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Physiol. Genomics 17: 122-129, 2004. First published March 2, 2004; doi:10.1152/physiolgenomics.00002.2003 Free Article
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Received 6 January 2003; accepted in final form 19 February 2004.
Physiological Genomics 17:122-129 (2004)
1094-8341/04 $5.00 © 2004 American Physiological Society

DNA microarray analysis of vitamin D-induced gene expression in a human colon carcinoma cell line

Richard J. Wood1, Laurie Tchack1, Giana Angelo1, Richard E. Pratt2 and Larry A. Sonna3,4

1 Mineral Bioavailability Laboratory, Jean Mayer United States Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston 02111
2 Cardiology Division and the Gene Array Technology Center, Brigham and Women’s Hospital/Harvard Medical School, Boston 02115
3 Thermal and Mountain Medicine Division, United States Army Research Institute of Environmental Medicine, Natick 01760
4 Division of Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital/Harvard Medical School, Boston, Massachusetts 02115


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The full extent to which 1,25-dihydroxyvitamin D3 affects gene expression in human intestinal epithelial cells is unknown. We used oligonucleotide arrays to catalog vitamin D-induced changes in gene expression in Caco-2 cells, a human colon carcinoma cell line. Five paired sets of Caco-2 cell cultures were subjected to either control conditions or 1,25-dihydroxyvitamin D (10–7 mol/l x 24 h), and RNA was analyzed on an Affymetrix cDNA array containing 12,625 human sequences. Only 13 sequences representing 12 distinct genes exhibited statistically significant changes in expression of twofold or greater and were also called as "present" or "marginal" by the array-reading software in all five experiments. Genes regulated by 1,25-dihydroxyvitamin D included two previously known genes (25-hydroxyvitamin D-24-hydroxylase and amphiregulin) and 10 genes (sorcin, Gem, adaptin-{gamma}, TIG1, CEACAM6, carbonic anhydrase XII, junB, ceruloplasmin, and two unidentified sequences) that were novel. We tested and independently confirmed the effect of 1,25-dihydroxyvitamin D on 11 of these genes by RT-PCR. Increased protein expression was tested and confirmed in two of the novel 1,25-dihydroxyvitamin D-regulated genes, ceruloplasmin and sorcin. The known function of these genes suggests that many of them could be involved in the antiproliferative effects of 1,25-dihydroxyvitamin D3.

high-density oligonucleotide arrays; calcitriol; Caco-2 cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
VITAMIN D IS PRODUCED in the body in the skin in response to sunlight. It also enters the body from absorption from the diet. Vitamin D is extensively metabolized but exerts its biological effects via 1,25-dihydroxyvitamin D3, an activated hormonal form of the vitamin that is the ligand for a nuclear vitamin D receptor (VDR). In partnership with a vitamin A receptor, retinoic acid X receptor (RXR), the VDR-RXR heterodimer acts as a nuclear transcriptional factor and initiates the process of vitamin D-dependent gene expression in various cell types throughout the body (22). 1,25-Dihydroxyvitamin D3 is recognized to have prodifferentiating and antiproliferative actions in different cell types, including the intestinal enterocyte (7). These physiological properties have spurred the development of various synthetic vitamin D analogs with possible anticancer and other therapeutic potential for humans (17, 18, 23, 33). In addition, a classic action of 1,25-dihydroxyvitamin D3 occurring in the intestine that is important to bone and mineral homeostasis involves the stimulation of calcium absorption (34). As a steroid hormone, 1,25-dihydroxyvitamin D3 acts as a ligand for the nuclear VDR to affect gene expression. However, the full extent to which 1,25-dihydroxyvitamin D3 influences gene expression in human intestinal epithelial cells is unknown.

