scholarly journals Reduced Calcium Sensing Receptor (CaSR) Expression Is Epigenetically Deregulated in Parathyroid Adenomas

2020 ◽  
Vol 105 (9) ◽  
pp. 3015-3024
Author(s):  
Priyanka Singh ◽  
Sanjay Kumar Bhadada ◽  
Divya Dahiya ◽  
Ashutosh Kumar Arya ◽  
Uma Nahar Saikia ◽  
...  
Keyword(s):  
Protein Expression ◽  
Parathyroid Adenoma ◽  
Histone Modifications ◽  
Chromatin Immunoprecipitation ◽  
Methylation Status ◽  
Sequencing Analysis ◽  
Calcium Sensing Receptor ◽  
Calcium Sensing ◽  
Bisulphite Sequencing ◽  
Parathyroid Adenomas

Abstract Aim Reduced calcium sensing receptor (CaSR) expression has been implicated in parathyroid tumorigenesis, but the underlying mechanism remains elusive. Accordingly, we aimed to explore the epigenetic changes (DNA methylation and histone modifications) involved in CaSR regulation in sporadic parathyroid adenomas and correlate epigenetic state with disease indices. Experimental Design Forty sporadic parathyroid adenomas and 10 control parathyroid tissues were studied. Real-time quantitative PCR (qPCR) for mRNA and immunohistochemistry for protein expression of CaSR were performed. The methylation status of the CaSR promoter 2 was determined by bisulphite sequencing analysis of sodium bisulphite-converted DNA. To determine the role of histone modifications in the CaSR regulation, chromatin immunoprecipitation-qPCR assay was performed. Results Real-time qPCR revealed reduced CaSR mRNA expression with a fold reduction of 0.12 (P < 0.0001) in parathyroid adenomas. Immunohistochemistry revealed reduced protein expression of CaSR in 90% (36/40) of adenomas. The promoter 2 region of CaSR displayed significant hypermethylation in 45% (18/40) of the adenomas compared with the controls (6.7%; 1 of 10) (P < 0.002). Bisulphite sequencing analysis revealed maximum methylated CpG at glial cell missing 2 binding site on the CaSR promoter 2 compared to other CpG sites. The methylation status of CaSR correlated directly with plasma intact parathyroid hormone levels in patients with parathyroid adenoma. With chromatin immunoprecipitation-qPCR analysis, H3K9me3 levels showed increased enrichment by 10-fold in adenomas and correlated with CaSR-mRNA expression (r = 0.61; P < 0.003). Treatment with 5-aza-2′deoxycytidine restored the expression of CaSR in a parathyroid cell line. Conclusion Our data suggest that hypermethylation and increased H3K9me3 of the CaSR promoter 2 are involved in silencing CaSR expression in sporadic parathyroid adenoma.

GCM2 Silencing in Parathyroid Adenoma is associated with Promoter Hypermethylation and Gain of Methylation on Histone 3

2021 ◽  
Author(s):  
Priyanka Singh ◽  
Sanjay Kumar Bhadada ◽  
Divya Dahiya ◽  
Uma Nahar Saikia ◽  
Ashutosh Kumar Arya ◽  
...  
Keyword(s):  
Protein Expression ◽  
Parathyroid Adenoma ◽  
Dna Methyltransferase ◽  
Promoter Hypermethylation ◽  
Epigenetic Alterations ◽  
Protein Levels ◽  
Histone 3 ◽  
Parathyroid Adenomas ◽  
Mrna And Protein Expression

Abstract Purpose Glial cells missing 2 (GCM2), a zinc finger-transcription factor, is essentially required for the development of parathyroid glands. We sought to identify if the epigenetic alterations in the GCM2 transcription are involved in the pathogenesis of sporadic parathyroid adenoma. In addition, we examined the association between promoter methylation and histone modifications with disease indices. Experimental design mRNA and protein expression of GCM2 were analyzed by RT-qPCR and immunohistochemistry in 33 adenomatous and 10 control parathyroid tissues. DNA methylation and histone methylation/acetylation of GCM2 promoter were measured by bisulfite sequencing and ChIP-qPCR. Additionally, we investigated the role of epigenetic modifications on GCM2 and DNA methyltransferase 1 (DNMT1) expression in PTH-C1 cells by treating with 5-aza 2’deoxycytidine (DAC) and BRD4770 and assessed for GCM2 mRNA and DNMT1 protein levels. Results mRNA and protein expression of GCM2 were lower in sporadic adenomatous than in control parathyroid tissues. This reduction correlated with hypermethylation (P<0.001) and higher H3K9me3 levels in GCM2 promoter (P<0.04) in adenomas. In PTH-C1 cells, DAC treatment resulted in increased GCM2 transcription and decreased DNMT1 protein expression, while cells treated with the BRD4770 showed reduced H3K9me3 levels but a non-significant change in GCM2 transcription. Conclusion These findings suggest the concurrent association of promoter hypermethylation and higher H3K9me3 with the repression of GCM2 expression in parathyroid adenomas. Treatment with DAC restored GCM2 expression in PTH-C1 cells. Our results showed a possible epigenetic landscape in the tumorigenesis of parathyroid adenoma and also that DAC may be promising avenues of research for parathyroid adenoma therapeutics.

