Gastroenterology Section, Imperial College School of Medicine, Hammersmith Campus, London W12 0NN, United Kingdom
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ABSTRACT |
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Calcium absorption in intestine and kidney involves transport through the apical membrane, cytoplasm, and basolateral membrane of the epithelial cells. Apical membrane calcium influx channels have recently been described in rabbit (epithelial calcium channel, ECaC) and rat (calcium transport protein, CaT1). We amplified from human duodenum a 446-base partial cDNA probe (ECAC2) having a predicted amino acid similarity of 97% to rat CaT1. Duodenum, but not ileum, colon, or kidney, expressed a 3-kb transcript. A larger transcript was also found in placenta and pancreas, and a different, faint transcript was found in brain. In duodenal biopsies from 20 normal volunteers, expression varied considerably but was not significantly correlated with vitamin D metabolites. This signal correlated with calbindin-D9k (r = 0.48, P < 0.05) and more strongly with the plasma membrane calcium ATPase PMCA1 (r = 0.83, P < 0.001). These data show that although individual variations in calcium channel transcripts are not vitamin D dependent, expression of genes governing apical entry and basolateral extrusion are tightly linked. This may account for some of the unexplained variability in calcium absorption.
intestine; absorption; 1,25-dihydroxycholecalciferol; brush-border membrane; calcium-adenosinetriphosphatase; calbindin-D9k; vitamin D
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INTRODUCTION |
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DIETARY CALCIUM IS ABSORBED in the intestine and is necessary for the development and maintenance of bone mineralization. Studies in humans have indicated that there is considerable variation in fractional calcium absorption among individuals (9). Although the active metabolite of vitamin D, 1,25-dihydroxycholecalciferol [1,25(OH)2D3], is clearly involved in calcium absorption, studies have suggested that much of the variability in human fractional calcium absorption is not caused by this or other known factors (1).
Active absorption of dietary calcium occurs principally in the proximal small intestine and necessitates separate steps of transport, first across the apical (brush border) membrane, then through the cytoplasm, and finally extrusion at the basolateral membrane of the enterocyte. Transepithelial transport of calcium also takes place in the placenta and in the kidney. In the intestine, the vitamin D-dependent calcium binding protein calbindin-D9k and the plasma membrane calcium-pumping ATPase 1b (PMCA1b) are thought to be the major molecules involved in the cytoplasm and basolateral membrane. We (13, 15) have characterized the molecular nature of both of these in human duodenum. Recently, we (25) studied the range and variability of their expression in duodenum, showing that there was significant, although weak, correlation of 1,25(OH)2D3 with calbindin-D9k expression but not with PMCA1b.
The molecular nature of the apical calcium influx channel has been unclear until recently. In 1999, Hoenderop and colleagues (11) identified a strong candidate for this channel in the rabbit. The RNA for this epithelial calcium channel (ECaC) was expressed in proximal small intestine, in the distal part of the nephron, and in placenta. Peng and colleagues (20) described a homologous channel in the rat. This was expressed principally in duodenum and cecum but not in kidney and was named CaT1 (for calcium transport protein). These two molecules, which have 75% amino acid identity, are unlike previously described calcium channels.
In this paper we report the amplification of a partial sequence of an apical calcium transport channel in humans using a strategy of mixed-primer PCR from duodenal cDNA. We have investigated the expression of this transcript in duodenum and its relationship with other critical factors involved in intestinal calcium absorption.
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EXPERIMENTAL PROCEDURES |
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Amplification of cDNA. PCR primers were based on regions of conserved amino acids in the rabbit ECaC (accession no. AJ133128) and rat CaT1 (accession no. AF160798) sequence. The forward primer, coding for GGPFHV between positions 1553 and 1568 of rat CaT1 cDNA, had the sequence 5'-GGNGGNCCNTTYCAYG and 256-fold degeneracy. The reverse primer, between positions 2014 and 2031, had 32-fold degeneracy and was the reverse complement of 5'-GCNATGATGGGNGAYAC coding for AMMGDT. The sequences of these primers are also conserved in human ECAC1, described subsequently (accession no. AJ271207; Ref. 17). Restriction sites for cloning were included.
cDNA was prepared from human duodenal RNA and amplified by PCR as described before (14). Products were cloned into pGEM3 (Promega-UK, Southampton, UK), and three clones were sequenced using standard methodology. Sequence comparisons used BLAST, GAP, and BESTFIT programs of the GCG package.Expression studies. Northern blotting was performed using total RNA samples prepared from duodenum, ileum, colon, and placenta (14, 26) and with poly(A)-enriched RNA on a multitissue Northern blot (Clontech, Palo Alto, CA). The cDNA probe amplified above was 32P-labeled, hybridized, and washed as previously described (14). Hybridization was performed in buffer containing 125 mM NaH2PO4, pH 7.2, 10% dextran sulfate, 5% SDS, 0.5% (wt/vol) dried milk powder, 25 µM aurin tricarboxylic acid, and 2.5 mM EDTA overnight at 60°C. Blots were washed for 1 h in 2% SDS, 0.25 M NaH2PO4, pH 7.2, and 1 mM EDTA and for 1 h in 30 mM NaCl, 2 mM NaH2PO4, and 0.2 mM EDTA with 0.2% SDS, also at 60°C.
