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4 Department of Molecular Nutrition, Kagawa Nutrition University, 3-9-21 Chiyoda, Sakado, Saitama 350-0288, Japan; and 5 Department of Pharmacology, Saitama Medical University, 38 Moro-Hongo, Moroyama, Iruma, Saitama 350-0492, Japan
* To whom correspondence should be addressed. E-mail: sakuma{at}eiyo.ac.jp.
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ABSTRACT |
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 TOP ABSTRACT IntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Introduction |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Nutrients and food materials that are thought to be effective in preventing osteoporosis include calcium (3), vitamin D (3), vitamin K (4), milk basic protein (5), soybean isoflavones (6), and fructooligosaccharides (FOS)6 (7). FOS are indigestible oligosaccharides that have been shown to increase calcium absorption (7). Many balance studies using rats have shown that FOS increase calcium absorption and appear to exert effects, mainly in the colon, based on changing calcium levels in luminal contents in the various segments of the gastrointestinal tract (8,9). Furthermore, when the cecum, the main area of FOS fermentation, was excised from rats, FOS did not promote calcium absorption (10), suggesting that instead of FOS themselves promoting calcium absorption, as undigested FOS reach the colon through the small intestine, fermentation products of FOS produced by intestinal bacteria promote calcium absorption. However, the details of the increased calcium absorption resulting from FOS ingestion have not been elucidated.
The present study focused on the genes involved in calcium absorption in the gastrointestinal tract and regulation of their expression. Calcium absorption by the gastrointestinal tract has 2 routes, transcellular and paracellular (3). The following molecules are reportedly involved with the transcellular route: transient receptor potential vanilloid type 6 (TRPV6/CaT1/ECaC2), expressed in the luminal side of small intestine mucosal epithelial cells for absorbing calcium into cells; calbindin-D9k, binding with calcium inside cells and transporting calcium; and plasma membrane calcium-ATPase 1b (PMCA1b), expressed in the basolateral side and transporting calcium from cells to blood (3). We reported that in the gastrointestinal tract of rats fed a FOS diet, levels of calbindin-D9k protein significantly changed in a segment-specific manner (8), and in the colon where calcium absorption is promoted, levels of calbindin-D9k protein correlated to the rate of calcium absorption and changes occurred at the transcriptional level (11). Furthermore, cDNA expression arrays were used to comprehensively analyze genetic changes in the gastrointestinal tract of rats fed the FOS diet (12).
The present study clarified changes in the mRNA expression of TRPV6, calbindin-D9k, and PMCA1b genes. Furthermore, SCFA were ascertained as direct mediators. A segment between –71 nucleotides (nt) and the translation start site of the TRPV6 gene was estimated to contain a positive responsive element to butyric acid.
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Materials and Methods |
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Preparation of intestinal samples and RNA isolation. After washing out the luminal contents with cold saline, mucosal cells were scraped from the colorectal segment with a glass slide. Total RNA and poly(A)+ RNA were prepared using Wako ISOGEN (Wako Pure Chemical) and Oligotex-dT30 super (Takara Shuzo), respectively, in accordance with the manufacturers' instructions.
Cell culture. Caco-2 human colon adenocarcinoma cells were obtained from American Type Culture Collection and used in experiments at passage 30–60. Cells were propagated and maintained in high-glucose (4500 mg/L D-glucose) DMEM (Sigma) supplemented with 100 mL/L fetal calf serum (Gibco BRL) and 0.1 mmol/L nonessential amino acids in a 5% CO2 and 95% air incubator at 37°C. Cells were passaged using Dulbecco's PBS containing 0.25% trypsin.
Cell treatments and RNA isolation. Cells were plated at 1.0 x105 cells/cm2 in 6-well plates in quadruplicate and grown for 24 h before the addition of SCFA to the culture medium, as indicated below. For dose response experiments, cells were cultured in DMEM supplemented with 0, 0.5, 1.0, 2.0, or 5.0 mmol/L of sodium butyrate for 12 h and levels of TRPV6 mRNA were measured. Four wells were used for each concentration. For time course experiments, cells were cultured with or without 2.0 mmol/L sodium butyrate. Levels of TRPV6 mRNA were measured 6, 12, 24, and 48 h after adding sodium butyrate. Again, 4 wells were used for each time. Sterile stock solutions of sodium salts of the SCFA (acetate, lactate, propionate, and butyrate) were prepared in PBS. The medium was changed every other day.
