11β-hydroxysteroid dehydrogenase 2 in rat Leydig cells:: Its role in blunting glucocorticoid action at physiological levels of substrate
11β-hydroxysteroid dehydrogenase 2 in rat Leydig cells:: Its role in blunting glucocorticoid action at physiological levels of substrate
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DOI：
10.1210/en.2005-0046
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发表时间：
2005-06-01
期刊：
影响因子：
4.8
通讯作者：
Hardy, MP
中科院分区：
文献类型：
--
作者：
Ge, RS;Dong, Q;Hardy, MP
皮质酮（CORT）通过抑制参与睾酮生物合成的蛋白质的表达，包括促类固醇急性调节蛋白和促类固醇酶，从而抑制间质细胞的类固醇生成。在大多数细胞中，细胞内糖皮质激素水平由11 β -羟基类固醇脱氢酶（11 β HSD）的两种已知亚型中的一种或两种控制：烟酰胺腺嘌呤二核苷酸磷酸还原依赖性低亲和力I型11 β HSD （11 β HSD1）氧化还原酶和烟酰胺腺嘌呤二核苷酸依赖性11 β HSD2高亲和力单向氧化酶。在间质细胞中，单独的11 β HSD1可能不足以防止糖皮质激素介导的抑制，因为它在基础浓度下对CORT的亲和力较低。如果也存在高亲和力的单向11 β HSD2，则可能对降低细胞内CORT水平至关重要。在本研究中，我们通过PCR扩增、免疫组织化学染色、酶组织化学、免疫沉淀和Western blotting发现大鼠间质细胞中存在11 β HSD2。Real-time PCR显示，与整个睾丸相比，这些细胞中11 β HSD2 mRNA的富集量为6倍，11 β HSD2的信息量比11 β HSD1少约1000倍。间质细胞质中11 β HSD2蛋白的弥漫性免疫荧光染色与其在光滑内质网中的定位一致。利用反义方法选择性地抑制11 β HSD1或11 β HSD2的活性：11 β HSD1的抑制使还原酶活性降低60%，氧化活性降低25%，而单独抑制11 β HSD2可抑制氧化酶活性50%。这表明，高亲和、低容量的11 β HSD2异构体（仅为低亲和异构体的千分之一）可能显著影响CORT水平。在CORT存在的情况下，抑制11 β HSD1或11 β HSD2均可显著降低睾酮的产生。这些数据表明，间质细胞中的I型和II型11 β HSD均发挥保护作用，反对过量CORT对睾酮产生的不利影响。
Corticosterone (CORT) suppresses Leydig cell steroidogenesis by inhibiting the expression of proteins involved in testosterone biosynthesis including steroidogenic acute regulatory protein and steroidogenic enzymes. In most cells, intracellular glucocorticoid levels are controlled by either or both of the two known isoforms of 11 beta-hydroxysteroid dehydrogenase (11 beta HSD): the nicotinamide adenine dinucleotide phosphate reduced-dependent low-affinity type I 11 beta HSD (11 beta HSD1) oxidoreductase and the nicotinamide adenine dinucleotide-dependent 11 beta HSD2 high-affinity unidirectional oxidase. In Leydig cells, 11 beta HSD1 alone may not be sufficient to prevent glucocorticoid-mediated suppression due to its low affinity for CORT at basal concentrations. The high-affinity unidirectional 11 beta HSD2, if also present, may be critical for lowering intracellular CORT levels. In the present study, we showed that 11 beta HSD2 is present in rat Leydig cells by PCR amplification, immunohistochemical staining, enzyme histochemistry, immunoprecipitation, and Western blotting. Real-time PCR showed a 6-fold enrichment of 11 beta HSD2 mRNA in these cells, compared with whole testis and that the amount of 11 beta HSD2 message was about 1000-fold lower, compared with 11 beta HSD1. Diffuse immunofluorescent staining of 11 beta HSD2 protein in the Leydig cell cytoplasm was consistent with its localization in the smooth endoplasm reticulum. 11 beta HSD1 or 11 beta HSD2 activities were selectively inhibited using antisense methodology: inhibition of 11 beta HSD1 lowered reductase activity by 60% and oxidation by 25%, whereas inhibition of 11 beta HSD2 alone suppressed oxidase activity by 50%. This shows that the high-affinity, low-capacity 11 beta HSD2 isoform, present at only one thousandth the level of the low-affinity isoform may significantly affect the level of CORT. The inhibition of either 11 beta HSD1 or 11 beta HSD2 significantly lowered testosterone production in the presence of CORT. These data suggest that both types I and II 11 beta HSD in Leydig cells play a protective role, opposing the adverse effects of excessive CORT on testosterone production.