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,1
* Wageningen Centre for Food Sciences (WCFS),
RIKILT, Institute of Food Safety, 6700 AE, Wageningen; and
NIZO Food Research, 6710 BA, Ede, the Netherlands.
1Correspondence: WCFS, Nutrition and Health, Bornsesteeg 45, P.O box 230, 6700 AE, Wageningen, the Netherlands. E-mail: jaap.keijer{at}wur.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: red meat • microarray • colon cancer
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. A high consumption of red meat has been linked to an elevated risk for colon cancer (2)
. Colon cancer is a disease of the colonic mucosa caused by a time-dependent accumulation of mutations in genes controlling the renewal of epithelial cells. Epithelial renewal is necessary for maintaining the integrity of the mucosa, repairing mucosal injury, and replenishing the specialized cells of the epithelium. It is a complicated process involving proliferation, migration, differentiation, and apoptosis of cells (3)
. Mutations in genes controlling the renewal processes are probably caused by endogenous processes and may not be caused by dietary mutagens (4
, 5)
. The presence of luminal irritants is regarded as a risk factor, since they damage colonic cells and thereby stimulate continuous regeneration of the epithelium, which increases risk for endogenous mutations. Such irritants can be formed by nutrients and metabolites that are not absorbed in the proximal gastrointestinal tract and thus determine the composition of the colonic contents.
In our previous work, we hypothesized that the association between red meat and colon cancer risk can be explained by the presence of heme in red meat (6
7
8
9)
. We found that heme is converted to a cytotoxic compound in the gut lumen that damages colonic mucosa. Cell damage leads to a compensatory hyperproliferation of mucosal cells, a risk marker for colon carcinogenesis. In a strictly controlled rat study, we demonstrated that dietary heme strongly increases the cytotoxicity of fecal water toward mammalian cells and stimulates proliferation of the colonic epithelium (7)
. These heme effects were observed at heme concentrations mimicking the human diet and were not found with dietary fat (6)
. To gain further insight into the molecular effects of dietary heme on colonic mucosa, we applied cDNA microarray technology, which allows measurement of the expression levels for many genes simultaneously (10
11
12
13)
. Overall, our data show the feasibility of cDNA array expression profiling to analyze gene-modulating effects of a single dietary component in an intact animal system and to characterize dietary heme-induced altered mRNA levels. Most striking was a 10-fold down-regulation of a novel gene, which we have further characterized in this manuscript.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The fatty acid composition of the blend of fat (80% palm fat and 20% corn oil) mimics the ratio of saturated to monounsaturated to polyunsaturated fatty acids in an average human Western diet. Minerals, vitamins, and choline as choline chloride were added to the diets according to the recommendations of the American Institute of Nutrition 1993 (14)
, except for potassium, which was added as tripotassium citrate. For one group the diet was supplemented with 0.5 mmol heme (Sigma, St. Louis, MO, USA) per kilogram diet. The rats consumed food and demineralized drinking water ad libitum. Food intake and body weights were recorded daily. Feces were collected daily during the last 4 days of the experiment and frozen at -20°C. Fecal water was prepared by reconstituting freeze-dried feces with appropriate amounts of double-distilled water to obtain the physiological osmolarity of 300 mosmol/L, as described previously (7)
. Fecal waters were stored at -20°C until analysis. On day 14, 16 randomly selected rats (8 from both diet groups) were used to determine colonic epithelial proliferation. The other rats (again 8 per group) were killed by CO2 inhalation, and the colon was removed and cut open longitudinally. Colonic contents were removed by rinsing in 154 mM KCl and subsequently mucosa was scraped off with a spatula. Scrapings were weighed, quickly frozen in liquid N2, and stored at -80°C for subsequent RNA extraction. The experimental protocol was approved by the animal welfare committee of Wageningen University, Wageningen, the Netherlands.
