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. 2019 Oct 11;9(1):14677.
doi: 10.1038/s41598-019-50988-2.

Retinoic Acid Receptor α as a Novel Contributor to Adrenal Cortex Structure and Function Through Interactions With Wnt and Vegfa Signalling

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Free PMC article

Retinoic Acid Receptor α as a Novel Contributor to Adrenal Cortex Structure and Function Through Interactions With Wnt and Vegfa Signalling

Rami M El Zein et al. Sci Rep. .
Free PMC article

Abstract

Primary 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.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Transcriptome analysis reveals two distinct subgroups of APA. (A) Hierarchical clustering using Euclidean distance and complete linkage of 48 APA and 11 control adrenals. Heat-map of the whole gene expression is displayed using black for low expression and green for high expression. (B) Principal Component Analysis in the correlation space over the entire transcriptome. The first two principal components and their associated inertia are displayed. (C) Singular-value-decomposition-initialized multidimensional scaling under the correlation space over all genes,. Kruskal error is indicated as “stress”. (D) Aldosterone synthase immunohistochemistry performed on adrenals from APA_A and APA_B. (E) Three groups (APA_A, APA_B and CA) are pair-wise compared to obtain a list of statistically significantly expressed candidate genes for each of the three comparisons. The common and comparison-specific genes are classified in seven different groups some of which are associated to a biological process. Venn-diagrams are displayed for statistically significantly differentially expressed candidate genes. The smaller Venn diagrams on the right-hand side of the figure represent the overlap for up-regulated and down-regulated genes. Bars are displayed next to the represented sets to indicate the relative proportion of the overlapping regions to the corresponding list of candidate genes. Figures displayed are the number of genes that populate each of the selected sets. (F) Expression of RARα was investigated by RT-qPCR on mRNA extracted from 6 control adrenals and 19 APA. Values are presented as the mean ± SEM; p values were calculated using a two-sided Mann-Whitney test. *p < 0.05. (G) RARα immunohistochemistry performed on control adrenal and in APA.
Figure 2
Figure 2
Rarα affects adrenal cortex morphology in 12 weeks old male and female mice. (A) Morphological characterization of adrenals from 12 weeks old Rarα+/+ and Rarα−/− mice. HES staining, Dab-2 immunofluorescence and Ki67 immunohistochemistry were performed on adrenal sections from the indicated group of mice. (B) Number of Dab-2 positive cells in the cortex was determined in 7 to 11 animals of each genotype and sex using an automated molecular imaging platform (Vectra, Perkin Elmer) and is expressed as a percentage of total number of cells in the entire cortex area. (C) Number of nuclei in the adrenal cortex was determined in 7 to 11 animals of each genotype and sex using an automated molecular imaging platform (Vectra, Perkin Elmer). (D) Relative proliferative index of adrenals from male and female Rarα+/+ and Rarα−/− mice. Ki67 positive cells were separately counted in the adrenal cortex in 5–6 animals per genotype and age. Values are presented as means ± SEM. *p<0.05; **p<0.01.
Figure 3
Figure 3
Impact of Rarα inactivation on adrenal steroidogenesis. (A) Expression of aldosterone synthase and 11β-hydroxylase in 12 weeks old male and female Rarα+/+ and Rarα−/− mice. (BF) Expression of steroidogenic genes in male and female Rarα+/+ and Rarα−/− mice. mRNA expression of Star (B), Cyp11a1 (C), Cyp11b1 (D) and Cyp11b2 (E) was assessed by RT-qPCR. RT-qPCR were performed on mRNA extracted from 6–11 adrenals from 12 weeks old male and female Rarα+/+ and Rarα−/− mice. (F,G) Measure of plasma aldosterone (F) and corticosterone (G) concentration by mass spectrometry in male mice. (H,I) Plasma renin concentration (PRC) (H) and aldosterone to renin ratio (I). Measure of plasma aldosterone, plasma corticosterone and plasma renin were done on 5–6 animals per group. Values are presented as the mean ± SEM; p values were calculated using a Mann-Whitney test or t-test. For correlation, Pearson calculations were performed. *p < 0.05; **p < 0.01.
Figure 4
Figure 4
