Received 2002-03-15 Accepted 2002-08-05

This work was supported by the National Basic Reseach Program (G1999054000) of China.

*Corresponding author. Tel: +86-21-25070308.

大鼠海马神经元内11β-HSD1和GR的共存及其意义

万顺伦, 廖茂瑶, 郝如松, 李召峰, 孙刚*

第二军医大学生理学教研室, 上海 200433

摘要: 本研究旨在探讨糖皮质激素代谢酶－11β-羟基类固醇脱氢酶Ⅰ型(11β-HSD1)和糖皮质激素受体(GR)在大鼠海马神经元内的共同分布及其意义。用免疫细胞化学方法研究显示, 海马神经元内不仅存在11β-HSD1免疫反应物质, 还存在GR免疫反应物质, 而且11β-HSD1与GR共存于同一个海马神经元内。用Western印迹杂交和薄层层析(TLC)方法研究表明, 地塞米松(DEX)可以促进11β-HSD1蛋白表达及其酶的活性。利用11β-HSD1基因启动子区序列构建的以CAT酶为报告基因的pBLCAT6质粒转染PC12细胞, 证实DEX能够促进CAT酶的表达。以上糖皮质激素的作用均可为GR受体阻断剂RU38486所阻断。结果提示: 糖皮质激素(GC)与GR结合后, 可以作用于与其共存的11β-HSD1基因启动子区, 使11β-HSD1表达增加, 从而使更多的GC代谢产物转化为有活性的GC。此机制可能与保证GC在海马神经元内与亲和力较低的GR结合有关。

关键词: 11β-羟基类固醇脱氢酶Ⅰ型; 糖皮质激素受体; 海马神经元

中图分类号: Q459

Colocalization of 11β-hydroxysteroid dehydrogenase typeⅠand glucocorticoid receptor and its significance

in the rat hippocampus

WAN Shun-Lun, LIAO Mao-Yao, HAO Ru-Song, LI Zhao-Feng , SUN Gang

*Department of Physiology , The Second Military Medical University , Shanghai 200433

Abstract: Glucocorticoids (GC) have a matrix function in the nervous system. The actions of GC on nervous system are regulated at both receptor and pre-receptor levels. There are two types of corticosteroid receptors, the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). At the receptor level, GC binds to both MR and GR, but with higher affinity for MR than GR. Changes in plasam corticosterone concentrations from basal up to peak levels(induced by stress or circadian variation) are accompanied by relatively small differences in MRs occupancy (range:70%~90%),while occupancy of GRs can vary considerably (from10%~90%). At the pre-receptor level, the amounts of GC binding to the receptors are determined by glucocorticoid binding proteins as well as 11β-hydroxysteroid dehydrogenases (11β-HSDs). There are two types of 11β-HSDs recognized in the body. 11β-HSD1 predominantly acts as a reductase in tact cells converting biologically inert 11-keto GC metabolites into biologically active GC. Thus this enzyme regenerates GC and amplifies GC's actions locally. 11β-HSD2 is an exclusive oxidase converting biologically active GC into its inert products thus attenuating GC's actions. Then the 11β-HSD1 reductase in the neuron may allow GC's activation of more GR in the brain. However, whether 11β-HSD1 and GR are co-localized in the same neuron has not been addressed. Moreover, sequence analysis of the cloned 11β-HSD1 gene revealed a sequence resembling GRE-like in the promoter region. There are evidences showing that GC regulates the expression of 11β-HSD1 both in vitro and vivo. But the mechanism of up-regulation of 11β-HSD1 by GC has not been clear either. This paper was designed to observe the colocalization of 11β-11β-HSD1 and GR, and its significance in the rat hippocampus. Immunocytochemical dual-staining showed that not only 11β-HSD1 but also GR immunoreactive substances were present in the cultured rat hippocampal neurons.Moreover,they were colocalized in the same hippocampal neuron. Synthetic glucocorticoid dexamethasone (DEX) up-regulated the protein expression and activity of 11β-HSD1 in the cultured hippocampal neurons, as determined by Western blot and thin layer chromatography (TLC) respectively. The transfection of PC12 cells with the plasmid containing promoter sequence of 11β-HSD1 gene and the reporter gene of CAT enzyme was conducted. DEX up-regulated the reporter gene expression in the system described above. The up-regulation of 11β-HSD1 and reporter gene expression induced by DEX were both blocked by GR antagonist RU38486. Our study suggests that the colocalization of 11β-HSD1 and GR in the hippocampus may be implicated in the up-regulation of 11β-HSD1 expression by glucocorticoids combining to its promoter region , which in turn produces more biologically active glucocorticoids necessary for the binding of low affinity of GR.

Key words: 11β-hydroxysteroid dehydrogenase typeⅠ; glucocorticoid receptor; hippocampus