Animals and study design
Six 12 to 15-week-old male vitamin D receptor knockout mice (VDR-KO; B6.129S4-VDRtm1Mbd>/J; Jackson Laboratory, Bar Habor, USA) and six age-matched male wildtype (WT) mice (C57BL/6 J, Charles River, Sulzfeld, Germany) were fed a rescue diet containing 2% calcium and 1.25% phosphorus. Other basal components of the diet were (in g/kg) casein (200), sucrose (200), lactose (200), starch (49.5), coconut fat (200), soybean oil (10), cholesterol (1.5), cellulose (50), DL-methionine (2), vitamin and mineral mixture (87), containing 1,000 IU vitamin D3. All mice were kept individually in a room controlled for temperature (22 ± 2°C), relative humidity (50–60%) and a 12-h light, 12-h dark cycle. All mice were allowed free access to food and water. The experimental procedures described followed established guidelines for the care and handling of laboratory animals and were approved by the council of Saxony-Anhalt (approval number: 42502-5-34 MLU).
Prior to killing by decapitation under light anesthesia with diethyl ether all mice were food deprived for 10 hours. Blood was collected into EDTA tubes. Plasma was obtained by centrifugation at 1,500 g for 20 min and stored at -20°C. The small intestine (from pylorus to ileocecal valve) was completely excised and washed several times with cold NaCl solution (0.9%). Intestinal mucosa was harvested by scraping the surface of the small intestine. Obtained mucosa samples were snap-frozen in liquid nitrogen and stored at -80°C. The liver was excised and samples for lipid extraction were snap-frozen and stored at -80°C as well.
For isoelectric focusing (IEF), protein extracts of small intestinal mucosa of each mouse were prepared. Therefore, 20 mg of frozen small intestinal mucosa were mechanically disrupted (MPI FastPrep®24-System, MP Biomedicals LLC, Illkirch Cedex, France) in 200 μl of 50 mM Tris–HCl buffer (pH 7.5) containing protease inhibitor cocktail (1:100, Roche, Mannheim Germany). Crude homogenates were centrifuged for 15 min (16,000 g, 4°C), the supernatants were collected and protein concentrations were determined according to Bradford . For subsequent fluorescence labeling and IEF, the samples were pooled group-wise at equal protein amounts and the resulting protein solutions were cleaned using the ReadyPrep™ 2D Cleanup Kit (Bio-Rad, Munich, Germany) according to the manufacturer’s protocol. The resulting protein precipitate was resuspended in a 2D compatible buffer (7 M Urea, 2 M thiourea, 4% CHAPS, 30 mM Tris–HCl, pH 8.5). Protein concentrations of the resuspended solutions were measured in dilution to discriminate urea interferences.
Protein pools were labeled with fluorescence dyes using the Refraction-2D Kit (NH DyeAGNOSTICS GmbH, Halle (Saale), Germany) according to the manufacturer’s instructions. The internal standard consisted of homoequivalent amounts of protein from VDR-KO and WT mice. For analytical gels, 5 μg of labeled protein per animal group along with 5 μg of the internal standard were pooled and mixed with DeStreak rehydration buffer (GE Healthcare, Munich, Germany) containing 0.5% carrier ampholytes (pH 4–7, SERVA Electrophoresis, Heidelberg, Germany) and added to immobilized pH gradient strips (pH 4–7, 7 cm, linear, Bio-Rad, Munich, Germany) for passive sample loading overnight at room temperature. Preparative gels were loaded with 300 μg of total protein that was spiked with labeled protein for the subsequent matching process with analytical gels.
First dimension IEF was run on a Protean IEF Cell (Bio-Rad, Munich, Germany) followed by a two step equilibration process using equilibration buffer (50 mM Tris–HCl (pH 8.8), 6 M Urea, 2% SDS, 30% glycerol, bromophenol blue) supplemented with 2% DTT (step 1) or 2.5% iodoacetamide (step 2), and incubating the stripes for 15 min, respectively. Thereafter, proteins were separated using linear SDS-PAGE (12.5%) and fixed (50% ethanol, 10% acetic acid) for 1 h. The samples were processed in six replicates. For preparative gels, the fixation step was omitted and the fluorescence signal was recorded directly before staining with colloidal Coomassie blue . Fluorescence signal acquisition was achieved using a Typhoon Trio laser scanner (GE Healthcare, Munich, Germany). Gel analysis was performed with the Delta2D software (Decodon, Greifswald, Germany). Protein spots that showed a regulation factor of at least 2 between the two groups were considered for further analysis.