scholarly journals The Calcium Sensing Receptor: From Understanding Parathyroid Calcium Homeostasis to Bone Metastases

2008 ◽  
Vol 90 (4) ◽  
pp. 271-277 ◽  
Author(s):  
Radu Mihai
Keyword(s):  
Breast Cancer ◽  
Bone Metastases ◽  
Intracellular Calcium ◽  
Calcium Homeostasis ◽  
Membrane Trafficking ◽  
Calcium Concentration ◽  
Fluorescent Microscopy ◽  
Calcium Sensing Receptor ◽  
Calcium Sensing ◽  
Parathyroid Adenomas

The cloning of the calcium sensing receptor (CaR) confirmed that parathyroid cells monitor extracellular calcium concentration ([Ca2+]ext) via a receptor-type mechanism. This lead to the hypothesis that abnormalities in the expression and/or function of the CaR could explain the biochemical abnormalities in primary hyperparathyroidism (PHPT). Cultured cells from parathyroid adenomas of patients operated for PHPT were used to monitor real-time changes in intracellular calcium concentration ([Ca2+]i) as measured by fluorescent microscopy using the Fura-2/AM dye. We found that CaR agonists trigger release of intracellular calcium pools and such responses are amplified by increasing the affinity of IP3 receptors. Using confocal microscopy to monitor membrane trafficking in living parathyroid cells labelled with the fluorescent dye FM1-43, we found that a decrease in [Ca2+]i rather than an absolute change in [Ca2+]ext is the main stimulus for exocytosis from human parathyroid cells. These data suggest that, in PHPT, a defective signalling mechanism from the CaR allows cells from parathyroid adenomas to maintain low [Ca2+]i with uninhibited PTH secretion in the face of hypercalcaemia. Over longer periods of time, CaR controls parathyroid proliferation via changes in tyrosine phosphorylation. We found that multiple proteins of molecular weight 20–65 kDa are phosphorylated within 10–60 min in response to CaR agonists. Further work demonstrated that high [Ca2+]i stimulates the expression of bcl-2 oncoprotein in cultured human parathyroid cells and that, in parathyroid adenomas, predominant expression of bcl-2 rather than bax oncoprotein might prevent apoptosis and explain the slow growth rate of these tumours. More recently, it became apparent that CaR stimulates cell proliferation in several cell types not involved in calcium homeostasis. Using archived histological material from 65 patients who died with metastatic breast cancer, we identified CaR expression predominantly in tumours from patients who developed bone rather than visceral metastases (35 of 49 versus 7 of 16; P < 0.01, chi-squared test). These data suggest that CaR expression has the potential to become a new biological marker predicting the risk of bone metastases in patients with breast cancer. A prospective study should investigate if patients with CaR-positive tumours are more likely to develop bone metastases and whether they could benefit more from prophylactic treatment with bisphosphonates or the newly developed CaR antagonists.

scholarly journals Genetic Alteration Profiles and Clinicopathological Associations in Atypical Parathyroid Adenoma

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Xinxin Mao ◽  
Yan Wu ◽  
Shuangni Yu ◽  
Jie Chen
Keyword(s):  
Parathyroid Adenoma ◽  
De Novo ◽  
Sequencing Analysis ◽  
Sequencing Data ◽  
Genomic Alterations ◽  
Parathyroid Tumors ◽  
Formalin Fixed Paraffin ◽  
Parathyroid Adenomas ◽  
Formalin Fixed Paraffin Embedded ◽  
Atypical Parathyroid Adenoma

Genomic aberrations associated with atypical parathyroid adenoma (AA) are poorly understood. Thus, herein, we sought to expand our current understanding of the molecular basis of atypical parathyroid adenomas. We analyzed 134 samples that had been surgically obtained from parathyroid tumors, including parathyroid carcinomas, atypical parathyroid adenomas, and parathyroid adenomas. The tumors were harvested from formalin-fixed, paraffin-embedded tissues. Fifteen tumor-related genes from recently published genome sequencing data were subjected to targeted sequencing analysis, and an average sequencing depth of 500x was achieved. Sixteen (16/50, 32%) AA tumors harbored at least one of the following genomic alterations: CDC73 (12, 24%), EZH2 (4, 8%), HIC1 (1, 2%), and CDKN2A (1, 2%). Our study identified, for the first time, a relatively high frequency of genomic alterations in patients with AA in a Chinese population. This suggests that AA arises de novo, rather than developing from a parathyroid adenoma. Altogether, these findings will improve our understanding of the malignant potential of parathyroid tumors at the molecular level.

