Retinoic acid receptor α as a novel contributor to adrenal cortex structure and function through interactions with Wnt and Vegfa signalling

International audiencePrimary aldosteronism (PA) is the most frequent form of secondary arterial hypertension. Mutations in different genes increase aldosterone production in PA, but additional mechanisms may contribute to increased cell proliferation and aldosterone producing adenoma (APA) development. We performed transcriptome analysis in APA and identified retinoic acid receptor alpha (RARα) signaling as a central molecular network involved in nodule formation. To understand how RARα modulates adrenal structure and function, we explored the adrenal phenotype of male and female Rarα knockout mice. inactivation of Rarα in mice led to significant structural disorganization of the adrenal cortex in both sexes, with increased adrenal cortex size in female mice and increased cell proliferation in males. Abnormalities of vessel architecture and extracellular matrix were due to decreased Vegfa expression and modifications in extracellular matrix components. On the molecular level, Rarα inactivation leads to inhibition of non-canonical Wnt signaling, without affecting the canonical Wnt pathway nor PKA signaling. Our study suggests that Rarα contributes to the maintenance of normal adrenal cortex structure and cell proliferation, by modulating Wnt signaling. Dysregulation of this interaction may contribute to abnormal cell proliferation, creating a propitious environment for the emergence of specific driver mutations in PA. Primary aldosteronism (PA) is the most common and curable form of secondary arterial hypertension, with prevalence estimations of up to 10% of cases in referred hypertensive patients, 4% of patients in primary care 1,2 and 20% of patients with resistant hypertension 3,4. Rapid diagnosis and treatment are important to prevent severe cardiovas-cular consequences of long term aldosterone exposure, which are independent of blood pressure levels and are du

The Warburg Effect Is Genetically Determined in Inherited Pheochromocytomas

The Warburg effect describes how cancer cells down-regulate their aerobic respiration and preferentially use glycolysis to generate energy. To evaluate the link between hypoxia and Warburg effect, we studied mitochondrial electron transport, angiogenesis and glycolysis in pheochromocytomas induced by germ-line mutations in VHL, RET, NF1 and SDH genes. SDH and VHL gene mutations have been shown to lead to the activation of hypoxic response, even in normoxic conditions, a process now referred to as pseudohypoxia. We observed a decrease in electron transport protein expression and activity, associated with increased angiogenesis in SDH- and VHL-related, pseudohypoxic tumors, while stimulation of glycolysis was solely observed in VHL tumors. Moreover, microarray analyses revealed that expression of genes involved in these metabolic pathways is an efficient tool for classification of pheochromocytomas in accordance with the predisposition gene mutated. Our data suggest an unexpected association between pseudohypoxia and loss of p53, which leads to a distinct Warburg effect in VHL-related pheochromocytomas

Different Somatic Mutations in Multinodular Adrenals With Aldosterone-Producing AdenomaNovelty and Significance

Frontal view of the Auditorium Building by Sullivan, from Grant Par

Genetic, Cellular, and Molecular Heterogeneity in Adrenals With Aldosterone-Producing Adenoma

International audienceAldosterone-producing adenoma (APA) cause primary aldosteronism—the most frequent form of secondary hypertension. Somatic mutations in genes coding for ion channels and ATPases are found in APA and in aldosterone-producing cell clusters. We investigated the genetic, cellular, and molecular heterogeneity of different aldosterone-producing structures in adrenals with APA, to get insight into the mechanisms driving their development and to investigate their clinical and biochemical correlates. Genetic analysis of APA, aldosterone-producing cell clusters, and secondary nodules was performed in adrenal tissues from 49 patients by next-generation sequencing following CYP11B2 immunohistochemistry. Results were correlated with clinical and biochemical characteristics of patients, steroid profiles, and histological features of the tumor and adjacent adrenal cortex. Somatic mutations were identified in 93.75% of APAs. Adenoma carrying KCNJ5 mutations had more clear cells and cells expressing CYP11B1, and fewer cells expressing CYP11B2 or activated β-catenin, compared with other mutational groups. 18-hydroxycortisol and 18-oxocortisol were higher in patients carrying KCNJ5 mutations and correlated with histological features of adenoma; however, mutational status could not be predicted using steroid profiling. Heterogeneous CYP11B2 expression in KCNJ5 -mutated adenoma was not associated with genetic heterogeneity. Different mutations were identified in secondary nodules expressing aldosterone synthase and in independent aldosterone-producing cell clusters from adrenals with adenoma; known KCNJ5 mutations were identified in 5 aldosterone-producing cell clusters. Genetic heterogeneity in different aldosterone-producing structures in the same adrenal suggests complex mechanisms underlying APA development

