All surgical procedures were approved by Utah State University Institutional Animal Use and Care Committee. White leghorn cockerels were obtained on the day of hatch (Privett Hatchery, Portales, NM) and raised on a commercially available vitamin D-replete diet (Nutrena Feeds, Murray, UT) generally for 3-7 weeks prior to experimentation. On the day of use, chicks were anesthetized with anhydrous ethyl ether (Fisher Scientific, Fair Lawn, NJ). The abdominal cavity was surgically opened and the duodenal loop was removed to ice-cold 0.9% saline solution and chilled for 15 min. The pancreas was excised from the duodenal loop and discarded. The duodenal loop was everted and rinsed three times in chilled saline solution.

The chick intestinal cells were isolated with citrate chelation media (96 mM NaCl, 27 mM Citrate Anhydrous, 1.5 mM KCl, 8 mM KH2PO4, 5.6 mM Na₂HPO₄, pH 5.0 - the acidic pH promotes viability and retention of morphology) [3,18]. The cells were collected by low speed centrifugation (500 x g, 5 min, 4ºC), and cell pellets were resuspended in a small volume of Gey’s balanced salt solution (GBSS, containing 119 mM NaCl, 4.96 mM KCl, 0.22 mM KH2PO4, 0.84 mM NaHPO₄, 1.03 mM MgCl2•6H₂O, 0.28 mM MgSO₄•7H₂O, 0.9 mM CaCl₂, pH 7.3). Aliquots of the cell suspension (0.4 ml) were pipetted into 35 mmplastic Petri dishes (Falcon, Scientific Products; Franklin Lakes, NJ) containing 1.5 ml of RPMI 1640 medium and antibiotics (100 units/ ml penicillin, 100 mg/ml streptomycin, Sigma Chemical Co, St. Louis, Mo). The cells were incubated overnight in the absence of serum at 37ºC with 5% CO₂/95% air to promote cell adhesion.

The following morning, media were replaced with 0.1% BSA in GBSS (GBSS-BSA, Bovine Serum Albumin, Sigma, St. Louis, MO) and cells treated either with vehicle (0.01% ethanol) or hormone for 15 sec to 60 min (15 sec, 30 sec, 7 min, 10 min, 15 min, 25 min, 30 min, 40 min, 50 min, 60 min). At the end of each time point, media were replaced with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA), 3% sucrose in PBS (phosphate buffered saline) and fixed for 30 min. After washing with 0.1% PBS-BSA, cells were incubated with 0.05% NaBH4 in PBS for 5 min to eliminate auto-fluorescence in the brush border, then washed and permeabilized with 0.15% Triton X-100 in PBS for 5 min for intracellular catalase activity, or without Triton X-100 for surface catalase activity. After washing with 0.1% PBSBSA, cells were overlaid with normal rabbit serum (JacksonImmuno Research, West Grove, PA) for 5 min and then washed. Coverslips were then overlaid with primary antibody Ab365 (a highly specific polyclonal antibody, Center for Integrated BioSystems, Logan, UT) for 30 min (1/1000 in 0.1% PBS-BSA), and then incubated for another 30 min (after addition of more PBS-BSA to prevent drying), washed, and then overlaid with fluorescently-tagged secondary antibody Alexa Fluor 594 (Jackson Immuno Research, West Grove, PA), excitation at 591 nm and emission at 614 nm; and Phalloidin (Sigma-Aldrich, St. Louis, MO) labeled with fluorescein isothiocyanate (with excitation at 495 nm and emission at 513 nm) for 30 min. Coverslips were then washed three times. After the final wash, the coverslips were placed over mounting media (10% 1 M Tris, 80% glycerol) on a microscope slide, and sealed for subsequent confocal microscopy analysis. A Nikon TE-200 microscope (BioRad) was used for confocal imaging. Images were collected with ZEN software, using a 60x oil immersion objective and further processed with ImageJ and Adobe Photoshop CS5.

The isolated intestinal cells described above were collected by centrifugation at 500×g, 5 min (4ºC), and resuspended in 30 ml of GBSS. 5 ml of the cell suspensions were combined with test substance in GBSS to give a final concentration of 0.01% ethanol, 6.5 nM 24R,25(OH)₂D₃ and 200 pM 24S,25(OH)₂D₃]. The cells were incubated for 10 minutes and 25 minutes, then 1 ml were removed to 10 ml of ice-cold PBS for cytoplasmic and nuclear extraction. The extraction procedure involves mixing with a series of detergents in the presence of protease inhibitors.

SDS-PAGE and Western blot analyses were used to determine immunoreactive levels of catalase in control and vitamin D treated cells. Protein was determined with the Bradford reagent (Bio-Rad, Hercules, CA) and then samples (5-30 µg/well) were separated on 8% (wt/vol) SDS-PAGE gels with 5% stacking gels. After separation on SDS-PAGE, proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P, Fisher Scientific) by the use of a TransBlot SD Semidry transfer cell (Bio-Rad.) and Western analyses were performed. To avoid nonspecific binding, the membrane was soaked for 1 hr at 37ºC in blocking solution (0.5% nonfat dry milk in phosphate buffered saline (PBS; 0.9% NaCl and 10 mM Na₂HPO₄, pH 7.4), followed by washing three times for 5 min each time with washing solution (0.1%(wt/vol) BSA in Tris-buffered saline (TBS; 0.9% NaCl in 20 mM Tris-HCl, pH 7.4), and incubation with primary antibody (Abcam Inc., Cambridge, MA) overnight at 4°C. After three additional washes, the membrane was incubated with secondary antibody (alkaline phosphatase-conjugated goat anti-rabbit IgG) in 1% BSA and 0.05% Tween 20 in TBS for 2 hr at room temperature and then washed as previously indicated. Immunoreactive bands were visualized with the chromogens 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium, and relative amounts of catalase were quantitated using Adobe Photoshop.

