Antagonistic functions of tetradecanoyl phorbol acetate-inducible-sequence 11b and HuR in the hormonal regulation of vascular endothelial growth factor messenger ribonucleic acid stability by adrenocorticotropin.: ACTH-induced VEGF expression

International audienceExpression of vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen and a potent angiogenic factor, is up-regulated by a variety of factors including hypoxia, growth factors, and hormones. In the adrenal cortex, regulation of VEGF expression by the pituitary hormone ACTH ensures the maintenance of the organ vasculature. We have previously shown that ACTH evokes a rapid and transient increase in VEGF mRNA levels in primary adrenocortical cells through transcription-independent mechanisms. We further demonstrated that the zinc finger RNA-binding protein Tis11b (tetradecanoyl phorbol acetate-inducible-sequence 11b) destabilizes VEGF mRNA through its 3'-untranslated region (3'-UTR) and that Tis11b is involved in the decay phase of ACTH-induced VEGF mRNA expression. In the present study, we attempted to determine the mechanisms underlying ACTH-elicited increase in VEGF mRNA levels in adrenocortical cells. We show that ACTH triggers an increase in the levels of the mRNA-stabilizing protein HuR in the cytoplasm and a concomitant decrease in the levels of HuR in the nucleus. This process is accompanied by an increased association of HuR with the nucleocytoplasmic shuttling protein pp32, indicating that ACTH induces HuR translocation from the nuclear to the cytoplasmic compartment. Leptomycin B, a specific inhibitor of CRM1-dependent nuclear export of pp32, significantly reduced ACTH-induced VEGF mRNA levels. Furthermore, RNA interference-mediated depletion of HuR in adrenocortical cells abrogated ACTH-induced VEGF mRNA expression. Finally, we show that Tis11b and HuR exert antagonistic effects on VEGF 3'-UTR in vitro. Although both proteins could bind simultaneously on VEGF 3'-UTR, Tis11b markedly decreases HuR-binding to this RNA sequence. Altogether, these results suggest that the RNA-stabilizing protein HuR is instrumental to ACTH-induced expression of VEGF mRNA and that the nuclear export of HuR is a rate-limiting step in this process. HuR appears to transiently stabilize VEGF transcripts after ACTH stimulation of adrenocortical cells, and Tis11b appears to subsequently trigger their degradation

Apport de la biopsie radioguidée dans le diagnostic histopathologique des tumeurs de l’enfant: expérience de l’Hôpital d’Enfant de Rabat

La biopsie radioguidée constitue une alternative à la biopsie chirurgicale invasive et à la cytologie pour le diagnostic des tumeurs pédiatriques. L'intérêt de notre étude est d'évaluer la valeur diagnostique des biopsies radioguidées examinées au laboratoire d'anatomopathologie de l'hôpital d'Enfants de Rabat (HER). L'étude a porté sur 78 biopsies radioguidées recueillies dans notre laboratoire entre janvier 2008 et décembre 2011. l'âge moyen des patients était de 5 ans et 10 mois avec une prédominance masculine  (65,4 %). La tumeur était abdominale dans 80 % des cas, thoracique dans 15 % cas, thoracique et abdominale dans 2,5% et sacrée dans 1,2%. Les biopsies étaient écho-guidées dans 90 % des cas et scannoguidées dans 10% des cas. Le diagnostic histopathologique était posé dans 89 % des cas.  L'immuno-histochimie a été indiquée dans 35 % des cas. Les diagnostics les plus fréquents étaient : tumeurs neuroblastiques (42 cas), lymphomes non hodgkiniens (10 cas), rhabdomyosarcomes (6 cas), autres (sarcome d'Ewing, néphroblastomes, tumeur  myofibroblastique inflammatoire, maladies de Hodgkin, leucémie aiguë, hépatoblastome et ostéosarcome). Dans notre série, la biopsie radioguidée a permis un diagnostic histopathologique certain dans 89 % des cas.Elle nécessite une étroite collaboration entre clinicien, radiologue et anatomopathologiste pour discuter son indication, afin de diminuer le nombre de biopsies peu ou non représentatives.Key words: Biopsie, radioguidée, tumeur, enfan

Gene expression profiling of human adrenocortical tumors using complementary deoxyribonucleic Acid microarrays identifies several candidate genes as markers of malignancy.

