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© 2007 American Society for Nutrition J. Nutr. 137:2359-2365, November 2007

Biochemical, Molecular, and Genetic Mechanisms

Conjugated Linoleic Acid Alters Global Gene Expression in Human Intestinal-Like Caco-2 Cells in an Isomer-Specific Manner1–3,

Eileen F. Murphy4, Guido J. Hooiveld5, Michael Muller5, Raffaele A. Calogero6 and Kevin D. Cashman4,*

4 Department of Food and Nutritional Sciences, and Department of Medicine, University College, Cork, Ireland; 5 Nutrition, Metabolism and Nutrigenomics Group, Division of Human Nutrition, Wageningen University, Wageningen 6700 EV, The Netherlands; and 6 Bioinformatics and Genomics Unit, Department of Clinical and Biological Sciences, University of Turin, Regione Gonzole 10, 10043 Orbassano, Italy

* To whom correspondence should be addressed. E-mail: k.cashman{at}ucc.ie.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Conjugated linoleic acid (CLA) exhibits isomer-specific effects on transepithelial calcium (Ca) transport as well as on cell growth in human intestinal-like Caco-2 cells. However, the molecular mechanisms of action are still unclear. Therefore, this study used a transcriptomic approach to help elucidate the molecular mechanisms underlying such isomer-specific effects. Caco-2 cells were treated with 80 µmol/L linoleic acid (control), 80 µmol/L trans-10, cis-12 CLA, or 80 µmol/L cis-9, trans-11 CLA for 12 d. Ca transport was measured radio-isotopically. RNA was isolated from the cells, labeled, and hybridized to the Affymetrix U133 2.0 Plus arrays (n = 3). Data and functional analysis was preformed using Bioconductor. Using a minimum fold-change criterion of 1.6 and a false discovery rate criterion of P-value ≤ 0.05, trans-10, cis-12 CLA altered the expression of 918 genes, whereas, cis-9, trans-11 CLA had no effect on gene expression. Gene ontology analysis revealed that trans-10, cis-12 CLA strongly modulated a number of processes inherently related to carcinogenesis, such as cell cycle, cell proliferation, and DNA metabolism. Trans-10, cis-12 CLA, but not cis-9, trans-11 CLA, increased transepithelial Ca transport in Caco-2 cells, which corresponded to changes in molecular mediators of paracellular (including claudin 2 and 4) and transcellular (calbindin D9k and vitamin D receptor) Ca transport. This microarray-based study highlighted a number of gene expression patterns of relevance to 2 important intestinal processes (carcinogenesis and Ca transport), which were modulated by trans-10, cis-12 CLA. These may help our mechanistic understanding of the role of CLA in promoting gut function and health.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Conjugated linoleic acid (CLA)7 is a collective term describing a mixture of positional and geometric isomers of linoleic acid with conjugated double bonds, which may be of cis or trans configuration at positions 9 and 11 or 10 and 12. The cis-9, trans-11-CLA isomer is the most abundant isomer in meat and dairy products, with lesser amounts of the trans-10, cis-12-CLA present (1). Both of these isomers appear in almost equal concentrations in most dietary supplements (2) and have been shown to possess biological activity (3). However, isomer-specific effects have been documented in relation to some biological processes (3–8).

Considerable attention over the last 10 y has focused on the possible beneficial effects of dietary CLA on body composition, lipoprotein metabolism, inflammation, and carcinogenesis (9–13). Intestinal epithelial cells, as the first interface with dietary CLA, are exposed to the highest concentration of CLA isomers in vivo. It is not surprising, therefore, that CLA has received research interest in relation to its effects on gut function and health. For example, numerous in vivo studies with experimental animals have shown that CLA inhibits carcinogenesis in the colon (13). In addition, CLA (in particular trans-10, cis-12 CLA) has been shown to inhibit cell growth and proliferation in a number of colon tumor cell lines, including Caco-2 cells derived from a human adenocarcinoma (5,14–19).

