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Endocrinology, doi:10.1210/en.2007-1655
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Endocrinology Vol. 149, No. 6 3196-3205
Copyright © 2008 by The Endocrine Society

Active Intestinal Calcium Transport in the Absence of Transient Receptor Potential Vanilloid Type 6 and Calbindin-D9k

Bryan S. Benn, Dare Ajibade, Angela Porta, Puneet Dhawan, Matthias Hediger, Ji-Bin Peng, Yi Jiang, Goo Taeg Oh, Eui-Bae Jeung, Liesbet Lieben, Roger Bouillon, Geert Carmeliet and Sylvia Christakos

Department of Biochemistry and Molecular Biology (B.S.B., D.A., A.P., P.D., S.C.), University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103; Institute of Biochemistry and Molecular Medicine (M.H.), University of Berne, CH-3012 Berne, Switzerland; Department of Medicine (J.-B.P., Y.J.), University of Alabama, Birmingham, Alabama 35294; Laboratory of Cardiovascular Genomics (G.T.O.), Ewha Woman’s University, Seoul 120-750, Korea; Laboratory of Veterinary Biochemistry and Molecular Biology (E.-B.J.), College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk 361-763, Korea; and Laboratory of Experimental Medicine and Endocrinology (L.L., R.B., G.C.), Katholieke Universiteit Leuven, Leuven B-3000, Belgium

Address all correspondence and requests for reprints to: Dr. Sylvia Christakos, Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School E609, 185 South Orange Avenue, Newark, New Jersey 07103. E-mail: christak{at}umdnj.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To study the role of the epithelial calcium channel transient receptor potential vanilloid type 6 (TRPV6) and the calcium-binding protein calbindin-D9k in intestinal calcium absorption, TRPV6 knockout (KO), calbindin-D9k KO, and TRPV6/calbindin-D9k double-KO (DKO) mice were generated. TRPV6 KO, calbindin-D9k KO, and TRPV6/calbindin-D9k DKO mice have serum calcium levels similar to those of wild-type (WT) mice (~10 mg Ca2+/dl). In the TRPV6 KO and the DKO mice, however, there is a 1.8-fold increase in serum PTH levels (P < 0.05 compared with WT). Active intestinal calcium transport was measured using the everted gut sac method. Under low dietary calcium conditions there was a 4.1-, 2.9-, and 3.9-fold increase in calcium transport in the duodenum of WT, TRPV6 KO, and calbindin-D9k KO mice, respectively (n = 8–22 per group; P > 0.1, WT vs. calbindin-D9k KO, and P < 0.05, WT vs. TRPV6 KO on the low-calcium diet). Duodenal calcium transport was increased 2.1-fold in the TRPV6/calbindin-D9k DKO mice fed the low-calcium diet (P < 0.05, WT vs. DKO). Active calcium transport was not stimulated by low dietary calcium in the ileum of the WT or KO mice. 1,25-Dihydroxyvitamin D3 administration to vitamin D-deficient null mutant and WT mice also resulted in a significant increase in duodenal calcium transport (1.4- to 2.0-fold, P < 0.05 compared with vitamin D-deficient mice). This study provides evidence for the first time using null mutant mice that significant active intestinal calcium transport occurs in the absence of TRPV6 and calbindin-D9k, thus challenging the dogma that TRPV6 and calbindin-D9k are essential for vitamin D-induced active intestinal calcium transport.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
VITAMIN D IS the principal factor that maintains calcium homeostasis and is also required for bone development and maintenance (1). Previous studies have shown that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] is the major controlling hormone of intestinal calcium absorption (2, 3). Because the body’s demand for calcium increases from a diet deficient in calcium, from growth, or from pregnancy, the synthesis of 1,25(OH)2D3 is increased, stimulating the rate of calcium absorption (2, 3). However, an understanding of the molecular mechanisms responsible for 1,25(OH)2D3-dependent intestinal calcium absorption remains incomplete. Active intestinal calcium transport is transcellular and believed to be comprised of three component 1,25(OH)2D3-regulated steps: entry across the brush border membrane, intracellular diffusion, and extrusion across the basolateral membrane (2, 3, 4). It is thought that the calcium-binding protein calbindin, which is induced by 1,25(OH)2D3 in the intestine, acts to facilitate the diffusion of calcium through the cell interior toward the basolateral membrane (2, 3, 4, 5). Supporting this hypothesis for a role of calbindin in intestinal calcium absorption are findings observed in the vitamin D receptor (VDR) knockout (KO) mice. In these mice, the major defect is in intestinal calcium absorption, indicating that the small intestine plays the major role in 1,25(OH)2D3 action on calcium homeostasis (6, 7, 8). The defect in intestinal calcium absorption is accompanied by a 50% reduction in the expression of calbindin-D9k (9). However, previous studies have noted that the induction of calbindin-D9k does not always correlate with an increase in intestinal calcium absorption (10, 11, 12), suggesting that 1,25(OH)2D3 has multiple effects at various control points in the intestinal cell (2, 3).

