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SummaryIn contrast with most cancers, adrenocortical carcinomas (ACC) are more frequent in women than men, but the underlying mechanisms of this sexual dimorphism remain elusive. Homozygous deletion of the negative WNT pathway regulator ZNRF3 is the most frequent alteration in ACC patients. Here, we show that Cre-mediated inactivation of Znrf3 in steroidogenic cells of the mouse adrenal cortex is associated with sexually dimorphic tumour progression. Indeed, although most knockout female mice develop metastatic carcinomas over an 18 month-time course, adrenal hyperplasia gradually regresses in male knockout mice. This male-specific regression is associated with induction of senescence and recruitment of macrophages, which differentiate as active phagocytes that clear-out senescent preneoplastic cells. Macrophage recruitment is also observed in female mice. However, it is delayed and dampened compared to males, which allows for tumour progression. Interestingly, testosterone treatment of female knockouts is sufficient to induce senescence, recruitment of phagocytic macrophages and regression of hyperplasia. We further show that although macrophages are present within adrenal tumours at 18 months, MERTKhigh active phagocytes are mostly found in indolent lesions in males but not in aggressive tumours in females. Consistent with our observations in mice, analysis of RNA sequencing data from the TCGA cohort of ACC shows that phagocytic macrophages are more prominent in men than women and associated with better prognosis. Altogether, these data establish that phagocytic macrophages prevent aggressive ACC development in male mice and suggest that they may play a key role in the unusual sexual dimorphism of ACC in patients.

International audience; Large-cell calcifying Sertoli cell tumors (LCCSCTs) are among the most frequent lesions occurring in male Carney complex (CNC) patients. Although they constitute a key diagnostic criterion for this rare multiple neoplasia syndrome resulting from inactivating mutations of the tumor suppressor PRKAR1A, leading to unrepressed PKA activity, LCCSCT pathogenesis and origin remain elusive. Mouse models targeting Prkar1a inactivation in all somatic populations or separately in each cell type were generated to decipher the molecular and paracrine networks involved in the induction of CNC testis lesions. We demonstrate that the Prkar1a mutation was required in both stromal and Sertoli cells for the occurrence of LCCSCTs. Integrative analyses comparing transcriptomic, immunohistological data and phenotype of mutant mouse combinations led to the understanding of human LCCSCT pathogenesis and demonstrated PKA-induced paracrine molecular circuits in which the aberrant WNT4 signal production is a limiting step in shaping intratubular lesions and tumor expansion both in a mouse model and in human CNC testes.

International audience; BACKGROUND: EZH2 is overexpressed and associated with poor prognosis in adrenocortical carcinoma (ACC) and its inhibition reduces growth and aggressiveness of ACC cells in culture. Although EZH2 was identified as the methyltransferase that deposits the repressive H3K27me3 histone mark, it can cooperate with transcription factors to stimulate gene transcription. METHODS: We used bioinformatics approaches on gene expression data from three cohorts of patients and a mouse model of EZH2 ablation, to identify targets and mode of action of EZH2 in ACC. This was followed by ChIP and functional assays to evaluate contribution of identified targets to ACC pathogenesis. RESULTS: We show that EZH2 mostly works as a transcriptional inducer in ACC, through cooperation with the transcription factor E2F1 and identify three positive targets involved in cell cycle regulation and mitosis i.e., RRM2, PTTG1 and ASE1/PRC1. Overexpression of these genes is associated with poor prognosis, suggesting a potential role in acquisition of aggressive ACC features. Pharmacological and siRNA-mediated inhibition of RRM2 blocks cell proliferation, induces apoptosis and inhibits cell migration, suggesting that it may be an interesting target in ACC. CONCLUSIONS: Altogether, these data show an unexpected role of EZH2 and E2F1 in stimulating expression of genes associated with ACC aggressiveness. British Journal of Cancer https://doi.org/10.1038/s41416-019-0538-y BACKGROUND Adrenocortical carcinomas (ACC) are endocrine malignancies associated with dismal prognosis. At diagnosis, 80% of patients present metastases reducing the 5 years survival rate below 30% for most series.

