1,25-Dihydroxyvitamin D3 increases the expression of the CaT1 epithelial calcium channel in the Caco-2 human intestinal cell line
© Wood et al; licensee BioMed Central Ltd. 2001
Received: 2 July 2001
Accepted: 17 August 2001
Published: 17 August 2001
The active hormonal form of vitamin D (1,25-dihydroxyvitamin D) is the primary regulator of intestinal calcium absorption efficiency. In vitamin D deficiency, intestinal calcium absorption is low leading to an increased risk of developing negative calcium balance and bone loss. 1,25-dihydroxyvitamin D has been shown to stimulate calcium absorption in experimental animals and in human subjects. However, the molecular details of calcium transport across the enterocyte are not fully defined. Recently, two novel epithelial calcium channels (CaT1/ECaC2 and ECaC1/CaT2) have been cloned and suggested to be important in regulating intestinal calcium absorption. However, to date neither gene has been shown to be regulated by vitamin D status. We have previously shown that 1,25-dihydroxyvitamin stimulates transcellular calcium transport in Caco-2 cells, a human intestinal cell line.
In the current study, we have demonstrated that Caco-2 cells express low but detectable levels of CaT1 mRNA in the absence of 1,25-dihydroxyvitamin D treatment. CaT1 mRNA expression is rapidly up regulated (4-fold increase at 4 h and 10-fold at 24 h) by treatment with 1,25-dihydroxyvitamin D (10-7 moles/L). Moreover, the increase in CaT1 mRNA expression preceded by several hours the vitamin D induction of calbindin D9K, a putative cytosolic calcium transport protein.
These observations are the first to demonstrate regulation of CaT1 expression by vitamin D and are consistent with a new model of intestinal calcium absorption wherein vitamin D-mediated changes in brush border membrane CaT1 levels could be the primary gatekeeper regulating homeostatic modulation of intestinal calcium absorption efficiency.
Low intestinal calcium absorption efficiency in humans is common and contributes to negative calcium balance and bone loss . Recent epidemiological evidence suggests that low fractional calcium absorption is a significant risk factor for future osteoporotic hip fracture . The primary homeostatic mechanism for altering calcium absorption in response to low dietary calcium intake and increased calcium requirements during growth is an increase in plasma 1,25-dihydroxyvitamin D, the active hormonal form of vitamin D produced in the kidney . 1,25-dihydroxyvitamin D is a seco-steroid that acts on gene transcription through a nuclear steroid receptor protein and also may have nongenomic actions mediated by a putative plasma membrane vitamin D receptor . Changes in vitamin D status primarily alter the rate of Ca absorption in the proximal small intestine. During transcellular calcium transport across the absorptive enterocyte, calcium must pass across an apical brush border membrane, then transverse the interior of the cell, and exit against an electrochemical gradient across the basolateral membrane. Changes in the rate of vectorial Ca transport in the intestine however must be accomplished without a major disruption in the free intracellular Ca concentration, which is used as an important intracellular signal in the enterocyte  as in other cells.
It has been known for over 30 years that vitamin D status influences the level of calbindin D, a cytosolic calcium-binding protein  believed to be the rate-limiting step in calcium absorption  by acting as an intracellular calcium ferry or chaperone. Calcium extrusion at the basolateral membrane is an active process involving a Ca-ATPase that may also be affected by vitamin D status . However, although it has been known for more than 20 years that the rate of Ca transport across the intestinal brush border membrane can be increased by 1,25-dihydroxyvitamin D , the identity of the calcium transport protein involved in Ca influx has not been ascertained. Recently, two novel, closely related, epithelial calcium channels, CaT1 (also known as ECaC2) and ECaC1 (also known as CaT2), have been cloned from rat  and rabbit  respectively and shown to mediate calcium influx when expressed in Xenopus oocytes. CaT1 and ECaC1 are found in several tissues, including 1,25-dihydroxyvitamin D-responsive epithelia in the intestine and kidney. These epithelial calcium channel genes are products of evolutionary local gene duplication found on chromosome 7 and represent a new family of Ca-selective ion channels involved in active Ca (re)absorption . Furthermore, it has been suggested that the control of the number of active channels at the cell surface is critical for regulation of active calcium transport and that the epithelial calcium channel could confer the rate-limiting step in transcellular calcium transport .