Angiogenesis in SDH and VHL-related PH/PGL.

<p>(A) CD34 immunohistochemistry was performed to evaluate angiogenesis in all samples. Diaminobenzidin was used as a chromogen for detection (brown labeling). Calibration bar: 200 µm. (B) Quantification of vascular density showing an increased number of blood vessels in SDH, and VHL tissues. Data are means±SEM. *p<0.05, **p<0.01, ***p<0.001. (C) Correlation between vascular density and SCCR enzymatic values for individual patients.</p

Microarray analysis of oxidative phosphorylation in hereditary PH/PGL.

<p>(A) Unsupervised hierarchical clustering analysis of the 68 samples according to the expression of 200 genes. Expression profiles are shown as a heat map indicating high (red) and low (blue) expression according to a log2-transformed scale. The higher bipartition allows to distinguish VHL (white) and SDH (grey) patients from RET and NF1 (black) patients. (B) Principal component analysis of the 68 samples according to the expression of 200 genes. Three groups are focused on, corresponding to the SDH (red), VHL (green) and RET/NF1 (blue) patients. PC1: principal component; PC2: principal component 2; PC3: principal component 3. (C) Mean values for genes expression between SDH, VHL, RET and NF1 tumors. Data are means±SEM, represented as relative to NF1 expression values. **p<0.01, ***p<0.001.</p

Decreased oxidative phosphorylation in SDH and VHL-related PH/PGL.

<p>(A) The abundance of proteins of mitochondrial complexes I (20 kDa subunit), II (SDHB and SDHA), III (Core 2) and IV (Cox II) is lower in PH/PGL from SDH and most VHL than from RET and tumor tissues. (B) SDHB immunohistochemistry performed on the adrenal adjacent to a RET-related PH and in RET, VHL and SDHD-mutated PH reveals a strong labeling in the adrenal compared to tumor cells (asterisks). In VHL PH/PGL tumor, expression of SDHB was reduced when compared to RET-related PH while it is absent in SDHD-related tumor. Note that vascular immunostaining was present in all samples (arrows). Calibration bar: 50 µm. (C) Individual values of SCCR activity reveal that low complex II+III enzymatic activity is associated with low protein abundance. (D–F) Mean values for mitochondrial complexes II+III, III and IV reveal a generalized decrease in respiration in SDH and VHL PH/PGL. Data are means±SEM. **p<0.01, ***p<0.001.</p

Microarray analysis of glycolysis in hereditary PH/PGL.

<p>(A) Unsupervised hierarchical clustering analysis of the 68 samples according to the expression of 38 genes. Expression profiles are shown as a heat map indicating high (red) and low (blue) expression according to a log2-transformed scale. The different mutations are localized in three distinct clusters: SDH (grey), VHL (white) and RET/NF1 (black). (B) Mean values for genes expression between SDH, VHL, RET and NF1 tumors. Data are means±SEM, represented as relative to NF1 expression values. **p<0.01, ***p<0.001.</p

Pseudohypoxia in SDH and VHL-related PH/PGL.

<p>(A) HIF-1α and HIF-2α immunohistochemistry were performed to evaluate activation of the hypoxic pathway in all samples. Histogreen was used as a chromogen for detection (blue labeling). Calibration bar: 100 µm. Microarray evaluation of HIF-1α (B) and HIF-2α (C) expression between SDH, VHL, RET and NF1 tumors. Data are means±SEM. ***p<0.001.</p