Corticosterone (C2505), Luteinizing hormone (L5259), bovine lipoprotein (L3626), Percoll (P1644) and DMEM-Ham's F12 (D2906) were purchased from Sigma-Aldrich Chemical (St. Louis, Missouri, USA). The antibodies for 11β-HSD2 (sc-20176) and β-actin (sc-130657) were obtained from Santa Cruz Biotechnology (Santa Cruz, California, USA). The Dual-Luciferase Reporter Assay System (E1910) was purchased from Promega Co. (Madison, Wisconsin, USA). [³H]CORT was kindly provided by Dr. Ren-Shan Ge (Population Council, New York, USA). The Male Sprague-Dawley rats (90 days of age) were purchased from the Animal Centre of the Chinese Academy of Sciences (Shanghai, China). The animals were killed by CO₂ asphyxiation for isolation of Leydig cells. Mouse Leydig tumor cell line mLTC-1 (CRL-2065) and rat Leydig tumor cell line R2C (CCL-97) were purchased from American Type Culture Collection (Manassas, Virginia, USA).

Adult Leydig cells were isolated from 90-day-old rats according to the procedure of Sriraman et al. 20, which is a modification of the procedure described by Klinefelter et al. 21. The decapsulated testis was subjected to collagenase digestion in a 50-mL plastic tube containing 10 mL medium with collagenase (600 units) and DNase (750 units). The tubes were placed in a shaking water bath with constant agitation (50 times/min) at 34°C for 15–20 min until the seminiferous tubules were separated. The enzyme action was terminated by adding excess medium. The tubules were allowed to settle by gravity and the medium, consisting of interstitial cells, was aspirated and filtered through a 100-μm nylon mesh. The filtrate was centrifuged at 250 × g for 10 min at 25°C, which yielded a crude interstitial pellet. The pellet obtained was suspended in 35 mL 55% isotonic Percoll with 750 units DNase in Oakridge tubes. The tubes were centrifuged at 20 000 × g for 1 h at 4°C. Percoll fractions corresponding to densities of 1.070–1.090 g/mL were collected and the cells present in this fraction were pelleted by centrifugation at 250 × g for 10 min at 25°C after diluting with 3–4 volumes of medium. The purities of isolated cell fractions were evaluated by histochemical staining for 3β-hydroxysteroid dehydrogenase activity, with 0.4 nmol/L etiocholanolone as the steroid substrate 22. The mean purity of Leydig cells was 85%.

Rat Leydig cells culture was conducted as previously described 23. Briefly, freshly isolated Leydig cells were seeded in 6-well plates, at a density of 4 × 10⁵ cells per well, and cultured for 24 h in phenol red-free DMEM/Ham's F12 medium supplemented with 1 mg/mL bovine lipoprotein in an incubator gassed with 5% O₂, 5% CO₂, at 34°C.

The mLTC-1 cells were cultured in DMEM medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS); R2C cells were cultured in Ham's F10 medium supplemented with 15% horse serum, 2.5% FBS. Both cell lines were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO₂.

Total RNA was extracted from cultured cells using an RNeasy Mini Kit (Qiagen, Valencia, California, USA) coupled with on-column DNase digestion with the RNase-Free DNase Set (Qiagen) according to the manufacturer's instructions. First strand cDNA synthesis was performed using Superscript II reverse transcriptase (Invitrogen, Shanghai, China) in a reaction using 2 μg of total RNA primed with random hexamers in a total reaction volume of 20 μL.

Real-time PCR was carried out according to Lee JJ and Widmaier EP with some modification 24. In brief, each PCR reaction contains 1 × Absolute TM QPCR SYBR Green Mix (Abgene, Epsom, UK), 0.3 μM primers and 2 μL cDNA with a total volume of 25 μL. The primers for 11β-HSD2 [GenBanK:NM_017081] (Forward: 5'-TGGCCAACTTGCCTAGAGAG; Reverse: 5'-TTCAGGAATTGCCCATGC) and 18S [GenBank:X03205] (Forward: 5'-CGCCGCTAGAGGTGAAATTC; Reverse: 5'-CCAGTCGGCATCGTTTATGG) are the same as described previously 24. Initially, the reaction mix was incubated at 95°C for 15 min to activate Thermo-Start^® DNA Polymerase (Abgene), then templates were amplified for 40 cycles (95°C for 15 s, 60°C for 1 min) on the Rotor-Gene 3000 system (Corbett Research, Sydney, NSW, Australia). Meanwhile, genomic DNA contamination was evaluated by real-time PCR of non-retrotranscribed RNA for each sample. Melting curve analysis was conducted immediately after completion of PCR as following: 95°C for 10 s, then 60°C for 10 s and continuous heating to 99°C at 0.5°C/s. The resulting PCR products were analyzed on a 2% agarose gel and cloned into pGEM-T Easy vector (Promega) followed by DNA sequencing to confirm the specificity of PCR. The relative quantitative analysis of 11β-HSD2 mRNA expression was performed using the delta-delta Ct method with 18S RNA as an internal control 25.