To determine the range of expression in duodenum, we used total RNA on blots previously prepared for the study of calbindin-D9k and PMCA1 (25). Blots of duodenal RNA were available for study from 20 subjects who had undergone normal endoscopy approved by the local Research Ethics Committee. RNA signals were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression, and 1,25(OH)2D and 25(OH)D levels in plasma had been determined. Results were quantified by phosphorimaging. Statistical analysis used the data analysis package of Microsoft Excel. ![]() |
RESULTS |
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Human calcium transporter cDNA probe.
Amplification of human duodenal cDNA with the primers based on
conserved regions of rabbit ECaC and rat CaT1 resulted in a single band
on electrophoresis that had the expected size of ~479 bp. This was
eluted, cloned, and sequenced. This sequence was obtained in at least
10 different clones, with no variation apart from a single nucleotide
difference, probably the result of infidelity in PCR. The sequence
matched several expressed sequence tags in the human database,
including W88570, AA447311, H18519, and T92755, but did not detect any
previously known human genes, although subsequently, a related human
epithelial calcium channel sequence, AJ271207, has been reported and
named ECAC1 (17). As shown in Fig.
1A, the human nucleotide
sequence we amplified had high identity (90%) to the
corresponding region of rat CaT1. It had lower identity with rabbit
ECaC (86%) and is clearly distinct from the other reported human
sequence (ECAC1), with which it has 91% identity in this region.
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Tissue expression.
The expression of human ECAC2 (CaT1) was investigated in the intestine
and other tissues by Northern blotting (Fig.
2). A single signal ~3 kb in size was
readily detectable in duodenum but not in ileum or colon, although the
quality of the placental total RNA was poor. On a multitissue
blot of poly(A)-enriched RNA, two bands, one the same size and another
larger transcript (>9 kb), were demonstrated in human placenta and
pancreas. Another faint transcript (~7.5 kb) could be detected in
brain. No expression could be found in kidney using this probe.
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Variation in duodenal expression.
To investigate vitamin D dependence and other correlates of expression,
we quantified levels of transcripts detected by this calcium channel
probe on Northern blots of RNA from the normal duodenum of 20 subjects.
Their details are described in Table 1.
Signals were obvious in all of these duodenal samples but varied
considerably (Fig. 3). After correction
for differences in RNA loading by measuring GAPDH expression, there was
a 10-fold range in expression between the highest and lowest values.
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DISCUSSION |
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The sequence we have amplified by RT-PCR from human duodenal RNA appears to be part of the human homologue of rat CaT1, which has been shown to have the expected properties of an apical membrane calcium channel implicated in calcium entry to the enterocyte during calcium absorption (20). In humans, as in rats, we have shown expression of the transcript in proximal small intestine and also in the placenta and pancreas, where related roles in calcium transport can be envisaged. In these latter tissues, a second, larger-sized transcript can be detected, raising the possibilities of alternative splicing or control of RNA stability. Low-level expression of a different-sized transcript in brain will also require further exploration to determine any role in this tissue.
The inability to detect this transcript in human kidney RNA suggests that, despite their high degree of identity in this region, the conditions used in the expression studies do not result in cross-hybridization with human ECAC1, the other member of this epithelial apical membrane calcium channel/transporter family. ECaC expression has been detected in the rabbit distal nephron as well as the duodenum (11) and, with the use of specific primers, has been shown in kidney, duodenum, and other tissues in humans (17). Our PCR data with mixed primers suggests that the CaT1-related ECAC2 sequence described here is of greater abundance than ECAC1 in duodenum, although this method is subject to considerable error. To determine accurately the relative abundance of these two genes in duodenum and elsewhere will require parallel Northern blots with unequivocally different probes, perhaps from the 3'-end of the cDNA. Further data regarding the human sequence and information about tissue and cellular expression in humans will clarify the relationships between these genes and how the functions of their various products might differ according to species or tissues.
