Regulation of the epithelial Ca2+ channels in small intestine as studied by quantitative mRNA detection

Monique van Abel,1 Joost G. J. Hoenderop,1 Annemiete W. C. M. van der Kemp,1 Johannes P. T. M. van Leeuwen,2 and René J. M. Bindels1

1Department of Cell Physiology, Nijmegen Center for Molecular Life Sciences, University Medical Center Nijmegen, P. O. Box 9101, 6500 HB Nijmegen, The Netherlands; and 2Department of Internal Medicine, Erasmus Medical Center Rotterdam, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands

Submitted 21 January 2003 ; accepted in final form 26 February 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The epithelial Ca2+ channels TRPV5 and TRPV6 are localized to the brush border membrane of intestinal cells and constitute the postulated rate-limiting entry step of active Ca2+ absorption. The aim of the present study was to investigate the hormonal regulation of these channels. To this end, the effect of 17{beta}-estradiol (17{beta}-E2), 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], and dietary Ca2+ on the expression of the duodenal Ca2+ transport proteins was investigated in vivo and analyzed using realtime quantitative PCR. Supplementation with 17{beta}-E2 increased duodenal gene expression of TRPV5 and TRPV6 but also calbindin-D9K and plasma membrane Ca2+-ATPase (PMCA1b) in ovariectomized rats. 25-Hydroxyvitamin D3-1{alpha}-hydroxylase (1{alpha}-OHase) knockout mice are characterized by hyperparathyroidism, rickets, hypocalcemia, and undetectable levels of 1,25(OH)2D3 and were used to study the 1,25(OH)2D3-dependency of the stimulatory effects of 17{beta}-E2. Treatment with 17{beta}-E2 upregulated mRNA levels of duodenal TRPV6 in these 1{alpha}-OHase knockout mice, which was accompanied by increased serum Ca2+ concentrations from 1.69 ± 0.10 to 2.03 ± 0.12 mM (P < 0.05). In addition, high dietary Ca2+ intake normalized serum Ca2+ in these mice and upregulated expression of genes encoding the duodenal Ca2+ transport proteins except for PMCA1b. Supplementation with 1,25(OH)2D3 resulted in increased expression of TRPV6, calbindin-D9K, and PMCA1b and normalization of serum Ca2+. Expression levels of duodenal TRPV5 mRNA are below detection limits in these 1{alpha}-OHase knockout mice, but supplementation with 1,25(OH)2D3 upregulated the expression to significant levels. In conclusion, TRPV5 and TRPV6 are regulated by 17{beta}-E2 and 1,25(OH)2D3, whereas dietary Ca2+ is positively involved in the regulation of TRPV6 only.

ECaC; CaT1; estrogen; vitamin D; dietary Ca2+


THE MAINTENANCE OF THE EXTRACELLULAR Ca2+ concentration is important for mammalian development and function. Intestinal Ca2+ absorption is a crucial control system in the regulation of Ca2+ homeostasis, because it facilitates the entry of dietary Ca2+ into the extracellular compartment (28).

The intestinal absorption of Ca2+ follows two pathways: a transcellular and a paracellular route (49). Paracellular transport is the passive, nonsaturable way of intestinal Ca2+ absorption, which occurs down an electrochemical gradient. Transcellular Ca2+ absorption takes place against an electrochemical gradient and, therefore, requires energy. This active Ca2+ transport is under the control of hormones in a Ca2+-dependent manner (7). 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the active form of vitamin D, is the primary regulator of active Ca2+ absorption. 1,25(OH)2D3 is synthesized from the inactive metabolite 25-hydroxyvitamin D3 by 25-hydroxyvitamin D3-1{alpha}-hydroylase (1{alpha}-OHase) in kidney. 1,25(OH)2D3 acts through nuclear vitamin D receptors (VDR), which are present within the enterocytes of the intestine (8, 27, 46). In addition, functional estrogen receptors have also been detected in small intestine (47). Arjmandi et al. (2) showed that 17{beta}-estradiol (17{beta}-E2) enhances the uptake of Ca2+ by intestinal cells in vitro. Furthermore, active intestinal Ca2+ absorption can be regulated by dietary Ca2+ intake. Active absorption of Ca2+ is increased after feeding a low-Ca2+ diet or under conditions of increased Ca2+ needs (7).

