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Population Council (R.-S.G., Q. D., E-m.N., C.M.S., D.O.H., J.F.C., M.P.H.) and The Rockefeller University (M.P.H., J.F.C.), New York, New York 10021; and Department of Pathology and Laboratory Medicine (S.A.L., D.J.M.), The Miriam Hospital, Brown University School of Medicine, Providence, Rhode Island 02906
Address all correspondence and requests for reprints to: Matthew P. Hardy, The Population Council, 1230 York Avenue, New York, New York 10021. E-mail: m-hardy{at}popcbr.rockefeller.edu.
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Introduction |
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The biological ligand of GR in rat is corticosterone (CORT). Intracellular levels of CORT are regulated by 11ß-hydroxysteroid dehydrogenase (11ßHSD), which has two known isoforms. Type I 11ßHSD (11ßHSD1) is an nicotinamide adenine dinucleotide phosphate (oxidized form) (NADP+)/nicotinamide adenine dinucleotide phosphate reduced (NADPH)-dependent oxidoreductase, catalyzing the interconversion of 11ß-hydroxyl steroids (such as CORT) and 11-keto steroids [such as 11-dehydrocorticosterone (11DHC) in rats]. 11ßHSD1 is a low-affinity, high-capacity enzyme with a Michaelis constant (Km) of 2 µM (18). Its direction of catalysis depends on the cell type and intracellular milieu (19). For example, when a plasmid containing the entire coding region of 11ßHSD1 was transiently transfected into two different cell lines, Chinese hamster ovary P-type (CHOP) and monkey kidney fibroblast (COS1), oxidative activity was observed to be predominant in the former, whereas reductive activity was higher in the latter (20). The catalytic direction of 11ßHSD1 is determined through the redox potential set by the NADP+ to NADPH cofactor ratio (21, 22). Recently it has been shown that the NADP+ to NADPH ratio is modulated by hexose-6-phosphate dehydrogenase activity (21, 22, 23, 24). Hexose-6-phosphate dehydrogenase catalyzes the synthesis of NADPH, thereby raising the intracellular level of the cofactor, and it is thought that this may favor the reductase activity of 11ßHSD1 observed in several tissues (21, 22, 23, 24). In contrast, type II 11ßHSD (11ßHSD2) is a unidirectional oxidase that inactivates CORT through conversion to 11DHC. 11ßHSD2 is a high-affinity, nicotinamide adenine dinucleotide (oxidized form) (NAD+)-dependent enzyme (with a Km of 
15 nM) (25) that is expressed in cells that are targets for mineralocorticoid receptor (MR) action (26). Due to the nonselective binding properties of the MR, the heterologous ligand CORT would normally be bound because it is present at concentrations that are up to 1000-fold higher relative to aldosterone (ALDO) (27, 28). In this context, 11ßHSD2 lowers the intracellular level of CORT, allowing ALDO to bind with specificity. The role of 11ßHSD2 in conferring MR binding selectivity is seen in humans with the condition of apparent mineralocorticoid excess caused by mutations in 11ßHSD2 and resulting in the indiscriminate binding of CORT to MR in the mineralocorticoid-sensitive tissue, kidney, leading to hypokalemic hypertension (26).
A number of studies has established that 11ßHSD1 is present in mammalian testis and the Leydig cells (29, 30, 31, 32). Although 11ßHSD2 has not been reported in mammalian testes thus far (rats and mice) (33, 34), it is abundant in fish Leydig cells (35). In addition, we have now confirmed that this isoform is also expressed in human testis samples along with 11ßHSD1 (our unpublished observations). In fish, 11ßHSD2 synthesizes 11ketotestosterone, using 11ß-hydroxytestosterone as substrate. Previously we reported that an 11ß-hydroxylase (Cyp11b1) is present in rat Leydig cells in which its function may be to form 11ß-hydroxytestosterone and other 11ß-hydroxyl androgens derivatives (36). In the present study, we investigated 11ßHSD isoform expression in rat Leydig cells and investigated whether 11ßHSD2 is coexpressed with 11ßHSD1. We report that 11ßHSD2 is present in the Leydig cell, at levels that are 1000-fold lower relative to 11ßHSD1. Despite a lower expression level, 11ßHSD2 may also play a protective role in blunting the suppressive effects of glucocorticoid on Leydig cell steroidogenesis due to its high affinity for glucocorticoid substrates.