Caco-2 cells are a human colon adenocarcinoma-derived cell line that spontaneously differentiate in culture into small intestine-like cells (15, 16, 47, 48) and have gained widespread use as a model of intestinal cell function, including studies related to the intestinal action of vitamin D (5, 12–14, 20, 44, 46)

The development of DNA microarray technologies has created the opportunity to investigate the effects of 1,25-dihydroxyvitamin D3 on the gene expression profile in various cell types, including the enterocyte. In the current study, we have studied the effect of 1,25-dihydroxyvitamin D3 on gene expression in well-differentiated Caco-2 cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

Cell culture.
Caco-2 cells (HTB37; American Type Culture Collection, Rockville, MD) were cultured at 37°C in a humid atmosphere consisting of 5% CO2 and 95% air. Cells were maintained in high-glucose (4.5 g/l glucose) Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 1 mM sodium pyruvate, 100 µM nonessential amino acids, 50 µg/l gentamicin sulfate, 100 U/l penicillin, 100 U/l streptomycin, and 2 mM glutamine at pH 7.2. Routine passage of the cells was done with the aforementioned DMEM-supplemented medium with 20% fetal bovine serum (FBS). Cells were seeded for the DNA microarray experiments in six-well plastic dishes and were fed with DMEM supplemented with 10% FBS every other day until ready for 24 h of hormone treatment on day 15 in culture.

1,25-Dihydroxyvitamin D3 exposure.
After 15 days in culture to allow for spontaneous differentiation of Caco-2 cells into a small intestine-like phenotype (13), cells were treated with DMEM growth media, as described above, supplemented with 5% FBS. The two treatment conditions used in these studies were either ethanol vehicle (0.1%) in control cultures or 10–7 mol/l 1,25-dihydroxyvitamin D3 (Biomol Research Laboratories, Plymouth Meeting, PA). The treated cells were incubated at 37°C for 24 h and harvested for total RNA. Five independent paired experiments were conducted using different batches of Caco-2 cells.

RNA isolation and quality assurance.
TriReagent and bromocholoropropane (Molecular Research Center, Cincinnati, OH) were used to isolate total RNA, per the manufacturer’s directions. The quality of the extracted RNA was assessed as recommended by Farrell (8). Samples were judged suitable for DNA array analysis only if the RNA was of a suitable yield, exhibited intact bands corresponding to the 18S and 28S subunits, and displayed no spurious peaks on an UV absorption spectrum in the range 230–320 nm. In addition, prior to submission of samples for DNA microarray analysis, RNA from each of the five studies was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) to ensure that a detectable vitamin D response had occurred at the level of mRNA expression, as judged by an increase in 25-dihydroxyvitamin D-24-hydroxylase (24-hydroxylase) expression (43) following 1,25-dihydroxyvitamin D3 exposure. Expression of 24-hydroxylase was normalized to that of the housekeeping gene GAPDH. In the absence of hormone, 24-hydroxylase was not expressed in Caco-2 cells, whereas this mRNA species was clearly expressed in cells exposed to 1,25-dihydroxyvitamin D3 (data not shown). Thus we were assured from these findings that 1,25-dihydroxyvitamin D3 was bioactive in all studies with our Caco-2 cells prior to undertaking the DNA microarray analysis.

GeneChip array hybridization.
Transcript profiling with Affymetrix GeneChips (Affymetrix, Santa Clara, CA) was performed using HG-U95Av2 chips containing ~12,600 sequences (representing ~11,300 unique GenBank accession numbers), as previously described (41).

Data analysis.
Data analysis was performed using Microsoft Excel, Microsoft Access, and SigmaStat 2.0 for Windows. The fold changes in gene expression reported by the Affymetrix software (MAS 4.0) in the paired experiments were used to determine whether a statistically significant change in expression had occurred, by computing geometric means and 95% confidence intervals as described in detail previously (41). Because the natural log of a onefold change in expression is equal to zero, a sequence was considered to have experienced a statistically significant change in expression if the 95% confidence intervals on the mean of the five sets of natural log transformed data excluded zero (i.e., the natural log of 1).

