Osteopathy and resistance to vitamin D toxicity in mice null for vitamin D binding protein (original) (raw)
Generation of DBP–/– mice. A fragment of the mDBP gene spanning exons 2–8 was isolated from a mouse 129 SV genomic library by cross-hybridization with a rat DBP cDNA probe (Fig. 1a). The 8.8-kb genomic insert was restriction-mapped and partially sequenced to confirm its identity and establish restriction sites for vector construction. A vector for homologous recombination was generated by cloning the PGK-neor cassette into the _Bam_HI site into DBP exon 5 and ligating a DTA cassette into intron 3 at the 5′ terminus of the targeting insert (Fig. 1a). Mouse ES cells were electroporated with the targeting insert, and surviving cells (i.e., those lacking the DTA cassette) were selected for resistance to G418. One hundred ninety-five surviving ES colonies were analyzed by Southern blotting (Fig. 1, b and c). A hybridization probe from exon 2 detected an 8.8-kb _Eco_RI fragment from the intact mouse DBP gene and a 5.7-kb _Eco_RI fragment from the disrupted locus. Of three ES cell clones with the disrupted DBP allele, a single colony, D1, was selected for subsequent use (Fig. 1c). D1 ES cells were expanded and microinjected into 3.5-day-old blastocytes from a C57Bl/6J mouse; five chimeric animals that ranged from 5% to almost 100% agouti coat color were obtained. The near-100% agouti chimeric animals produced only agouti offspring, 56% of which carried the DBP– allele. DBP−/+ animals were intercrossed; of the initial 177 offspring, 25% were wild type (DBP+/+), 49% were heterozygous (DBP+/–), and 26% were homozygous (DBP–/–) for the disrupted locus. This distribution, consistent with normal Mendelian inheritance, suggested that DBP–/– mice were of normal viability. Intercrosses of DBP–/– progeny resulted in normal frequency and sizes of litters, indicating normal fertility and fecundity of the DBP–/– males and females. DBP–/– mice appeared grossly normal, had growth curves identical to wild-type littermates, and showed no abnormalities after necropsy and histologic examination of all major organs (data not shown).
Targeted disruption of the mouse DBP locus. (a) A fragment of mouse genomic DNA containing exons 4–8 of the DBP gene was used to design the targeting vector. A PGK-promoter/neomycin phosphotransferase cassette was inserted at the _Bam_HI (M) site in exon 5 to disrupt the mDBP gene and provide for positive selection. A DTA cassette was ligated to the 5′ _Hin_dIII site for selection against random integration. (b) Restriction enzyme mapping distinguished the intact from the disrupted DBP allele. A mouse DBP exon 2 probe, located outside of the targeting vector itself, hybridized to an 8.8-kb _Eco_RI (R) fragment from the native DBP allele and a 5.7-kb _Eco_RI fragment from the disrupted mDBP allele. Restriction sites are: S, _Sal_I; H, _Hin_dIII; B, _Bgl_II; R, _Eco_RI; C, _Cla_I; M, _Bam_HI. (c) The targeting vector (a) was transfected into ES cells, G418 selection was applied, and surviving clones were analyzed by Southern blotting. Analysis of 8 representative ES cell lines among the 65 examined is shown. All eight clones contained the native 8.8-kb mouse DBP _Eco_RI fragment, and one clone (D1) also contained the 5.7-kb fragment, indicative of successful homologous recombination. DBP, vitamin D binding protein; DTA, diphtheria toxin A; ES, embryonic stem.
