Hormonal Regulation of Oxidative and Reductive Activities of 11 -Hydroxysteroid Dehydrogenase in Rat Leydig Cells

Abstract

We have proposed that the 11␤-hydroxysteroid dehydrogenase (11␤-HSD) of Leydig cells protects against glucocorticoid-induced inhibition of testosterone (T) production. However, Leydig cells express type I 11␤-HSD, which has been shown to be reductive in liver parenchymal cells. Because reduction would have the opposite effect of activating glucocorticoid, the present study was designed to determine: 1) whether Leydig cell 11␤-HSD is primarily oxidative or reductive; and 2) whether oxidative and reductive activities are separately modified by known regulators of Leydig cell steroidogenic function. Leydig cells and liver parenchymal cells were purified from mature male Sprague-Dawley rats (250 g BW), and 11␤-HSD oxidative and reductive activities were measured using radiolabeled substrates and TLC of triplicate media samples from 1-h incubations immediately after cell isolation. Enzyme activities also were examined in purified Leydig cells at the end of 3 days of culture in vitro in the presence of LH (10 ng/ml), dexamethasone (DEX, 100 nM), T (50 nM), or epidermal growth factor (EGF, 50 ng/ml). In confirmation of previous reports, the reductive activity of 11␤-HSD was predominant over oxidation in liver parenchymal cells. In contrast, 11␤-HSD oxidative activity prevailed over reduction in Leydig cells by a ratio of 2:1. The activities of 11␤-HSD also were analyzed in Leydig cells that were purified 7 days after endogenous glucocorticoid levels were suppressed by adrenalectomy (ADX). Oxidative activity declined in Leydig cells after ADX (22.53 Ϯ 1.12 pmol/h⅐10 6 cells, mean Ϯ SEM vs. 31.47 Ϯ 1.48 pmol/h⅐10 6 cells in sham-operated controls, P Ͻ 0.05), whereas there was no change in reductive activity. This indicated that physiologically active corticosterone is involved in maintaining the predominance of 11␤-HSD oxidation. When enzyme activities were analyzed in Leydig cells after 3 days of hormonal treatment in vitro, oxidation and reduction were observed to change in opposing directions. Culture of Leydig cells from sham-operated control rats with either LH, T, or EGF resulted in declines in oxidative activity from 33.35 Ϯ 0.77 to 28.24 Ϯ 1.93, 27.30 Ϯ 0.96, and 24.13 Ϯ 1.02 pmol/ h⅐10 6 cells (x Ϯ SE), respectively. However, EGF stimulated 11␤-HSD reductive activity in cultured Leydig cells from both control (from 18.97 Ϯ 1.10 to 27.16 Ϯ 0.71 pmol/h⅐10 6 cells and ADX rats (from 16.51 Ϯ 0.75 to 23.56 Ϯ 0.84 pmol/h⅐10 6 cells). Among the hormonal treatments, only DEX increased oxidative activity and simultaneously decreased reductive activity in Leydig cells from ADX rats. This increase accentuated the predominance of oxidative activity in Leydig cells, with a ratio of oxidative to reductive activity of 4:1 after DEX treatment, compared with 2:1 in controls that were untreated. We conclude that 11␤-HSD activity in Leydig cells is primarily oxidative. Moreover, oxidation and reduction are regulated separately by hormones. (Endocrinology 138: 156 -161, 1997)

Protein Kinase C Increases 11β‐Hydroxysteroid Dehydrogenase Oxidation and Inhibits Reduction in Rat Leydig Cells

“…LH, the primary hormone required for maintenance of Leydig cell steroidogenesis, has been shown to suppress 11␤HSD oxidative activity and increase reductive activity (Gao et al, 1997). Given that LH increases calcium flux within Leydig cells, we infer that its regulation of 11␤HSD activities is calcium-dependent.…”

“…The effects of the PKC and calcium-dependent kinase pathways on 11␤HSD oxidative and reductive activities could be exerted directly or indirectly. An indirect mechanism is seen, for example, in the case of testosterone itself, which has been shown to decrease 11␤HSD oxidative activity and increase reductive activity (Gao et al, 1997). Both PKC and calcium signaling have been shown to regulate testosterone production (Janzen et al, 1976;Nikula et al, 1987;Gudermann et al, 1992b).…”

“…To date, two distinct forms of 11␤HSD have been identified: type I 11␤HSD (11␤HSD-I), which was first purified from rat liver and later cloned, has both oxidative and reductive activities (Lakshmi and Monder, 1988;Agarwal et al, 1989); and Type II 11␤HSD (11␤HSD-II), first identified in kidney and later cloned, is exclusively oxidative with a high affinity for glucocorticoids (Rusvai and Naray-FejesToth, 1993;Albiston et al, 1994;Zhou et al, 1995). Leydig cells have been shown to express only 11␤HSD-I and not 11␤HSD-II (Phillips et al, 1989;Li et al, 1996;Gao et al, 1997;Ge et al, 1997a). In Leydig cells, 11␤HSD-I catalyzes the interconversion of active glucocorticoid, corticosterone, and its biologically inert metabolite, 11-dehydrocorticosterone.…”

“…Luteinizing hormone (LH) and glucocorticoid each induce opposing changes in the oxidative versus reductive activities of 11␤HSD in Leydig cells (Gao et al, 1997). Because these hormones regulate Leydig cell function through more than one intracellular signal transduction pathway, including the cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA; Cooke, 1996), calcium (Janzen et al, 1976;Gudermann et al, 1992a,b), protein kinase C (PKC; Nikula et al, 1987;Majercik and Puett, 1991), and tyrosine protein kinase (Wuerther et al, 1995), 11␤HSD oxidation and reduction may be separately controlled.…”

Local cortisol activation is involved in EGF-induced immunosuppression

“…The 11b-HSD1 isoform is predominantly a reductase that catalyses conversion of cortisone/11-DHC to cortisol/corticosterone. 5,6 The 11b-HSD2 isoform catalyses inactivation of cortisol/corticosterone to cortisone/11-DHC. 7 Circulating cortisone is converted to active cortisol in tissue through 11b-HSD1 in cells.…”