the Notch1 deficient mice results in lesions of the eye surface, we showed that Notch1 is essential for wound-healing of the cornea. In absence of Notch1 the stem cells of the corneal epithelium are no longer able to differentiate in the corneal fate but instead repair the wound by differentiating into skin-like epidermis. This result indicated that Notch1 is essential for the differentiation of corneal stem cells specifically implicated in corneal wound-healing. Moreover, we showed that CRBP1 expression in the corneal epithelium was lost in the absence of Notch1, possibly being at the origin of plaque formation.

Triiodothyronine stimulation of oligodendroglial differentiation and myelination. A developmental study.

The biochemical development of rotation-mediated aggregating brain cell cultures was studied in a serum-free chemically defined medium in the presence (complete medium) or the absence of triiodothyronine (T3). The expression of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) and myelin basic protein (MBP), two myelin components, was temporally dissociated in brain cell aggregating cultures grown in a complete medium. CNP increased from day 8 and reached a plateau around day 25. MBP accumulated rapidly from the third until the fourth week in culture. The total protein content increased gradually until day 25. The activity of ornithine decarboxylase (ODC) used as an index of cell growth and differentiation, showed two well-defined peaks of activity. The first peak reached a maximum at day 6 and correlated with both the highest DNA content and the peak of [3H]-thymidine incorporation. The second peak of ODC activity (from day 19 to 35) coincided with the differentiation of oligodendrocytes. These results confirm that aggregating fetal rat brain cells cultured in a serum-free chemically defined medium undergo extensive differentiation. Addition of T3 to the culture medium doubled the CNP activity by day 16. In contrast, MBP was only slightly increased by day 16, reaching at 25 and 35 days 8 to 10-fold higher values than the untreated cultures. When T3 was removed between day 16 and 25, CNP decreased almost to control values and MBP failed to accumulate. Moreover, when T3 was reintroduced into the medium (between day 25 and 35), CNP activity was restored and MBP content was partially corrected. T3 treatment produced a concentration-dependent increase in ODC activity which was observed only around day 19. The first peak of ODC activity observed at culture day 6 was independent of the presence of T3. These results obtained in brain cell cultures emphasize the direct effect of T3 on myelination.

Calcineurin inhibitors activate the proto-oncogene Ras and promote protumorigenic signals in renal cancer cells.

The development of cancer is a major problem in immunosuppressed patients, particularly after solid organ transplantation. We have recently shown that calcineurin inhibitors (CNI) used to treat transplant patients may play a critical role in the rapid progression of renal cancer. To examine the intracellular signaling events for CNI-mediated direct tumorigenic pathway(s), we studied the effect of CNI on the activation of proto-oncogenic Ras in human normal renal epithelial cells (REC) and renal cancer cells (786-0 and Caki-1). We found that CNI treatment significantly increased the level of activated GTP-bound form of Ras in these cells. In addition, CNI induced the association of Ras with one of its effector molecules, Raf, but not with Rho and phosphatidylinositol 3-kinase; CNI treatment also promoted the phosphorylation of the Raf kinase inhibitory protein and the downregulation of carabin, all of which may lead to the activation of the Ras-Raf pathway. Blockade of this pathway through either pharmacologic inhibitors or gene-specific small interfering RNA significantly inhibited CNI-mediated augmented proliferation of renal cancer cells. Finally, it was observed that CNI treatment increased the growth of human renal tumors in vivo, and the Ras-Raf pathway is significantly activated in the tumor tissues of CNI-treated mice. Together, targeting the Ras-Raf pathway may prevent the development/progression of renal cancer in CNI-treated patients.

Increased connexin43 expression in human saphenous veins in culture is associated with intimal hyperplasia.

