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Endocrinology Vol. 138, No. 6 2435-2442
Copyright © 1997 by The Endocrine Society

ARTICLES

Identification of a Kinetically Distinct Activity of 11ß-Hydroxysteroid Dehydrogenase in Rat Leydig Cells1

Ren-Shan Ge, Hui-Bao Gao2, Vijaya L. Nacharaju, Glen L. Gunsalus and Matthew P. Hardy

The Population Council (R.-S.G., H.-B.G, G.L.G., M.P.H.) and Rockefeller University (M.P.H.), 1230 York Avenue, New York, New York 10021; and Department of Obstetrics and Gynecology, State University of New York Health Science Center-Brooklyn (V.L.N.), Brooklyn, New York 11203

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Leydig cells are susceptible to direct glucocorticoid-mediated inhibition of testosterone biosynthesis but can counteract the inhibition through 11ß-hydroxysteroid dehydrogenase (11ß-HSD), which oxidatively inactivates glucocorticoids. Of the two isoforms of 11ß-HSD that have been identified, type I is an NADP(H)-dependent oxidoreductase that is relatively insensitive to inhibition by end product and carbenoxolone (CBX). The type I form has been shown to be predominantly reductive in liver parenchymal cells and other tissues. In contrast, type II, which is postulated to confer specificity in mineralocorticoid receptor (MR)-mediated responses, acts as an NAD-dependent oxidase that is potently inhibited by both end product and CBX. The identity of the 11ß-HSD isoform in Leydig cells is uncertain, because the protein in this cell is recognized by an anti-type I 11ß-HSD antibody, but the activity is primarily oxidative, more closely resembling type II. The goal of the present study was to determine whether the kinetic properties of 11ß-HSD in Leydig cells are consistent with type I, type II, or neither. Leydig cells were purified from male Sprague-Dawley rats (250 g), and 11ß-HSD was evaluated in Leydig cells by measuring rates of oxidation and reduction, cofactor preference, and inhibition by end product and CBX. Leydig cells were assayed for type I and II 11ß-HSD and MR messenger RNAs (mRNAs), and for type I 11ß-HSD protein. Leydig cell 11ß-HSD had bidirectional catalytic activity that was NADP(H)-dependent. This is consistent with the hypothesis that type I 11ß-HSD is present in rat Leydig cells. However, unlike the type I 11ß-HSD in liver parenchymal cells, the Leydig cell 11ß-HSD was predominantly oxidative. Moreover, analysis of kinetics revealed two components, the first being low a Michaelis-Menten constant (Km) NADP-dependent oxidative activity with a Km of 41.5 ± 9.3 nM and maximum velocity (Vmax) of 7.1 ± 1.2 pmol · min · 106 cells. The second component consisted of high Km activities that were consistent with type I: NADP-dependent oxidative activity with Km of 5.87 ± 0.46 µM and Vmax of 419 ± 17 pmol · min · 106 cells, and NADPH-dependent reductive activity with Km of 0.892 ± 0.051 µM and Vmax of 117 ± 6 pmol · min · 106 cells. The results for end product and CBX inhibition were also inconsistent with a single kinetic activity in Leydig cells. Type I 11ß-HSD mRNA and protein were both present in Leydig cells, whereas type II mRNA was undetectable. We conclude that the low Km NADP-dependent oxidative activity of 11ß-HSD in Leydig cells does not confirm to the established characteristics of type I and may reside in a new form of this protein. We also demonstrated the presence of the mRNA for MR in Leydig cells, and the low Km component could allow for specificity in MR-mediated responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GLUCOCORTICOIDS ACTING through the glucocorticoid receptor induce a variety of responses in cells, including proliferation, differentiation, and apoptosis (reviewed in 1 . Pathological conditions such as stress and Cushing’s disease that elevate circulating levels of the active, reduced form of glucocorticoids lead to depressed serum testosterone (2, 3). Suppression of endogenous glucocorticoid production increases the steroidogenic capacity of Leydig cells (4). Leydig cells contain glucocorticoid receptors and are the primary target of glucocorticoid action in the testis (5, 6, 7). Because glucocorticoid-induced suppression of testosterone production is mediated by glucocorticoid receptors (8, 9), the intracellular concentration of glucocorticoid within the Leydig cell should determine the magnitude of its effect. In this regard, 11ß-hydroxysteroid dehydrogenase (11ß-HSD) is an enzymatic determinant of glucocorticoid action because this enzyme can mediate the intracellular concentration of active glucocorticoid.