Peptide hormones and other cell surface-acting signal molecules trigger increases in the concentration of calcium ions inside the cell by release of calcium from stores within the cell, or activation of Ca++ ion channels in the plasma membrane allowing calcium to enter from outside the cell. One of the most common mechanisms that regulate entry of calcium is by capacitative or store-operated calcium entry . The epithelial Ca-release activated channel, ICRAC, is a highly calcium-selective plasma membrane ion channel that is activated on lowering of either intracellular calcium concentration or intracellular calcium stores. The CaT1 channel has been recently expressed in mammalian CHO cells and patch clamp studies have shown it to have the characteristics of ICRAC. Nearly ubiquitous, store-operated calcium entry is essential in a wide variety of cellular processes including muscle contraction, protein secretion, metabolism, cell differentiation, cell division and apoptosis .
Intestinal calcium absorption is increased after feeding a low calcium diet or under conditions of increased calcium needs, such as during growth, via a parathyroid hormone (PTH)-vitamin D mediated process . A lowering of serum calcium levels stimulates the secretion of PTH that then activates the renal conversion of inactive 25-hydroxyvitamin D to the active hormonal metabolite 1,25-dihydroxyvitamin D. At the intestine, 1,25-dihydroxyvitamin D, acting through a nuclear receptor, stimulates transcription of vitamin D-mediated genes, including calbindin D, involved in calcium transport thereby acting as the primary hormonal regulator of active intestinal calcium transport . It is thus likely that any putative key molecular player in calcium absorption, such as CaT1 or ECaC1, should be a prime target for hormonal regulation by 1,25-dihydroxyvitamin D. However, to date, neither CaT1 nor ECaC1 expression has been shown to be related to serum 1,25-dihydroxyvitamin D or vitamin D status [10, 16].
We have developed [17–22] a unique human intestinal cell culture model of vitamin D-mediated transepithelial Ca transport using Caco-2 cells, a colon adenocarcinoma cell line. This cell line spontaneously differentiates in culture and expresses biochemical and morphological characteristics of well-differentiated small intestine-like enterocytes [17, 23]. We have previously shown that Caco-2 cells possess a classical nuclear vitamin D receptor  that directly mediates vitamin D-dependent gene expression , and exhibits a high degree of specificity for various vitamin D metabolites and analogs [20, 21]. Most importantly, in response to short-term (<24 h) 1,25-dihydroxyvitamin D treatment, Caco-2 cells increase transepithelial calcium transport  and calbindin D9K expression , a cytosolic calcium binding protein. Kinetic analysis of Ca transport across Caco-2 monolayers grown on permeable filter supports indicated that 1,25-dihydroxyvitamin D treatment increased the Vmax of Ca transport , which would be consistent with a vitamin D-dependent increase in the number of calcium transporters.
In the current paper, we have used the Caco-2 cell culture model to demonstrate that CaT1 mRNA is preferentially expressed in the human enterocyte compared to ECaC1, and for the first time demonstrate that 1,25-dihydroxyvitamin D treatment can cause a rapid and marked increase in CaT1 mRNA expression in the enterocyte. These observations suggest a likely important role for this calcium transporter in vitamin D-dependent calcium absorption.
Results and Discussion
CaT1 is expressed in Caco-2 cells
CaT1 mRNA is regulated by 1,25-dihydroxyvitamin D
1,25-dihydroxyvitamin D causes a rapid and marked up regulation of CaT1 expression
The rapid time course of induction and sensitivity of CaT1 mRNA response to 1,25-dihydroxyvitamin D treatment supports the idea that CaT1 could play a role as a gatekeeper of intestinal calcium absorption. We have previously reported that it takes from 12–16 h following 1,25-dihydroxyvitamin D treatment of Caco-2 cells to first appreciate an increase in saturable transcellular calcium transport . This temporal response is similar to the 8–16 h observed for the achievement of maximal rates of intestinal calcium absorption in vivo following 1,25-dihydroxyvitamin D treatment of vitamin D-deficient chickens . Future studies of the time course of CaT1 protein expression and localization of this protein to the brush border will be necessary to confirm this point, but must await the development of a suitable CaT1 antibody.