Verification of DBP-null state. Loss of expression from the disrupted DBP allele was confirmed by analysis of mRNA and protein. Northern blots of total liver RNA from DBP+/+, DBP+/–, and DBP–/– mice were generated. The expected 1.8-kb DBP mRNA was detected in the DBP+/+ liver, present, but at reduced levels, in DBP+/– liver, and totally absent from DBP–/– liver (Fig. 2a). Western blots of sera from animals of each genotype confirmed the reduction of DBP in DBP+/– mice and the total absence in DBP–/– mice (Fig. 2b). Analysis of DBP content in sera by radial immunodiffusion readily distinguished between the levels in the wild-type and carrier mice and confirmed the absence of immunoreactive DBP in DBP–/– mouse serum (Fig. 2c). The simple radial immunodiffusion assay was subsequently used for genotyping. The profiles of total serum proteins as determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) were otherwise identical in DBP–/– and DBP+/+ sera (not shown). Serum saturation binding analysis using tracer 25(OH)[3H]D3was performed to detect any bioactive fragments of DBP that might be produced by the mutant locus. No binding activity was detectable in the DBP–/– serum. The affinity constants were essentially identical in wild-type and heterozygous sera (_K_a = 1.0 × 10−9 M and 0.9 × 10−9 M) and comparable to that previously reported for hDBP (5 × 10−8 M). From the Scatchard plot, the maximum binding capacity of the DBP+/+ serum was estimated as 570 μg/l and for DBP+/– serum, 380 μg/l (67% of wild-type) (Fig. 2d). When sera were preincubated with 25(OH)[3H]D3 and subjected to a native gel electrophoresis (32), the DBP band was absent from DBP–/– sera and detected at reduced levels in DBP+/– sera compared with that of DBP+/+ animals (data not shown). These data demonstrated a complete absence of expression of immunologically or biologically detectable DBP in the DBP–/– mice. The data also suggested that heterozygotes have a minor upregulation of protein expression from the intact DBP allele.
Functional inactivation of the mouse DBP locus by homologous recombination. (a) An autoradiogram of the Northern blot analysis of total RNA from livers of DBP+/+, DBP+/–, and DBP–/– mice hybridized with rat DBP cDNA is shown. The full-length 1.8-kb mDBP mRNA was detected in wild-type mice, present at diminished levels in heterozygous mice, and totally absent in mice homozygous for DBP gene activation. Balanced RNA loading was confirmed by the ethidium bromide staining of 18S rRNA (bottom). (b) Western analysis for serum DBP in 12 mice representing each of the three genotypes is shown. The antibody was a cross-reacting, polyclonal rabbit antiserum to rat DBP. The presence and relative levels of the 58-kDa DBP paralleled the mRNA levels in panel a. (c) Semiquantitative radial immunodiffusion analysis of sera from mice of all three genotypes using the polyclonal antiserum confirmed the absence of DBP in DBP–/– mice. (d) Serum saturation binding analysis using tracer 25(OH)[3H]D3 in the presence of increasing concentrations of cold 25(OH)D3is shown. The data were further analyzed by Scatchard plotting (inset). 25(OH)[3H]D3, 25(OH)[26,(27)-methyl-3H]vitamin D3.
DBP−/− mice are vitamin D–depleted. The consequences of the DBP-null mutation on vitamin D levels and calcium homeostasis were determined. Serum levels of calcium, phosphorous, alkaline phosphatase, PTH, 25(OH)D, and 1,25(OH)2D were initially measured in DBP–/– and DBP+/+ littermates maintained on standard laboratory chow containing vitamin D. On this standard diet, DBP–/– mice had significantly lower total serum levels of 25(OH)D and 1,25(OH)2D than DBP+/+ mice (Fig. 3, a and b). Heterozygotes fell within the intermediate range in both assays. No significant differences in serum calcium, phosphorous, or alkaline phosphatase were observed between wild-type and DBP–/– littermates on a vitamin D–replete diet (Table 1), and the levels of PTH were equivalent for these two groups (Fig. 3c). Therefore, the low total serum sterol levels in the DBP–/– mice coexisted in equilibrium with adequate intracellular concentrations of 1,25(OH)2D under steady-state conditions, based on analysis of the PTH response.
Low serum 25(OH)D and 1,25(OH)2D levels and secondary hyperparathyroidism after mild dietary vitamin D deficiency in DBP–/– mice. Groups of DBP+/+, DBP+/–, and DBP–/– mice were fed either standard (vitamin D+) (a–c) or vitamin D–deficient (vitamin D–) (d–f) diets for 4 weeks. Serum 25(OH)D (a and d), 1,25(OH)2D (b and e), and PTH (c and f) levels were determined. Data displayed are the mean + SEM from 10 animals. The differences between DBP+/+ and DBP–/– groups were statistically significant in a, b (**P < 0.001), and f (*P < 0.01). PTH, parathyroid hormone.
