LIVER-TYPE 11-BETA-HYDROXYSTEROID DEHYDROGENASE CDNA ENCODES REDUCTASE BUT NOT DEHYDROGENASE-ACTIVITY IN INTACT MAMMALIAN COS-7 CELLS
LIVER-TYPE 11-BETA-HYDROXYSTEROID DEHYDROGENASE CDNA ENCODES REDUCTASE BUT NOT DEHYDROGENASE-ACTIVITY IN INTACT MAMMALIAN COS-7 CELLS
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DOI：
10.1677/jme.0.0130167
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发表时间：
1994-10-01
影响因子：
3.5
通讯作者：
SECKL, JR
中科院分区：
文献类型：
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作者：
LOW, SC;CHAPMAN, KE;SECKL, JR
11 β-羟基类固醇脱氢酶（11 β-HSD）催化皮质酮代谢为惰性11-脱氢皮质酮，从而阻止糖皮质激素进入其他非选择性肾盐皮质激素受体（MR），在体内产生醛固酮选择性。至少存在两种11 β-HSD的同种型。已从大鼠肝脏中纯化了一种异构体（11 β-HSD 1），并从大鼠肝脏文库中克隆了编码cDNA。将大鼠11 β-HSD，cDNA转染到具有盐皮质激素表型的两栖动物细胞中编码11 β-还原酶活性（惰性11-脱氢皮质酮的活化），表明11 β-HSD 1不具有保护肾MR免受糖皮质激素暴露的必要特性。这种功能可能存在于第二个11 β-HSD同种型中。11 β-HSD 1与糖皮质激素受体（GR）共定位，并可调节糖皮质激素对该受体类型的进入。为了检测完整哺乳动物细胞中11 β-HSD 1活性的主要方向，以及11 β-HSD在调节糖皮质激素进入GR中的可能作用，我们将大鼠11 β-HSD 1 cDNA转染入哺乳动物肾脏衍生的细胞系统（COS-7），该系统几乎没有内源性11 β-HSD活性或mRNA表达。HSD cDNA表现出11 β-脱氢酶活性的剂量相关性增加。与此相反，完整的细胞没有转换皮质酮11-脱氢皮质酮超过24小时，但显示出明确的剂量相关的11 β-还原酶活性，明显的4小时内添加11-脱氢皮质酮的培养基。为了证明这反映了功能性细胞内糖皮质激素的变化，COS-7细胞用编码GR的表达载体和糖皮质激素诱导型MMTV-LTR荧光素酶报告基因构建体共转染，有或没有11 β-HSD。在存在或不存在11 β-HSD的情况下，皮质酮诱导MMTV-LTR荧光素酶表达。11-脱氢皮质酮在不存在11 β-HSD的情况下没有活性，但在存在11 β-HSD的情况下诱导MMTV-LTR荧光素酶活性。这些结果表明，大鼠11 β-HSD 1在完整的哺乳动物细胞中可以专门作为还原酶。因此，在体内的一些组织中，11 β-HSD 1可以通过重新激活惰性糖皮质激素来调节配体与GR的接触。
11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) catalyses the metabolism of corticosterone to inert 11-dehydrocorticosterone, thus preventing glucocorticoid access to otherwise non-selective renal mineralocorticoid receptors (MRs), producing aldosterone selectivity in vivo. At least two isoforms of 11 beta-HSD exist. One isoform (11 beta-HSD1) has been purified from rat liver and an encoding cDNA cloned from a rat liver library. Transfection of rat 11 beta-HSD, cDNA into amphibian cells with a mineralocorticoid phenotype encodes 11 beta-reductase activity (activation of inert 11-dehydrocorticosterone) suggesting that 11 beta-HSD1 does not have the necessary properties to protect renal MRs from exposure to glucocorticoids. This function is likely to reside in a second 11 beta-HSD isoform. 11 beta-HSD1 is co-localized with glucocorticoid receptors (GRs) and may modulate glucocorticoid access to this receptor type. To examine the predominant direction of 11 beta-HSD1 activity in intact mammalian cells, and the possible role of 11 beta-HSD in regulating glucocorticoid access to GRs, we transfected rat 11 beta-HSD1 cDNA into a mammalian kidney-derived cell system (COS-7) which has little endogenous 11 beta-HSD activity or mRNA expression.Homogenates of COS-7 cells transfected with increasing amounts of 11 beta-HSD cDNA exhibited a dose-related increase in 11 beta-dehydrogenase activity. In contrast, intact cells did not convert corticosterone to 11-dehydrocorticosterone over 24 h, but showed a clear dose-related 11 beta-reductase activity, apparent within 4 h of addition of 11-dehydrocorticosterone to the medium. To demonstrate that this reflected a change in functional intracellular glucocorticoids, COS-7 cells were co-transfected with an expression vector encoding GR and a glucocorticoid-inducible MMTV-LTR luciferase reporter construct, with or without 11 beta-HSD. Corticosterone induced MMTV-LTR luciferase expression in the presence or absence of 11 beta-HSD. 11-Dehydrocorticosterone was without activity in the absence of 11 beta-HSD, but induced MMTV-LTR luciferase activity in the presence of 11 beta-HSD. These results indicate that rat 11 beta-HSD1 can behave exclusively as a reductase in intact mammalian cells. Thus in some tissues in vivo, 11 beta-HSD1 may regulate ligand access to GRs by reactivating inert glucocorticoids.