In addition to the role of 1,25(OH)2D3 on transcellular movement of calcium, 1,25(OH)2D3 is also known to increase the rate of calcium entry into the intestinal cell (2, 3). However, only recently have the apical calcium channels in 1,25(OH)2D3-responsive epithelia been identified, suggesting a mechanism for calcium entry. In duodenum and jejunum, transient receptor potential vanilloid type 6 (TRPV6) has been shown to be colocalized with calbindin-D9k (13, 14, 15). Using 1,25(OH)2D3-depleted mice or mice lacking VDR, it has been shown that the expression of TRPV6 is regulated by 1,25(OH)2D3 (16, 17). In VDR KO mice, TRPV6 was found to be more markedly decreased in the intestine than calbindin-D9k, suggesting that the expression of TRPV6 may be a rate-limiting step in the process of vitamin D-dependent intestinal calcium absorption (17). In other studies, a similar regulation of TRPV6 and calbindin-D9k was observed (16). Both are markedly induced at weaning [the time of onset of active intestinal calcium transport and intestinal responsiveness to 1,25(OH)2D3], under low dietary calcium conditions, and after 1,25(OH)2D3 injection of vitamin D-deficient mice (16). After a single injection of 1,25(OH)2D3, TRPV6 mRNA is rapidly induced before the increase in calcium absorption (16). Although these previous studies are suggestive, the role of TRPV6 and calbindin-D9k in vivo in 1,25(OH)2D3-mediated transcellular calcium absorption and the functional relationship between TRPV6 and calbindin-D9k are not known. The generation of TRPV6 KO mice, calbindin-D9k KO mice, and TRPV6/calbindin-D9k double KO (DKO) mice makes possible for the first time in vivo studies of the role of TRPV6 and calbindin-D9k in 1,25(OH)2D3-regulated intestinal calcium absorption.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
45Ca (39.49 mCi/ml), {gamma}-[32P]deoxy-ATP (300 Ci/mmol), polyvinylidene difluoride membranes, prestained protein molecular weight markers, and chemiluminescent detection system were obtained from NEN Life Science Products (Boston, MA). Antiserum against purified rat calbindin-D28k was prepared as previously described (18). Antiserum against rat calbindin-D9k was obtained from Swant Swiss Antibodies (Bellinzona, Switzerland). Chemically synthesized 1,25(OH)2D3 was provided by Dr. M. Uskokovic of Hoffmann-La Roche (Nutley, NJ). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise specified below.

Animals
The TRPV6 KO mice and calbindin-D9k KO mice were generated as previously described by Bianco et al. (19) and Lee et al. (20), respectively. The TRPV6/calbindin-D9k DKO mice were generated by breeding TRPV6 KO female mice with calbindin-D9k KO male mice for generation of double-heterozygous mice, which were subsequently bred to obtain the TRPV6/calbindin-D9k DKO mice. Mice were maintained in a virus and parasite-free barrier facility and exposed to a 12-h light, 12-h dark cycle. Food and water were given ad libitum. Both TRPV6 and calbindin-D9k KO mice have the same background strain (B6/129). Initial studies were done using littermates. KO mice were subsequently backcrossed for multiple generations (six or greater). All of the animal experiments conducted were approved by the University of Medicine and Dentistry of New Jersey Animal Care and Use Committee.

Experimental design
For in vivo experiments, mice were fed a standard rodent chow diet (Rodent Laboratory Chow 5001; Ralston Purina Co., St. Louis, MO) ad libitum from birth and were killed at 3 months of age. Blood was collected, and serum was prepared for analysis of calcium and phosphorus levels. Tissues were harvested and RNA and protein were isolated as previously described (16, 21, 22) for PCR and Northern and Western blot analyses. Duodenum and ileum of mice under the same protocol were also used for the determination of intestinal calcium transport using the everted gut sac assay. TRPV6 is present in duodenum and jejunum, but not in ileum (14). The highest levels of TRPV6 are in the duodenum. Thus, duodenum was used for these studies and ileum was used for comparison.

For dietary studies, 4-wk-old mice were fed either a high-calcium (1% Ca, 2200 IU vitamin D2/kg; Teklad diet 92309) or low-calcium (0.02% Ca, 2200 IU vitamin D2/kg; Teklad diet 86162) diet for 4 wk. Blood was collected. The serum 1,25(OH)2D3 levels of the high-calcium-fed mice were WT, 62.6 ± 10; TRPV6 KO, 115.8 ± 25; calbindin-D9k KO, 84.5 ± 14; and DKO, 169 ± 13 pg/ml. Tissues were harvested, and RNA and protein were isolated as previously described (16, 21, 22). Duodenum and ileum of mice under the same protocol were also used for the determination of intestinal calcium transport using the everted gut sac assay.

In another study, 12-wk-old mice were fed a 0.8% strontium, 0.02% calcium, vitamin D-deficient diet (Teklad diet 00562) for 7 d to inhibit endogenous renal synthesis of 1,25(OH)2D3 and to ensure functional vitamin D deficiency (23, 24). The serum calcium levels of the strontium-fed mice were less than 8.7 mg/dl. Vitamin D-deficient mice were then randomly separated into two groups and injected with either vehicle or 1,25(OH)2D3 three times over the next 48 h (48, 24, and 6 h before being killed; 100 ng/100 g body weight in 0.1 ml of a 9:1 mix of propylene glycol and ethanol) before termination. The three-dose protocol was used to study both short-term and long term effects of 1,25(OH)2D3 administration. The average serum calcium of 1,25(OH)2D3-treated mice was 10.2 ± 0.2 mg/dl. Null mutant and WT mice responded similarly to 1,25(OH)2D3 treatment (there was no significant difference among all 1,25(OH)2D3-treated groups (P > 0.1, comparison of multiple group means). Blood was collected. Tissues were harvested, and RNA and protein were isolated as previously described (16, 21, 22). Duodenum and ileum of mice under the same protocol were also used for determination of intestinal calcium transport using the everted gut sac assay.

