Adrenal cortex physiology relies on functional zonation, essential for production of aldosterone by outer zona glomerulosa (ZG) and glucocorticoids by inner zona fasciculata (ZF). The cortex undergoes constant cell renewal, involving recruitment of subcapsular progenitors to ZG fate and subsequent lineage conversion to ZF identity. Here we show that WNT4 is an important driver of WNT pathway activation and subsequent ZG differentiation and demonstrate that PKA activation prevents ZG differentiation through WNT4 repression and WNT pathway inhibition. This suggests that PKA activation in ZF is a key driver of WNT inhibition and lineage conversion. Furthermore, we provide evidence that constitutive PKA activation inhibits, whereas partial inactivation of PKA catalytic activity stimulates β-catenin-induced tumorigenesis. Together, both lower PKA activity and higher WNT pathway activity lead to poorer prognosis in adrenocortical carcinoma (ACC) patients. These observations suggest that PKA acts as a tumour suppressor in the adrenal cortex, through repression of WNT signalling.

Adrenal glands are zonated endocrine organs that are essential in controlling body homeostasis. How zonation is induced and maintained and how renewal of the adrenal cortex is ensured remain a mystery. Here we show that capsular RSPO3 signals to the underlying steroidogenic compartment to induce β-catenin signaling and imprint glomerulosa cell fate. Deletion of RSPO3 leads to loss of SHH signaling and impaired organ growth. Importantly, Rspo3 function remains essential in adult life to ensure replenishment of lost cells and maintain the properties of the zona glomerulosa. Thus, the adrenal capsule acts as a central signaling center that ensures replacement of damaged cells and is required to maintain zonation throughout life.

No abstract

Adrenal cortex steroids are essential for body homeostasis, and adrenal insufficiency is a life-threatening condition. Adrenal endocrine 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 phosphodiesterases 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.

Chronic exposure to high circulating levels of glucocorticoids has detrimental effects on health, including metabolic abnormalities, as exemplified in Cushing’s syndrome (CS). Magnetic resonance imaging (MRI) studies have found volumetric changes in gray and white matter of the brain in CS patients during the course of active disease, but also in remission. In order to explore this further, we performed MRI-based brain volumetric analyses in the AdKO mouse model for CS, which presents its key traits. AdKO mice had reduced relative volumes in several brain regions, including the corpus callosum and cortical areas. The medial amygdala, bed nucleus of the stria terminalis, and hypothalamus were increased in relative volume. Furthermore, we found a lower immunoreactivity of myelin basic protein (MBP, an oligodendrocyte marker) in several brain regions but a paradoxically increased MBP signal in the male cingulate cortex. We also observed a decrease in the expression of glial fibrillary acidic protein (GFAP, a marker for reactive astrocytes) and ionized calcium-binding adapter molecule 1 (IBA1, a marker for activated microglia) in the cingulate regions of the anterior corpus callosum and the hippocampus. We conclude that long-term hypercorticosteronemia induced brain region-specific changes that might include aberrant myelination and a degree of white matter damage, as both repair (GFAP) and immune (IBA1) responses are decreased. These findings suggest a cause for the changes observed in the brains of human patients and serve as a background for further exploration of their subcellular and molecular mechanisms.

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.

BackgroundLKB1 is an evolutionary conserved kinase implicated in a wide range of cellular functions including inhibition of cell proliferation, regulation of cell polarity and metabolism. When Lkb1 is inactivated in the liver, glucose homeostasis is perturbed, cellular polarity is affected and cholestasis develops. Cholestasis occurs as a result from deficient bile duct development, yet how LKB1 impacts on biliary morphogenesis is unknown.Methodology/Principal FindingsWe characterized the phenotype of mice in which deletion of the Lkb1 gene has been specifically targeted to the hepatoblasts. Our results confirmed that lack of LKB1 in the liver results in bile duct paucity leading to cholestasis. Immunostaining analysis at a prenatal stage showed that LKB1 is not required for differentiation of hepatoblasts to cholangiocyte precursors but promotes maturation of the primitive ductal structures to mature bile ducts. This phenotype is similar to that obtained upon inactivation of Notch signaling in the liver. We tested the hypothesis of a functional overlap between the LKB1 and Notch pathways by gene expression profiling of livers deficient in Lkb1 or in the Notch mediator RbpJκ and identified a mutual cross-talk between LKB1 and Notch signaling. In vitro experiments confirmed that Notch activity was deficient upon LKB1 loss.ConclusionLKB1 and Notch share a common genetic program in the liver, and regulate bile duct morphogenesis.

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.