Wang Qian, Zhang Ping, Gao Hui-Bao
Department of Biochemistry and Molecular Biology, Shanghai Jiao Tong University School of Medicine, 280 South Chong Qing Road, Shanghai 200025, PR China.
Reprod Biol Endocrinol. 2009 May 4;7:39. doi: 10.1186/1477-7827-7-39.
Leydig cells are the primary source of testosterone in male vertebrates. The biosynthesis of testosterone in Leydig cells is strictly dependent on luteinizing hormone (LH). On the other hand, it can be directly inhibited by excessive glucocorticoid (Corticosterone, CORT, in rats) which is beyond the protective capability of 11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) and type 2 (11beta-HSD2; encoded by gene Hsd11b2 in rats) in Leydig cells. Our previous study found that LH increases 11beta-HSD1 expression in rat Leydig cells, but the effect of LH on the expression and activity of 11beta-HSD2 is not investigated yet.
The Leydig cells were isolated from male Sprague-Dawley rats (90 days of age). After Leydig cells were incubated either for 24 h with various concentrations of LH (2.5, 5, 10 and 20 ng/mL) or for different time periods (2, 8, 12 and 24 h) with 20 ng/mL LH, the mRNA expression of 11beta-HSD2 was measured by real-time PCR. 11beta-HSD2 protein levels in Leydig cells were assayed by Western Blot and 11beta-HSD2 enzyme activity was determined by calculating the ratio of conversion of [3H]CORT to [3H]11-dehydrocorticosterone by 24 h after stimulation with 20 ng/ml LH. Four reporter gene plasmids containing various lengths of Hsd11b2 promoter region were constructed and transfected into mouse Leydig tumor cells to investigate the effect of LH on Hsd11b2 transcription. A glucocorticoid-responsive reporter gene plasmid, GRE-Luc, was constructed. To evaluate influence of LH on intracellular glucocorticoid level, rat Leydig cells were transfected with GRE-Luc, and luciferase activities were measured after incubation with CORT alone or CORT plus LH.
We observed dose- and temporal-dependent induction of rat 11beta-HSD2 mRNA expression in Leydig cells subject to LH stimulation. The protein and enzyme activity of 11beta-HSD2 and the luciferase activity of reporter gene driven by promoter regions of Hsd11b2 were increased by LH treatment. LH decreased the glucocorticoid-induced luciferase activity of GRE-Luc reporter gene.
The results of the present study suggest that LH increases the expression and enzyme activity of 11beta-HSD2, and therefore enhances capacity for oxidative inactivation of glucocorticoid in rat Leydig cells in vitro.
睾丸间质细胞是雄性脊椎动物睾酮的主要来源。睾丸间质细胞中睾酮的生物合成严格依赖于促黄体生成素（LH）。另一方面，过量的糖皮质激素（大鼠体内为皮质酮，CORT）可直接抑制其合成，而睾丸间质细胞中的11β-羟基类固醇脱氢酶1型（11β-HSD1）和2型（11β-HSD2；大鼠中由基因Hsd11b2编码）无法对其进行有效保护。我们之前的研究发现，LH可增加大鼠睾丸间质细胞中11β-HSD1的表达，但LH对11β-HSD2表达及活性的影响尚未见研究报道。
从90日龄雄性Sprague-Dawley大鼠分离睾丸间质细胞。将睾丸间质细胞分别用不同浓度的LH（2.5、5、10和20 ng/mL）孵育24小时，或用20 ng/mL LH孵育不同时间段（2、8、12和24小时），然后通过实时PCR检测11β-HSD2的mRNA表达。采用蛋白质免疫印迹法检测睾丸间质细胞中11β-HSD2蛋白水平，通过计算20 ng/ml LH刺激24小时后[3H]CORT转化为[3H]11-脱氢皮质酮的转化率来测定11β-HSD2酶活性。构建含有不同长度Hsd11b2启动子区域的四种报告基因质粒，并转染至小鼠睾丸间质细胞瘤细胞中，以研究LH对Hsd11b2转录的影响。构建糖皮质激素反应性报告基因质粒GRE-Luc。为评估LH对细胞内糖皮质激素水平的影响，将GRE-Luc转染至大鼠睾丸间质细胞，分别单独用CORT或CORT加LH孵育后测定荧光素酶活性。
我们观察到LH刺激的大鼠睾丸间质细胞中11β-HSD2 mRNA表达呈剂量和时间依赖性诱导。LH处理可增加11β-HSD2的蛋白和酶活性以及由Hsd11b2启动子区域驱动的报告基因的荧光素酶活性。LH降低了GRE-Luc报告基因的糖皮质激素诱导的荧光素酶活性。
本研究结果表明，LH可增加11β-HSD2的表达和酶活性，从而增强体外培养的大鼠睾丸间质细胞中糖皮质激素的氧化失活能力。