Colonic epithelial proliferation and cytolytic activity of fecal water
For determination of proliferation, the rats were injected i.p. with [methyl-3H]thymidine (Amersham International, Amersham, UK; specific activity 25 Ci/mmol; dose 100 µCi/kg body weight) in 154 mM KCl on day 14 of the diet. After 2 h, rats were killed and incorporation of [methyl-3H]thymidine/µg DNA was determined as described before (7)
. Cytolytic activity of fecal water was quantified by in vitro erythrocyte lysis assays, according to previously described methods (7)
. In short, 5 or 10 µL fecal water was mixed with saline to a volume of 80 µL. After preincubation for 5 min at 37°C, 20 µL of a washed human erythrocyte suspension was added (final hematocrit 5%) and samples were incubated for 15 min at 37°C. Simultaneously, erythrocytes were incubated in 154 mM NaCl (0% hemolysis) and in double-distilled water (100% hemolysis). Hemolysis was determined by potassium release measured with an inductively coupled plasma atomic emission spectrometer (Varian, Leersum, the Netherlands). Cytotoxicity was expressed as the area under the hemolytic curve relative to the maximal area (at 100% lysis).
RNA isolation and labeling
Rat mucosal scrapings were homogenized in liquid N2 and total RNA (totRNA) was isolated using TRIzol reagent (Invitrogen, San Diego, CA, USA) according to the protocol. Glycogen (10 µg) was added as a carrier in precipitation steps. TotRNA was purified using RNeasy columns (Qiagen, Chatsworth, CA, USA); DNase treatment was not included. Concentrations were determined spectrophotometrically at A260nm and all samples were checked on 1% TAE/agarose gels. For hybridization of cDNA microarrays, RNA from rat colon scrapings was labeled with Cy-5 and a standard reference sample, consisting of a pool of RNA extracted from scrapings from small intestine and colon, was labeled with Cy-3. To generate Cy-5- or Cy-3-labeled cDNA probes, 20 µg totRNA was mixed with 2 µg T21 primer (Isogen) in a final volume of 13.5 µL and heated for 3 min at 65°C (RNA denaturation) and 10 min at 25°C (primer annealing). To synthesize cDNA, 1x first strand buffer (Invitrogen), 10 mM DTT, 0.5 mM dATP, 0.5 mM dGTP, 0.5 mM dTTP, 0.04 mM dCTP, 0.04 mM Cy5-dCTP (or Cy3-dCTP), 15 U RNaseOUT (Invitrogen), and 150 U Superscript II Reverse Transcriptase (Invitrogen) were added to a final volume of 25 µL. The reaction was incubated for 2 h at 37°C. Purification, precipitation, and denaturation of the labeled cDNA were performed as described (15
, 16)
. Before hybridization, the Cy-3- and Cy-5-labeled samples were mixed 1:1 (v/v).
Construction of a subtractive rat colon cDNA library
A subtractive library of cDNA sequences from rat colon was made in order to generate cDNA sequences to be put on the microarray. totRNA, isolated from scrapings of rats that were fed Westernized diets supplemented with dietary calcium, was used as starting material for the subtractive hybridization to ensure that the generated population of messengers is representative for healthy conditions. totRNA isolated from mucosal scrapings of eight rats was pooled and treated with DNase. cDNA synthesis from 1 µg of totRNA was performed using the SMART PCR cDNA Synthesis kit (Clontech, Palo Alto, CA, USA). The cDNA populations were digested with RsaI to create blunt-ended cDNA molecules suitable for adaptor ligation. Adaptor ligation and subtraction were performed according to the protocol of the PCR-Select cDNA Subtraction kit (Clontech). For subtraction, adaptor ligated PCR fragments were used as the 'tester' population and cDNA generated from our control group of rats (fed diets without supplemental calcium or heme) was used as 'driver'. The subtraction procedure was performed to enrich for sequences that are differentially expressed between tester and driver population. The final pool of PCR products was purified using Qiaquick columns (Qiagen). To enable cloning, AmpliTaq (Perkin-Elmer, Norwalk, CT, USA) was used to create an A overhang on both ends of the PCR products; 50 ng of the A-tailed PCR products was used for ligation into the pGEM-T Easy vector and subsequent transformation into Escherichia coli XL-2 Blue supercompetent cells (Stratagene, San Diego, CA, USA). Cells were plated on LB-agar plates containing 100 µg/mL ampicillin, 80 µg/mL X-gal, and 0.5 mM IPTG. Colonies were grown overnight at 37°C. White colonies were selected by a colony picker (Flexys) and inoculated in 200 µL LB medium containing 36 mM K2HPO4, 13.2 mM KH2PO4, 1.7 mM natrium citrate, 0.4 mM MgSO4-7H2O, 6.8 mM ammonium sulfate, 4.4% (vol/vol) glycerol, and 100 µg/mL ampicillin in 96-well plates. After overnight growth, bacterial stocks were stored at -80°C.