There are also various lines of experimental evidence to suggest that CLA enhances the efficiency of intestinal absorption of calcium (Ca) (6,20). In the Caco-2 cell model, which is useful for predicting Ca absorption in human subjects (21), chronic exposure (2–3 wk) to CLA (in particular trans-10, cis-12 CLA) significantly increased the rate of paracellular and transcellular Ca transport across the epithelial monolayers (20).

Although the molecular mechanisms of action of CLA on intestinal cellular processes, including Ca absorption and cell growth and differentiation, are not understood, it is clear that the CLA isomers are highly bioactive compounds and are capable of regulating, either directly or indirectly, a number of processes in the intestine and other tissues. Many of these effects are likely to be mediated at the level of the transcriptome. Thus, the availability of techniques such as microarray technology, which allows the investigation of the effects of substances such as CLA on gut function by measuring the expression of thousands of genes simultaneously, offers the potential to gain valuable insights into the possible mechanisms of action. House et al. (22) recently used this approach to examine the effect of the trans-10, cis-12 CLA isomer on gene expression in a polygenic obese line of mice and revealed a number of candidate genes involved in adipose delipidation.

Therefore, to better understand the molecular mechanisms by which CLA affects intestinal cells, we compared the effects of 2 common dietary-derived isomers of CLA, namely trans-10, cis-12 and cis-9, trans-11 CLA, on global gene expression (54,000 probe sets) in human intestinal-like Caco-2 cells using microarray technology. In addition to investigating the effects on global gene expression, particular emphasis was given to the analysis of the effects of CLA isomers on gene expression patterns of relevance to processes involved in Ca absorption and carcinogenesis due to the available functional data for modulatory effects of CLA on these processes [Ca transport (23,24) and carcinogenesis (25–30)].


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
    Materials. 45Ca (as 45Ca in an aqueous solution of CaCl2, with a specific activity of 1.85 MBq/mg Ca) was purchased from Nensure. The cis-9, trans-11 and trans-10, cis-12 isomers of CLA were purchased from Cayman Chemicals. The HU133A 2.0 Plus arrays and associated reagents were purchased from Affymetrix. All other reagents were purchased from Sigma Aldrich Ireland.

    Conditions of cell culture and cell treatments. The human colon adenocarcinoma cell line, Caco-2, was purchased from the American Type Culture Collections. Cells were maintained as described previously (6). For Ca transport and gene expression experiments, cells were seeded at a density of 6 x 104/cm2 into Transwell (Costar) inserts and 6-well culture plates (Costar), respectively. Cells were cultured in complete culture medium containing 10% fetal bovine serum for the first 2 d and then with media containing either 80 µmol/L linoleic acid, 80 µmol/L trans-10, cis-12 isomer of CLA, or 80 µmol/L cis-9, trans-11 isomer of CLA (as the nonesterified forms of the fatty acids) for an additional 12 d. In addition, for the purposes of the Ca transport studies only, cells were treated with vehicle or 10 nmol/L 1, 25-dihydroxycholecalciferol (positive control) for 24 h prior to Ca transport experiments. The concentration of CLA (80 µmol/L) was chosen on the basis of the earlier estimate of typical luminal concentrations in the human small intestine of an individual with a daily dietary CLA intake of ~150 mg (6). Linoleic acid was chosen as the control fatty acid, because it is an essential fatty acid and the parent of the CLA isomers. All compounds were added to complete culture medium prior to addition to the cells. In all studies, at least 3 wells were examined per treatment. Experiments were repeated 3 times.