The 12- to 14-month-old mice were also fed either a high-calcium (1% Ca; Teklad diet 92309) or low-calcium (0.02% Ca; Teklad diet 86162) diet for 4 wk. Duodenum and ileum of these mice were used for determination of intestinal calcium transport using the everted gut sac assay.

Intestinal calcium transport
Intestinal calcium transport was determined by the everted gut sac assay. A 5-cm segment of duodenum was removed from the mice proximally to the pyloric junction or a 5-cm segment of ileum was removed from the mice distally to the cecum. A blunt-ended 23-gauge needle was passed into the lumen of the intestinal segment and the distal end of the segment was tied to the end of the needle. The intestinal segment was everted by rolling the proximal end slowly along the needle. The intestinal segment was then filled with 400 µl transport buffer [125 mM NaCl, 10 mM fructose, 11.3 mM HEPES, and 0.25 mM CaCl2 (pH 7.4), at 37 C]. The 23-gauge needle was pulled out, the knot of the second ligation was tied, and the sacs were incubated in flasks containing 10 ml transport buffer containing 45CaCl2 (20,000 cpm/ml) and kept in a water bath at 37 C for 1 h aerated continuously with 95% O2/5% CO2 (a plateau of response under conditions that stimulated active intestinal calcium transport was observed at 2 h). At the end of the incubation, sacs were removed from the flask, gently blotted dry, and cut open, and the internal fluid from each sac as well as samples from the external incubation medium were assayed in triplicate for 45Ca using a scintillation counter. The active accumulation of 45Ca in the inside (serosal) fluid was expressed as a ratio of the final concentration of 45Ca inside/outside. The fold stimulation of intestinal calcium transport observed in these studies in WT mice under low dietary calcium conditions or after 1,25(OH)2D3 administration is consistent with previously reported studies (25, 26). Paracellular calcium transport, which occurs at higher concentrations of calcium and is unsaturable, is not measured by this assay. The everted gut sac assay selectively measures active intestinal calcium transport because the calcium transport is against a concentration gradient, occurs at low concentrations of calcium, and is saturable. Calcium transport measured by the everted gut sac assay is sensitive to intestinal segment, 1,25(OH)2D3 treatment, and dietary calcium (25, 27, 28).

Northern blot analysis
Total RNA was prepared with guanidinium isothiocyanate, and 20 µg total RNA was analyzed by Northern blot for calbindin-D9k and calbindin-D28k gene expression as previously described (16, 21, 22). All membranes were stripped and rehybridized to [32P]β-actin cDNA. The relative OD obtained using the test probes was divided by the relative OD obtained after probing with β-actin to normalize for sample variation.

Preparation of [32P]cDNA probes
A 1.2-kb mouse calbindin-D28k cDNA and a 170-base mouse calbindin-D9k cDNA were obtained by restriction endonuclease digestion of the respective plasmid preparations as previously described (16, 21, 22). The β-actin cDNA was purchased from CLONTECH Laboratories Inc. (Palo Alto, CA). 32P-labeled cDNA probes were prepared using the High Prime DNA Labeling Kit (Roche Diagnostics, Indianapolis, IN) according to the manufacturer’s instructions.

Western blot analysis
For Western blot analysis, postmitochondrial supernatants were prepared and analyzed for protein concentration by the Bradford method (29), and 50 µg protein was analyzed by Western blot for calbindin-D9k and calbindin-D28k protein expression as previously described (16). All membranes were stripped and reprobed with β-actin antibody. The relative optical density (OD) obtained using the test antibody was divided by the relative OD obtained after probing with β-actin to normalize for sample variation.

Mouse genotyping
Genotyping of TRPV6 KO mice and calbindin-D9k KO mice was performed as previously described (19, 20).

RT-PCR analysis
Total RNA was prepared with guanidinium isothiocyanate. After determining the integrity of total RNA by electrophoresis, 2 µg total RNA was reverse transcribed using Superscript III Reverse Transcriptase (Invitrogen) according to the manufacturer’s protocol. Transient receptor potential vanilloid type 5 (TRPV5), TRPV6, PMCA1b, or GAPDH mRNA levels were assessed by semiquantitative RT-PCR. For each primer set, PCR cycle numbers were chosen so that the amplification was in the linear range of amplification efficiency. Primers and annealing temperatures (Ta) were as follows: TRPV5 forward, 5'-ATTGACGGACCTGCCAATTACAGAG-3', and reverse, 5'-TCAACCCGTAAGAACCAACGGTC-3' (Ta = 60 C); TRPV6 forward, 5'-ATCGATGGCCCTGCGAACT-3', and reverse, 5'-CAGAGTAGAGGCCATCTTGTTGCTG-3' (Ta = 60 C); PMCA1b forward, 5'-GTTCTAGTGGTCGCCGTGCC-3', and reverse, 5'-CCGCGATACACGTAAGGCCG-3' (Ta = 60 C); and GAPDH forward, 5'-TCACCATCTTCCAGGAGCG-3', and reverse, 5'-CTGCTTCACCACCTTCTTGA-3' (Ta = 60 C).