Serum calcium, phosphorous, and alkaline phosphatase in wild-type and DBP–/– mice on standard and vitamin D–deficient diets
The mice were next stressed by placing them on a vitamin D–deficient diet for four to six weeks. The effects of this diet on calcium and vitamin D homeostasis were compared between the DBP–/– and wild-type littermates. After four weeks without exogenous vitamin D, the 25(OH)D and 1,25(OH)2D levels decreased in the wild-type mice to levels comparable to those of the DBP–/– animals, the lowest detection limits of these assays (Fig. 3, d and e). At this point, both groups became hypophosphatemic and alkaline phosphatase levels increased slightly (Table 1). Serum calcium was maintained at the same level in the DBP+/+ and DBP–/– groups. Although no differences between the two groups were noted in serum PTH levels while on standard diet (Fig. 3c), there was a doubling in the mean PTH levels in the DBP–/– group on the vitamin D–deficient diet (P < 0.05; Fig. 3f). Thus, the DBP–/– mice were more sensitive to dietary vitamin D deficiency than their normal littermates, selectively manifesting secondary hyperparathyroidism.
Bone histomorphometric analyses demonstrated an increased sensitivity of the bone in DBP–/– mice to vitamin D deficiency. Skeletal radiographs showed no discernible differences between DBP–/– and DBP+/+ mice (data not shown). Qualitative bone histological examination and quantitative histomorphometric analyses were carried out to detect bony abnormalities at a higher level of resolution. The bones of age- and sex-matched DBP–/– and DBP+/+ littermates were studied on standard diets and after eight weeks on vitamin D–deficient diets. The following end points were analyzed: femoral lengths, trabecular bone volume/tissue volume (BV/TV) in two planes, osteoid surface/bone surface (OS/BS), osteoclast surface/bone surface (OcS/BS), osteoclast number/bone surface (OCN/BS), mineralizing surface/bone surface (MS/BS), mineral apposition rate (MAR), and osteoid thickness (OTh). On standard diets, there were no significant differences between the DBP+/+ and DBP–/– groups in any of these end points (data not shown), nor was there any distinguishable difference in osteoclast numbers.
On the vitamin D–deficient diet, BV/TV was significantly lower in both the DBP+/+ and DBP–/– groups compared with values on the standard diet (8% vs. 4%, P < 0.01 for DBP+/+ groups; 12% vs. 4%, P < 0.05 for DBP–/– groups). This shared decrease confirmed the effectiveness of the vitamin D–deficient diet in altering bone metabolism without regard to the DBP status.
The remaining end points demonstrated a markedly increased and selective sensitivity of the DBP–/– mice to bone changes caused by vitamin D deficiency. OS/BS of the DBP–/– group was significantly higher on the vitamin D–deficient diet (from 3.3% to 8.8%, P < 0.001, male animals only; data not shown), whereas the OS/BS in the DBP+/+ group was not influenced by diet. A comparison between the two groups after vitamin D deprivation showed the net result of this differential effect (Fig. 4c; P < 0.05). The accentuated thickening of the osteoid seams in the DBP–/– mice, reflecting osteoblast activity and mineralization, could be appreciated qualitatively in a visual comparison of the Masson's trichrome–stained sections (Fig. 4, a and b; arrowhead). This selective effect in the DBP–/– mice was confirmed quantitatively by measurement of the mean OTh (Fig. 4d; P < 0.001). A significant and selective difference between the DBP–/– and DBP+/+ mice was also noted in the lower MAR and MS/BS in the DBP–/– mice (Fig. 4, e and f; P < 0.05 and P < 0.01, respectively). When fed a vitamin D–replete diet, neither DBP+/+ nor DBP–/– mice had skeletal abnormalities. However, when fed a vitamin D–deficient diet, the DBP–/– group demonstrated abnormally high osteoblastic activity, with undermineralization of the newly synthesized matrix compared with the matched DBP+/+ group. Thus, the DBP–/– mice demonstrated an increased sensitivity to dietary vitamin D deprivation by developing hypovitaminosis D osteopathy.