La glande corticosurrenale produit les corticosteroides necessaires a l’homeostasie hydrominerale et metabolique de l’organisme. Elle s’organise, chez la souris, en deux regions concentriques, les zones glomerulee (zG) et fasciculee (zF), renouvelees a partir de progeniteurs situes en peripherie capsulaire. Le renouvellement centripete et le maintien de la zonation corticale, dependent d’un equilibre entre les signalisations WNT/β-catenine et ACTH/PKA assurant le recrutement puis la differenciation des progeniteurs, en zG puis zF. La SUMOylation est une modification post-traductionnelle reversible, essentielle au developpement embryonnaire et qui, dans la surrenale affiche un gradient centripete decroissant dont la fonction est inconnue. Afin d’etudier l’impact d’une hyperSUMOylation dans la differenciation endocrine, nous avons genere un modele de souris deficiente pour Senp2 (codant une deSUMOylase) dans le cortex. La perte de Senp2 induit, des quatre semaines, une insuffisance glucocorticoide, provoquee par une hypoplasie fasciculee sans atteinte de la zG mais accompagnee d’un exces de progeniteurs. Cette hypoplasie est associee a une stimulation de l’apoptose a la jonction zG-zF. Par ailleurs, la β-catenine, normalement limitee a la membrane des cellules glomerulees est retrouvee dans les noyaux des cellules fasciculees tandis que la reponse a l’ACTH est diminuee. L’hyperSUMOylation affecterait donc le renouvellement homeostatique de la zF en alterant l’equilibre des signalisations ACTH/PKA et WNT/β-catenine et en stimulant une apoptose precoce des cellules fasciculee. La SUMOylation constitue un mecanisme central des reponses cellulaires adaptatives aux stress, nos resultats suggerent qu’elle fait partie des mecanismes retenus par l’evolution pour preserver l’organisation fonctionnelle du cortex surrenal.

SUMOylation is a highly conserved and dynamic post-translational mechanism primarily affecting nuclear programs for adapting organisms to stressful challenges. Alteration of SUMOylation cycles leads to severe developmental and homeostatic defects and malignancy, but signals coordinating SUMOylation are still unidentified. The adrenal cortex is a zonated endocrine gland that controls body homeostasis and stress response. Here, we show that in human and in mouse adrenals, SUMOylation follows a decreasing centripetal gradient that mirrors cortical differentiation flow and delimits highly and weakly SUMOylated steroidogenic compartments, overlapping glomerulosa, and fasciculata zones. Activation of PKA signaling by acute hormonal treatment, mouse genetic engineering, or in Carney complex results in repression of small ubiquitin-like modifier (SUMO) conjugation in the inner cortex by coordinating expression of SUMO pathway inducers and repressors. Conversely, genetic activation of canonical wingless-related integration site signaling maintains high SUMOylation potential in the outer neoplastic cortex. Thus, SUMOylation is tightly regulated by signaling pathways that orchestrate adrenal zonation and diseases.-Dumontet, T., Sahut-Barnola, I., Dufour, D., Lefrancois-Martinez, A.-M., Berthon, A., Montanier, N., Ragazzon, B., Djari, C., Pointud, J.-C., Roucher-Boulez, F., Batisse-Lignier, M., Tauveron, I., Bertherat, J., Val, P., Martinez, A. Hormonal and spatial control of SUMOylation in the human and mouse adrenal cortex.

International audience; Adrenal cortex steroids are essential for body homeostasis, and adrenal insufficiency is a life-threatening condition. Adrenal endo-crine activity is maintained through recruitment of subcapsular progenitor cells that follow a unidirectional differentiation path from zona glomerulosa to zona fasciculata (zF). Here, we show that this unidirectionality is ensured by the histone methyltransferase EZH2. Indeed, we demonstrate that EZH2 maintains adrenal steroidogenic cell differentiation by preventing expression of GATA4 and WT1 that cause abnormal dedifferentiation to a progenitor-like state in Ezh2 KO adrenals. EZH2 further ensures normal cortical differentiation by programming cells for optimal response to adrenocorticotrophic hormone (ACTH)/PKA signaling. This is achieved by repression of phospho-diesterases PDE1B, 3A, and 7A and of PRKAR1B. Consequently, EZH2 ablation results in blunted zF differentiation and primary glucocorticoid insufficiency. These data demonstrate an all-encompassing role for EZH2 in programming steroidogenic cells for optimal response to differentiation signals and in maintaining their differentiated state. adrenal | differentiation | progenitors | PKA signaling | EZH2

