Aromatase (Cyp19) expression is up-regulated by targeted disruption of Dax1

DAX-1 [dosage-sensitive sex reversal, adrenal hypoplasia congenita (AHC) critical region on the X chromosome, gene 1] is an orphan nuclear receptor that represses transcription by steroidogenic factor-1 (SF-1), a factor that regulates expression of multiple steroidogenic enzymes and other genes involved in reproduction. Mutations in the human DAX1 gene (also known as AHC) cause the X-linked syndrome AHC, a disorder that is associated with hypogonadotropic hypogonadism also. Characterization of Dax1-deficient male mice revealed primary testicular defects that included Leydig cell hyperplasia (LCH) and progressive degeneration of the germinal epithelium, leading to infertility. In this study, we investigated the effect of Dax1 disruption on the expression profile of various steroidogenic enzyme genes in Leydig cells isolated from Dax1-deficient male mice. Expression of the aromatase (Cyp19) gene, which encodes the enzyme that converts testosterone to estradiol, was increased significantly in the Leydig cells isolated from mutant mice, whereas the expression of other proteins (e.g., StAR and Cyp11a) was not altered. In in vitro transfection studies, DAX-1 repressed the SF-1-mediated transactivation of the Cyp19 promoter but did not inhibit the StAR or Cyp11a promoters. Elevated Cyp19 expression was accompanied by increased intratesticular levels of estradiol. Administration of tamoxifen, a selective estrogen-receptor modulator, restored fertility to the Dax1-deficient male mice and partially corrected LCH, suggesting that estrogen excess contributes to LCH and infertility. Based on these in vivo and in vitro analyses, aromatase seems to be a physiologic target of Dax-1 in Leydig cells, and increased Cyp19 expression may account, in part, for the infertility and LCH in Dax1-deficient mice.

BDNF but not NT-4 is required for normal flexion reflex plasticity and function

Neurotrophins can directly modulate the function of diverse types of central nervous system synapses. Brain-derived neurotrophic factor (BDNF) might be released by nociceptors onto spinal neurons and mediate central sensitization associated with chronic pain. We have studied the role of BDNF and neurotrophin-4 (NT-4), both ligands of the trkB tyrosine kinase receptor, in synaptic transmission and reflex plasticity in the mouse spinal cord. We used an in vitro spinal cord preparation to measure monosynaptic and polysynaptic reflexes evoked by primary afferents in BDNF- and NT-4-deficient mice. In situ hybridization studies show that both these neurotrophins are synthesized by sensory neurons, and NT-4, but not BDNF, also is expressed by spinal neurons. BDNF null mutants display selective deficits in the ventral root potential (VRP) evoked by stimulating nociceptive primary afferents whereas the non-nociceptive portion of the VRP remained unaltered. In addition, activity-dependent plasticity of the VRP evoked by repetitive (1 Hz) stimulation of nociceptive primary afferents (termed wind-up) was substantially reduced in BDNF-deficient mice. This plasticity also was reduced in a reversible manner by the protein kinase inhibitor K252a. Although the trkB ligand NT-4 is normally present, reflex properties in NT-4 null mutant mice were normal. Pharmacological studies also indicated that spinal N-methyl-d-aspartate receptor function was unaltered in BDNF-deficient mice. Using immunocytochemistry for markers of nociceptive neurons we found no evidence that their number or connectivity was substantially altered in BDNF-deficient mice. Our data therefore are consistent with a direct role for presynaptic BDNF release from sensory neurons in the modulation of pain-related neurotransmission.

Altered potassium balance and aldosterone secretion in a mouse model of human congenital long QT syndrome

The voltage-dependent K+ channel responsible for the slowly activating delayed K+ current IKs is composed of pore-forming KCNQ1 and regulatory KCNE1 subunits, which are mutated in familial forms of cardiac long QT syndrome. Because KCNQ1 and KCNE1 genes also are expressed in epithelial tissues, such as the kidneys and the intestine, we have investigated the adaptation of KCNE1-deficient mice to different K+ and Na+ intakes. On a normal K+ diet, homozygous kcne1−/− mice exhibit signs of chronic volume depletion associated with fecal Na+ and K+ wasting and have lower plasma K+ concentration and higher levels of aldosterone than wild-type mice. Although plasma aldosterone can be suppressed by low K+ diets or stimulated by low Na+ diets, a high K+ diet provokes a tremendous increase of plasma aldosterone levels in kcne1−/− mice as compared with wild-type mice (7.1-fold vs. 1.8-fold) despite lower plasma K+ in kcne1−/− mice. This exacerbated aldosterone production in kcne1−/− mice is accompanied by an abnormally high plasma renin concentration, which could partly explain the hyperaldosteronism. In addition, we found that KCNE1 and KCNQ1 mRNAs are expressed in the zona glomerulosa of adrenal glands where IKs may directly participate in the control of aldosterone production by plasma K+. These results, which show that KCNE1 and IKs are involved in K+ homeostasis, might have important implications for patients with IKs-related long QT syndrome, because hypokalemia is a well known risk factor for the occurrence of torsades de pointes ventricular arrhythmia.

