Cell culture. Osteosarcoma ROS 17/2.8 cells were cultured according to previously published protocols. Briefly, ROS 17/2.8 cells (kindly provided by M.C. Farach-Carson, University of Delaware) were grown in DMEM/Ham’s F-12 (50:50, v/v) supplemented with 10% fetal bovine serum and antibiotics. Cell cultures were kept in a humidified atmosphere containing 5% CO2 at 37°C. For patch-clamp experiments, cells were used within the first 4 days after each passage. Primary osteoblast cultures were established from 3-5 day old mouse calvaria as published before. Stripped parietal bones were fragmented (~1 mm2), washed several times in sterile HEPES buffer, and transferred to 35 mm Falcon culture dishes. Typically, osteoblasts migrated from the bone fragments and settled on the bottom of culture dishes after 1-2 days. Mature osteoblasts showing a typical polyhedral shape were used for patch-clamp recordings within the first 2-4 weeks in culture.
Vertebrate animals. We used the Tokyo VDR KO mice generated by targeted ablation of exon 2 at the University of Tokyo, and kindly provided by S. Kato. Heterozygous (+/-) mice were bred to generate (-/-) homozygous VDR KO mice, which were raised on a normal diet. Wild type heterozygous (+/-) and ho- mozygous (+/+) siblings were used as controls. Animals were genotyped using a 1-cm tail sample at 21-30 days of age. Newborn and adult mice were euthanized by decapitation and cervical dislocation, respectively, to isolate calvaria. Primary cells from bone samples were kept in culture for up to 2 months.
Electrophysiology. Whole-cell patch-clamp recordings were performed with a HEKA EPC-9 amplifier (ALA Scientific Instruments Inc., Westbury, NY) on individual osteoblasts. Patch pipettes of about 2 MQ were fabricated with a DMZ Universal micropipette puller from Drummond capillaries (Drum- mond Scientific Co., Broomall, PA), coated with Sylgard elastomer (Dow Corning Corp., Midland, MI) to reduce capacitative transients, and fire-polished. Ion channel activities were recorded in a bath (external) solution composed of (in mM): 150 TEA-Cl, 3.1 KCl, 20 BaCl2, 1 MgCl2, 4 NaHCO3, 10 Hepes, 20 sucrose, pH 7.4 (adjusted with TEA-OH). The pipette (internal) solution consisted of (in mM): 150 CsCl, 15 NaCl, 4 MgCl2, 5 EGTA, 10 HEPES, pH 7.4 (adjusted with NaOH). Whole-cell capacitance values were measured using the software-based lock-in implementation of Pulse (v.8, HEKA EPC- 9). We applied a sine wave with a frequency of 500 Hz and peak amplitude of 20 mV, which was superimposed on a holding potential of 0 mV. Whole-cell capacitance was continuously monitored for 10-20 min, sampling every 0.1-1 sec. Microscopy. Rat osteosarcoma ROS 17/2.8 cells were cultured on cover slips in 35 mm Petri-dishes for 48 hours. Cytoplasmic calcium signals were visualized in live, single osteoblasts with a laser scanning confocal Leica TCS SP2 Microscope (Leica Microsystems, Inc., Exton, PA). Cells were loaded with the calcium sensitive dye Fluo 3-AM (5 jM, Molecular Probes), for 30 min at 30°C. The raise in cytoplasmic Ca2+ concentration was detected as an increase in fluorescence intensity 1-3 min after the addition of 5 nM 1,25D to the bath.
For immunocytochemistry observations, ROS 17/2.8 cells were fixed with 3.7% (v/v) formaldehyde at room temperature for 20 min, and permeabilized with ice-cold ethanol for 5 min. We used a primary antibody against VDR (D-6, Santa Cruz Biotechnology CA) and a FITC-conjugated anti-mouse secondary antibody (Sigma Immunochemicals St. Louis, MO). Im- munostaining was visualized with an Olympus IX50 inverted fluorescence microscope. For SEM observations, cells were grown on glass cover slips for 5 days and fixed with 4% formalin (Sigma) in 0.1 M Na cacodylate buffer, for 1 hour at room temperature. Coverslips were then washed 3 times with 0.1 M sodium cacodylate. Samples were incubated with 1% osmium tetroxide in the same buffer, for 1 hour at room temperature. Dehydration was performed in an ethanol series (30%, 50%, 70%, 80%, 95%, and 100%), and samples were critical point dried. Samples were then covered by a thin gold/palladium layer, and observed on a Philips SEM XL-30 microscope.
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