International audience; The adrenocortical gland undergoes structural and functional remodelling in the fetal and postnatal periods. After birth, the fetal zone of the gland undergoes rapid involution in favor of the definitive cortex, which reaches maturity with the emergence of the zona reticularis(zR) at the adrenarche. The mechanisms underlying the adrenarche, the process leading to pre-puberty elevation of plasma androgens in higher primates, remain unknown, largely due to lack of any experimental model. By following up fetal and definitive cortex cell lines in mice, we showed that activation of protein kinase A (PKA) signaling mainly impacts the adult cortex by stimulating centripetal regeneration, with differentiation and then conversion of the zona fasciculata into a functional zR. Animals developed Cushing syndrome associated with primary hyperaldosteronism, suggesting possible coexistence of these hypersecretions in certain patients. Remarkably, all of these traits were sex-dependent: testicular androgens promoted WNT signaling antagonism on PKA, slowing cortical renewal and delaying onset of Cushing syndrome and the establishment of the zR in male mice, this being corrected by orchidectomy. In conclusion, zR derives from centripetal conversion of the zona fasciculata under cellular renewal induced by PKA signaling, determining the size of the adult cortex. Finally, we demonstrated that this PKA-dependent mobilization of cortical progenitors is sexually dimorphic and could, if confirmed in humans, account for female preponderance in adrenocortical pathologies.

Adrenocortical carcinoma (ACC) is a rare cancer with poor prognosis. Pan-genomic analyses identified p53/Rb and WNT/β-catenin signaling pathways as main contributors to the disease. However, isolated β-catenin constitutive activation failed to induce malignant progression in mouse adrenocortical tumors. Therefore, there still was a need for a relevant animal model to study ACC pathogenesis and to test new therapeutic approaches. Here, we have developed a transgenic mice model with adrenocortical specific expression of SV40 large T-antigen (AdTAg mice), to test the oncogenic potential of p53/Rb inhibition in the adrenal gland. All AdTAg mice develop large adrenal carcinomas that eventually metastasize to the liver and lungs, resulting in decreased overall survival. Consistent with ACC in patients, adrenal tumors in AdTAg mice autonomously produce large amounts of glucocorticoids and spontaneously activate WNT/β-catenin signaling pathway during malignant progression. We show that this activation is associated with downregulation of secreted frizzled related proteins (Sfrp) and Znrf3 that act as inhibitors of the WNT signaling. We also show that mTORC1 pathway activation is an early event during neoplasia expansion and further demonstrate that mTORC1 pathway is activated in ACC patients. Preclinical inhibition of mTORC1 activity induces a marked reduction in tumor size, associated with induction of apoptosis and inhibition of proliferation that results in normalization of corticosterone plasma levels in AdTAg mice. Altogether, these data establish AdTAg mice as the first preclinical model for metastatic ACC.

International audience; : Primary pigmented nodular adrenocortical disease (PPNAD) is associated with inactivating mutations of the PRKAR1A tumor suppressor gene that encodes the regulatory subunit R1α of the cAMP-dependent protein kinase (PKA). In human and mouse adrenocortical cells, these mutations lead to increased PKA activity, which results in increased resistance to apoptosis that contributes to the tumorigenic process. We used in vitro and in vivo models to investigate the possibility of a crosstalk between PKA and mammalian target of rapamycin (mTOR) pathways in adrenocortical cells and its possible involvement in apoptosis resistance. Impact of PKA signaling on activation of the mTOR pathway and apoptosis was measured in a mouse model of PPNAD (AdKO mice), in human and mouse adrenocortical cell lines in response to pharmacological inhibitors and in PPNAD tissues by immunohistochemistry. AdKO mice showed increased mTOR complex 1 (mTORC1) pathway activity. Inhibition of mTORC1 by rapamycin restored sensitivity of adrenocortical cells to apoptosis in AdKO but not in wild-type mice. In both cell lines and mouse adrenals, rapid phosphorylation of mTORC1 targets including BAD proapoptotic protein was observed in response to PKA activation. Accordingly, BAD hyperphosphorylation, which inhibits its proapoptotic activity, was increased in both AdKO mouse adrenals and human PPNAD tissues. In conclusion, mTORC1 pathway is activated by PKA signaling in human and mouse adrenocortical cells, leading to increased cell survival, which is correlated with BAD hyperphosphorylation. These alterations could be causative of tumor formation.