Following experimental treatment, cells were washed 3 times with ice-cold PBS. Cells were harvested from each plate and total RNA was isolated. RNA preparation was conducted as described by Favaloro et al. (14) with some modification. Appropriate amounts of homogenate were centrifuged at 2000 x g; 5 min, then cell pellets were retrieved and resuspended in lysis buffer [0.14 mol/L NaCl, 1.5 mmol/L MgCl2, 10 mmol/L Tris-HCl (pH 8.6), 0.5% NP-40, 1.0 x 106 units/L RNase inhibitor]. The suspension was vortexed for 10 s, then the cell suspension was underlaid with an equal volume of lysis buffer containing sucrose (24% wt:v) and NP-40 (1%). Centrifugation was performed at 10,000 x g for 20 min at 4°C, then the cytoplasmic layer was recovered. To this upper layer was added an equal volume of 2x proteinase K buffer [0.2 mol/L Tris-HCl (pH 7.5), 25 mmol/L EDTA, 0.3 mol/L NaCl, 2% wt:v SDS], and proteinase K was further added to a final concentration of 0.2 g/L. After incubation at 37°C for 30 min, the solution was phenol chloroform extracted and ethanol precipitated. The pellet was dissolved in the buffer [50 mmol/L Tris-HCl (pH 7.5), 1 mmol/L EDTA], with addition of MgCl2, RNase inhibitor and RNase-free DNase I were added to a final concentration 10 mmol/L, 1.0 x 106 units/L and 2.0 x 104 units/L, respectively. After incubation at 37°C for 30 min to stop this reaction, EDTA and SDS were added to final concentrations of 10 mmol/L and 0.2%, respectively. This solution was phenol chloroform extracted and ethanol precipitated.
Real-time RT-PCR. First-strand cDNA synthesis and real-time RT-PCR were performed using the TaqMan Reverse Transcription Reagent (Applied Biosystems), TaqMan Universal PCR Master Mix (Applied Biosystems), and Power SYBR Green PCR Master Mix (Applied Biosystems). Primer and probe for rat calbindin-D9k (assay ID Rn00560940_m1), rat TRPV6 (assay ID Rn00586673_m1), and human TRPV6 (assay ID Hs00367960_m1) were obtained from TaqMan gene expression assays (Applied Biosystems). The following primers were used in real-time RT-PCR using SYBR Green for rat PMCA1b sense, 5'-CAC CGT ACT TCA CTT GGG CAA T-3', antisense, 5'-GGC AGG TCA TCC AGA TAC CTG TA-3'. PCR was performed in accordance with the instructions from the manufacturer. Analysis was conducted using an ABI PRISM 7700 Sequence Detector (Applied Biosystems). Gene expression was quantified using the comparative threshold cycle method (15). Target mRNA expression was normalized to that of 18s ribosomal RNA.
Cloning of rat TRPV6 genomic gene. A rat phage genomic library (Clontech Laboratories) was screened using digitoxigenin-labeled rat TRPV6 cDNA. The rat intestinal cDNA library was prepared by amplification of rat intestinal poly(A)+ RNA using RT, as described previously (16). To produce the hybridization probe, rat TRPV6 cDNA was amplified from the rat intestinal cDNA library using primers comprising bases 14–34 and 2793–2824 according to the published rat TRPV6 cDNA sequence (GenBank accession no. AF160798). Digitoxigenin labeling, hybridization, washing, and detection were performed according to the instructions of the manufacturer (Roche Diagnostics). The DNA insert of the positive phage clone was then subcloned into the pBluescript II KS+ vector (Invitrogen) after digestion with restriction enzymes and then sequenced. Sequencing was conducted using a BigDye Termination v3.1 Cycle Sequencing kit (Applied Biosystems) and Applied Biosystems 3730 x 1 DNA Analyzer (Applied Biosystems).
To isolate the rat TRPV6 gene, we searched for conserved elements between ours and other published TRPV6 sequences (GenBank accession nos. AF336378 for mouse, AY225461 for human, and AF453325 for bovine). Sequences were aligned using CLUSTAL W software (17).
The sequence of the region from –278 to +321 revealed the presence of potential recognition sites for trans-factors in a search using P-Mach v.1.0 software (BIOBASE GmbH) (18) and TRANSFAC (19).