The importance of the hormones involved in Ca2+ homeostasis is reflected by severe disorders. For example, mutations in the genes encoding for 1{alpha}-OHase or VDR result in pseudovitamin D-deficiency rickets (VDDR-1) and hereditary hypocalcemic vitamin D-resistant rickets (VDDR-2), respectively (26, 30). High oral doses of Ca2+ can prevent the concomitant bone pathology (21). Furthermore, estrogen deficiency in postmenopausal women results in a negative Ca2+ balance and osteoporosis. This is often associated with intestinal malabsorption, which is corrected by estrogen therapy (16). On the basis of these data, it is obvious that active Ca2+ absorption in the small intestine plays an indispensable role in Ca2+ homeostasis and bone mineralization.

Active Ca2+ absorption is localized to the duodenum and can be described in three sequential cellular steps: entry, intracellular diffusion, and extrusion (49). The Ca2+-binding protein calbindin-D-9K is involved in intracellular diffusion of Ca2+. It binds Ca2+ and moves it from the brush border membrane to the basolateral site of the duodenal cell. In this respect, calbindin serves as both a Ca2+ carrier and a cytosolic Ca2+ buffer (18, 34). The extrusion of Ca2+ across the basolateral membrane from the enterocyte is mediated by the plasma membrane Ca2+-ATPase PMCA1b (10). The molecular nature of the apical Ca2+ entry channel was elusive until the identification of the epithelial Ca2+ channels ECaC1 and ECaC2 (24, 39). These two Ca2+ channels represent a new family of Ca2+-selective ion channels belonging to the superfamily of transient receptor potential (TRP) channels. The TRP family can be divided by sequence homology in several subfamilies (31). ECaC1 and ECaC2 are members of the TRP-Vanilloid (TRPV) subfamily and have, therefore, been renamed into TRPV5 and TRPV6, respectively (32). Both channels are expressed in several tissues including the small intestine, in which they are localized to the brush border membrane of intestinal absorptive cells (22, 53). Importantly, it has been postulated that these channels form the rate-limiting step in transcellular Ca2+ (re)absorption (23).

The regulation of TRPV5 and TRPV6 in duodenum may shed new light on hormone-controlled Ca2+ metabolism. Primary or secondary involvement of one or both epithelial Ca2+ channels can be expected in several pathological situations, such as VDDR and osteoporosis. Therefore, the present study was designed to investigate the regulation of TRPV5 and TRPV6 as the entry channels of active Ca2+ absorption in duodenum. To this end, the effects of 17{beta}-E2, 1,25(OH)2D3, and dietary Ca2+ on the expression of these duodenal Ca2+ transport proteins were investigated in vivo and analyzed using real-time quantitative PCR.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals. Twenty-five virgin female Wistar rats (Hsd/Cpd: Wu, SPF-bred by Harlan, CPB, Zeist, The Netherlands) were subjected to a bilateral ovariectomy or sham operation. Thereafter, rats received daily 17{beta}-E2 (Sigma, St. Louis, MO) or vehicle (gelatin, mannitol) added to the pelleted food. Sham-operated animals (Sham, n = 5) served as controls. Ovariectomized animals were given either the vehicle alone (OVX, n = 5) or 2 x 32 (OVX + E2L, n = 5), 2 x 125 (OVX + E2M, n = 5), or 2 x 500 µg 17{beta}-E2/day (OVX + E2H, n = 5). Treatment was started immediately after ovariectomy and lasted for 7 days.

