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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
ECaC; CaT1; estrogen; vitamin D; dietary Ca2+
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-hydroylase (1
-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
-estradiol (17
-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-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-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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1-OHase knockout mice were generated by Dardenne and colleagues
(14) through inactivation of
the 1
-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
-E2 supplementation: using Alzet
osmotic minipumps (model 1007D). Eight male 1
-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
-E2/day for 7 days;
2) Ca2+ supplementation: eight 1
-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
-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 58.
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-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.
|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Subsequently, we investigated whether 17-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
-E2 compared with untreated OVX animals
(Fig. 1A). TRPV5 gene
expression was also upregulated by 17
-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).
|
These observations led us to study the influence of 17-E2
treatment on duodenal TRPV5 and TRPV6 expression in 1
-OHase knockout
mice to investigate the involvement of 1,25(OH)2D3.
Serum 17
-E2 levels were not detectable in the male mice but
rose to 67 pg/ml after treatment with 17
-E2. Interestingly,
after treatment with 17
-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
-E2 (Fig.
2).
|
|
In two following experiments, the 1-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
-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
-OHase
knockout mice. In addition, after treatment with either 17
-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.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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-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
-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-OHase plays a
pivotal role in maintaining Ca2+ homeostasis
(52). It was demonstrated by
Stumpf et al. (44) that
17
-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
-E2
on 1
-hydroxylase activity and 1,25(OH)2D3
synthesis (1,
1113,
16,
20). So far, conclusive in
vivo data for a direct effect of 17
-E2, independent of
1,25(OH)2D3, on intestinal Ca2+
absorption are lacking.
Dardenne et al. (14)
generated 1-OHase knockout mice by targeted inactivation of the
1
-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
-E2 on intestinal Ca2+ transport
independent of 1,25(OH)2D3. Treatment with
17
-E2 increased serum Ca2+ levels to
subnormal concentrations. Furthermore, 17
-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
-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
-E2
acts directly on duodenum to promote active Ca2+
absorption.
In the 1-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
-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
-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-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-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
-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-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 |
---|
This work was supported by grants from the Dutch Organization of Scientific Research (Zon-Mw 902.18.298, Zon-Mw 016.006.001).
![]() |
FOOTNOTES |
---|
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|