Design and manufacturing of rat colon microarrays
The microarrays used for this study were spotted with 2304 cDNA fragments, which can be divided into the following three groups:
1) 30 control sequences (5 sets of 6 genes each): each set contained five luciferase clones (acc. no. M15077) that were arrayed as positive controls, including a 5' end clone (bp 304–978), the middle part (bp 961–1608), the 3' end (bp 1590–2252), and two cDNAs nearly full length (bp 304–2252); intron sequences were excluded. Luciferase mRNA (Promega, Madison, WI, USA) was spiked into all rat RNA samples to check the quality of the RNA labeling reaction. One Salmonella gene fragment (410 bp) was arrayed as a negative control. Each set of six control genes was printed at five different positions on each array.
2) 55 known rat cDNAs (Research Genetics, Huntsville, AL, USA), including cDNAs that represent genes selected for functional relevance in cell turnover, differentiation and carcinogenesis. The identity of these clones was confirmed by resequencing.
3) 2219 randomly selected uncharacterized cDNAs from our rat colon cDNA library (consisting of 
40.000 clones), generated by subtractive hybridization. cDNA fragments were produced by PCR amplification of bacterial stocks using the following primers: 5'-AGGCGATTAAGTTGGGTAAC-3' and 5'-GTGTGAAATTGTTATCCGCT-3'. To check the quality of the PCR-reactions and the length of the amplified fragments, 1 µL of each product was run on 1% agarose/TBE gel. Amplified products were purified using L-50 fine Sephadex. Eluates were evaporated in a speedvac and pellets were dissolved in 10 µL 5x SSC.
cDNAs were printed on silylated slides (CEL & Associates, Los Angeles, CA, USA), using a PixSys 7500 arrayer (Cartesian Technologies, USA) as described (15)
. After printing, microarrays were allowed to dry at room temperature for at least 2 days. Free aldehyde groups were blocked with NaBH4 according to Schena et al. (11)
.
Microarray hybridization and scanning
Prehybridization of the arrays was carried out as described previously (15)
. Hybridization was performed in a Gene frame (Westburg, the Netherlands) in a volume of 25 µL. Arrays were hybridized overnight at 42°C in humid hybridization chamber. After hybridization the arrays were washed and dried as described (15)
. Arrays were scanned using a confocal laser scanner ScanArray 3000 (General Scanning Inc., Watertown, MA, USA). Scans were made with a pixel resolution of 10 micron, a laser power of 90%, a PMT voltage of 80% for the Cy-3 scans, and a PMT voltage of 70% for the Cy-5 scans.