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Materials and Methods |
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-hydroxyandrostenedione, and 17-androstenedione, purchased from Steraloids, were both used as negative controls in the enzyme histochemical staining for 11ßHSD. Male Sprague Dawley rats (250–300 g) were purchased from Charles River Laboratories (Wilmington, MA).
Cell isolation
Sprague Dawley rats, 90 d old, were killed by asphyxiation with CO2. Testes were removed for sectioning or purification of Leydig cells. The animal protocol was approved by the Institutional Animal Care and Use Committee of the Rockefeller University (protocol 91200). Leydig cells were purified from rats as described previously (38). Purities of Leydig cell fractions were evaluated by histochemical staining for 3ß-hydroxysteroid dehydrogenase activity, with 0.4 mM etiocholanolone as the steroid substrate (39). More than 95% adult Leydig cells were intensely stained.
Primer selection
All primers in this study were chosen using a sequence analysis software package (Primer 3, Whitehead Institute for Biomedical Research, Cambridge, MA) following guidelines for internal stability (40). Forward and reverse primers were in different exons to minimize the effects of possible DNA contamination. For 11ßHSD1 (41), the forward primer was 5'-GAAGAAGCATGGAGGTCAAC (exon 3), the reverse primer was 5'-GCAATCAGAGGTTGGGTCAT (exon 4), and the amplicon length was 133 bp. For 11ßHSD2 (42), the forward primer was 5'-CGTCACTCAAGGGGACGTAT (exon 3), the reverse primer was 5'-CGTCACTCAAGGGGACGTAT (exon 4), and the amplicon length was 144 bp. For the internal standard, primers to ribosomal protein S16 were as described previously (43), and the amplicon length was 118 bp. Oligonucleotides were synthesized by Biosource International (Camarillo, CA).
RT-PCR and amplicon confirmation
First-strand cDNAs synthesized using total RNA from purified adult rat Leydig cells or total RNA from adult rat testis, as described previously (7), were used as templates for PCR. Buffer conditions for standard amplification were: 50 mM KCl, 10 mM Tris-HCl (pH 9.0 at 25 C), 1.0% Triton X-100, 1.5 mM MgCl2, 50 µM dATP, deoxycytidine triphosphate, dGTP, and deoxythymidine triphosphate, and each primer was present at 100 nM. The thermal cycle parameters were 94 C for 15 sec (denaturing), 65 C for 30 sec (annealing), and 72 C for 15 sec (extension) for 30 cycles. Products were analyzed on a 2% agarose gel alongside a 100-bp sizing ladder to confirm the specificity of the reactions.
PCR products were directly inserted into linearized pCR2.1 vectors with unmatched 3'-deoxythymidine residues using the Original TA cloning kit (Invitrogen, San Diego, CA). Plasmids were purified using the QIAprep Spin miniprep kit (QIAGEN Inc., Valencia, CA). Samples were submitted to The Rockefeller University Protein DNA Technology Center for automated sequence analysis.
Real-time PCR quantitation
Real-time PCR was carried out in a 25-µl volume using a 96-well plate format using the SYBR Green PCR core reagents purchased from Applied Biosystems (Foster City, CA). Primer titration was performed and the concentration of 300 nM was selected. Fluorescence was detected on an ABI 7700 system (PE Applied Biosystems). Each sample was run in triplicate, in parallel with no template controls.
Histochemical staining
Cryostat sections of adult rat testes were prepared at a thickness of 8 µm. Histochemical staining for 11ßHSD was performed as described previously (44). Sections were incubated with 5 µM steroid substrate in a mixture of the tetrazolium dye containing either NADP+ or NAD+. After 60 min at room temperature in a dark humidified chamber, sections were washed with PBS and fixed with 10% formalin. The sections were mounted in 50% glycerol for microscopy.