5' Rapid amplification of cDNA ends analysis and RNase mapping analyses. Using rat organ intestine full-length cDNA (Seegene), 5' Rapid amplification of cDNA ends (5'-RACE) analysis was performed according to the protocol of the manufacturer. The first PCR was conducted using this cDNA as a template and the 5'-RACE primer 1, which was TRPV6 specific (5'-TGA GCC CAG GAC TCT CGT CTG TGG AAC C-3'). Amplification of cDNA was conducted for 1 min at 94°C and 35 cycles of 1 min at 94°C, 1 min at 58°C, and 1 min at 72°C, followed by a final extension for 10 min at 72°C. The resulting amplicons were subjected to nested PCR with the TRPV6-specific 5'-RACE primer 2 (5'-GCT GCT CTT AGG AGT CCG AAG AGG-3'). Amplification was performed under the same conditions. The resulting PCR products were then cloned using a TOPO TA Cloning kit for Sequencing (Invitrogen) and sequenced using a BigDye Termination v3.1 Cycle Sequencing kit (Applied Biosystems) and a 3730 x 1 DNA Analyzer (Applied Biosystems).
The riboprobe used for RNase mapping covered all 3 transcription start sites that were detected by 5'-RACE analysis and was prepared by subcloning the PvuII-NcoI fragment of our rat TRPV6 clone into the plasmid pSPT18 (Roche Diagnostics). RNase mapping was then performed by mixing the riboprobe with 10 µg of total RNA prepared from rat proximal (upper half) small intestine or from the colorectal segment. Hybridization and RNase treatment were performed as described previously (20).
Plasmids and transfections.
The plasmid pGL3/rTRPV6–2600 containing the Sau3A I-NcoI fragment (–2600 to +293 nt) of the rat TRPV6 gene was inserted into a promoterless luciferase reporter gene (pGL3 Basic vector; Promega). The NcoI site was located at the translation start site. Subsequently, a series of truncated promoter fragments were generated and subcloned into the pGL3-basic vector using the natural restriction sites present in the TRPV6 gene (HindIII, nt position –1442; XbaI, nt position –753; EcoRV, nt position –154; PvuII, nt position –71; DraI, nt position –26; SacI, nt position +42). These were designated pGL3/rTRPV6–1442, pGL3/rTRPV6–753, pGL3/rTRPV6–154, pGL3/rTRPV6–71, pGL3/rTRPV6–154 to –71, pGL3/rTRPV6–154(
–71 to –26), and pGL3/rTRPV6+42.
For transfection experiments, 1.0 x105 cells per well were then split and replated in 6-well plates in quadruplicate. On d 2 of culture, this medium was replaced with serum-free Opti-MEM (Gibco BRL). Cells were then transfected with the constructed 0.4 µg appropriate luciferase reporter plasmids or controls using Lipofectamine 2000 (Gibco BRL) according to the instructions of the manufacturer. The reporter phRL-SV40 (0.04 µg per well) was cotransfected for normalization. Culture medium was changed 3 h after transfection from serum-free Opti-MEM to DMEM containing 10% fetal calf serum. After 12 h of transfection, medium was then removed and replaced by a medium containing either the solvent or sodium salts of butyrate (2.0 mmol/L) or 1,25-(OH)2cholecalciferol(3) (10 nmol/L). At 36 h after transfection, luciferase activity of the cell lysates was determined using a Dual Luciferase Reporter Assay System (Promega) and firefly luciferase activity was corrected for Renilla luciferase activity from the phRL-SV40 plasmid.
Statistical analysis. Results are expressed as means ± SEM. All data were analyzed using SPSS 15.0J for Windows software. Statistical analyses were performed using Student's t test for effects of FOS in rats. Dose response experiments (Fig. 1A) were tested by 1-way ANOVA with post hoc Dunnett's test for comparing groups to the control without sodium butyrate. Time course experiments were tested using Student's t test for comparing identical times (Fig. 1B). For transfection analysis (Fig. 2), data were tested by 1-way ANOVA with post hoc Dunnett's test using pGL3 basic plasmid-transfected cells without any addition as control.
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Results |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Effects of SPCFA on levels of TRPV6 mRNA in Caco-2 cells. Compared with cells incubated with 0 mmol/L of sodium butyrate, TRPV6 mRNA levels were higher (P < 0.05), peaking at 2.0 mmol/L of sodium butyrate (Fig. 1A). Levels of TRPV6 mRNA increased at 6 h after adding sodium butyrate (P < 0.05) and peaked at 12 h (Fig. 1B). Compared with levels of TRPV6 mRNA without SCFA (100%), levels were 4013% higher with sodium butyrate (P < 0.05) and 588% higher with sodium propionate (P < 0.05) (Table 1).