OBJECTIVE: Intimal hyperplasia is a vascular remodelling process that occurs after a vascular injury. The mechanisms involved in intimal hyperplasia are proliferation, dedifferentiation, and migration of medial smooth muscle cells towards the subintimal space. We postulated that gap junctions, which coordinate physiologic processes such as cell growth and differentiation, might participate in the development of intimal hyperplasia. Connexin43 (Cx43) expression levels may be altered in intimal hyperplasia, and we therefore evaluated the regulated expression of Cx43 in human saphenous veins in culture in the presence or not of fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity. METHODS: Segments of harvested human saphenous veins, obtained at the time of bypass graft, were opened longitudinally with the luminal surface uppermost and maintained in culture for 14 days. Vein fragments were then processed for histologic examination, neointimal thickness measurements, immunocytochemistry, RNA, and proteins analysis. RESULTS: Of the four connexins (Cx37, 40, 43, and 45), we focused on Cx43 and Cx40, which we found by real-time polymerase chain reaction to be expressed in the saphenous vein because they are the predominant connexins expressed by smooth muscle cells and endothelial cells. After 14 days of culture, histomorphometric analysis showed a significant increase in the intimal thickness as observed during the process of intimal hyperplasia. A time-course analysis revealed a progressive upregulation of Cx43 to reach a maximal increase of sixfold to eightfold at both transcript and protein levels after 14 days in culture. In contrast, the expression of Cx40, abundantly expressed in the endothelial cells, was not altered. Immunofluorescence showed a large increase in Cx43 within smooth muscle cell membranes of the media layer. The development of intimal hyperplasia in vitro was decreased in presence of fluvastatin and was associated with reduced Cx43 expression. CONCLUSIONS: These data show that Cx43 is increased in vitro during the process of intimal hyperplasia and that fluvastatin could prevent this induction, supporting a critical role for Cx43-mediated gap-junctional communication in the human vein during the development of intimal hyperplasia. CLINICAL RELEVANCE: Stenosis due to intimal hyperplasia is the most common cause of failure of venous bypass grafts. To better understand the development of intimal hyperplasia, we used an ex vivo organ culture model to study saphenous veins harvested from patients undergoing a lower limb bypass surgery. In this model, the morphologic and functional integrity of the vessel wall is maintained and significant intimal hyperplasia development occurs after 14 days in culture. We have postulated that gap junctions, which coordinate physiologic processes such as cell growth and differentiation, may participate in the development of intimal hyperplasia. Indeed, intimal hyperplasia consists of proliferation and migration of smooth muscle cells into the subendothelial space. Intercellular communication is responsible for the direct transfer of ions and small molecules from one cell to the other through gap-junction channels found at cell-cell appositions. No study to date has evaluated whether gap junctional communication is involved in the process of intimal hyperplasia in humans. This assertion was investigated by using the aforementioned organ culture model of intimal hyperplasia in human saphenous veins, and our data support a critical role for Cx43-mediated gap junctional communication in human vein during the development of intimal hyperplasia.

Dexamethasone stimulates the biochemical differentiation of fetal forebrain cells in reaggregating cultures.

The influence of dexamethasone on the development of neurons and oligodendrocytes was studied in serum-free, aggregating rat brain cell cultures. Synaptogenesis and myelination occur in this culture system. The concentration of myelin basic protein and the activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase were used as oligodendroglia and myelin markers. Choline acetyltransferase and acetylcholinesterase served as neuronal markers, glutamine synthetase reflected astrocyte differentiation, while ornithine decarboxylase served as a general marker for cell growth and maturation. This study showed that dexamethasone stimulated the differentiation of cholinergic neurons and astrocytes. The effect of dexamethasone on oligodendroglial differentiation and myelination depended on the stage of development: during the early phase of myelination dexamethasone had a stimulatory effect, whereas at a later stage it showed a significant inhibition.

Coculture of bladder urothelial and smooth muscle cells on small intestinal submucosa: potential applications for tissue engineering technology.

