Fujii Hiroko, Tamamori-Adachi Mimi, Uchida Kousuke, Susa Takao, Nakakura Takashi, Hagiwara Haruo, Iizuka Masayoshi, Okinaga Hiroko, Tanaka Yuji, Okazaki Tomoki
Department of General Medicine, National Defense Medical College, Tokorozawa City, Saitama, Japan; Department of Biochemistry, Teikyo University School of Medicine, Tokyo, Japan.
Department of Biochemistry, Teikyo University School of Medicine, Tokyo, Japan.
PLoS One. 2014 Oct 21;9(10):e110543. doi: 10.1371/journal.pone.0110543. eCollection 2014.
The ectopic expression of the glucose-dependent insulinotropic polypeptide receptor (GIPR) in the human adrenal gland causes significant hypercortisolemia after ingestion of each meal and leads to Cushing's syndrome, implying that human GIPR activation is capable of robustly activating adrenal glucocorticoid secretion. In this study, we transiently transfected the human GIPR expression vector into cultured human adrenocortical carcinoma cells (H295R) and treated them with GIP to examine the direct link between GIPR activation and steroidogenesis. Using quantitative RT-PCR assay, we examined gene expression of steroidogenic related proteins, and carried out immunofluorescence analysis to prove that forced GIPR overexpression directly promotes production of steroidogenic enzymes CYP17A1 and CYP21A2 at the single cell level. Immunofluorescence showed that the transfection efficiency of the GIPR gene in H295R cells was approximately 5%, and GIP stimulation enhanced CYP21A2 and CYP17A1 expression in GIPR-introduced H295R cells (H295R-GIPR). Interestingly, these steroidogenic enzymes were also expressed in the GIPR (-) cells adjacent to the GIPR (+) cells. The mRNA levels of a cholesterol transport protein required for all steroidogenesis, StAR, and steroidogenic enzymes, HSD3β2, CYP11A1, CYP21A2, and CYP17A1 increased 1.2-2.1-fold in GIP-stimulated H295R-GIPR cells. These changes were reflected in the culture medium in which 1.5-fold increase in the cortisol concentration was confirmed. Furthermore, the levels of adenocorticotropic hormone (ACTH) receptor and ACTH precursor proopiomelanocortin (POMC) mRNA were upregulated 2- and 1.5-fold, respectively. Immunofluorescence showed that ACTH expression was detected in GIP-stimulated H295R-GIPR cells. An ACTH-receptor antagonist significantly inhibited steroidogenic gene expression and cortisol production. Immunostaining for both CYP17A1 and CYP21A2 was attenuated in cells treated with ACTH receptor antagonists as well as with POMC siRNA. These results demonstrated that GIPR activation promoted production and release of ACTH, and that steroidogenesis is activated by endogenously secreted ACTH following GIP administration, at least in part, in H295R cells.
葡萄糖依赖性促胰岛素多肽受体（GIPR）在人肾上腺中的异位表达会导致每餐进食后出现显著的高皮质醇血症，并引发库欣综合征，这意味着人GIPR的激活能够强有力地激活肾上腺糖皮质激素的分泌。在本研究中，我们将人GIPR表达载体瞬时转染到培养的人肾上腺皮质癌细胞（H295R）中，并用GIP处理它们，以研究GIPR激活与类固醇生成之间的直接联系。使用定量RT-PCR分析，我们检测了类固醇生成相关蛋白的基因表达，并进行了免疫荧光分析，以证明在单细胞水平上，强制过表达GIPR直接促进类固醇生成酶CYP17A1和CYP21A2的产生。免疫荧光显示，GIPR基因在H295R细胞中的转染效率约为5%，GIP刺激增强了导入GIPR的H295R细胞（H295R-GIPR）中CYP21A2和CYP17A1的表达。有趣的是，这些类固醇生成酶也在与GIPR（+）细胞相邻的GIPR（-）细胞中表达。在GIP刺激的H295R-GIPR细胞中，所有类固醇生成所需的胆固醇转运蛋白StAR以及类固醇生成酶HSD3β2、CYP11A1、CYP21A2和CYP17A1的mRNA水平增加了1.2 - 2.1倍。这些变化反映在培养基中，其中皮质醇浓度证实增加了1.5倍。此外，促肾上腺皮质激素（ACTH）受体和ACTH前体阿黑皮素原（POMC）mRNA的水平分别上调了2倍和1.5倍。免疫荧光显示，在GIP刺激的H295R-GIPR细胞中检测到ACTH表达。ACTH受体拮抗剂显著抑制类固醇生成基因的表达和皮质醇的产生。在用ACTH受体拮抗剂以及POMC siRNA处理的细胞中，CYP17A1和CYP21A2的免疫染色减弱。这些结果表明GIPR激活促进了ACTH的产生和释放，并且在给予GIP后，类固醇生成至少部分是由内源性分泌的ACTH在H295R细胞中激活的。
