CYP11B2 Expression Research Articles

Lysophosphatidic Acid-Mediated GPR35 Signaling in CX3CR1+ Macrophages Regulates Intestinal Homeostasis

Single-nucleotide polymorphisms in the gene encoding G protein-coupled receptor 35 (GPR35) are associated with increased risk of inflammatory bowel disease. However, the mechanisms by which GPR35 modulates intestinal immune homeostasis remain undefined. Here, integrating zebrafish and mouse experimental models, we demonstrate that intestinal Gpr35 expression is microbiota dependent and enhanced upon inflammation. Moreover, murine GPR35+ colonic macrophages are characterized by enhanced production of pro-inflammatory cytokines. We identify lysophosphatidic acid (LPA) as a potential endogenous ligand produced during intestinal inflammation, acting through GPR35 to induce tumor necrosis factor (Tnf) expression in macrophages. Mice lacking Gpr35 in CX3CR1+ macrophages aggravate colitis when exposed to dextran sodium sulfate, which is associated with decreased transcript levels of the corticosterone-generating gene Cyp11b1 and macrophage-derived Tnf. Administration of TNF in these mice restores Cyp11b1 expression and intestinal corticosterone production and ameliorates DSS-induced colitis. Our findings indicate that LPA signals through GPR35 in CX3CR1+ macrophages to maintain TNF-mediated intestinal homeostasis.

Incomplete Pattern of Steroidogenic Protein Expression in Functioning Adrenocortical Carcinomas.

Autonomous steroid secretion is a common feature of adrenocortical carcinomas (ACC), although not always clinically evident owing to inefficient steroidogenesis with increased release of steroid precursors. Our study aim was to analyze the expression profile of four key proteins involved in the steroidogenesis cascade, in different adrenocortical tumors. Expression of proteins involved in steroidogenesis, namely steroidogenic acute regulatory protein (StAR), 11β-hydroxylase (CYP11B1), aldosterone synthase (CYP11B2) and 17α-hydroxylase (CYP17A1), were analyzed by immunohistochemistry in ACC (n = 14), adenomas presenting with Cushing’s syndrome (ACAc) (n = 11) and clinically non-functioning adenomas (ACAn) (n = 15). A percentage of the stained area for each protein was analyzed using ImageJ software for computerized morphometric quantification. CYP11B1, StAR and CYP17A1 expression were significantly lower in ACC when compared to ACAc. In addition, ACC presented co-staining cells for CYP11B1 and CYP11B2. CYP11B1 was the steroidogenic enzyme with the most discriminative power to distinguish ACC from ACAc, with a sensitivity of 100%, specificity of 92%, and an expression higher than 4.44%, indicating the presence of a cortisol secreting adenoma. ACC depicts an incomplete pattern of steroidogenic protein expression, with decreased CYP11B1 and CYP17A1, which could explain the predominant secretion of steroid precursors.

Pathogenesis and treatment of primary aldosteronism.

Early diagnosis and appropriate treatment of primary aldosteronism, the most frequent cause of secondary hypertension, are crucial to prevent deleterious cardiovascular outcomes. In the past decade, the discovery of genetic abnormalities responsible for sporadic and familial forms of primary aldosteronism has improved the knowledge of the pathogenesis of this disorder. Mutations in genes encoding ion channels and pumps lead to increased cytosolic concentrations of calcium in zona glomerulosa cells, which triggers CYP11B2 expression and autonomous aldosterone production. Improved understanding of the mechanisms underlying the disease is key to improving diagnostics and to developing and implementing targeted treatments. This Review provides an update on the genetic abnormalities associated with sporadic and familial forms of primary aldosteronism, their frequency among different populations and the mechanisms explaining excessive aldosterone production and adrenal nodule development. The possible effects and uses of these findings for improving the diagnostics for primary aldosteronism are discussed. Furthermore, current treatment options of primary aldosteronism are reviewed, with particular attention to the latest studies on blood pressure and cardiovascular outcomes following medical or surgical treatment. The new perspectives regarding the use of targeted drug therapy for aldosterone-producing adenomas with specific somatic mutations are also addressed.

Modified expression of vitamin D receptor and CYP27B1 in denervation-induced muscle atrophy

The vitamin D pathway is related to the mass and function of skeletal muscles. Several studies have demonstrated the role of vitamin D receptor (VDR) and CYP27B1 in skeletal muscles, suggesting that these proteins may regulate skeletal muscles and their function. However, it remains unclear whether the expression of VDR and CYP27B1 is modified in skeletal muscle atrophy. We investigated whether denervation-induced muscle atrophy is associated with altered expression of VDR and CYP27B1 in murine skeletal muscles. Skeletal muscles were excised from C57BL/6J mice, 3 and 7 days after the mice underwent denervation surgery. Denervation induced muscle atrophy and enhanced the expression of MuRF1 and Atrogin-1 in the gastrocnemius and soleus. The protein expression of VDR was increased in the denervated gastrocnemius; in contrast, denervation decreased the protein expression of CYP27B1 in the gastrocnemius and soleus. These results suggest that denervation-induced muscle atrophy is associated with changes in the expression of vitamin D-related proteins in murine skeletal muscles.

