Cells and reagents
Cell line mLTC-1 (mouse Leydig tumor cell) was purchased from American Type Culture Collection (Cat. No. CRL-2065). CORT was purchased from Sigma Chemical Co. QuickChange Site-Directed Mutagenesis Kit was purchased from Stratagene Inc (La Jolla. CA, Cat. No.200518). Qiagen Plasmid Maxiprep Kit was obtained from Qiagen (Santa Clarita, CA, Cat. No.12162) The Dual-Luciferase Reporter Assay System was purchased from Promega Co (Madson, WI, Cat. No.E1910). The Fluo-3/AM was purchased from Sigma Co. Specific antibody for FasL was obtained from Oncogene (Cambridge, MA, Cat. No. PC78). The anti-NFAT2 was obstained from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, Cat. No. SC-1149).
Isolation of primary rat Leydig cells
Adult Leydig cells were isolated from the 90-day-old rats according to the procedure of Sriraman et al. , which is a modification of the procedure described by Klinefelter et al. . The decapsulated testes were 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 and constantly agitated (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 down by centrifugation at 250 × g for 10 min at 25°C after diluting it with 3–4 volumes of the medium. The purities of the isolated cell fractions were evaluated by histochemical staining for 3β-hydroxysteroid dehydrogenase activity, with 0.4 nm etiocholanolone as the steroid substrate . The enrichment of the Leydig cells was up to a purity of 85% on average.
RNA isolation and RT-PCR
Total RNA was prepared from mLTC-1 cells using the Trizol Reagent (Gibco, Cat. No.15596-026) according to the manufacturer's instruction. First strand cDNA synthesis was performed using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, Cat. No. A3500) in a reaction using 2 μg of total RNA primed with random hexamers in a total reaction volume of 20 μl. Following first strand synthesis, 10% of the reaction volume was used as a DNA template for amplification by polymerase chain reaction (PCR). For detection of NFAT2, the forward and reverse primers were 5'-GCCCTGACCACCGATAGCAC-3' and 5'-GCTGCCTTCCGTCTCATAGTG-3', respectively. PCR was performed for 30–32 cycles with denaturing at 94°C for 1 min, annealing at 58°C for 1 min and extension at 72°C for 1 min, in a GeneAmp PCR System 9600 (PerkinElmer, Norwalk, CT, USA). For detection of FasL, specific oligonucleotide primers were used as follows: forward primers; 5'-ATTGGCACCATCTTTACT-3' and reverse primers; 5'-CCTTAGAATCTGTTTGTCC-3'). PCR was performed for 28–30 cycles with denaturing at 95°C for 50s, annealing at 58°C for 45s and extension at 72°C for 50s. The signal intensities of the amplified fragments of NFAT2 and FasL were normalized to β-actin using a densitometer. Amplified PCR products were separated by agarose gel electrophoresis and detected by ethidium bromide staining. For amplification of NFAT2, the forward and reverse primers were 5'- CCG GAA TTC GAC ATG ACG GGG CTG GAG CAG GAC CCG GAG -3'and 5'- CGC GGA TCC TCG TAA ATA AAA CAC CCT AGA AAG TTG AAA-3', respectively. PCR was performed for 34 cycles with denaturing at 94°C for 50s, annealing at 60°C for 45s and extension at 72°C for 120s. The NFAT2 cDNA was purified by QIAquick PCR Purification Kit.
Western blot analysis
mLTC-1 and primary rat Leydig 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 by the Bradford dye-binding assay using bovine serum albumin as a standard. Aliquots of cell extracts containing equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis on 8% gels using the Laemmli buffering system, then proteins were transferred to Immuno-Blot polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA) 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). Blots were exposed to primary antibodies for 1 h at room temperature, multiply washed with Tris-buffered saline with 0.1% Tween 20 (TBST), treated with secondary antibody (HRP-conjugated) for 1 h, and followed by a final series of washed with TBST. Primary (goat polyclonal anti-NFAT2 or rabbit polyclonal anti-FasL) and secondary antibodies were prepared in blocking buffer. Signals were detected with an Enhanced Chemiluminescence kit (ECL Pierce, Rockford, IL) and analyzed using Adobe Photoshop 6.0 software.
The following sequences specific for mouse NFAT2 gene were used: NFAT2 siRNA394 (Sense: 5'-GGA GGU GGA AGA CGU ACU UTT-3'; Antisense: 5'-AAG UAC GUC UUC CAC CUC CTT-3') and NFAT2 siRNA1232 (Sense: 5'-CCG UCA CAU UCU GGU CCA UTT-3'; Antisense: AUG GAC CAG AAU GUG ACG GTT-3'). The Lipofectamine 2000 was used to deliver siRNA (100 pmol) into cultured mLTC-1 cells (5 × 104) in 6-well plates according to the manufacturer's protocol. The double-strand scrambled siRNA (5'-UUC UCC GAA CGU GUC ACG UTT-3') were used as negative control. The cultures were incubated for six hours and then washed and incubated in fresh medium. All siRNA were obtained from Shanghai Genepharma Co. Inc. (Shanghai, China).