Microarray data analysis
TIFF images generated by the scanner were analyzed with the software package ArrayVision (Imaging Research, St. Catharines, Ont., Canada). Average spot intensities were collected and further processed in Microsoft Excel. Data analysis and normalization were performed as follows: two different correction factors were used to normalize hybridization data. At first the Cy-3-labeled reference samples, which were exactly the same in each hybridization, were used to correct for 1) differences in amount of spotted DNA and 2) differences in hybridization conditions within one array or between arrays. For each spot, the Cy5/Cy3 ratio was multiplied by the median of all Cy-3 signals. The second normalization was performed to correct for 1) differences in the amount of labeled totRNA and 2) differences in the labeling efficiency of the samples. The Cy5 signal of each spot was divided by the median of all Cy5 signals of the array. This value was multiplied by the median of all medians of all arrays. Cy-3 and Cy-5 values equal to or below the background value (intensity of array surrounding area) were excluded from data analysis. Principal component analysis was performed using the software package GeneMaths version 1.5 (Applied Maths, Sint-Martens-Latem, Belgium).
Sequencing and BLAST searches
For DNA sequence analysis, we used the CEQ DCTS kit (Beckman, Fullerton, CA, USA) according to the protocol of the supplier. One microliter of the bacterial stock solution was taken as starting material. Sequences were matched to the GenBank databases, using BLAST-n, BLAST-x, and BLAST-p.
Real-time RT-PCR
mRNA was isolated from totRNA extracts using the Dynabeads mRNA purification kit (Dynal, Great Neck, NY, USA); 500 ng mRNA was used for cDNA synthesis using SuperScriptTM Preamplification System for first strand synthesis (Invitrogen). Each cDNA was diluted (1:25, 1:250, and 1:1000) and aliquots were stored at -20°C. Primers used for PCR are shown in Table 1
. For each reaction, 5 µL of the cDNA dilution was added to 15 µL pre-mix containing 0.5 µM of each primer, 3 mM MgCl2, 2 µL 5x LightCyclerTM-FastStart DNA Master SYBR Green I. The sample was incubated at 95°C for10 min (denaturation), followed by 45 amplification cycles (0 s 95°C, 5 s 60°, 18 s 72°C (slope=20°C/s for all incubation steps) using the LightCyclerTM (Roche, Nutley, NJ, USA). After each cycle, fluorescence was measured at 72°C with an excitation wavelength of 470 nm and emission at 530 nm. PCR products were subjected to melting curve analysis; 0 s 95°C, 10 s 55°C (both with a slope of 20°C/s), 0 s 95°C (with a slope of 0.1°C/s). Each sample was determined in duplicate. Analysis of LightCycler data was performed using the 
Ct method, as described previously (17)
. The rat with the lowest relative expression was used as calibrator.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The heme-fed rats show a slightly reduced gain in body weight (3.1 vs. 3.8 g/day) without a concomitant decrease in food intake. The daily fecal output was slightly increased compared with the control group. The feces of heme-fed rats were softer whereas feces from control rats appeared normal. This may reflect a disturbance of the absorption or secretion function of the colon. Addition of heme to the rat diet resulted in a 1.7-fold increase of the proliferative rate of rat colonocytes (Table 2)
. The cytotoxic activity of rat fecal water was strongly increased (43-fold), resulting in 60% lysis of erythrocytes. These results are consistent with previous observations by Sesink et al. (7)
.
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Hybridization of rat colon-specific microarrays
We generated a rat colon-specific microarray to investigate the effect of dietary heme on colon gene expression levels. The size of the cDNA fragments used for printing on the arrays ranges from 300 to 1300 bp, with an average size of 600 bp. In a control hybridization experiment using a fluorescent probe corresponding to the vector sequence flanking cDNA inserts, we ascertained that all spots on the array contain abundant cDNAs. From rat mucosal scrapings, 300 µg of totRNA could be isolated on average (1.8 µg totRNA/mg scraping). In a typical hybridization experiment with Cy-5-labeled rat colonic RNA, signals above background value were obtained from 1915 spots (83% of the cDNAs) on average. With the reference RNA sample, consisting of a large pool of Cy-3-labeled cDNAs synthesized from RNA isolated from scrapings from small intestine and colon, signals were obtained from 98% of the spots. The reference RNA, labeled in a single reaction, was cohybridized on each array. The signal intensities were similar for each array and used for data normalization. All RNA samples were spiked with luciferase mRNA to check for the efficiency of the reverse transcription reaction as part of the labeling protocol. Strong signals were obtained with all luciferase fragments, establishing efficient cDNA synthesis. Signals from the negative controls, being Salmonella-derived cDNA, were always close to the background value.