Transcriptome analysis of Clarias magur brain and gonads suggests neuro-endocrine inhibition of milt release from captive GnRH-induced males

Transcriptome analysis of Clarias magur brain and gonads at preparatory, mature, 6 and 16 h post-GnRH injection (hpi) stages yielded 9.5 GB data with 39,738 contigs. Sequences of 45 reproductive genes were identified for the first time in C. magur along with unique and differentially expressed genes. The expression of 20 genes was validated by qRT-PCR. Upregulation of Cyp11A1, Cyp17A1 and FTZF1 genes in the 16hpi testis accompanied by the 17β-HSD3 expression indicates testosterone (T) synthesis in response to LH surge, while reduced expression of CYP11B1 suggests a high T: 11-KT ratio. It is evident by the gene expression analysis that the inhibitory neurotransmitter GABA, altered T: 11-KT, increased testicular bile acids, and oxytocin-like neuropeptide in the male brain, appear to be involved in arresting the pulsatile motion of testicular smooth muscles. The work generates important leads for an effective induced breeding strategy for silurid catfish.

Transcriptional activity of vitamin D receptor in human periodontal ligament cells is diminished under inflammatory conditions.

BackgroundAlthough vitamin D3 deficiency is considered as a risk factor for periodontitis, supplementation during periodontal treatment has not been shown to be beneficial to date. Human periodontal ligament cells (hPDLCs) are regulated by vitamin D3 and play a fundamental role in periodontal tissue homeostasis and inflammatory response in periodontitis. The aim of this study is to investigate possible alterations of the vitamin D3 activity in hPDLCs under inflammatory conditions.MethodsCells isolated from six different donors were treated with either 1,25(OH)2D3 (0 to 10 nM) or 25(OH)D3 (0 to 100 nM) in the presence and absence of ultrapure or standard Porphyromonas gingivalis lipopolysaccharide (PgLPS), Pam3CSK4, or interferon‐γ for 48 hours. Additionally, nuclear factor (NF)‐κB inhibition was performed with BAY 11‐7082. The bioactivity of vitamin D in hPDLCs was assessed based on the gene expression levels of vitamin D receptor (VDR)‐regulated genes osteocalcin and osteopontin. Additionally, VDR and CYP27B1 expression levels were measured.ResultsThe vitamin D3‐induced increase of osteocalcin and osteopontin expression was significantly decreased in the presence of standard PgLPS and Pam3CSK4, which was not observed by ultrapure PgLPS. Interferon‐y had diverse effects on the response of hPDLCs to vitamin D3 metabolites. NF‐kB inhibition abolished the effects of standard PgLPS and Pam3CSK4. Standard PgLPS and Pam3CSK4 increased VDR expression in the presence of vitamin D3. CYP27B1 expression was not affected by vitamin D3 and inflammatory conditions.ConclusionsThis study indicates that the transcriptional activity of VDR is diminished under inflammatory conditions, which might mitigate the effectiveness of vitamin D3 supplementation during periodontal treatment.

Local expression profiles of vitamin D-related genes in airways of COPD patients

Treatment of Chronic Obstructive Pulmonary Disease (COPD) is based on bronchodilation, with inhaled corticosteroids or azithromycin associated when frequent exacerbations occur. Despite the proven benefits of current treatment regimens, the need for new interventions in delineated subgroups remains. There is convincing evidence for oral vitamin D supplementation in reducing exacerbations in COPD patients severely deficient for circulating vitamin D. However, little is known about local vitamin D metabolism in the airways and studies examining expression of the vitamin D receptor (VDR), the activating enzyme (CYP27B1) and inactivating enzyme (CYP24A1) of vitamin D in lung tissue of COPD patients are lacking. Therefore, the expression and localization of key enzymes and the receptor of the vitamin D pathway were examined in tissue of 10 unused donor lungs and 10 COPD explant lungs. No differences in the expression of CYP27B1 and CYP24A1 were found. Although protein expression of VDR was significantly lower in COPD explant tissue, there was no difference in downstream expression of the antimicrobial peptide cathelicidin. Whereas CYP27B1 and CYP24A1 were present in all layers of the bronchial epithelium, VDR was only expressed at the apical layer of a fully differentiated bronchial epithelium with no expression in vascular endothelial cells. By contrast, CYP24A1 expression was highly present in lung endothelial cells suggesting that systemic vitamin D can be inactivated before reaching the epithelial compartment and the tissue immune cells. These data support the idea of exploring the role of vitamin D inhalation in patients with COPD.