Serum analysis
Serum concentrations of calcium and phosphorus were determined using Sigma Diagnostic reagents. Serum intact PTH levels were measured using the two-site immunoradiometric assay (Immunotopics, San Clemente, CA). Serum 1,25(OH)2D3 determinations were performed using a commercially available RIA kit (Immunodiagnostic Systems, Ltd., Boldon, UK).

Statistical analysis
Data were analyzed by ANOVA for multiple-group comparisons and by the Student’s t test for two-group analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characteristics of the TRPV6 and calbindin-D9k single and double KO mice
To verify null mutation, tissues were analyzed by RT-PCR and Northern and Western blotting. No signals (TRPV6 or calbindin-D9k) were detected in mice homozygous for the targeted mutation. TRPV6/calbindin-D9k DKO mice lacked expression of both TRPV6 and calbindin-D9k in the intestine and kidney (data not shown).

When fed a standard rodent chow diet, TRPV6 KO mice, calbindin-D9k KO mice, and TRPV6/calbindin-D9k DKO mice have serum calcium levels similar to those of WT mice (~10 mg/dl; Fig. 1Go). In the TRPV6 KO and the TRPV6/calbindin-D9k DKO mice, serum PTH levels were significantly increased compared with WT mice (Fig. 1Go; P < 0.05 compared with WT). The increase in PTH in the TRPV6 KO mouse was previously reported and consistent with an observed 9.6% decrease in femoral bone density (19). In the calbindin-D9k KO mice, however, serum PTH levels were not significantly different from the levels observed in WT mice (Fig. 1Go). Serum phosphorus concentrations were not significantly different in the null mutant mice compared with WT mice (WT, 8.2 ± 0.3; TRPV6 KO, 8.5 ± 0.2; calbindin-D9k KO, 8.9 ± 0.4; DKO, 8.3 ± 0.5 mg/dl; n = 7–13; P > 0.1 compared with WT; P > 0.1 comparison of multiple group means).


Figure 1
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  		FIG. 1. Concentration of calcium and PTH in the serum of WT, TRPV6 KO, calbindin-D9k KO, and TRPV6/calbindin-D9k DKO mice at 3 months of age. Each value represents the mean ± SEM for male mice (n = 7–23 mice per group; *, P < 0.05 compared with WT). Similar findings were observed using female mice (data not shown). Body weights for the mice at 3 months of age were as follows: WT, 25.4 ± 0.7 g; TRPV6 KO, 24.6 ± 1.1 g; calbindin-D9k KO, 24.7 ± 1.5 g; DKO, 26.3 ± 1.2 g (n = 7–23 mice per group; P > 0.1 compared with WT).

 
Compensatory or reciprocal mechanisms
TRPV5 mRNA is absent in intestine (Fig. 2AGo), and TRPV5 mRNA is not altered significantly in renal samples of TRPV6 KO mice, calbindin-D9k KO mice, and TRPV6/calbindin-D9k DKO mice compared with WT mice (Fig. 2AGo, middle panel; P > 0.1). Thus, TRPV5 mRNA expression is not altered in the absence of TRPV6 or calbindin-D9k or in the absence of both TRPV6 and calbindin-D9k.


Figure 2
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  		FIG. 2. Possible compensatory or reciprocal mechanisms involving vitamin D target genes/proteins in the null mutant mice. A, Left panel, Representative RT-PCR analysis of TRPV6 and TRPV5 mRNA expression in intestine and kidney of WT and null mutant male mice; right panel, quantitation of TRPV5 and TRPV6 mRNA expression (note no induction of TRPV5 expression in the intestine and no change in renal TRPV5 expression in null mutant mice; n = 5–9 per group). B, Left panel, Representative RT-PCR analysis of TRPV5 and TRPV6 mRNA expression in WT and calbindin-D9k KO male mice; right panel, quantitative analysis. Note the induction of TRPV6 mRNA in the intestine under low dietary calcium conditions (0.02%, LC) and no difference in the levels of TRPV6 mRNA under high dietary calcium (1%, HC) or low dietary calcium conditions between WT and calbindin-D9k KO mice. All values are reported as the mean ± SEM (n = 5–6 per group). *, Significantly different from the respective HC group at P < 0.05. C, Left, Representative Northern blot of calbindin-D9k mRNA (upper panel) and summary of densitometric scans of Northern blot analysis of calbindin-D9k mRNA levels in intestine and kidney of WT and TRPV6 KO mice (lower panel); right, representative Western blot (upper panel) and summary of densitometric scans of Western blots (lower panel) of calbindin-D9k in intestine and kidney of WT and TRPV6 KO mice. Note in the TRPV6 KO mice, there is no compensatory increase in calbindin-D9k. All values are reported as mean ± SEM (n = 8–9 per group; WT vs. TRPV6 KO, calbindin-D9k mRNA, or calbindin-D9k protein in intestine and kidney, P > 0.1).

 
To determine whether there is reciprocal compensation between TRPV6 and calbindin-D9k in the TRPV6 KO mice and the calbindin-D9k KO mice, levels of TRPV6 mRNA and calbindin-D9k mRNA and protein were examined. There was no significant increase in TRPV6 mRNA in either the intestine or kidney of calbindin-D9k KO mice compared with WT (Fig. 2AGo). Furthermore, under low dietary calcium conditions, TRPV6 mRNA was induced similarly in calbindin-D9k KO mice compared with WT mice, indicating that there was not a compensatory increase in TRPV6 mRNA in the calbindin-D9k KO mice (Fig. 2BGo). Also, levels of calbindin-D9k mRNA or protein were not significantly different in TRPV6 KO mice compared with WT mice (Fig. 2CGo).

