The Role of the Androgen Receptor Cofactor p44/WDR77 in Astrocyte Activation

Astrogliosis is induced by neuronal damage and is also a pathological feature of the major aging-related neurodegenerative disorders. The mechanisms that control the cascade of astrogliosis have not been well established. In a previous study, we identified a novel androgen receptor (AR)-interacting protein (p44/WDR77) and found that it plays a critical role in the control of proliferation and differentiation of prostate epithelial cells. In the present study, we found that deletion of the p44 gene in the mouse brain caused accelerated aging with dramatic astrogliosis. The p44/WDR77 is expressed in astrocytes and loss of p44/WDR77 expression in astrocytes leads to astrogliosis. Our results reveal a novel role of p44/WDR77 in astrocytes, which may explain the well-documented role of androgens in suppression of astrogliosis. While many of detailed mechanisms of astrocyte activation remain to be elucidated, a number pathways have been implicated in astrocyte activation including p21Cip1 and the NF-kB pathway. Astrocytic activation induced by p44/WDR77 gene deletion was associated with a significant increase of p21Cip1 expression and NF-kB activation characterized by p65 nuclear localization. We found that down-regulation of p21Cip1 expression inhibited astrocyte activation induced by the p44/WDR77 deletion and was accompanied by a decreased p65 nuclear localization. While p21Cip1 role in astrocyte activation and NF-kB activation is not well understood, studies of other cell cycle regulators have implicated cell cycle control systems as modulators of astrocyte activation, thus p21Cip1 could induce secondary effect to induce p65 nuclear localization. However, p65 knockdown completely relieved the inhibition of astrocyte growth induced by the p44/WDR77 deletion, while p21Cip1 knockdown only partially recovered this inhibition. Thus, NF-kB activity performs additional regulatory actions not mediated by p21Cip1. These analyses imply that p4/WDR77 suppresses astrocyte activation through modulating p21Cip1 expression and NF-kB activation.

Quantitation of C4a and C4b in systemic lupus erythematosus patients

The fourth component of human complement (C4) exists in blood as two major forms or isotypes which differ in their biochemical and functional properties. Because C4A preferentially transacylates onto amino groups, it has been postulated that this isotype is more important in the clearance of immune complexes. Patients having systemic lupus erythematosus (SLE), an autoimmune disease, have an increased incidence of C4A null genes and presumably decreased levels of C4A. Currently accepted methods for the detection of C4, however, cannot accurately quantitate C4A and C4B. Thus, their role in disease susceptibility and activity has not been studied. A novel immunoassay, which utilized heat-aggregated IgG to activate and capture C4, was developed for accurate quantitation of total C4, C4A and C4B by monoclonal antibody conjugates. Higher mean total C4 values were found in a healthy Black control population when compared to White controls. This appeared to be due to an increase in C4B. In SLE patients, mean total C4 levels were significantly lower than controls regardless of disease activity. Serial patient studies showed that the ratio of C4A:C4B remained relatively constant. When the patient group was compared to controls based on C4 null gene status, the mean levels of C4A were identical while C4B was decreased in the patients. This suggests that the common HLA-B8, Dr3 C4A*Q0 gene deletion found in SLE patients may also adversely affect genetic control of the C4B genes. Furthermore, low levels of C4A cannot fully account for disease development in SLE patients having C4A null genes. ^