International audienceThe aim of this study was to identify predictor sets of genes whose over- or underexpression in human sporadic adrenocortical tumors would help to identify malignant vs. benign tumors and to predict postsurgical metastatic recurrence. For this, we analyzed the expression of 230 candidate genes using cDNA microarrays in a series of 57 well-characterized human sporadic adrenocortical tumors (33 adenomas and 24 carcinomas). We identified two clusters of genes (the IGF-II cluster containing eight genes, including IGF-II, and the steroidogenesis cluster containing six genes encoding steroidogenic enzymes plus eight other genes) whose combined levels of expression appeared to be good predictors of malignancy. This predictive value was as strong as that of the pathological score of Weiss. The analysis of the population of carcinomas (13 tumors) for genes whose expression would be strongly different between recurring and nonrecurring tumors allowed identification of 14 genes meeting these criteria. Among these genes, there are probably new markers of tumor evolution that will deserve additional validation on a larger scale. Taken together, these results show that the parallel analysis of the expression levels of a selected group of genes on microgram quantities of tumor RNA (a quantity that can be obtained from fine needle aspirations) appears as a complementary method to histopathology for the diagnosis and prognosis of evolution of adrenocortical carcinomas

microRNAs as Potential Biomarkers in Adrenocortical Cancer: Progress and Challenges

International audienceAdrenocortical carcinoma (ACC) is a rare malignancy with poor prognosis and limited therapeutic options. Over the last decade, pan-genomic analyses of genetic and epigenetic alterations and genome-wide expression profile studies allowed major advances in the understanding of the molecular genetics of ACC. Besides the well-known dysfunctional molecular pathways in adrenocortical tumors, such as the IGF2 pathway, the Wnt pathway, and TP53, high-throughput technologies enabled a more comprehensive genomic characterization of adrenocortical cancer. Integration of expression profile data with exome sequencing, SNP array analysis, methylation, and microRNA (miRNA) profiling led to the identification of subgroups of malignant tumors with distinct molecular alterations and clinical outcomes. miRNAs post-transcriptionally silence their target gene expression either by degrading mRNA or by inhibiting translation. Although our knowledge of the contribution of deregulated miRNAs to the pathogenesis of ACC is still in its infancy, recent studies support their relevance in gene expression alterations in these tumors. Some miRNAs have been shown to carry potential diagnostic and prognostic values, while others may be good candidates for therapeutic interventions. With the emergence of disease-specific blood-borne miRNAs signatures, analyses of small cohorts of patients with ACC suggest that circulating miRNAs represent promising non-invasive biomarkers of malignancy or recurrence. However, some technical challenges still remain, and most of the miRNAs reported in the literature have not yet been validated in sufficiently powered and longitudinal studies. In this review, we discuss the current knowledge regarding the deregulation of tumor-associated and circulating miRNAs in ACC patients, while emphasizing their potential significance in pathogenic pathways in light of recent insights into the role of miRNAs in shaping the tumor microenvironment

The Acute Regulation of Mineralocorticoid Biosynthesis: Scenarios for the StAR System

International audienceThe zona glomerulosa cell of the adrenal cortex produces mineralocorticoids in response to physiological stimuli (angiotensin II and extracellular K(+)) activating the Ca(2+) messenger system. The mechanisms underlying the generation of the Ca(2+) signal have been analyzed extensively and recent developments have contributed to bridging the gap between intracellular signals and activation of the biological function. This article summarizes the current knowledge on the intracellular targets of the Ca(2+) messenger, obtained mainly in bovine glomerulosa cells. Ca(2+) appears to exert a dual effect, both at the intramitochondrial level and at the nuclear level, where it activates steroidogenic acute regulatory protein (StAR) gene transcription

Characterization of the 3 beta-hydroxysteroid dehydrogenase activity associated with bovine adrenocortical mitochondria.

International audienceThe enzymatic activity of 3 beta-hydroxysteroid dehydrogenase (3 beta HSD/I) constitutes an essential step in the biosynthesis of active steroid hormones such as progesterone, mineralo- and gluco-corticoids, estrogens, and androgens. Its subcellular localization in steroidogenic tissues is usually considered to be mainly microsomal; however, 3 beta HSD/I activity is also present in mitochondrial preparations. In the present study, the distribution of 3 beta HSD/I in bovine adrenocortical subcellular preparations has been reexamined, and the catalytic properties of the enzyme present in the various cell compartments have been characterized. About 30% of the total 3 beta HSD/I was found to remain tightly associated with highly purified mitochondrial preparations. The preferred substrate of the mitochondrial enzyme was pregnenolone. Examination of submitochondrial preparations revealed that 3 beta HSD/I was associated with both the inner membrane and a particulate fraction that sediments in a density gradient between inner and outer membranes. The specific activity of the enzyme was at its highest in this intermediate density fraction, which exhibited the properties of mitochondrial intermembrane contact sites. Taken together, these observations suggest that these contact sites may represent a supramolecular organization of biological significance in adrenocortical cell steroidogenic functions. Such intermembrane fusion sites would facilitate the access of cholesterol to the inner membrane in which cholesterol side-chain cleavage cytochrome P-450 is located as well as the rapid transformation of its reaction product (i.e. pregnenolone) to progesterone by 3 beta HSD/I. Such a submitochondrial organization opens new possibilities in the understanding of the regulation of adrenocortical differentiated functions

