Supplemental Digital Content is available in the text. The endoplasmic reticulum (ER) plays a pivotal role in syntheses of proteins and steroid hormones and regulation of intracellular Ca2+ 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.

中文翻译：

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内质网伴侣 Calmegin 在醛固酮生成腺瘤中上调并与醛固酮生成相关

补充数字内容在文本中可用。内质网 (ER) 在蛋白质和类固醇激素的合成以及细胞内 Ca2+ 水平的调节中起关键作用。我们旨在研究醛固酮腺瘤 (APA) 中的 ER 相关基因，并阐明它们对醛固酮生成的影响。使用无功能的肾上腺皮质腺瘤 (n=5) 和 APA (n=19) 进行靶向 288 个 ER 相关基因的微阵列分析。对 13 个无功能的肾上腺皮质腺瘤和 48 个 APA 样本进行了免疫组织化学和定量聚合酶链反应分析。对人肾上腺皮质癌 (HAC15) 细胞进行了功能研究，其中一些细胞是使用慢病毒进行基因改造的。相对于无功能的肾上腺皮质腺瘤，ER 伴侣钙蛋白 (CLGN) 是 APA 中表达最高的 ER 相关基因。定量聚合酶链反应分析显示，与无功能的肾上腺皮质腺瘤相比，APA 中的 CLGN 上调了 9.5 倍。影响醛固酮生成的不同 APA 基因型之间没有差异。免疫组织化学分析显示 CLGN 在 APA 和醛固酮产生细胞簇中强烈表达。HAC15 细胞中血管紧张素 II 刺激或 KCNJ5 T158A 过表达不影响 CLGN mRNA 水平。HAC15 细胞中的 CLGN 过表达增加了醛固酮水平，但不刺激 CYP11B2 mRNA 水平。使用 RNA 测序结果的通路和基因本体分析表明，tRNA 氨酰代谢是 CLGN 过表达细胞中最丰富的通路。CYP11B2（醛固酮合酶）和 HSD3B2（3 β-羟基类固醇脱氢酶/δ 5-> 4-异构酶 2 型）蛋白表达在 CLGN 过表达细胞中更为丰富。在携带 KCNJ5 突变的 HAC15 细胞中使用 CRISPR/Cas9（成簇规则间隔短回文重复序列/成簇规则间隔短回文重复序列相关 9）方法的 CLGN 敲低不影响醛固酮的产生。总而言之，CLGN 被上调并通过 APA 中 CYP11B2 的翻译调节与醛固酮产生相关。4-异构酶 2) 蛋白表达在 CLGN 过表达细胞中更为丰富。在携带 KCNJ5 突变的 HAC15 细胞中使用 CRISPR/Cas9（成簇规则间隔短回文重复序列/成簇规则间隔短回文重复序列相关 9）方法的 CLGN 敲低不影响醛固酮的产生。总而言之，CLGN 被上调并通过 APA 中 CYP11B2 的翻译调节与醛固酮产生相关。4-异构酶 2) 蛋白表达在 CLGN 过表达细胞中更为丰富。在携带 KCNJ5 突变的 HAC15 细胞中使用 CRISPR/Cas9（成簇规则间隔短回文重复序列/成簇规则间隔短回文重复序列相关 9）方法的 CLGN 敲低不影响醛固酮的产生。总而言之，CLGN 被上调并通过 APA 中 CYP11B2 的翻译调节与醛固酮产生相关。