Two isoforms of 11ß-HSD have been identified. Type I is a NADP(H)-dependent oxidoreductase that is relatively insensitive to inhibition by end product and carbenoxolone (CBX), a synthetic derivative of glycyrrhytenic acid that potently inhibits 11ß-dehydrogenation (9, 10, 11). Type I 11ß-HSD has a Michaelis-Menten constant (Km) in the micromolar range (9, 10, 11) and, when purified from rat liver, has an apparent molecular mass of 34 kDa (12). The complementary DNAs (cDNAs) encoding this isoform have been cloned from human, rat, and mouse (13, 14, 15). Type II is a NAD-dependent unidirectional oxidase that is profoundly inhibited by both end product and CBX, and has a Km in the nanomolar range (11, 16). Type II enzyme purified from human placenta has an apparent molecular mass of 40 kDa (17). The cDNAs encoding type II isoform have been cloned from human, rat, mouse, sheep, and rabbit (18, 19, 20, 21, 22). One hypothesized role of type II 11ß-HSD is to confer specificity in mineralocorticoid receptor (MR)-mediated responses in the kidney (11, 16). The concentration of corticosterone (CORT) in the blood is normally 100-fold higher than aldosterone and, because MRs can bind to CORT (23), nonspecific activation of MR-mediated responses would occur without the metabolism of glucocorticoid by the type II enzyme. However, type II 11ß-HSD messenger RNA (mRNA) has not been detected in the rat testis by Northern blot or in situ hybridization (19, 24, 25), nor has the existence of gonadal MRs been established. In contrast, the presence of type I 11ß-HSD protein has been demonstrated in testis by immunocytochemistry, where it is expressed uniquely in Leydig cells (26). Because the Km of type I is high, 2–5 µM, and circulating levels of free CORT vary between 0.5 and 100 nM (27), it seems unlikely that an enzyme with a Km in the µM range could efficiently decrease glucocorticoid levels to the very low levels needed to eliminate glucocorticoid receptor binding. In fact, in tissues such as liver and lung, type I 11ß-HSD functions predominantly as a reductase in vivo (28, 29, 30), thereby increasing glucocorticoid action (29, 30). Furthermore, reductase activity has been shown to predominate in all cell lines transfected with type I 11ß-HSD cDNA (31, 32, 33).

If 11ß-HSD in Leydig cells were primarily reductive, the resulting activation of glucocorticoids might inhibit testosterone production thereby preventing or delaying sexual maturation. However, testosterone production and type I 11ß-HSD immunoreactivity within Leydig cells increase in parallel during puberty (26), indicating that the enzyme does not facilitate glucocorticoid activation. The resolution of this apparent paradox will be achieved only by determining the characteristics of 11ß-HSD in Leydig cells. The goal of the present study was to characterize 11ß-HSD in Leydig cells and determine whether its properties are consistent with the type I, type II, or neither isoforms. Because the reductive activity of type I 11ß-HSD is unstable (10, 12) and greatly reduced by homogenization, leading to predominance of oxidative activity (30, 32, 34, 35), the present study measured enzyme activities in intact Leydig cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
\[1,2,6,7-N-3H\] CORT (specific activity 88 Ci/mmol) was purchased from Dupont-New England Nuclear (Boston, MA). CORT and 11-dehydrocorticosterone (11-DHC) were purchased from Steraloids (Wilton, NH). NADP+, NAD+, NADPH, and NADH were purchased from Sigma Chemical Co. (St. Louis, MO). 11-dehydro\[1,2,6,7-N-3H\]corticosterone ([3H]11-DHC) was prepared from [3H]CORT as described earlier (36). Polyclonal anti-type I 11ß-HSD antiserum (No. 56–127) was generated by immunizing animals with purified type I 11ß-HSD from rat liver (37). TLC plates (Polygram Silica Gel/UV254) were obtained from Brinkmann Instruments (Westbury, NY).