    Transepithelial Ca transport studies. The method used for determining Ca transport across the 14-d-old Caco-2 cell monolayers in this study has been described in detail previously (6). In brief, the transport buffer contained 1.2 mmol/L CaCl2 and 45Ca (with an activity of 148 mBq/L). Transport of 45Ca over 60 min was assessed by measuring nmol·L transported–1·min–1·well during the 30- to 60-min time interval. Fluorescein transport after 60 min across the monolayer was used to measure paracellular (diffusional) transport across the Caco-2 monolayer. The amount of 45Ca appearing in the basolateral buffer was expressed as a percentage of the total 45Ca applied to the upper chamber. This represented total transepithelial 45Ca transport (i.e. by both the paracellular and transcellular transport routes) and was expressed as percent per hour. By subtracting the percentage of fluorescein transport per hour from the total 45Ca transport (%/h), the amount of 45Ca crossing the Caco-2 cell monolayer by the transcellular (active) route was calculated and expressed as %/h. In all studies, at least 3 wells were examined per treatment. Experiments were repeated 3 times.

    RNA isolation, processing, and microarray analysis. After experimental treatments, total RNA was extracted from the Caco-2 cells, quantified, and processed to biotinylated complementary RNA, as described previously (8). Biotinylated complementary RNA was fragmented and each sample was hybridized to an Affymetrix HU133 2.0 Plus array (54,645 probe sets) at 42°C for 17 h, then washed, stained, and scanned to generate digitized image data files following the standard Affymetrix protocol.

    Microarray data analysis. Three biological replicas were generated for each experimental condition (80 µmol/L linoleic acid, 80 µmol/L trans-10, cis-12 CLA, and 80 µmol/L cis-9, trans-11 CLA). Microarray quality control and statistical validation were performed using Bioconductor (31). For a detailed method of the statistical analysis performed, please see Supplemental Information. Background correction, normalization, and probe set intensities were obtained by means of GCRMA (32). The number of genes evaluated was reduced by applying an interquartile (IQR) filter to remove the nonsignificant probe sets (i.e. not expressed and those not changing) (33). To assess differential expression, we used an empirical Bayes method (34) together with a false discovery rate (FDR) correction of the P-value (35). The fold-change threshold was defined, taking advantage of the absence of significant differences in gene expression between cis-9, trans-11 CLA- and linoleic acid-treated cells, as the mean of cis-9, trans-11 CLA vs. linoleic acid fold-changes plus 2 SD |log2(fold-change)|0.70 (equivalent to an absolute fold-change of 1.6). Thus, the list of differentially expressed genes was generated using an FDR ≤ 0.05 (35) together with an absolute fold-change (fold-change) threshold of 1.6 (i.e. |log2(fold-change)|≥0.7) and further refined by a grading scale (i.e. grade A) developed by Affymetrix (36) and by selecting those probe sets mapping to unique Entrez Gene identifiers (37). Principal component analysis (PCA) (38) was used to investigate the overall behavior of the 3 experimental conditions. The data discussed in this publication have been deposited in the NCBI Gene Expression Omnibus (39) and are accessible through GEO Series accession number GSE6518.

    Bioinformatic data analysis. Gene ontology (GO) provides a restricted vocabulary as well as clear indications of the relationships between biological terms and genes (40). In this study, to identify enriched GO terms in the set of differentially expressed genes, genes were categorized according to 2 independent ontologies for gene products using EASE (41): 1) biological process; and 2) molecular function after partitioning the differentially expressed genes into those up- and downregulated upon treatment with trans-10, cis-12 CLA.

Gene Set Enrichment Analysis (GSEA) is a computational method that determines whether an a priori defined set of genes shows significant, concordant differences between 2 biological states (e.g. phenotypes). The method derives its power by focusing on gene sets, i.e. groups of genes that share common biological function, chromosomal location, or regulation. In this study, GSEA was used to identify pathways enriched by the trans-10, cis-12 isomer (42), particularly in relation to those of relevance to carcinogenesis and Ca transport, as the central interests of this study. It exploits the idea that alterations in gene expression might manifest at the level of biological pathways rather than at the level of individual genes. GSEA provides an enrichment score (ES) that measures the degree of enrichment of the pathway at the top (upregulated in trans-10, cis-12 CLA vs. linoleic acid) or bottom (downregulated in trans-10, cis-12 CLA vs. linoleic acid). A nominal P-value was used to assess the significance of the ES. All statistics were based on 1000 iterations.