Reproducibility of hybridizations
To verify the reproducibility of the hybridization experiments, we made two sets of Cy-5-labeled probes from the same batch of totRNA for all animals and hybridized them to separate arrays. The normalized Cy-5 data of each array were transformed onto a log scale and plotted against those of the duplicate array. Figure 1
is a representation of a typical duplicate hybridization experiment. Scatter plot analysis of the duplicates of all animals showed that only 1.4 ± 1.1% of the spots differed > twofold in signal intensity, on average, indicating good reproducibility. Without normalization of the Cy-5 data, the signal intensities of 20.2 ± 17.8% spots differed > twofold. This emphasizes the benefits of using a reference sample as a normalizer in data analysis. In further analysis, the signal intensities from duplicate hybridizations were averaged for every spot.
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Variance of gene expression between animals
To analyze the effect of heme on gene expression, it is important that differences in gene expression due to heme intake are distinguished from differences in gene expression due to animal-to-animal variability. In scatter plots as well as in direct visual observation of the hybridized arrays, it appeared that within both diet groups the animals showed similar expression profiles with the exception of three animals. These three animals (E33 of the control group and F54 and F62 of the heme group) showed a completely divergent expression profile and showed signal intensities that differed substantially from those of the other members of the group. According to Grubbs et al. (18)
, an outlier test was applied to decide whether these outlying observations are significant and should be rejected. In this test, it is computed whether the highest and lowest value of the group differ unreasonably from the others in the group (tested at a 1% significance level). In the control group, 290 values where significant outliers (13%); all these outlying values belonged to rat E33. Based on this result, rat E33 was rejected. The heme group contained two rats with suspect values, which complicates the calculation somewhat. At first, the outliers were tested for the data set in which rat F54 was left out. A total of 442 significant outliers were observed, which all belonged to rat F54. Alternatively, in case rat F62 was left out, 303 outliers were observed and 295 of them belonged to rat F62. Based on these calculations, both animals were rejected.
An alternative way to study the variation between expression profiles of all individual rats is principal component analysis (PCA), a technique to reduce the dimensionality of the data set (19)
. PCA produces a 3-dimensional plot in which the rats are spread according to their relatedness. The plot confirmed that rat E33, F54, and F62 are outliers (data not shown). We could not identify the reason for their divergent gene expression profile. There were no visual abnormalities with these three rats.
Identification of differentially expressed genes
To identify genes that are differentially expressed in rat colonic epithelium after heme intake, we compared the average hybridization signals of the six rats of the heme group with the average signals of the seven control rats. Scatter plot analysis of the log transformed mean data revealed that the majority of the dots lie relatively close to the diagonal line of identity, representing genes that are expressed equally in the two mRNA populations (Fig. 2
). In the scatter plot, one particular group of dots (encircled in Fig. 2
) falls very far from the line of identity and shows at least 10-fold difference in expression level between both diet groups. Because this is an unprecedented effect of diet on gene expression, we concentrated on the identification of these cDNAs.