Isolation and purification of liver parenchymal cells
Isolation of liver parenchymal cells was performed according to Pertoft and Smedsrød (38). Livers of male rats were perfused in situ with a calcium-free buffer, then dispersed by a solution containing 0.05% collagenase, and parenchymal cells were purified by density gradient centrifugation in Percoll. The purity of parenchymal cells in the final suspension was assessed by judging the uniformity of cell size in hematocytometer counts and was typically over 95%.

Isolation and purification of Leydig cells
Leydig cells were isolated from testes by centrifugal elutriation and Percoll density gradient centrifugation, according to the method of Klinefelter et al. (39). The purity of cells isolated using this method was evaluated by histochemical staining for 3ß-HSD activity, with 0.4 mM etiocholanolone as the steroid substrate (40). Enrichment of Leydig cells was typically to 92–96% purity.

Assay of 11ß-HSD enzymatic activity
11ß-HSD oxidative activity was determined by measuring the rate of conversion of CORT to 11-DHC. The reaction mixture (0.5 ml) was prepared in phenol red-free Leydig cell medium (DMEM-Ham’s F12, D-2906; Sigma Chemical Co.) that contained 0.2 µCi [3H]CORT and unlabeled CORT at a final concentration ranging from 1 nM to 20 µM, and 0 or 200 µM NADP+ or NAD+. The 11ß-HSD reductive activity of 11ß-HSD was determined similarly, except that [3H]11-DHC and unlabeled 11-DHC was used as the substrate and NADPH or NADH as cofactor. When enzyme activities were assayed in the presence of cofactors, intact cells were first incubated with cofactors for 45 min, and the substrate was then added. The pH of the reaction mixture was 7.2, which is appropriate for intact Leydig cells. Replicate samples were preincubated for 10 min at 34 C (the physiological temperature for Leydig cells) in a shaking water bath, and the reactions were initiated by adding cell suspension containing 0.2 x 106 liver parenchymal cells or Leydig cells, or 20–50 µg Leydig cell microsomes to each tube. A preliminary study showed that conversions of CORT to 11-DHC and 11-DHC to CORT were linear with respect to cell number (0.1–0.3 x 106 cells) and time of incubation (0–5 min). Therefore, the reaction time was fixed at 5 min for determination of initial velocities. Reactions were terminated by placing the reaction mixture on ice and adding ice-cold ethyl acetate. Steroids were then extracted with ethyl acetate, and the organic layer was dried under nitrogen. The steroid residues were chromatographed on TLC plates in chloroform/methanol (9:1), and the radioactivity was measured with a Bioscan System Scanner (Bioscan Inc., Washington, DC). The percentage of conversion of CORT to 11-DHC and 11-DHC to CORT was calculated by dividing the radioactive counts identified as 11-DHC (or CORT, respectively) by the total counts associated with CORT plus 11-DHC.

Determination of kinetic constants
Kinetic analysis was performed by fitting initial velocity data as a function of substrate concentration to a one or two component Michaelis-Menten equation of the form:

where S is substrate concentration and KmL, VmaxL, KmH, and VmaxH are the parameters for the low and high Km components, respectively. Vmax is defined for any given amount of enzyme as the initial velocity at starting concentration of substrate. Km is defined as the concentration of substrate when initial velocity is equal to one half Vmax. Parameters and their estimated standard deviations were determined by weighted nonlinear least squares curve fitting using the SAAM II optimizer (41). The appropriate model was selected by assessing goodness of fit to each equation form using multiple criteria including the principal of parsimony (Akaike information criterion and Schwarz criterion) and F tests for comparison between models (42).

Northern blot for type I and II 11ß-HSD mRNA
Northern blotting for type I 11ß-HSD mRNA was performed as described by Agarwal et al. (14). Poly(A) RNAs (6 µg) were electrophoresed on 0.78% agarose gels in the presence of 2.2 M formaldehyde. Full-length type I rat 11ß-HSD cDNA and type II sheep 11ß-HSD cDNA (kindly provided by Dr. White) was radioactively labeled with [32P]deoxycytidine triphosphate using a random priming kit (Boehringer Mannheim, Indianapolis, IN). During hybridization with the probes, stringent washes were performed at 60 C in 15 mM NaCl, 1.5 mM sodium citrate, 0.1% SDS.