Exon involvement in the regulation of MuSVts110 RNA splicing

MuSVts110 is a conditionally defective mutant of Moloney murine sarcoma virus which undergoes a novel tmperature-dependent splice event at growth temperatures of 33$\sp\circ$C or lower. Relative to wild-type MuSV-124, MuSVts110 contains a 1487 base deletion spanning from the 3$\sp\prime$ end of the p30 gag coding region to just downstream of the first v-mos initiation codon. As a result, the gag and mos genes are fused out of frame and no v-mos protein is expressed. However, upon a shift to 33$\sp\circ$C or lower, a splice event occurs which removes 431 bases, realigns the gag and mos genes, and allows read-through translation of a P85gag-mos transforming protein. Interestingly, while the cryptic splice sites utilized in MuSVts110 are present and unaltered in MuSV-124, they are never used. Due to the 1487 base deletion, the MuSV-124 intron was reduced from 1919 to 431 bases suggesting that intron size might be involved in the activation of these cryptic splice sites in MuSVts110. Since the splicing phenotype of the MuSVts110 equivalent (TS32 DNA) which contains the identical 1487 base deletion introduced into otherwise wild-type MuSV-124 DNA, was indistinguishable from authentic MuSVts110, it was concluded that this deletion alone is responsible for activation of the cryptic splice sites used in MuSVts110. These results also confirmed that thermodependent splicing is an intrinsic property of the viral RNA and not due to some cellular defect. Furthermore, analysis of gag gene deletion and frameshift MuSVts110 mutants demonstrated that viral gag gene proteins do not play a role in regulation of MuSVts110 splicing. Instead, cis-acting viral sequences appear to mediate regulation of the splice event.^ Our initial observation that truncation of the MuSVts110 transcript, leaving only residual amounts of the flanking exon sequences, completely abolished splicing activity argued that exon sequences might participate in the regulation of the splice event.^ Analysis of exon sequence involvement has also identified cis-acting sequences important in the thermodependence of the splice event. Data suggest that regulation of the MuSVts110 splice event involves multiple interactions between specific intron and exon sequences and spliceosome components which together limit splicing activity to temperatures of 33$\sp\circ$C or lower while simultaneously restricting splicing to a maximum of 50% efficiency. (Abstract shortened with permission of author.) ^

Transcriptional regulation of the invariant chain associated with MHC class II molecules

The invariant chain associated with the major histocompatibility complex (MHC) class II molecules is a non-polymorphic glycoprotein implicated in antigen processing and class II molecule intracellular transport. Class II molecules and invariant chain (In) are expressed primarily by B lymphocytes and antigen-presenting cells such as macrophages and can be induced by interferon gamma (IFN-$\gamma$) in a variety of cell types such as endothelial cells, fibroblasts, and astrocytes. In this study the cis-acting sequences involved in the constitutive, tissue-specific, and IFN-$\gamma$ induced expression of the human In gene were investigated and nuclear proteins which specifically bound these sequences were identified.^ To define promoter sequences involved in the regulation of the human In gene, 790 bp 5$\sp\prime$ to the initiation of transcription were subcloned upstream of the gene encoding chloramphenicol acetyl transferase (CAT). Transfection of this construct into In expressing and non-expressing cell lines demonstrated that this 790 bp In promoter sequence conferred tissue specificity to the CAT gene. Deletion mutants were created in the promoter to identify sequences important for transcription. Three regulatory regions were identified $-$396 to $-$241, $-$241 to $-$216, and $-$216 to $-$165 bp 5$\sp\prime$ to the cap site. Transfection into a human glioblastoma cell line, U-373 MG, and treatment with IFN-$\gamma$, demonstrated that this 5$\sp\prime$ region is responsive to IFN-$\gamma$. An IFN-$\gamma$ response element was sublocalized to the region $-$120 to $-$61 bp. This region contains homology to the interferon-stimulated response element (ISRE) identified in other IFN responsive genes. IFN-$\gamma$ induces a sequence-specific DNA binding factor which binds to an oligonucleotide corresponding to $-$107 to $-$79 bp of the In promoter. This factor also binds to an oligonucleotide corresponding to $-$91 to $-$62 of the interferon-$\beta$ gene promoter, suggesting this factor may be member of the IRF-1/ISGF2, IRF-2, ICSBP family of ISRE binding proteins. A transcriptional enhancer was identified in the first intron of the In gene. This element, located in a 2.6 kb BamHI/PstI fragment, enhances the IFN-$\gamma$ response of the promoter in U-373 MG. The majority of the In enhancer activity was sublocalized to a 550 bp region $\sim$1.6 kb downstream of the In transcriptional start site. ^

Regulation of phospholipid biosynthesis in {\it Saccharomyces cerevisiae\/} by CDP-diacylglycerol synthase