Immunocytochemistry
One testis from each animal was used for immunohistochemistry (Vectastain, Elite, ABC kit, PK-6101; Vector Laboratories, Inc., Burlingame, CA) according to the manufacturer’s instructions. The primary antibodies were as follows: rabbit polyclonal anti-11ßHSD1 antibody (29), rabbit polyclonal antibody of anti-11ßHSD2 (catalog no. sc-20176 from Santa Cruz Biotechnology, Santa Cruz, CA). Endogenous peroxidase was blocked with 0.5% H2O2 in methanol for 30 min. The sections were then incubated with anti-11ßHSD1 (diluted 1:1000) or anti-11ßHSD2 (diluted 1:200) antibodies for 1 h at room temperature. The antibody-antigen complexes were visualized with diaminobenzidine (peroxidase substrate kit, SK-4100, Vector Laboratories). The sections were counterstained with Mayer’s hematoxylin, dehydrated in graded concentrations of alcohol, and coverslipped with resin (Permount, SP15–100; Fisher Scientific Co., Fair Lawn, NJ). Control sections were incubated with nonimmune rabbit IgG using the same working dilution as the primary antibody.
Immunofluorescent staining was performed using Leydig cells that were grown on microscope cover glasses. Cells were fixed with 4% formaldehyde, washed with PBS, and permeabilized with 0.1% (wt/vol) Saponin detergent in PBS + 10% normal serum. Nonspecific binding was blocked by incubation with 10% normal serum before addition of the primary antibody. Cells were incubated with anti-11ßHSD1 (diluted 1:1000) or anti-11ßHSD2 (diluted 1:200) antibodies for 1 h at room temperature. Cells were then incubated with Alex488-conjugated second antibody for 1 h. Afterward, the cells were counterstained with 4',6'-diamino-2-phenylindole and mounted. The slides were examined under a Nikon fluorescence microscope with a filter suitable for selectively detecting the fluorescence of fluorescein isothiocyanate (green).
Immunoprecipitation of microsomal protein
Microsomal preparations of Leydig cells were prepared as described previously (32). To protect against proteases, Halt proteinase inhibitor (catalog no.78410; Pierce Biotechnology, Rockford, IL) was added. The immunoprecipitation procedure was described as previously (45). In brief, Leydig cell microsomes were incubated for 1 h with primarily antibodies to 11ßHSD1 or 11ßHSD2 at1:500 dilution at 4 C for 1 h. Agarose-conjugated IgA/G beads were added to the supernatants, and the mixtures were incubated for 1 h at 4 C. Bound immune complexes were washed three times with PBS. The pellets were resuspended and protein contents were measured. The immunoaffinity-purified proteins were used for measurement of 11ß HSD activities.
Cell culture and antisense treatment
Purified Leydig cells (0.2 ± 106 per well) were cultured in 12-well plates for 24 h in media described previously (46) and then exposed to 3 µM concentrations of antisense phosphorothioate oligonucleotides to 11ßHSD1 or 11ßHSD2 for 2 d in vitro (47). 11ßHSD antisense treatment is known to affect vascular contractile response and glucocorticoid metabolism (47). Sense oligonucleotides served as negative control. At the end of 2 d, 11ßHSD activity was assessed as previously described (32). In brief, medium was removed and the cells were incubated with 25 or 500 nM 3H-CORT or 3H-11DHC in 0.5 ml phenol-red-free DMEM at 34 C for 15–60 min at 15-min intervals. The media were harvested for measurement of substrate and product amounts as described in the next section.
11ßHSD assay
11ßHSD activity assay tubes contained 25 nM (within the Km range for 11ßHSD2) or 500 nM (within the Km range for 11ßHSD1). 3H-CORT (88 Ci/mmol; DuPont-NEN Life Science Products) or 3H-11DHC (7) and the reactions were initiated with addition of immunoprecipitated protein with and without cofactors [NAD+, NADP+, nicotinamide adenine dinucleotide (reduced) (NADH), or NADPH at final concentrations of 0.5 mM]. The reactions were stopped by adding 2 ml ice-cold ethyl acetate. The steroids were extracted, and the organic layer was dried under nitrogen. The steroids were separated chromatographically on thin-layer plates in chloroform and methanol (90:10), and the radioactivity was measured using a scanning radiometer (System AR2000, Bioscan Inc., Washington, DC). The percentage conversion of CORT to 11DHC and 11DHC to CORT was calculated by dividing the radioactive counts identified as 11DHC (or CORT, respectively) by the total counts associated with CORT plus 11DHC.