Type β1 transforming growth factor is an inhibitor of 3β-hydroxysteroid dehydrogenase isomerase in mouse adrenal tumor cell line Y1

International audienceIn the Y1 mouse adrenal tumor cell line, the 3 beta-hydroxysteroid dehydrogenase isomerase enzyme (3 beta-HSD) which catalyzes the transformation of 3 beta-hydroxy-5-ene steroids to 3-keto-4-ene steroids is active. The effect of type beta 1 transforming growth factor (TGF beta 1), a potent modulator of adrenocortical differentiated functions, on the 3 beta-HSD enzyme was studied. Four isoforms of 3 beta-HSD yielding proteins of different mobility on SDS-PAGE were previously detected in the mouse; whereas only one form was present in the mouse adrenal, we detected two isoforms in the Y1 cells. An inhibition of the basal enzymatic activity was observed after TGF beta 1 treatment which was correlated with a decrease in 3 beta-HSD protein (both isoforms) and mRNA levels

Organization of 3β-hydroxysteroid dehydrogenase/isomerase and cytochrome P450scc into a catalytically active molecular complex in bovine adrenocortical mitochondria

International audienceWe have previously reported the co-localization [Cherradi et al., Endocrinology 134 (1994) 1358-1364] of 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD) and cytochrome P450scc (cyt. P450scc) in the inner membrane and in the intermembrane contact sites of adrenocortical mitochondria. This observation raises the question of a possible functional association between the two proteins. Isolated bovine adrenocortical mitochondria are able to convert cholesterol to progesterone without the need of exogenous cofactors. An association of 3 beta-HSD and cyt. P450scc is observed during the purification of 3 beta-HSD from mitochondria. The behaviour of 3 beta-HSD on a column of Heparin-Sepharose is modified by the presence of cyt. P450scc. Immunoprecipitations from mitochondria with either anti-cyt. P450scc or anti 3 beta-HSD antibodies result in a co-precipitation of the two proteins. Both proteins engaged in these immunocomplexes are catalytically active. The interaction was further demonstrated by the surface plasmon resonance method using purified components. An affinity demonstrated by the surface plasmon resonance method using purified components. An affinity constant of 0.12 microM between 3 beta-HSD and P450scc was obtained. These observations suggest that P450scc and 3 beta-HSD may associate into a molecular complex in the mitochondrial compartment and may constitute a functional steroidogenic unit, thus opening new possibilities in the regulation of the production of progesterone and its flow in the adrenocortical cell

Dual subcellular localization of the 3β-hydroxysteroid dehydrogenase isomerase: Characterization of the mitochondrial enzyme in the bovine adrenal cortex

International audienceThe enzyme 3 beta-hydroxysteroid dehydrogenase isomerase (3 beta-HSD/I) is an essential step in the biosynthesis of steroid such as progesterone, mineralo- and gluco-corticoids, estrogens and androgens in steroidogenic tissues. It is considered to be mainly localized in microsomes; however, 3 beta-HSD/I activity has also been described to be associated with mitochondrial preparations. In this study, we examined the subcellular distribution of 3 beta-HSD/I in bovine adrenocortical tissue and we characterized the catalytic properties of the enzyme present in the various cell compartments. About 30% of the total 3 beta-HSD/I activity was found to remain tightly associated with the purified mitochondrial pellet. The 3 beta-HSD/I and 3-ketoreductase activities were found in microsomes as well as in mitochondria. The 3 beta-HSD/I associated with the mitochondrial fraction did not require addition of exogenous NAD+. When the pyridine nucleotide was reduced following addition of substrates of the tricarboxylic acids cycle, the mitochondrial 3 beta-HSD/I activity decreased, suggesting that the enzyme utilizes NAD+ available from the matrix space. By contrast, the microsomal enzyme was inactive in the absence of exogenous NAD+. Submitochondrial fractionation disclosed that 3 beta-HSD/I was associated (i) with the inner membrane and (ii) with a particulate fraction sedimenting in a density gradient between inner and outer membranes. This fraction was characterized as contact sites between the two membranes. 3 beta-HSD/I specific activity was much higher in this fraction than in the inner mitochondrial membrane. Altogether, these observations suggest that these mitochondrial intermembrane contact sites may represent a special organization of functional significance, facilitating both the access of cholesterol to the inner membrane where cytochrome P-450scc is located and the rapid transformation of its product, pregnenolone, to progesterone, through 3 beta-HSD/I activity