Amine-containing phospholipid synthesis in Saccharomyces cerevisiae starts with the conversion of CDP-diacylglycerol (CDP-DAG) and serine to phosphatidylserine (PS) while phosphatidylinositol (PI) is formed from CDP-DAG and inositol (derived from inositol-1-phosphate). In this study a gene (CDS1) encoding CDP-DAG synthase in S. cerevisiae was isolated and identified. The CDS1 gene encodes the majority, if not all, of the synthase activity, and is essential for cell growth. Overexpression of the CDS1 gene resulted in an elevation in the apparent initial rate of synthesis and also steady-state level of PI relative to PS in both wild type yeast and the cds1 mutant. Down-regulation of CDS1 expression resulted in an inositol excretion phenotype and an opposite effect on the above phospholipid synthesis in the cds1 mutant. This regulation of phospholipid biosynthesis is mediated by changes of the phospholipid biosynthetic enzymes via a mechanism independent of the expression of the INO2-OPI1 regulatory genes. Reduction in the level of CDP-DAG synthase activity resulted in an increase in PS synthase activity which followed a similar change in the CHO1/PSS (encodes PS synthase) mRNA level. INO1 (encodes inositol-1-phosphate synthase) mRNA also increased but only after CDP-DAG synthase activity fell below the wild type level. PI synthase activity followed the decrease of the CDP-DAG synthase activity, but there was no parallel change in the level of PIS1 mRNA. A G$\sp{305}$/A$\sp{305}$ point mutation within the CDS1 gene which causes the cdg1 phenotype was identified. A human cDNA clone encoding CDP-DAG synthase activity was characterized by complementation of the yeast cds1 null mutant. ^

The generation of neutrophils in the bone marrow is controlled by autophagy.

Autophagy has been demonstrated to have an essential function in several cellular hematopoietic differentiation processes, for example, the differentiation of reticulocytes. To investigate the role of autophagy in neutrophil granulopoiesis, we studied neutrophils lacking autophagy-related (Atg) 5, a gene encoding a protein essential for autophagosome formation. Using Cre-recombinase mediated gene deletion, Atg5-deficient neutrophils showed no evidence of abnormalities in morphology, granule protein content, apoptosis regulation, migration, or effector functions. In such mice, however, we observed an increased proliferation rate in the neutrophil precursor cells of the bone marrow as well as an accelerated process of neutrophil differentiation, resulting in an accumulation of mature neutrophils in the bone marrow, blood, spleen, and lymph nodes. To directly study the role of autophagy in neutrophils, we employed an in vitro model of differentiating neutrophils that allowed modulating the levels of ATG5 expression, or, alternatively, intervening pharmacologically with autophagy-regulating drugs. We could show that autophagic activity correlated inversely with the rate of neutrophil differentiation. Moreover, pharmacological inhibition of p38 MAPK or mTORC1 induced autophagy in neutrophilic precursor cells and blocked their differentiation, suggesting that autophagy is negatively controlled by the p38 MAPK-mTORC1 signaling pathway. On the other hand, we obtained no evidence for an involvement of the PI3K-AKT or ERK1/2 signaling pathways in the regulation of neutrophil differentiation. Taken together, these findings show that, in contrast to erythropoiesis, autophagy is not essential for neutrophil granulopoiesis, having instead a negative impact on the generation of neutrophils. Thus, autophagy and differentiation exhibit a reciprocal regulation by the p38-mTORC1 axis.

Phenotype and genotype in 103 patients with tricho-rhino-phalangeal syndrome.

Tricho-rhino-phalangeal syndrome (TRPS) is characterized by craniofacial and skeletal abnormalities, and subdivided in TRPS I, caused by mutations in TRPS1, and TRPS II, caused by a contiguous gene deletion affecting (amongst others) TRPS1 and EXT1. We performed a collaborative international study to delineate phenotype, natural history, variability, and genotype-phenotype correlations in more detail. We gathered information on 103 cytogenetically or molecularly confirmed affected individuals. TRPS I was present in 85 individuals (22 missense mutations, 62 other mutations), TRPS II in 14, and in 5 it remained uncertain whether TRPS1 was partially or completely deleted. Main features defining the facial phenotype include fine and sparse hair, thick and broad eyebrows, especially the medial portion, a broad nasal ridge and tip, underdeveloped nasal alae, and a broad columella. The facial manifestations in patients with TRPS I and TRPS II do not show a significant difference. In the limbs the main findings are short hands and feet, hypermobility, and a tendency for isolated metacarpals and metatarsals to be shortened. Nails of fingers and toes are typically thin and dystrophic. The radiological hallmark are the cone-shaped epiphyses and in TRPS II multiple exostoses. Osteopenia is common in both, as is reduced linear growth, both prenatally and postnatally. Variability for all findings, also within a single family, can be marked. Morbidity mostly concerns joint problems, manifesting in increased or decreased mobility, pain and in a minority an increased fracture rate. The hips can be markedly affected at a (very) young age. Intellectual disability is uncommon in TRPS I and, if present, usually mild. In TRPS II intellectual disability is present in most but not all, and again typically mild to moderate in severity. Missense mutations are located exclusively in exon 6 and 7 of TRPS1. Other mutations are located anywhere in exons 4-7. Whole gene deletions are common but have variable breakpoints. Most of the phenotype in patients with TRPS II is explained by the deletion of TRPS1 and EXT1, but haploinsufficiency of RAD21 is also likely to contribute. Genotype-phenotype studies showed that mutations located in exon 6 may have somewhat more pronounced facial characteristics and more marked shortening of hands and feet compared to mutations located elsewhere in TRPS1, but numbers are too small to allow firm conclusions.