    RT and quantitative PCR analysis. Single-stranded cDNA was synthesized from 1 µg total RNA as described previously (8). Quantitative RT-PCR (qRT-PCR) was performed on a MyIQ thermal cycler (Bio-Rad) using Platinum Taq DNA polymerase (Invitrogen) and SYBR green (Molecular Probes). Primer sequences used in the real-time qRT-PCR were chosen based on the sequences available in GenBank. Primers were designed using Beacon Designer software (version 2) and Primer 3 (43) to generate a PCR amplification product of 100–200 bp. All samples were analyzed in duplicate and normalized to cyclophilin (as a constitutively expressed control gene).

    Statistical analysis of Ca transport and qRT-PCR data. For the transepithelial Ca transport studies and gene expression studies, data for all variables were normally distributed and allowed for parametric tests of significance. Data are presented as means and SEM. Treatment effects on Ca transport were assessed by 1-way ANOVA with variation attributed to treatment compound: linoleic acid; cis-9, trans-11, and trans-10, cis-12 CLA isomers; 1,25-dihydroxycholecalciferol; and vehicle (control) (44). To follow-up the ANOVA, all pairs of means were compared by Tukey's multiple comparison test (44). Differences in gene expression (via qRT-PCR) between CLA-treated (cis-9, trans-11 and trans-10, cis-12 CLA isomers, separately) and linoleic acid-treated cells were assessed by unpaired Student's t tests (44).


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
    Global gene expression profile in CLA-treated cells. Of the 54,645 probe sets on the HU133 2.0 Plus array, 6417 (11%) were found to remain after applying the IQR filter of 0.3. Linear model analysis indicated that the number of probe sets with apparently altered expression from Caco-2 cells treated with the cis-9, trans-11 CLA relative to linoleic acid (the parent and control fatty acid) was less than expected by random chance. Conversely, using a minimum fold-change criterion of 1.6 and a FDR criterion of P-value 0.05, 1417 probe sets were differentially expressed in Caco-2 cells treated with the trans-10, cis-12 CLA vs. linoleic acid. Further refinement by grading scale (i.e. grade A) developed by Affymetrix (36) and for probe sets mapping to unique Entrez Genes identifiers showed that 918 Entrez Genes identifiers were regulated by trans-10, cis-12 CLA treatment relative to linoleic acid. Among these Entrez Genes identifiers, 406 (44.2%) were upregulated and 512 (55.8%) were downregulated.

As a complimentary approach to our statistical analysis, PCA analysis (38) was used to investigate the overall behavior of the 3 experimental groups in the IQR filtered subset. The first component accounted for the greatest part of the variability within the dataset (95.8%). Therefore, the differences between the samples were graphically represented using the first 2 components of the PCA analysis only (Supplemental Fig. 1). Consistent with the linear model analysis, the effect of the trans-10, cis-12 CLA isomer on gene expression was very different to that of linoleic acid, whereas the effect of the cis-9, trans-11 CLA isomer seemed to be very similar to that observed with linoleic acid (Supplemental Fig. 1).

    GO analysis of the effects of trans-10, cis-12 CLA on gene expression profiles. To investigate the global effects of trans-10, cis-12 CLA on gut cell biology, we categorized the genes according to GO. When the differentially expressed genes from trans-10, cis-12 CLA-treated Caco-2 cells were partitioned into those up- and downregulated, among the GO biological process terms found to be enriched with upregulated genes included lipid metabolism, cell proliferation, organic acid metabolism, cell differentiation, and alcohol metabolism (Table 1). On the other hand, GO terms enriched with genes downregulated by trans-10, cis-12 CLA included, among others, cell cycle, DNA metabolism, cell proliferation, and amine metabolism (Table 2). Analysis of the molecular function category showed that a wide variety of GO terms were found to be enriched in both the up- and downregulated list of differentially expressed genes (Tables 1 and 2). Catalytic and transcription factor activities were enriched with the upregulated genes, whereas DNA binding and cytoskeleton terms were enriched with downregulated genes. In general, the trans-10, cis-12 CLA isomer altered a number of genes involved in cell growth and metabolism.


