Does Genotype-Specific Phenotype in Patients with Multiple Endocrine Neoplasia Type 2 Occur as Current Guidelines Predict?

The clinical manifestation of multiple endocrine neoplasia type 2 (MEN2) in terms of developing medullary thyroid cancer (MTC), pheochromocytoma (PCC), and/or primary hyperparathyroidism (PHPT) is related to the respective pathogenic variant of the RET proto-oncogene. The aim of this study is to retrospectively analyze the individual, genotype-dependent clinical manifestations of a large cohort of MEN2 patients. By comparing their clinical profile with currently existing evidence-based knowledge, an optimal therapy and prevention strategy in terms of prophylactic thyroidectomy and clinical follow-up could be ensured. This is a retrospective single-center study of 158 MEN2 patients who were diagnosed and/or surgically treated at a tertiary referral care center between 1990 and 2022. All participants were categorized according to their pathogenic variant of the RET proto-oncogene. Subsequently, the clinical manifestation of the disease and its time of occurrence was documented. Our analysis showed results in line with existing studies, except for a considerably lower-than-predicted occurrence of PCC in patients with V804M/L mutations. This study supports the current recommendation regarding the pathogenic variant-dependent management of this rare cancer-associated syndrome.

The Interplay of Adipokines, Body Composition and Glucose Homeostasis in Pregnant Women with a History of RYGB Operation.

Roux-en-Y gastric bypass operations (RYGB-OP) and pregnancy alter glucose homeostasis and the adipokine profile. This study investigates the relationship between adipokines and glucose metabolism during pregnancy post-RYGB-OP. (1) Methods: This is a post hoc analysis of a prospective cohort study during pregnancy in 25 women with an RYGB-OP (RY), 19 women with obesity (OB), and 19 normal-weight (NW) controls. Bioimpedance analysis (BIA) was used for metabolic characterization. Plasma levels of adiponectin, leptin, fibroblast-growth-factor 21 (FGF21), adipocyte fatty acid binding protein (AFABP), afamin, and secretagogin were obtained. (2) Results: The phase angle (φ) was lower in RY compared to OB and NW. Compared to OB, RY, and NW had lower leptin and AFABP levels, and higher adiponectin levels. φ correlated positively with leptin in RY (R = 0.63, p < 0.05) and negatively with adiponectin in OB and NW (R = -0.69, R = -0.69, p < 0.05). In RY, the Matsuda index correlated positively with FGF21 (R = 0.55, p < 0.05) and negatively with leptin (R = -0.5, p < 0.05). In OB, FGF21 correlated negatively with the disposition index (R = -0.66, p < 0.05). (3) Conclusions: The leptin, adiponectin, and AFABP levels differ between RY, OB, and NW and correlate with glucose metabolism and body composition. Thus, adipokines might influence energy homeostasis and maintenance of cellular health during pregnancy.

Novel mutations of the calcium-sensing receptor impede differential diagnosis of primary hyperparathyroidism and familial hypocalciuric hypercalcemia.

BACKGROUND: Familial hypocalciuric hypercalcemia 1 (FHH1) is an autosomal dominant disorder caused by inactivating mutations in the calcium-sensing receptor (CaSR) gene, commonly leading-in contrast to primary hyperparathyroidism (PHPT)-to asymptomatic hypercalcemia. It is important to establish the correct diagnosis, as surgery may be curative in PHPT, but most likely ineffective in FHH. The study aims to evaluate patients with FHH1, initially misinterpreted as PHPT and some even undergone surgery. METHODS: CaSR-genotyping was conducted, various biochemical parameters including twenty-four-hour urinary Ca excretion (24hU CE) and the calculated relation of urinary Ca clearance to creatinine clearance (CCCR), type of surgery and 1-year follow-up data of fourteen patients with proven FHH1 were evaluated retrospectively. RESULTS: Genetic analysis revealed a total of nine novel heterozygous variants in the CaSR gene in our study population. Six of fourteen patients (42.9%) underwent surgery for initially suspected PHPT, showing normalized biochemical parameters immediately after surgery. In 1-year follow-up, however, five of six operated patients (83.3%) showed normal parathyroid hormone (PTH), but elevated serum calcium levels. In contrast, only one of the operated patients (16.7%) presented both PTH and serum calcium in the normal range. Histology showed adenoma in three (50%), hyperplasia in two (33.3%), and normal parathyroid tissue in one (16.7%) of the patients. CONCLUSIONS: We discovered novel heterozygous variants in the CaSR gene, which considerably impede differential diagnosis of PHPT and FHH1. Furthermore, our results indicate that parathyroid surgery fails to provide long-term benefits for patients with FHH1 and suspected PHPT, even though this coincidence seems to exist.