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Supplemental Figure 2 compares our TRPV6 sequence with other published sequences. The transcription initiation site for rat TRPV6 is located in the region from –31 to +4, a region with 100% homology among rat, mouse, and human sequences.
The sequence of the region from –278 to +321 revealed the presence of potential recognition sites for trans-factors such as cellular reticuloendotheliosis (c-Rel), signal transducer and activator of transcription x (STATx), orphan nuclear receptor 
-1 (ROR-1), ecotropic viral integration-1 (Evi-1), cAMP response element binding protein/activating transcription factor (CREB/ATF), nuclear factor-
B (NF-B), hepatocyte nuclear factor-41 (HNF-41), Adenovirus E2 (E2) (Supplemental Fig. 3).
Analysis of the butyric acid-responsive element of the TRPV6 gene.
When 1,25-(OH)2cholecalciferol was added, luciferase activities were mostly comparable to the vector alone, but when sodium butyrate was added, luciferase activities were observed with pGL3/rTRPV6–2633, pGL3/rTRPV6–1442, pGL3/rTRPV6–753, pGL3/rTRPV6–154, and pGL3/rTRPV6–71 plasmids (Fig. 2). Luciferase activity of pGL3/rTRPV6–154 to –71, pGL3/rTRPV6–154(–71 to –26), and pGL3/rTRPV6+42 plasmids were comparable to that of the vector alone (Fig. 2). We thus estimated that the segment between –71 nt and the translation start site contains a positive responsive element to butyric acid.
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Discussion |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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FOS are indigestible oligosaccharides that cause increased calcium absorption in the rat colorectum (8,9), but the mechanisms underlying this absorption have not been clarified. At 2 d after feeding rats the FOS diet, TRPV6 and calbindin-D9k mRNA levels in the colorectum were significantly higher than controls. Studies have shown that vitamin D upregulates transcription of TRPV6, calbindin-D9k, and PMCA1b (3). Each gene has been reported to include a vitamin D receptor (VDR)-binding sequence (21–23), but differing results have been obtained using cultured cells and transgenic mice and many theories have been proposed regarding the location of the VDR-binding sequences in the genome (22,24). Expression of these 3 genes is not completely eliminated in VDR-knockout mice and, furthermore, levels of expression reveal change with a high-calcium diet (25). VDR thus appears to be involved in regulating expression of the 3 genes along with other regulatory factors.
The intake of indigestible oligosaccharides allows bacteria in the large intestinal lumen to produce SCFA and significantly lowers intestinal lumen pH. Low pH reportedly increases calcium absorption in the large intestine (26). However, the addition of SCFA to cultured Caco-2 cells without changing pH reportedly increased paracellular permeability (27). As a result, low pH alone is not responsible for high calcium absorption.
Many studies have reported that SCFA, mainly butyric acid, alter gene expressions. Because the intake of indigestible oligosaccharides lowers the onset of colon carcinoma, several studies have investigated the effects of butyric acid, an SCFA, on Caco-2 cells as human colonic epithelial cells. Butyric acid suppresses proliferation of colon carcinoma cells by increasing expression of p21/WAF1/Cip1, a transcription factor that negatively regulates the cell cycle (28). The antiproliferative effects of butyric acid have also been shown in studies using Caco-2 cells, demonstrating increased expression of VDR and 25-hydroxyvitamin cholecalciferol-1-hydroxylase (29,30). Butyric acid is widely known to inhibit histone deacetylase (31), a suppressor of gene expression, and is thus thought to promote transcription. However, butyric acid does not promote transcription of all genes and instead displays gene specificities. Moreover, butyric acid suppresses transcription of some genes, such as monocyte chemoattractant protein 1 (32). As a result, butyric acid activities cannot be explained by inhibition of histone deacetylase alone, suggesting the existence of unknown transcription regulation mechanisms.
The expression of many intestine-specific genes is regulated by these transcription factors, such as cdx-2, HNF-1 families, GATA families, and C/EBP families (33–35). A study using Caco-2 cells reported that butyric acid increases expression of cdx-2 (36). In the present study, our analysis using Caco-2 cells showed that butyric acid increased levels of TRPV6 mRNA, which could have involved cdx-2. However, cdx-2 expression was not increased by the addition of butyric acid (data not shown). In searching for putative regulatory elements, no consensus sequences for cdx-2, HNF-1 families, GATA families, or C/EBP families were found in the region between –71 nt and the translation start site, the region we estimated to contain the positive responsive element to butyric acid in the TRPV6 gene (Supplemental Fig. 3). These findings suggest the involvement of unknown factors.