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A novel rat gene down-regulated by dietary heme
A reliable sequence was obtained for 13 out of 14 differentially expressed cDNAs; the sequence of 1 clone could not be obtained due to contamination of the bacterial stock. Sequence identification of these strongly down-regulated cDNAs showed that they all represent the same gene. By sequence alignment we deduced the total nucleotide sequence of the mRNA (Fig. 3
). Two cDNA clones with identical sequence include the poly-A-tail of the cDNA and represent the 3' part of the mRNA. The other 11 cDNAs, with inserts in two orientations, represent the upstream part of the mRNA. Comparison with the genomic rat sequence shows that these two sequences are adjacent to each other. An RsaI restriction site is present at the junction of both fragments, which explains why the gene is represented in two parts, since the cDNA subtraction procedure involves a RsaI restriction step. In the genomic sequence, 30 bp upstream of the start point of our cDNA, we found a TATA box, a sequence element typical of the mammalian site for transcription initiation. The total mRNA sequence contains a 657 bp ORF, encoding a protein of 219 amino acids. Blast searches revealed that the cDNA has not yet been reported in public databases. The sequence shows highest similarity (84%) to a functionally unknown mouse gene isolated from mouse colon (BC024348).
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We performed a control hybridization experiment to investigate whether other clones on the array correspond to the sequence of the novel gene. We hybridized the rat colon microarray with a fluorescent 60 mer based on the cDNA sequence of the gene (indicated in Fig. 3
). None of the other clones hybridized with this probe. Differences in hybridization intensities we observed between the 14 cDNAs are within the limit of experimental variation and most likely are due to the fact that the cDNAs differ in length and represent different parts of the gene.
The novel gene encodes a pentraxin
Blast searches in GenBank databases showed that the identified sequence produces significant alignments with pentraxins from rat, mouse, human, and several lower organisms and contains a conserved domain typical for the family of pentraxins. The gene was therefore identified as a novel pentraxin and baptized Mucosal pentraxin, Mptx. Pentraxins are a family of 25 kDa proteins that form a pentameric structure. The consensus pentraxin domain comprehends 206 a.a. and is preceded by a signal peptide, a signal for entering the secretory pathway. The best-known pentraxins are C-reactive protein (CRP) and serum amyloid P component (SAP), both serum proteins involved in immunological responses. Mptx shows 44 and 47% identity to rat CRP and SAP, respectively. Based on sequence similarity to SAP and CRP, it is likely that Mptx contains a 19 a.a. signal sequence. The SignalP World Wide Web server (www.cbs.dtu.dk/Services/SignalP) also predicts the presence of a secretory signal peptide in Mptx and indicates position 19-20 as the most likely cleavage site. A notable difference between the a.a. sequence of Mptx and the conserved pentraxin domain could be detected as a lack of 4 amino acid residues at position 196-199. This gap is present in Mptx but also in the unidentified mouse colonic pentraxin, which is 84% identical to rat Mptx.
Genomic organization of Mptx and homologues
The Mptx sequence was checked against the rat genome database, which consists of 68 unordered pieces of Rattus Norvegicus genomic DNA. In the output, the Mptx gene showed homology to two different genomic contigs, which indicates that Mptx consists of two exons. The putative exon-intron boundaries conform to the GT-AG rule for eukaryotic split genes (20)
. The rat sap gene, encoding the pentraxin serum amyloid P component, is organized in exactly the same way on chromosome 1, with an intron splice site at the same position as in Mptx. The genomic organization of crp is not known. Comparison of the uncharacterized mouse gene BC024348 to the mouse genome revealed that this gene is organized in a similar way on mouse chromosome 1. The two exons are interrupted by a 1556 nt. intron. Mptx shows significant similarity to human cDNA XM_060355.2, which is most likely a human homologue of Mptx. The uncharacterized human sequence was identified to contain a pentraxin domain, and the encoded protein shows 58% similarity to Mptx.