International audience; Adrenal Cortex Carcinoma (ACC) is an aggressive tumour with poor prognosis. Common alterations in patients include constitutive WNT/β-catenin signalling and overexpression of the growth factor IGF2. However, the combination of both alterations in transgenic mice is not sufficient to trigger malignant tumour progression, suggesting that other alterations are required to allow development of carcinomas. Here, we have conducted a study of publicly available gene expression data from three cohorts of ACC patients to identify relevant alterations. Our data show that the histone methyltransferase EZH2 is overexpressed in ACC in the three cohorts. This overexpression is the result of deregulated P53/RB/E2F pathway activity and is associated with increased proliferation and poorer prognosis in patients. Inhibition of EZH2 by RNA interference or pharmacological treatment with DZNep inhibits cellular growth, wound healing and clonogenic growth and induces apoptosis of H295R cells in culture. Further growth inhibition is obtained when DZNep is combined with mitotane, the gold-standard treatment for ACC. Altogether, these observations suggest that overexpression of EZH2 is associated with aggressive progression and may constitute an interesting therapeutic target in the context of ACC.

In the adrenal gland, adrenocorticotropin (ACTH) acting through the cAMP protein kinase (PKA) transduction pathway is the main regulator of genes involved in glucocorticoid synthesis. The prolactin (PRL) receptor is expressed in the adrenal cortex of most mammals, but experimental proof that PRL ensures direct control on glucocorticoid synthesis in rodents remains elusive. To unravel the physiological importance of PRL in adrenocortical functions, we measured steroidogenic capacity of Prlr-deficient mice (Prlr−/−) and explored the influence of JAK/STAT signaling, the major PRL transduction pathway, on the steroidogenic activity of adrenocortical cell cultures. We demonstrate that lack of Prlr does not affect basal (nor stress-induced) corticosterone levels in mice. PRL triggers JAK2/STAT5-dependent transcription in adrenal cells, but this does not influence corticosterone release. In contrast, pharmacological or siRNA-mediated inhibition of JAK2 reveals its essential role in both basal and ACTH/cAMP-induced steroidogenesis. We demonstrate that nuclear JAK2 regulates the amount of active transcription factor CREB (cAMP response element-binding protein) through tyrosine phosphorylation and prevention of proteasomal degradation, which in turn leads to transcriptional activation of the rate-limiting steroidogenic Star gene. Hence, we describe a novel link between PKA and JAK2 by which nuclear JAK2 signaling controls adrenal steroidogenesis by increasing the stability of CREB.

Here, we show that three enzymes belonging to the 1B group of the aldo-keto reductase (AKR) superfamily, i.e., human placental aldose reductase (AKR1B1), mouse kidney aldose reductase (AKR1B3) and mouse vas deferens protein (AKR1B7), catalyse the reduction of prostaglandin (PG) H(2), a common intermediate of various prostanoids, to form PGF(2alpha) in the presence of NADPH. AKR1B1, AKR1B3 and AKR1B7 displayed higher affinities for PGH(2) (K(m) = 1.9, 9.3 and 3.8 microM, respectively) and V(max) values (26, 53 and 44 nmol/min/mg protein, respectively) than did the human lung PGF(2alpha) synthase (AKR1C3; 18 microM and 4 nmol/min/mg protein, respectively). The PGF(2alpha) synthase activity of AKR1B1 and AKR1B3 was efficiently inhibited by two AKR inhibitors, tolrestat (K(i) = 3.6 and 0.26 microM, respectively) and sorbinil (K(i) = 21.7 and 0.89 microM, respectively), in a non-competitive or mixed-type manner, whereas that of AKR1B7 was not sensitive to these inhibitors (K(i) = 9.2 and 18 mM, respectively). These data provide a molecular basis for investigating novel functional roles for AKR1B members and PGF(2alpha) as mediators of physiological and pathological processes in mammalian organisms.