Molecular Mechanisms of Vascular Disease in Patients with Rare Variants in MYH11

Thoracic aortic aneurysms and dissections (TAAD) are the primary disease affecting the thoracic ascending aorta, with an incidence rate of 10.4/100,000. Although about 20% of patients carry a mutation in a single gene that causes their disease, the remaining 80% of patients may also have genetic factors that increase their risk for developing TAAD. Many of the genes that predispose to TAAD encode proteins involved in smooth muscle cell (SMC) contraction and the disease-causing mutations are predicted to disrupt contractile function. SMCs are the predominant cell type in the ascending aortic wall. Mutations in MYH11, encoding the smooth muscle specific myosin heavy chain, are a rare cause of inherited TAAD. However, rare but recurrent non-synonymous variants in MYH11 are present in the general population but do not cause inherited TAAD. The goal of this study was to assess the potential role of these rare variants in vascular diseases. Two distinct variants were selected: the most commonly seen rare variant, MYH11 R247C, and a duplication of the chromosomal region spanning the MYH11 locus at 16p13.1. Genetic analyses indicated that both of these variants were significantly enriched in patients with TAAD compared with controls. A knock-in mouse model of the Myh11 R247C rare variant was generated, and these mice survive and reproduce normally. They have no structural abnormalities of the aorta or signs of aortic disease, but do have decreased aortic contractility. Myh11R247C/R247C mice also have increased proliferative response to vascular injury in vivo and increased proliferation of SMCs in vitro. Myh11R247C/R247C SMCs have decreased contractile gene and protein expression and are dedifferentiated. In fibroblasts, myosin force generation is required for maturation of focal adhesions, and enhancers of RhoA activity replace enhancers of Rac1 activity as maturation occurs. Consistent with these previous findings, focal adhesions are smaller in Myh11R247C/R247C SMCs, and there is decreased RhoA activation. A RhoA activator (CN03) rescues the dedifferentiated phenotype of Myh11R247C/R247C SMCs. Myh11R247C/R247C mice were bred with an existing murine model of aneurysm formation, the Acta2-/- mouse. Over time, mice carrying the R247C allele in conjunction with heterozygous or homozygous loss of Acta2 had significantly increased aortic diameter, and a more rapid accumulation of pathologic markers. These results suggest that the Myh11 R247C rare variant acts as a modifier gene increasing the risk for and severity of TAAD in mice. In patients with 16p13.1 duplications, aortic MYH11 expression is increased, but there is no corresponding increase in smooth muscle myosin heavy chain protein. Using SMCs that overexpress Myh11, we identified alterations in SMC phenotype leading to excessive protein turnover. All contractile proteins, not just myosin, are affected, and the proteins are turned over by autophagic degradation. Surprisingly, these cells are also more contractile compared with wild-type SMCs. The results described in this dissertation firmly establish that rare variants in MYH11 significantly affect the phenotype of SMCs. Further, the data suggests that these rare variants do increase the risk of TAAD via pathways involving altered SMC phenotype and contraction. Therefore, this study validates that these rare genetic variants alter vascular SMCs and provides model systems to explore the contribution of rare variants to disease.