Glypican-4 in pregnancy and its relation to glucose metabolism, insulin resistance and gestational diabetes mellitus status.

Glypican-4 (GPC-4) is an adipokine that enhances insulin receptor signaling. Plasma concentrations were found to be elevated in patients with prediabetes but reduced in type 2 diabetes mellitus. No study on Glypican-4 in pregnancy and pregnancy-related insulin resistance has been published yet. GPC-4 levels were investigated in 59 overweight women throughout their pregnancy at the Medical University of Vienna. GPC-4 levels, fasting insulin, fasting glucose, estradiol, liver and renal parameters, and markers of bone development were assessed before the < 21st week of gestation (GW), and at GW 35-37. GPC-4 levels increased from < 21 GW (mean = 2.38 pg/ml, SD = 0.68 pg/ml) to GW 35-37 (mean = 2.96 pg/ml, SD = 0.77 pg/ml, p < 0.001). At the same time, GPC-4 levels correlated negatively with estimated glomerular filtration rate (eGFR), serum protein and serum albumin levels and were positively related to creatinine and uric acid levels at GW 35-37. Concerning glucose metabolism, GPC-4 levels were inversely related to ISSI-2, fasting insulin and HOMA-IR, however, not significantly different between women with normal glucose tolerance (NGT) and GDM (p = 0.239). In conclusion, GPC-4 levels rose significantly during pregnancy, correlated negatively with fasting insulin and HOMA-IR but might not be related to gestational diabetes mellitus status.

Effects of pharmacological calcimimetics on colorectal cancer cells over-expressing the human calcium-sensing receptor.

The calcium-sensing receptor (CaSR) is a ubiquitously expressed multifunctional G protein-coupled receptor. Several studies reported that the CaSR plays an anti-inflammatory and anti-tumorigenic role in the intestine, and that it is down-regulated during colorectal carcinogenesis. We hypothesized that positive allosteric CaSR modulators (type II calcimimetics) selectively targeting the intestinal cells could be used for the treatment of intestinal pathologies. Therefore, the aim of this study was to determine the effect of pharmacological stimulation of CaSR on gene expression in vitro and on tumor growth in vivo. We stably transduced two colon cancer cell lines (HT29 and Caco2) with lentiviral vectors containing either the CaSR fused to GFP or GFP only. Using RNA sequencing, RT-qPCR experiments and ELISA, we determined that CaSR over-expression itself had generally little effect on gene expression in these cells. However, treatment with 1 µM of the calcimimetic NPS R-568 increased the expression of pro-inflammatory factors such as IL-23α and IL-8 and reduced the transcription of various differentiation markers in the cells over-expressing the CaSR. In vivo, neither the presence of the CaSR nor p.o. treatment of the animals with the calcimimetic cinacalcet affected tumor growth, tumor cell proliferation or tumor vascularization of murine HT29 xenografts. In summary, CaSR stimulation in CaSR over-expressing cells enhanced the expression of inflammatory markers in vitro, but was not able to repress colorectal cancer tumorigenicity in vivo. These findings suggest potential pro-inflammatory effects of the CaSR and type II calcimimetics in the intestine.

Parathyroid hormone induces a browning program in human white adipocytes.

