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

Abstract

The endoplasmic reticulum (ER) plays a pivotal role in syntheses of proteins and steroid hormones and regulation of intracellular Ca 2+ 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 ( CLGN ) was the most highly expressed ER-associated gene in APAs relative to nonfunctioning adrenocortical adenomas. Analysis with quantitative polymerase chain reaction revealed CLGN 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 CLGN mRNA levels. CLGN overexpression in HAC15 cells increased aldosterone levels but did not stimulate CYP11B2 mRNA levels. Pathway and gene ontology analyses using RNA sequencing results showed that tRNA aminoacyl metabolism was the most enriched pathway in CLGN -overexpressing cells. CYP11B2 (aldosterone synthase) and HSD3B2 (3 beta-hydroxysteroid dehydrogenase/delta 5->4-isomerase type 2) protein expression were more abundant in CLGN -overexpressing cells. CLGN 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 KCNJ5 mutation did not affect aldosterone production. To summarize, CLGN was upregulated and associated with aldosterone production via translational regulation of CYP11B2 in APAs.

Update on Genetics of Primary Aldosteronism

“…Depolarization activates voltage-gated Ca 2+ channels and intracellular calcium signaling and promotes the transcription of aldosterone synthase ( CYP11B2 ), resulting in overproduction of aldosterone ( Figure 1 ). Furthermore, some key molecules such as VSNL1, CALN1, GSTA1, NPNT, and CLGN have been detected in APA, and their functions in aldosterone production have been elucidated [ 14 , 15 , 16 , 17 , 18 ]. Epigenetic regulation of CYP11B2 has also been indicated in APA [ 19 , 20 , 21 , 22 ].…”

The landscape of molecular mechanism for aldosterone production in aldosterone-producing adenoma

“…Entire intracellular mechanisms of aldosterone production are still complex. In addition, recent basic studies suggested that mRNA translation and translation via miRNA also participate aldosterone regulation in APA [19,[45][46][47], however these have not been fully elucidated. Significant more work including basic studies on the regulation of aldosterone biosynthesis will be necessary for the development of medical therapies for primary aldosteronism and aldosterone-producing adenomas.…”

“…CLGN, calmegin, is the highest expressed genes among ER related genes in APA in comparison to NFA by transcriptome analysis [19]. CLGN over-expressing adrenal cells increased CYP11B2 protein levels and aldosterone production, but not CYP11B2 transcription [19].…”

“…expression in APA, and separate from CYP11B1 expression [8]. The antibody is also useful for western blotting, and thus it is utilized in basic studies focused on CYP11B2 expression using cell culture [19]. The expressions of steroidogenic enzymes except for CYP11B2 were influenced by the detection method and adrenal reference tissue, and the difference appeared to be slight if any.…”

“…CLGN, calmegin, is the highest expressed genes among ER related genes in APA in comparison to NFA by transcriptome analysis [19]. CLGN over-expressing adrenal cells increased CYP11B2 protein levels and aldosterone production, but not CYP11B2 transcription [19]. The RNA sequencing analysis using CLGN overexpressing adrenal cells revealed that tRNA metabolic process and aminoacyl-tRNA biosynthesis were the most enriched pathways, indicating translation activation [19].…”