Expression of adenoviral E3 transgenes in β cells prevents autoimmune diabetes

The adenovirus (Ad) genome contains immunoregulatory and cytokine inhibitory genes that are presumed to function in facilitating acute infection or in establishing persistence in vivo. Some of these genes are clustered in early region 3 (E3), which contains a 19-kDa glycoprotein (gp19) that inhibits the transport of selected class I major histocompatibility complex (MHC) molecules out of the endoplasmic reticulum. In addition, the E3 region contains three protein inhibitors of the cytolytic function of tumor necrosis factor α (TNF-α). Because type I autoimmune diabetes destroys islets by mechanisms that involve class I MHC and TNF-α, we investigated whether the entire cassette of Ad E3 genes might prevent the onset of diabetes in a well studied lymphocytic choriomeningitis viral (LCMV) murine model of virus-induced autoimmune diabetes. In this model, a LCMV polypeptide (either glycoprotein or nucleoprotein) expressed as a transgene in the islets is a target for autoimmune destruction of β cells after LCMV infection. In this scenario the LCMV-induced immune response is directed not only against the virus but also against the LCMV transgenes expressed in the β cells. Our experiments demonstrated a very efficient prevention of this LCMV-triggered diabetes by the Ad E3 genes. This resulted from the inhibition of target cell recognition by a fully competent and LCMV-primed immune system. Unlike the results from the β-2 microglobulin gene deletion experiments, our approach shows that selective regulation at the level of the target cell is sufficient to prevent autoimmune diabetes without disrupting the function of the systemic immune response. Although the Ad genes in these experiments were provided as transgenes, recent experiments may permit the introduction of such genes through the use of viral vectors. Although the decrease in class I MHC in islets by Ad genes was demonstrated in these in vivo studies, the relative importance of this process and the control of TNF-α cytolysis must await further genetic dissection of the introduced Ad genes.

RNA Polymerase Subunits Encoded by the Plastid rpo Genes Are Not Shared with the Nucleus-Encoded Plastid Enzyme1

Plastid genes in photosynthetic higher plants are transcribed by at least two RNA polymerases. The plastid rpoA, rpoB, rpoC1, and rpoC2 genes encode subunits of the plastid-encoded plastid RNA polymerase (PEP), an Escherichia coli-like core enzyme. The second enzyme is referred to as the nucleus-encoded plastid RNA polymerase (NEP), since its subunits are assumed to be encoded in the nucleus. Promoters for NEP have been previously characterized in tobacco plants lacking PEP due to targeted deletion of rpoB (encoding the β-subunit) from the plastid genome. To determine if NEP and PEP share any essential subunits, the rpoA, rpoC1, and rpoC2 genes encoding the PEP α-, β′-, and β"-subunits were removed by targeted gene deletion from the plastid genome. We report here that deletion of each of these genes yielded photosynthetically defective plants that lack PEP activity while maintaining transcription specificity from NEP promoters. Therefore, rpoA, rpoB, rpoC1, and rpoC2 encode PEP subunits that are not essential components of the NEP transcription machinery. Furthermore, our data indicate that no functional copy of rpoA, rpoB, rpoC1, or rpoC2 that could complement the deleted plastid rpo genes exists outside the plastids.

A TRP homolog in Saccharomyces cerevisiae forms an intracellular Ca2+-permeable channel in the yeast vacuolar membrane

The molecular identification of ion channels in internal membranes has made scant progress compared with the study of plasma membrane ion channels. We investigated a prominent voltage-dependent, cation-selective, and calcium-activated vacuolar ion conductance of 320 pS (yeast vacuolar conductance, YVC1) in Saccharomyces cerevisiae. Here we report on a gene, the deduced product of which possesses significant homology to the ion channel of the transient receptor potential (TRP) family. By using a combination of gene deletion and re-expression with direct patch clamping of the yeast vacuolar membrane, we show that this yeast TRP-like gene is necessary for the YVC1 conductance. In physiological conditions, tens of micromolar cytoplasmic Ca2+ activates the YVC1 current carried by cations including Ca2+ across the vacuolar membrane. Immunodetection of a tagged YVC1 gene product indicates that YVC1 is primarily localized in the vacuole and not other intracellular membranes. Thus we have identified the YVC1 vacuolar/lysosomal cation-channel gene. This report has implications for the function of TRP channels in other organisms and the possible molecular identification of vacuolar/lysosomal ion channels in other eukaryotes.