Where noted in this paper, sequences that showed a statistically significant change in expression were filtered by two post hoc criteria. First, sequences were excluded if they were called as "absent" by the array reading software in any of the control samples (for downregulated genes) or in any of the samples obtained from cells exposed to 1,25-dihydroxyvitamin D3 (for upregulated genes). Second, sequences were excluded if the change in geometric mean expression was less than twofold.

Confirmatory RT-PCR.
RT-PCR was used to confirm the expression of vitamin D-dependent genes found by transcriptional profiling of Caco-2 cells. To prepare cDNA, total RNA was first reverse transcribed using an oligo dT primer (Invitrogen, Grand Island, NY). The cDNA samples were then amplified by PCR using AmpliTaq (Applied Biosystems) and specific PCR primers (Table 1). The PCR products were electrophoretically separated on an 8% TBE gel (Invitrogen, Carlsbad, CA). The PCR products on the gels were examined under UV light, the images were digitally captured (Gel Doc 2000; Bio-Rad, Hercules, CA), and the density of each band was measured using Quantity One software (Bio-Rad). GAPDH, a housekeeping gene, was used for normalizing the density of the RT-PCR product bands. For the purpose of confirmation of 1,25-dihydroxyvitamin D responsiveness of individual genes, RNA from all five experiments was pooled prior to analysis.


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  		Table 1. List of specific PCR primers used to confirm 1,25-dihydroxyvitamin D-regulated gene expression in Caco-2 cells

 
Confirmation of 1,25-dihydroxyvitamin D-induced protein expression in Caco-2 cells.
Two of the novel 1,25-dihydroxyvitamin D-regulated genes (ceruloplasmin and sorcin) identified by the microarray analysis were chosen for independent confirmation of increased protein expression in response to 1,25-dihydroxyvitamin D3 treatment. Caco-2 cells after 15 days in culture were treated with either ethanol vehicle control or 10–7 mol/l 1,25-dihydroxyvitamin D3 for 24 h. Cells were then scraped into ice-cold RIPA buffer containing 1 mM PMSF, aprotinin, and protease inhibitor cocktail (Sigma, St. Louis, MO). Cells were then sonicated for 10 s and centrifuged at 14,000 rpm for 15 min, and the whole cell homogenate supernatant was collected. Proteins were quantitated using the Bio-Rad method. The samples were diluted into SDS loading buffer and boiled for 5 min. Equal volumes were loaded on an 8% Tris-glycine gel and separated for 90 min at 170 V and then transferred to a nitrocellulose membrane. The membrane was blocked for 60 min with 5% milk in PBST (phosphate-buffered saline + Triton X-100), washed (three times for 5 min each) in PBST, and incubated in 5% milk in PBST for 60 or 90 min with either an anti-ceruloplasmin antibody (Dako) at 1:1,000 or sorcin monoclonal antibody (Zymed Laboratories) at 1:2,000. The membranes were then washed (three times for 10 min each) and incubated with an anti-rabbit horseradish peroxidase (HRP)-linked secondary antibody (Bio-Rad) at 1:2,000 for the ceruloplasmin assay or anti-mouse HRP-linked secondary antibody at 1:3,000 for the sorcin assay. The membrane was washed with PBST (three times for 10 min each) and developed with ECL Plus (Amersham) and exposed to X-ray film. The images were digitally captured (Gel Doc 2000; Bio-Rad), and the density of each band was measured using Quantity One software (Bio-Rad). Statistical differences between vehicle and 1,25-dihydroxyvitamin D treatments in mean density of the bands were determined from three paired studies by paired t-test using Prism 4 statistical software (GraphPad Software, San Diego CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 

Transcriptional profiling of 1,25-dihydroxyvitamin D regulated genes in Caco-2 cells.
Caco-2 cells were treated with 1,25-dihydroxyvitamin D3 (100 nM x 24 h), and RNA from five independent paired experiments was subjected to transcriptional profiling with an oligonucleotide microarray containing ~12,600 sequences. The oligonucleotide array identified an average of 5,167 ± 82 (mean ± SE, n = 5) sequences as "present" or "marginal" under control (ethanol vehicle alone) conditions and 5,119 ± 233 sequences in the cells exposed to 1,25-dihydroxyvitamin D3. This difference was not statistically significant (P = 0.87 by paired t-test). Using much stricter expression criteria (a present or marginal call in all five of experiments performed), we found that 3,668 sequences were expressed under control conditions and 3,371 sequences were expressed in the cells exposed to 1,25-dihydroxyvitamin D3.