PURPOSE: Small intestinal submucosa is a xenogenic, acellular, collagen rich membrane with inherent growth factors that has previously been shown to promote in vivo bladder regeneration. We evaluate in vitro use of small intestinal submucosa to support the individual and combined growth of bladder urothelial cells and smooth muscle cells for potential use in tissue engineering techniques, and in vitro study of the cellular mechanisms involved in bladder regeneration. MATERIALS AND METHODS: Primary cultures of human bladder urothelial cells and smooth muscle cells were established using standard enzymatic digestion or explant techniques. Cultured cells were then seeded on small intestinal submucosa at a density of 1 x 105 cells per cm.2, incubated and harvested at 3, 7, 14 and 28 days. The 5 separate culture methods evaluated were urothelial cells seeded alone on the mucosal surface of small intestinal submucosa, smooth muscle cells seeded alone on the mucosal surface, layered coculture of smooth muscle cells seeded on the mucosal surface followed by urothelial cells 1 hour later, sandwich coculture of smooth muscle cells seeded on the serosal surface followed by seeding of urothelial cells on the mucosal surface 24 hours later, and mixed coculture of urothelial cells and smooth muscle cells mixed and seeded together on the mucosal surface. Following harvesting at the designated time points small intestinal submucosa cell constructs were formalin fixed and processed for routine histology including Masson trichrome staining. Specific cell growth characteristics were studied with particular attention to cell morphology, cell proliferation and layering, cell sorting, presence of a pseudostratified urothelium and matrix penetrance. To aid in the identification of smooth muscle cells and urothelial cells in the coculture groups, immunohistochemical analysis was performed with antibodies to alpha-smooth muscle actin and cytokeratins AE1/AE3. RESULTS: Progressive 3-dimensional growth of urothelial cells and smooth muscle cells occurred in vitro on small intestinal submucosa. When seeded alone urothelial cells and smooth muscle cells grew in several layers with minimal to no matrix penetration. In contrast, layered, mixed and sandwich coculture methods demonstrated significant enhancement of smooth muscle cell penetration of the membrane. The layered and sandwich coculture techniques resulted in organized cell sorting, formation of a well-defined pseudostratified urothelium and multilayered smooth muscle cells with enhanced matrix penetration. With the mixed coculture technique there was no evidence of cell sorting although matrix penetrance by the smooth muscle cells was evident. Immunohistochemical studies demonstrated that urothelial cells and smooth muscle cells maintain the expression of the phenotypic markers of differentiation alpha-smooth muscle actin and cytokeratins AE1/AE3. CONCLUSIONS: Small intestinal submucosa supports the 3-dimensional growth of human bladder cells in vitro. Successful combined growth of bladder cells on small intestinal submucosa with different seeding techniques has important future clinical implications with respect to tissue engineering technology. The results of our study demonstrate that there are important smooth muscle cell-epithelial cell interactions involved in determining the type of in vitro cell growth that occurs on small intestinal submucosa. Small intestinal submucosa is a valuable tool for in vitro study of the cell-cell and cell-matrix interactions that are involved in regeneration and various disease processes of the bladder.

Development of an Fn14 agonistic antibody as an anti-tumor agent.

TWEAK, a TNF family ligand with pleiotropic cellular functions, was originally described as capable of inducing tumor cell death in vitro. TWEAK functions by binding its receptor, Fn14, which is up-regulated on many human solid tumors. Herein, we show that intratumoral administration of TWEAK, delivered either by an adenoviral vector or in an immunoglobulin Fc-fusion form, results in significant inhibition of tumor growth in a breast xenograft model. To exploit the TWEAK-Fn14 pathway as a therapeutic target in oncology, we developed an anti-Fn14 agonistic antibody, BIIB036. Studies described herein show that BIIB036 binds specifically to Fn14 but not other members of the TNF receptor family, induces Fn14 signaling, and promotes tumor cell apoptosis in vitro. In vivo, BIIB036 effectively inhibits growth of tumors in multiple xenograft models, including colon (WiDr), breast (MDA-MB-231), and gastric (NCI-N87) tumors, regardless of tumor cell growth inhibition response observed to BIIB036 in vitro. The anti-tumor activity in these cell lines is not TNF-dependent. Increasing the antigen-binding valency of BIB036 significantly enhances its anti-tumor effect, suggesting the contribution of higher order cross-linking of the Fn14 receptor. Full Fc effector function is required for maximal activity of BIIB036 in vivo, likely due to the cross-linking effect and/or ADCC mediated tumor killing activity. Taken together, the anti-tumor properties of BIIB036 validate Fn14 as a promising target in oncology and demonstrate its potential therapeutic utility in multiple solid tumor indications.

