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Angiogenesis contributes in multiple ways to disease progression in tumors and reduces treatment efficiency. Molecular therapies targeting Vegf signaling combined with chemotherapy or other drugs exhibit promising results to improve efficacy of treatment. Dopamine has been recently proposed to be a novel safe anti-angiogenic drug that stabilizes abnormal blood vessels and increases therapeutic efficacy. Here, we aimed to identify a treatment to normalize tumoral vessels and restore normal blood perfusion in tumor tissue with a Vegf receptor inhibitor and/or a ligand of dopamine G protein-coupled receptor D2 (D2R). Dopamine, via its action on D2R, is an endogenous effector of the pituitary gland, and we took advantage of this system to address this question. We have used a previously described Hmga2/T mouse model developing haemorrhagic prolactin-secreting adenomas. In mutant mice, blood vessels are profoundly altered in tumors, and an aberrant arterial vascularization develops leading to the loss of dopamine supply. D2R agonist treatment blocks tumor growth, induces regression of the aberrant blood supply and normalizes blood vessels. A chronic treatment is able to restore the altered balance between pro- and anti-angiogenic factors. Remarkably, an acute treatment induces an upregulation of the stabilizing factor Angiopoietin 1. An anti-Vegf therapy is also effective to restrain tumor growth and improves vascular remodeling. Importantly, only the combination treatment suppresses intratumoral hemorrhage and restores blood vessel perfusion, suggesting that it might represent an attractive therapy targeting tumor vasculature. Similar strategies targeting other ligands of GPCRs involved in angiogenesis may identify novel therapeutic opportunities for cancer.

There are well-recognized sex differences in many pituitary endocrine axes, usually thought to be generated by gonadal steroid imprinting of the neuroendocrine hypothalamus. However, the recognition that growth hormone (GH) cells are arranged in functionally organized networks raises the possibility that the responses of the network are different in males and females. We studied this by directly monitoring the calcium responses to an identical GH-releasing hormone (GHRH) stimulus in populations of individual GH cells in slices taken from male and female murine GH-eGFP pituitary glands. We found that the GH cell network responses are sexually dimorphic, with a higher proportion of responding cells in males than in females, correlated with greater GH release from male slices. Repetitive waves of calcium spiking activity were triggered by GHRH in some males, but were never observed in females. This was not due to a permanent difference in the network architecture between male and female mice; rather, the sex difference in the proportions of GH cells responding to GHRH were switched by postpubertal gonadectomy and reversed with hormone replacements, suggesting that the network responses are dynamically regulated in adulthood by gonadal steroids. Thus, the pituitary gland contributes to the sexually dimorphic patterns of GH secretion that play an important role in differences in growth and metabolism between the sexes.

Our view of anterior pituitary organization has been altered with the recognition that folliculo-stellate (FS) and somatotroph cell populations form large-scale three-dimensional homotypic networks. This morphological cellular organization may optimize communication within the pituitary gland promoting coordinated pulsatile secretion adapted to physiological needs. The aim of this study was to identify the molecules involved in the formation and potential functional organization and/or signaling within these cell–cell networks. Here, we have focused on one class of cell adhesion molecules, the cadherins, since β-catenin has been detected in the GH cell network. We have characterized, by qPCR and immunohistochemistry, their cellular expression and distribution. We have also examined whether their expression could be modulated during pituitary tissue remodeling. The mouse anterior pituitary has a restricted and cell-type specific repertoire of cadherin expression: cadherin-11 is exclusively expressed in TSH cells; N-cadherin displays a ubiquitous expression pattern but with different levels of expression between endocrine cell types; E-cadherin is restricted to homotypic contacts between FS cells; while cadherin-18 is expressed both in somatotrophs and FS cells. Thus, each cell type presents a defined combinatorial expression of different subsets of cadherins. This cell-type specific cadherin expression profile emerges early during development and undergoes major changes during postnatal development. These results suggest the existence within the anterior pituitary of cell–cell contact signaling based on a defined pattern of cadherin expression, which may play a crucial role in cellular recognition during the formation and fate of pituitary cell homotypic networks.