Energy dissipation through the promotion of brown adipose tissue (BAT) or browning of white adipose tissue has recently evolved as novel promising concept in the fight against metabolic disease. New evidence suggests that hormones can contribute to the thermogenic programming of adipocytes through paracrine or endocrine actions. Recent studies in rodents identified parathyroid hormone (PTH) and PTH-related peptide as mediators of energy wasting in cachexia models due to adipocyte browning. However, the effects of PTH on human adipocyte thermogenesis and metabolic activity are unknown. Here we isolated subcutaneous white adipocyte precursor cells (APCs) from human donors followed by stimulation with recombinant PTH. Our data show that acute and chronic PTH administration in primary in vitro differentiated human subcutaneous adipocytes induces a molecular thermogenic program with increased mitochondrial activity and oxidative respiratory capacity. PTH also enhances hormone sensitive lipase activity and lipolysis in human adipocytes which may contribute to the observed thermogenic effects. In summary, we demonstrate here that PTH is a novel mediator of human adipocyte browning, suggesting a hitherto unknown endocrine axis between the parathyroid gland and adipose tissue in humans.

Ultralong TE In Vivo 1 H MR Spectroscopy of Omega-3 Fatty Acids in Subcutaneous Adipose Tissue at 7 T.

BACKGROUND: Omega-3 (n-3) fatty acids (FA) play and important role in neural development and other metabolic diseases such as obesity and diabetes. The knowledge about the in vivo content and distribution of n-3 FA in human body tissues is not well established and the standard quantification of FA is invasive and costly. PURPOSE: To detect omega-3 (n-3 CH3 ) and non-omega-3 (CH3 ) methyl group resonance lines with echo times up to 1200 msec, in oils, for the assessment of n-3 FA content, and the n-3 FA fraction in adipose tissue in vivo. STUDY TYPE: Prospective technical development. POPULATION: Three oils with different n-3 FA content and 24 healthy subjects. FIELD STRENGTH/SEQUENCE: Single-voxel MR spectroscopy (SVS) with a point-resolved spectroscopy (PRESS) sequence with an echo time (TE) of 1000 msec at 7 T. ASSESSMENT: Knowledge about the J-coupling evolution of both CH3 resonances was used for the optimal detection of the n-3 CH3 resonance line at a TE of 1000 msec. The accuracy of the method in oils and in vivo was validated from a biopsy sample with gas chromatography analysis. STATISTICAL TESTS: SVS data were compared to gas chromatography with the Pearson correlation coefficient. RESULTS: T2 relaxation times in oils were assessed as follows: CH2 , 65 ± 22 msec; CH3 , 325 ± 7 msec; and n-3 CH3 , 628 ± 34 msec. The n-3 FA fractions from oil phantom experiments (n = 3) were in agreement with chromatography analysis and the comparison of in vivo obtained data with the results of chromatography analysis (n = 5) yielded a significant correlation (P = 0.029). DATA CONCLUSION: PRESS with ultralong-TE can detect and quantify the n-3 CH3 signal in vivo at 7 T. LEVEL OF EVIDENCE: 1 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2019;50:71-82.

Nutritional and Pharmacological Targeting of the Calcium-Sensing Receptor Influences Chemically Induced Colitis in Mice.

