Experimental procedures

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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