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  		TABLE 1 Two independent GO (molecular function and biological process) for gene products overrepresented (P < 0.01) for upregulated genes in Caco-2 cells treated with trans-10, cis-12 CLA

 

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  		TABLE 2 Two independent GO (molecular function and biological process) for gene products overrepresented (P < 0.01) for downregulated genes in Caco-2 cells treated with trans-10, cis-12 CLA

 
    Effect of trans-10, cis-12 CLA on carcinogenesis-related gene expression. Of the various biological processes strongly modulated by the trans-10, cis-12 CLA isomer, a number are known to be inherently related to carcinogenesis, such as cell cycle, cell proliferation, and DNA metabolism (Tables 1 and 2). Using only those genes "present" (as defined by applying IQR filter) in these 3 processes (cell cycle, cell proliferation, and DNA metabolism), ~22, 19, and 21% of genes, respectively, were significantly overrepresented for downregulated genes in the Caco-2 cells treated with trans-10, cis-12 CLA. Of the 918 CLA modulated genes, 67 were found to be associated with cell cycle (GO:0007049) (Supplemental Table 1), 45 with DNA metabolism (GO:0006259) (Supplemental Table 2), and 56 with cell proliferation (GO:0008283) (Supplemental Table 3), accounting in total for ~16% of the regulated genes. GSEA identified an enrichment for downregulated genes by the trans-10, cis-12 CLA isomer in the KEGG cell cycle pathway (normalized ES = 1.98; FDR = 0.02) (Supplemental Fig. 2).

    Effect of CLA isomers on functional Ca transport. As expected, treatment of Caco-2 cell monolayers with 10 nmol/L 1,25-dihydroxycholecalciferol (positive control) increased (P < 0.05) the total transepithelial and transcellular Ca transport, but not paracellular Ca transport, relative to that in control (vehicle only-treated) monolayers (data not shown). Treatment of Caco-2 cells with 80 µmol/L linoleic acid (control fatty acid treatment) did not affect the rate of Ca transport (total, transcellular, or paracellular) relative to that in control (vehicle only-treated) monolayers (data not shown).

Treatment of Caco-2 cells with 80 µmol/L cis-9, trans-11 CLA for 12 d did not affect the rate of Ca transport (total, transcellular, or paracellular) relative to those treated with 80 µmol/L linoleic acid (Fig. 1). Treatment of Caco-2 cells with 80 µmol/L trans-10, cis-12 CLA for 12 d increased total transepithelial (P < 0.0001), transcellular (P < 0.0001), and paracellular Ca transport (P = 0.002) relative to those treated with 80 µmol/L linoleic acid (Fig. 1).


Figure 1
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  		FIGURE 1  Effect of 80 µmol/L linoleic acid (LA), 80 µmol/L cis-9, trans-11 CLA (c9, t11), and 80 µmol/L trans-10, cis-12 CLA (t10, c12) on Ca transport in Caco-2 cells. Values are means ± SEM, n = 9 or 10. Means for a variable without a common letter differ, P < 0.002.

 
    The effect of CLA on Ca transport-related gene expression. Because the process of transcellular Ca transport is not available as a pathway or GO category, known key genes, which encode mediators of this process, were selected. Microarray analysis showed that calbindin D9k and vitamin D receptor (VDR) gene expression in Caco-2 cells exposed to trans-10, cis-12 CLA was significantly upregulated relative to that in linoleic acid-treated cells (Table 3). However, expression of a number of other genes that have a central role in the process of transcellular Ca absorption, including transient receptor potential channel, subfamily V, member 6 (TRPV6), and TRPV5 (also referred to as calcium transport channel and epithelial calcium channel, respectively, and the basolateral plasma membrane calcium ATPase isoform 1b) were unaffected by trans-10, cis-12 CLA treatment, at least at the transcriptional level.