Calcium-sensing receptor expression and signalling in human parathyroid adenomas and primary hyperplasia

2000 ◽  
Vol 52 (3) ◽  
pp. 339-348 ◽  
Author(s):  
Sabrina Corbetta ◽  
Giovanna Mantovani ◽  
Andrea Lania ◽  
Stefano Borgato ◽  
Leonardo Vicentini ◽  
...  
Keyword(s):  
Receptor Expression ◽  
Calcium Sensing Receptor ◽  
Calcium Sensing ◽  
Parathyroid Adenomas

Decreased expression of calcium-sensing receptor messenger ribonucleic acids in parathyroid adenomas

Surgery ◽  
1998 ◽  
Vol 124 (6) ◽  
pp. 1094-1099 ◽  
Author(s):  
Filip Farnebo ◽  
Anders Höög ◽  
Kerstin Sandelin ◽  
Catharina Larsson ◽  
Lars-Ove Farnebo
Keyword(s):  
Calcium Sensing Receptor ◽  
Ribonucleic Acids ◽  
Calcium Sensing ◽  
Parathyroid Adenomas

scholarly journals OR07-06 The Roles of GNAQ and GNA11 in Calcium-Sensing Receptor (CaSR) Signalling

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Anna K Gluck ◽  
Mark Stevenson ◽  
Sara Falcone ◽  
Asuka Inoue ◽  
Gerda E Breitwieser ◽  
...  
Keyword(s):  
Protein Expression ◽  
G Protein ◽  
Extracellular Calcium ◽  
Luciferase Reporter ◽  
Reporter Construct ◽  
Calcium Sensing Receptor ◽  
Luciferase Reporter Construct ◽  
Response Element ◽  
Calcium Sensing

Abstract The G-protein subunits Gα 11 and Gα q, which share &gt;90% peptide sequence identity and are encoded by the GNA11 and GNAQ genes, respectively, mediate signalling by the calcium-sensing receptor (CaSR), a class C G-protein coupled receptor (GPCR) that regulates extracellular calcium (Ca2+e) homeostasis. Germline Gα 11 inactivating and activating mutations cause familial hypocalciuric hypercalcaemia type-2 (FHH2) and autosomal dominant hypocalcaemia type-2 (ADH2), respectively, but such Gα q mutations have not been reported. We therefore investigated the DiscovEHR cohort database, which has exomes from 51,289 patients with matched phenotyping data, for such GNAQ mutations. The DiscovEHR cohort was examined for rare GNAQ variants, which were transiently expressed in CaSR-expressing HEK293A Gα q/11 knockout cells, and their effects on CaSR-mediated intracellular calcium (Ca2+i) release and MAPK activity, in response to increasing concentrations of extracellular calcium were assessed using a nuclear factor of activated T-cells response element (NFAT-RE) luciferase reporter construct and a serum response element (SRE) luciferase reporter construct, respectively. Responses were compared to those of wild-type (WT), inactivating FHH2-associated GNA11 mutations (Leu135Gln and Phe220Ser), and engineered GNAQ mutations that were equivalent to the FHH2-causing GNA11 mutations. Gα q/11 protein expression was confirmed by Western blot analysis. Six rare missense GNAQ variants (Arg19Trp, Ala110Val, Gln299His, Ala302Ser, Ala331Thr, Val344Ile) were identified in DiscovEHR individuals, all of whom had mean plasma calcium values in the normal range (8.30–10.00 mg/dL). Functional characterisation of all six Gα q variants showed no significant difference to WT Gα q responses, thereby indicating that these variants are unlikely to be disease-causing mutations. In addition, the FHH2-causing GNA11 mutations (Leu135Gln and Phe220Ser) had significantly reduced responses, compared to WT Gα 11; however, this could be compensated by WT Gα q. GNAQ Leu135Gln and Phe220Ser, in contrast to their Gα 11 counterparts, showed no differences in protein expression or signalling responses when compared to WT Gα q. Our study, which provides mechanistic insights into the differences between Gα q and Gα 11, indicates that Gα q, unlike Gα 11, does not play a major role in the pathogenesis of FHH2 or ADH2.