A statistically significant change in expression between the control and hormone-exposed cells was found in 648 sequences. Of these, 372 (57%) showed an increase in expression, and 276 showed a decrease in expression. However, less than half of these sequences (111 increased and 123 decreased) were identified as present or marginal in all five experiments under control conditions (for the decreased genes) or in cells exposed to hormone (for the increased genes). Moreover, although the change in gene expression in response to hormone treatment was found to be statistically significant in these genes, the relative fold change in expression caused by 1,25-dihydroxyvitamin D was fairly modest for the vast majority of these sequences. Of these 234 sequences of potential interest that were identified as present/marginal and statistically significant, only 13 (i.e., 6%), representing 12 different genes, showed a mean change in expression of twofold or greater. Eleven of these vitamin D-regulated genes were upregulated, and one was downregulated. The vitamin D-regulated genes and their fold changes in response to 1,25-dihydroxyvitamin D3 as determined by the Affymetrix software are listed in Table 2. Of note, this list includes both sequences previously known to be vitamin D regulated (24-hydroxylase and amphiregulin), as well as 10 novel sequences whose responsiveness to 1,25-dihydroxyvitamin D3 has not, to the best of our knowledge, been reported previously.


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  		Table 2. Effect of 1,25-dihydroxyvitamin D3 on gene expression by human Caco-2 cells

 
Effect of 1,25-dihydroxyvitamin D3 on housekeeping gene expression.
To confirm that the changes in expression noted in Table 2 were not part of a nonspecific change in gene expression to 1,25-dihydroxyvitamin D3 exposure, we also examined the effect of hormone treatment on the expression of a number of important control sequences, including ß-actin, GAPDH, several ribosomal proteins, and a replication-independent histone (Table 3). We found no statistically significant effect of 1,25-dihydroxyvitamin D3 exposure on any of these control sequences.


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  		Table 3. Effect of 1,25-dihydroxyvitamin D3 exposure on the expression of important control sequences in Caco-2 cells

 
Confirmatory RT-PCR analysis of vitamin D-responsive genes.
Pooled RNA from the five independent experiments was used in confirmatory RT-PCR studies on 11 genes identified by the oligonucleotide array experiments as being significantly, substantially, and consistently affected by 1,25-dihydroxyvitamin D3 exposure (Table 2). The confirmatory PCR findings are illustrated in Fig. 1. In all cases tested, PCR independently confirmed the findings of the oligonucleotide array. However, the correlation between actual fold changes in gene expression determined by the Affymetrix software (Table 2) and RT-PCR (Fig. 1) was quite low and not statistically significant (data not shown).



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  		Fig. 1. Confirmatory PCR for 1,25-dihydroxyvitamin D-regulated genes found by Affymetrix GeneChip transcriptional profiling. RNA was pooled from the five independent paired experiments used in the microarray transcriptional profiling analysis and subjected to RT-PCR using gene-specific primers (Table 1) to confirm 1,25-dihydroxyvitamin D-responsive gene expression.

