immunoprecipitation-with-an-anti-epitope-tag-affinity-gel-to-study

Immunoprecipitation with an Anti-Epitope Tag Affinity Gel to Study Protein-Protein Interactions

Protein-protein interactions (PPIs) play a pivotal role in biological phenomena, such as cellular organization, intracellular signal transduction, and ...

Hiroshima University

Elevated midnight cortisol levels induced by non-suppressed ACTH levels may lead to false-positive results for hypercortisolism in patients with adrenal incidentaloma. We investigated whether plasma ACTH-associated high midnight serum cortisol levels are correlated with other endocrinological findings with respect to hypothalamic-pituitaryadrenal function or hypercortisolism status.

Measurement of midnight ACTH levels is useful for the evaluation of midnight cortisol levels.

The endoplasmic reticulum (ER) plays a pivotal role in syntheses of proteins and steroid hormones and regulation of intracellular Ca level. We aimed to investigate ER-associated genes in aldosterone-producing adenomas (APAs) and clarify their effect on aldosterone production. Microarray analysis targeting 288 ER-associated genes was conducted using nonfunctioning adrenocortical adenomas (n=5) and APAs (n=19). Immunohistochemistry and quantitative polymerase chain reaction analyses were performed with 13 nonfunctioning adrenocortical adenoma and 48 APA samples. Functional studies were performed with human adrenocortical carcinoma (HAC15) cells, some of which were genetically modified using lentiviruses. The ER chaperone calmegin () was the most highly expressed ER-associated gene in APAs relative to nonfunctioning adrenocortical adenomas. Analysis with quantitative polymerase chain reaction revealed  to be 9.5-fold upregulated in APAs relative to nonfunctioning adrenocortical adenomas. There were no differences among different APA genotypes affecting aldosterone production. Immunohistochemistry analysis revealed that CLGN was strongly expressed in APAs and aldosterone-producing cell clusters. Angiotensin II stimulation or KCNJ5 T158A overexpression in HAC15 cells did not affect  mRNA levels.  overexpression in HAC15 cells increased aldosterone levels but did not stimulate  mRNA levels. Pathway and gene ontology analyses using RNA sequencing results showed that tRNA aminoacyl metabolism was the most enriched pathway in -overexpressing cells. CYP11B2 (aldosterone synthase) and HSD3B2 (3 beta-hydroxysteroid dehydrogenase/delta 5->4-isomerase type 2) protein expression were more abundant in -overexpressing cells.  knockdown using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) method in HAC15 cells that carry the  mutation did not affect aldosterone production. To summarize,  was upregulated and associated with aldosterone production via translational regulation of CYP11B2 in APAs.

Endoplasmic Reticulum Chaperone Calmegin Is Upregulated in Aldosterone-Producing Adenoma and Associates With Aldosterone Production.

Angiotensin-converting enzyme 2 expression is not induced by the renin-angiotensin system in the lung.

The intracellular molecular mechanisms underlying the genotype of cortisol-producing adenoma (CPA) have not been fully determined. We analyzed gene expressions in CPA and the human adrenocortical cell line (HAC15 cells) with PRKACA mutation. Clustering analysis using a gene set associated with responses to cAMP revealed the possible differences between PRKACA mutant CPAs and GNAS and CTNNB1 mutant CPAs. The levels of STAR, CYP11A1, CYP17A1, CYP21A2, and FDX1 transcripts and cortisol levels per unit area in PRKACA mutant CPAs were significantly higher than those in GNAS mutant CPAs. PRKACA mutations led to an increase in steroidogenic enzyme expression and cortisol production in HAC15 cells. Transcriptome analysis revealed differences between PRKACA mutant CPAs and GNAS and CTNNB1 mutant CPAs. Cortisol production in PRKACA mutant CPAs is increased by the cAMP-PKA signaling pathway-mediated upregulation of steroidogenic enzymes transcription. The intracellular molecular mechanisms underlying these processes would be notably important in PRKACA mutant CPAs.

Genotype-specific cortisol production associated with Cushing's syndrome adenoma with PRKACA mutations.

The molecular mechanisms by which  mutation-mediated cell proliferation or tumorigenesis in aldosterone-producing adenomas (APAs) have not been elucidated. First, we investigated whether the APA-associated  L104R mutation stimulated cell proliferation. Second, we aimed to clarify the molecular mechanisms by which the  mutation-mediated cell proliferated. We performed transcriptome analysis in APAs with  mutation.  L104R mutation were modulated in human adrenocortical carcinoma (HAC15) cells (ATP1A1-mutant cells), and we evaluated cell proliferation and molecular signaling events. Transcriptome and immunohistochemical analysis showed that Na/K-ATPase (NKA) expressions in  mutated APA were more abundant than those in non-functioning adrenocortical adenoma or  mutated APAs. The significant increase of number of cells, amount of DNA and S-phase population were shown in ATP1A1-mutant cells. Fluo-4 in ATP1A1-mutant cells were significantly increased. Low concentration of ouabain stimulated cell proliferation in ATP1A1-mutant cells. ATP1A1-mutant cells induced Src phosphorylation, and low concentration of ouabain supplementation showed further Src phosphorylation. We demonstrated that NKAs were highly expressed in  mutant APA, and the mutant stimulated cell proliferation and Src phosphorylation in ATP1A1-mutant cells. NKA stimulations would be a risk factor for the progression and development to an  mutant APA.