The calcium-sensing receptor (CaSR) is the main regulator of extracellular Ca2+ homeostasis. It has diverse functions in different tissues, including the intestines. Intestine-specific knockout of the CaSR renders mice more susceptible to dextran sulphate sodium (DSS)-induced colitis. To test our hypothesis that the CaSR reduces intestinal inflammation, we assessed the effects of nutritional and pharmacological agonists of the CaSR in a colitis model. We treated female Balb/C mice with dietary calcium and protein (nutritional agonists of the CaSR) or pharmacological CaSR modulators (the agonists cinacalcet and GSK3004774, and the antagonist NPS-2143; 10 mg/kg), then induced colitis with DSS. The high-protein diet had a strong pro-inflammatory effect-it shortened the colons (5.3 ± 0.1 cm vs. 6.1 ± 0.2 cm normal diet, p < 0.05), lowered mucin expression and upregulated pro-inflammatory cytokines, such as interferon-γ, (4.2-fold, p < 0.05) compared with the normal diet. Cinacalcet reduced mucin expression, which coincided with an increase in tumor necrosis factor-α (4.4-fold, p < 0.05) and IL-6 (4.9-fold, p < 0.05) in the plasma, compared with vehicle. The CaSR antagonist, NPS-2143, significantly reduced the cumulative inflammation score compared with the vehicle control (35.3 ± 19.1 vs. 21.9 ± 14.3 area under the curve, p < 0.05) and reduced infiltration of inflammatory cells. While dietary modulation of the CaSR had no beneficial effects, pharmacological inhibition of the CaSR may have the potential of a novel add-on therapy in the treatment of inflammatory bowel diseases.

Determinants of growth differentiation factor 15 in patients with stable and acute coronary artery disease. A prospective observational study.

BACKGROUND: Growth differentiation factor 15 (GDF-15) is a member of the transforming growth factor ß family and has been associated with inflammation, cancer, aging, diabetes mellitus (DM) and atherosclerosis. Determinants of GDF-15 have been investigated in several conditions. We aimed to investigate determinants of GDF-15 plasma levels in patients with angiographically proven coronary artery disease (CAD). METHODS: Four hundred and seventy three consecutive patients with CAD were investigated between May 2009 and February 2011. Patients were separated into those with stable CAD (SCAD) and with ST-elevation and non-ST-elevation myocardial infarction (STEMI and NSTEMI). Blood samples for determination of GDF-15 were obtained before coronary angiography. Determinant of GDF-15 levels were analyzed by logistic regression analysis in unadjusted and adjusted models. Study endpoints were cardiovascular death (CV-death), myocardial infarction, unstable angina, unplanned revascularization, stent thrombosis and stroke assessed at a mean follow-up of 188 (177.2-243) days. RESULTS: Overall median and (25-27th percentile) GDF-15 level was 1212.8 pg/ml (833.2-1957 pg/ml). GDF-15 was significantly higher in STEMI compared to SCAD and NSTEMI groups (P < 0.0001). In a multivariate regression analysis advanced age, DM, acute hyperglycemia (AHG), CRP and chronic kidney disease (CKD) were independent predictors of elevated GDF-15 levels (P < 0.05). Receiver operating curve analysis of GDF-15 for prediction of CV-death showed an area under the curve of 0.852 with a confidence interval of 0.745-0.960, P < 0.0001. The estimated cut-off was 2094.6 pg/ml with a sensitivity of 76 % and specificity of 80 %. CONCLUSION: In patients with CAD undergoing PCI with stent implantation, GDF-15 is determined by advanced age, acute and chronic hyperglycemia, inflammation and CKD. GDF-15 is a valuable predictor of CV-death in a population of CAD patients after PCI.

Active vitamin D potentiates the anti-neoplastic effects of calcium in the colon: A cross talk through the calcium-sensing receptor.

