Luteinizing hormone induces expression of 11beta-hydroxysteroid dehydrogenase type 2 in rat Leydig cells

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Figure 1

Effect of LH on expression of mRNA for 11β-HSD2. Leydig cells were treated with increasing concentrations of LH for 24 h (A). The other cells were treated with LH for 2–24 h (B). At end of treatment, total cellular RNA was isolated, and the steady-state level of 11β-HSD2 mRNA was assessed by relative quantitative RT-PCR, as described in Methods. The expressions of mRNA for 11β-HSD2 in Leydig cells treated with 2.5, 5, 10, or 20 ng/ml LH for 24 h, or 20 ng/mL LH for 8, 12 or 24 h are significantly higher compared to that in intact Leydig cells. Each data point is expressed as percentage of control. Asterisks denote significant differences, compared with Control at P < 0.05.

Figure 2

Effect of LH on expression of 11β-HSD2 protein. R2C cells were transfected with pcDNA3.0 (lane 1) or pcDNA-hsd11b2 (lane 2); Leydig cells were treated with vehicle (PBS for LH, lane 3) or 20 ng/ml LH for 24 h (lane 4). At end of treatment, levels of 11β-HSD2 protein were determined by western blot analysis as described in Methods.

Figure 3

Effect of LH on 11β-HSD2 promoter activity. A series of luciferase reporter gene driven by rat 11β-HSD2 promoter region were transfected into mLTC-1 cells. After overnight incubation, the mLTC-1 cells were treated with 20 ng/lm LH for 24 h. The cells were lysed and assayed for luciferase activity. The luciferase activity was compared with the normalized activity of the transfected but not treated cells. These data are representative of at least three independent experiments and are normalized to an internal Renilla control. Asterisks indicate differences between groups control versus LH treatment are statistically significant at P < 0.05.

Figure 4

Effect of LH on intracellular glucocorticoid concentration. Leydig cells were transfected transiently with a GRE-Luc reporter plasmid, and intracellular glucocorticoid concentration was estimated by GR-mediated transcription of GRE-Luc. Leydig cells were treated with CORT (50 nM), LH (20 ng/mL) or CORT plus LH as described in Methods. Dissimilar superscripts indicate significant differences between groups (P < 0.05).