Levels of duodenal PMCA1b mRNA and renal calbindin-D28k mRNA and protein were also not significantly altered in the TRPV6 KO, calbindin-D9k KO, and TRPV6/calbindin-D9k DKO mice compared with WT mice (data not shown).

Intestinal calcium transport
Active intestinal calcium transport by the mouse duodenum was measured by the everted gut sac assay. Using this assay, no change in active intestinal calcium transport in the duodenum was observed between WT and TRPV6 KO, calbindin-D9k KO, and TRPV6/calbindin-D9k DKO mice fed a standard rodent chow diet (P > 0.1, comparison of multiple group means, data not shown). Because the chow diet is high in calcium (~1% calcium), under these conditions, active duodenal calcium transport is not stimulated, and therefore differences may not be observed.

Under low dietary calcium (0.02%) conditions, there was a 4.1-, 2.9-, and 3.9-fold increase in calcium transport in the duodenum of WT, TRPV6 KO, and calbindin-D9k KO mice, respectively, compared with mice under high dietary calcium (1%) conditions (Fig. 3AGo, P < 0.05 compared with HC; P > 0.1, WT vs. calbindin-D9k KO; and P < 0.05, WT vs. TRPV6 KO on the low-calcium diet). Furthermore, the TRPV6/calbindin-D9k DKO mice displayed a 2.1-fold induction in active calcium transport under low dietary calcium conditions (P < 0.05 compared with HC; P < 0.05, WT vs. TRPV6/calbindin-D9k DKO). Active calcium transport was not stimulated by low dietary calcium in the ileum of the WT or null mutant mice (data not shown). An induction of TRPV6 and calbindin-D9k mRNA levels in the duodenum of WT mice under low dietary calcium conditions compared with mice fed a high-calcium diet was also observed (Fig. 3AGo, right panel). Although intestinal calcium absorption was decreased in the 2-month-old TRPV6 KO and TRPV6/calbindin-D9k DKO mice under low dietary calcium conditions, active intestinal calcium transport is still possible under these conditions in the absence of TRPV6 and calbindin-D9k.


Figure 3
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  		FIG. 3. Active intestinal calcium transport in the duodenum of mice fed high- (1%) or low- (0.02%) calcium diet. A, Calcium transport was measured using everted intestinal sacs formed from the duodenum of 2-month-old mice that had been fed a high-calcium (HC) or low-calcium (LC) diet from 4 wk of age. Data are expressed relative to levels seen in WT high-calcium mice (HC WT = 1). Values represent the mean ± SEM (n = 8–22 per group; *, significantly different from the respective high-calcium group at P < 0.05,; P > 0.1, WT vs. calbindin-D9k KO; and +, P < 0.05 WT vs. TRPV6 KO and TRPV6/calbindin-D9k DKO on the low-calcium diet). Right panel, Representative RT-PCR analysis of TRPV6 mRNA expression and Northern blot analysis for calbindin-D9k mRNA. Under low dietary calcium conditions, serum PTH was similarly elevated in all groups of mice (WT, 77.6 ± 11; TRPV6 KO, 85 ± 12; calbindin-D9k KO, 100 ± 12; DKO, 100.5 ± 20 pg/ml; P > 0.1 compared with WT; P > 0.1, comparison of multiple group means). 1,25(OH)2D3 serum levels were also similarly elevated in all groups of mice under low dietary calcium conditions (WT, 427 ± 77; TRPV6 KO, 414 ± 62; calbindin-D9k KO, 403 ± 65; DKO, 457 ± 46 pg/ml; P > 0.1 compared with WT; P > 0.1, comparison of multiple group means). Also, under low dietary calcium conditions, serum calcium levels were similar in all groups of mice (WT, 10.2 ± 0.2; TRPV6 KO, 9.8 ± 0.2; calbindin-D9k KO, 10.1 ± 0.2; DKO, 10.4 ± 0.4 mg/dl; P > 0.1 compared with WT; P > 0.1, comparison of multiple group means). B, Calcium transport was measured using everted intestinal sacs from the duodenum of 12- to 14-month-old mice that had been fed a high- or low-calcium diet for 4 wk. Data are relative to WT 12- to 14-month-old mice fed the high-calcium diet (aged high-calcium WT = 1; n = 6–18 per group).

 
When 12- to 14-month-old WT, TRPV6 KO, or calbindin-D9k KO mice were placed on a low-calcium diet (0.02%) for 4 wk, the increase in active calcium transport in response to low dietary calcium was significantly reduced compared with calcium transport in the corresponding 2-month-old group fed low dietary calcium (Fig. 3BGo; P < 0.05 compared with the corresponding group fed the low calcium diet in Fig. 3AGo). There was no significant difference among all groups of 12- to 14-month-old mice in duodenal calcium transport in response to low dietary calcium (P > 0.1).