Osteocalcin may participate to the bone-parathyroid crosstalk through activation of the calcium-sensing receptor in human parathyroid adenomas

2020 ◽  
Author(s):  
Chiara Verdelli ◽  
Tavanti Giulia Stefania ◽  
Riccardo Maggiore ◽  
Gilberto Mari ◽  
Veronica Sansoni ◽  
...  
Keyword(s):  
Calcium Sensing Receptor ◽  
Calcium Sensing ◽  
Parathyroid Adenomas

scholarly journals Decrease in vitamin D receptor and calcium-sensing receptor in highly proliferative parathyroid adenomas

2003 ◽  
pp. 403-411 ◽  
Author(s):  
S Yano ◽  
T Sugimoto ◽  
T Tsukamoto ◽  
K Chihara ◽  
A Kobayashi ◽  
...  
Keyword(s):  
Vitamin D ◽  
Vitamin D Receptor ◽  
Serum Levels ◽  
Clinical Severity ◽  
Regression Analyses ◽  
Calcium Sensing Receptor ◽  
Calcium Sensing ◽  
Strong Positive Relationship ◽  
Parathyroid Adenomas ◽  
Single Regression

OBJECTIVE: A significant decrease in vitamin D receptor (VDR) and calcium-sensing receptor (CaSR) protein expression has been demonstrated recently in parathyroid (PT) adenomas. In this study, we investigated the relationships between the proliferative activity of parathyroid glands (PTGs) and the expression of VDR as well as CaSR, and compared it with the clinical severity in patients with primary hyperparathyroidism (1 degrees HPT). DESIGN: Seven patients with 1 degrees HPT were included in this study. Four patients with thyroid carcinoma served as controls. METHODS: Immunohistochemical staining was performed on serial sections of PTGs with specific antibodies against CaSR, VDR, and Ki67. Areas examined in each section were selected at random in relation to the gland size. The number of Ki67-positive cells was expressed as a labeling index (LI; positive cells per 1000 PT cells). The expression of CaSR and VDR was semi-quantitatively analyzed based on the intensity of staining. After averages of the scores from all areas were calculated, CaSR and VDR scores, and Ki67 LI were assigned to each gland for use in statistical analyses. RESULTS: In PT adenomas, scores of VDR and CaSR were markedly lower than in normal PTGs (P<0.01), while the proportion of Ki67-positive cells in PT adenomas was significantly higher than in normal PTGs (P<0.01). Single regression analyses revealed that Ki67 LI was positively correlated with serum levels of intact parathyroid hormone and Ca, and PTG weight (R=0.70, P<0.05, R=0.78, P<0.01 and R=0.84, P<0.05 respectively). Ki67 LI was negatively correlated with CaSR and VDR scores (R=-0.78, P<0.01 and R=-0.72, P<0.05 respectively). Moreover, there was a strong positive relationship between CaSR and VDR expression (R=0.95, P<0.001). CONCLUSIONS: Marked decreases in VDR and CaSR expression could, at least in part, be responsible for the high proliferation of PT cells and the pathological progression of 1 degree HPT.

Double jeopardy: a patient’s tale of two concurrent hypercalcaemic syndromes

2020 ◽  
Vol 13 (8) ◽  
pp. e237036
Author(s):  
Aditi Sharma ◽  
Fatima Bahowairath ◽  
Chukwuma Uduku ◽  
Julia E Ostberg
Keyword(s):  
Serum Calcium ◽  
Receptor Gene ◽  
Caucasian Woman ◽  
Sequencing Analysis ◽  
Calcium Sensing Receptor ◽  
Clearance Ratio ◽  
X Ray ◽  
Calcium Sensing ◽  
Sestamibi Scan ◽  
Calcium Sensing Receptor Gene

Primary hyperparathyroidism (PHPT) is the most common cause of parathyroid hormone (PTH) dependent hypercalcaemia, however there are few reported cases of its co-occurrence in patients with familial hypocalciuric hypercalcaemia (FHH). This case highlights the challenges in managing a rare case of dual pathology. A 49-year-old Caucasian woman with symptoms of hypercalcaemia presented with an adjusted serum calcium of 2.77 mmol/L and PTH of 11.5 pmol/L. Neck ultrasound and sestamibi scan were concordant with a left lower parathyroid adenoma, and a preoperative dual-energy X-ray absorptiometry scan confirmed osteopenia. Parathyroidectomy resulted in a PTH reduction from 11.5 pmol/L to 2.7 pmol/L. Interestingly, her lowest pre-operative adjusted serum calcium of 2.67 mmol/L remained unchanged 14 months post-parathyroidectomy. Twenty-four hours urine calcium:creatinine clearance ratio performed postoperatively was low and sequencing analysis of the calcium-sensing receptor gene confirmed the coexistence of FHH. Although surgery is not indicated in FHH, parathyroidectomy may help reduce hypercalcaemia and its associated complications if there is coexistent PHPT.

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