Involvement of Protein Kinase C and Cyclic Adenosine: MATERIALS AND METHODS
Dibutyryl cAMP ((Bu)2cAMP), U73122, 22(R)-hydroxycholesterol, H8Q, PMA, hCG, and Waymouth MB 752/1 medium were obtained from Sigma (St. Louis, MO). Fetal bovine serum (FBS), gentamicin, and horse serum were purchased from Invitrogen Life Technologies (Carlsbad, CA). GF-10Q203X (Bisindolylmaleimide I) and Go6983 were purchased from Calbiochem (San Diego, CA).
Cell Culture and Animals
R2C cells were grown in Waymouth MB 752/1 medium supplemented with 15% horse serum and 2.5% (v/v) FBS in the presence of 0.4% (v/v) gentamicin. MA-10 cells were grown in Waymouth MB 752/1 medium supplemented with 15% (v/v) heat-inactivated horse serum in the presence of 0.4% (v/v) gentamicin. Dr. Pulak R. Manna (Texas Tech University Health Sciences Center, Lubbock, TX) kindly provided the mLTC-1 cells. Primary cultures of immature rat Leydig cells were isolated as previously described. Experimental protocols using animals were approved by the Institutional Animal Care and Use Committee at Morehouse School of Medicine-Atlanta University Center, where these studies were conducted. The cell lines and primary Leydig cell cultures were incubated in 5% CO2 at 37°C.
Western Blot Analysis
Cells were cultured in 6-well plates in triplicate, incubated with different inhibitors for 15 min, and followed with stimulating or nonstimulating treatments. Total cell lysates were prepared in lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 2 mM PMSF, 2 |xg/ml of aprotinin, and 1% [v/v] Nonidet P-40) and assayed for protein content after sonication. Equal amounts of protein were analyzed by 12.5% SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were probed with specific antibodies recognizing total StAR (tot-StAR), cytochrome P450 17a-hydroxylase/C17-20 lyase (CYP17A1; herein called CYP17; kind gifts from Dr. Walter L. Miller, University of California, San Francisco, CA), phospho-StAR (P-StAR; generated against a peptide conjugated to keyhole limpet hemocyanin corresponding to amino acids 1Q0-1QQ of mouse StAR with Ser194 phosphorylated; obtained from Be-thyl Laboratories, Montgomery, TX), phospho-Ser/Thr PKA substrate (Cell Signaling Technology Inc., Beverly, MA), phospho-cAMP responsive element binding (P-CREB; Cell Signaling Technology) ortotal-CREB (tot-CREB; Cell Signaling Technology). A second rabbit anti-porcine CYP17 antibody was obtained from Dr. Dale B. Hales (University of Illinois at Chicago, Chicago, IL) [2Q]. After incubation with secondary antibodies (anti-mouse-horseradish peroxidase [HRP]-conjugated or anti-rab-bit-HRP conjugated), antibody binding was determined using enhanced chemiluminescence (Perkin-Elmer Life Sciences, Boston, MA) and exposure to x-ray film (Marsh Bio Products, Inc., Rochester, NY). The integrated optical density of the protein bands was quantified using the BioImage Visage 2000 (BioImage Corp., Ann Arbor, MI).
Northern Blot Analysis
Northern blot analysis for Star was performed as previously described. Briefly, total RNA was extracted from cells using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA). Total RNA (10 |xg) was loaded on a 1% agarose gel containing 5.2% (v/v) formaldehyde. Northern blot analysis of Star mRNAs was performed using the nonradioactive, North2South Hybridization kit (Pierce, Rockford, IL). Blots were hybridized with biotin-labeled mouse Star cDNA probes and exposed to x-ray film (Marsh Bio Products) for different lengths of time. After stripping the membrane, the biotin-labeled 18S (Rnl8s) cDNA probes were hybridized and analyzed for use as a loading control.
Semiquantitative Reverse Transcription-Polymerase Chain Reaction
Reverse transcription-polymerase chain reaction (RT-PCR) was performed using total RNA as described. Briefly, 2 |xg of total RNA was used for the RT reaction using avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI) at 37°C for 1.5 h. PCR reactions were performed using 2 |xl of RT reaction products with the addition of the PCR master mix containing dNTP, Taq DNA polymerase (Promega), [a-32P] dCTP (2 |xCi of 3000 Ci/mmol), and specific oligonucleotide primers for Star (forward, 5 ‘-TTCAAGCTGTGTGCTGGAAGCTCCTA-3′ [1641 base pairs]; reverse, 5 ‘-TTAACACTGGGCCTCAGAGGCAGGGC-3′ [830-855 base pairs]). The primers that served as a reaction control were for ribosomal protein L1Q mRNA (Rpl19) (forward, 5′-GAAATCGCCA-ATGCCAACTC-3′; reverse, 5′-TCTTAGACCTGCGAGCCTCA-3′). The expected sizes of Star and Rpl19 PCR products were 840 and 400 base pairs, respectively. Optimum conditions made during the exponential phase of the amplification procedure were established to assure measurements.
Dual Luciferase Reporter Assay
Various segments of the mouse Star promoter (—Q66, —254, —151, and —110) were previously cloned into the pGL3-Basic vector (Promega), thereby controlling expression of a reporter gene encoding firefly luciferase. MA-10 cells were plated in 12-well plates (1 X 105 cells/well) and transfected on the next day with 0.4 |xg of individual constructs using Effectene reagent (Qiagen, Valencia, CA) following previously optimized conditions in accordance with the manufacturer’s instructions. To correct for variations in transfection efficiency, 10 ng of pRL-SV40 encoding Renilla luciferase was cotransfected as a normalization control. On the second day of transfection, the medium was changed. On the third day, the cells were thoroughly washed and treated with various stimuli for 6 h in serum-free Waymouth media. To determine the level of luciferase activity, cells were washed and lysed with 100 |xl of passive lysis buffer provided with the Dual-Luciferase Reporter assay system (Promega) using a TD-20/20 Luminometer (Turner Designs, Sunnyvale, CA) following the manufacturer’s instructions. Firefly luciferase activity was measured by addition of 50 |xl of Luciferase Assay Reagent II to 10 |xl of cell lysates. Stop & Glo Reagent (50 |xl) was added to quench firefly luciferase activity and measure Renilla luciferase activity. Normalized relative luciferase units or activity was calculated as the ratio of firefly luciferase:Renilla luciferase activity obtained from the same cell lysates.
Steroid production was assessed with radioimmunoassay. Cells were cultured in duplicate, thoroughly washed, and treated in serum-free culture media for each treatment. The medium was recovered and progesterone or testosterone concentration was measured as described earlier. In most cases, steroid levels were normalized using total cellular protein or cell number and expressed as picograms per micrograms (pg/^g) of protein or nanograms per cell number
Each experiment was repeated at least three times using triplicate samples within each experiment. The data are expressed as the mean ± SEM. Statistical analyses were performed using the Student f-test (two-sample assuming unequal variances), and P values less than 0.05 were accepted as significantly different.