International audience; Aldose reductases (AKR1B) are widely expressed oxidoreductases whose physiological function remains elusive. Some isoforms are genuine prostaglandin F2α (PGF2α) synthases, suggesting they might influence adipose homeostasis because PGF2α inhibits adipogenesis. This was shown by Akr1b7 gene ablation in the mouse, which resulted in increased adiposity related to a lower PGF2α content in fat. Yet humans have no ortholog gene for Akr1b7, so the role of aldose reductases in human adipose homeostasis remains to be explored. We analyzed expression of genes encoding human and mouse aldose reductase isoforms in adipose tissues and differentiating adipocytes to assess conserved mechanisms regulating PGF2α synthesis and adipogenesis. The Akr1b3 gene encoded the most abundant isoform in mouse adipose tissue, whereas Akr1b7 encoded the only isoform enriched in the stromal vascular fraction. Most mouse aldose reductase gene expression peaked in early adipogenesis of 3T3-L1 cells and diminished with differentiation. In contrast with its mouse ortholog Akr1b3, AKR1B1 expression increased throughout differentiation of human multipotent adipose-derived stem cells, paralleling PGF2α release, whereas PGF2α receptor (FP) levels collapsed in early differentiation. Pharmacological inhibition of aldose reductase using Statil altered PGF2α production and enhanced human multipotent adipose-derived stem adipocyte differentiation. As expected, the adipogenic effects of Statil were counteracted by an FP agonist (cloprostenol). Thus, in both species aldose reductase-dependent PGF2α production could be important in early differentiation to restrict adipogenesis. PGF2α antiadipogenic signaling could then be toned down through the FP receptor or aldose reductases down-regulation in human and mouse cells, respectively. Our data suggest that aldose reductase inhibitors could have obesogenic potential.

Adrenocortical carcinoma (ACC) is a highly aggressive cancer. Although surgical resection remains the treatment of choice in localized ACC, clinical management of metastatic disease is ineffective. It is thus essential to identify the actors of malignant ACC progression to allow development of more effective therapeutic strategies. Deregulation of epigenetic factors is frequently involved in tumor progression. We have recently shown that the histone methyltransferase EZH2 is the most deregulated epigenetic histone modifier in ACC. EZH2 is over expressed downstream of TP53/RB1/E2F1 and is associated with tumor proliferation and poor prognosis. Pharmacological inhibition of EZH2 inhibits H295R cells growth and aggressive behaviour and induces apoptosis. This suggests that EZH2 is involved in malignant progression and could represent an interesting therapeutic target. Expression of the pro-apoptotic factor NOV/CCN3 is decreased in ACC, which is correlated with poor prognosis. Our molecular analyses show that EZH2 inhibition by siRNA or pharmacological treatment with DZNep increases expression of NOV/CCN3, suggesting that EZH2 over expression may favour malignant progression in ACC by inhibition of apoptosis stimulators. NOV has previously been identified as a negative target of the nuclear receptor SF1 in ACC cells, although the molecular mechanisms underlying this inhibition were unidentified. Interestingly, in prostate cancer, NOV expression is inhibited by the androgen receptor, through recruitment of EZH2 and deposition of the H3K27me3 mark. We are now conducting a combination of transfection and ChIP experiments to evaluate a similar cooperation between SF1 and EZH2 to repress NOV expression and block apoptosis in ACC.