 Mutant in Aldosterone-Producing Adenoma Leads to Cell Proliferation.

DNA methylation alteration is tissue-specific and play a pivotal role in regulating gene transcription during cell proliferation and survival. We aimed to detect genes regulated by DNA methylation, and then investigated whether the gene influenced cell proliferation or survival in adrenal cells. DNA methylation and qPCR analyses were performed in nonfunctioning adrenocortical adenoma (NFA, n = 12) and aldosterone-producing adenoma (APA, n = 35) samples. The VDR gene promoter was markedly hypomethylated in APA with ATP1A1 mutation, and the promoter methylation levels showed a significant inverse association with the transcripts in APA. ATP1A1 mutation led to VDR transcription in HAC15 cells, and VDR suppression abrogated ATP1A1 mutation-mediated cell proliferation in HAC15 cells. We demonstrated that APA with ATP1A1 mutation showed entire hypomethylation in the VDR promoter and abundant VDR mRNA and protein expression. VDR suppression abrogated ATP1A1 mutation-mediated cell proliferation in HAC15 cells. Abundant VDR expression would be essential for ATP1A1 mutation-mediated cell proliferation.

Hypomethylation associated vitamin D receptor expression in ATP1A1 mutant aldosterone-producing adenoma.

DNA methylation and demethylation regulate the transcription of genes. DNA methylation-associated gene expression of adrenal steroidogenic enzymes may regulate cortisol production in cortisol-producing adenoma (CPA). We aimed to determine the DNA methylation levels of all genes encoding steroidogenic enzymes involved in CPA. Additionally, the aims were to clarify the DNA methylation-associated gene expression and evaluate the difference of CPA genotype from others using DNA methylation data. Twenty-five adrenal CPA and six nonfunctioning adrenocortical adenoma (NFA) samples were analyzed. RNA sequencing and DNA methylation array were performed. The methylation levels at 118 methylation sites of the genes were investigated, and their methylation and mRNA levels were subsequently integrated. Among all the steroidogenic enzyme genes studied, CYP17A1 gene was mainly found to be hypomethylated in CPA compared to that in NFA, and the Benjamini-Hochberg procedure demonstrated that methylation levels at two sites in the CYP17A1 gene body were statistically significant. PRKACA mutant CPAs predominantly exhibited hypomethylation of CYP17A1 gene compared with the GNAS mutant CPAs. Inverse associations between CYP17A1 methylation in three regions of the gene body and its mRNA levels were observed in the NFAs and CPAs. In applying clustering analysis using CYP17A1 methylation and mRNA levels, CPAs with PRKACA mutation were differentiated from NFAs and CPAs with a GNAS mutation. We demonstrated that CPAs exhibited hypomethylation of the CYP17A1 gene body in CPA, especially in the PRKACA mutant CPAs. Methylation of CYP17A1 gene may influence its transcription levels.

Association of DNA methylation with steroidogenic enzymes in Cushing's adenoma.

Hypertension imposes substantial health and economic burden worldwide. Primary aldosteronism (PA) is one of the most common causes of secondary hypertension, causing cardiovascular events at higher risk compared with essential hypertension. However, the germline genetic contribution to the susceptibility of PA has not been well elucidated.

Genetic Risk of Primary Aldosteronism and Its Contribution to Hypertension: A Cross-Ancestry Meta-Analysis of Genome-Wide Association Studies.

Beige adipocytes are inducible thermogenic adipocytes used for anti-obesity treatment. Beige adipocytes rapidly lose their thermogenic capacity once external cues are removed. However, long-term administration of stimulants, such as PPARγ and β-adrenergic receptor agonists, is unsuitable due to various side effects. Here, we reported that PPARα pharmacological activation was the preferred target for maintaining induced beige adipocytes. Pemafibrate used in clinical practice for dyslipidemia was developed as a selective PPARα modulator (SPPARMα). Pemafibrate administration regulated the thermogenic capacity of induced beige adipocytes, repressed body weight gain, and ameliorated impaired glucose tolerance in diet-induced obese mouse models. The transcriptome analysis revealed that the E-twenty-six transcription factor ELK1 acted as a cofactor of PPARα. ELK1 was mobilized to the  transcription regulatory region with PPARα and modulated its expression by pemafibrate. These results suggest that selective activation of PPARα by pemafibrate is advantageous to maintain the function of beige adipocytes.

Ryuta Baba

Department of Molecular and Internal Medicine,
Graduate School of Biomedical & Health Sciences,
Department of Molecular and Internal Medicine, Graduate School of Biomedical & Health Sciences

Hiroshima University

Immunoprecipitation with an Anti-Epitope Tag Affinity Gel to Study Protein-Protein Interactions

Ryuta Baba

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Molecular and Internal Medicine,Graduate School of Biomedical & Health Sciences,Department of Molecular and Internal Medicine, Graduate School of Biomedical & Health Sciences

Hiroshima University

Immunoprecipitation with an Anti-Epitope Tag Affinity Gel to Study Protein-Protein Interactions

Department of Molecular and Internal Medicine,
Graduate School of Biomedical & Health Sciences,
Department of Molecular and Internal Medicine, Graduate School of Biomedical & Health Sciences