11β-hydroxysteroid dehydrogenase 2 in rat Leydig cells:: Its role in blunting glucocorticoid action at physiological levels of substrate

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.