The aldose reductase (human AKR1B1/mouse Akr1b3) has been the focus of many research because of its role in diabetic complications. The starting point of these alterations is the massive entry of glucose in polyol pathway where it is converted into sorbitol by this enzyme. However, the issue of aldose reductase function in non-diabetic condition remains unresolved. Aldose reductase-like enzymes (AKR1B10, Akr1b7 and Akr1b8) are highly related isoforms often co-expressed with bona fide aldose reductase, making functional analysis of one or the other isoform a challenging task. AKR1B/Akr1b members share at least 65% protein identity and the general ability to reduce many redundant substrates such as aldehydes provided from lipid peroxidation, steroids and their by-products and xenobiotics in vitro. Based on these properties, AKR1B/Akr1b are generally considered as detoxifying enzymes. Considering that divergences should be more informative than similarities to help understanding their physiological functions, we chose to review specific hallmarks of each human/mouse isoforms by focusing on tissue distribution and specific mechanisms of gene regulation. Indeed, although the aldose reductase shows ubiquitous expression, aldose reductase-like proteins exhibit tissue-specific patterns of expression. We focused on 3 organs where certain isoforms are enriched, the adrenal gland, enterohepatic and adipose tissues and tried to connect recent enzymatic and regulation data with endocrine and metabolic functions of these organs. We presented recent mouse models showing unsuspected physiological functions in the regulation of glucido-lipidic metabolism and adipose tissue homeostasis. Beyond the widely accepted idea that AKR1B/Akr1b are detoxification enzymes, these recent reports provide growing evidences that they are able to modify or generate signal molecules. This conceptually shifts this class of enzymes from unenviable status of scavenger to upper class of messengers.

Prostaglandin F(2alpha) (PGF(2alpha)), represses ovarian steroidogenesis and initiates parturition in mammals but its impact on adrenal gland is unknown. Prostaglandins biosynthesis depends on the sequential action of upstream cyclooxygenases (COX) and terminal synthases but no PGF(2alpha) synthases (PGFS) were functionally identified in mammalian cells. In vitro, the most efficient mammalian PGFS belong to aldo-keto reductase 1B (AKR1B) family. The adrenal gland is a major site of AKR1B expression in both human (AKR1B1) and mouse (AKR1B3, AKR1B7). Thus, we examined the PGF(2alpha) biosynthetic pathway and its functional impact on both cortical and medullary zones. Both compartments produced PGF(2alpha) but expressed different biosynthetic isozymes. In chromaffin cells, PGF(2alpha) secretion appeared constitutive and correlated to continuous expression of COX1 and AKR1B3. In steroidogenic cells, PGF(2alpha) secretion was stimulated by adrenocorticotropic hormone (ACTH) and correlated to ACTH-responsiveness of both COX2 and AKR1B7/B1. The pivotal role of AKR1B7 in ACTH-induced PGF(2alpha) release and functional coupling with COX2 was demonstrated using over- and down-expression in cell lines. PGF(2alpha) receptor was only detected in chromaffin cells, making medulla the primary target of PGF(2alpha) action. By comparing PGF(2alpha)-responsiveness of isolated cells and whole adrenal cultures, we demonstrated that PGF(2alpha) repressed glucocorticoid secretion by an indirect mechanism involving a decrease in catecholamine release which in turn decreased adrenal steroidogenesis. PGF(2alpha) may be regarded as a negative autocrine/paracrine regulator within a novel intra-adrenal feedback loop. The coordinated cell-specific regulation of COX2 and AKR1B7 ensures the generation of this stress-induced corticostatic signal.

Primary pigmented nodular adrenocortical disease (PPNAD) is a rare form of bilateral hyperplasia leading to high morbidity due to pituitary-independent hypercortisolism and Cushing’s syndrome. PPNAD may be either sporadic or regarded as the most frequent endocrine manifestation of Carney’s complex (CNC), an autosomic dominant multiple neoplasia syndrome characterized by cardiac myxomas, spotty skin pigmentation and endocrine overactivity [1]. Both isolated PPNAD and CNC have been associated with null mutations in PRKAR1A, a gene encoding the type 1 αregulatory subunit (RIα) of the cAMP-dependent protein kinase (PKA) [2 and 3]. Tumor-specific loss of heterozygosity within the chromosomal region harboring PRKAR1A is observed in CNC patients and isolated PPNAD suggesting that PRKAR1A is a potential tumor suppressor gene [4]. Because general homozygous loss of Prkar1a is lethal in early mouse embryos [5], adrenal-specific knockout was required to demonstrate tumor suppressor activity. Therefore, we produced mice with Prkar1a gene inactivation in adrenocortical cells by mating Prkar1a floxed mice with the Akr1b7 -Cre mouse line, a novel Cre expressing line we developed to allow specific gene ablation in the steroidogenic lineage of the adrenals without affecting the gonads [6]. Adrenal cortex-specific Prkar1a knockout mice (AdKO) develop pituitary-independent Cushing’s syndrome and evident signs of deregulated adrenocortical cells differentiation and proliferation. These defects lead to improper maintenance and expansion of foetal adrenal cells in adult adrenals and establishment of pretumoral conditions. Our data provide the first in vivo evidence that the absence of RIα subunit of PKA is sufficient to explain autonomous adrenal hyperactivity and bilateral hyperplasia observed in PPNAD. They also strongly suggest that deregulated PKA activity positively affects the maintenance of foetal characters in adult glands.