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  		TABLE 3 Confirmation of microarray expression by quantitative qRT-PCR

 
Using GSEA, we identified an enrichment for upregulated genes by the trans-10, cis-12 CLA isomer in the KEGG tight junction pathway (Supplemental Fig. 3) (normalized ES = 1.70; FDR = 0.20). A number of genes encoding proteins that have a role in the tight junction between neighboring intestinal [(Caco-2) cells were found to be significantly up- (such as claudin 2, claudin 4, Ras-related protein Rab-3B, myosin heavy polypeptide 14, and related RAS viral (r-ras) oncogene homolog)] and downregulated [erythrocyte membrane protein band 4.1-like 2 and cyclin-dependent kinase (CDK) 4] by treatment with trans-10, cis-12 CLA relative to that in linoleic acid-treated cells. As noted previously, treatment of Caco-2 cells with the cis-9, trans-11 CLA isomer did not affect gene expression, including those involved in Ca transport, relative to that in linoleic acid-treated cells.

    Confirmation of microarray expression by qRT-PCR. The microarray-based expression data for a select number of genes, differentially regulated by trans-10, cis-12 CLA, were validated using real-time qRT-PCR. These included: the 2 genes found to be the most highly regulated, i.e. ubiquitin thiolesterase and sucrase isomaltase; genes involved in Ca transport (calbindin D9k, VDR, and claudin-4) for which the corresponding functional data are reported; PPAR{gamma}, which has been previously shown to be upmodulated by CLA treatment of colonocytes (45); and ATP-binding cassette, sub-family A, member 1, as a representative of a novel CLA-regulated gene observed in this study. There was a good concordance between the microarray and real-time qRT-PCR-based expression data for all 7 genes (Table 3). In addition, real-time qRT-PCR confirmed that for these genes, gene expression did not differ between the cis-9, trans-11 CLA- and linoleic acid-treated cells (Table 3).


   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
In this study, the lack of effect of cis-9, trans-11 CLA on global gene expression is consistent with the apparent lack of effect of this isomer on various intestinal-related biological processes (4–6,16–19,46,47). In contrast, the trans-10, cis-12 isomer had a major impact on global gene expression in Caco-2 cells (with 918 genes differentially expressed), which at least fits reasonably well with the apparent multiple intestinal effects attributed to this isomer (4–6,16–19,46,47). In particular, the processes of cell cycle, DNA metabolism, lipid metabolism, and cell proliferation were regulated by the trans-10, cis-12 isomer, which supports the isomer-specific effects observed at the cellular level reported in several studies of CLA-treated intestinal cells (15,17–19,46,47). Other processes regulated by the trans-10, cis-12 isomer included amino acid metabolism, organic acid metabolism, alcohol metabolism, and cytoskeleton organization and biogenesis. These wide-ranging effects are worthy of more in-depth investigation and offer the potential for future research that may link them to functional outcomes. In this study, we decided to pay particular attention to gene expression profiles for a limited number of biological processes of relevance to intestinal cell function, namely carcinogenesis and transepithelial Ca transport, for which, as mentioned previously, in vivo evidence supports a modulatory effect of CLA. This is important because, despite the in vivo observations of apparent beneficial effects of CLA on these processes, the underlying molecular mechanisms are as yet unclear.