 
1,25-Dihydroxyvitamin D-induced protein expression in Caco-2 cells.
As further confirmation of the validity of the microarray analysis of 1,25-dihydroxyvitamin D-induced gene expression, we assessed the change in protein concentration (Fig. 2) of two of the novel 1,25-dihydroxyvitamin D-regulated genes, ceruloplasmin and sorcin, which had commercial antibodies available. Treatment of Caco-2 cells for 24 h with 1,25-dihydroxyvitamin D3 caused no change in expression of the GAPDH control protein (Fig. 2C). However, hormone treatment was associated with a clear increased expression of a prominent predicted human ceruloplasmin band of ~130–135 kDa on Western blot (Fig. 2A). Densitometric evaluation of this band indicated that 1,25-dihydroxyvitamin D treatment increased ceruloplasmin protein expression (corrected for GAPDH expression) by 210 percent (0.532 ± 0.259 vs. 1.650 ± 0.005, P < 0.02 paired t-test). Western blot of control and 1,25-dihydroxyvitamin D-treated cells with the sorcin primary monoclonal antibody resulted in the identification of two bands (Fig. 2C). We have consistently found these two bands in Caco-2 cells on several different Western blots and with different batches of Caco-2 cells. Both the lower band (~20.5 kDa) and the upper band (~22 kDa) are close in apparent molecular mass to the predicted molecular mass of human sorcin of 21,676 Da. There was no significant change in the density of the upper band in response to hormone treatment. However, the density of the lower molecular weight species putative sorcin band (corrected for GAPDH protein expression) was significantly increased 124% by 1,25-dihydroxyvitamin D3 (0.354 ± 0.114 vs. 0.792 ± 0.082, P < 0.037, paired t-test).



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  		Fig. 2. 1,25-dihydroxyvitamin D-induced changes in protein expression in Caco-2 cells. As a further confirmation of the validity of the Affymetrix GeneChip findings in Caco-2 cells, we assessed the change in protein concentration of two of the novel 1,25-dihydroxyvitamin D-regulated genes (ceruloplasmin and sorcin). Three paired studies of Caco-2 cells were conducted. Cells (15 days in culture) were treated with ethanol (vehicle control; lanes 1–3) or 10–7 mol/l 1,25-dihydroxyvitamin D [+1,25(OH)2D; lanes 4–6] for 24 h, and relative protein concentration for ceruloplasmin (A) and sorcin (C) was determined by Western blot. The last lane of the gel was run with MagicMark (Invitrogen, Carlsbad, CA) molecular mass markers. As shown in A, 1,25-dihydroxyvitamin D treatment caused a significant (P < 0.02, paired t-test) twofold increase (corrected for GAPDH expression) in a ~130-kDa ceruloplasmin protein band. The loading control protein GAPDH (B) was not affected by hormone treatment. The sorcin monoclonal anti-human sorcin primary antibody recognizes two protein species in Caco-2 cells Western blots (C) near the predicted molecular mass (21.7 kDa) of sorcin. A higher molecular mass species (~22 kDa) was not significantly affected by hormone treatment. However, the minor ~20.5-kDa molecular mass band recognized by the sorcin antibody was significantly (P < 0.037) increased by 1.24-fold in response to hormone treatment.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
It is well established that vitamin D affects intestinal function, particularly the ability to absorb dietary calcium. More recently, it has become increasingly apparent that 1,25-dihydroxyvitamin D3, the active hormonal form of the vitamin, has pleiotropic effects on processes other than vitamin D metabolism and calcium homeostasis (31). The present study confirmed the regulation of two previously identified vitamin D-responsive genes [24-hydroxylase (11, 43) and amphiregulin (1)] and identified 10 genes not previously known to be vitamin D responsive. The known functional roles of some, but not all, of these genes is consistent with the reported influence of 1,25-dihydroxyvitamin D3 on cellular proliferation and differentiation.