Lightcycler-based real-time RT-PCR
Finally, we used LightCycler-based real-time PCR to validate expression levels of the gene Mptx. Mptx expression levels of five control animals and six heme-fed animals as well as average values are shown in Fig. 4
. To normalize the PCRs for the amount of RNA added to the reverse transcription reactions, the gene aldolase was used as internal control gene. This gene was detected at similar expression levels in all rats in our microarray experiments and therefore identified as a valid reference. We chose not to use the gene GAPDH, commonly used as a reference gene, since our results indicated that mRNA levels are modulated by dietary heme. Moreover, continuing reports emphasize that GAPDH expression is not constant and therefore inappropriate as a normalizer (21)
. In Fig. 4
the data show the fold change in gene expression relative to rat F46, which has the lowest expression and designated as a calibrator. As Fig. 4
shows, real-time PCR confirms the results of our array experiments, showing strong down-regulation of Mptx in response to dietary heme, although with a higher magnitude (factor 18 on average). There is considerable variation in Mptx gene expression levels between individual rats of the control group. The differences are likely to be caused by variation in the metabolic response to diet between individuals, as can be expected in an in vivo system. Variation might be due to some heterogeneity of the samples, which is inevitable with colon scrapings.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Heme is thought to be play a role in the association between red meat and colon cancer risk observed in epidemiologic studies. Heme is involved in formation of a luminal cytotoxic factor (7)
, which causes epithelial damage. To restore damage done to cells and regenerate the epithelial monolayer, increased cell proliferation and/or reduced apoptosis can be expected. In agreement with reports by Sesink et al. (6
7
8
9)
, our results show that heme increases the cytotoxicity of fecal water and elevates the proliferative rate of rat colonic mucosa. Our results show for the first time that this heme-induced increase in cell turnover leads to a 10-fold down-regulation of the Mptx gene.
To identify heme-regulated genes, an expression microarray was produced containing a limited set of genes consisting of >2000 cDNAs from a subtractive rat colon cDNA library. This array was used to find heme-regulated genes by comparing gene expression in colonic mucosa of heme-fed rats and control rats. Although manufacturers offer a number of premade arrays nowadays, dedicated expression arrays offer the advantage to study those genes typically expressed in a particular tissue, such as colonic mucosa in our study. Furthermore, novel genes can be identified that have not yet been reported in public databases. To emphasize this point, we also used our RNA samples to hybridize commercial rat gene chips from Affymetrix (Genechip Rat U34A) containing 7000 full-length sequences and 1000 EST clusters from the UniGene database and Clontech Atlas Rat 1.0 arrays, containing 1081 cDNA fragments. However, none of these genes was up- or down-regulated > threefold (data not shown). These commercial arrays do not contain the Mptx gene. Therefore, the strong effect on Mptx observed on our arrays is highly remarkable.
On our cDNA arrays, >80% of cDNAs gave signals at sufficient levels for detection whereas a similar mRNA sample gave signals for only 40% of the spots on Clontech Atlas Rat 1.0 arrays. The redundancy of cDNA clones on our expression array was not determined. Expression profiles were analyzed in duplicate for all individual rats. The high reproducibility of our results is, in our opinion, due to normalization using the Cy-3-labeled internal standard sample. In published expression profiling work, the reproducibility often is not given because RNA used for probe preparation is mostly generated from pooled samples, since the amount of mRNA generated from tissue samples is usually low. In contrast, our experimental design allows for analysis of gene expression changes and the magnitude of these changes across all individual rats of the population.
Three of 16 rats showed completely divergent expression patterns. These three rats showed dissimilar expression levels for the majority of the evaluated genes, possibly due to a different genetic background of the outbred animals. Excluding these animals precluded these effects from being falsely attributed to diet effects. These results emphasize the advantage of expression profiling of individual subjects of a population instead of using pooled samples. Nevertheless, inclusion of these rats did not alter the heme-induced down-regulation of Mptx observed in our study.
Real-time quantitative RT-PCR measurements confirmed the effect of heme on Mptx expression, although with a higher magnitude. We recently used RT-PCR measurements to analyze Mptx expression in colon samples from rats that were fed dietary heme in a completely independent but similar rat feeding experiment. We again confirmed the strong down-regulation of Mptx (by a factor of 8). Magnitude differences between array and RT-PCR experiments have been reported before and may be attributable to limitations in the sensitivity of both methods (21)
. The large effect of dietary heme on colonic expression of Mptx is unprecedented in in vivo dietary exposure studies. In a comparable rat study in which modulation of intestinal gene expression in response to zinc deficiency was studied, only small expression changes were reported (22)
. For all other genes on our array, only moderate expression changes were observed between both diet groups. Still, these moderate changes might be important in colonic mucosa in response to dietary heme. Their significance remains to be determined in future.