L’objectif est de definir la sequence d’apparition des complications cataboliques et anaboliques du syndrome de Cushing ACTH-independant (CS) developpe par un modele murin et d’evaluer l’efficacite d’un antagoniste selectif du recepteur aux glucocorticoides (GR). Grâce aux souris AdKOv2 invalidees pour le gene Prkar1a dans le cortex surrenalien, nous avons evalue les consequences cataboliques du CS sur le muscle (poids relatif des gastrocnemien et soleaire) et l’os (microarchitecture en microtomographie et expression des marqueurs osteoclastiques/blastiques en RTqPCR). L’evaluation des troubles anaboliques a ete suivie par des tests de resistance a l’insuline et l’analyse de marqueurs neoglucogeniques hepatiques (Pepck, G6pc). Enfin, la composition corporelle a ete caracterisee en EchoMRI. Des l’apparition du CS, WT et AdKOv2 sont reparties en trois bras de traitements – vehicule, Mifepristone, CT8 (antagoniste specifique du GR) – administres quotidiennement par injection intraperitoneale pendant 4 semaines. Les mâles AdKOv2 developpent un CS franc des 1 mois, associe a un exces de mortalite, un retard de croissance, et une amyotrophie, puis une osteoporose plus tardive a 1,5 mois (p Le modele AdKOv2 developpe, des le stade du CS subclinique, l’ensemble des comorbidites, a l’exception de l’osteoporose, et constitue ainsi un support d’etude preclinique pour cibler les comorbidites preferentiellement accessibles aux traitements.

Les dysplasies micronodulaires pigmentees du cortex surrenalien (PPNAD) sont des tumeurs benignes rares caracteristiques du complexe de Carney. Ces dysplasies sont associees a des mutations inactivatrices du gene PRKAR1A conduisant a la perte de la sous-unite regulatrice R1α de la PKA, a son activation constitutive et a l’installation d’un syndrome de Cushing ACTH-independant (CS). Un modele murin de PPNAD developpe au laboratoire, et comportant une invalidation du gene Prkar1a ciblee dans le cortex surrenalien, a revele les caracteristiques fœtales de ces tumeurs qui se developpent au detriment du cortex definitif adulte. Elles pourraient resulter de deux processus neoplasiques : l’un initie dans des progeniteurs corticosurrenaliens fœtaux et l’autre provenant de la differenciation fœtale de cellules du cortex adulte. Nous avons utilise une approche genetique de lignage cellulaire afin de tracer chez les souris, l’origine de ces tumeurs apres invalidation de Prkar1a soit dans les progeniteurs du cortex definitif soit dans ceux du cortex fœtal. Alors que l’invalidation de Prkar1a dans ces derniers ne conduit a aucune anomalie endocrine ni tumorale, son invalidation dans le cortex definitif conduit a un CS accompagne d’une hyperplasie. Celle-ci provient d’une expansion centrifuge de cellules du cortex profond cumulant des caracteristiques fœtales et de cellules de la zone reticulee normalement absente chez la souris. En conclusion, les populations cellulaires contribuant aux PPNAD proviennent de la differenciation fœto-reticulee de cellules du cortex adulte sous l’impulsion de la signalisation PKA. Les mecanismes sous-jacents restent a elucider mais pourraient contribuer a l’adrenarche propre aux grands primates.