Abstract D114: VIitamin D signaling of immune-related genes in diverse prostate cancer cancer cell lines

Abstract BACKGROUND: African American (AA) men are disproportionately affected by both prostate cancer (PCa) and vitamin D deficiency compared with European American (EA) men. Vitamin D deficiency is linked to increased PCa aggressiveness and mortality. Inflammation also plays an important role in PCa pathogenesis and progression, and expression of immune-related in genes in PCa tissues differs significantly between AAs and EAs. While evidence linking vitamin D and immune response to PCa remains scarce, previous studies demonstrated that vitamin D has anti-inflammatory effects in prostate tissue and mediates immune-related gene expression. Therefore, we hypothesize that vitamin D deficiency in AA men may alter the expression of immune-related genes associated with prostatic inflammation and PCa progression. METHODS: This study examined the effects of vitamin Don the expression of vitamin D- and immune-related genes. Weexposed a racially diverse panel of PCa cell lines—MDA-PCa-2b (AA), RC77T/E (AA), PC3 (EA), 22Rv1 (EA), and DU145 (EA)—to various concentrations (2.5 nM, 10 nM, 50 nM) of 25-hydroxyvitamin D (25(OH)D) and 1,25 dihydroxyvitamin D (1,25(OH)2D) for 24 hours, and quantified transcript levels and protein expression of VDR, RXRa, RXRb, LRP2, CYP27B1, GDF15, GFRAL, TNFa, IL6, IL8, and IGFBP3 with qPCR and immunoblotting. RESULTS:All cell lines expressed basal levels of VDR, RXRa, and RXRbconfirming the function of vitamin D signaling within PCa cells. Of the downstream immune-related genes tested, we observed a trend demonstrating that vitamin D treatment induced the greatest upregulation in either transcript and/or protein levels among the AA PCa cell lines RC77T/E and/or MDA-PCa-2b. In particular, LRP2, GDF15, TNFa, IL6, IL8, and IGFBP3 significantly increased in AA PCa cell lines exposed to either 25(OH)D or 1,25(OH)2D. CONCLUSIONS: These findings provide insight into a potential differential regulation of immune-related pathways by vitamin D in AA men compared to EA men with PCa. Further studies are warranted to better define the immune profile in prostatic tissue in response to vitamin D deficiency in AA men, and the downstream inflammatory effects on the microenvironment and tumor aggressiveness. Citation Format: Madhavi Bathina, Leanne Woods-Burnham, Mya S Walker, K Sean Kimbro, Rick A Kittles. VIitamin D signaling of immune-related genes in diverse prostate cancer cancer cell lines [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr D114.

OR34-02 Somatic Transmembrane Domain Mutations of a Cell Adhesion Molecule, CADM1, Cause Primary Aldosteronism by Preventing Gap Junction Communication Between Adrenocortical Cells

Primary Aldosteronism (PA) is the commonest curable cause of hypertension. Whole exome sequencing (WES) in 2011 and 2013 identified common somatic mutations in genes regulating membrane polarisation in 60–80% of aldosterone-producing adenomas (APA). We undertook WES on 39 consecutive APAs in search of further variants. 1 APA revealed a somatic mutation (Val380Asp) within the single transmembrane domain of Cell Adhesion Molecule 1 (CADM1). An adjacent mutation (Gly379Asp) was discovered on WES from a PA patient in Munich.Both short and long isoforms (442 & 453 residues) of wild-type (WT) and both mutant CADM1 genes were cloned into lentivirus vectors and each transduced into adrenocortical (H295R) cells to assess its effect on aldosterone secretion and other parameters. Previous studies in pancreatic islet cells suggested a role of CADM1 in regulating gap junction (GJ) communication. To assess this we microinjected single WT or mutant H295R cells with the GJ permeable dye calceinAM and counted the dye-positive cells after 1 hour. The effect of inhibiting or silencing GJs in H295R cells using peptide gap27 or a Dharmacon smartpool was assessed. H295R cells were also co-transfected with WT or mutant CADM1 and the GJ protein CX43, tagged with the mApple fluorophore. These were mixed with cells transfected with CX43-Venus, allowing confocal visualisation of GJ formation. Protein modelling was undertaken to determine whether Asp in the intramembranous domain changes angulation of CADM1.All mutant isoforms had consistently different effects, shown as a range compared to WT. Cells transduced with mutant CADM1 showed 3-6-fold increase in aldosterone secretion (p<0.01) and 10-20-fold increase in CYP11B2 expression (p<0.001) compared to WT. Dye transfer assays showed paucity of dye transfer between neighbouring mutant CADM1 cells, while calcein passed easily through GJs in WT cells. CX43 inhibition increased aldosterone secretion 2-fold (p<0.01), and CYP11B2 expression 3 to 8-fold (<0.001). Knock-down of GJ proteins increased aldosterone secretion 1.5-fold (p<0.01) and CYP11B2 expression 1.7-fold (p<0.001).Protein modelling showed mutations to increase the angle of ectodomains to cell membrane, from 49o in WT cells, to 62o and 90o in Gly379Asp and Val380Asp respectively; increasing inter-cell distance from 21.2nm to 24.7 and 27.9nm. Mixing of Venus and mApple-tagged CX43 transfected cells showed fewer intact GJ channels in cells co-transfected with mutant compared to WT CADM1 [mutant 42/291 (14.4%) VS WT 68/212 (32.1%) p<0.001].The CADM1 mutations shows the importance of membrane proteins in aldosterone regulation to extend beyond ion channels and transporters. A key role may be to bring opposing CX43 hemichannels close enough to form GJ channels, permitting the oscillating Ca2+ currents which regulate aldosterone in intact adrenal slices.