Bone mineralization defect after mild dietary vitamin D deficiency in DBP–/– mice. Sections of femurs from age- and sex-matched groups of vitamin D–deficient (vitamin D−) DBP+/+ and DBP–/– mice were stained with Masson's trichrome. Representative photomicrographs from a DBP+/+ (a) and a DBP–/– (b) mouse are shown. Osteoid seams were characteristically thicker in the DBP–/– group (arrowhead). This difference was not observed in mice fed vitamin D–sufficient chow. (c–f) Quantitative histomorphometric analyses of mice on vitamin D–deficient diets demonstrated significant abnormalities in OS/BS (male animals, n = 5; trend similar among females, but not significant; c) and osteoid thickness (both sexes compared, n = 10; d) in DBP–/– mice. The bones of mice from both groups were labeled by two injections of the fluorochrome (calcein) at a 7-day interval, and bone sections were subjected to quantitative histomorphometric analyses to determine the amount of mineralization during this period. The MAR (both sexes compared, n = 7, 8; e) and the MS/BS (both sexes compared, n = 6; f) were indicative of a quantitative mineralization defect in DBP–/– mice. DBP+/+ and DBP–/– mice maintained on normal diets showed no significant differences in any of these parameters (not shown). MAR, mineral apposition rate; MS/BS, mineralizing surface/bone surface; OS/BS, osteoid surface/bone surface.
DBP increases the serum half-life of vitamin D and 25(OH)D. DBP is the main carrier of vitamin D in the serum and as such has been proposed to play a role in plasma clearance of 25(OH)D (33). To directly establish the role of DBP in the clearance and distribution of 25(OH)D, DBP+/+ and DBP–/– mice were subjected to four weeks of vitamin D deprivation sufficient to generate serum 25(OH)D deficiency in both groups (Fig. 3, d and e). This period was followed by intravenous injection of tracer amounts of 25(OH)[3H]D3 previously incubated with homologous serum (see Methods). Animals were sacrificed over short (40 minutes) and long (24 hours) time courses, and plasma was collected and counted. In DBP+/+ mice, 25(OH)[3H]D3 was gradually cleared from plasma with ∼15% of the injected isotope still present after 24 hours (Fig. 5a). In contrast, the half-life of 25(OH)[3H]D3 in the plasma of the DBP–/– mice was markedly shortened, with a very low level detected in the circulation several minutes after injection (Fig. 5a and inset).
[![Accelerated clearance of 25(OH)[3H]D3 from the plasma of DBP–/– mice. (a) 2](https://dm5migu4zj3pb.cloudfront.net/manuscripts/5000/5244/small/JCI9905244.f5.gif)
](/articles/view/5244/figure/5)Figure 5
Accelerated clearance of 25(OH)[3H]D3 from the plasma of DBP–/– mice. (a) 25(OH)[3H]D3 was preincubated with aliquots of serum from either DBP+/+ or DBP–/– mice, and these were injected intravenously into mice of homologous genotype. Plasma was sampled at the indicated times after injection, and tritium counts were obtained. Data were normalized to the calculated total plasma volume and expressed as a percentage of total cpm injected. Data for the time interval from 0 to 24 h represent the mean ± SEM of five replicate experiments, and data in the inset depict the mean ± SEM of four experiments examining the 0–40-min time interval. (b) 25(OH)[3H]D3 was preincubated with aliquots of DBP+/+ or DBP–/– serum and injected intravenously into mice of the same respective genotype. Urine was collected for 24 h using metabolic cages and was counted. Data are the mean ± SEM of six determinations (P < 0.01). (c) Aliquots of plasma from one of the 0–40-min studies in a were extracted in organic solvent and analyzed by TLC. Data were expressed as a percentage of total cpm chromatographed. The percentage of cpm migrating in the 25(OH)D region (left) and the polar region (right) of the chromatograph for each time point were plotted.
The fate of the rapidly cleared 25(OH)[3H]D3 from the plasma of the DBP–/– mice was investigated. Urine was collected from the DBP+/+ and DBP–/– animals for 24 hours after intravenous injection of the tracer 25(OH)[3H]D3. Threefold more isotope was excreted into the urine of the DBP–/– group than the wild-type controls (Fig. 5b). Total urine 25(OH)D content was specifically quantified by radioimmunoassay (RIA) as well. A 2.4-fold greater clearance was observed over a 24-hour period in the DBP–/– mice (DBP+/+, 149 pg/24 hours; DBP–/–, 361 pg/24 hours). Aliquots of plasma harvested at each time point of the short time course (Fig. 5a and inset) were extracted and fractionated by TLC. The percentage of total plasma tritium migrating at either the 25(OH)D position or at the more polar end of the chromatography media was plotted (Fig. 5c). Ninety-five percent of the injected 25(OH)[3H]D3 remained in the 25-hydroxylated form, even at 40 minutes, in the wild-type animal, whereas only 46% remained in this form in the DBP–/– animals. In the DBP–/– mouse, 13% of the plasma tritium migrated as more polar metabolites at 40 minutes in contrast to 2% in the wild-type animal. These data suggested that in the absence of DBP, the injected 25(OH)D was being more rapidly metabolized and excreted in the urine in the absence of DBP.