Blast search in a database containing fragments of the rat genome revealed that the transcript of MPtx most likely originates from two exons. The rat genome is not entirely sequenced yet, which leaves the possibility that additional genomic homologues are present. However, in the human and mouse genome only a single copy was identified.
Mptx shows high homology to the family of pentraxins. This family consists of proteins that contain a carboxyl-terminal pentraxin domain (a highly conserved region of 200 a.a.), preceded by a signal peptide, a signal for entering the secretory pathway (23)
. Pentraxins are generally 25 kDa and are characterized by a discoid arrangement of five noncovalently bound subunits. They have been recognized in human, mouse, rat, hamster, and several lower organisms, but their function is still unclear. They show calcium-dependent binding to a variety of ligands, such as carbohydrates, membrane phospholipids, chromatin, bacterial lipopolysaccharide, and extracellular matrix components such as laminins (24
25
26
27
28)
.
CRP and SAP, the earliest described pentraxins, have been reported to be produced in liver, brain, heart, and arteries. They are acute phase proteins whose concentrations in the blood increase strongly upon infection or trauma. Although their exact functions are unclear, it is believed that pentraxins play a role in inflammation (25)
. Pentraxins were shown to be induced by inflammatory cytokines and to interact with complement components to activate the classical complement cascade as part of the host defense system. It was hypothesized that they are mainly involved in recognition and clearance of self-antigens released from apoptotic and necrotic cells (26
, 27)
. CRP and SAP were shown to bind to phagocytic receptors, such as Fcg-receptor, to mediate phagocytosis of autoantigens in order to protect against development of autoimmunity (28)
.
MPtx and its mouse homologue are the first pentraxins reported that are expressed in colonic tissue. In line with the reported activities of pentraxins, MPtx might play a role in the recognition and clearance of dying cells in colon. We speculate that continuous intake of dietary heme results in a new steady state in colonic cell turnover in which apoptosis might be inhibited in order to limit cell loss. It is remarkable that MPtx is expressed at high levels in healthy animals and that intake of heme, which exerts cytolytic activity in colon, induces down-regulation of MPtx. This suggests that Mptx might represent a marker for diet-induced stress of colonic mucosa. To further investigate this, we are now studying the effects of dietary calcium on expression of Mptx. We earlier reported that dietary heme-induced colonic cytotoxicity and epithelial hyperproliferation are inhibited by dietary calcium (9)
. Preliminary results show threefold down-regulation of Mptx in animals given dietary heme and calcium, which indicates that dietary calcium also inhibits the heme-induced down-regulation of Mptx (data not shown).
In summary, our results support the utility of cDNA microarray technology to study levels of gene expression in response to nutrition in an intact animal system. A new rat gene, named MPtx, could be identified that is >10-fold down-regulated in response to dietary heme and encodes a pentraxin-like protein. MPtx might be involved in dealing with damaged cells in colonic mucosa. Therefore, it might represent a molecular marker for diet-induced stress of mammalian colonic mucosa. To strengthen this supposition, the response of MPtx expression to other dietary compounds is currently under investigation.
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ACKNOWLEDGMENTS |
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Received for publication October 29, 2002. Accepted for publication March 10, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ct method. Methods 25,402-408[CrossRef][Medline]
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  C. van der Meer-van Kraaij, E. Kramer, D. Jonker-Termont, M.B. Katan, R. van der Meer, and J. Keijer Differential gene expression in rat colon by dietary heme and calcium Carcinogenesis, January 1, 2005; 26(1): 73 - 79. [Abstract] [Full Text] [PDF]
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