Vitamin E content in fish oil emulsion does not prevent lipoperoxidative effects on human colorectal tumors.

OBJECTIVE: The anticancer action exerted by polyunsaturated fatty acid peroxidation may not be reproduced by commercially available lipid emulsions rich in vitamin E. Therefore, we evaluated the effects of fish oil (FO) emulsion containing α-tocopherol 0.19 g/L on human colorectal adenocarcinoma cells and tumors. METHODS: HT-29 cell growth, survival, apoptosis, and lipid peroxidation were analyzed after a 24-h incubation with FO 18 to 80 mg/L. Soybean oil (SO) emulsion was used as an isocaloric and isolipidic control. In vivo, nude mice bearing HT-29 tumors were sacrificed 7 d after an 11-d treatment with intravenous injections of FO or SO 0.2 g ∙ kg(-1) ∙ d(-1) FO or SO to evaluate tumor growth, necrosis, and lipid peroxidation. RESULTS: The FO inhibited cell viability and clonogenicity in a dose-dependent manner, whereas SO showed no significant effect compared with untreated controls. Lipid peroxidation and cell apoptosis after treatment with FO 45 mg/L were increased 2.0-fold (P < 0.01) and 1.6-fold (P = 0.04), respectively. In vivo, FO treatment did not significantly affect tumor growth. However, immunohistochemical analyses of tumor tissue sections showed a decrease of 0.6-fold (P < 0.01) in the cell proliferation marker Ki-67 and an increase of 2.3-fold (P = 0.03) in the necrotic area, whereas malondialdehyde and total peroxides were increased by 1.9-fold (P = 0.09) and 7.0-fold (P < 0.01), respectively, in tumors of FO-treated compared with untreated mice. CONCLUSION: These results suggest that FO but not SO has an antitumor effect that can be correlated with lipid peroxidation, despite its vitamin E content.