In addition to low dietary calcium, 1,25(OH)2D3 administration to vitamin D-deficient mice also resulted in a significant increase in duodenal calcium transport. A 2.0-fold induction in active intestinal calcium absorption was observed for WT, TRPV6 KO, and calbindin-D9k KO mice after 1,25(OH)2D3 administration (Fig. 4Go). No difference among these groups in 1,25(OH)2D3-stimulated calcium transport was observed (P > 0.1). The TRPV6/calbindin-D9k DKO mice displayed a 1.4-fold induction in active calcium transport after 1,25(OH)2D3 administration [P < 0.05 compared with vitamin D-deficient DKO mice, and P < 0.05 compared with WT injected with 1,25(OH)2D3]. It is possible that duodenal calcium transport in response to 1,25(OH)2D3 may be more sensitive to the lack of both TRPV6 and calbindin-D9k than to the absence of either TRPV6 or calbindin-D9k alone. An induction of TRPV6 and calbindin-D9k mRNA levels was observed in the duodenum of WT mice after 1,25(OH)2D3 administration (Fig. 4Go, right panel). Thus, despite the absence of both TRPV6 and calbindin-D9k, 1,25(OH)2D3-stimulated active intestinal calcium transport occurred.


Figure 4
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  		FIG. 4. 1,25(OH)2D3-stimulated calcium transport in the duodenum of WT and null mutant mice. Calcium transport was measured using everted intestinal sacs from the duodenum of 12-wk-old mice made 1,25(OH)2D3 deplete by feeding a 0.8% strontium diet for 7 d. Mice were then injected with 1,25(OH)2D3 (+D3) or vehicle (–D3) 48, 24, and 6 h before termination (ip, 100 ng/100 g body weight per injection). Values represent the mean ± SEM [n = 6–16 per group; *, P < 0.05 for 1,25(OH)2D3-treated (+D3) compared with the respective-deficient (–D3) mice; +, P < 0.05 compared with WT +D3]. This graph (as well as Fig. 3Go) represents data from both male and female mice. The number of male and female mice was balanced to increase the power to detect genotype vs. treatment differences. Right panel, Representative RT-PCR analysis of TRPV6 mRNA expression and Northern blot analysis for calbindin-D9k mRNA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Active calcium transport involves the saturable transcellular absorption of calcium in the mammalian and avian duodenum. The established three-step model of active intestinal calcium absorption postulates a critical role for TRPV6 in the entry of calcium from the intestinal lumen into the absorptive cells of the duodenum and for intestinal calbindin as a calcium-binding protein thought to shuttle calcium from the apical to the basolateral side of the intestinal cell for extrusion into the blood. Until recently, the exact in vivo role of both TRPV6 and calbindin-D9k in 1,25(OH)2D3-mediated transcellular calcium absorption and the functional relationship between TRPV6 and calbindin-D9k has been a matter of debate. The generation of TRPV6 KO mice, calbindin-D9k KO mice, and TRPV6/calbindin-D9k DKO mice made possible in vivo studies examining the role of TRPV6 and calbindin-D9k in 1,25(OH)2D3-regulated intestinal calcium absorption. This study is the first to examine active calcium transport in these KO animals and the first study in which TRPV6/calbindin-D9k DKO mice were generated. Our findings indicate that active intestinal calcium transport occurs in the absence of TRPV6 and calbindin-D9k, suggesting a need for reevaluating the concept that TRPV6 and calbindin are required for stimulation of active intestinal calcium absorption.

Previous studies examining calcium absorption in intestine of null mutant mice employed the oral gavage assay, which measured the contribution of both transcellular and paracellular calcium absorption to total duodenal calcium absorption (17, 19, 30). The oral gavage assay does not allow calculation of the contribution of active calcium absorption in the duodenum (17). To measure active transcellular calcium transport in the mouse duodenum, we performed the everted gut sac assay. A significant induction in active intestinal calcium transport was observed for TRPV6 KO, calbindin-D9k KO, and TRPV6/calbindin-D9k DKO mice under low dietary calcium conditions or after 1,25(OH)2D3 administration to vitamin D-deficient mice (Figs. 3AGo and 4Go). Thus, our data suggest that other key factors are involved in the process of active transcellular calcium absorption. Previous studies proposed a role for TRPV6 as the rate-limiting step in the process of 1,25(OH)2D3-mediated intestinal calcium absorption (17, 19, 31). However, other studies indicated that transcellular calcium absorption occurs in the presence of low TRPV6 mRNA levels in the mouse duodenum (12). Although the level of intestinal calcium transport in the TRPV6 KO mice under low dietary calcium conditions was reduced compared with WT mice, a significant 2.9-fold increase in active intestinal calcium transport still occurred compared with TRPV6 KO mice fed the high-calcium diet (Fig. 3AGo). Similarly, 1,25(OH)2D3-treated TRPV6 KO mice also showed a significant induction in active intestinal calcium transport compared with vitamin D-deficient mice (Fig. 4Go). Thus, TRPV6 may not be the rate-limiting factor in the process of transcellular calcium absorption, or its function in this process in the KO mouse may be compensated, at least in part, by a currently unknown factor.