Western blot analysis of 11ßHSD2
Leydig cells were homogenized and boiled in equal volumes of sample loading buffer, a Tris-Cl buffer (pH 6.8) containing 20% glycerol, 5% sodium dodecyl sulfate, 3.1% dithiothreitol, and 0.001% bromophenol blue. Homogenized samples (25 µg protein) of liver, kidney, and adult Leydig cells (ALC) were electrophoresed on 10% polyacrylamide gels containing sodium dodecyl sulfate (7). Proteins were electrophoretically transferred onto nitrocellulose membranes, and after 30 min exposure to 10% nonfat milk to block nonspecific binding, the membranes were incubated with a 1:1000 dilution of a rabbit polyclonal antitype 11ßHSD2 antibody. The membranes were then washed and incubated with a 1:2000 dilution of goat antirabbit antiserum that was conjugated to horseradish peroxidase. The washing step was repeated, and immunoreactive bands were visualized by chemiluminescence using a kit (ECL, Amersham, Arlington Heights, IL).
Statistics
Each experiment was repeated four times. Data were subjected to analysis by one-way ANOVA followed by Duncan multiple comparisons testing to identify significant differences between groups (48). All data are expressed as means ± SEM. Differences were regarded as significant at P < 0.05.
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). The PCR product of 11ßHSD2 was sequenced and found to be identical with the known rat 11ßHSD2 (GenBank accession no. 755573).
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). The expression level of 11ßHSD2 was approximately 1000-fold lower, compared with 11ßHSD1 (Fig. 2).
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). Frozen sections of rat testis were examined for the presence of 11ßHSD1 and 11ßHSD2 by immunohistology procedures using specific antibodies. This showed that 11ßHSD1 was exclusively present in Leydig cells (Fig. 4E). 11ßHSD2 was localized to interstitial areas of the testis and occasional vascular endothelial cells (Fig. 4H). Immunofluorescent labeling of living purified adult rat Leydig cells showed 11ßHSD2 diffusely in cytoplasmic areas, consistent with localization in the smooth endoplasmic reticulum (49) (Fig. 4K).
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). Dye conversion in the presence of CORT and NAD+ also occurred specifically in Leydig cells (Fig. 4J), consistent with the presence of the 11ßHSD2 isoform. An alternative substrate for rat 11ßHSDs is androstene-11ß-ol-3,17-dione, and this produced the same results (not shown). Initiating the reactions with NADP+ and NAD+ in the absence of substrate produced no dye (Fig. 4B), and neither did incubation with androstene-16ß-ol-3,17-dione (not shown). Both NADP+ and NAD+-dependent 11ßHSD activities were inhibited by carbenoxolone, an inhibitor of both 11ßHSD1 and 11ßHSD2 (not shown). To obtain protein for biochemical assays in vitro, specific antibodies were used for immunoprecipitation of 11ßHSDs from Leydig cell microsome preparations. The enzymatic conversion of 11ß hydroxyl steroids by the extracted proteins was measured in the presence of NAD+, NADP+, NADPH, or NADH as cofactor. The protein purified using antibody to 11ßHSD1 assayed with CORT as substrate produced a 30% conversion in the presence of NADP+, whereas the conversion in the presence NAD+ was the same (<5%) as when no cofactor was added (Fig. 5A). The same protein assayed with 11DHC as substrate produced a 15% conversion in the presence of NADPH, whereas the conversion in the presence of NADH was the same (<5%) as observed in the absence of added cofactor (Fig. 5B). This was expected because 11ßHSD1 is known to have oxidase activity requiring NADP+ and reductase activity requiring NADPH. The protein purified using antibody to 11ßHSD2 assayed with CORT as substrate produced a 15% conversion in the presence of NAD+, whereas the conversion rates in the presence and absence of NADP+ did not differ, i.e. both were 6%. No NADH-dependent activity was detected in the presence of 11DHC (Fig. 5D).