The effect of vitamin D hormone exposure on gene expression in Caco-2 cells appears to be fairly selective rather than global. The total number of genes whose expression was affected by vitamin D was not large enough to produce a net change in gene expression, as determined either by the Affymetrix software expression calls (5,167 ± 82 before and 5,119 ± 233 after vitamin D exposure, P = 0.87), or by the number of sequences that showed a statistically significant difference in expression as a result of vitamin D exposure (648, or 5.1% of the total number of sequences on the array, which would be expected from random chance when a P value of 0.05 is taken as statistically significant). Thus the vitamin D exposure applied produced relatively narrow changes in gene expression, rather than the widespread changes that have been observed in other cell lines after exposures to stressors such as heat shock (41) and hypoxia (40). Importantly, the observation that the number of genes affected by vitamin D exposure was small provided an important rationale to apply post hoc filtering to the data set, so as to limit the list of candidate vitamin D-responsive genes to those whose changes in expression would be less likely to have occurred by random chance alone. Accordingly, we applied two filtering criteria, based on consistency of sequence expression calls and the magnitude of the observed change (>=2-fold). By our strict filtering criteria, only 13 sequences (representing 12 genes) were identified as being differentially expressed in response to vitamin D. The success of this approach is illustrated by the fact that we were able to confirm the vitamin D-responses of 11 of these genes (IMAGE 649765 EST was not tested) on subsequent analysis by RT-PCR.

In addition, we assessed changes in protein expression of two of these 1,25-dihydroxyvitamin D-regulated genes (sorcin and ceruloplasmin), which had available commercial antibodies. A clear change in ceruloplasmin expression was evident on Western blots in response to hormone treatment. Changes in sorcin expression were more complex due to the recognition of two reactive protein bands (~22 and ~20.5 kDa) of approximately the correct predicted size of sorcin (21.7 kDa). Of the two bands we noted on Western blot, only the slightly lower molecular weight band was clearly responsive (124% increase) to vitamin D hormone treatment. Others have reported multiple molecular weight species of sorcin in rat heart and isolated cardiac myocytes (~22 kDa and ~18 kDa) or only a single lower band (~18 kDa) in HEK 293 human embryonic kidney cells (29). Additional research will be needed to specifically identify the difference between these two sorcin antibody-reactive protein species. It should be noted, however, that these two forms of sorcin appear to be characteristic of Caco-2 cells because we have found them consistently expressed in different experiments and with different batches of Caco-2 cells.

An important limitation of this study is that our confirmed set of vitamin D-regulated genes in Caco-2 cells probably represents an underestimate of the total number of genes affected by vitamin D status in the enterocyte, for several reasons. First, even though we were able to examine the expression of ~11,500 different human genes with the Affymetrix U95 oligonucleotide microarrays, this is probably only one-third or less of the total human genome. Second, we measured changes in mRNA expression at only a single time point (24 h) and at only one 1,25-dihydroxyvitamin D3 concentration (10 –7 mol/l). Third, our use of strict post hoc filtering, which was applied to help distinguish independently verifiable findings from false-positive results, necessarily excluded from the final list genes that are affected by vitamin D but did not meet our filter criteria. One example of this is calbindin D9k, a well-known vitamin D-regulated gene in the intestine (4) that is upregulated by 1,25-dihydroxyvitamin D3 in Caco-2 cells (12). In our experiment, calbindin D9k showed a statistically significant mean change in expression of 3.1-fold in response to 1,25-dihydroxyvitamin D3 but was identified by the Affymetrix software as present or marginal after vitamin D exposure in only two of the five experiments (suggesting a low absolute level of expression) and was thus excluded from our final list of vitamin D-responsive genes. Analysis of calbindin D expression by RT-PCR, using the same RNA from the five microarray experiments, confirmed that calbindin D was indeed vitamin D responsive but that the absolute level of expression in some of the experiments was quite low even after vitamin D exposure (data not shown). Despite this limitation, we believe strict post hoc filtering is valuable in the analysis of microarray data when the primary objective of the experiment is the efficient discovery of novel responses to a given stimulus, as was the case in this study. A further limitation of this study is that independent evidence, such as antisense studies, was not presented to demonstrate the specific functional involvement of a given gene in the cellular actions of 1,25-dihydroxyvitamin D3. Moreover, with the exception of 24-hydroxylase, it is not known whether the identified novel target genes have a vitamin D response element in their promoters. Finally, because we chose in this initial microarray study to investigate changes in gene expression only at 24 h after treatment, some of the treatment effects on specific target genes may have been missed or are indirect secondary effects of 1,25-dihydroxyvitamin D3. Investigation of earlier time points following treatment could be a useful strategy in the future to identify genes directly stimulated by 1,25-dihydroxyvitamin D3.