1{alpha}-OHase knockout mice were generated by Dardenne and colleagues (14) through inactivation of the 1{alpha}-OHase gene. Three different experiments were performed, using these homozygous knockout mice as a vitamin D-deficient model, to study the effect of: 1) 17{beta}-E2 supplementation: using Alzet osmotic minipumps (model 1007D). Eight male 1{alpha}-OHase knockout mice, 9 wk of age, were randomized in two groups. Control mice received vehicle solution alone [15% (vol/vol) ethanol, 50% (vol/vol) DMSO], and the supplemented group received an infusion dose of 10 µg 17{beta}-E2/day for 7 days; 2) Ca2+ supplementation: eight 1{alpha}-OHase knockout mice were equally divided into two groups and were fed either a normal diet [1.1% (wt/wt) Ca2+, 0.8% (wt/wt) phosphorus, 0% (wt/wt) lactose] from ages 3 to 8 wk or received a Ca2+-enriched diet [2% (wt/wt) Ca2+, 1.25% (wt/wt) phosphorus, 20% (wt/wt) lactose; Harlan Tekland, Madison, WI]; 3) 1,25(OH)2D3 supplementation: eight 1{alpha}-OHase knockout mice received either 1,25(OH)2D3 or vehicle injections intraperitoneally from ages 3 to 8 wk. From weeks 3 to 4, mice were daily injected intraperitoneally with 1,25(OH)2D3 repletions of 500 and 100 pg/g body wt daily in weeks 5–8.

At the end of the treatment periods, animals were killed and blood and duodenum tissue samples were taken. The animal ethics board of the University Medical Center Nijmegen approved all animal experimental procedures.

Analytical procedures. Serum Ca2+ concentrations were analyzed using a colorimetric assay kit as described previously (5). Serum 17{beta}-E2 was measured by an extraction procedure using diethyl ether followed by radioimmunoassay (DPC, Los Angeles, CA) (13).

RNA isolation and quantitative PCR. Total RNA from duodenal mucosa was isolated using TRIzol reagent (GIBCOBRL, Life Technologies, Breda, The Netherlands) according to the manufacturer's protocol. RNA was treated with DNAse to prevent contamination of genomic DNA and finally resuspended in diethylpyrocarbonate-treated milliQ. Total RNA (2 µg) was subjected to reverse transcription using Moloney Murine Leukemia Virus reverse transcriptase (GIBCO-BRL) as described previously (22). Expression levels of duodenal TRPV5, TRPV6, calbindin-D9K, and PMCA1b mRNA were quantified by real-time quantitative PCR, using the ABI Prism 7700 Sequence Detection System (PE Biosystems, Rotkreuz, Switzerland). With the use of standard curves, the amount of copy numbers of the target genes in each sample was calculated and expressed as a ratio to the hypoxanthineguanine phosphoribosyl transferase gene. Primers and probes targeting the genes of interest were designed using Primer Express software (Applied Biosystems, Foster City, CA) and are listed in Table 1.


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Table 1. Sequences of primers and Taqman probes for real-time quantitative PCR

 

Statistical analysis. Values are expressed as means ± SE. Statistical significance was determined by ANOVA followed by contrast analysis according to Fisher. In the case of only two experimental groups, statistical significance was determined using the Mann-Whitney U-test. Differences in means with P values <0.05 were considered statistically significant. All analyses were performed using the Statview Statistical Package (Power PC version 4.51, Berkeley, CA) on a Macintosh computer.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
OVX Wistar rats were used as a model of estrogen deficiency. Ovariectomy was confirmed by the reduced serum 17{beta}-E2 levels compared with Sham-operated animals (Table 2). Correction of this deficiency by supplementation with 17{beta}-E2 resulted in a dose-responsive increase with significantly higher serum 17{beta}-E2 levels in OVX + E2H rats (Table 2). Importantly, 17{beta}-E2 treatment reduced serum Ca2+ levels, resulting in a slight but significantly lower serum Ca2+ concentration in the OVX + E2H group (Table 2).