RT-PCR analysis for type I and II 11ß-HSD and MR mRNA levels
The procedure for RT-PCR was as previously described (4). In brief, total RNA (400 ng/sample) was reverse transcribed with avian myeloblastosis virus reverse transcriptase in the presence of random hexamer primers at 42 C for 75 min. After adding the primer pairs for 11ß-HSD, or MR and ribosomal protein S16 (RPS16), PCR was initiated by Taq DNA polymerase in the presence of [32P]deoxycytidine triphosphate, and proceeded for 35 cycles with an annealing temperature of 48 C. The PCR primer sequences for type I 11ß-HSD were those published by Agarwal et al. (43). The RT-PCR product (696 bp in size) was confirmed by restriction enzyme analysis after digestion with Fok I. PCR primer sequences for type II 11ß-HSD were chosen from those described by Leckie et al. (44). The RT-PCR product (658 bp) was confirmed by restriction enzyme analysis after digestion with Fok I and PstI. The primer sequences for MR were described by Todd-Turla et al. (45). The RT-PCR product (381 bp) was sequenced, and the sequence of the 381-bp product had 100% identity with the published sequence of rat MR. The primers for the internal control, RPS16, were those described previously (46).

Detection of 11ß-HSD protein by Western blot
All tissue homogenates were prepared and transferred to nitrocellulose membranes according to the method described by Monder and Lakshmi (37). The membrane was blocked in 10% fat-free milk for 1 h at room temperature. The membrane was then incubated in 10% fat-free milk containing 1:1500 dilution of normal rabbit serum or polyclonal anti-type I 11ß-HSD antiserum (No. 56–127). The membrane was washed four times in PBST (10 mM sodium phosphate, 0.15 M NaCl, and 0.05% Tween-20; pH 7.4), and incubated for 1 h in a 1:2000 dilution of goat antirabbit antiserum that was conjugated to horseradish peroxidase, and immunoactove bands were visualized by chemiluminenscence (ECL, Amersham, Arlington Heights, IL).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cofactor preference of 11ß-HSD in Leydig cells
Cofactor preference is one criterion for identifying 11ß-HSD: the type I isoform is NADP(H)-dependent, whereas the type II isoform is NAD-dependent (9, 10, 11, 16, 17, 18, 19, 20, 21, 22). The effects of NADP+ and NAD+ on 11ß-HSD oxidative activity, and NADPH and NADH on reductive activity were tested in Leydig cell microsomes at 25 and 500 nM substrate concentrations, using [3H]CORT or [3H]11-DHC. NADP+ increased 11ß-HSD oxidative activity at both low and high concentrations of CORT (P < 0.001). NAD+ slightly increased 11ß-HSD oxidative activity at 500 nM CORT. However, it was less effective than NADP+ (Fig. 1aGo). Similarly, NADPH increased 11ß-HSD reductive activity at both low and high concentrations of 11-DHC, and NADH did not (Fig. 1bGo). The cofactor preference of the enzyme was also determined using intact Leydig cells. The results were similar to those obtained using Leydig cell microsomes (data not shown). This indicated that Leydig cells contain NADP(H)-dependent oxidoreductase activities that are similar to type I and dissimilar to NAD-dependent type II.



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  		Figure 1. Cofactor preference of 11ß-HSD oxidative and reductive activities in Leydig cells. Leydig cell microsomes (20 µg protein) were incubated with 25 or 500 nM [3H]CORT in absence of added cofactors (No cofactor) or with 200 µM NAD+ or NADP+. a, Oxidative activity. b, Reductive activity in Leydig cell microsomes (50 µg protein) was measured similarly, except with 11-DHC instead of CORT and, respectively, NADH, or NADPH instead of NAD+ or NADP+. Values are means ± SEM (n = 6). *, P < 0.05, ***, P < 0.001 compared with control (no cofactor).