Transcription regulation in male germ cells can involve specialised mechanisms and testis-specific paralogues of the general transcription machinery. Here we describe TAF7L, a germ-cell-specific paralogue of the TFIID subunit TAF7. TAF7L is expressed through most of the male germ-cell differentiation programme, but its intracellular localisation is dynamically regulated from cytoplasmic in spermatogonia and early spermatocytes to nuclear in late pachytene spermatocytes and haploid round spermatids. Import of TAF7L into the nucleus coincides with decreased TAF7 expression and a strong increase in nuclear TBP expression, which suggests that TAF7L replaces TAF7 as a TFIID subunit in late pachytene spermatocytes and in haploid cells. In agreement with this, biochemical experiments indicate that a subpopulation of TAF7L is tightly associated with TBP in both pachytene and haploid cells and TAF7L interacts with the TFIID subunit TAF1. We further show that TAF3, TAF4 and TAF10 are all strongly expressed in early spermatocytes, but that in contrast to TBP and TAF7L, they are downregulated in haploid cells. Hence,different subunits of the TFIID complex are regulated in distinct ways during male germ-cell differentiation. These results show for the first time how the composition of a general transcription factor such as TFIID and other TAF-containing complexes are modulated during a differentiation programme highlighting the unique nature of the transcription regulatory machinery in spermatogenesis.

International audience; The BTB/POZ domain is an evolutionarily conserved protein-protein interaction domain present in the N-terminal region of numerous transcription factors involved in development, chromatin remodeling, and human cancers. This domain is involved in homomeric and heteromeric associations with other BTB/POZ domains. The Drosophila BTB/POZ proteins Bric ?rac 1 (BAB1) and Bric ?rac 2 (BAB2) are developmentally regulated transcription factors which are involved in pattern formation along the proximo-distal axis of the leg and antenna, in the morphogenesis of the adult ovaries, and in the control of sexually dimorphic characters. We have identified partners of the BAB1 protein by using the two-hybrid system. The characterization of one of these proteins, called BIP2 for BAB Interacting Protein 2, is presented. BIP2 is a novel Drosophila TATA-box Protein Associated Factor (TAF(II)), also named dTAF(II)155. We show that the BTB/POZ domains of BAB1 and BAB2 are sufficient to mediate a direct interaction with BIP2/dTAF(II)155. This provides a direct link between these BTB/POZ transcription factors and the basal transcriptional machinery. We discuss the implications of the interaction between a BTB/POZ domain and a TAF(II) for the molecular mechanisms of transcriptional control mediated by BTB/POZ transcription factors.

The RNA polymerase II transcription factor TFIID comprises the TATA binding protein (TBP) and a set of TBP-associated factors (TAF(II)s). TFIID has been extensively characterized for yeast, Drosophila, and humans, demonstrating a high degree of conservation of both the amino acid sequences of the constituent TAF(II)s and overall molecular organization. In recent years, it has been assumed that all the metazoan TAF(II)s have been identified, yet no metazoan homologues of yeast TAF(II)47 (yTAF(II)47) and yTAF(II)65 are known. Both of these yTAF(II)s contain a histone fold domain (HFD) which selectively heterodimerizes with that of yTAF(II)25. We have cloned a novel mouse protein, TAF(II)140, containing an HFD and a plant homeodomain (PHD) finger, which we demonstrated by immunoprecipitation to be a mammalian TFIID component. TAF(II)140 shows extensive sequence similarity to Drosophila BIP2 (dBIP2) (dTAF(II)155), which we also show to be a component of Drosophila TFIID. These proteins are metazoan homologues of yTAF(II)47 as their HFDs selectively heterodimerize with dTAF(II)24 and human TAF(II)30, metazoan homologues of yTAF(II)25. We further show that yTAF(II)65 shares two domains with the Drosophila Prodos protein, a recently described potential dTAF(II). These conserved domains are critical for yTAF(II)65 function in vivo. Our results therefore identify metazoan homologues of yTAF(II)47 and yTAF(II)65.