SAT-224 Recurrent Co-Driver Mutation in CTNNB1-Mutant Aldosterone-producing Adenomas (APA), Causing Reversible Hypertension in Puberty, Pregnancy or Menopause

Background: Three patients with a syndrome of LH/HCG-activated primary aldosteronism in pregnancy or menopause carrying somatic CTNNB1 mutations were reported four years ago (Teo et al. NEJM 2015). This proved but half the story. Diagnosis of an APA in a 12-year old boy with severe hypertension revealed an apparently essential co-driver mutation. Methods: WES of tumour and blood was performed in the pubertal boy. Candidate genes were Sanger sequenced in other APAs from GB/Ireland, and France with known or suspected CTNNB1 mutations. LHCGR, GNRHR and CYP11B2 expression were measured in all available patients’ APAs and the adjacent adrenal gland (AAG) by RT-PCR. RNA and gDNA from the zona glomerulosa (ZG) of the proband’s AAG were collected by laser capture microdissection for Sanger sequencing of GNA11 and CTNNB1. Function of mutant genes was assessed by measurement of aldosterone production and LHCGR expression by immunofluorescence (IFC) in NCI-H295R adrenocortical cells and primary human APA cells. Results: The proband’s APA contained a p.(S45F) somatic mutation in CTNNB1, and a p.(Q209P) somatic mutation of the GTPase-activating residue (Q209) in GNA11. Mutations of Q209, to P or H, were also found in six other GB/Irish patients with previously identified mutations of CTNNB1 (S33C, G34R, T41A, S45F, or S45P). All seven patients remain normotensive 2-12 years post-adrenalectomy, including some with long-standing pre-operative hypertension. Four of the 13 French patients with CTNNB1 exon 3 mutant APAs have somatic mutation of Q209 of either GNA11 (n=3) or GNAQ. In comparison with their own AAG, the GB/Irish double mutant APAs showed an increase in expression of LHCGR, CYP11B2 and GNRHR by 32-166, 158-18980, and 1174-6642 fold, respectively. All four French double-mutants had >10 fold higher LHCGR than APAs with single mutations of CTNNB1 or other genes. Hyperplasia of ZG was observed in the ZG of the boy’s AAG but no APCC was detected. Homozygous or heterozygous Q209P mutation of GNA11 was detected in multiple ZG samples in RNA and/or gDNA but WT in CTNNB1 exon 3. H295R cells (CTNNB1 S45P) were GNA11 WT. Overexpression of GNA11 Q209 mutation increased aldosterone secretion to 465% of GNA11 WT overexpressing cells (n=6, P<0.001) and CYP11B2 expression was also increased several-fold. Smaller increases were seen in primary human adrenal cells after double-transfection by GNA11 and CTNNB1 mutants (n=3, P<0.001). This also caused membrane expression of LHCGR, visualised by IFC. Conclusions: APAs with double mutation of GNA11/GNAQ Q209 and CTNNB1 have a distinct phenotype, in which hypertension is triggered by high LH or HCG, and cured in all cases by adrenalectomy. GNA11/Q mediates the aldosterone response to ANGII, and the Q209 codon is analogous to the Q227 of GNAS, mutated in McCune Albright. Mosaicism for GNA11 may cause ZG hyperplasia.

SUN-LB93 Mineralocorticoid Receptor Mediates Sex-Dependent Anticontractile Effect of Perivascular Adipose Tissue in Obese Mice