Although our findings indicate that TRPV6 is not essential for 1,25(OH)2D3-mediated intestinal calcium absorption, the decrease observed in active calcium transport under low dietary calcium conditions in the absence of TRPV6 suggests that TRPV6 is part of the calcium absorptive process. In these studies, TRPV6 KO mice showed a decrease compared with WT in active intestinal calcium transport under low dietary conditions but not in response to 1,25(OH)2D3 administration, suggesting that, under the conditions used in these experiments, calcium transport under low dietary calcium is more sensitive to the lack of TRPV6. TRPV6 expression has been shown to be regulated by 1,25(OH)2D3 levels and dietary calcium conditions (16, 17). Furthermore, transcriptional regulation of TRPV6 by 1,25(OH)2D3 through its binding to the VDR/retinoid X receptor at vitamin D response elements located at –2.1 and –4.3 kb relative to the start site of transcription has been recently reported (32). These findings suggest that TRPV6 plays a role in regulation of calcium homeostasis by 1,25(OH)2D3.

TRPV6 has been reported to interact with other proteins that may modulate its function and thus may also play a role in calcium entry. Calmodulin interacts with TRPV6 at the carboxyl-terminal region (33, 34, 35), and this interaction has been shown to be a dynamic association that facilitates the rapid inactivation of TRPV6 (35). Also, previous studies have shown that constitutive trafficking of TRPV6 to the plasma membrane and TRPV6 channel activity involve the interaction of the C-terminal region of the channel with the S100A10-annexin 2 protein complex (36). Furthermore, Rab11a has been shown to play a role in recycling TRPV6 to the plasma membrane (37). An additional physiological modulator of TRPV6 is the PDZ (postsynaptic density-95, disc large, zona occludens-1) domain-containing protein PDZK2, which may function as a scaffold protein, allowing the coupling of TRPV6, through PDZ domain 4 interaction, with other factors to regulate TRPV6 at the apical membrane (38, 39, 40, 41). Thus, it is possible that these TRPV6-associated proteins represent novel components of 1,25(OH)2D3-regulated intestinal calcium transport that specifically influence calcium entry mechanisms.

Although a decrease in active calcium transport under low dietary calcium conditions was observed using TRPV6 KO mice, calbindin-D9k KO mice do not differ from WT mice in their ability to actively transport calcium in the intestine either under low dietary calcium conditions or after 1,25(OH)2D3 administration (Figs. 3AGo and 4Go). Calbindin-D9k KO mice were able to maintain normal serum calcium levels, and serum PTH levels were not significantly different from WT mice (Fig. 1Go). These data are consistent with previously published reports that examined the role of calbindin-D9k in intestinal calcium absorption using an inbred 129/OlaHsd line that was homozygous for a mutant calbindin-D9k (42). Calbindin-D9k KO mice were generated by injecting the E14.11C ES cell subline, which contained a frameshift deletion in calbindin-D9k resulting in a 22-nucleotide deletion in exon III of the calbindin-D9k gene, into C57BL/6 host blastocytes (42). In comparison with the calbindin-D9k KO mice used in our study, in which a neomycin-resistant cassette replaced exon I and part of exon II, calbindin-D9k KO mice generated by Kutuzova et al. (42, 43) using the oral gavage assay showed no change in 1,25(OH)2D3-mediated intestinal calcium absorption and in serum calcium levels compared with WT. These studies support an alternative pathway for intestinal calcium absorption and intracellular calcium diffusion that does not require intestinal calbindin.

Although previous studies suggested that both calbindin-D9k in mammalian intestine and calbindin-D28k in chick intestine play an essential role as shuttle proteins that function to facilitate cytosolic calcium diffusion, Turnbull et al. (44) showed that active calcium transport was unaffected in developing teeth in the absence of calbindin-D28k, thus similarly challenging the notion of calbindin as an essential calcium ferry. Furthermore, our data are consistent with previous studies using 1,25(OH)2D3 analogs that noted that the induction of calbindin-D9k does not always correlate with an increase in intestinal calcium absorption, suggesting that other vitamin D-dependent factors are important in the intestinal absorptive process (10, 11). Impaired intestinal calcium absorption has also been observed in VDR KO mice in which calbindin-D9k mRNA and protein levels were repressed compared with WT mice but not significantly decreased enough to account for the dramatic reduction in the percentage of calcium absorbed (12). Thus, it appears from our data that calbindin-D9k is not essential for active intestinal calcium absorption, or it may be compensated for by another factor. Calbindin may also have another role in the intestine, for example as a modulator of calcium channel activity and/or as an intracellular calcium buffer.

With age, the ability of the intestine to adapt to low dietary calcium conditions by increasing intestinal calcium absorption, calbindin-D9k, and TRPV6 decreases (25, 45, 46). Aged rats also show a significant reduction in their ability to convert 25OHD3 to the active vitamin D compound 1,25(OH)2D3 (47). Furthermore, the capacity of 1,25(OH)2D3 to increase intestinal calcium absorption decreases with age (27). Previous reports have also shown that although serum calcium levels do not change significantly with age, the expression of duodenal calbindin-D9k and TRPV6 declines with age in parallel with transepithelial calcium transport (25, 45, 46). In our studies, we found a similar decline in intestinal calcium transport in response to low calcium with age in the null mutant and WT mice (Fig. 3BGo). This result may be due, in part, to the low levels of TRPV6 expression in the duodenum with age (46) and the decrease in the capacity of the adult kidney to produce 1,25(OH)2D3 as well as the reported decreased responsiveness of the adult duodenum to 1,25(OH)2D3 (27, 47). Components other than TRPV6 and calbindin may also be unresponsive to 1,25(OH)2D3 with age, thus preventing maximal active intestinal calcium absorption in response to low calcium.