The role of DBP in determining the kinetics and hepatic uptake of vitamin D has received less study. In vivo, it is known that some vitamin D is presented to the liver on low-density lipoprotein (LDL) and chylomicron remnants, and some redistributes to other plasma carriers, including DBP, albumin, and other lipoproteins (33). To determine the importance of DBP in vitamin D serum transport and metabolism, DBP+/+ and DBP–/– mice were fed vitamin D–deficient diets for four weeks. Each mouse was then injected intravenously with [3H]vitamin D3 that had been preincubated in homologous serum. These animals were sacrificed over a period of 40 minutes, plasma was collected, and livers were homogenized. The clearance of [3H]vitamin D3 from the DBP–/– plasma was substantially more rapid than from the DBP+/+ plasma (Fig. 6a). A reciprocal pattern of [3H]vitamin D3 uptake was seen in the livers. Between 10 and 20 minutes after injection, tritium counts were 14–15-fold higher in the livers of the DBP–/– animals than in the DBP+/+ controls (Fig. 6b). By 40 minutes after injection, the level of tritium in the DBP–/– livers fell to a value indistinguishable from the DBP+/+ controls. This pattern suggested that the accelerated transfer of [3H]vitamin D3 from the serum to the liver of the DBP–/– mice was followed by egress of isotope from the liver. This accelerated entry into the liver appeared to be an effect specific to this organ because there was less isotope detected in the kidney parenchyma of DBP–/– animals at all time points (not shown).
[![Accelerated entry of serum [3H]vitamin D into the liver and its conversion](https://dm5migu4zj3pb.cloudfront.net/manuscripts/5000/5244/small/JCI9905244.f6.gif)
](/articles/view/5244/figure/6)Figure 6
Accelerated entry of serum [3H]vitamin D into the liver and its conversion to polar metabolites. [3H]vitamin D3 was preincubated with aliquots of DBP+/+ or DBP–/– serum and injected intravenously into mice in the respective groups. Plasma samples (a) and livers (b) were harvested at the indicated times after injection, and tritium counts were obtained. Data were expressed as a percentage of total cpm injected normalized to total plasma volume (P < 0.05 at 20 and 40 min; a) or per gram of liver (b), and represent the mean ± SEM from three independent experiments. (c) Aliquots of plasma from the 1-min time point in a were extracted and subjected to TLC. The position of a 25(OH)D standard was localized by ultraviolet visualization, and the percentage of total chromatographed cpm migrating in the 25(OH)D region was plotted. (d) The percentage of total chromatographed cpm migrating in the polar region was plotted. The data are the mean ± SEM of two to three independent experiments.
The accelerated rate of transfer of vitamin D3 from the serum to the liver in the DBP–/– mice might be paralleled by the hepatocyte-mediated 25-hydroxylation to 25(OH)D or conversion of the vitamin D to water-soluble inactive esters that could then be excreted (34). To determine which pathway was being used, aliquots of plasma from the one-minute time points of each [3H]vitamin D3 kinetic experiment were extracted and subjected to TLC. One minute after injection, a mean of 32% of the total plasma tritium migrated at the 25(OH)[3H]D position on thin-layer chromatographic analysis in the wild-type animals compared with 7.5% in the DBP–/– animals (Fig. 6c). At the same time point, almost three times more polar metabolites were detected in DBP–/– mice than in DBP+/+ mice (29% and 11%, respectively). Thus, the accelerated entry of vitamin D into the liver in the absence of DBP appeared to result in the shunting of a substantial proportion of the substrate into an inactivating pathway or pathways. The relative contributions of liver and other tissues to this pathway cannot be determined from the present data.