Targeted expression of heme oxygenase-1 prevents the pulmonary inflammatory and vascular responses to hypoxia

Chronic hypoxia causes pulmonary hypertension with smooth muscle cell proliferation and matrix deposition in the wall of the pulmonary arterioles. We demonstrate here that hypoxia also induces a pronounced inflammation in the lung before the structural changes of the vessel wall. The proinflammatory action of hypoxia is mediated by the induction of distinct cytokines and chemokines and is independent of tumor necrosis factor-α signaling. We have previously proposed a crucial role for heme oxygenase-1 (HO-1) in protecting cardiomyocytes from hypoxic stress, and potent anti-inflammatory properties of HO-1 have been reported in models of tissue injury. We thus established transgenic mice that constitutively express HO-1 in the lung and exposed them to chronic hypoxia. HO-1 transgenic mice were protected from the development of both pulmonary inflammation as well as hypertension and vessel wall hypertrophy induced by hypoxia. Significantly, the hypoxic induction of proinflammatory cytokines and chemokines was suppressed in HO-1 transgenic mice. Our findings suggest an important protective function of enzymatic products of HO-1 activity as inhibitors of hypoxia-induced vasoconstrictive and proinflammatory pathways.

Virus-mediated gene transfer into hippocampal CA1 region restores long-term potentiation in brain-derived neurotrophic factor mutant mice.

Long-term potentiation (LTP) has been shown to be impaired in mice deficient in the brain-derived neurotrophic factor (BDNF) gene, as well as in a number of other knockout animals. Despite its power the gene-targeting approach is always fraught with the danger of looking at the cumulative direct and indirect effects of the absence of a particular gene rather than its immediate function. The re-expression of a specific gene at a selective time point and at a specific site in gene-defective mutants presents a potent procedure to overcome this limitation and to evaluate the causal relationship between the absence of a particular gene and the impairment of a function in gene-defective animals. Here we demonstrate that the re-expression of the BDNF gene in the CA1 region almost completely restores the severely impaired LTP in hippocampal slices of BDNF-deficient mice. The results therefore provide strong evidence for the direct involvement of BDNF in the process of LTP.

Leukocyte lipid body formation and eicosanoid generation: cyclooxygenase-independent inhibition by aspirin.

Lipid bodies, cytoplasmic inclusions that develop in cells associated with inflammation, are inducible structures that might participate in generating inflammatory eicosanoids. Cis-unsaturated fatty acids (arachidonic and oleic acids) rapidly induced lipid body formation in leukocytes, and this lipid body induction was inhibited by aspirin and nonsteroidal antiinflammatory drugs (NSAIDs). Several findings indicates that the inhibitory effect of aspirin and NSAIDs on lipid body formation was independent of cyclooxygenase (COX) inhibition. First, the non-COX inhibitor, sodium salicylate, was as potent as aspirin in inhibiting lipid body formation elicited by cis-fatty acids. Second, cis-fatty acid-induced lipid body formation was not impaired in macrophages from COX-1 or COX-2 genetically deficient mice. Finally, NSAIDs inhibited arachidonic acid-induced lipid body formation likewise in macrophages from wild-type and COX-1- and COX-2-deficient mice. An enhanced capacity to generate eicosanoids developed after 1 hr concordantly with cis-fatty acid-induced lipid body formation. Arachidonic and oleic acid-induced lipid body numbers correlated with the enhanced levels of leukotrienes B4 and C4 and prostaglandin E2 produced after submaximal calcium ionophore stimulation. Aspirin and NSAIDs inhibited both induced lipid body formation and the enhanced capacity for forming leukotrienes as well as prostaglandins. Our studies indicate that lipid body formation is an inducible early response in leukocytes that correlates with enhanced eicosanoid synthesis. Aspirin and NSAIDs, independent of COX inhibition, inhibit cis-fatty acid-induced lipid body formation in leukocytes and in concert inhibit the enhanced synthesis of leukotrienes and prostaglandins.