 
Prevalence of oxidative and reductive activities of 11ß-HSD
The preferred reaction direction of catalysis is a second criterion distinguishing the type I and type II 11ß-HSD isoforms. In the case of type I, reductive activity exceeds oxidative activity in intact liver parenchymal cells (34), whereas type II 11ß-HSD is almost entirely oxidative (16). 11ß-HSD oxidative activity was measured in the presence of 25 nM CORT with NADP+ as cofactor, and reductive activity was measured in the presence of 25 nM 11-DHC with NADPH as cofactor. 11ß-HSD in intact Leydig cells showed both oxidative and reductive activities, but oxidation was predominant (Fig. 2aGo). Liver parenchymal cells, however, had high rates of reductive activity that greatly exceeded oxidation (Fig. 2bGo). This corroborated earlier reports that 11ß-HSD in liver cells is predominantly reductive (15, 29, 30, 34). The predominance of the oxidative direction of 11ß-HSD in Leydig cells indicated that the enzyme is kinetically distinct from the type I form found in liver and other tissues.



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  		Figure 2. 11ß-HSD activities in intact Leydig cells and liver parenchymal cells. Oxidative (conversion of CORT to 11-DHC, •) and reductive (11-DHC to CORT, {circ}) activities of 11ß-HSD were measured in 0.2 x 106 freshly purified Leydig cells (a) and liver parenchymal cells (b) with 25 nM CORT or 11-DHC. Values are means ± SEM (n = 6 in Leydig cells, n = 3 in liver parenchymal cells. *, P < 0.05 compared with reductive activity at same time point.

 
Kinetic characteristics
Reaction velocity showed a uniform coefficient of variation (typically 7–10%) over the range of substrate concentrations used; therefore, the data were weighted according to the fractional standard deviation and fit to the one and two component Michaelis-Menten models.

With oxidative activity (using CORT as substrate) the two component model consistently gave the better fit (P < 0.01), yielding parameter estimates of KmL = 41.5 ± 9.3 nM and VmaxL = 7.1 ± 1.2 pmol · min · 106 cells for the low Km component and KmH = 5.87 ± 0.46 µM and VmaxH = 419 ± 17 pmol · min · 106 cells for the high Km component (Fig. 3aGo).



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  		Figure 3. Reaction velocity as a function substrate concentration for 11ß-HSD oxidative and reductive activities in freshly isolated Leydig Cells. Intact Leydig cells (0.2 x 106) were incubated with varying concentrations of CORT as substrate (10 nM to 20 µM) in presence of 200 µM NADP+ (a, oxidative) or 11-DHC as substrate (1 nM to 5 µM) in presence of 200 µM NADPH (b, reductive). Data are presented as means ± SEM (n = 3–5). Solid lines are from best fit parameter estimates; dashed line represents computer-generated decomposition in low and high Km components. a, Data for low substrate concentrations show resolution into two components, low (VL) and high (VH). The KmL associated with VL = 41.5 nM and the KmH associated with VH = 5.87 µM. Inset shows data and fitted curve for high concentrations of substrate.

 
In contrast, the kinetic data using 11-DHC as substrate showed Leydig cell 11ß-HSD to have a single component NADPH-dependent reductive activity with Km = 0.892 ± 0.051 µM and Vmax = 117.4 ± 6 pmol · min · 106 cells (Fig. 3bGo). The Km values for reductive activity and high Km component of oxidative activity of 11ß-HSD (KmH = 5.87 µM) in Leydig cells were comparable with previous observations of type I 11ß-HSD in liver (9, 10). These results suggested that, in addition to type I 11ß-HSD, Leydig cells contain a low Km NADP-dependent oxidase.

Effects of end product on Leydig cell 11ß-HSD oxidative and reductive activities
Type II 11ß-HSD can be distinguished from type I because it is significantly inhibited by the end product (11, 16). The effects of end products, 11-DHC or CORT, respectively, on 11ß-HSD oxidative or reductive activity were observed in this study to determine whether Leydig cell 11ß-HSD was susceptible to end product inhibition. At 500 nM CORT or 11-DHC, where most 11ß-HSD activity is attributable to 11ß-HSD reductase and the high Km component of 11ß-HSD oxidase, both 11ß-HSD oxidative (Fig. 4aGo) and reductive activities (Fig. 4bGo) were sharply activated by end product in a concentration-dependent manner. In contrast, at 25 nM CORT, where most 11ß-HSD oxidative activity is attributable to the low Km component, oxidative activity showed slight activation by 11-DHC (Fig. 4aGo). Because inhibition did not occur at either substrate concentration, these results were inconsistent with type II activity, and suggested that the low and high Km 11ß-HSD activities in Leydig cells each respond differently to end product.