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); when Leydig cells were treated with 11ßHSD2 antisense and 25 nM substrate, the 11ßHSD oxidative activity declined by 50% (Fig. 6B). Treatment of Leydig cells with 100 nM CORT resulted in a 24% decline in LH-stimulated testosterone production. Testosterone production was lowered further in the presence of 100 nM CORT by treatment with 11ßHSD1 antisense (50%) or 11ßHSD2 antisense (60%) oligonucleotides to levels that were equivalent to cells that were treated with 500 nm CORT alone (Fig. 7).
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Discussion |
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Previously 11ßHSD2 was not detected in rat Leydig cells by Northern blotting and immunohistochemical staining (32, 50). However, the present study shows that sensitive detection methods are needed to reveal the presence of this isoform: 11ßHSD2 mRNA levels were only 1:1000, compared with 11ßHSD1 (Fig. 2). Despite its lower signal intensity, several lines of evidence, including partial sequencing, real-time PCR, immunohistochemical and enzyme histochemical staining, immunoprecipitation, and cofactor preference assays all confirmed that 11ßHSD2 mRNA is present in the Leydig cell. The antisense experiments showed further that 11ßHSD2 antisense specifically inhibited 11ßHSD oxidase activity at 25 nM glucocorticoid substrate, which is within the Km range for this isoform.
The expression level of 11ßHSD2 was lower in Leydig cells, compared with 11ßHSD1 (Fig. 2). In assessing contributions of the two isoforms to enzymatic activity in whole cells, their kinetic parameters are relevant: 11ßHSD2 is a high-affinity oxidase with a Km of approximately 15 nM (26), compared with the micromolar Km of 11ßHSD1. Seen in this way, the two 11ßHSD isoforms, in combination, may play an important physiological role in metabolizing CORT in the physiological or stressful range of glucocorticoid concentrations (250 nM). We still do not fully understand the potentially beneficial effects of basal glucocorticoid activity. There is an abundance of evidence that Leydig cells express glucocorticoid receptors (10, 11) and that elevated glucocorticoid concentrations during stress cause decrease testosterone production. During pubertal development, however, glucocorticoid may play a supportive, rather than an inhibitory role. Basal glucocorticoid levels are low at birth, and increases in glucocorticoid activity are thought to be involved in the acquisition of LH sensitivity in differentiating Leydig cells (51). Similarly, Parthasarathy et al. (52) have shown that administration of an inhibitor of glucocorticoid synthesis, metyrapone, decreases glucose oxidation, 17ßHSD activity, and testosterone production in Wistar rat Leydig cells. We postulate that basal levels of glucocorticoid activity govern the rate of energy metabolism in Leydig cells, thereby influencing steroidogenic function in puberty. The primarily reductive 11ßHSD activity in Leydig cells before puberty may reflect this physiological arrangement (7). Adrenalectomy results in changes in androgen synthesis (14), providing further evidence that Leydig cells are affected by basal levels of glucocorticoid exposure, although interpretation of such experiments is presently complicated by the fact that removal of the adrenal eliminates both mineralocorticoid and glucocorticoid as well as adrenal medullary hormones. In adult Leydig cells, 11ßHSD2 may significantly contribute to the overall 11ßHSD oxidase activity and inactivate glucocorticoids.
The physiological function of 11ßHSD2 in Leydig cells may be similar to the role it plays in other tissues. In the placenta, for example, 11ßHSD2 modulates glucocorticoid action by inactivating maternal cortisol, which might otherwise have adverse effects on the fetus (53). The major action of 11ßHSD2 in the kidney is to allow ALDO binding to MR (54), which is necessary for maintenance of salt balance. The 100- to 1000-fold excess of CORT, compared with ALDO, in circulation would result in CORT binding to most of the MR because this receptor has approximately the same affinity for these two glucocorticoids (27, 42). It is currently thought that through 11ßHSD2 oxidation, intracellular concentrations of CORT are lowered in kidney to a sufficient extent that the MR binds ALDO. We previously detected the expression of MR in Leydig cells (32) and that ALDO may stimulate testosterone production by an MR-mediated mechanism, an effect inhibited by the specific antimineralocorticoid, RU28318 (55). We postulate that Leydig cells express this metabolic pathway for the same purpose: to lower CORT levels such that ALDO is available for MR binding. A role of 11ßHSD2 activity in the GR pathway is also suggested by our data because glucocorticoid hormone inhibits expression of steroidogenic enzyme genes and steroidogenic acute regulatory protein after binding the GR in Leydig cells (14, 15, 16). We demonstrated that, when 11ßHSD was inhibited by 11ßHSD2 antisense oligomer treatment, the inhibitory potency of CORT on testosterone production increased (Fig. 6). 11ßHSD2 antisense inhibited the 11ßHSD oxidative activity by almost 50%, lowering the rate at which CORT is converted into inactive 11DHC. MRs have also been shown to be present in Leydig cells (32), and ALDO stimulates testosterone production (Ref. 55 and manuscript in preparation). Further experiments are now necessary to better understand the interaction between 11ßHSD2 activity, MR occupancy by ALDO, and CORT and the control of testosterone biosynthesis in Leydig cells by glucocorticoid.