It should also be noted that in our experience the absolute mean fold change in vitamin D-induced gene expression determined on the microarray (0.46- to 5.9-fold, Table 2) was of no value in predicting fold change as determined by semiquantitative RT-PCR analysis (0.46 to 18.1-fold, Fig. 1). In this analysis, changes in 24-hydroxylase expression were excluded, because in Caco-2 cells this vitamin D-responsive gene is not expressed in the absence of 1,25-dihydroxyvitamin D3 treatment, so a calculation of fold change is meaningless.

The known functional roles of the genes identified as being affected by vitamin D exposure are consistent with established physiological effects of 1,25-dihydroxyvitamin D3 on the enterocyte, including vitamin D metabolism, calcium homeostasis, and cell proliferation and differentiation. Two of these genes, 24-hydroxylase and amphiregulin, have previously been shown to be upregulated by 1,25-dihydroxyvitamin D3 in human cells (1, 11, 43). The 24-hydroxylase enzyme participates in vitamin D metabolism via a negative feedback loop wherein this enzyme adds a hydroxyl group to the C-24 position of 1,25-dihydroxyvitamin D3, which reduces its affinity for the VDR and initiates its catabolism. Amphiregulin, a member of the epidermal growth factor family, is regulated by 1,25-dihydroxyvitamin D3 in human tongue squamous cell carcinoma (SCC25) and human breast cancer cell lines (1) Amphiregulin is the most commonly expressed ligand for the epidermal growth factor receptor and is believed to function as an autocrine growth factor (6). It is also believed to be involved in vitamin D-mediated growth inhibition in SCC25 and breast cancer cell lines (1) and might therefore play a role in the known antiproliferative action of 1,25-dihydroxyvitamin D3 in Caco-2 cells (13, 19).

In addition to amphiregulin, several other vitamin D-regulated genes identified in this study have been found to influence cell proliferation or differentiation in at least one experimental system. TIG1 (tazarotene-induced gene 1, also known as retinoic acid receptor responder 1 or RARRES1) is a gene whose expression in skin is increased by the drug tazarotene, a synthetic ligand for retinoic acid receptor ß- and {gamma}-isoforms (32). TIG1 may be an important tumor suppressor gene in prostate cells (25). JunB is a member of the Jun subfamily of transcription factors that associate with other basic region-leucine zipper proteins, such as those in the Fos subfamily, to regulate AP-1 activity (38, 39) and thereby affect cell proliferation. JunB negatively regulates AP-1 activity and cell proliferation in malignant mouse keratinocytes (9). Gem is a Ras-related GTP-binding protein that may act as a regulatory protein to modulate cell proliferation (28). Interestingly, Gem can bind the calcium binding protein calmodulin (10), and a recent study (3) has found that Gem inhibits high-voltage-activated calcium channel activity by interacting directly with the ß-subunit of the channel and reducing {alpha}1-subunit expression at the plasma membrane. It has been suggested (3) that Gem may inhibit cell proliferation by reducing calcium-mediated cell growth. Sorcin, a calcium-binding protein originally identified in multidrug-resistant cells (30), is widely distributed among mammalian tissues and modulates ryanodine receptor function and intracellular calcium release. Sorcin associates with the {alpha}1-pore-forming subunit of voltage-dependent L-type calcium channels and may mediate interchannel communication between L-type calcium channels in the plasma membrane and intracellular calcium-release channels (29). Given that both sorcin and Gem are upregulated by 1,25-dihydroxyvitamin D3 in Caco-2 cells, we speculate that these may work together in a coordinated fashion to affect calcium channel activity and promote the antiproliferative effect of 1,25-dihydroxyvitamin D3 in Caco-2 cells (13). CEACAM6 is a cell surface glycoprotein associated with a differentiated cell phenotype. Increasing levels of CEACAM6 have been observed as normal colonocytes differentiate and migrate up colonic crypt walls (2) and in Caco-2 cells as they spontaneously differentiate in culture (21). Thus six of the genes identified in this study, amphiregulin, TIG1, junB, Gem, sorcin, and CEACAM6, have known cellular roles that would be consistent with the reported (13, 20) growth-suppressing effects of 1,25-dihydroxyvitamin D3 in Caco-2 cells