Total RNA was prepared from rat kidney tissue using the Trizol Reagent (Invitrogen) according to the manufacturer's instruction. First strand cDNA synthesis was performed using Superscript II reverse transcriptase (Invitrogen) in a reaction using 2 μg of total RNA primed with random hexamers in a total reaction volume of 20 μl. For amplification of rat 11β-HSD2 [GenBanK:NM_017081], the forward and reverse primers were 5'-GAC GGT ACC CGA GTA TCC CTC CCA C-3' and 5'-ACG CTC GAG TCT CCT GCT GAA ACA CCT A-3', respectively. PCR was performed for 30 cycles with denaturing at 94°C for 30 s, annealing at 58°C for 30 s and extension at 72°C for 90 s. The 11β-HSD2 cDNA was purified by QIAquick PCR Purification Kit.

Rat genomic DNA was isolated from tail tissue using DNAeasy kit (Qiagen). Four DNA fragments corresponding to different lengths of rat gene Hsd11b2 promoter region [GenBank:NC_005118] were generated using their respective upstream primers and the common downstream primer 5'-cccaagcttagtgcagaggaacaccagcctg-3' in PCR using platinum Pfx high fidelity polymerase (Invitrogen) and 50 ng of genomic DNA template. The upstream primers, 5'-cggggtaccggaatcaatgggtttagaaaag-3', 5'-cggggtaccagcaagagaccttgatgtctg-3', 5'-cggggtaccggcggggcgggggggcacctgc-3' and 5'-cggggtaccggcgctttataagctgggtcc-3' correspond to constructs -1740/+45,-756/+45,-138/+45 and -35/+45, respectively (+1 designates the transcription start site). The resultant fragments were gel-purified, cloned into pGEM-T Easy vector (Promega) and sequenced for fidelity. The four inserts were then subcloned into pGL3-Basic luciferase vector (Promega) after digestion with Kpn I and Hind II.

11β-HSD2 cDNA sequences were digested with Kpn I and Xho II, and ligated into the Kpn I/Xho I site of pcDNA3.0 plasmids (Invitrogen) to construct the 11β-HSD2 expression vector, named pcDNA-hsd11b2.

A luciferase reporter gene plasmid, GRE-Luc was constructed as described previously 26, which transcriptional activity is subject to intracellular glucocorticoid level. Briefly, An oligonucleotide (5'-tatataacgcgt

tgtacaggatgttct

tgtacaggatgttct

R2C cells were seeded in 24 well culture plates (5 × 10⁵/well) in fresh medium for 24 h. Then, 1.0 μg of pcDNA-hsd11b2 was transiently transfected into R2C cells using Lipofectamine™ 2000 reagent (Invitrogen) according to the supplier's protocol. After another 48 h of culture, the expression of 11β-HSD2 protein was identified by Western blotting. R2C cells transfected with pcDNA3.0 vector served as control.

Primary rat Leydig cells and transfected R2C cells were lysed in Ripa buffer (1% NP-40, 0.1% SDS, 0.5% DOC, 150 mM NaCl, 10 mM Tris-HCl, and PMSF mixture) at 4°C for 30 min. Protein concentrations were determined BCA protein assay reagent (Pierce Chemical, Rockford, Illinois, USA). Aliquots of cell extracts containing equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis on 12% gels using the Laemmli buffering system, then proteins were transferred to ImmunoBlot polyvinylidene difluoride membranes (Bio-Rad, Hercules, California, USA) and blocked by rocking for 1 h at room temperature in blocking buffer (Tris-buffered saline with 0.1% Tween 20 and 5% nonfat dry milk). Afterwards, the membranes were incubated with a 1:1000 dilution of a rabbit polyclonal antibody for 11β-HSD2 for 1 h, then repeatedly washed and incubated with a 1:10000 dilution of goat anti-rabbit antibody that was conjugated to horseradish peroxidase. Signals were detected with an Enhanced Chemilluminescence kit (Pierce Chemical). Beta-actin was used as internal control.

