LGR5 Activates Noncanonical Wnt Signaling and Inhibits Aldosterone Production in the Human Adrenal.

CONTEXT: Aldosterone synthesis and cellularity in the human adrenal zona glomerulosa (ZG) is sparse and patchy, presumably due to salt excess. The frequency of somatic mutations causing aldosterone-producing adenomas (APAs) may be a consequence of protection from cell loss by constitutive aldosterone production. OBJECTIVE: The objective of the study was to delineate a process in human ZG, which may regulate both aldosterone production and cell turnover. DESIGN: This study included a comparison of 20 pairs of ZG and zona fasciculata transcriptomes from adrenals adjacent to an APA (n = 13) or a pheochromocytoma (n = 7). INTERVENTIONS: Interventions included an overexpression of the top ZG gene (LGR5) or stimulation by its ligand (R-spondin-3). MAIN OUTCOME MEASURES: A transcriptome profile of ZG and zona fasciculata and aldosterone production, cell kinetic measurements, and Wnt signaling activity of LGR5 transfected or R-spondin-3-stimulated cells were measured. RESULTS: LGR5 was the top gene up-regulated in ZG (25-fold). The gene for its cognate ligand R-spondin-3, RSPO3, was 5-fold up-regulated. In total, 18 genes associated with the Wnt pathway were greater than 2-fold up-regulated. ZG selectivity of LGR5, and its absence in most APAs, were confirmed by quantitative PCR and immunohistochemistry. Both R-spondin-3 stimulation and LGR5 transfection of human adrenal cells suppressed aldosterone production. There was reduced proliferation and increased apoptosis of transfected cells, and the noncanonical activator protein-1/Jun pathway was stimulated more than the canonical Wnt pathway (3-fold vs 1.3-fold). ZG of adrenal sections stained positive for apoptosis markers. CONCLUSION: LGR5 is the most selectively expressed gene in human ZG and reduces aldosterone production and cell number. Such conditions may favor cells whose somatic mutation reverses aldosterone inhibition and cell loss.This work was supported by MJB is an NIHR Senior Investigator NF-SI-0512–10 052; LHS holds a British Heart Foundation PhD studentship FS/11/35/28871; JZ holds a Cambridge Overseas Trust Scholarship; AEDT is funded by the Wellcome Trust Translational Medicine and Therapeutics program 085 686/Z/08/A, and by Singapore A* program; EABA was supported by the Austin Doyle Award (Servier Australia); LHS, JZ and EABA were additionally supported by the NIHR Cambridge Biomedical Research Centre; GM are funded by MRC Programme Grants RDAG/287 and SKAG/001 awarded to Ashok Venkitaraman.This is the author accepted manuscript. The final version is available from the Endocrine Society via http://dx.doi.org/10.1210/jc.2015-173

DACH1, a zona glomerulosa selective gene in the human adrenal, activates transforming growth factor-β signaling and suppresses aldosterone secretion.

Common somatic mutations in CACNAID and ATP1A1 may define a subgroup of smaller, zona glomerulosa (ZG)-like aldosterone-producing adenomas. We have therefore sought signature ZG genes, which may provide insight into the frequency and pathogenesis of ZG-like aldosterone-producing adenomas. Twenty-one pairs of zona fasciculata and ZG and 14 paired aldosterone-producing adenomas from 14 patients with Conn's syndrome and 7 patients with pheochromocytoma were assayed by the Affymetrix Human Genome U133 Plus 2.0 Array. Validation by quantitative real-time polymerase chain reaction was performed on genes >10-fold upregulated in ZG (compared with zona fasciculata) and >10-fold upregulated in aldosterone-producing adenomas (compared with ZG). DACH1, a gene associated with tumor progression, was further analyzed. The role of DACH1 on steroidogenesis, transforming growth factor-β, and Wnt signaling activity was assessed in the human adrenocortical cell line, H295R. Immunohistochemistry confirmed selective expression of DACH1 in human ZG. Silencing of DACH1 in H295R cells increased CYP11B2 mRNA levels and aldosterone production, whereas overexpression of DACH1 decreased aldosterone production. Overexpression of DACH1 in H295R cells activated the transforming growth factor-β and canonical Wnt signaling pathways but inhibited the noncanonical Wnt signaling pathway. Stimulation of primary human adrenal cells with angiotensin II decreased DACH1 mRNA expression. Interestingly, there was little overlap between our top ZG genes and those in rodent ZG. In conclusion, (1) the transcriptome profile of human ZG differs from rodent ZG, (2) DACH1 inhibits aldosterone secretion in human adrenals, and (3) transforming growth factor-β signaling pathway is activated in DACH1 overexpressed cells and may mediate inhibition of aldosterone secretion in human adrenals.The work was funded by a National Institute for Health Research (NIHR) Senior Investigator Award (NF-SI-0512-10052) to M.J. Brown, the Wellcome Trust (085687/Z/08/A), and the NIHR Cambridge Biomedical Research Centre (Cardiovascular). J. Zhou and E.A.B. Azizan were supported by The Cambridge Commonwealth, European & International Trust. J. Zhou was also supported by the Sun Hung Kai Properties–Kwoks’ Foundation.This is the final version of the article. It first appeared from the American Heart Association via http://dx.doi.org/10.1161/HYP.000000000000002