Obesity, a condition of excessive fat mass and subclinical inflammation, reached epidemic proportions with higher prevalence in women compared to men worldwide. Expansion of the perivascular adipose tissue (PVAT) is observed in obesity and clinical studies indicate a positive correlation between PVAT amount and body mass index. PVAT, a fat depot surrounding most of the vessels, modulates vascular function by releasing PVAT-derived factors such as adipokines that exert anticontractile effect in health individuals. Despite sexual dimorphism on PVAT morphology, it is still unknown whether or not there is sex differences in the PVAT modulating vascular function in the setting of obesity. Aldosterone-mineralocorticoid receptor (MR) signaling pathway has been demonstrated to be adipogenic and proinflammatory in classical fat depots and treatment with MR antagonists (A) might reverse vascular dysfunction and remodeling in obese models, especially in female sex. Therefore, we aimed to evaluate the anticontractile effect of PVAT in male and female obese mice and hypothesized that MR signaling would be involved in possible sex differences in PVAT dysfunction in obesity. Male and female C57Bl6/J mice were fed a chow or a high-fat diet (HFD, 60% energy from fat) for 20 weeks. At the last 4 weeks of HFD, female and male mice were treated with the MRA spironolactone (Spi, 100 mg/kg/day). HFD feeding significantly increased body weight and visceral adipose tissue, which was not modified by Spi treatment in both sexes. Resistance mesenteric arteries were isolated with or without PVAT and mounted in a wire myograph to evaluate vascular contractile responses. Lean male and female mice PVAT had an anticontractile effect in the response to phenylephrine that was greater in females than males. The anticontractile effect of PVAT was significantly impaired in obese females but not modified in males. HFD-induced dysfunctional PVAT was prevented by Spi treatment in females. Next, we evaluated the protein expression of aldosterone-synthase CYP11B2, serum and glucocorticoid-regulated kinase 1 (SGK1), and epithelial sodium channel subunits (ENaCs) in isolated mesenteric PVAT of lean and obese male and female mice. There was an increased expression of CYP11B2, SGK1 and ENaCs only in obese female PVAT. Protein expression of adiponectin, a major PVAT-released adipokine was also increased in female mesenteric PVAT. In conclusion, the findings suggest sexual dimorphism in PVAT function in health and in obesity. Although anticontractile role of PVAT was exacerbated in lean female mice, female sex was more susceptible to develop PVAT dysfunction in the setting of obesity which was prevented by MR blockade. HFD-induced PVAT dysfunction in females was associated with increased expression of SGK1 and ENaCs. Therefore, data suggest MR activation as a mechanism mediating sex differences in PVAT dysfunction.FAPESP, CAPES.

SAT-306 Muscarinic and Adrenergic Receptor Cooperativity in a Human Adrenocortical Carcinoma Cell Line

The role of autonomic receptors in the regulation of the adrenal cortex is poorly understood. We recently showed that activation of M3 muscarinic receptors stimulates intracellular calcium oscillations, aldosterone production, and expression of CYP11B2 (1). The present study explores the relationship between muscarinic and adrenergic receptors in corticosteroid production. Using live-cell fluorescence imaging of HAC15 adrenocortical cells with the calcium-sensitive probe Fluo-4, we have shown that stimulation of adrenergic receptors with the endogenous, non-selective adrenergic agonist norepinephrine (10μM) enhances intracellular Ca2+ oscillations caused by the cholinergic agonist carbachol (1μM). However, Ca2+ is not affected by norepinephrine alone. Adrenergic enhancement of carbachol-induced Ca2+ oscillations is blocked by the ⍺ adrenergic receptor antagonist phentolamine, but not by the β adrenergic receptor antagonist propanolol. Specifically, ⍺2 and β2 antagonists (such as yohimbine and butoxamine, respectively) significantly suppressed the norepinephrine effect, but ⍺1 and β1 antagonists (such as tamsulosin and metoprolol, respectively) had no effect. RT qPCR identified ⍺2A receptors as the most abundant adrenergic receptor in HAC15 cells. Saturation experiments using 3H-NMS and 3H-Rauwolscine confirmed the presence of muscarinic M2 and M3 receptors as well as ⍺2A receptors. Using competition radiolabeled binding assays we explored the cooperation between M2/M3 and ⍺2A adrenergic receptors. Our results suggest that autonomic regulation of intracellular Ca2+ depends on an interplay of M3 and ⍺2A receptors. Additional experiments will use ELISA methods to determine the functional impact of autonomic receptor cooperativity on steroid synthesis and secretion.

SAT-LB23 Paraneoplastic Hypercalcemia in a PTH Producing Adrenocortical Carcinoma - a Rare and Deadly Condition

