Familial Glucocorticoid Receptor Haploinsufficiency by Non-Sense Mediated mRNA Decay, Adrenal Hyperplasia and Apparent Mineralocorticoid Excess

Primary glucocorticoid resistance (OMIM 138040) is a rare hereditary disease that causes a generalized partial insensitivity to glucocorticoid action, due to genetic alterations of the glucocorticoid receptor (GR). Investigation of adrenal incidentalomas led to the discovery of a family (eight affected individuals spanning three generations), prone to cortisol resistance, bilateral adrenal hyperplasia, arterial hypertension and hypokalemia. This phenotype exacerbated over time, cosegregates with the first heterozygous nonsense mutation p.R469[R,X] reported to date for the GR, replacing an arginine (CGA) by a stop (TGA) at amino-acid 469 in the second zinc finger of the DNA-binding domain of the receptor. In vitro, this mutation leads to a truncated 50-kDa GR lacking hormone and DNA binding capacity, devoid of hormone-dependent nuclear translocation and transactivation properties. In the proband's fibroblasts, we provided evidence for the lack of expression of the defective allele in vivo. The absence of detectable mutated GR mRNA was accompanied by a 50% reduction in wild type GR transcript and protein. This reduced GR expression leads to a significantly below-normal induction of glucocorticoid-induced target genes, FKBP5 in fibroblasts. We demonstrated that the molecular mechanisms of glucocorticoid signaling dysfunction involved GR haploinsufficiency due to the selective degradation of the mutated GR transcript through a nonsense-mediated mRNA Decay that was experimentally validated on emetine-treated propositus' fibroblasts. GR haploinsufficiency leads to hypertension due to illicit occupation of renal mineralocorticoid receptor by elevated cortisol rather than to increased mineralocorticoid production reported in primary glucocorticoid resistance. Indeed, apparent mineralocorticoid excess was demonstrated by a decrease in urinary tetrahydrocortisone-tetrahydrocortisol ratio in affected patients, revealing reduced glucocorticoid degradation by renal activity of the 11β-hydroxysteroid dehydrogenase type 2, a GR regulated gene. We propose thus that GR haploinsufficiency compromises glucocorticoid sensitivity and may represent a novel genetic cause of subclinical hypercortisolism, incidentally revealed bilateral adrenal hyperplasia and mineralocorticoid-independent hypertension

Identification of polymorphisms in the human 11beta-hydroxysteroid dehydrogenase type 2 gene promoter: functional characterization and relevance for salt sensitivity

Reduced activity of 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) plays a role in essential hypertension and the sensitivity of blood pressure to dietary salt. Nonconservative mutations in the coding region are extremely rare and do not explain the variable 11beta-HSD2 activity. We focused therefore on the 5'-regulatory region and identified and characterized the first promoter polymorphisms. Transfections of variants G-209A and G-126A into SW620 cells reduced promoter activity and affinity for activators nuclear factor 1 (NF1) and Sp1. Chromatin immunoprecipitation revealed Sp1, NF1, and glucocorticoid receptor (GR) binding to the HSD11B2 promoter. Dexamethasone induced expression of mRNA and activity of HSD11B2. GR and/or NF1 overexpression increased endogenous HSD11B2 mRNA and activity. GR complexes cooperated with NF1 to activate HSD11B2, an effect diminished in the presence of the G-209A variant. When compared to salt-resistant subjects (96), salt-sensitive volunteers (54) more frequently had the G-209A variant, higher occurrence of alleles A4/A7 of polymorphic microsatellite marker, and higher urinary ratios of cortisol to cortisone metabolites. First, we conclude that the mechanism of glucocorticoid-induced HSD11B2 expression is mainly mediated by cooperation between GR and NF1 on the HSD11B2 promoter and, second, that the newly identified promoter variants reduce activity and cooperation of cognate transcription factors, resulting in diminished HSD11B2 transcription, an effect favoring salt sensitivity

Role of DNA methylation in the tissue-specific expression of the CYP17A1 gene for steroidogenesis in rodents