Pregnancy, Primary Aldosteronism, and Adrenal CTNNB1 Mutations.

This is a metadata record relating to an article that cannot be shared due to publisher copyright.Recent discoveries of somatic mutations permit the recognition of subtypes of aldosterone-producing adenomas with distinct clinical presentations and pathological features. Here we describe three women with hyperaldosteronism, two who presented in pregnancy and one who presented after menopause. Their aldosterone-producing adenomas harbored activating mutations of CTNNB1, encoding β-catenin in the Wnt cell-differentiation pathway, and expressed LHCGR and GNRHR, encoding gonadal receptors, at levels that were more than 100 times as high as the levels in other aldosterone-producing adenomas. The mutations stimulate Wnt activation and cause adrenocortical cells to de-differentiate toward their common adrenal-gonadal precursor cell type. (Funded by grants from the National Institute for Health Research Cambridge Biomedical Research Centre and others.).AEDT is supported by Agency for Science, Technology and Research (A*STAR) Singapore and the Wellcome Trust (085686/Z/08/A); SG and LHS are supported by the British Heart Foundation (FS/14/75/31134; FS/11/35/28871); EABA was supported by the Tunku Abdul Rahman Centenary Fund (St Catharine's College, Cambridge, UK) and the Austin Doyle Award (Servier Australia). JZ was supported by the Cambridge Overseas Trust Scholarship. MG is supported by the NIHR Cambridge Biomedical Research Centre (Metabolic), and MB is supported by the MRC (U105192713). The work was funded by the NIHR Cambridge Biomedical Research Centre (Cardiovascular) and an NIHR Senior Investigator award (NF-SI-0512-10052) to MJB

Description and performance of track and primary-vertex reconstruction with the CMS tracker

A description is provided of the software algorithms developed for the CMS tracker both for reconstructing charged-particle trajectories in proton-proton interactions and for using the resulting tracks to estimate the positions of the LHC luminous region and individual primary-interaction vertices. Despite the very hostile environment at the LHC, the performance obtained with these algorithms is found to be excellent. For tbar t events under typical 2011 pileup conditions, the average track-reconstruction efficiency for promptly-produced charged particles with transverse momenta of p(T) > 0.9GeV is 94% for pseudorapidities of |η| < 0.9 and 85% for 0.9 < |η| < 2.5. The inefficiency is caused mainly by hadrons that undergo nuclear interactions in the tracker material. For isolated muons, the corresponding efficiencies are essentially 100%. For isolated muons of p(T) = 100GeV emitted at |η| < 1.4, the resolutions are approximately 2.8% in p(T), and respectively, 10μm and 30μm in the transverse and longitudinal impact parameters. The position resolution achieved for reconstructed primary vertices that correspond to interesting pp collisions is 10–12μm in each of the three spatial dimensions. The tracking and vertexing software is fast and flexible, and easily adaptable to other functions, such as fast tracking for the trigger, or dedicated tracking for electrons that takes into account bremsstrahlung