Epidemiological studies suggest an inverse correlation between dietary calcium (Ca(2+)) and vitamin D intake and the risk of colorectal cancer (CRC). It has been shown in vitro that the active vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25-D3) can upregulate expression of the calcium-sensing receptor (CaSR). In the colon, CaSR has been suggested to regulate proliferation of colonocytes. However, during tumorigenesis colonic CaSR expression is downregulated and we hypothesized that the loss of CaSR could influence the anti-tumorigenic effects of Ca(2+) and vitamin D. Our aim was to assess the impact of CaSR expression and function on the anti-neoplastic effects of 1,25-D3 in colon cancer cell lines. We demonstrated that in the healthy colon of mice, high vitamin D diet (2500 IU/kg diet) increased expression of differentiation and apoptosis markers, decreased expression of proliferation markers and significantly upregulated CaSR mRNA expression, compared with low vitamin D diet (100 IU/kg diet). To determine the role of CaSR in this process, we transfected Caco2-15 and HT29 CRC cells with wild type CaSR (CaSR-WT) or a dominant negative CaSR mutant (CaSR-DN) and treated them with 1,25-D3 alone, or in combination with CaSR activators (Ca(2+) and NPS R-568). 1,25-D3 enhanced the anti-proliferative effects of Ca(2+) and induced differentiation and apoptosis only in cells with a functional CaSR, which were further enhanced in the presence of NPS R-568, a positive allosteric modulator of CaSR. The mutant CaSR inhibited the anti-tumorigenic effects of 1,25-D3 suggesting that the anti-neoplastic effects of 1,25-D3 are, at least in part, mediated by the CaSR. Taken together, our data provides molecular evidence to support the epidemiological observation that both, vitamin D and calcium are needed for protection against malignant transformation of the colon and that their effect is modulated by the presence of a functional CaSR. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.

miR-135b- and miR-146b-dependent silencing of calcium-sensing receptor expression in colorectal tumors.

Studies have shown that the calcium-sensing receptor (CaSR) mediates the antitumorigenic effects of calcium against colorectal cancer (CRC). Expression of the CaSR in colorectal tumors is often reduced. We have reported previously that silencing of CaSR in CRC is caused in part by methylation of CaSR promoter 2 and loss of histone acetylation. We investigated the impact of aberrant microRNA expression on loss of CaSR expression. A microarray study in two Caco-2 subclones (Caco2/AQ and Caco2/15) that have similar genetic background, but different CaSR expression levels (Caco2/AQ expressing more CaSR than Caco2/15), identified 22 differentially expressed microRNAs that potentially target the CaSR. We validated these results by performing gain- and loss-of-function studies with the top candidates: miR-9, miR-27a, miR-135b, and miR-146b. Modulation of miR-135b or miR-146b expression by mimicking or inhibiting their expression regulated CaSR protein levels in two different colon cancer cell lines: Caco2/AQ (moderate endogenous CaSR expression) and HT29 (low endogenous CaSR levels). Inhibition of miR-135b and miR-146b expression led to high CaSR levels and significantly reduced proliferation. In samples of colorectal tumors we observed overexpression of miR-135b and miR-146b, and this correlated inversely with CaSR expression (miR-135b: r = -0.684, p < 0.001 and miR-146b: r = -0.448, p < 0.001), supporting our in vitro findings. We demonstrate that miR-135b and miR-146b target the CaSR and reduce its expression in colorectal tumors, reducing the antiproliferative and prodifferentiating actions of calcium. This provides a new approach for finding means to prevent CaSR loss, developing better treatment strategies for CRC.

The role of proto-oncogene GLI1 in pituitary adenoma formation and cell survival regulation.

The Hedgehog (Hh) pathway is an important regulator of early tissue patterning and stem cell propagation. It was found to be aberrantly activated in numerous types of human cancer and might be relevant in cancer stem cells. The identification of adult stem cells in the pituitary raised the question if tumor-initiating cells and Hh signaling are involved in pituitary adenoma formation. The present study aimed at the evaluation of Hh signaling in relation to stem cell and cell cycle markers in 30 human pituitary adenomas and in cultured murine adenoma cells. Therefore, expression levels of components of the Hh pathway, stem cell marker SOX2, cell cycle regulator tumor-protein 53 (TP53), proliferation marker Ki67 (MKI67) and superoxide dismutase 1 (SOD1) were evaluated in 30 human pituitary adenomas in comparison to control tissue. Modulation of cell function and target gene expression by the inhibition and activation of the Hh pathway were studied in murine adenoma cells. We show that transcription factor glioma-associated oncogene 1 (GLI1) is overexpressed in 87% of all pituitary adenomas. The expression of GLI1 significantly correlated with that of SOX2, TP53, MKI67 and SOD1. Inhibition of GLI1 resulted in the downregulation of the above genes and severe cell death in mouse adenoma cells. On the other hand, activation of the Hh pathway increased cell viability and target gene expression. In conclusion, our findings point toward an alternative, ligand-independent Hh pathway activation with GLI1 playing a major role in the cell survival of pituitary adenoma cells.