Here, we isolated the rat genomic TRPV6 gene and used this to analyze the butyric acid-responsive element. Between our rat genomic clone and AF160798, 11 base mismatches were identified at the 5' terminus of cDNA, attributable to a linker sequence in AF160798. In addition, there was a single-base mismatch at –97 nt from the translation start site in the 5' noncoding region, which was thought to be due to a racial difference between the Sprague-Dawley rats and Norway rats (AF160798). In the present study, adenine at –293 nt from the translation start site was considered as the transcription start site. Reporter assay also showed activity of an expression plasmid, pGL3/rTRPV+42, including +42 nt downstream of the transcription start site that we determined was comparable to that of pGL3 basic. Furthermore, activity of pGL3 basic was comparable to pGL3/rTRPV6–154 to –71 and pGL3/rTRPV6–154(–71 to –26) plasmids. We thus determined that adenine at –267 nt from the translation start site is the transcription start site.
According to Meyer et al. (37,21) studies on the 1,25-(OH)2cholecalciferol-responsive element of TRPV6, the sequence was located at –2 and –4 kb for mice and –2.1 and –4.3 kb for humans. Our analysis of rat genes should have included the 1,25-(OH)2cholecalciferol-responsive element, because the sequence up to –2.6 kb was used. However, with pGL3/rTRPV6–2633 (shown in Fig. 2), activity of the reporter gene was mostly comparable to that of vector-only pGL3 basic. Meyer et al. (37) reported that the rat TRPV6 gene had the 1,25-(OH)2cholecalciferol-responsive element in a comparable site based on sequence analysis, but no empirical data support this point. Whether this difference is due to different sites of the 1,25-(OH)2cholecalciferol-responsive element or a difference between LS180 cells in Meyer et al. (37) and Caco-2 cells in the present study is unclear.
Studies have been conducted on butyric acid-responsive elements examining AGCAAGCTCCAA for the mouse calbindin-D28k gene (38) and 2 Sp1 sites at –82 and –69 nt for the p21/WAF1/Cip1 gene (28). Furthermore, with the p21/WAF1/Cip1 gene, transcription is promoted by Sp3, a transcription factor acting through the 2 Sp1 sites. There was no sequence resembling the butyric acid-responsive element for the 2 genes between –71 nt and the translation start site of the TRPV6 gene, which we estimated as the region containing the positive responsive element to butyric acid. No similarities in base sequence of the butyric acid-responsive element were seen between the calbindin-D28k gene and the p21/WAF1/Cip1 gene. With TRPV6, transcription appeared to be accelerated via a new sequence.
In conclusion, after rats were fed the FOS diet, calcium absorption was accelerated by increasing gene expression of colorectal TRPV6 and calbindin-D9k, which are involved in intestinal calcium absorption. The results of the analysis using cultured Caco-2 cells suggested that SCFA, which is produced by intestinal bacteria, increased gene expression of TRPV6. This study is the first to our knowledge to suggest that factors besides VDR are involved in regulating gene expression of TRPV6. We believe that SCFA does not directly act on the genes but instead transmits signals into cells via some receptor. G protein-coupled receptor 43 is an SCFA receptor expressed in the small intestine but only in enteroendocrine cells expressing peptide YY, not in absorptive epithelial cells, thus suggesting the existence of unidentified receptors (39).
To date, activated vitamin D has been thought to play the central role in calcium absorption, but the present findings indicate that SCFA are also involved in increasing calcium absorption.
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FOOTNOTES |
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2 Author disclosures: A. Fukushima, Y. Aizaki, and K. Sakuma, no conflicts of interest.
3 Supplemental Figures 1–3 are available with the online posting of this paper at jn.nutrition.org.
6 Abbreviations used: FOS, fructooligosaccharide; HNF, hepatocyte nuclear factor; nt, nucleotide; PMCA1b, plasma membrane calcium-ATPase 1b; 5'-RACE, 5' rapid amplification of cDNA ends; TRPV6, transient receptor potential vanilloid type 6; VDR, vitamin D receptor.
Manuscript received 11 July 2008. Initial review completed 21 August 2008. Revision accepted 18 October 2008.
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LITERATURE CITED |
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