The CYP17A1 gene is the qualitative regulator of steroidogenesis. Depending on the presence or absence of CYP17 activities mineralocorticoids, glucocorticoids or adrenal androgens are produced. The expression of the CYP17A1 gene is tissue as well as species-specific. In contrast to humans, adrenals of rodents do not express the CYP17A1 gene and have therefore no P450c17 enzyme for cortisol production, but produce corticosterone. DNA methylation is involved in the tissue-specific silencing of the CYP17A1 gene in human placental JEG-3 cells. We investigated the role of DNA methylation for the tissue-specific expression of the CYP17A1 gene in rodents. Rats treated with the methyltransferase inhibitor 5-aza-deoxycytidine excreted the cortisol metabolite tetrahydrocortisol in their urine suggesting that treatment induced CYP17 expression and 17alpha-hydroxylase activity through demethylation. Accordingly, bisulfite modification experiments identified a methylated CpG island in the CYP17 promoter in DNA extracted from rat adrenals but not from testes. Both methyltransferase and histone deacetylase inhibitors induced the expression of the CYP17A1 gene in mouse adrenocortical Y1 cells which normally do not express CYP17, indicating that the expression of the mouse CYP17A1 gene is epigenetically controlled. The role of DNA methylation for CYP17 expression was further underlined by the finding that a reporter construct driven by the mouse -1041 bp CYP17 promoter was active in Y1 cells, thus excluding the lack of essential transcription factors for CYP17 expression in these adrenal cells

Insulin, CCAAT/enhancer-binding proteins and lactate regulate the human 11β-hydroxysteroid dehydrogenase type 2 gene expression in colon cancer cell lines.

11β-Hydroxysteroid dehydrogenases (11beta-HSD) modulate mineralocorticoid receptor transactivation by glucocorticoids and regulate access to the glucocorticoid receptor. The isozyme 11beta-HSD2 is selectively expressed in mineralocorticoid target tissues and its activity is reduced in various disease states with abnormal sodium retention and hypertension, including the apparent mineralocorticoid excess. As 50% of patients with essential hypertension are insulin resistant and hyperinsulinemic, we hypothesized that insulin downregulates the 11beta-HSD2 activity. In the present study we show that insulin reduced the 11beta-HSD2 activity in cancer colon cell lines (HCT116, SW620 and HT-29) at the transcriptional level, in a time and dose dependent manner. The downregulation was reversible and required new protein synthesis. Pathway analysis using mRNA profiling revealed that insulin treatment modified the expression of the transcription factor family C/EBPs (CCAAT/enhancer-binding proteins) but also of glycolysis related enzymes. Western blot and real time PCR confirmed an upregulation of C/EBP beta isoforms (LAP and LIP) with a more pronounced increase in the inhibitory isoform LIP. EMSA and reporter gene assays demonstrated the role of C/EBP beta isoforms in HSD11B2 gene expression regulation. In addition, secretion of lactate, a byproduct of glycolysis, was shown to mediate insulin-dependent HSD11B2 downregulation. In summary, we demonstrate that insulin downregulates HSD11B2 through increased LIP expression and augmented lactate secretion. Such mechanisms are of interest and potential significance for sodium reabsorption in the colon

Lactate accumulation in the media upon insulin stimulation and insulin-dependent down-regulation of 11beta-HSD2 activity.

<p>(<i>A</i>) Dose-response effect of insulin on L-lactate production in cultured HT-29 cells after 24 h incubation. The concentration in lactate found in the media of HT-29 cells after 24 h of culture is reported above the bars (Mean +/− SEM). (<i>B</i>) 11beta-HSD2 activity in cultured HT-29 and HCT116 cells exposed to exogenous L-lactate for 3 h. (<i>C</i>) 24 h L-lactate production in cultured HT-29 cells exposed to DCA alone or in combination with insulin. (<i>D</i>) 11beta-HSD2 activity in cultured HT-29 cells exposed to DCA alone or in combination with insulin.</p

Schematic representation of the insulin pathway and its regulation by sustained insulin stimulation in HT-29.

<p>mRNAs were quantified 24 h after insulin (10⁻⁷ M) treatment using RT² Profiler PCR Arrays PAHS-30C. Up-regulated transcripts are shown in red and down-regulated transcripts are shown in green.</p

Insulin-dependent regulation of the 11beta-HSD2 protein level and role of the CCAAT/enhancer-binding protein (C/EBP) family.