The calcium-sensing receptor: A promising target for prevention of colorectal cancer.

The inverse correlation between dietary calcium intake and the risk of colorectal cancer (CRC) is well known, but poorly understood. Expression of the calcium-sensing receptor (CaSR), a calcium-binding G protein-coupled receptor is downregulated in CRC leading us to hypothesize that the CaSR has tumor suppressive roles in the colon. The aim of this study was to understand whether restoration of CaSR expression could reduce the malignant phenotype in CRC. In human colorectal tumors, expression of the CaSR negatively correlated with proliferation markers whereas loss of CaSR correlated with poor tumor differentiation and reduced apoptotic potential. In vivo, dearth of CaSR significantly increased expression of proliferation markers and decreased levels of differentiation and apoptotic markers in the colons of CaSR/PTH double knock-out mice confirming the tumor suppressive functions of CaSR. In vitro CRC cells stably overexpressing wild-type CaSR showed significant reduction in proliferation, as well as increased differentiation and apoptotic potential. The positive allosteric modulator of CaSR, NPS R-568 further enhanced these effects, whereas treatment with the negative allosteric modulator, NPS 2143 inhibited these functions. Interestingly, the dominant-negative mutant (R185Q) was able to abrogate these effects. Our results demonstrate a critical tumor suppressive role of CaSR in the colon. Restoration of CaSR expression and function is linked to regulation of the balance between proliferation, differentiation, and apoptosis and provides a rationale for novel strategies in CRC therapy.

Levothyroxine replacement in hypothyroid humans reduces myocardial lipid load and improves cardiac function.

CONTEXT: Hypothyroidism is a common endocrine disorder frequently accompanied by alterations in lipid metabolism, such as hypercholesterolemia and high circulating triglycerides, both risk factors for nonischemic cardiomyopathy. Rodent studies suggest that the hypothyroid state promotes cardiac lipid retention by increasing lipid uptake into cardiomyocytes while reducing fatty acid oxidation. Furthermore, increased cardiac lipid load has been linked to cardiac dysfunction. OBJECTIVE: Dyslipidemia and hypothyroidism frequently coexist; thus, we hypothesized that overt hypothyroidism causes cardiac lipid deposition and ultimately cardiac dysfunction. DESIGN: An interventional prospective study with balanced within-subject comparison. PARTICIPANTS/SETTING/INTERVENTION: Ten patients recruited at an academic center, who underwent a thyroidectomy due to differentiated thyroid carcinoma, were examined 4 weeks postoperatively in the overtly hypothyroid state, right before radioiodine therapy, and 6-8 weeks after initiation of levothyroxine replacement. MAIN OUTCOME PARAMETERS: We measured cardiac lipid content and function in vivo before and after levothyroxine treatment using electrocardiogram-gated (1)H-magnetic resonance spectroscopy and imaging. RESULTS: Levothyroxine therapy reduced cardiac lipid content in nine of the 10 patients (0.35 ± 0.09 vs 0.22 ± 0.06 % water signal; P = .008; n = 10) and improved cardiac index (2 ± 0.2 vs 2.4 ± 0.1 L/min/m(2); P = .047) when comparing the hypothyroid with the euthyroid state, independent of changes in liver fat content (7.5 ± 3.2 vs 7.1 ± 2.6% magnetic resonance spectroscopy signal; P = .60) or body weight. CONCLUSION: Here we show that levothyroxine treatment reduces lipid accumulation in the heart and increases cardiac output in overtly hypothyroid patients. These results could in part explain the increased risk of death and heart failure in hypothyroid patients.

Calcium-sensing receptor silencing in colorectal cancer is associated with promoter hypermethylation and loss of acetylation on histone 3.