The toxic response to high-level vitamin D administration was decreased in the absence of DBP. The profile of serum clearance and hepatic modification of vitamin D seen in the DBP–/– mouse suggested that DBP was necessary for efficient hepatic 25-hydroxylation and subsequent activation. This observation would suggest that the biologic activity of administered vitamin D would be significantly blunted in DBP–/– mice. This result would contradict the prediction from the free-hormone hypothesis that the lack of serum DBP in the DBP–/– mouse would accentuate the effects of a given dose of vitamin D due to absence of its putative buffering action (13). To differentiate between these two predictions, the effect of a sublethal dose (1000 IU/g body weight) of vitamin D3 was compared between wild-type and DBP-deficient mice. There were no significant differences in weight loss between the two groups over a one-week period of observation after delivery of vitamin D (data not shown). At day 7, the animals were sacrificed, serum was collected, and kidneys were harvested. The large dose of vitamin D resulted in the expected increase in serum calcium levels in the DBP+/+ mice compared with control mice injected with vehicle only. Remarkably, the DBP–/– mice responded to the same dose of vitamin D with a significantly less pronounced increase in serum calcium (Fig. 7a); von Kossa staining of kidneys confirmed the greater effect of the vitamin D dose on serum calcium levels in the DBP+/+ mice by demonstrating correspondingly more significant accumulation of calcium deposits in the cortices of the DBP+/+ kidneys (Fig. 7, b_–_e). Thus, the lack of serum DBP resulted in relative protection of the DBP–/– mice from the toxicity of high-dose vitamin D administration.
Relative resistance to vitamin D3 toxicity demonstrated by DBP–/– mice. DBP+/+ and DBP–/– mice were injected with toxic doses of vitamin D or with vehicle alone. (a) Serum calcium levels were determined 7 days after injection, and results are expressed as a percentage of the serum calcium in the vehicle-injected control groups. The differences were significant in both comparisons: DBP+/+ group (P < 0.001) and DBP–/– group (P < 0.01) compared with vehicle (not shown), and the calcium increase in the DBP+/+ group compared with the DBP–/– group (P < 0.05, shown). Seven days after injection, kidney sections were fixed and stained with von Kossa to detect calcium deposits. Representative kidney sections from DBP+/+ mice injected with vehicle (b) or vitamin D (c), and DBP–/– mice injected with vehicle (d) or vitamin D (e) are shown (×10). The arrows (c and e) point to calcium deposits in the renal cortex, and these regions are shown in the insets (×20).
1,25(OH)2D-dependent gene induction is more rapid in the absence of DBP. DBP–/– mice were more susceptible to vitamin D deficiency and were relatively resistant to vitamin D toxicity (Figs. 3 and 7). This observation could imply that the bioactivity of 1,25(OH)2D is in some way dependent on DBP. The importance of DBP to the delivery of biologically active 1,25(OH)2D to target tissues and the activation of gene expression was tested. Induction of calbindin-D9K mRNA in the proximal small intestine is dependent on 1,25(OH)2D. The steady-state levels of proximal intestinal calbindin-D9K mRNA were determined in vitamin D–deficient DBP+/+ and DBP–/– mice 24 hours after intravenous injection of 1,25(OH)2D. Before injection (t = 0), proximal intestinal levels of calbindin-D9K mRNA were identical (Fig. 8, a and b). Immediately after injection of 1,25(OH)2D, there was a transient depression in normalized calbindin-D9K mRNA levels in both groups. This period was followed by a progressive increase to a peak value and a subsequent fall toward baseline values. The overall maximal levels at the peak of induction were similar in both groups, although the timing was different. In the DBP+/+ animals, calbindin-D9K mRNA levels reached the maximum response 12 hours after 1,25(OH)2D injection. This kinetic response was in agreement with that previously reported in wild-type mice by others (35). In contrast, the calbindin-D9K mRNA levels in the DBP–/– mice reached a similar maximal response at eight hours (Fig. 8, a and b). These kinetic differences were statistically significant and reproducible over four experiments. Thus, DBP slowed the kinetics but not the overall level of calbindin-D9K gene expression in response to induction by 1,25(OH)2D.
Accelerated activation of calbindin-D9K gene expression by 1,25(OH)2D in DBP–/– mice. (a) Vitamin D–deficient DBP+/+ and DBP–/– mice were injected intravenously with 50 ng 1,25(OH)2D3. Animals were sacrificed at the indicated times after injection, and RNA was isolated from the most proximal centimeter of small intestine. The RNA was analyzed by Northern blots hybridized with 32P-labeled calbindin-D9K and [32P]rpL32 (loading control) probes. A representative autoradiogram is shown. (b) Relative band intensities were quantitated by PhosphorImager and were normalized for RNA loading. The data presented are expressed as a percentage of the maximal calbindin-D9K mRNA levels in response to 1,25(OH)2D3. The mean ± SEM from four independent experiments is shown.