The extent of heterocellular communication mediated by gap junctions is predictive of bystander tumor cytotoxicity in vitro.

Herpes simplex virus thymidine kinase (HSV-tk)/ganciclovir (GCV) viral-directed enzyme prodrug gene therapy causes potent, tumor-selective cytotoxicity in animal models in which HSV-tk gene transduction is limited to a minority of tumor cells. The passage of toxic molecules from HSV-tk+ cells to neighboring HSV-tk- cells during GCV therapy is one mechanism that may account for this "bystander" cytotoxicity. To investigate whether gap junction-mediated intercellular coupling could mediate this bystander effect, we used a flow cytometry assay to quantitate the extent of heterocellular coupling between HSV-tk+ murine fibroblasts and both rodent and human tumor cell lines. Bystander tumor cytotoxicity during GCV treatment in a coculture assay was highly correlated (P < 0.001) with the extent of gap junction-mediated coupling. These findings show that gap junction-mediated intercellular coupling contributes to the in vitro bystander effect during HSV-tk/GCV therapy and that retroviral transduction of tumor cells is not required for bystander cytotoxicity.

Nitrogen regulation of protein–protein interactions and transcript levels of GlnK PII regulator and AmtB ammonium transporter homologs in Archaea

Gene homologs of GlnK PII regulators and AmtB-type ammonium transporters are often paired on prokaryotic genomes, suggesting these proteins share an ancient functional relationship. Here, we demonstrate for the first time in Archaea that GlnK associates with AmtB in membrane fractions after ammonium shock, thus, providing a further insight into GlnK-AmtB as an ancient nitrogen sensor pair. For this work, Haloferax mediterranei was advanced for study through the generation of a pyrE2-based counterselection system that was used for targeted gene deletion and expression of Flag-tagged proteins from their native promoters. AmtB1-Flag was detected in membrane fractions of cells grown on nitrate and was found to coimmunoprecipitate with GlnK after ammonium shock. Thus, in analogy to bacteria, the archaeal GlnK PII may block the AmtB1 ammonium transporter under nitrogen-rich conditions. In addition to this regulated protein–protein interaction, the archaeal amtB-glnK gene pairs were found to be highly regulated by nitrogen availability with transcript levels high under conditions of nitrogen limitation and low during nitrogen excess. While transcript levels of glnK-amtB are similarly regulated by nitrogen availability in bacteria, transcriptional regulators of the bacterial glnK promoter including activation by the two-component signal transduction proteins NtrC (GlnG, NRI) and NtrB (GlnL, NRII) and sigma factor σN (σ54) are not conserved in archaea suggesting a novel mechanism of transcriptional control.

Rôles de la protéine tyrosine phosphatase DEP-1 dans l'angiogenèse, la perméabilité vasculaire et la progression tumorale

