Kobuke Kazuhiro, Oki Kenji, Gomez-Sanchez Celso E, Gomez-Sanchez Elise P, Ohno Haruya, Itcho Kiyotaka, Yoshii Yoko, Yoneda Masayasu, Hattori Noboru
From the Department of Molecular and Internal Medicine, Graduate School of Biomedical and Health Sciences, Hiroshima University, Japan (K.K., K.O., H.O., K.I., Y.Y., M.Y., N.H.); Division of Endocrinology, G.V. (Sonny) Montgomery VA Medical Center, Jackson, MS (C.E.G.-S., E.P.G.-S.); and University of Mississippi Medical Center, Jackson (C.E.G.-S., E.P.G.-S.).
Hypertension. 2018 Jan;71(1):125-133. doi: 10.1161/HYPERTENSIONAHA.117.10205. Epub 2017 Nov 6.
Aldosterone production is initiated by angiotensin II stimulation and activation of intracellular Ca signaling. In aldosterone-producing adenoma (APA) cells, the activation of intracellular Ca signaling is independent of the renin-angiotensin-aldosterone systems. The purpose of our study was to clarify molecular mechanisms of aldosterone production related to Ca signaling. Transcriptome analysis revealed that the gene encoding calneuron 1 had the strongest correlation with (aldosterone synthase) among genes encoding Ca-binding proteins in APA. modulation and synthetic or fluorescent compounds were used for functional studies in human adrenocortical carcinoma (HAC15) cells. expression was 4.4-fold higher in APAs than nonfunctioning adrenocortical adenomas. CALN1 expression colocalized with CYP11B2 expression as investigated using immunohistochemistry in APA and zona glomerulosa of male rats fed by a low-salt diet. CALN1 expression was detected in the endoplasmic reticulum (ER) by using GFP-fused CALN1, CellLight ER-RFP, and the corresponding antibodies. -overexpressing HAC15 cells showed increased Ca in the ER and cytosol fluorescence-based studies. Aldosterone production was potentiated in HAC15 cells by expression, and dose-responsive inhibition with TMB-8 showed that CALN1-mediated Ca storage in ER involved sarcoendoplasmic reticulum calcium transport ATPase. The silencing of decreased Ca in ER, and abrogated angiotensin II- or KCNJ5 T158A-mediated aldosterone production in HAC15 cells. Increased CALN1 expression in APA was associated with elevated Ca storage in ER and aldosterone overproduction. Suppression of CALN1 expression prevented angiotensin II- or KCNJ5 T158A-mediated aldosterone production in HAC15 cells, suggesting that CALN1 is a potential therapeutic target for excess aldosterone production.
醛固酮的产生由血管紧张素II刺激和细胞内钙信号激活引发。在醛固酮分泌性腺瘤（APA）细胞中，细胞内钙信号的激活独立于肾素-血管紧张素-醛固酮系统。我们研究的目的是阐明与钙信号相关的醛固酮产生的分子机制。转录组分析显示，在APA中编码钙神经元1的基因与编码钙结合蛋白的基因中，与（醛固酮合酶）的相关性最强。钙调蛋白调节以及合成或荧光化合物用于人肾上腺皮质癌（HAC15）细胞的功能研究。在APA中，其表达比无功能肾上腺皮质腺瘤高4.4倍。使用免疫组织化学在喂食低盐饮食的雄性大鼠的APA和肾小球带中进行研究，发现CALN1表达与CYP11B2表达共定位。通过使用绿色荧光蛋白融合的CALN1、内质网红色荧光蛋白标记物（CellLight ER-RFP）和相应抗体，在内质网（ER）中检测到CALN1表达。基于荧光的研究表明，过表达CALN1的HAC15细胞在内质网和细胞质中的钙增加。CALN1表达使HAC15细胞中的醛固酮产生增强，用TMB-8进行的剂量反应抑制表明CALN1介导的内质网钙储存涉及肌浆内质网钙转运ATP酶。CALN1沉默降低了内质网中的钙，并消除了血管紧张素II或KCNJ5 T158A介导的HAC15细胞中的醛固酮产生。APA中CALN1表达增加与内质网钙储存增加和醛固酮过量产生相关。抑制CALN1表达可防止血管紧张素II或KCNJ5 T158A介导的HAC15细胞中的醛固酮产生，表明CALN1是醛固酮产生过多的潜在治疗靶点。