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  		Figure 4. Effect of end product on 11ß-HSD oxidative and reductive activities in Leydig cells. Intact Leydig cells (0.2 x 106) were incubated with 25 nM [3H]CORT or 500 nM [3H]11-DHC (a) at a range of concentrations of 11-DHC (25 nM to 5 µM) in presence of 200 µM NADP+ for 5 min to determine effect of 11-DHC on oxidative activity. Aliquots containing 0.2 x 106 intact Leydig cells were incubated with 500 nM [3H]11-DHC (b) at various concentrations of CORT (25 nM to 5 µM) in presence of 200 µM NADPH for 5 min to determine effect of CORT on reductive activity. Values are means ± SEM (n = 6). *, P < 0.05 compared with group with 25 nM CORT at same concentration of 11-DHC.

 
Inhibition of 11ß-HSD oxidative and reductive activities by CBX
CBX is a known inhibitor of type I and II 11ß-HSD (11, 16). Type II is more sensitive to inhibition by CBX than type I (11). The effects of CBX on 11ß-HSD oxidative and reductive activities were examined to determine the potency of CBX inhibition. The 1 µM concentration of CBX inhibited oxidative activity by 56% at either 25 or 500 nM CORT (Fig. 5aGo), but had no effect on reductive activity (Fig. 5bGo). At 25 nM CORT, 100 nM CBX significantly inhibited 11ß-HSD oxidative activity (Fig. 5aGo). The sensitivity of 11ß-HSD oxidative activity to inhibition by CBX indicated that the oxidative activity could reside in a separate from the protein that catalyzes 11ß-HSD reduction.



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  		Figure 5. Effect of CBX on 11ß-HSD oxidative and reductive activities in Leydig cells. Intact Leydig cells (0.2 x 106) were incubated with 25 nM [3H]CORT or 500 nM [3H]CORT (panel a) at a range of concentrations of CBX (100 nM to 100 µM) in presence of 200 µM NADP+ for 5 min to determine effect of CBX on oxidative activity. Intact Leydig cells (0.2 x 106) were incubated with 25 nM [3H]11-DHC (b) at various concentrations of CBX (100 nM to 100 µM) in presence of 200 µM NADPH for 5 min to determine effect of CBX on reductive activity. Values are means ± SEM (n = 6). *, P < 0.05 compared with 500 nM CORT at same concentration of CBX.

 
11ß-HSD mRNA levels in Leydig cells
The characterization of enzyme activity indicated that Leydig cells contained type I rather than type II 11ß-HSD. The identity of 11ß-HSD isotypes was evaluated further by Northern blotting. A single 1.7-kilobase (kb) band was demonstrated in Leydig cells with a type I 11ß-HSD cDNA probe, corresponding to the known molecular size of type I 11ß-HSD mRNA (Fig. 6Go). Type II 11ß-HSD mRNA was not detected (data not shown). When the more sensitive RT-PCR method was used to detect type I and type II 11ß-HSD, Leydig cells and liver contained equivalent levels of type I mRNA (Fig. 6Go). In contrast, type II mRNA was not detected in liver (negative control) or Leydig cells, whereas a 658-bp PCR product was detected in the positive control, kidney. This further confirmed that type II 11ß-HSD is not expressed in Leydig cells.



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  		Figure 6. Northern blotting and RT-PCR analysis of type I and type II 11ß-HSD mRNA. Poly(A) RNAs (6 µg) from Leydig cells were used for Northern blotting. Total RNAs (2 µg) from each group were reverse transcribed and cDNA products were subsequently amplified in presence of primer pairs for type I 11ß-HSD, type II 11ß-HSD, and RPS16 by 35 cycles of PCR and detected by autoradiography. A 1.7-kb type I 11ß-HSD mRNA species was detected in Leydig cells (left). RT-PCR products for type I, type II, and RPS16 were 696, 658, and 148 bp, respectively. RT-PCR analysis showed type I mRNA in liver, kidney, and Leydig cells (center), and RT-PCR showed type II 11ß-HSD in kidney but not in liver and Leydig cells (right).

