A HAMP promoter bioassay system for identifying chemical compounds that modulate hepcidin expression
A HAMP promoter bioassay system for identifying chemical compounds that modulate hepcidin expression
复制标题
DOI：
10.1016/j.exphem.2015.01.005
复制
发表时间：
2015-05-01
影响因子：
2.6
通讯作者：
Takaori-Kondo, Akifumi
中科院分区：
文献类型：
--
作者：
Kawabata, Hiroshi;Uchiyama, Tatsuki;Takaori-Kondo, Akifumi
铁调素是系统性铁稳态的中心调节剂;铁调素表达的失调引起各种铁代谢紊乱，包括遗传性血色素沉着症和炎症性贫血。为了鉴定调节hepcidin表达的分子，我们通过用表达质粒稳定转染Hep 3B肝癌细胞，建立了hepcidin基因（HAMP）启动子活性的生物测定系统，其中EGFP连接到2.5kb的人HAMP启动子。白细胞介素6、骨形态发生蛋白6（BMP-6）和制瘤素M（HAMP启动子的特征性刺激物）强烈增强了这些细胞的绿色荧光强度。Dorsomorphin、肝素和氯化钴，已知的铁调素表达抑制剂，显著抑制绿色荧光强度，并且当用BMP-6刺激细胞时，这些抑制作用更加突出。利用这个系统，我们筛选了1，280种具有生物活性的小分子，并发现了几种hepcidin表达的候选抑制剂。阿扑吗啡、苯扎米尔、依托泊苷、CGS-15943、kenpaullone和吴茱萸次碱（均为10 μ mol/L）显著抑制Hep 3B细胞的hepcidin mRNA表达而不影响细胞活力。CGS-15943是这些细胞中BMP-6诱导的hepcidin-25分泌的最强抑制剂。我们的结论是，我们新开发的hepcidin启动子生物测定系统是有用的，用于识别和评估化合物，调节hepcidin的表达。版权所有（C）2015 Elsevier Inc. ISEH -国际实验血液学学会。
Hepcidin is the central regulator of systemic iron homeostasis; dysregulation of hepcidin expression causes various iron metabolic disorders, including hereditary hemochromatosis and anemia of inflammation. To identify molecules that modulate hepcidin expression, we developed a bioassay system for hepcidin gene (HAMP) promoter activity by stable transfection of Hep3B hepatoma cells with an expression plasmid in which EGFP was linked to a 2.5kb human HAMP promoter. Interleukin 6, bone morphogenetic protein 6 (BMP-6), and oncostatin M, well-characterized stimulators of the HAMP promoter, strongly enhanced the green fluorescence intensity of these cells. Dorsomorphin, heparin, and cobalt chloride, known inhibitors of hepcidin expression, significantly suppressed green fluorescence intensity, and these inhibitory effects were more prominent when the cells were stimulated with BMP-6. Employing this system, we screened 1,280 biologically active small molecules and found several candidate inhibitors of hepcidin expression. Apomorphine, benzamil, etoposide, CGS-15943, kenpaullone, and rutaecarpine (all at 10 mu mol/L) significantly inhibited hepcidin mRNA expression by Hep3B cells without affecting cell viability. CGS-15943 was the strongest suppressor of BMP-6-induced hepcidin-25 secretion in these cells. We conclude that our newly developed hepcidin promoter bioassay system is useful for identifying and evaluating compounds that modulate hepcidin expression. Copyright (C) 2015 Published by Elsevier Inc. on behalf of ISEH - International Society for Experimental Hematology.