Cell Culture and Transfection
Chinese hamster ovary (CHO)-K1 (ATCC CCL-61) were grown in Iscove's modified Dulbecco's medium (IMDM; Sigma-Aldrich, St. Louis, MO), 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were transiently transfected for 24 h with cDNA constructs in complete medium using LipofectAMINE 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Stably expressing cells were generated by transfection with the ORP1L/pcDNA3.1 construct using LipofectAMINE 2000 followed by geneticin G418-sulfate selection (400 μg/ml IMDM) (Invitrogen) according to Invitrogen's protocol.
Isolation of Monocytes and Their Differentiation into Macrophages
Mononuclear cells were isolated from blood of healthy normolipidemic donors (thrombopheresis residues). Monocytes isolated by Ficoll gradient centrifugation were suspended in RPMI 1640 medium containing 40 mg/ml gentamicin, 0.05% glutamine (Sigma-Aldrich), and 10% of pooled human serum. Differentiation of monocytes into macrophages occurred spontaneously by adhesion of cells to the culture dishes. Mature monocyte-derived macrophages were used for experiments after 8 d of culture, whereas experiments in monocytes were done using cells isolated after 45 min of adherence to the plastic dish in RPMI 1640 medium containing gentamicin and glutamine.
A full-length ORP1L cDNA was created by combining overlapping ORP1L 5′-end and 3′-end cDNA fragments. One microgram of total RNA from human fetal brain was reverse transcribed with the Superscript II enzyme (Invitrogen) according to the manufacturer's instructions by using primers ORP1N-R (5′-TCCAGTGCTTCTGACAAGATTTTG-3′) and ORP1L-R (5′-ACTCGGATCCGTAGATTAGCCAAACACCCTGAC-3′) for the 5′-end and 3′-end fragments, respectively. The 5′-end cDNA fragment was amplified using ORP1N-R cDNA as template. The 3′ end was amplified using ORP1L-R cDNA as template. All polymerase chain reactions (PCR) were carried out with 2.5 U of Pfu Turbo DNA-polymerase (Stratagene, La Jolla, CA) in a reaction volume of 20 μl containing 2–5 μl of template cDNA, 10× cloned Pfu DNA polymerase reaction buffer, 200 μM dNTPs, 10 pmol of each primer, and 10% dimethyl sulfoxide. The thermal cycling protocol applied for all reactions consisted of denaturation at 94°C for 5 min followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 3 min, and a final extension at 72°C for 10 min. The PCR products were cloned into pcDNA3.1 (Invitrogen) and transformed into Escherichia coli JM109. The cloned sequences were verified with a cycle-sequencing kit (BIGDYE; Applied Biosystems, Foster City, CA) and an automated ABI377 sequencer. The N-terminal fragment was subsequently ligated using a unique XbaI site in the overlap region into the C-terminal construct, generating the full-length ORP1L construct. The full-length ORP1L/pcDNA3.1 plasmid was used as template for the amplification of all truncated constructs except the ankyrin repeat (ANK) + PHD construct, which was generated by removing a 3′-end fragment by XbaI digestion of ORP1L. Oligonucleotide primers for the generation of cDNA constructs are listed in Table . EGFP-Rab5 and -Rab7 expression constructs were kindly provided by Dr. Marino Zerial (Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany) and Dr. Angela Wandinger-Ness (Department of Pathology, University of New Mexico Health Sciences Center, Albuquerque, NM).
Oligonucleotide primer sequences for amplification of ORP1 cDNAs
A glutathione S
-transferase–ORP1 fusion protein corresponding to amino acids 428–950 in the ORP1L protein was expressed in E. coli
BL21(DE3), purified by affinity chromatography on glutathione-Sepharose 4B (Pharmacia AB, Uppsala, Sweden), and used for immunization of HsdRIVM ELCO rabbits according to a standard protocol. The antibody (R250) was affinity purified in two steps. First, the ORP1 antiserum (1:5 dilution in phosphate-buffered saline [PBS]) was absorbed using glutathione-Sepharose 4B carrying glutathione S
-transferase–ORP2 (Laitinen et al., 2002
). Then, the unbound fraction was incubated with a Sepharose 4B column (Pharmacia AB) to which ORP1 (aa 428–950) had been coupled. The antibodies were eluted with 0.2 M glycine pH 2.8, neutralized and dialyzed against PBS. This antibody reacts with both ORP1S and ORP1L. A multiple antigenic peptide (Invitrogen) spanning amino acids 118–137 of ORP1L (not present in ORP1S) was used for raising antibodies (R279) specific for ORP1L, which were affinity purified using a Sepharose 4B column with covalently coupled immunizing peptide. Expression of ORP1 inserts in vector pcDNA4/HisMax was detected with mouse monoclonal antibody against the Xpress epitope (Invitrogen).