We were able to study the variability of expression of the transcripts detected with our probe in normal duodenal RNA from a range of subjects to test the hypothesis that expression was affected by 1,25(OH)2D levels. Our data suggest that there is no vitamin D dependence of expression, at least at the population level. Using the same experimental samples, we (25) were previously able to show that calbindin-D9k transcripts correlate with 1,25(OH)2D. It is interesting to note that the strength of this relationship in humans is weak, at both the RNA and protein levels (23), and that the presence of a vitamin D-responsive element in the calbindin-D9k gene remains debatable (2, 22). However, expression of the calcium channel signal and calbindin-D9k are also significantly correlated in our samples. It will be of interest to see whether this relates to similar transcriptional control of these genes by factors expressed in duodenum or whether calcium entering the cell through apical channels may be affecting calbindin-D9k transcript levels independently of 1,25(OH)2D. In the vitamin D receptor knockout mouse, there is still some expression of intestinal calbindin-D9k (27), and posttranscriptional effects of calcium on calbindin-D9k RNA stability have been described (6).
The lack of vitamin D dependence of expression of the human apical calcium channel is also similar to that suggested for rat CaT1 by the results of feeding different levels of dietary calcium (20). In the chicken, using ion microscopic imaging, Chandra and colleagues (4) showed that calcium entry at the apical membrane was unchanged in vitamin D-deficient chicks but that transport away from this site was impaired in this species because of lower vitamin D-dependent calbindin-D28k concentrations. In contrast, preliminary data in the vitamin D knockout mouse (24) reported greatly reduced expression of ECaC, indicating that, in this species at least, one of the apical calcium channel genes may be vitamin D dependent.
Probably the most interesting finding in the present study is the strength of the correlation of the expression of calcium channel and PMCA1 transcripts. First, this shows that there is sufficient precision in our experimental methods to detect physiologically relevant associations in variations in gene expression in endoscopic biopsies. More importantly, this points to fundamental mechanisms controlling the activities of calcium channels and PMCA1. It makes considerable sense for the expression of genes regulating calcium entry and exit from the cell to be closely regulated. PMCA1 appears to have the properties expected of the major basolateral membrane calcium pump involved in calcium absorption. Although it is expressed in most if not all tissues, it is highly expressed in duodenum in humans as in other species (8, 14, 15). The structure of the PMCA1 gene ATP2B1 has been studied (10), but the control of its tissue-specific expression is not yet understood. Identification of the promoter of the human ECAC2 and further studies of PMCA1 will be needed to show how the expression of these genes is tightly regulated.
The large variation in expression of epithelial calcium channel transcripts may be an important factor explaining some of the considerable variability in calcium absorption among individuals. Low fractional calcium absorption has recently been shown to be related to an increase in the frequency of osteoporotic hip fractures in subjects on low-calcium diets (7); calcium supplementation can improve bone mineralization (5). The reasons for much of the variability in fractional calcium absorption have remained unclear (1). The identification of these human calcium channels, and subsequently the control of their expression, may help resolve the differences in calcium absorption and help target therapy.
High intestinal expression of apical calcium channels could result in absorptive hypercalciuria, leading to renal calculi (19). The lack of expression of this CaT1 homologue, ECAC2, in renal tissues could be important in this. Several steps in the pathophysiology of absorptive hypercalciuria have been suggested. It is likely that multiple genetic changes can produce this phenotype, but it is intriguing to note that a relationship of hypercalciuria and high red blood cell ATPase activity has been reported (3). How this finding is linked to our discovery of closely correlated calcium channel and PMCA1 expression remains to be determined.
Recently, a locus for absorptive hypercalciuria has been mapped in three families by linkage studies to chromosome 1q23.3-q24 (21). Another locus at 4q33-qter has been suggested in two unrelated patients (16). PMCA1 is located at chromosome 12q21-23 (18), and mapping of a genomic clone derived from our cDNA sequence by fluorescent in situ hybridization has suggested a locus for ECAC2 at chromosome 7q34-7q35 (Barley et al., manuscript in preparation). Human ECAC1 has been mapped to 7q31.1-7q31.2 (17). Thus it seems unlikely that genetic variation of apical calcium channels is directly responsible for hypercalciuria in these families. However, the factor(s) regulating expression of these calcium channels and PMCA1 could be found at these loci.
The identification of the partial sequence of a human homologue of a gene that is likely to be responsible for calcium entry at the apical membrane in intestine and the associations with its expression have suggested several fruitful areas for further study. In particular, the lack of association with vitamin D in a healthy population, the wide variability, and the closely regulated coexpression with PMCA1 may help explain many of the gaps in our current knowledge relating to the physiology of calcium absorption.
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ACKNOWLEDGEMENTS |
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We acknowledge the contributions to the original study of Lisa Lowery and Barbara Mawer. We thank James Scott for providing some materials and for discussion.
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FOOTNOTES |
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The partial cDNA sequence reported here has been assigned the GenBank accession number AJ277909 and the gene symbol ECAC2.
Address for reprint requests and other correspondence: J. R. F. Walters, Gastroenterology Section, Imperial College School of Medicine, Hammersmith Campus, London W12 0NN, UK (E-mail: julian.walters{at}ic.ac.uk).
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.
Received 26 May 2000; accepted in final form 31 August 2000.
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