Because in the absence of both TRPV6 and calbindin-D9k, normal serum calcium levels were maintained and stimulated active calcium transport occurred, the question that remains is the identification of other novel factors involved in intestinal calcium absorption. In vivo studies have reported that GH is a determinant of active intestinal calcium absorption (48, 49). The effect of GH has been proposed to be mediated by IGF-I (49). In addition, in vitro studies have shown that IGF-I can increase transcellular calcium transport across monolayers of Caco-2 cells (49). Although we found that serum levels of IGF-I were not altered under our experime conditions (high vs. low calcium; 1,25(OH)2D3 deplete vs. 1,25(OH)2D3 treated or among the different groups (WT and different KO mice; unpublished observation), IGF-I mRNA was significantly induced in the duodenum of DKO mice under low dietary calcium conditions (1.5-fold, P < 0.05 compared with intestinal IGF-I mRNA in WT HC mice; Benn, B. S., and S. Christakos, preliminary results). These preliminary results suggest that IGF-I release from the liver was not altered in response to dietary calcium or 1,25(OH)2D3 and that there may be a differential response of the small intestine and the liver to low dietary calcium. A previous study has also indicated a differential response of the small intestine and liver to nutritional treatment (no change in serum IGF-I but an increase in IGF-I mRNA and protein in the intestine), suggesting a direct local effect of IGF-I in the intestine (50). Although more studies (including examination of changes in intestinal IGF-I protein, in IGF-binding proteins, and in intestinal IGF-I receptors in all the different groups of mice) are needed, it is possible that IGF-I may be a factor that contributes, in part, to the active calcium absorption observed in these studies. It is also possible that other calcium channels and other calcium-binding proteins may be involved in intestinal calcium absorption. Although calcium is absorbed more rapidly from the duodenum than the jejunum and ileum, calcium absorption occurs in each segment of the intestine (51). Most recently, the L-type calcium channel isoform Cav1.3 was reported in low concentrations in duodenum and in high concentrations in the distal jejunum and proximal ileum (52). Cav1.3 was localized to the apical membrane of the intestine (52). It is of interest that the calcium-binding protein sorcin, which has been reported to bind to and to modulate the activity of L-type calcium channels (53, 54), is present in highest concentrations in the jejunum (Ajibade, D., and S. Christakos, unpublished observation). Whether or not sorcin or Cav1.3 has a role in vitamin D-regulated calcium absorption remains to be determined. However, because we are now only beginning to understand the multiple factors involved in intestinal calcium absorption, it is indeed possible that calcium channels and calcium-binding proteins other than TRPV6 and calbindin, respectively, are involved in 1,25(OH)2D3-mediated intestinal calcium absorption.

Although not investigated in this study, 1,25(OH)2D3-mediated paracellular transport of calcium may have contributed to the normalization of serum calcium in the null mutant mice. Gene array studies from the DeLuca lab (55) indicated that in the duodenum, 1,25(OH)2D3 down-regulates cadherin-17 (important in cell to cell contact) and aquaporin 8 (a tight junction channel), suggesting that 1,25(OH)2D3, by regulating these proteins, can route calcium absorption through the paracellular path. In preliminary results, we found that there was a significant 1.3- to 1.4-fold decrease (P < 0.05) in cadherin-17 mRNA in response to low dietary calcium in the duodenum of both WT and DKO mice (Benn, B. S., and S. Christakos, unpublished observation). These findings suggest that transjunctional movement of calcium occurs in a regulated fashion and that down-regulation of cadherin 17 may contribute to the changes in serum calcium in the KO mice. Thus, to provide new insight into vitamin D-regulated intestinal calcium absorption, future studies examining different regions of the intestine as well as novel 1,25(OH)2D3-regulated proteins involved in both transcellular and paracellular calcium absorption are needed.

In summary, this study demonstrates, for the first time using null mutant mice, TRPV6- and calbindin-D9k-independent regulation of active intestinal calcium absorption, thus challenging the dogma of the need for TRPV6 and calbindin-D9k for 1,25(OH)2D3-induced active intestinal calcium transport and necessitating the further analysis of candidate genes induced by 1,25(OH)2D3 in the intestine of the TRPV6/calbindin-D9k DKO mice. Dysregulation of intestinal calcium transport can contribute to age-related bone disease. Understanding the factors involved in active intestinal calcium absorption may lead to the development of drugs that may have clinical benefit by specifically influencing various steps in the process of intestinal calcium absorption.


   Acknowledgments
 
We gratefully acknowledge the assistance of Xiaorong Peng and Kopal Dhawan in certain aspects of this investigation. We also appreciate helpful discussions with Drs. Felix Bronner, Richard Wood, and Joseph Feher.


   Footnotes
 
This work was supported by the National Institutes of Health Grants AG297512 to B.B., DK38961 to S.C., and DK072154 to J.-B.P.

Disclosure Statement: The authors of this manuscript have nothing to disclose.

First Published Online March 6, 2008

Abbreviations: DKO, Double knockout; KO, knockout; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; Ta, annealing temperature; TRPV6, transient receptor potential vanilloid type 6; TRPV 5, transient receptor potential vanilloid type 5; VDR, vitamin D receptor; WT, wild type.

Received November 30, 2007.

Accepted for publication February 26, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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