<p>(<i>A</i>) Concentration-dependent effects of insulin on 11beta-HSD2, C/EBP alpha, and C/EBP beta protein levels. HT-29 cells were cultured for 24 h without and with increasing concentrations of insulin (10⁻⁹–10⁻⁵ M), then harvested for Western blotting to evaluate expression of 11beta-HSD2, C/EBP alpha, C/EBP beta. (<i>B</i>) Concentration-dependent effects of insulin on C/EBP alpha, C/EBP beta, and C/EBP delta mRNA expression. HT-29 cells were treated like in (A). The level of C/EBP alpha (open circles), C/EBP beta (open squares), and C/EBP delta (filled triangles) mRNA was measured using qRT-PCR with S18 as internal control. Expression levels in treated cells were normalized to untreated controls (100%). Representative data for at least three independent experiments. The relative intensity was determined by densitometric scanning. The ratio of relative densities of 11beta-HSD2 to beta-actin in cells cultured in the abscence of hormone was considered as 100% (control). The ratio of relative densities of nuclear extract proteins to HDAC in cells cultured without hormone was considered as 100% (control). * LIP was undetectable in the control samples, so the LAP/LIP ratio was not calculated. (<i>C, D</i>) Silencing of C/EBP alpha (<i>C</i>) and C/EBP beta (<i>D)</i> was performed using siRNA. The expression of C/EBP alpha, C/EBP beta (left panel) and HSD11B2 (right panel) mRNA was measured using qRT-PCR.</p

Sustained insulin treatment diminished the 11beta-HSD2 expression and activity in HT-29 cells.

<p>(<i>A</i>) 11beta-HSD2 activity was measured by ³H-cortisol/cortisone conversion assay in colonic cell lines 24 h after incubation with insulin (10⁻¹¹–10⁻⁷ M). The activity measured for HCT116 in absence of insulin was set as 100%. (<i>B</i>) Dose-response effect of insulin (10⁻⁹–10⁻⁵ M) on HSD11B2 mRNA (gray bars) and activity (curve) in HT-29 cells treated for 24 h. (<i>C</i>) Time-dependent effect of insulin (10⁻⁷ M) on HSD11B2 mRNA (gray bars) and activity (curve) in HT-29 cells. (<i>D</i>) Time-dependent effect of insulin (10⁻⁷ M) on 11beta-HSD2 protein level.</p

Binding of C/EBP alpha/beta on human HSD11B2 promoter.

<p><i>(A) Nuclear proteins isolated from HT-29 cells bind to identified C/EBP alpha/beta sites.</i>4 µg of nuclear extracts isolated from insulin treated (for the indicated period of time, 10⁻⁷ M) or untreated HT-29 cells were incubated with radiolabeled probe encompassing the consensus C/EBP alpha/beta site in the presence or absence of non-radiolabeled (100×) competitor probe (cons C/EBP alpha/beta or mut C/EBP alpha/beta) (lanes1–7). Arrows indicate C/EBP alpha/beta / DNA shifts (C1, C2, C3) separated from free probe by gel electrophoresis. The complex C3 is formed in presence of radiolabeled −198 C/EBP alpha/beta probe (lanes 14–17) while complex C2 is formed in presence of radiolabeled −4362 C/EBP alpha/beta probe (lane 22–25). <i>(B) Nuclear proteins isolated from HT-29 cells bind to the consensus SP1 site.</i> Nuclear extracts isolated from insulin treated (for the indicated period of time, 10⁻⁷ M) or untreated HT-29 cells were incubated with radiolabeled probe encompassing the consensus SP1 site with and without non-radiolabeled (100X) competitor probe (cons SP1, lane 5or mut SP1, lane 6). The arrow indicates SP1/DNA shifts separated from free probe by gel electrophoresis. The complex intensity increased modestly with insulin treatment. The specific shift was abolished by the cold cons SP1 probe (lane 5) while not affected when mut SP1 probe was used as competitor (lane 6). <i>(C) Chromatin immunoprecipitation (ChIP) analysis of C/EBP alpha and C/EBP beta during insulin stimulation in HT-29 cells.</i> ChIPs were performed from untreated (W/O) and insulin induced (1–24 h) HT-29 cells using antibodies specific for C/EBP alpha (middle panel) and C/EBP beta (bottom panel), a no-antibody control (NO). The precipitated chromatin was analyzed using primers specific for the human HSD11B2 promoter. The DNA fragments were amplified with PCR primers to detect a 210 bp fragment containing the potential −177 and −198 C/EBP sites within the HSD11B2 promoter. Input chromatin is represented in upper panel.</p