Genomic DNA was isolated from C57BL6 using DNAeasy kit (Qiagen, Valencia, CA, Cat. No.69504). A 689 bp PCR fragment corresponding to the promoter region of FasL (-618- +71) was generated using the upstream primer 5'-CGGGGTACCGTACCTCAGTTTTCATCTGGTGACCAGAAG-3' and the downstream primer 5'-CCCAAGCTTGCACCCAGCCCCAGGAAA GG-3' in a PCR using platinum Pfx high fidelity polymerase (Invitrogen, Cat.No. 11708013) and 50 ng of genomic DNA template. The resultant 689 bp fragment was gel-purified, and cloned into pGEM ®-T Easy vector (Promega, Cat.No. A1360) and sequenced for orientation and fidelity. This plasmid (pGEM-689pCD95L) was served as the source of the insert for further plasmid constructions. To make the -618 to +71 luciferase reporter plasmid, the 689 bp insert was excised with Kpn I and Hind III and ligated into the Kpn I/Hind III site of pGL3 Basic luciferase vector (Promega, Cat.No.E1751). To make the 5'truncation mutants, shorter fragments were generated using new upstream primers, and the common downstream primer was used to make the -618 to +71 insert. For the -258 to +71 fragment, the upstream primer 5'-CGGGGTACCCAGGCAAGCCTGGTTTACCAGC C-3' and the common downstream were used. To generate the -201 to +71 fragment, the upstream primer 5'-CGGGGTACCCGAAGACTTGTCGTCAGAA ATTTC-3' was used. The -167 to +71 fragment was produced using 5'-CGGGGTACCCTTCCTGGGGTT GCTGTGAGC- 3'. The -138 to +71 fragment was produced using 5'-CGGGGTACCGCTTCTCAGCTTC A ATGCAAG-3'. The -67 to +71 fragment was produced by using the upstream primer 5' -CCCAAGCTT GCACCCAGCCCCAGGAAAGG-3'. The resultant fragment were subcloned into the polylinker site of pGEM-T vector and sequenced for orientation and fidelity. Luciferase reporter constructs were produced by excising the various fragments with Kpn I and Hind III and then subcloning the fragments into the polylinker site of pGL3-basic.
NFAT2 cDNA sequences were digested with EcoRI and BamHI, and ligated into the EcoRI/BamHI site of pEGFP-C1 plasmids to construct the NFAT2-GFP fusion protein expression vector.
Mutant construct was generated by using the QuickChange Site-Directed Mutagenesis Kit. The mutation was verified by DNA sequencing. Mutant of the NFAT site in FasL promoter (FasL-P272m) were synthesized by Sangon Co.: 5'-GTCGTCAGAAATTTCTGGGAATC AACTTCCTGGGGTTGCTGTGAG -3'.
Transient transfections and luciferase reporter assays
For transfection analysis, luciferase assays were performed according to the manufacturer's instruction using the Lipofectamine™ 2000 reagent (Invitrogen Co. Cat. No.11668-019). 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, 0.1 μg of pRL SV40 reporter plasmid, and 2 μg of Lipofectamine™ 2000. Thirty-six hours following transfection, the cells were treated with 100 nM CORT. Following the treatment, the Dual Luciferase Assay was performed by lysing the cells in Passive Lysis Buffer, and reading the relative light units of one-fifth the lysate with both the firefly substrate and the renilla substrate using a luminometer (Monolight 2010, Analytical Luminescence Laboratory, SanDiego, CA). Each transfection was performed in triplicate and in a minimum of three independent experiments.
Confocal microscopy analysis of NFAT2-GFP nuclear translocation
mLTC-1 were transiently transfected with NFAT2-GPF expression vector. At 36 h after transfection, cells were treated with 100 nM CORT, or 100 nM CORT plus 100 ng/ml CsA for 12 h. NFAT2-GFP expression and localization was monitored by Confocal microscopy.
Chromatin immunoprecipitation assay
The chromatin immunoprecipitation (ChIP) assay was performed according to the manufacturer's protocol. mLTC, cultivated in DMEM containing 10% bovine calf serum, were incubated with 100 nM CORT for 12 h, cross-linked by formaldehyde for 10 minutes at 37°C. The reaction was stopped with 0.125 M glycine. Cells were washed using cold PBS containing protease inhibitors. DNA were sheared by sonicate lysate to lengths between 200–1000 bp. Cell samples were centrifuged for 10 minutes at 13,000 rpm at 4°C and supernatant stored at -80°C as chromatin extracts. For chip analysis, pre-cleared the sample with salmon sperm DNA/protein agarose for 30 min at 4°C. Lysates were subjected to immunoprecipitation with anti-NFAT2 antibodies (Santa Cruz Biotechnology) and non-specific IgG (Santa Cruz) were added individually and incubated at 4°C overnight with rotation. Bound and input chromatin samples were placed in 0.5% (wt/vol) SDS and incubated at 65°C for 4 hours to reverse the formaldehyde cross-linking. DNA was further purified by phenol-chloroform extraction and ethanol precipitated using glycogen as an inert carrier. Of the obtained DNA, 2 μl was used in a 20-μl PCR reaction (Taq DNA Polymerase; Qiagen) using the following primers: FasL-F, 5'-CAGTTAGCACAGAGACGCCAAT-3'; FasL-R, 5' -TGCTTCTCTGTGAGACACCCAC-3'.
mLTC-1 cells were treated with 100 nM CORT or 100 nM CORT plus 100 ng/ml CsA for 12 h followed by detection of apoptosis. The annexin-V was used to assess apoptosis. For the annexin-V assay, the mLTC-1 cells were incubated with 10 μL propidium iodide (PI) and 5 μL FITC-annexin-V (BD Biosciences, Franklin Lakes, NJ, USA) at room temperature for 15 min. Then, the cells were analyzed on FACS. Annexin-V binds to those cells that express phosphotidylserine on the outer layer of the cell membrane, and PI stains the cellular DNA of cells with a compromised cell membrane. This allows for the discrimination between live cells (unstained with either fluorochrome) from apoptotic cells (stained only with annexin-V) and necrotic cells (stained with both annexin-V and PI).
All experiments were performed in triplicate. The Student's t-test was used to determine the statistical significance of the data obtained. P < 0.05 or P < 0.01 was taken to represent a statistically significant difference between group mean.