Background: Hypercalcemia is a commonly encountered paraneoplastic manifestation of certain cancers with or without endocrine differentiation. However, the association between adrenocortical carcinoma (ACC) with paraneoplastic hypercalcemia is very rare, and therefore little is known about the cause and its relevance in the disease. Clinical Case: A 40-year-old woman presented in the hospital with a 5-month history of progressive flank pain with unintentional weight loss of 6 kg. MRI revealed a mass of 9x8.1x4.8 cm of the right adrenal gland with inhomogeneous contrast enhancement. Biochemical investigations provided evidence of endogenous hypercortisolism (24-hour urinary cortisol excretion [490 µg, n<236 µg/l], 1mg dexamethasone suppression test [199 nmol/l, n<50 nmol/l], ACTH [28 ng/l, n<61 ng/l]) although the patient did not show any specific clinical sign of overt hypercortisolism. In addition, laboratory testing revealed an exceptionally high plasma level of calcium [max 3.67 mmol/l (albumin-corrected)] and low phosphate [min 0.26 mmol/l] in the setting of low PTH [6.4 ng/l, n>15 ng/l] and PTHrP levels [<0.50 pmol/l]. However, subsequent dilution unmasked a highly elevated PTH concentration of 2171.5 ng/l with persistent low PTHrP levels, indicating false low values due to a hook effect in the initial measurement. Levels of 1,25-dihydroxy vitamin D and 25-hydroxy vitamin D were in the normal range. A PET-CT provided no indications of metabolically active (osseous) metastases. After correction of the serum calcium towards tolerable values, the tumor was removed by open en bloc adrenalectomy. Histologic evaluation confirmed an ACC (TNM pT4 pN1 (2/3), L1, V1, high grade) despite missing immunohistochemically expression of classical adrenal markers (diagnosis of exclusion). Supplemental quantitative RT-PCR studies support the diagnosis of ACC by detecting significant SF-1 and CYP11B2 expression in the tumor cells. Further analyses provided evidence that the mRNA expression of PTH, but not PTHrP, was moderately increased in the ACC sample compared to NCI H295R cells. Upon tumor resection, serum calcium levels swiftly normalized indicating the tumor as the sole source of PTH secretion. Despite initiation of adjuvant mitotane- and salvage chemo-therapy, the patient died 3 months later upon of a massive tumor relapse with a recurrence of severe hypercalcemia. Conclusion: This case demonstrates paraneoplastic hypercalcemia in a PTH producing ACC. PTH may induce hypercalcemia, impair adrenal steroid synthesis and act as an autocrine growth factor in ACC, as described in few individual cases for PTHrp producing ACC [1]. This suggests a poor prognosis for this rare entity.1. Rizk-Rabin, M., et al., Differential Expression of Parathyroid Hormone-Related Protein in Adrenocortical Tumors: Autocrine/Paracrine Effects on the Growth and Signaling Pathways in H295R Cells. 2008.

OR34-07 Prospective Multicentre Study Comparing 11C-metomidate PET CT with Adrenal Vein Sampling (AVS) in the Detection of Unilateral Aldosterone-Producing Adenomas (APAs)

Approximately 50% of Primary Aldosteronism (PA) cases are unilateral, potentially curable by adrenalectomy. However far fewer patients progress to surgery, partly due to difficulties in identifying unilateral disease. AVS is the current criterion standard method for lateralisation. However it is an invasive procedure, technically difficult to perform, and only available in few specialist centres. MATCH is a prospective, multicentre study comparing the diagnostic accuracy of AVS with 11C-metomidate PET-CT, a non-invasive functional scan (ClinicalTrials.gov Identifier NCT02945904).Patients fulfilling Endocrine Society criteria for PA undergo both investigations in random order. At a multidisciplinary meeting PET-CT results are scored first, followed by AVS. Patients are offered surgery if one or both investigations indicate unilateral disease. Each investigation will be re-scored by an independent, blinded endpoints committee, without knowledge of other investigations or outcomes in the same patient. Hierarchical primary outcomes are the change in aldosterone renin ratio (ARR) and average home SBP readings. If no superiority is observed for either investigation, non-inferiority of PET-CT will be tested. MATCH is powered to detect 25% superiority, or non-inferiority within a margin of 18%. Factors predicting cure will be assessed as secondary outcomes. These include BP response to aldosterone antagonists, correlation of standardised uptake value (SUV) max ratio of adenoma to adjacent normal adrenal, and phenotyping / genotyping of tumours.Target recruitment of 140 patients has been achieved. Interesting observations to date include a high prevalence of hypokalaemia (73%), reflective of our referral base and inclusion criteria. The surgery rate is also high at 66%, consistent with finding frequent patients in whom only one investigation yields a positive result. The following case illustrates such a patient. A slim 45-year-old lady with PA and failed ONDST had inconclusive AVS (selectivity index in right adrenal vein 2.6). PET-CT revealed a 29mm metomidate-avid left adrenal nodule (SUVmax ratio 1.52, >1.25 suggestive of unilateral disease). Left adrenalectomy was recommended based on PET-CT, and achieved biochemical and clinical cure. However she required hydrocortisone replacement for 14 months. Her relatively low right adrenal vein cortisol, despite successful cannulation, was attributed to contralateral suppression by co-secretion of cortisol from her adenoma. This was confirmed by finding high CYP11B1 and CYP11B2 mRNA expression in her tumour, typical of a KCNJ5 mutation, confirmed as L168R on Sanger sequencing.PA is a high risk subset of hypertension. Under-treatment has serious public health consequences. 11C-Metomidate PET CT has the potential to simplify the investigation pathway and allow more patients to receive potentially curable treatment.