The possible connection of some of the novel 1,25-dihydroxyvitamin D-upregulated genes identified in Caco-2 cells in this study with the known functional effects of 1,25-dihydroxyvitamin D3 is less evident or even apparently paradoxical. For example, adaptin-{gamma} is a component of the Golgi-derived AP-1 clathrin adaptor complex and is expressed ubiquitously (36). Adaptor proteins of the clathrin coat are classically viewed as mediating the sorting of cargo protein passengers into clathrin-coated pits and the recruitment of clathrin into the budding area of the donor membrane, and recent evidence suggests that AP-1 can interact with microtubules in the cell (35). How these actions relate to 1,25-dihydroxyvitamin D3 function is presently unknown. Likewise, ceruloplasmin is a well-known systemic copper transport protein produced primarily in the liver. Ceruloplasmin has a proposed role in angiogenesis (37) and has been observed to be over expressed in renal cancer (42) and other human cancer cells (27). How local ceruloplasmin production in enterocytes induced by 1,25-dihydroxyvitamin D specifically influences 1,25-dihydroxyvitamin D-mediated actions in the enterocyte will require additional study. Carbonic anhydrase XII is a recently discovered member of the {alpha}-carbonic anhydrase gene family (45) and has a suggested role in von Hippel-Lindau gene-mediated carcinogenesis (24). It is normally found in the large intestine in humans, is dramatically expressed in colorectal tumors (26), and may create a favorable microclimate for tumor invasion by acidifying the immediate extracellular milieu surrounding cancer cells (24). Increased expression of carbonic anhydrase XII in response to 1,25-dihydroxyvitamin D3 treatment would presumably favor tumor growth, which would be paradoxical in the face of a general antiproliferative effect of 1,25-dihydroxyvitamin D3.

In summary, exposure of Caco-2 cells to 1,25-dihydroxyvitamin D3 induces changes in gene expression in a select number of genes. Two of these vitamin D-induced genes (24-hydroxylase and amphiregulin) have been previously reported to be regulated by vitamin D and function in vitamin D catabolism and cell growth, respectively. Our study also identified a number of novel 1,25-dihydroxyvitamin D-regulated genes. The known functions of some of these genes suggest these may be instrumental in cellular calcium homeostasis and the antiproliferative effects of 1,25-dihydroxyvitamin D3 in Caco-2 cells. The roles of some other genes that we identified to be regulated by the hormonal form of vitamin D remain more ambiguous.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This material is based upon work supported by the U.S. Department of Agriculture, under agreement No. 58-1950-4-401. Any opinions, findings, conclusion, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the United States Department of Agriculture, and should not be construed as an official United States Department of the Army position, policy, or decision, unless so designated by other documentation. Approved for public release; distribution unlimited.


   ACKNOWLEDGMENTS
 
Editor P. S. Meltzer served as the review editor for this manuscript submitted by Editor R. E. Pratt.


   FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: R. J. Wood, Mineral Bioavailability Laboratory, USDA HNRCA at Tufts Univ., 711 Washington St., Boston, MA 02111 (E-mail: richard.wood{at}tufts.edu).

10.1152/physiolgenomics.00002.2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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