Altered surfactant function and structure in SP-A gene targeted mice.

The surfactant protein A (SP-A) gene was disrupted by homologous recombination in embryonic stem cells that were used to generate homozygous SP-A-deficient mice. SP-A mRNA and protein were not detectable in the lungs of SP-A(-/-) mice, and perinatal survival of SP-A(-/-) mice was not altered compared with wild-type mice. Lung morphology, surfactant proteins B-D, lung tissue, alveolar phospholipid pool sizes and composition, and lung compliance in SP-A(-/-) mice were unaltered. At the highest concentration tested, surfactant from SP-A(-/-) mice produced the same surface tension as (+/+) mice. At lower concentrations, minimum surface tensions were higher for SP-A(-/-) mice. At the ultrastructural level, type II cell morphology was the same in SP-A(+/+) and (-/-) mice. While alveolar phospholipid pool sizes were unperturbed, tubular myelin figures were decreased in the lungs of SP-A(-/-) mice. A null mutation of the murine SP-A gene interferes with the formation of tubular myelin without detectably altering postnatal survival or pulmonary function.

Skin wounds and severed nerves heal normally in mice lacking tenascin-C.

A large number of functions have been demonstrated for tenascin-C by antibody perturbation assays and in vitro cell culture experiments. However, these results contrast sharply with the lack of any apparent phenotype in mice with a genetic deletion of tenascin-C. A possible explanation for the lack of phenotype would be expression of some altered but functional tenascin-C in the mutant. We report the generation of an independent tenascin-C null mouse and conclude that the original tenascin-C knockout, which is genetically very similar to ours, is also a true null. As found previously, the absence of tenascin-C has no influence on development, adulthood, life span, and fecundity. We have studied in detail two models of wound healing. After axotomy, the regeneration of the sciatic nerve is not altered without tenascin-C. During healing of cutaneous wounds, deposition of collagen I, fibulin-2, and nidogen is identical in mutant and wild-type mice. In contrast. fibronectin appears diminished in wounds of tenascin-C-deficient mice. However, the lack of tenascin-C together with the reduced amount of fibronectin has no influence on the quality of the healing process.

Long-term culture of lymphohematopoietic stem cells.

Pluripotent hematopoietic stem cells (PHSCs) show self-renewal and give rise to all blood cell types. The extremely low number of these cells in primary hematopoietic organs and the lack of culture systems that support proliferation of undifferentiated PHSCs have precluded the study of both the biology of these cells and their clinical application. We describe here cell lines and clones derived from PHSCs that were established from hematopoietic cells from the fetal liver or bone marrow of normal and p53-deficient mice with a combination of four growth factors. Most cell lines were Sca-1+, c-Kit+, PgP-1+, HSA+, and Lin- (B-220-, Joro 75-, 8C5-, F4/80-, CD4-, CD8-, CD3-, IgM-, and TER 119-negative) and expressed three new surface markers: Joro 177, Joro 184, and Joro 96. They did not synthesize RNA transcripts for several genes expressed at early stages of lymphocyte and myeloid/erythroid cell development. The clones were able to generate lymphoid, myeloid, and erythroid hematopoietic cells and to reconstitute the hematopoietic system of irradiated mice for a long time. The availability of lymphohematopoietic stem cell lines should facilitate the analysis of the molecular mechanisms that control self-renewal and differentiation and the development of efficient protocols for somatic gene therapy.

Identification, sequence, and expression of caveolin-2 defines a caveolin gene family.

Caveolin, a 21- to 24-kDa integral membrane protein, is a principal component of caveolae membranes. Caveolin interacts directly with heterotrimeric guanine nucleotide binding proteins (G proteins) and can functionally regulate their activity. Here, an approximately 20-kDa caveolin-related protein, caveolin-2, was identified through microsequencing of adipocyte-derived caveolin-enriched membranes; caveolin was retermed caveolin-1. Caveolins 1 and 2 are similar in most respects. mRNAs for both caveolin-1 and caveolin-2 are most abundantly expressed in white adipose tissue and are induced during adipocyte differentiation. Caveolin-2 colocalizes with caveolin-1, indicating that caveolin-2 also localizes to caveolae. However, caveolin-1 and caveolin-2 differ in their functional interactions with heterotrimeric G proteins, possibly explaining why caveolin-1 and -2 are coexpressed within a single cell.

Early-onset multifocal inflammation in the transforming growth factor beta 1-null mouse is lymphocyte mediated.