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Table 2. Effects of OVX and 17{beta}-E2 supplementation on serum parameters in female Wistar rats

 

Subsequently, we investigated whether 17{beta}-E2 treatment altered the expression of genes encoding Ca2+ transport proteins involved in duodenal transcellular Ca2+ absorption. With the use of real-time quantitative PCR, a more than sevenfold increase in TRPV6 mRNA levels was observed in OVX rats supplemented with the highest dose of 17{beta}-E2 compared with untreated OVX animals (Fig. 1A). TRPV5 gene expression was also upregulated by 17{beta}-E2, although detection levels were lower and differences between the various groups were less pronounced than for TRPV6 (Fig. 1B). In addition, upregulation of both Ca2+ channels was accompanied by an increase in expression of the other Ca2+ transport proteins, namely calbindin-D9K (9-fold) and PMCA1b (2-fold; Fig. 1, C and D).



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Fig. 1. Effects of ovariectomy (OVX) and 17{beta}-estradiol (17{beta}-E2) supplementation on mRNA expression levels of Ca2+ transport proteins in duodenum of rats. With the use of real-time quantitative PCR, duodenal expression of transient receptor potential (TRP)-vanilloid (TRPV) 6 (A), TRPV5 (B), calbindin-D9K (C), and plasma membrane Ca2+-ATPase (PMCA1b; D) of the different experimental groups were measured and presented as a ratio to hypoxanthine-guanine phosphoryibosyl transferase (HPRT) expression. Sham, sham-operated; E2L, supplemented with 2 x 32 µg 17{beta}-E2/day; E2M, supplemented with 2 x 125 µg 17{beta}-E2/day; E2H, supplemented with 2 x 500 µg 17{beta}-E2/day. Data are presented as means ± SE (n = 5). *P < 0.05 vs. OVX and OVX + E2L; {ddagger}P < 0.05 vs. Sham, OVX, and OVX + E2L; {dagger}P < 0.05 vs. OVX, OVX + E2L, and OVX + E2M; #P < 0.05 vs. OVX.

 

These observations led us to study the influence of 17{beta}-E2 treatment on duodenal TRPV5 and TRPV6 expression in 1{alpha}-OHase knockout mice to investigate the involvement of 1,25(OH)2D3. Serum 17{beta}-E2 levels were not detectable in the male mice but rose to 67 pg/ml after treatment with 17{beta}-E2. Interestingly, after treatment with 17{beta}-E2, serum Ca2+ levels significantly increased from a hypocalcemic state to subnormal concentrations of 2.03 ± 0.12 mM (Table 3). Analysis of gene expression in duodenum revealed a 12-fold increase in TRPV6 mRNA after treatment with 17{beta}-E2 (Fig. 2).


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Table 3. The effect of 17{beta}-E2, 1,25(OH)2D3, and high dietary Ca2+ on serum Ca2+ levels in 1{alpha}-OHase knockout mice

 


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Fig. 2. Effect of 17{beta}-E2 supplementation on the mRNA expression level of TRPV6 in duodenum of 25-hydroxyvitamin D3-1{alpha}-hydroxylase (1{alpha}-OHase) knockout mice. Duodenal expression of TRPV6 assessed by real-time quantitative PCR analysis is presented as a ratio to HPRT expression. Control, 1{alpha}-OHase knockout mice; 17{beta}-E2, 1{alpha}-OHase knockout mice supplemented with 10 µg17{beta}-E2/day. Data are presented as means ± SE (n = 4). *P < 0.05 vs. control.

 

In two following experiments, the 1{alpha}-OHase knockout mice were used to study the influence of 1,25(OH)2D3 itself and dietary Ca2+ on gene expression levels of the Ca2+ transport proteins. Inactivation of the 1{alpha}-OHase gene in the knockout mice resulted in severe hypocalcemia with serum Ca2+ concentrations as low as 1.20 mM. Supplementation with 1,25(OH)2D3 or a high dietary Ca2+ intake normalized serum Ca2+ concentrations (Table 3). Subsequently, analysis of gene expression showed an increase in mRNA levels of TRPV6 after high dietary Ca2+ intake (Fig. 3A). 1,25(OH)2D3 supplementation also upregulated the expression of this transcript, but to a much higher degree (Fig. 4A). In addition, high dietary Ca2+ stimulated the expression of calbindin-D9K significantly (Fig. 3B), whereas PMCA1b levels were not significantly changed (P > 0.1; Fig. 3C). Supplementation with 1,25(OH)2D3 significantly upregulated the expression of both calbindin-D9K (Fig. 4B) and PMCA1b (Fig. 4C). Detection of TRPV5 mRNA in duodenum was below detection limits in the 1{alpha}-OHase knockout mice. In addition, after treatment with either 17{beta}-E2 or high dietary Ca2+, expression of TRPV5 mRNA levels could also not be detected. Interestingly, supplementation with 1,25(OH)2D3 upregulated the expression of TRPV5 mRNA to significant levels.