CHO-K1 cells were fixed with 4% paraformaldehyde, 250 mM HEPES pH 7.4 for 30 min and permeabilized for 30 min with 0.05% Triton X-100 in PBS. Nonspecific binding of antibodies was blocked with 10% FBS/PBS for 30 min, after which cells were incubated with ORP1 antiserum or anti-Xpress antibody in 5% FBS/PBS for 30 min at 37°C. The bound primary antibodies were visualized with tetramethylrhodamine B isothiocyanate (TRITC)-conjugated anti rabbit or TRITC-conjugated anti-mouse secondary antibody (Immunotech, Marseille, France). Cells were mounted in Mowiol (Calbiochem, San Diego, CA) containing 50 mg/ml 1,4-diazocyclo-[2,2,2]octane (Sigma-Aldrich). The specimens were analyzed with a TCS SP laser scanning confocal microscope (Leica, Wetzlar, Germany). In some experiments, cells were treated with 0.05% saponin, 80 mM PIPES, 5 mM EGTA, and 1 mM MgCl2 pH 6.8, for 1 min on ice before fixation.
Cells cultured in complete IMDM medium containing G418-sulfate were fixed for 1 h at room temperature with 2.5% glutaraldehyde in 0.1 M phosphate buffer pH 7.2, postfixed with osmium tetroxide, dehydrated, and embedded in EMBed 812 (Electron Microscopy Sciences, Fort Washington, PA). Sections were cut horizontally and viewed with an EX200 electron microscope (JOEL, Tokyo, Japan).
Analysis of ORP1 Membrane Association
CHO-K1 cells were grown on 10-cm culture dishes and transfected with cDNA constructs for 24 h, after which cells were washed with PBS and scraped in 0.8 ml of 250 mM sucrose; 140 mM KCl; 10 mM HEPES pH 7.4; and 25 μg/ml each of chymostatin, leupeptin, antipain, and pepstatin A. The cells were disrupted by repeated passage through a 21-gauge needle and nuclei were pelleted by centrifugation for 10 min at 500 × g. Sucrose was added to the postnuclear supernatant to a final concentration of 2 M in a total volume of 2.0 ml, which was overlaid with 1.6 ml of 1.7 M sucrose and 0.4 ml of 0.8 M sucrose, 140 mM KCl, and 10 mM HEPES pH 7.4. The gradients were centrifuged in a SW 60 Ti rotor at 37 000 rpm for 20.5 h at 10°C. Fractions of 0.8 ml were collected, and proteins were precipitated with trichloroacetic acid and analyzed by Western blotting.
Proteins were resolved in 12.5% SDS-polyacrylamide gels and transferred to Hybond-C extra nitrocellulose (Amersham Biosciences, Piscataway, NJ). Nonspecific binding of antibodies was blocked with, and all antibody incubations were carried out in, 5% fat-free powdered milk in 10 mM Tris-HCl pH 7.4, 150 mM NaCl, and 0.05% Tween 20. The bound antibodies were visualized with horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad) and the enhanced chemiluminescence system ECL (Amersham Biosciences).
Isolation and Quantitation of Total RNA
Total RNA was isolated from freshly isolated monocytes (day 0) and differentiating macrophages (days 4 and 8) with TRIzol reagent (Invitrogen). For tissue-specific expression analysis, seven human total RNA samples were purchased from Stratagene: fetal brain (male, 17 wk), adult liver (male, 73 yr), adult kidney (male, 56 yr), adult skeletal muscle (male, 17 yr), adult heart (male, 52 yr), adult colon (male, 69 yr), and adult lung (male, pooled donors 40, 51, 56, and 68 yr). Contaminating genomic DNA was removed from RNA samples by DNase treatment. The RNA quantity was determined with the RiboGreen RNA quantitation kit (Molecular Probes, Eugene, OR) and a 1420 multilabel counter (PerkinElmer Wallac, Turku, Finland).
Quantitation of ORP mRNA Expression by Real-Time PCR
Before first-strand cDNA synthesis, contaminating genomic DNA was eliminated as described previously with the following modifications (Huang et al., 1996
). DNase treatment was performed in a total volume of 10 μl containing 1 μg of total RNA, 50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl2
, 1 U of RQ1 RNase-free DNase I, 5 μM random hexamers (Applied Biosystems), and 12 U of RNasin ribonuclease inhibitor. The reaction mixture was incubated at 37°C for 30 min, whereafter the DNase I was heat inactivated at 75°C for 5 min. The first-strand cDNA synthesis was initiated by adding a 10-μl reaction mixture containing 50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl2
, 20 mM dithiothreitol, 0.8 mM dNTP mix, 12 U of RNasin ribonuclease inhibitor, and 200 U of SuperScript II RNase H−
reverse transcriptase (Invitrogen). After incubation for 1 h at 42°C, the reaction was stopped by keeping the samples at 70°C for 15 min.
