Expression of 11β-Hydroxylase in Rat Leydig Cells

11β-Hydroxy (11β-OH) derivatives of certain steroids function as inhibitors of 11β-hydroxysteroid dehydrogenase isoform 1 (11βHSD1), an enzyme expressed in Leydig cells that catalyzes the reversible oxidation of biologically active glucocorticoids to inactive 11-dehydro metabolites. 11β-Hydroxylase is an adrenal enzyme responsible for glucocorticoid biosynthesis, catalyzing 11β-hydroxylation of steroids and thus producing 11β-OH-steroid derivatives. The aims of the present study were 1) to examine whether 11β-hydroxylase is expressed in testis, 2) to define the biochemical characteristics of the testicular form of this enzyme, and 3) to establish whether 11β-hydroxylated steroids inhibit Leydig cell 11βHSD1 activities. 11β-Hydroxylase mRNA was detected in purified rat Leydig cells by RT-PCR. Sequencing confirmed that the PCR products had 100% identity with the published rat adrenal enzyme cDNA sequence. Immunohistochemistry and Western blot analysis using a mouse monoclonal antibody confirmed the expression of 11β-hydroxylase protein in Leydig cells. Moreover, 11β-hydroxylase activity, synthesis of corticosterone from 11-deoxycorticosterone, was measurable in Leydig cells, and the Km and maximum velocity values were 7.28 ± 0. 92 μm and 1.13 ± 0.04 μmol/106 cell·h, respectively. When assayed in Leydig cells, several 11β-hydroxylated steroids were efficient inhibitors of 11βHSD1 dehydrogenase activity, whereas other 11-keto compounds were effective as inhibitors of oxidoreductase activity. These results provide the first direct evidence that rat Leydig cells express 11β-hydroxylase, which may be involved in the regulation of glucocorticoid metabolism within the testis through local biosynthesis of endogenous inhibitors of 11βHSD1.