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Fig. 3. Effect of dietary Ca2+ on the mRNA expression levels of Ca2+ transport proteins in duodenum of 1{alpha}-OHase knockout mice. Duodenal expression of TRPV6 (A), calbindin-D9K (B), and PMCA1b (C) of the different experimental groups assessed by real-time quantitative PCR analysis is presented as a ratio to HPRT expression. Control, 1{alpha}-OHase knockout mice; calcium, 1{alpha}-OHase knockout mice on a Ca2+-enriched diet [2% (wt/wt) Ca2+ from ages 3–8 wk]. Data are presented as means ± SE (n = 4). *P < 0.05 vs. control.

 


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Fig. 4. Effect of 1,25(OH)2D3 supplementation on the mRNA expression levels of Ca2+ transport proteins in duodenum of 1{alpha}-OHase knockout mice. Duodenal expression of TRPV6 (A), calbindin-D9K (B), and PMCA1b (C) of the different experimental groups assessed by real-time quantitative PCR analysis is presented as a ratio to HPRT expression. Control, 1{alpha}-OHase knockout mice; 1,25(OH)2D3, 1{alpha}-OHase knockout mice supplemented with 1,25(OH)2D3 (500 pg/g body wt daily in weeks 3 and 4 and 100 pg/g body wt daily in weeks 5–8). Data are presented as means ± SE (n = 4). *P < 0.05 vs. control.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present study demonstrated that duodenal TRPV5 and TRPV6 mRNA levels are both upregulated by 17{beta}-E2 and 1,25(OH)2D3, whereas dietary Ca2+ is positively involved in the regulation of TRPV6 mRNA only. Moreover, the expression of genes encoding the other known duodenal Ca2+ transport proteins is upregulated concomitantly, which will facilitate Ca2+ absorption optimally.

OVX rats were used as an animal model of estrogen deficiency in postmenopausal women (29). Ovariectomy did not affect mRNA expression levels of the various Ca2+ transport proteins in duodenum, which agrees with the measured unchanged serum Ca2+ levels 1 wk after OVX. Theoretically, the loss of function of 17{beta}-E2 could be compensated by other mechanisms such 1,25(OH)2D3 within a 7-day period. In contrast, a significant upregulation of TRPV5 and TRPV6 mRNA expression was observed after estrogen replacement therapy in OVX rats. These increased mRNA levels were accompanied by upregulated mRNA levels of both calbindin-D9K and PMCA1b. However, upregulation of the genes encoding for the Ca2+ transport proteins was accompanied by decreased serum Ca2+ levels after 17{beta}-E2 treatment. Several studies in human subjects observed this effect of estrogen treatment on serum Ca2+ levels (16, 41). It has been suggested that this fall in Ca2+ is transitory, due to increased Ca2+ requirements of the estrogen-deficient animals. Correction of estrogen deficiency results in decreased bone resorption and increased formation, causing a slight fall in serum Ca2+ concentration (40).

Because 1,25(OH)2D3 is the primary hormone involved in the regulation of Ca2+ absorption, it has been suggested that the effects of estrogen on intestinal absorption of Ca2+ are indirectly mediated by 1,25(OH)2D3 (40). In the kidney, production of 1,25(OH)2D3 by 1{alpha}-OHase plays a pivotal role in maintaining Ca2+ homeostasis (52). It was demonstrated by Stumpf et al. (44) that 17{beta}-E2 was retained in the cell nuclei of proximal tubules, where the synthesis of 1,25(OH)2D3 takes place. Conflicting data are presented concerning the effect of 17{beta}-E2 on 1{alpha}-hydroxylase activity and 1,25(OH)2D3 synthesis (1, 1113, 16, 20). So far, conclusive in vivo data for a direct effect of 17{beta}-E2, independent of 1,25(OH)2D3, on intestinal Ca2+ absorption are lacking.