CaT1 induction precedes the increase in calbindin D9k mRNA expression
Given what is already known about the characteristics of the epithelial calcium channels and calbindin D, it is a plausible hypothesis that CaT1 and calbindin D could work in tandem to maintain high rates of calcium absorption. Studies in human duodenal biopsy specimens have found a significant correlation between CaT1 mRNA and calbindin D mRNA expression . Thus, as a working hypothesis, we propose that during times of dietary calcium stress, or during periods of high calcium demand, CaT1 expression in the enterocyte is increased by a vitamin D-dependent mechanism. This regulatory genomic response of the enterocyte would result in an increase in the brush border membrane content of CaT1 calcium influx channels that could function as a gatekeeper mechanism to augment the rate of calcium entry across the apical membrane . However, given that there is a Ca-dependent negative feedback mechanism to limit calcium entry through the channel by maintaining the channel in the closed position [14, 15] there must be a way to remove calcium from the vicinity of the cytosolic face of the channel to optimize and sustain calcium influx rates. The coordinated vitamin D-dependent induction of the cytosolic calcium-binding protein calbindin D could serve this function when the need for transcellular calcium flux is high by acting directly or indirectly as a molecular sponge and selectively removing calcium ions from the CaT1 channel and/or to buffer increases in intracellular calcium, thereby preventing premature inactivation of this ICRAC channel . Calbindin D could also play its traditionally conceived role as a calcium chaperone protein to facilitate the transfer of calcium through the cytosol  and perhaps delivery of Ca++ to the basolateral ATP-dependent calcium pump for extrusion out of the enterocyte .
These observations are the first to demonstrate regulation of CaT1 expression by 1,25-dihydroxyvitamin D in the human enterocyte and are consistent with a new model of intestinal calcium absorption wherein vitamin D-mediated changes in brush border membrane CaT1 levels could be the primary gatekeeper regulating homeostatic modulation of intestinal calcium absorption efficiency.
Materials and Methods
Conditions of cell culture
Caco-2 cells (HTB 37, ATCC, Rockville, MD) were propagated and maintained as described previously . Caco-2 cells were studied between passages 30–40. Cells were seeded into six-well dishes (35-mm diameter; Costar, Cambridge, MA) and grown for 15 days. Cell culture medium, nutrients and antibiotics were purchased from BioWhittaker (Walkersville, MD). FBS was purchased from HyClone Laboratories (Logan, Utah). Chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) and 1,25-dihydroxyvitamin D was purchased from Biomol Research Laboratories (Plymouth Meeting, PA).
In time-course studies, cells were treated with 100 nanomol/L 1,25-dihydroxyvitamin D diluted in DMEM + 5% FBS for 0, 2 h, 4 h, 8 h and 24 h. All treatments ended at the 24 h time point. Control treatments were treated with ethanol vehicle alone. In dose-response studies, cells were treated for 24 h with 0,1,10, or 100 nanomol/L 1,25-dihydroxyvitamin D diluted in DMEM + 5% FBS. Control cells were treated with ethanol vehicle.