In this study we confirmed that 11ßHSD1 is an oxidoreductase with equivalent oxidative and reductive activities when measured in intact cells after culture (Fig. 4). The results indicate that 11ßHSD reductive activity is catalyzed by 11ßHSD1 because the 11ßHSD reductase was suppressed after 11ßHSD1 antisense oligonucleotide treatment. However, 11ßHSD1 antisense oligomer also inhibited 11ßHSD oxidase activity. The oxidative activities attributable to 11ßHSD1 and -2 inactivate CORT, thereby ameliorating the adverse effects of glucocorticoid exposure. In the current experiments, when 11ßHSD1 was inhibited by an antisense oligomer, the potency of the CORT-mediated inhibition of testosterone production was enhanced (Fig. 6). Recent data suggest that the catalytic direction of 11ßHSD1 as an oxidase or reductase is regulated by the endogenous NADP+/NADPH redox potential (21, 22, 23, 24). In many tissues 11ßHSD1 behaves as a predominant reductase (56), but the redox potential in Leydig cells established by the steroidogenic milieu favors stronger oxidative activity. Moreover, enzymes such as 17-hydroxylase, type 3 17ß-hydroxysteroid dehydrogenase, and 5-reductase and 3-hydroxysteroid dehydrogenase are present in Leydig cells and use NADPH as their cofactor. Accordingly, NADP+ is generated as a byproduct of the testosterone biosynthesis, in turn stimulating oxidative activity in 11ßHSD1, promoting very strong oxidative catalysis. A similar potential redox pair between 11ßHSD1 and hexose-6-phosphate dehydrogenase has been suggested (21, 22, 23, 24). The testing of this hypothesis will be essential for a complete understanding of the kinetics of the two isoforms of 11ßHSD when they are coexpresssed in a single cell type.
In conclusion, rat Leydig cells were observed to coexpress 11ß-HSD1 and 11ßHSD2. In addition to11ßHSD1 dehydrogenase activity, 11ßHSD2 may also protect Leydig cells from suppressive effects exerted by glucocorticoid and allow homologous MR activation. Further experiments are needed to better understand the role(s) of 11ßHSD1, which possesses both oxidative and reductive activities, and 11ßHSD2, which possesses dehydrogenase activity, in CORT inactivation. Additional studies will also be necessary to determine which receptor mechanisms are used by each of the 11ßHSD isoforms in the control of Leydig cell testosterone production.
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Footnotes |
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First Published Online March 10, 2005
Abbreviations: ALDO, Aldosterone; CORT, corticosterone; 11DHC, 11-dehydrocorticosterone; GR, glucocorticoid receptor; 3H-CORT, [1,2-3H]corticosterone; 3H-11DHC, [1,2, 3H]11dehydrocorticosterone; 11ßHSD, 11ß-hydroxysteroid dehydrogenase; Km, Michaelis constant; MR, mineralocorticoid receptor; NAD+, nicotinamide adenine dinucleotide (oxidized form); NADH, nicotinamide adenine dinucleotide (reduced); NADP+, nicotinamide adenine dinucleotide phosphate (oxidized form); NADPH, nicotinamide adenine dinucleotide phosphate reduced.
Received January 13, 2005.
Accepted for publication March 2, 2005.
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