L’angiogenèse et l’augmentation de la perméabilité vasculaire sont des éléments clés pour la croissance et la progression tumorale. Par conséquent, de nombreux efforts sont déployés à comprendre les mécanismes moléculaires impliqués dans la formation et le remodelage des vaisseaux sanguins de manière à identifier de nouvelles cibles thérapeutiques potentielles. De cette optique, les travaux de cette thèse se sont concentrés sur la protéine tyrosine phosphatase DEP-1, initialement identifiée comme un régulateur négatif de la prolifération et de la phosphorylation du VEGFR2 lorsque fortement exprimée dans les cellules endothéliales. Toutefois, en utilisant une approche d’ARNi, il a été démontré que via sa capacité à déphosphoryler la tyrosine inhibitrice de Src (Y529), DEP-1 était également un régulateur positif de l’activation de Src dans les cellules endothéliales stimulées au VEGF. Puisque Src joue un rôle central dans la promotion de l’angiogenèse et la perméabilité vasculaire, nous avons en plus démontré que DEP-1 était un promoteur de ces fonctions in vitro et que la tyrosine phosphorylation de sa queue C-terminale, permettant l’interaction et l’activation de Src, était requise. Les travaux de recherche présentés dans cette thèse démontrent dans un premier temps à partir d’une souris Dep1 KO, dont le développement ne présente aucun phénotype apparent, que la perte de l’expression de DEP-1 se traduit en une inhibition de l’activation de Src et de l’un de ses substrats, la VE-Cadherine, en réponse au VEGF chez la souris adulte. Nos résultats démontrent donc, pour la première fois, le rôle primordial de DEP-1 dans l’induction de la perméabilité vasculaire et de la formation de capillaires in vivo. Conséquemment, la croissance tumorale et la formation de métastases aux poumons sont réduites due à une inhibition de leur vascularisation ce qui se traduit par une diminution de la prolifération et une augmentation de l’apoptose des cellules cancéreuses. De façon intéressante, l’expression élevée de DEP-1 dans les vaisseaux sanguins tumoraux de patientes atteintes du cancer du sein corrèle avec une vascularisation accrue de la tumeur. En plus du rôle de DEP-1 dans la réponse angiogénqiue à l’âge adulte, nos travaux ont également démontré le rôle important de DEP-1 lors de la vascularisation de la rétine, un modèle in vivo d’angiogenèse développementale. Dans ce contexte, DEP-1 inhibe la prolifération des cellules endothéliales et limite leur bourgeonnement et la complexification du réseau vasculaire rétinien en permettant l’expression adéquate du Dll4, un régulateur crucial de l’organisation de la vascularisation développementale. Cette expression du Dll4 découlerait de la stabilisation de la β-caténine par l’inactivation de la GSK3β, un régulateur important de la dégradation de la β-caténine, en réponse au VEGF selon la voie de signalisation VEGFR2-Src-PI3K-Akt-GSK3β. Ainsi, ces travaux identifient DEP-1 comme un régulateur important de l’organisation vasculaire rétinienne. Les rôles positifs de DEP-1 dans les cellules endothéliales découlent principalement de sa capacité à lier et activer la kinase Src. En plus de contribuer à la réponse angiogénique, Src est également un oncogène bien caractérisé notamment pour sa contribution au programme invasif des cellules cancéreuses mammaires. Les travaux de cette thèse illustrent que DEP-1 est préférentiellement exprimée dans les cellules cancéreuses mammaires invasives et qu’il régule l’activation de Src, de voies de signalisation invasives et, par le fait même, de l’invasivité de ces cellules in vitro et in vivo. De façon intéressante, ces observations corrèlent avec des données cliniques où l’expression modérée de DEP-1 est associée à un mauvais pronostic de survie et de rechute. Ces résultats démontrent donc, pour la première fois, le rôle positif de DEP-1 dans l’activation de Src au niveau des cellules endothéliales et des cellules cancéreuses mammaires ce qui permet la régulation du bourgeonnement endothélial, de la perméabilité vasculaire, de l’angiogenèse normale et pathologique en plus de l’invasion tumorale.

Cardiotrophin-like cytokine/cytokine-like factor 1 is an essential trophic factor for lumbar and facial motoneurons in vivo

The ciliary neurotrophic factor alpha-receptor(CNTFRalpha) is required for motoneuron survival during development, but the relevant ligand(s) has not been determined. One candidate is the heterodimer formed by cardiotrophin-like cytokine (CLC) and cytokine-like factor 1 (CLF). CLC/CLF binds to CNTFRalpha and enhances the survival of developing motoneurons in vitro; whether this novel trophic factor plays a role in neural development in vivo has not been tested. We examined motor and sensory neurons in embryonic chicks treated with CLC and in mice with a targeted deletion of the clf gene. Treatment with CLC increased the number of lumbar spinal cord motoneurons that survived the cell death period in chicks. However, this effect was regionally specific, because brachial and thoracic motoneurons were unaffected. Similarly, newborn clf -/- mice exhibited a significant reduction in lumbar motoneurons, with no change in the brachial or thoracic cord. Clf deletion also affected brainstem motor nuclei in a regionally specific manner; the number of motoneurons in the facial but not hypoglossal nucleus was significantly reduced. Sensory neurons of the dorsal root ganglia were not affected by either CLC treatment or clf gene deletion. Finally, mRNA for both clc and clf was found in skeletal muscle fibers of embryonic mice during the motoneuron cell death period. These findings support the view that CLC/CLF is a target-derived factor required for the survival of specific pools of motoneurons. The in vivo actions of CLC and CLF can account for many of the effects of CNTFRalpha on developing motoneurons.