After rat Leydig cells were isolated and cultured for 24 h, cells were treated with 20 ng/mL LH for 24 h. Then, Leydig cell homogenates were prepared with the method described by Arcuri F et al 29. In short, cell cultures were rinsed three times with 0.1 M phosphate buffer solution, harvested with a rubber scraper and pelleted by centrifugation at 1000 × g for 5 min at 4°C. Cell pellets were suspended in ice-cold sodium phosphate buffer 0.1 M (pH 7.5), 0.2% (v/v) of Triton X-100, and sonicated for 15 s on ice. Protein concentrations were determined by BCA protein assay reagent (Pierce Chemical). The 11β-HSD2 enzyme activity assay was performed according to the protocol of Ge RS et al. with some modification 16. Briefly, 0.5 mL reaction mixture was prepared in phenol red-free medium that contained 2 nM [³H]CORT and 23 nM unlabeled CORT, obtaining a final concentration of 25 nM of CORT. The reactions were initiated by the addition of 50 μg protein with NAD⁺ at a final concentration of 0.5 mM, then performed in a shaking water bath (100 strokes/min) at 34°C for 1 h, and finally stopped by adding 2 ml ice-cold ethyl acetate. Tubes were vortexed and centrifuged at 10,000 rpm for 5 min to separate the upper organic layer, which was dried under nitrogen. Samples were resuspended in 100 μl of methanol. The steroids were separated chromatographically on thin-layer plates in chloroform and methanol (90:10). Bands were visualized using I₂ vapor and scraped into scintillation fluid, and the radioactivity was measured using a Beckman LS65000 liquid scintillation counter (Beckman Coulter, Fullerton, USA). The percentage conversion of corticosterone to 11-dehydrocorticosterone was calculated by dividing the radioactive counts identified as 11-dehydrocorticosterone by the total counts associated with corticosterone plus 11-dehydrocorticosterone.

To determine the effect of LH on transcription of 11β-HSD2 gene, four luciferase reporter genes driven by different lengths of 11β-HSD2 promoter fragment were transiently transfected into mLTC-1. Briefly, the cells were seeded into 24-well plates at a density of 5 × 10⁵ cells/well and incubated at 37°C overnight. Cells were then placed in DMEM with 10% fetal bovine serum containing 1 μg of luciferase plasmid, 0.01 μg of pRL-TK (Promega) reporter plasmid, and 2 μg of Lipofectamine™ 2000 reagent (Invitrogen) according to the supplier's protocol. Twelve hours following transfection, the cells were treated with 20 ng/ml LH for 24 h. Following the treatment, Dual Luciferase Assay was performed by lysing the cells in Passive Lysis Buffer (Promega) and reading the relative light units of one-fifth the lysate with both the firefly substrate and the renilla substrate using a luminometer (Berthold Lumat LB9507, Bad Wildbad, Germany). Each transfection was performed in triplicate and in three independent experiments.

Given that both isoforms of 11β-HSD are expressed in rat Leydig cell and the inhibitory effect of LH on the net oxidative activity of 11β-HSD1 17, a glucocorticoid-responsive reporter gene, GRE-Luc, was employed to investigate the level of active intracellular glucocorticoid. The rationale is that the transcriptional activity of GRE-Luc which contains two copies of a consensus GRE upstream of the luciferase gene is subject to the extent of GR activation, i.e. intracellular glucocorticoid concentration set by the total oxidative activity of 11β-HSD1 and 11β-HSD2, thus the effect of LH on the transcriptional activity of GRE-Luc is indicative of the oxidative capacity of Leydig cells for glucocorticoid. Rat Leydig cells were isolated and cultured in 24 well culture plates (1 × 10⁶/well) in fresh medium for 48 h. Then, 1.0 μg of GRE-Luc and 0.01 μg of pRL-TK (Promega) reporter plasmid were transiently transfected into the Leydig cells using Lipofectamine™ 2000 reagent (Invitrogen). After another 48 h of culture, the cells were incubated in the presence or absence of LH (20 ng/mL) for 24 h. Subsequently, the cells pretreated with LH were treated with CORT (50 nM) plus LH (20 ng/mL) and the other cells not pretreated with LH were treated with CORT (50 nM) or LH (20 ng/mL) for 24 h. At the end of all treatments, Dual Luciferase Assays were performed as described above. The transfected cells treated with vehicles (PBS for LH; DMSO for CORT) served as controls.

Results are presented as means ± SE of three independent experiments. Statistical analyses of 11β-HSD2 mRNA data and Luciferase assay data were performed using one-way ANOVA followed by Student-Newman-Kuels test. 11β-HSD2 protein data was analyzed by a standard Student's t-test. Significance was set at P < 0.05. Calculations were performed using SPSS software version 10.0 (Chicago, USA).

As shown in Fig. 1A, a concentration-dependent increase in 11β-HSD2 mRNA was found with a maximal effect at 20 ng/ml. Treatment of Leydig cells for 24 h with 20 ng/ml LH led to a significant increase in levels of mRNA. (Fig. 1B). Fig. 2 showed treatment of Leydig cells for 24 h with 20 ng/ml LH increased the level of 11β-HSD2 protein compared with Control.

To further investigate the effect of LH on the expression of 11β-HSD2, the transcriptional activities of different lengths of rat gene Hsd11b2 promoter region were analyzed. LH treatment induced a two-fold increase in the activity of -1740/+45 construct. It was also shown that the -138/+45 region is crucial for the constitutive and LH-induced transactivation of 11β-HSD2 (Fig. 3).

LH significantly increased 11β-HSD2 activity (34.84 ± 2.98%) compared with control cells (15.17 ± 5.22%), p <0.05.

As shown in Fig. 4, 50 nM CORT (within the physiological concentration of CORT in rats) significantly increased the luciferase activity in Leydig cells and LH reduced glucocorticoid-activated transcriptional response of GRE-Luc.