Although growth hormone (GH)- and prolactin (PRL)-secreting pituitary adenomas are considered benign, in many patients, tumour growth and/or invasion constitute a particular challenge. In other tumours, progression relies in part on dysfunction of intercellular adhesion mediated by the large family of cadherins. In the present study, we have explored the contribution of cadherins in GH and PRL adenoma pathogenesis, and evaluated whether this class of adherence molecules was related to tumour invasiveness. We have first established, by quantitative polymerase chain reaction and immunohistochemistry, the expression profile of classical cadherins in the normal human pituitary gland. We show that the cadherin repertoire is restricted and cell-type specific. Somatotrophs and lactotrophs express mainly E-cadherin and cadherin 18, whereas N-cadherin is present in the other endocrine cell types. This repertoire undergoes major differential modification in GH and PRL tumours: E-cadherin is significantly reduced in invasive GH adenomas, and this loss is associated with a cytoplasmic relocalisation of cadherin 18 and catenins. In invasive prolactinomas, E-cadherin distribution is altered and is accompanied by a mislocalisation of cadherin 18, β-catenin and p120 catenin. Strikingly, de novo expression of N-cadherin is present in a subset of adenomas and cells exhibit a mesenchymal phenotype exclusively in invasive tumours. Binary tree analysis, performed by combining the cadherin repertoire with the expression of a subset of known molecular markers, shows that cadherin/catenin complexes play a significant role in discrimination of tumour invasion.

International audience; Traumatic brain injury is a leading cause of hypopituitarism, which compromises patients' recovery, quality of life, and life span. To date, there are no means other than standardized animal studies to provide insights into the mechanisms of posttraumatic hypopituitarism. We have found that GH levels were impaired after inducing a controlled cortical impact (CCI) in mice. Furthermore, GHRH stimulation enhanced GH to lower level in injured than in control or sham mice. Because many characteristics were unchanged in the pituitary glands of CCI mice, we looked for changes at the hypothalamic level. Hypertrophied astrocytes were seen both within the arcuate nucleus and the median eminence, two pivotal structures of the GH axis, spatially remote to the injury site. In the arcuate nucleus, GHRH neurons were unaltered. In the median eminence, injured mice exhibited unexpected alterations. First, the distributions of claudin-1 and zonula occludens-1 between tanycytes were disorganized, suggesting tight junction disruptions. Second, endogenous IgG was increased in the vicinity of the third ventricle, suggesting abnormal barrier properties after CCI. Third, intracerebroventricular injection of a fluorescent-dextran derivative highly stained the hy-pothalamic parenchyma only after CCI, demonstrating an increased permeability of the third ventricle edges. This alteration of the third ventricle might jeopardize the communication between the hypothalamus and the pituitary gland. In conclusion, the phenotype of CCI mice had similarities to the posttraumatic hypopituitarism seen in humans with intact pituitary gland and pituitary stalk. It is the first report of a pathological status in which tanycyte dysfunctions appear as a major acquired syndrome. (Endocrinology 155: 1887-1898, 2014)

International audience; The mammalian circadian system is composed of multiple peripheral clocks that are synchronized by a central pacemaker in the suprachiasmatic nuclei of the hypothalamus. This system keeps track of the external world rhythms through entrainment by various time cues, such as the light-dark cycle and the feeding schedule. Alterations of photoperiod and meal time modulate the phase coupling between central and peripheral oscillators. In this study, we used real-time quantitative PCR to assess circadian clock gene expression in the liver and pituitary gland from mice raised under various photoperiods, or under a temporal restricted feeding protocol. Our results revealed unexpected differences between both organs. Whereas the liver oscillator always tracked meal time, the pituitary circadian clockwork showed an intermediate response, in between entrainment by the light regimen and the feeding-fasting rhythm. The same composite response was also observed in the pituitary gland from adrenalectomized mice under daytime restricted feeding, suggesting that circulating glucocorticoids do not inhibit full entrainment of the pituitary clockwork by meal time. Altogether our results reveal further aspects in the complexity of phase entrainment in the circadian system, and suggest that the pituitary may host oscillators able to integrate multiple time cues.

Growth hormone (GH) exerts its actions via coordinated pulsatile secretion from a GH cell network into the bloodstream. Practically nothing is known about how the network receives its inputs in vivo and releases hormones into pituitary capillaries to shape GH pulses. Here we have developed in vivo approaches to measure local blood flow, oxygen partial pressure, and cell activity at single-cell resolution in mouse pituitary glands in situ. When secretagogue (GHRH) distribution was modeled with fluorescent markers injected into either the bloodstream or the nearby intercapillary space, a restricted distribution gradient evolved within the pituitary parenchyma. Injection of GHRH led to stimulation of both GH cell network activities and GH secretion, which was temporally associated with increases in blood flow rates and oxygen supply by capillaries, as well as oxygen consumption. Moreover, we observed a time-limiting step for hormone output at the perivascular level; macromolecules injected into the extracellular parenchyma moved rapidly to the perivascular space, but were then cleared more slowly in a size-dependent manner into capillary blood. Our findings suggest that GH pulse generation is not simply a GH cell network response, but is shaped by a tissue microenvironment context involving a functional association between the GH cell network activity and fluid microcirculation.