The calcium-sensing receptor (CaSR) is suggested to mediate the antiproliferative effects of calcium in colon. However, in colorectal cancer (CRC) the expression of the CaSR is silenced and the underlying mechanisms leading to its loss are poorly understood. We investigated whether loss of the CaSR expression in colorectal tumors is caused by DNA hypermethylation and imbalance of transcriptionally permissive/repressive histone alterations. We observed significantly lower CaSR mRNA expression (n = 65, p < 0.001) in colorectal tumors compared with the adjacent mucosa from the same patient. Immunofluorescence staining confirmed downregulation of the CaSR protein also. The CaSR promoter was methylated to a greater extent in tumors compared with adjacent mucosa as determined by bisulfite sequencing (n = 20, p < 0.01) and by pyrosequencing (n = 45, p < 0.001), and methylation correlated inversely with mRNA expression (n = 20, ρ = -0.310, p < 0.05 and n = 45, ρ = -0.588, p < 0.001). Treatments with 5-aza-2'-deoxycytidine (DAC), a DNA methyltransferase inhibitor and/or with two different histone deacetylase inhibitors, trichostatin A (TSA) or suberoylanilide hydroxamic acid (SAHA) restored the expression of CaSR in colon cancer cells. Restored CaSR expression in Coga1A and HT29 cells was functional. Inhibition of lysine-specific demethylase 1 (LSD1) to prevent demethylation of mono- and dimethylated H3K4, increased CaSR expression only marginally. Our data show that hypermethylation of the CaSR promoter and H3K9 deacetylation, but not H3K4me2 demethylation are important factors that cause silencing of the CaSR in colorectal cancer.

A Case of "Late-Onset" Idiopathic Infantile Hypercalcemia Secondary to Mutations in the CYP24A1 Gene.

OBJECTIVE: Mutations in the 24-hydroxylase gene, CYP24A1, have recently been reported to cause idiopathic infantile hypercalcemia (IIH), a rare disease presenting in the first year of life that is characterized by increased sensitivity to vitamin D, leading to severe symptomatic hypercalcemia. METHODS: We present a case report and review the relevant literature. RESULTS: A 24-year-old Caucasian man presented with repetitive signs of nephrolithiasis since the age of 18 years, hypercalciuria (17.1 mmol/24 h), slightly elevated serum calcium concentration (2.64 mmol/L), and inappropriately high levels of 1,25-dihydroxyvitamin D (101 pg/mL) in combination with suppressed levels of circulating parathormone (7.9 pg/mL). Exogenous vitamin D intoxication as well as granulomatous disease or malignancy were excluded. Genetic analysis revealed a loss-of-function mutation in CYP24A1. Of note, our patient denied any prior clinical signs of impaired calcium homeostasis during childhood. CONCLUSION: Here, we describe the exceptional case of a patient with hypercalciuria and recurrent nephrolithiasis secondary to mutations in CYP24A1, without any signs of IIH in childhood, indicating that the phenotypic spectrum includes mild "late-onset" disease that becomes symptomatic in adolescence. Therefore, reduced CYP24A1 activity should be considered as a possible reason for recurrent nephrolithiasis in adults.

Regulation of the calcium-sensing receptor expression by 1,25-dihydroxyvitamin D3, interleukin-6, and tumor necrosis factor alpha in colon cancer cells.

Anti-proliferative effects of calcium in the colon are mediated, at least in part, via the calcium-sensing receptor (CaSR), a vitamin D target gene. The expression of CaSR decreases during colorectal tumor progression and the mechanisms regulating its expression are poorly understood. The CaSR promoter harbors vitamin D elements responsive to 1,25-dihydroxyvitamin D3 (1,25D3) and NF-κB, STAT, and SP1 binding sites accounting for responsiveness to proinflammatory cytokines. Therefore, in the current study we investigated the impact of 1,25D3, tumor necrosis factor alpha (TNFα), and interleukin (IL)-6 on CaSR expression in a differentiated (Caco2/AQ) and in a moderately differentiated (Coga1A) colon cancer cell line. 1,25D3 induced CaSR expression in both cell lines. Treatment with TNFα was accompanied by a 134-fold induction of CaSR in Coga1A (p<0.01). In Caco2/AQ cells the expression of CaSR was upregulated also by IL-6 (3.5-fold). Our data demonstrated transcriptional and translational activation of the CaSR by 1,25D3, TNFα, and IL-6 in a time- and cell line-dependent manner. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.