 
Identification of type I 11ß-HSD isoforms in Leydig cells by Western blot
To characterize the 11ß-HSD in Leydig cells, freshly isolated Leydig cell homogenates were separated by SDS-PAGE, transferred to nitrocellulose membrane, and probed with an anti-type I 11ß-HSD antiserum (No. 56–127). As shown in Fig. 7Go, highly immunoreactive type I 11ß-HSD (34 kDa) bands were seen in liver, kidney, and Leydig cells, demonstrating that all three tissues have type I protein. However, in addition to the 34-kDa band, two fainter bands of approximately 40 and 68 kDa were observed in Leydig cell and kidney homogenates.



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  		Figure 7. Western blot analysis of type I 11ß-HSD antigen in different tissues using anti-type I 11ß-HSD antiserum. Tissue homogenates (15 µg) were mixed with 10 µl SDS-containing sample buffer and immediately electrophoresed.

 
MR mRNA in Leydig cells
The low Km type II 11ß-HSD effectively catalyzes the oxidative inactivation of glucocorticoids at low substrate concentrations (16). It is, therefore, believed to be essential in conferring specificity to MR-mediated processes, because MR binds to both CORT (a glucocorticoid) and aldosterone (a mineralocorticoid). It is unknown whether Leydig cells have MR. The low Km NADP-dependent 11ß-HSD component in Leydig cells and the type II isoform have similar Kms, and the former might serve the same role as the latter in mediating responses to mineralocorticoid. Therefore, the mRNA for MR was assayed by RT-PCR in liver, kidney, and Leydig cells. A 381-bp band was observed in Leydig cells and kidney (positive control) but not in liver (negative control). Sequencing of the 381-bp PCR product confirmed that the mRNA sequence detected in Leydig cells belongs to the published sequence for rat MR (Fig. 8Go). This indicated that Leydig cells are capable of responding to mineralocorticoid.



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  		Figure 8. RT-PCR detection of MR mRNA in Leydig cells. Total RNAs (2 µg) from liver, kidney, and Leydig cells were reverse transcribed and cDNA products were subsequently amplified by 35 cycles of PCR in presence of primer pairs for MR and RPS16 and detected by autoradiography. RT-PCR products for MR and RPS16 were 381 and 148 bp, respectively. The 381-bp PCR product for MR was sequenced to confirm MR cDNA identity. mRNA for MR was detected in Leydig cells and kidney (positive control) but not in liver (negative control).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrated that, like the type I form in other tissues, 11ß-HSD in Leydig cells is an NADP(H)-dependent oxidoreductase. The detection of both type I mRNA and protein in Leydig cells establishes unequivocally that this isotype is expressed. Furthermore, kinetic analysis revealed both a high Km oxidative activity (Km 5.87 µM) and a high Km reductive activity (Km of 0.89 µM), which were similar to previously published data on type I 11ß-HSD in rat and sheep liver (10, 11). These results corroborate an earlier study showing that rat Leydig cells contain type I 11ß-HSD (26). However, there are major differences that clearly set the enzyme activity in Leydig cells apart from the type I form as it has been described in liver. Under physiological conditions, 11ß-HSD in Leydig cells behaved predominantly as an oxidase. The difference between Leydig cells and liver parenchymal cells in the favored direction of the reaction did not result from the different endogenous cofactor levels, because sodium azide, a metabolic inhibitor, decreases cellular NADP+/NADPH ratio by 12-fold in intact liver parenchymal cells but has no effects on 11ß-HSD oxidative and reductive activities or their ratio (34). The kinetic analysis demonstrated that Leydig cells contain a low Km NADP-dependent oxidative activity (with a Km of 42 nM) in addition to the expected high Km activities. Unlike type I oxidoreductase, the low Km component of 11ß-HSD oxidative activity exhibits profound inhibition by CBX, a well-known inhibitor of type II 11ß-HSD, at concentrations of CORT in the range of the low Km component (25 nM). Taken together, these results indicate that Leydig cells contain a low Km 11ß-HSD oxidase different from the type I described previously in other tissues. It is possible that the low Km oxidase in Leydig cells represents a new isoform, similar to the Km (Km = 40 nM) NADP-dependent unidirectional 11ß-HSD oxidase found in the choriocarcinoma cell line JEG-3 (47).