In initial experiments, catalase localization by confocal microscopy was determined by adding hormones before primary antibody Ab365. Chick intestinal cells were cultured in petri dishes, treated with 0.01% ethanol, 6.5 nM 24R,25(OH)₂D₃, 200 pM 24S,25(OH)₂D₃, or 300 pM 1,25(OH)₂D₃, concentrations that have been shown to be equivalent to circulating levels [19], for selected times (15 sec, 30 sec, 7 min, 10min, 15 min, 25 min, 30 min, 40 min, 50 min, 60 min), and fixed for confocal microscopy. Red staining depicts Alexa Fluor 594 fluorescence, which is indicative of catalase localization on the cell surface. Figure 1A shows shorter time points and Figure 1B shows longer time points. Control cells treated with 0.01% ethanol for selected times revealed obvious surface staining, with a low level inside the nucleus. These observations in this and subsequent experiments were reproduced in triplicate experiments. Figure 2 depicts results from experiments in which cells were treated with 24R,25(OH)₂D₃ for shorter (Figure 2A) and longer (Figure 2B) time points, in which there were obvious increases in nuclear staining (indicated by yellow arrows) as well as surface staining relative to controls. Figure 3 depicts results from experiments in which cells were treated with 24S, 25(OH)2D₃. for shorter (Figure 3A) and longer (Figure 3B) time points, in which there were slight increases in nuclear staining relative to controls, but to a much lesser extent compared to 24R,25(OH)₂D₃ treatment. Figure 4 depicts results from experiments in which cells were treated with 1, 25(OH)2D₃ for shorter (Figure 4A) and longer (Figure 4B) time points, in which there were no increases in nuclear staining nor surface staining relative to controls, since 1,25(OH)₂D₃ does not compete with 24R,25(OH)₂D₃ for binding to catalase. In a previous study, it was found that 24R,25(OH)₂D₃ is capable of decreasing phosphate absorption after a 1-h injection in vivo and 24S,25(OH)₂D₃ is capable of increasing phosphate absorption after a 5-h injection in vivo, suggesting that the inhibitory effect might be mainly performed by 24R,25(OH)₂D₃ [20]. One possible gene regulatory mechanism could be catalase binding to STAT3 [21]. Further study is required to see if STAT3 is a binding partner of catalase.

The question was raised as to the origin of the nuclear staining. In order to answer that, additional confocal microscopy experiments were undertaken to determine whether cell surface catalase was the source of steroid-mediated nuclear redistribution. In these experiments, primary antibody was first added to cells for 30 min, and subsequently incubated with vehicle, 1,25(OH)₂D₃, 24S,25(OH)₂D₃, or 24R,25(OH)₂D₃. Figure 5 depicts control cells treated with 0.01% ethanol for shorter (Figure 5A) and longer (Figure 5B) time points revealed obvious surface staining, but little inside the nucleus. As shown in Figure 6A, B, nuclear redistribution after 24R,25(OH)₂D₃ treatment did not occur, suggesting that ligand binding to cell surface catalase induced redistribution to the nucleus, but was blocked by the antibody. Similarly, treatment of cells with antibody first inhibited the effects of 24S,25(OH)₂D₃ (Figure 7A, and 7B), and as expected, no effect of 1,25(OH)₂D₃ treatment was found (Figure 8A and 8B).

As a further analysis, catalase staining intensity inside the nucleus was quantified with ImageJ software. Figure 9 depicts comparisons among ‘hormone first treatments’—in which cells were treated with hormone prior to antibody. There was a significant increase in the intensity the 24R,25(OH)₂D₃ treated group, while the other groups stayed the same. Figure 10 depicts a comparison among cells treated with antibody prior to hormone, or ‘antibody first treatments’. There was no obvious increase in the intensity among the four groups, indicating that the nuclear redistribution mediated by 24R,25(OH)₂D₃ is indeed from cell surface catalase.

An independent approach was taken to verify these findings. In these experiments, cells were treated with either vehicle, 24S,25(OH)₂D₃, or 24R,25(OH)₂D₃ for 10 and 25 min, collected by centrifugation, and resuspended in homogenization buffer for subcellular fractionation. Aliquots of P1 (nuclei, brush borders, and unbroken cells), P2 (peroxisomes, lysosomes, mitochondria, Golgi, and basal lateral membranes, and S2 (microsomes and cytosol), were subjected to SDSpolyacrylamide gel electrophoresis, followed by Western blotting. The results reproducibly showed more nuclear redistribution after 24R,25(OH)₂D₃ treatment compared to 24S,25(OH)₂D₃ (Figure 11). One possible explanation might be there was a higher concentration of 24R,25(OH)₂D₃ in the incubations than 24S,25(OH)₂D₃. However, the fact that redistribution from cell surface and organelles to the nucleus following 24S,25(OH)₂D₃ was detected by Western analysis indicates that confocal microscopy is the less sensitive method. The results shown in Figure 11 also indicate that the commercially available antibody is less specific than Ab 365 [17].