RESUMO
Sheehan's syndrome (SS) is a rare but well-characterized cause of hypopituitarism. Data on skeletal health is limited and on microarchitecture is lacking in SS patients. PURPOSE: We aimed to explore skeletal health in SS with bone mineral density (BMD), turnover, and microarchitecture. METHODS: Thirty-five patients with SS on stable replacement therapy for respective hormone deficiencies and 35 age- and BMI-matched controls were recruited. Hormonal profile and bone turnover markers (BTMs) were measured using electrochemiluminescence assay. Areal BMD and trabecular bone score were evaluated using DXA. Bone microarchitecture was assessed using a second-generation high-resolution peripheral quantitative computed tomography. RESULTS: The mean age of the patients was 45.5 ± 9.3 years with a lag of 8.3 ± 7.2 years prior to diagnosis. Patients were on glucocorticoid (94%), levothyroxine (94%), and estrogen-progestin replacement (58%). None had received prior growth hormone (GH) replacement. BTMs (P1NP and CTX) were not significantly different between patients and controls. Osteoporosis (26% vs. 16%, p = 0.01) and osteopenia (52% vs. 39%, p = 0.007) at the lumbar spine and femoral neck (osteoporosis, 23% vs. 10%, p = 0.001; osteopenia, 58% vs. 29%, p = 0.001) were present in greater proportion in SS patients than matched controls. Bone microarchitecture analysis revealed significantly lower cortical volumetric BMD (vBMD) (p = 0.02) at the tibia, with relative preservation of the other parameters. CONCLUSION: Low areal BMD (aBMD) is highly prevalent in SS as compared to age- and BMI-matched controls. However, there were no significant differences in bone microarchitectural measurements, except for tibial cortical vBMD, which was lower in adequately treated SS patients.
Assuntos
Doenças Ósseas Metabólicas , Hipopituitarismo , Osteoporose , Feminino , Humanos , Adulto , Pessoa de Meia-Idade , Densidade Óssea , Osteoporose/diagnóstico por imagem , Hipopituitarismo/diagnóstico por imagem , Hipopituitarismo/tratamento farmacológico , Tomografia Computadorizada por Raios X , Tíbia/diagnóstico por imagem , Rádio (Anatomia) , Absorciometria de Fóton/métodosRESUMO
The continuous cell line of epithelial human parathyroid cells has been proven difficult. Previously, PTH-C1 cell line was only established rat parathyroid tissue cell line known to express the parathyroid hormone-related peptide (Pthrp) gene. The paucity of continuous cell line of human parathyroid cells secreting parathyroid hormone (PTH) has imposed hurdle in in vitro assessment of the mechanisms involved in the control of parathyroid cell function and proliferation. The primary cell cultures of human parathyroid cells were derived from parathyroid adenoma tissue biopsy (n = 5). The cells were subsequently subcultured to maintained primary subclones. Karyotyping analysis was performed to analyze the genotypic identity of derived subclones. The expression of calcium-sensing receptor (CaSR) and intact parathyroid hormone (iPTH) were analyzed using immunocytochemistry and immunofluorescence. In the present study, we have used a defined condition medium to generate the continuous culture of human parathyroid cells derived from patients with parathyroid adenoma due to primary hyperparathyroidism. The subcultured primary subclones were maintained epithelial and polygonal morphology, doubling time of approximately 25 h, displaying a diploid chromosome number, and secretion of PTH. This cell line produces PTH and expresses the calcium-sensing receptor (CaSR) known to be involved in parathyroid function. Altogether these findings indicate the uniqueness of the human parathyroid cell line as an in vitro model for cellular and molecular studies on parathyroid physiopathology.
RESUMO
CONTEXT: Primary hyperparathyroidism (PHPT) results from the hypersecretion of parathyroid hormone from parathyroid tumors. A transcription factor, namely Paired box1 (PAX1), is active in parathyroid gland development. OBJECTIVE: We aimed to study potential epigenetic-mediated mechanism of PAX1 gene in sporadic parathyroid adenomas. METHODS: In parathyroid adenomas tissues, we analyzed the DNA methylation via bisulfite-specific polymerase chain reaction (BSP) and histone modifications via chromatin immunoprecipitation in regulating the differential expression of PAX1. RESULTS: The results showed that mRNA and protein expression of PAX1 was significantly reduced in parathyroid adenomas. Bisulfite sequencing demonstrated hypermethylation in the promoter region of PAX1 (35%; 14/40) and lower levels of histone 3 lysine 9 acetylation (H3K9ac) were observed on the promoter region of PAX1 (6-fold; P < .004) in parathyroid adenomas. Furthermore, upon treatment with a pharmacologic inhibitor, namely 5'aza-2 deoxycytidine, in rat parathyroid continuous cells, we found re-expression of PAX1 gene. CONCLUSION: Our study not only reveals expression of PAX1 is epigenetically deregulated but also paves a way for clinical and therapeutic implications in patients with PHPT.
Assuntos
Adenoma/genética , Carcinogênese/genética , Epigênese Genética/genética , Fatores de Transcrição Box Pareados/genética , Neoplasias das Paratireoides/genética , Acetilação/efeitos dos fármacos , Adenoma/patologia , Adenoma/terapia , Adolescente , Adulto , Idoso , Animais , Carcinogênese/efeitos dos fármacos , Estudos de Casos e Controles , Linhagem Celular Tumoral , Metilação de DNA/efeitos dos fármacos , Decitabina/farmacologia , Decitabina/uso terapêutico , Epigênese Genética/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Voluntários Saudáveis , Código das Histonas/efeitos dos fármacos , Humanos , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/uso terapêutico , Masculino , Pessoa de Meia-Idade , Fatores de Transcrição Box Pareados/metabolismo , Glândulas Paratireoides/patologia , Glândulas Paratireoides/cirurgia , Neoplasias das Paratireoides/patologia , Neoplasias das Paratireoides/terapia , Paratireoidectomia , Regiões Promotoras Genéticas/genética , Ratos , Adulto JovemRESUMO
CONTEXT: Glial cells missing 2 (GCM2), a zinc finger-transcription factor, is essentially required for the development of the parathyroid glands. OBJECTIVE: We sought to identify whether the epigenetic alterations in 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. METHODS: Messenger RNA (mRNA) and protein expression of GCM2 were analyzed by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry in 33 adenomatous and 10 control parathyroid tissues. DNA methylation and histone methylation/acetylation of the GCM2 promoter were measured by bisulfite sequencing and chromatin immunoprecipitation-qPCR. Additionally, we investigated the role of epigenetic modifications on GCM2 and DNA methyltransferase 1 (DNMT1) expression in parathyroid (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 (Pâ <â .001) and higher H3K9me3 levels in the GCM2 promoter (Pâ <â .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 nonsignificant 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 a promising avenue of research for parathyroid adenoma therapeutics.
Assuntos
Adenoma/genética , Metilação de DNA/genética , Histonas/genética , Proteínas Nucleares/genética , Neoplasias das Paratireoides/genética , Fatores de Transcrição/genética , Adulto , Estudos de Casos e Controles , Linhagem Celular Tumoral , Epigênese Genética/genética , Feminino , Inativação Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Glândulas Paratireoides/metabolismo , Regiões Promotoras Genéticas/genética , RNA Mensageiro/metabolismoRESUMO
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 (Pâ <â 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) (Pâ <â 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 (râ =â 0.61; Pâ <â 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.