Reverse transcription polymerase chain reaction (RT-PCR)
Following experimental treatments, cells were harvested and total RNA was isolated according to manufacturer's instructions (Tri Reagent, Molecular Research Center, Cincinnati, OH). One microgram of total RNA was made into a cDNA library by a reverse transcription process using the following conditions: 0.1 ug of oligothymidine, 100 U of reverse transcriptase, 5 ug bovine serum albumin (BSA), 10 U RNAsin (Promega, Madison WI) in a final volume of 10 ul buffer containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 0.5 mM each of CTP, ATP, GTP, TTP, incubated for 2 h at 37°C, and denatured at 95°C for 10 minutes. The cDNA solution containing 0.1 μg equivalent RNA was analyzed by PCR for GAPDH, human calbindin D9K, and human CaT/ECaC. Primers sets for the epithelial calcium channel were as follows: sense primer 5'TGAACCTGGTGCGCGCACTGC3' (GenBank accession number AJ271207.1 nucleotides 480–500) and antisense primer 5'CCCAGGGAGTCCTGGGCCCGGA3' (nucleotides 657–678). The PCR primers used for GAPDH and calbindin D were as previously described . PCR conditions used were: denature at 94°C for 40 s, annealing at 55°C for 50 s with an extension at 72°C for 50 s. All reactions were run within the linear range of number of cycles to product formed. GAPDH was amplified for 21 cycles, CaT1 for 28 cycles and calbindin D9K for 28 cycles. A blank PCR was run using water for each gene tested. PCR products were electrophoresed on a 2% agarose gel containing ethidium bromide. Relative amounts of amplified PCR product from each experimental condition were visualized under ultraviolet light and digitized with the Gel Doc 2000 gel documentation system (Bio-Rad Laboratories, Hercules, CA). Relative amounts of product were estimated by digital densitometry using Quantity One (version 4) quantitation software (Bio-Rad). CaT1 and calbindin D9K mRNA expression were normalized relative to the expression of GAPDH mRNA, a constitutively expressed gene whose levels were not affected by 1,25-dihydroxyvitamin D treatment.
Sequencing and RFLP analysis to establish the identity of CaT1 in Caco-2 Cells
PCR products from the epithelial calcium channel were generated with the primers described above, subjected to electrophoresis on a 1% low-melting-point agarose gel, and purified for sequence verification. The DNA sequencing facility at Tufts University sequenced the PCR product to identify whether the amplified PCR product from Caco-2 cells was either CaT1 or the closely related ECaC1 sequence. Because of the high nucleotide similarity of CaT1 and ECaC1 (97%), an additional identification step based on restriction fragment length polymorphism (RFLP) analysis was also conducted to confirm the identity of CaT1 in Caco-2 cells. PCR products were generated using the following primer set: sense primer (5'TGACATCTGAGCTCTATGAGGGT3', GenBank AF365927 nucleotides 545–567) and antisense primer 5'CCCAGGGAGTCCTGGGCCCGGA3' (nucleotides 782–803). These primers yield a 259 bp PCR product with a specific restriction site that makes it possible to clearly distinguish between CaT1 and ECaC1 using the restriction enzymes Bgl1 (New England Biolabs). In this PCR amplified product, there is no predicted Bgl1 restriction enzyme site in the corresponding ECaC1 sequence. In contrast, the presence of this Bgl1 cut site in the CaT1 sequence will result in two smaller bands (≅ 180 and ≅ 80 bp) after digestion with the endonuclease. The endonuclease digested and undigested PCR samples were electrophoresed on a 2% agarose gel and the bands were visualized with ethidium bromide staining. The size of the cut bands was estimated with standard 50 pb markers to identify whether the PCR product amplified from Caco-2 cells was being cut into the appropriately sized bands with Bgl1.
Data are reported as mean ± the standard error of the mean (SEM). Experiments were repeated from 3 to 10 times. Treatment effects were evaluated by ANOVA with a post hoc Tukey test for individual treatment comparisons (SYSTAT version 9, SAS Institute, Cary NC) where applicable.
This work was supported from funds from the United States Department of Agriculture, Agricultural Research Service cooperative agreement 1950-51520-006-00D. The contents of the publication do not necessarily reflect the views or policies of the US Department of Agriculture, nor does mention of trade names, commercial products, or organizations imply endorsement by the US government.
Some of the data presented here were reported in preliminary form at the annual meeting of the federation of American Societies for Experimental Biology (Experimental Biology 2001) in Orlando FL (FASEB J 2001; 15: A59).
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