Dardenne et al. (14) generated 1{alpha}-OHase knockout mice by targeted inactivation of the 1{alpha}-OHase gene. These knockout mice express the same clinical phenotype as patients with VDDR-1, characterized by hyperparathyroidism, hypocalcemia, rickets, and undetectable levels of 1,25(OH)2D3. These mice represent an ideal animal model in which to study the role of 17{beta}-E2 on intestinal Ca2+ transport independent of 1,25(OH)2D3. Treatment with 17{beta}-E2 increased serum Ca2+ levels to subnormal concentrations. Furthermore, 17{beta}-E2 treatment was associated with an upregulation of duodenal TRPV6 mRNA expression. The observations that functional estrogen receptors are present within the enterocytes (47) and that 17{beta}-E2 enhances the uptake of Ca2+ by intestinal cells in vitro (2) are suggestive of a direct role in Ca2+ absorption. Together, these findings provide further evidence that 17{beta}-E2 acts directly on duodenum to promote active Ca2+ absorption.

In the 1{alpha}-OHase knockout mice, high dietary Ca2+ intake increased the expression levels of the genes encoding Ca2+ transport proteins, which was accompanied by normalization of serum Ca2+ levels. Under physiological conditions, Ca2+ acts via a negative feedback mechanism that eventually leads to suppression of 1{alpha}-OHase activity and production of 1,25(OH)2D3, which decreases expression of the Ca2+-transporting proteins and active Ca2+ absorption (6). However, this study suggests that in the absence of 1{alpha}-OHase activity, and thus circulating 1,25(OH)2D3, Ca2+ supplementation can increase the expression level of duodenal Ca2+ transport proteins. The mechanism that underlies this vitamin D-independent Ca2+-regulated pathway is not known. Previous studies have shown that cAMP- and serum-response elements can function as a Ca2+-response element (CaRE) in the control of gene expression (19, 43). Recently, a new Ca2+-responsive transcription factor was discovered in neuronal cells that contributes to Ca2+-stimulated gene expression of the brain-derived neurotrophic factor (BDNF) through a CaRE found in the promoter of the BDNF gene (45). Moreover, in the promoter region of calbindin-D28K, a Ca2+-sensitive transcriptional regulatory mechanism, named Purkinje cell element, was identified, which may play a key role in setting the Ca2+-buffering capacity of Purkinje cells (3). Likewise, Ca2+-response elements and/or transcription factors could be involved in the Ca2+-mediated regulation of gene expression found in our study.

Interestingly, high dietary Ca2+ intake, using VDR knockout mice, resulted in a decreased expression of both TRPV5 and TRPV6 and a reduction in calbindin-D9K and PMCA1b expression (48). The VDR is a nuclear receptor and acts as a ligand-activated transcription factor. On activation by 1,25(OH)2D3, the VDR can alter the rate of gene expression. However, 1,25(OH)2D3 can also activate second-messenger pathways mediated by cell surface receptors (33, 35). Furthermore, previous studies (33, 36) have shown that this nongenomic effect of 1,25(OH)2D3 can stimulate intestinal Ca2+ transport, a process called transcaltachia. VDR knockout mice have elevated 1,25(OH)2D3 levels, which decrease after high dietary Ca2+ intake (48). It is conceivable that this influences the intestinal expression of Ca2+ transport proteins and Ca2+ absorption through 1,25(OH)2D3-mediated nongenomic pathways, which could possibly explain the differential findings between VDR and 1{alpha}-OHase knockout mice after high dietary Ca2+ intake.