Rarα affects vessel architecture and extra cellular matrix composition. (A) Hierarchical clustering of samples using the 243 differentially expressed genes in adrenals from 12 weeks old Rarα+/+ and Rarα−/− male animals (4 animals per group). (B) Volcano plot showing the differentially expressed genes in 12 weeks old Rarα+/+ and Rarα−/− animals. The x-axis is the fold change between the two conditions; the adjusted p value based on –log10 is reported on the y-axis. Genes significantly different are highlighted as green (down-regulated in Rarα−/− mice) or red (up-regulated in Rarα−/− mice) dots. (C, D, E) The expression of genes involved in angiogenesis, Vegfa (C) and Vegfc (D) and in hypoxia, Hif1α (E) was investigated by RT-qPCR on mRNA extracted from 6–11 adrenals from 12 weeks old male and female Rarα+/+ and Rarα−/− mice. (F) The vascular architecture was assessed by Sirius red staining and podocalyxin immunofluorescence and extra cellular matrix integrity by laminin β1 immunofluorescence. Values are presented as the mean ± SEM; p values were calculated using a Mann-Whitney test. **p < 0.01.
Figure 5
Figure 5
Rarα inactivation alters Wnt signaling pathway in 12 weeks old male mice. (A) Expression of β-catenin was evaluated by immunofluorescence in 12 weeks old male and female Rarα+/+ and Rarα−/− mice. (B) Expression and phosphorylation of β-catenin in response to Rarα invalidation. Proteins were extracted from total adrenal and submitted to western blot analysis. Phosphorylation/dephosphorylation in activating (pS552 and pS675) and inactivating (pT41/S45) residues and total expression of β-catenin was investigated. (CF) Quantification of β-catenin expression (C) and of phospho-specific signals in inactivating (D) and activating (E,F) residues was performed in Rarα+/+ and Rarα−/− adrenal. (G-J) The expression of Wnt4 (G), Tcf3 (H), Lef1 (I) and Axin2 (J) was investigated by RT-qPCR on mRNA extracted from 6 to 11 adrenals from Rarα+/+ and Rarα−/− male and female mice. Values are presented as mean ± SEM; p values were calculated using a Mann-Whitney test or t-test. *p < 0.05; **p < 0.01.
Figure 6
Figure 6
Adrenal cortex disorganization persists with aging in Rarα−/− mice. (A) Morphological characterization of adrenals from 52 weeks old male and female Rarα+/+ and Rarα−/− mice. HES staining, Dab-2, aldosterone synthase and 11β-hydroxylase immunofluorescence and Ki67 immunohistochemistry were performed. (B) Number of Dab-2 positive cells in the cortex was determined in 3 to 9 animals of each genotype and sex using an automated molecular imaging platform (Vectra, Perkin Elmer) and is expressed as a percentage of total number of cells in the entire cortex area. (C) Relative proliferative index of adrenals from male and female Rarα+/+ and Rarα−/− mice. Ki67 positive cells were separately counted in the adrenal cortex in 5–6 animals per genotype. (D) Number of nuclei in the adrenal cortex was determined in 3 to 9 animals of each genotype and sex using an automated molecular imaging platform (Vectra, Perkin Elmer). (EH) Expression of steroidogenic genes in male and female Rarα+/+ and Rarα−/− mice. mRNA expression of Star (E), Cyp11a1 (F), Cyp11b1 (G) and Cyp11b2 (H) was assessed by RT-qPCR. RT-qPCR were performed on mRNA extracted from 6–8 adrenals from 52 weeks old male and female Rarα+/+ and Rarα−/− mice. (I,J) Measure of plasma aldosterone (I) and corticosterone (J) concentration by mass spectrometry in male mice. (K,L) Plasma renin concentration (PRC) and aldosterone to renin ratio. Measure of plasma aldosterone, plasma corticosterone and plasma renin were done on 5–6 animals per group. Values are presented as means ± SEM. *p < 0.05; **p < 0.01.
Figure 7
Figure 7
Proposed model for the role of Rarα in adrenocortical development. (A) Rarα regulates the expression of genes involved in the regulation of Wnt non-canonical pathway (Wnt4, Tcf3, Lef1…), angiogenesis (Vegfa) and Extra Cellular Matrix integrity (Fibronectin1, Collagen 3α1…) contributing to the organization of the adrenal cortex. Wnt4 activated pathway contributes to the differentiation of ZG cells, Vegfa to normal angiogenesis and Fibronectin1 and Collagen 3α1 being components of the Extra Cellular Matrix. (B) Model in which a homeostatic equilibrium between Rarα, Wnt and Vegf signaling pathways is required for the normal development of the vasculature and Extra Cellular Matrix structure, leading to normal adrenal cortex organization.

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