Differential phosphoproteomics of fragmenting yeast vacuoles

Many organelles exist in an equilibrium of fragmentation into smaller units and fusion into larger structures, which is coordinated with cell division, the increase in cell mass, and envi¬ronmental conditions. In yeast cells, organelle homeostasis can be studied using the yeast vacuole (lysosome) as a model system. Yeast vacuoles are the main compartment for degrada¬tion of cellular proteins and storage of nutrients, ions and metabolites. Fission and fusion of vacuoles can be induced by hyper- and hypotonic shock in vivo, respectively, and have also been reconstituted in vitro using isolated vacuoles. The conserved serine/threonine kinase TOR (target of rapamycin) is a central nutrient sensor and regulates cell growth and metabolism. In yeast, there are two TOR proteins, Torlp and Tor2p, which are part of larger protein complexes, TORCI and TORC2. Only TORCI is rapamycin-sensitive. Disregulation of TOR signaling is linked to a multitude of diseases in humans, e.g. cancer, neurodegenerative diseases and metabolic syndrome. It has been shown that TORCI localizes to the vacuole membrane, and recent findings of our laboratory demonstrated that TORCI positively regulates vacuole fragmentation. This suggests that the fragmentation machinery should contain target proteins phosphorylated by TORCI. I explored the rapamycin-and fission-dependent vacuolar phosphoproteome during frag¬mentation, using a label-free mass-spectrometry approach. I identified many vacuolar factors whose phosphorylation was downregulated in a TORCI- and fission-dependent manner. Among them were known protein complexes that are functionally linked to fission or fusion, like the HOPS, VTC and FAB1 complexes. Hence, TORCI-dependent phosphorylations might positively regulate vacuole fission. Several candidates were chosen for detailed microscopic analysis of in vivo vacuole frag-mentation, using deletion mutants. I was able to identify novel factors not previously linked to fission phenotypes, e.g. the SEA complex, Pib2, and several vacuolar amino acid transporters. Transport of neutral and basic amino acids across the membrane seems to control vacuole fission, possibly via TORCI. I analyzed vacuolar fluxes of amino acids in wildtype yeast cells and found evidence for a selective vacuolar export of basic amino acids upon hyperosmotic stress. This leads me to propose a model where vacuolar export of amino acids is necessary to reshape the organelle under salt stress. - Le nombre et la taille de certaines organelles peut être déterminé par un équilibre entre la fragmentation qui produit des unités plus petites et la fusion qui génère des structures plus larges. Cet équilibre est coordonné avec la division cellulaire, l'augmentation de la masse cellulaire, et les conditions environnementales. Dans des cellules de levure, l'homéostasie des organelles peut être étudié à l'aide d'un système modèle, la vacuole de levure (lysosome). Les vacuoles constituent le principal compartiment de la dégradation des protéines et de stockage des nutriments, des ions et des métabolites. La fragmentation et la fusion des vacuoles peuvent être respectivement induites par un traitement hyper- ou hypo-tonique dans les cellules vivantes. Ces processus ont également été reconstitués in vitro en utilisant des vacuoles isolées. La sérine/thréonine kinase conservée TOR (target of rapamycin/cible de la rapamycine) est un senseur de nutriments majeur qui régule la croissance cellulaire et le métabolisme. Chez la levure, il existe deux protéines TOR, Torlp et Tor2p, qui sont les constituants de plus grands complexes de protéines, TORCI et TORC2. TORCI est spécifiquement inhibé par la rapamycine. Une dysrégulation de la signalisation de TOR est liée à une multitude de maladies chez l'homme comme le cancer, les maladies neurodégénératives et le syndrome métabolique. Il a été montré que TORCI se localise à la membrane vacuolaire et les découvertes récentes de notre laboratoire ont montré que TORCI régule positivement la fragmentation de la vacuole. Ceci suggère que le mécanisme de fragmentation doit être contrôlé par la phosphorylation de certaines protéines cibles de TORCI. J'ai exploré le phosphoprotéome vacuolaire lors de la fragmentation, en présence ou absence de rapamycine et dans des conditions provoquant la fragmentation des organelles. La méthode choisie pour réaliser la première partie de ce projet a été la spectrométrie de masse différentielle sans marquage. J'ai ainsi identifié plusieurs facteurs vacuolaires dont la phosphorylation est régulée d'une manière dépendante de TORCI et de la fragmentation. Parmi ces facteurs, des complexes protéiques connus qui sont fonctionnellement liées à fragmentation ou la fusion, comme les complexes HOPS, VTC et FAB1 ont été mis en évidence. Par conséquent, la phosphorylation dépendante de TORCI peut réguler positivement la fragmentation des vacuoles. Plusieurs candidats ont été choisis pour une analyse microscopique détaillée de la fragmentation vacuolaire in vivo en utilisant des mutants de délétion. J'ai été en mesure d'identifier de nouveaux facteurs qui n'avaient pas été encore associés à des phénotypes de fragmentation tels que les complexes SEA, Pib2p, ainsi que plusieurs transporteurs vacuolaires d'acides aminés. Le transport des acides aminés à travers la membrane semble contrôler la fragmentation de la vacuole. Puisque ces transporteurs sont phosphorylés par TORCI, ces résultats semblent confirmer la

Jonctions communicantes et sécrétion [Gap junctions and secretion]

The emergence of multicellular organisms has necessitated the development of mechanisms for interactions between adjacent and distant cells. A consistent feature of this network is the expression of gap junction channels between the secretory cells of all glands so far investigated in vertebrates. Here, we reviewed the distribution of the gap junctions proteins, named connexins, in a few mammalian glands, and discussed the recent evidence pointing to the participation of these proteins in the functioning of endocrine and exocrine cells. Specifically, available data indicate the importance of gap junctions for the proper control of glucose-induced insulin secretion. Understanding the functions of beta-cell connexins are crucial for the engineering of surrogate cells, which is necessary for implementation of a replacement cell therapy in diabetic patients.