Differential regulation of aldosterone synthase and 11β-hydroxylase transcription by steroidogenic factor-1
Differential regulation of aldosterone synthase and 11β-hydroxylase transcription by steroidogenic factor-1
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DOI：
10.1677/jme.0.0280125
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发表时间：
2002-04-01
影响因子：
3.5
通讯作者：
Rainey, WE
中科院分区：
文献类型：
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作者：
Bassett, MH;Zhang, Y;Rainey, WE
羟化酶（hCYP11B1）和醛固酮合成酶（hCYP11B2）是密切相关的同工酶，分别在皮质醇和醛固酮的产生中起着不同的作用。醛固酮合成酶催化醛固酮生物合成的最后一步，仅在正常肾上腺的肾小球带中表达。11 p -羟化酶催化皮质醇生成的最终反应，并在束状带中表达较高水平。引起这些基因差异表达的机制尚不清楚。在此，我们展示了孤儿受体甾体生成因子-1 （SF-1）在调节人类(h) CYP11B1和hCYP11B2中的不同作用。用含有hCYP11B1、hCYP11B2、人17 α -羟化酶（hCYP17）、人胆固醇侧链切割（hCYP11A1）或小鼠(m) cyp11b2 (mcyp11b2) 5′侧区域的荧光素酶报告基因构建物瞬时转染人NCI-H295R （H295R）或小鼠Y-1细胞。共转染编码SF-1的载体增加了hCYP11B1、hCYP11A1和hCYP17构建体的表达，但抑制了hCYP11B2报告基因的活性。小鼠、牛和人SF-1不能增加hCYP11B2在H295R细胞中的转录。hCYP11B2和mcyp11b2启动子结构同样受到人SF-1的抑制。在小鼠Y-1细胞中，报告基因hCYP11B2和mcyp11b2的表达与hCYP11B1构建体相比非常低，提示该肾上腺细胞模型可能不适合CYP11B2的研究。电泳迁移率转移实验表明，SF-1与hCYP11B1和hCYP11B2的一个元件相互作用。然而，这种被称为Ad4的元素的突变并不能阻止血管紧张素11或福斯克林对hCYP11B2的激动剂刺激，而是阻断了hCYP11B1的活性。在一些（但不是全部）遗传连锁分析报告中，hCYF11B2 Ad4元件（-344C/T）中自然存在的单核苷酸多态性与心血管疾病有关。在这里，我们已经证明了这种多态性影响了SF-1在电泳迁移率转移试验中的结合，其中C等位基因与SF-1的结合比T等位基因更强烈。然而，当含有这些等位基因的hCYP11B2构建体被转染到H295R细胞中时，激动剂刺激的表达和两种报告基因构建体对人类SF-1共表达的反应没有差异。综上所述，这些数据表明SF-1和Ad4元件不是肾上腺hCYP11B2基因表达的主要调节因子。迄今为止，hCYP11B2是第一个不受SF-1正调控的类固醇羟化酶基因。
11beta-Hydroxylase (hCYP11B1) and aldosterone synthase (hCYP11B2) are closely related isozymes with distinct roles in cortisol and aldosterone production respectively. Aldosterone synthase catalyzes the final step in aldosterone biosynthesis and is expressed only in the zona glomerulosa of the normal adrenal. 11 P-Hydroxylase catalyzes the final reaction in the production of cortisol and is expressed at higher levels in the zona fasciculata. The mechanisms causing differential expression of these genes are not well defined. Herein, we demonstrate contrasting roles for the orphan receptor steroidogenic factor-1 (SF-1) in the regulation of human (h) CYP11B1 and hCYP11B2. Human NCI-H295R (H295R) or mouse Y-1 cells were transiently transfected with luciferase reporter constructs containing 5'-flanking regions of hCYP11B1, hCYP11B2, human 17alpha-hydroxylase (hCYP17), human cholesterol side-chain cleavage (hCYP11A1) or mouse (m) cyp11b2 (mcyp11b2). Co-transfection of vectors encoding SF-1 increased expression of hCYP11B1, hCYP11A1 and hCYP17 constructs, but inhibited hCYP11B2 reporter activity. Murine, bovine and human SF-1 were unable to increase transcription of hCYP11B2 in H295R cells. Both hCYP11B2 and mcyp11b2 promoter constructs were inhibited similarly by human SF-1. In mouse Y-1 cells, reporter expression of hCYP11B2 and mcyp11b2 was very low compared with hCYP11B1 constructs, suggesting that this adrenal cell model may not be appropriate for studies of CYP11B2. Electrophoretic mobility shift assay demonstrated that SF-1 interacted with an element from both hCYP11B1 and hCYP11B2. However, mutation of this element, termed Ad4, did not prevent agonist stimulation of hCYP11B2 by angiotensin 11 or forskolin but blocked activity of hCYP11B1. In some, but not all, reports of genetic linkage analysis, a naturally occurring single nucleotide polymorphism within the Ad4 element of hCYF11B2 (-344C/T) has been associated with cardiovascular disease. Herein, we have demonstrated that this polymorphism influenced binding of SF-1 in electrophoretic mobility shift assays, with the C allele binding SF-1 more strongly than the T allele. However, when hCYP11B2 constructs containing these alleles were transfected into H295R cells, there was no difference in agonist-stimulated expression or the response of either reporter construct to co-expression with human SF-1. Taken together, these data suggest that SF-1 and the Ad4 element are not major regulators of adrenal hCYP11B2 gene expression. Thus far, hCYP11B2 is the first steroid hydroxylase gene which is not positively regulated by SF-1.