Leydig cells contained a low Km NADP-dependent 11ß-HSD oxidase having a Km of 42 nM, which is 141 times lower than the high Km component of type I seen in this study. The low Km 11ß-HSD oxidase probably contributes to the predominance of 11ß-HSD oxidative over reductive activity in Leydig cells at the physiological range of free glucocorticoids. The capacity of the low Km oxidase was as high as that reported for type II in renal cortical collecting duct cells (48), which confers the specificity in MR-mediated responses in that cell type (49). Leydig cells were discovered to contain the mRNA for MR in the present study. Therefore, the predominance of 11ß-HSD oxidation over reduction could function to confer specificity in MR-mediated responses in Leydig cells.

The low Km component of 11ß-HSD oxidase in Leydig cells and type II 11ß-HSD have similar Km’s (16, 48, 50). However, the low Km oxidative activity was not attributable to type II, as demonstrated by the absence of a signal for type II mRNA in Leydig cells. This confirmed earlier studies in which type II mRNA was not seen in rat testis (19, 24, 25). Further evidence that type II is absent in Leydig cells comes from our examination of the cofactor preference of the enzyme. Type II 11ß-HSD is an NAD-dependent oxidase, whereas 11ß-HSD in Leydig cells is NADP-dependent. Finally, type II 11ß-HSD exhibits end product inhibition in kidney (11, 16), whereas the 11ß-HSD oxidative activity of Leydig cells was activated, not inhibited by 11-DHC. Similar end product activation has also been observed in another dehydrogenase, 17ß-hydroxysteroid dehydrogenase (51, 52, 53). Taken together, these results indicate that the low Km 11ß-HSD oxidative activity present in Leydig cells is not attributable to type II.

The results of the present study are consistent with earlier data on the heterogeneity of 11ß-HSD in rat tissues (37, 54). When rat tissues were screened using antisera (Nos. 56–125, 56–126, and 56–127) to purified type I rat liver 11ß-HSD, a 34-kDa band that is typical of type I 11ß-HSD was detected in liver, kidney, testis, and lung. Four other bands were also observed, depending on the tissue, with a 26-kDa band in brain, 40- and 68-kDa bands in kidney, and a 47-kDa band in spleen (37, 54). The 26-kDa band protein has proven to be a truncated type I 11ß-HSD protein without enzyme activities (55, 56). The 68-kDa band has been detected previously in Western blots of purified type I 11ß-HSD protein obtained from liver, and was postulated to be a dimer of the 34-kDa species (37). The nature of other bands has not been further characterized. Heterogeneity of type I 11ß-HSD in rat kidney likely results from multiple transcripts (57) that use alternate promoters (58). Rat Leydig cells were found to contain, like the kidney, 40- and 68-kDa species in addition to the characteristic 34-kDa protein. The molecular mass of purified type II 11ß-HSD is 40 kDa (17). Because anti-type I 11ß-HSD antibody has not been observed to cross-react with the type II (18, 59), the 40-kDa band in Leydig cells represents another protein. One interpretation is that kinetically distinct, low Km oxidase in Leydig cells might be translated form a variant of type I mRNA, using an alternative promoter. However, the full-length type I rat cDNA probe hybridized with only a single 1.7-kb mRNA that is known to correspond to type I 11ß-HSD. Therefore, the most likely explanation of these results is that a separate isoform of 11ß-HSD, not derived from type I, exists in Leydig cells.

In conclusion, the presence of a low Km 11ß-HSD oxidative activity and MR mRNA in rat Leydig cells was demonstrated. The enzyme activity of this low Km component existed at low endogenous concentrations of CORT and, consequently, could function to allow specific mineralocorticoid-mediated responses and to protect Leydig cells from the suppressive action of CORT on testosterone production.


   Acknowledgments
 
The technical assistance of Ms. Chantal Manon Sottas is gratefully acknowledged. We thank Dr. Perrin C. White (University of Texas Southwestern Medical School) for the type I and type II 11ß-HSD cDNA.


   Footnotes
 
1 This work was supported in part by Contraceptive Research and Development Program of the United States Agency for International Development (to R.-S.G.), the Rockefeller Foundation (to H.-B.G.), and NIH Grant HD-33000 (to M.P.H.). Back

2 Current address: Laboratory of Reproductive Biology, Shanghai Second Medical University, 280 South Chongqing Road, Shanghai 200025, People’s Republic of China. Back

Received December 12, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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