Real-time PCR was carried out with the Amplifluor universal amplification and detection system (Intergen, Purchase, NY). The PCR reaction was carried out in a volume of 20 μl containing 25–50 ng of cDNA, 20 mM Tris-HCl pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTP mix, 1 U of Platinum Taq DNA polymerase (Invitrogen), 10 pmol of Amplifluor uniprimer, 10 pmol of untailed specific primer, and 1–2 pmol of tailed specific primer. Each sample was run in triplicate. In addition to primers for 11 ORPs, OSBP, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes, specific primers were designed to detect the two alternatively spliced ORP1 variants (Table and Figure ). The PCR amplification and fluorescence signal detection were performed with the ABI Prism 7700 sequence detection system (Applied Biosystems). PCR was initiated with denaturation at 94°C for 4 min, followed by 50 cycles of denaturation at 94°C for 15 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s. The fluorescence signal was recorded during the annealing step.
Primers used for quantitation of ORP mRNA expression levels
Figure 1 Detection of the two ORP1 mRNA variants encoding ORP1L and ORP1S. The numbered boxes in the middle denote exons of the ORP1 gene. The bars consisting of black and gray boxes represent the two mRNAs; black indicates translated and gray nontranslated regions. (more ...)
To determine ORP mRNA expression levels during monocyte-macrophage differentiation, real-time PCR efficiency (E) values were determined for each target gene. Serial dilutions of cDNA corresponding to 100, 50, 25, 12.5, and 6.25 ng of total RNA were amplified as described above. Threshold cycle (CT
) values were standardized by using the same calibrator cDNA during all runs. The slope was determined by plotting CT
values against the logarithm of cDNA mass. E values for each target gene were calculated with the equation [E = 10(−1/slope)
] (Table ). The relative expression ratio (R) of the target gene was calculated as follows: R = (Etarget
). The expression levels were normalized for the total amount of RNA.
Tissue-specific Expression of ORP1L and ORP1S
Two DNA calibrators were generated for the quantitation of ORP1L and ORP1S expression in different tissues. ORP1L- and ORP1S-specific DNA templates were PCR amplified with the following primer pairs: ORP1L-F (5′-TGATTGCCTTAATCTCTTCACC-3′)/ORP1-R2 (5′-GCTTCCAGGGATAATGAATACACT-3′) and ORP1S-F (5′-GGTCCTCGGATCTGGCCCA-3′)/ORP1-R2 (Figure ). After PCR amplification, the ORP1L standard (1243 base pairs, predicted molecular mass of 768 kDa) and the ORP1S standard (1229 base pairs, predicted molecular mass of 759 kDa) were run in a 1% agarose gel, excised, and purified with QIAquick gel extraction columns (QIAGEN, Valencia, CA). Concentrations of DNA fragments were determined with the Picogreen dsDNA quantitation kit (Molecular Probes) and a 1420 multilabel counter (PerkinElmer Wallac). Concentrations of DNA standards and tissue-specific cDNA samples were optimized before quantification of ORP1 variants. Serially diluted DNA standards were used to generate a calibration curve. DNA standards (20, 4, 0.8, 0.16, and 0.032 fg of double-stranded DNA) and tissue samples (50 ng of total RNA transcribed into cDNA as described above) were PCR amplified with three sets of primers: ORP1L-F/ORP1-R (for detection of ORP1L mRNA), ORP1S-F/ORP1-R (for detection of ORP1S mRNA), and ORP1LS-F/ORP1-R (for detection of both ORP1L and ORP1S mRNA) (Figure ).
The transactivation assays were carried out essentially as described previously (Willy et al., 1995
). Twenty-four hours before transfection, COS-1, human embryonic kidney (HEK) 293, or HepG2 cells were seeded on 24-well plates in DMEM supplemented with 10% fetal calf serum at 5 × 104
cells/well. Transfection mixtures contained 50 ng of the luciferase reporter plasmid and 25 ng of LXRα and/or retinoid X receptor (RXR) expression plasmids, or 10 ng of a chimeric Gal4 construct containing the ligand-binding domain of LXRα, and (50–100 ng) of ORP1/pcDNA4HisMax expression plasmid. The expression plasmid for β-galactosidase, pSVβ-gal (50 ng), was added for normalization. Cells were transfected for 2 h by lipofection by using RPR-120535B (Aventis, Strasbourg, France) in serum-free medium. The medium was subsequently replaced by DMEM containing 0.2% fetal calf serum and cells were treated for 36 h with 10 μM 22(R
)-HC]; SigmaAldrich) or 0.1 μM synthetic LXR agonist T0901317 (Tularik, San Francisco, CA). Cell extracts were prepared and assayed for luciferase activity. Data were normalized using the β-galactosidase activity.