Protein identification by ESI-QTOF-MS/MS-analysis
Protein spots were excised from Coomassie-stained gels, washed, and digested with trypsin (Promega, Mannheim, Germany) in 10 μl of 10 mM ammonium bicarbonate (pH 8.0) overnight at 37°C. Peptides were extracted from gel pieces and injected into a nanoACQUITY UPLC system (Waters Co., Eschborn, Germany). 2 μl were injected via “microliter pickup” mode and desalted on-line through a symmetry C18 180 μm × 20 mm precolumn. The peptides were separated on a 100 μm × 100 mm analytical RP column (1.7 μm BEH 130 C18, Waters Co., Eschborn, Germany) using a typical UPLC gradient from 3.0 to 33.0% over 15 min and a flow rate of 600 nl/min. The mobile phases used were 0.1% formic acid in water and 0.1% formic acid in acetonitrile. The column was connected to a SYNAPT® G2 HDMS-mass spectrometer (Waters Co, Eschborn, Germany). The data were acquired in LC/MSE mode switching between low and elevated energy on alternate scans. Subsequent correlation of precursor and product ions can then be achieved using both retention and drift time alignment. Using ProteinLynx Global SERVER 2.5.2 data were processed and searched against the SwissProt database specified for Mus musculus using Mascot search engine of Matrix Science .
Western blot analysis
Standard western blot procedure was performed as described earlier . Small intestinal mucosa samples were prepared as described above and 30 μg of protein of each individual mouse were resolved by electrophoresis in 12% SDS-PAGE gels. Membranes were incubated with anti-67 kDa laminin receptor antibody (1:1000; Abcam, Cambridge, UK; ab133645) and anti-GAPDH antibody (1:1000; Cell Signaling, Boston MA, USA; #5174S) respectively and subsequently detected with secondary HRP-conjugated antibody (1:1000; anti-rabbit IgG, Cell Signaling) using ECL Prime western blotting detection reagent (GE Healthcare, Munich, Germany).
Real-time detection RT-PCR analysis
Total RNA was isolated from aliquots of mice small intestinal mucosa by peqGold Trifast™ reagent (Peqlab, Erlangen, Germany) according to the manufacturer’s protocol. cDNA synthesis and determination of mRNA abundance by real-time detection PCR (Rotor-Gene 6000, Corbett Research, Mortlake, Australia) were performed as described previously . Calculation of the relative mRNA concentrations was performed according to . PCR data were normalized to the reference genes hypoxanthine guanine phosphoribosyl transferase (HPRT) and peptidylprolyl isomerase A (Ppia). The following target-specific primers were used for real-time PCR analysis: peptidylprolyl isomerase A (NM_008907, Ppia), for, 5′-GTGGTCTTTGGGAAGGTGAA-3′, rev, 5′-TTACAGGACATTGCGAGCAG-3′; 37/67 kDa laminin receptor (NM_011029, Rpsa), for, 5′-CTTGACGTCCTGCAGATGAA-3′, rev, 5′-GGATTCTCGATGGCAACAAT-3′ and matrix metalloproteinase (MMP)-2 (NM_008610.2, Mmp2), for, 5′-GAGATCTTCTTCTTCAAGGAC-3′, rev, 5′-AATAGACCCAGTACTCATTCC-3′, β-actin (NM_007393, Actb), for, 5′- ACGGCCAGGTCATCACTATTG -3′, rev, 5′- CACAGGATTCCATACCCAAGAAG -3′. All other primer pairs were purchased from Sigma-Aldrich (http://www.kicqstart-primers-sigmaaldrich.com).
Liver lipids were extracted using a mixture of n-hexane and isopropanol (3:2, v:v) . Aliquots of lipid extracts were dried and dissolved in a small volume of Triton X-100 . Concentrations of total cholesterol and triglycerides were determined as described previously .
Data are presented as means ± standard deviation (s.d.). Means of VDR-KO and WT mice were compared by Student’s t-test using the Minitab Statistical software, version 13 (Minitab, State College, USA). Means were considered significantly different at p < 0.05.