Increased copy-number and not DNA hypomethylation causes overexpression of the candidate proto-oncogene CYP24A1 in colorectal cancer.

In colorectal cancer (CRC) the vitamin D catabolizing enzyme 1,25-dihydroxyvitamin D 24-hydroxylase (CYP24A1) is overexpressed with a potentially significant, positive impact on the catabolism of 1,25-dihydroxyvitamin D3 (1,25-D3 ). However, the underlying mechanism of CYP24A1 overexpression is poorly understood. In the present study, we investigated possible causes including hypomethylation of the CYP24A1 promoter, amplification of the CYP24A1 gene locus (20q13.2), and altered expression of CYP24A1-specific transcription factors. We quantified CYP24A1 gene copy-number, performed bisulfite sequencing of the CYP24A1 promoter to assess DNA methylation, and measured mRNA expression of CYP24A1, 25-hydroxyvitamin D 1α-hydroxylase (CYP27B1), vitamin D receptor (VDR) and retinoid X receptor (RXR). We found that 77 (60%) out of 127 colorectal tumors showed increased CYP24A1 gene copy-number and that more than 6 copies of CYP24A1 correlated positively with CYP24A1 mRNA expression suggestive of a causal relationship. No differences in CYP24A1 promoter methylation were found between tumor tissue and adjacent mucosa from the same patient or between tissues with high or low mRNA expression, thus excluding DNA hypomethylation as a possible cause of CYP24A1 overexpression in CRC. Furthermore, mRNA expression of several factors involved in replication licensing positively correlated with CYP24A1 mRNA expression, raising the possibility that CYP24A1 overexpression might favor increased proliferation in tumors by suppressing local 1,25-D3 levels. We conclude that high copy-number gain is a key determinant of CYP24A1 overexpression in CRC. Other postulated causes of CYP24A1 overexpression including promoter hypomethylation and enhanced VDR and/or RXR expression do not appear to be involved.

Opposite effects of serum- and glucocorticoid-regulated kinase-1 and glucocorticoids on POMC transcription and ACTH release.

Serum- and glucocorticoid-regulated kinase-1 (SGK1) is a glucocorticoid early-response gene; its function, however, has been elucidated mainly in the context of mineralocorticoid signaling. Here, we investigate the expression and function of SGK1 in the pituitary gland, one of the primary glucocorticoid targets. SGK1 is expressed in the human pituitary gland and colocalizes to ACTH. The AtT-20 murine corticotroph cell line was used for functional experiments. Glucocorticoids upregulated SGK1 mRNA and protein levels, parallel to decreasing proopiomelanocortin (POMC) transcription and ACTH release. Dexamethasone-induced changes in SGK1 protein were abolished by the steroid receptor antagonist RU-486 and reduced by the inhibition of PI 3-kinase with LY-294002. SGK1 overexpression increased CREB- and activator protein-1-dependent transcription, POMC transcription, and ACTH secretion but did not influence intracellular cAMP levels. SGK1 overexpression and corticotropin-releasing hormone had additive effects on POMC promoter activity but not on ACTH secretion. SGK1 knockdown by RNA interference decreased POMC promoter activity, demonstrating the importance of SGK1 for basal POMC signaling. In summary, SGK1 is strongly stimulated by glucocorticoids in pituitary corticotrophs; however, its effects on POMC transcription are antagonistic to the classical inhibitory glucocorticoid action, suggesting a cell-regulated counterregulatory mechanism to potentially detrimental glucocorticoid effects.