OR19-01 IHC-Guided Capture of Adrenocortical Zones from Archival Formalin-Fixed, Paraffin-Embedded Adrenal Tissue Allows the Study Of RNA Expression Profiles

Introduction: Characterization of human adrenal RNA has relied on fresh tissue, or laser capture from OCT-embedded frozen tissue. These approaches have allowed identification of differences between zones of the adrenal cortex, but the need for fresh or frozen material has limited access for most researchers and has limited the number of samples that can be studied. In order to expand tissue availability and researcher access, we examined the use of archival formalin-fixed, paraffin-embedded (FFPE) adrenals for the capture and analysis of RNA from each adrenal zone.Methods: 13 normal adrenals were obtained from deceased renal donors. Following FFPE, tissue serial sections were examined for zonal histology. Immunohistochemistry (IHC) was used to guide the capture of specific adrenal zones from FFPE tissue. CYP11B2, CYP17A1, and CYB5A IHC was used to visualize the zona glomerulosa (ZG), zona fasciculata (ZF), and zona reticularis (ZR) respectively. Serial unstained sections were then dissected under a dissection microscope based on IHC-guided visualization of the ZG, ZF, and ZR. Following capture of zones and isolation of RNA, quantitative PCR (qPCR) was used to examine zonal expression of transcripts for the IHC markers. Using housekeeping genes for normalization, specific zonal markers identified successfully isolated zones.Results: Adrenals with clear zonal expression of CYP11B2 (ZG), HSD3B2 (ZG/ZF), and CYB5A (ZR) were included for study. Based on marker inclusion criteria, 3 ZG, 3 ZF, 3 ZR, and 3 medulla samples were studied. Using these samples, confirmatory markers of ZG (VSNL1), ZF (CYP17A1), ZR (SLC27A2), and medulla (TH) were analyzed by qPCR. Levels of these transcripts segregated as expected in the adrenal zones.Discussion: The use of archival normal and pathologic adrenal tissue for RNA analysis would increase the number of samples available to many research groups. Our data demonstrates the ability to capture and use FFPE tissue for the capture and analysis of RNA. Future comparison of normal adrenal zones to adrenal tumor phenotypes will improve our understanding of tumor biology and provide potential genetic markers for targeted therapeutics.

SAT-LB90 Angiotensin II and ACTH Receptor Expression in Aldosterone-Producing Adenomas

Background: The mechanisms leading to elevated aldosterone synthesis in aldosterone-producing adenomas (APAs) remain an area of active research. Aldosterone-driver somatic gene mutations that allow inappropriate intracellular calcium entrance have been identified in most APAs. Cell-based studies of such mutations indicate that responses to physiologic stimuli, such as angiotensin II or ACTH, are increased. Little is known, however, regarding possible variations in response to hormonal stimuli between APAs with different aldosterone-driver mutations. Herein, we analyzed the transcript expression of the ACTH receptor (MC2R), the melanocortin 2 receptor accessory protein (MRAP) and the type 1 angiotensin II receptor (AGTR1) in APAs with known aldosterone-driver somatic mutations. Methods: RNA was isolated from normal adrenal glands (n=8), and from APAs with mutations in: KCNJ5 (n=14), ATP1A1 (n=14), CACNA1D (n=14), and ATP2B3 (n=5). The gene expressions of MC2R, MRAP, AGTR1 and aldosterone synthase (CYP11B2) were quantified using qPCR and normalized to β-actin. Results: All APA mutation groups had significantly higher transcript levels of CYP11B2, MC2R and AGTR1 as compared to whole normal adrenals. While MRAP and AGTR1 transcripts were comparable between tumor mutation groups, MC2R expression was significantly lower in KCNJ5-mutated APAs compared to other APAs. Overall, CYP11B2 expression demonstrated positive correlations with MC2R (R=0.728, p<0.0001) and AGTR1 (R=0.397, p=0.006) in APAs. These correlations were strongest in APAs harboring ATP1A1 mutations, and weakest in KCNJ5-mutated APAs. Conversely, CYP11B2 did not correlate with MC2R and AGTR1 in the normal adrenals. Conclusions: ACTH and angiotensin II receptors are expressed in all APAs, regardless of the underlying aldosterone-driver somatic mutations. Further research to clarify the effects of ACTH and posture on aldosterone production from APAs could provide additional insight into developing diagnostic and subtyping tools for primary aldosteronism.