The genomic response of 1,25(OH)2D3 is demonstrated by the significant increase in mRNA levels of TRPV6, calbindin-D9K, and PMCA1b after supplementation with 1,25(OH)2D3 in the 1{alpha}-OHase knockout mice. Furthermore, repletion with 1,25(OH)2D3 normalized serum Ca2+ concentrations. In agreement with our findings, previous studies (9, 46, 50) have shown that 1,25(OH)2D3 stimulates the expression level of calbindins and affects Ca2+ extrusion at the basolateral membrane of duodenal cells. 1,25(OH)2D3 has also been shown to stimulate transcellular Ca2+ transport in the human intestinal cell line Caco-2 and to increase the expression of calbindin-D9K in these cells (15, 17). Recently, Wood et al. (51) demonstrated that expression of TRPV6 in Caco-2 cells is upregulated by 1,25(OH)2D3. Besides TRPV6, also TRPV5 is expressed as apical Ca2+ channel in duodenum (22, 53). However, mRNA expression levels of this latter Ca2+ channel are hundredfolds lower in duodenum. Several other studies (4, 38, 51) reported that TRPV5 expression could not be detected in Caco-2 cells and human intestinal tissue. In our 1{alpha}-OHase knockout mice, the expression of duodenal TRPV5 is also below detection limits. Only after supplementation with 1,25(OH)2D3, TRPV5 mRNA reaches a detectable level in duodena of these knockout mice. Together, these findings indicate that 1,25(OH)2D3 is a significant regulator of both epithelial Ca2+ channels in duodenum and support the idea that 1,25(OH)2D3 stimulates active intestinal Ca2+ absorption by increasing the rate of Ca2+ influx across the intestinal brush border membrane (42, 49). In addition to TRPV6, which is abundantly present in duodenum, TRPV5 can also be strongly upregulated and could play an important role in intestinal Ca2+ absorption. The generation of TRPV5 and TRPV6 knockout mice will further substantiate the importance of these channels in Ca2+ homeostasis in general and, in particular, their role in Ca2+ absorption.

Similar to TRPV5 and TRPV6, calbindin-D9K and PMCA1b mRNA levels are also upregulated after different supplementations in rat and mouse. The activity of the epithelial Ca2+ channels is controlled by a Ca2+-dependent feedback mechanism (25, 37). Therefore, to facilitate Ca2+ transport, it is important to maintain a low intracellular Ca2+ environment. By the upregulation of the buffering and extrusion mechanisms, this requirement is fulfilled. Moreover, upregulation of expression levels of the genes encoding the intestinal Ca2+ transport proteins was accompanied by normalization of the serum Ca2+ concentration. Together, these findings underline the intimate relationship among apical influx, cytosolic diffusion, and basolateral efflux systems in transcellular Ca2+ transport, which could contribute to increased Ca2+ absorption and ultimately normalization of serum Ca2+ levels.

In conclusion, the present study demonstrated that 17{beta}-E2 and 1,25(OH)2D3 are both positively involved in the regulation of duodenal TRPV5 and TRPV6, whereas dietary Ca2+ has a stimulatory effect on the expression of TRPV6 only. This regulation substantiates the possible role of these channels in the pathogenesis of hormone-regulated Ca2+-disorders, such as osteoporosis or VDDR. Future research should aim to further unravel the mechanisms controlling the activity of TRPV5 and TRPV6, which may lead to new insights regarding Ca2+ homeostasis-related disorders.


    ACKNOWLEDGMENTS
 
We thank Organon Nederland for donating duodenal tissue samples from the ovariectomized rat study and Drs. R. St-Arnaud and O. Dardenne for providing the 1{alpha}-OHase knockout mice.

This work was supported by grants from the Dutch Organization of Scientific Research (Zon-Mw 902.18.298, Zon-Mw 016.006.001).


    FOOTNOTES
 

Address for reprint requests and other correspondence: R. J. M. Bindels, 160 Cell Physiology, Univ. Medical Center Nijmegen, P. O. Box 9101, NL-6500 HB Nijmegen, The Netherlands (E-mail: r.bindels{at}ncmls.kun.nl).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 DISCUSSION
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