We have generated transgenic mice with somatotroph-specific expression of a modified influenza virus ion channel, (H37A)M2, leading to ablation of GH cells with three levels of severity, dependent on transgene copy number. GH-M2(low) mice grow normally and have normal-size pituitaries but 40-50% reduction in pituitary GH content in adult animals. GH-M2(med) mice have male-specific transient growth retardation and a reduction in pituitary GH content by 75% at 42 d and 97% by 100 d. GH-M2(high) mice are severely dwarfed with undetectable pituitary GH. The GH secretory response of GH-M2(low) and GH-M2(med) mice to GH-releasing peptide-6 and GHRH was markedly attenuated. The content of other pituitary hormones was affected depending on transgene copy number: no effect in GH-M2(low) mice, prolactin and TSH reduced in GH-M2(med) mice, and all hormones reduced in GH-M2(high) mice. The effect on non-GH hormone content was associated with increased macrophage invasion of the pituitary. Somatotroph ablation affected GH cell network organization with limited disruption in GH-M2(low) mice but more severe disruption in GH-M2(med) mice. The remaining somatotrophs formed tight clusters after puberty, which contrasts with GHRH-M2 mice with a secondary reduction in somatotrophs that do not form clusters. A reduction in pituitary beta-catenin staining was correlated with GH-M2 transgene copy number, suggesting M2 expression has an effect on cell-cell communication in somatotrophs and other pituitary cell types. GH-M2 transgenic mice demonstrate that differing degrees of somatotroph ablation lead to correlated secondary effects on cell populations and cellular network organization.

International audience; An increase in circulating catecholamine levels represents one of the mechanisms whereby organisms cope with stress. In the periphery, catecholamines mainly originate from the sympathoadrenal system. As we reported, in addition to the central control through cholinergic innervation, a local gap junction-delineated route between adrenal chromaffin cells contributes to catecholamine exocytosis. Here, we investigated whether this intercellular communication is modified when the hormonal demand is increased as observed during cold stress. Our results show that in cold exposed rats, gap-junctional communication undergoes a functional plasticity, as evidenced by an increased number of dye-coupled cells. Of a physiological interest is that this upregulation of gap-junctional coupling results in the appearance of a robust electrical coupling between chromaffin cells that allows the transmission of action potentials between coupled cells. This enhancement of gap-junctional communication parallels an increase in expression levels of connexin36 (Cx36) and connexin43 (Cx43) proteins. Both transcriptional and posttranslational mechanisms are involved because Cx36 transcripts are increased in stressed rats and the expression of the scaffolding protein zonula occludens-1, known to interact with both Cx36 and Cx43, is also upregulated. Consistent with an upregulated coupling extent in stressed rats, the cytosolic Ca(2+) concentration rises triggered in a single cell by an iontophoretic application of nicotine occur simultaneously in several neighboring cells. These results describe for the first time a functional plasticity of junctional coupling between adult chromaffin cells that should be crucial for adaptation to stress or sensitization to subsequent stressors.