Transforming growth factor beta 1 (TGF beta 1)-null mice die fro complications due to an early-onset multifocal inflammatory disorder. We show here that cardiac cells are hyperproliferative and that intercellular adhesion molecule 1 (ICAM-1) is elevated. To determine which phenotypes are primarily caused by a deficiency in TGF beta 1 from those that are secondary to inflammation, we applied immunosuppressive therapy and genetic combination with the severe combined immunodeficiency (SCID) mutation to inhibit the inflammatory response. Treatment with antibodies to the leukocyte function-associated antigen 1 doubled longevity, reduced inflammation, and delayed heart cell proliferation. TGF beta 1-null SCID mice displayed no inflammation or cardiac cell proliferation, survived to adulthood, and exhibited normal major histocompatibility complex II (MHC II) and ICAM-1 levels. TGF beta 1-null pups born to a TGF beta 1-null SCID mother presented no gross congenital heart defects, indicating that TGF beta 1 alone does not play an essential role in heart development. These results indicate that lymphocytes are essential for the inflammatory response, cardiac cell proliferation, and elevated MHC II and ICAM-1 expression, revealing a vital role for TGF beta 1 in regulating lymphocyte proliferation and activation, which contribute to the maintenance of self tolerance.

Vitronectin is not essential for normal mammalian development and fertility.

Vitronectin (VN) is an abundant glycoprotein present in plasma and the extracellular matrix of most tissues. Though the precise function of VN in vivo is unknown, it has been implicated as a participant in diverse biological processes, including cell attachment and spreading, complement activation, and regulation of hemostasis. The major site of synthesis appears to be the liver, though VN is also found in the brain at an early stage of mouse organogenesis, suggesting that it may play an important role in mouse development. Genetic deficiency of VN has not been reported in humans or in other higher organisms. To examine the biologic function of VN within the context of the intact animal, we have established a murine model for VN deficiency through targeted disruption of the murine VN gene. Southern blot analysis of DNA obtained from homozygous null mice demonstrates deletion of all VN coding sequences, and immunological analysis confirms the complete absence of VN protein expression in plasma. However, heterozygous mice carrying one normal and one null VN allele and homozygous null mice completely deficient in VN demonstrate normal development, fertility, and survival. Sera obtained from VN-deficient mice are completely deficient in "serum spreading factor" and plasminogen activator inhibitor 1 binding activities. These observations demonstrate that VN is not essential for cell adhesion and migration during normal mouse development and suggest that its role in these processes may partially overlap with other adhesive matrix components.

Equine severe combined immunodeficiency: a defect in V(D)J recombination and DNA-dependent protein kinase activity.

V(D)J rearrangement is the molecular mechanism by which an almost infinite array of specific immune receptors are generated. Defects in this process result in profound immunodeficiency as is the case in the C.B-17 SCID mouse or in RAG-1 (recombination-activating gene 1) or RAG-2 deficient mice. It has recently become clear that the V(D)J recombinase most likely consists of both lymphoid-specific factors and ubiquitously expressed components of the DNA double-strand break repair pathway. The deficit in SCID mice is in a factor that is required for both of these pathways. In this report, we show that the factor defective in the autosomal recessive severe combined immunodeficiency of Arabian foals is required for (i) V(D)J recombination, (ii) resistance to ionizing radiation, and (iii) DNA-dependent protein kinase activity.

Mammary tumor suppression by transforming growth factor beta 1 transgene expression.

In cell culture, type alpha transforming growth factor (TGF-alpha) stimulates epithelial cell growth, whereas TGF-beta 1 overrides this stimulatory effect and is growth inhibitory. Transgenic mice that overexpress TGF-alpha under control of the mouse mammary tumor virus (MMTV) promoter/enhancer exhibit mammary ductal hyperplasia and stochastic development of mammary carcinomas, a process that can be accelerated by administration of the chemical carcinogen 7,12-dimethylbenz[a]anthracene. MMTV-TGF-beta 1 transgenic mice display mammary ductal hypoplasia and do not develop mammary tumors. We report that in crossbreeding experiments involving the production of mice carrying both the MMTV-TGF-beta 1 and MMTV-TGF-alpha transgenes, there is marked suppression of mammary tumor formation and that MMTV-TGF-beta 1 transgenic mice are resistant to 7,12-dimethylbenz[a]anthracene-induced mammary tumor formation. These data demonstrate that overexpression of TGF-beta 1 in vivo can markedly suppress mammary tumor development.

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.