Vitamin D status influences transcriptional levels of RANKL and inflammatory biomarkers which are associated with activation of PBMC

Vitamin D status is involved in the risk of many chronic diseases including cancer, inflammatory and autoimmune disease. The RANK/RANKL/OPG system is also implicated in the orchestration of immune functions.We aimed to investigate the expression of RANKL, OPG and markers of inflammation and immune activation in peripheral blood mononuclear cells (PBMC) from healthy subjects with different 25(OH)D3 plasma levels.The 25(OH)D3 plasma concentrations were assessed by HPLC. The gene expression was evaluated by qRT-PCR.The expression of CYP27B1 was lower in subjects with 25(OH)D3 levels below 50 nmol/L (deficiency) than subjects with both insufficient and sufficient levels of 25(OH)D3. In subjects with deficiency, we observed the up-regulation of RANKL, TNF-α, IFN-γ, ICAM and LFA-1, and a reduction of the anti-inflammatory cytokines IL-13 and IL-4 in comparison to other subjects. Finally, we found a negative correlation between RANKL mRNA levels and 25(OH)D3 and between 25(OH)D3 and ICAM mRNA levels. A positive correlation between ICAM and RANKL was observed.Our results give evidence of the modulatory effects of circulating 25(OH)D3 levels on gene expression of biomarkers of immune activation in PBMC, suggesting the possible use of PBMC as ex-vivo model to characterize molecular mechanisms of immune/inflammatory response in hypovitaminosis conditions.

Primary Aldosteronism in the Elderly.

The clinical spectrum and knowledge of the molecular mechanisms underlying primary aldosteronism (PA), the most frequent form of endocrine hypertension, has evolved over recent years. In accordance with the Endocrine Society guidelines and in light of the growing evidence showing adverse cardiovascular outcomes, it is expected that a progressively wider population of patients affected by hypertension will be screened for PA, including the elderly. A systematic search of PubMed was undertaken for studies related to the renin-angiotensin-aldosterone system (RAAS), PA, and adrenal histopathology in the elderly population. Several studies showed an age-dependent decrease in the activity of RAAS, together with a progressive decrease of the aldosterone response to sodium intake, particularly after the sixth decade of life. The positive correlation between age and serum aldosterone during liberal sodium intake over serum aldosterone during sodium restriction is paralleled by histological changes in adrenal aldosterone synthase (CYP11B2) expression patterns. Immunohistochemical studies showed a progressive loss of the continuous expression of CYP11B2 in the adrenal zona glomerulosa with aging and a concomitant increase of aldosterone-producing cell clusters, which might be responsible for relatively autonomous aldosterone production. Additionally, following PA confirmation and subtype diagnosis, older age is correlated with a lower benefit after adrenalectomy for unilateral PA. Accumulating evidence suggests that RAAS physiology and regulation show age-related changes. Further studies may investigate to what extent these variations might affect the diagnostic workup of patients affected by PA.

Immunohistochemistry of the Human Adrenal CYP11B2 in Normal Individuals and in Patients with Primary Aldosteronism.

The CYP11B2 enzyme is the terminal enzyme in the biosynthesis of aldosterone. Immunohistochemistry using antibodies against CYP11B2 defines cells of the adrenal ZG that synthesize aldosterone. CYP11B2 expression is normally stimulated by angiotensin II, but becomes autonomous in primary hyperaldosteronism, in most cases driven by recently discovered somatic mutations of ion channels or pumps. Cells expressing CYP11B2 in young normal humans form a continuous band beneath the adrenal capsule; in older individuals they form discrete clusters, aldosterone-producing cell clusters (APCC), surrounded by non-aldosterone producing cells in the outer layer of the adrenal gland. Aldosterone-producing adenomas may exhibit a uniform or heterogeneous expression of CYP11B2. APCC frequently persist in the adrenal with an aldosterone-producing adenoma suggesting autonomous CYP11B2 expression in these cells as well. This was confirmed by finding known mutations that drive aldosterone production in adenomas in the APCC of clinically normal people. Unilateral aldosteronism may also be due to multiple CYP11B2-expressing nodules of various sizes or a continuous band of hyperplastic ZG cells expressing CYP11B2. Use of CYP11B2 antibodies to identify areas for sequencing has greatly facilitated the detection of aldosterone-driving mutations.

Older Age Is Associated with Decreased Levels of VDR, CYP27B1, and CYP24A1 and Increased Levels of PTH in Human Parathyroid Glands.

Parathyroid glands contain the vitamin D receptor (VDR) and 25-hydroxyvitamin D-1α-hydroxylase (CYP27B1) and 24-hydroxylase (CYP24A1), which catalyze the production and degradation of 1,25-dihydroxyvitamin D [1,25(OH)2D], respectively. Previous studies have shown that the serum level of intact parathyroid hormone (iPTH) increases with age. We hypothesized that the expression of CYP27B1 or VDR in parathyroid glands decreases with age, which might account for the increased serum levels of iPTH due to decreased suppression of parathyroid hormone (PTH) secretion by 1,25(OH)2D in older people. To test this hypothesis, we examined relative expression levels of VDR, CYP27B1, CYP24A1, and PTH in specimens from parathyroid glands unintentionally removed during thyroidectomy for 70 patients varying in age from 10 to 70 years. The results showed that there was an inverse correlation between age and VDR, CYP27B1, and CYP24A1 expression (p < 0.05). A significant positive correlation between PTH expression levels and age was also observed (p < 0.05). These data indicate that older age is associated with decreased levels of VDR, CYP27B1, and CYP24A1 and increased levels of PTH in human parathyroid glands.