The intestinal homeostasis maintenance involves a permanent crosstalk between the epithelium, the microbiota and the immune system. ISC are responsible for the intestine renewal and regeneration, but they can also cause intestinal tumors. The Sox9 transcription factor is an interesting candidate as a key regulator of intestinal homeostasis because of its specific expression in ISC, Paneth cells and tuft cells. In addition, Sox9 is essential for the differentiation of Paneth cells since the loss of Sox9 in the mouse embryo (model Sox9LoxP / LoxP, Villin-Cre) leads to the absence of Paneth cells. First, we analysed the function of Sox9 in the adult intestinal epithelium using the inducible mouse model: Sox9LoxP / LoxP; Villin-CreERT2. We demonstrated that the deletion of Sox9 in adult Paneth cells leads to structural and functional alterations of Paneth cells, which induce alterations of bacterial diversity (dysbiosis). Dysbiosis is "sensed" by tuft cells that initiate a type 2 immune response. This study revealed the key role of Sox9 in adult Paneth cells to regulate intestinal homeostasis, thus preventing the establishment of a proinflammatory microbiota. Tuft cells, via their sensing function, are able to modulate mucosal immunity in response to a dysbiosis and thus participate in the formation of a vicious circle. In addition, we studied the biology of ISC, by integrating the contribution of Paneth cells properties that participate in the establishment of the niche. We analysed the properties of stem cells in a healthy context or during tumor initiation. Our data indicate that in a healthy context, Sox9 is required for the regulation of ISC fate, namely the balance between ISC self-renewal and differentiation. The mechanisms regulated by Sox9 involve cellular metabolism, a key player in the stem cells fate. Our work shows that an intact niche maintenance is necessary to control ISC fate. The deletion of Sox9 alters mitochondrial integrity and promotes mitochondrial ROS production that could modulate the ISC fate toward a differentiated state. In parallel, we demonstrated that Sox9 deletion concomitant with the acquisition of an initiating event such as the loss of function of the tumor suppressor gene Apc, affects the CSC and their cellular metabolism. The evaluation of the role of the Sox9 transcription factor in the control of metabolic homeostasis will provide a better understanding of the regulatory mechanisms in ISC biology, and eventually new therapeutic strategies targeting CSC might be proposed.; Le maintien de l’homéostasie intestinale met en jeu un dialogue entre l’épithélium, le microbiote et le système immunitaire. Les CSI assurent le renouvellement et la régénération de l’intestin en cas de lésions mais elles peuvent être également à l’origine des tumeurs intestinales. Le facteur de transcription Sox9 est un candidat intéressant comme régulateur clé de l’homéostasie intestinale car il est exprimé dans les CSI, les cellules de Paneth et les cellules tuft. De plus, Sox9 est indispensable à la différenciation des cellules de Paneth puisque l’inactivation de Sox9 chez l’embryon (modèle murin Sox9LoxP/LoxP ; Villin-Cre) conduit à une absence de cellules de Paneth. Au cours de ma thèse, nous avons dans un premier temps déterminé la fonction de Sox9 dans l’épithélium intestinal adulte à l’aide du modèle murin inductible : Sox9LoxP/LoxP ; Villin-CreERT2. Ainsi nous avons démontré que la délétion de Sox9 dans les cellules de Paneth conduit à des altérations structurales et fonctionnelles de ces dernières, qui induisent une altération de la biodiversité d’espèces bactériennes (dysbiose). La dysbiose est « sentie » par les cellules tuft qui initient une réponse immunitaire de type 2. Cette étude a révélé le rôle clé de Sox9 dans les cellules de Paneth adultes pour réguler l’homéostasie intestinale, en prévenant l’établissement d’un microbiote pro-inflammatoire. Les cellules tuft, via leur fonction de « sensing », sont capables en réponse à une dysbiose de moduler l’immunité mucosale et participent ainsi à la formation d’un cercle vicieux délétère. De plus, nous nous sommes intéressés à la biologie des CSI, en intégrant la contribution des propriétés des cellules de Paneth qui participent à l’établissement de la niche. Nous avons étudié les propriétés des cellules souches dans un contexte sain ou au cours de l’initiation tumorale. L’ensemble de nos données indiquent qu’en contexte sain, Sox9 est requis pour la régulation du destin cellulaire des CSI, c’est à dire l’équilibre entre l’auto-renouvellement des CSI et leur différenciation cellulaire. Les mécanismes régulés par Sox9 mettent en jeu le métabolisme cellulaire, un acteur clé du destin des cellules souches. Nos travaux montrent également que le maintien d’une niche intacte est nécessaire au contrôle du devenir des CSI. La délétion de Sox9 altère l’intégrité mitochondriale et favorise la production de ROS mitochondriaux qui pourrait moduler le destin des CSI vers un état différencié. En parallèle, nous avons mis en évidence que l’invalidation de Sox9 après l’acquisition d’un événement initiateur tel que la perte de fonction du gène suppresseur de tumeur Apc, affecte de façon majeure le destin des CSC vers un état souche ainsi que leur métabolisme cellulaire. L’évaluation du rôle du facteur de transcription Sox9 dans le contrôle de l’homéostasie métabolique permettra de mieux comprendre les mécanismes de régulation de la biologie des CSI, et de proposer à terme de nouvelles stratégies thérapeutiques ciblant les CSC.