In this study, GSEA analysis showed that the cell cycle pathway was strongly downregulated by the trans-10, cis-12 isomer. At an individual gene level within the cell cycle pathway, the transcription of a number of genes was up- [CDK inhibitor 1A (p21) and cell division cycle 2B] and downregulated (CDK2, CDK4, stratifin) by trans-10, cis-12 CLA. p21 inhibits the activity of cyclin-CDK2 or -CDK4 complexes and functions as a negative regulator of cell cycle progression at G1. These molecular findings in Caco-2 cells are consistent with findings in HT-29 cells, another colon cell line, which showed that trans-10, cis-12 CLA and not cis-9, trans-11 CLA induces G1 arrest through increased p21 protein levels and reduced activity of CDK4 and CDK2 (15,19). p21 also interacts with proliferating cell nuclear antigen and plays a regulatory role in S phase DNA replication and DNA damage repair. Thus, the transcriptional upmodulation of p21 may be a key component of the antiproliferative effect of the trans-10, cis-12 isomer observed in several studies using colon cancer cells (15,19,48). Consistent with the inhibition of cell cycle activity, a number of genes related to DNA synthesis were downregulated by trans-10, cis-12 CLA.

CLA induces apoptosis in a variety of cell systems (5,18). Activating transcription factor 3 and nonsteroidal antiinflammatory drug activating gene-1, 2 important pro-apoptotic factors (49,50), were upregulated by treatment of Caco-2 cells with trans-10, cis-12 CLA in this study. These findings are consistent with the recent findings of Lee et al. (47) that showed trans-10, cis-12 CLA, but not cis-9, trans-11 CLA, stimulated nonsteroidal antiinflammatory drug activating gene-1 expression by the activation of activating transcription factor 3 in apoptotic colon cancer cells. Our isomer-specific molecular findings would support and help explain observations at the cellular level whereby trans-10, cis-12 CLA, but not cis-9, trans-11 CLA, reduces cell growth (5,15,18,47), decreases DNA synthesis (5,18), and induces apoptosis (5,18) in colon cancer cell lines. The trans-10, cis-12 CLA isomer altered the expression of a number of nuclear receptors (such as PPAR{gamma} and VDR), which may underpin such wide-ranging effects of CLA on colon carcinogenesis; however, further investigation is warranted. Such studies should ideally be conducted in proliferating Caco-2 cells, which may be more appropriate than the differentiated cells in this study that have a small intestine phenotype. In addition, because the treatment was for 12 d, the use of time points may help delineate the direct and indirect effects of trans-10, cis-12 CLA on gene expression in Caco-2 cells.

In addition to highlighting gene expression patterns of relevance to carcinogenesis, the microarray analysis showed that a number of genes involved in transcellular and paracellular Ca transport (which together determine the overall rate of Ca transport across the intestine) were modulated by trans-10, cis-12 CLA, but not cis-9, trans-11 CLA. These isomer-specific effects are consistent with those reported on functional transepithelial Ca transport in Caco-2 cells in this and previous studies (6,8). The results of transcriptional profiling indicate that trans-10, cis-12 CLA upregulated the expression of calbindin D9k and the VDR, which are key genes encoding important mediators of transcellular Ca transport. This is the first study, to our knowledge, that shows that calbindin D9k can be altered at the transcription level by a nutritional factor other than 1,25-dihydroxycholecalciferol, which is the primary regulator of intestinal Ca absorption efficiency (51). Trans-10, cis-12 CLA did not affect other key genes involved in transcellular Ca transport (e.g. TRPV6, TRPV5, and the basolateral plasma membrane calcium ATPase isoform 1b). These observations suggest that CLA may enhance the vitamin D-mediated process of Ca transport. A possible interaction between CLA and 1,25-dihydroxycholecalciferol is currently under investigation that will provide more information on how trans-10, cis-12 CLA may enhance the vitamin D-mediated process of Ca transport.

The microarray data showed that the trans-10, cis-12 CLA-mediated increase in paracellular Ca transport was associated with an enrichment of genes upregulated by this isomer in the KEGG tight junction pathway. In particular, the trans-10, cis-12 isomer of CLA upregulated the transcription of members of the claudin family (e.g. claudin 2 and claudin 4), which encode protein components of the tight junction complex between neighboring intestinal cells. The claudins form ion-selective pores within the tight junction strands (52) and thus could be prime candidates in regulating paracellular Ca transport. However, the exact role of the individual claudin proteins within the functionality of the tight junction has still to be fully elucidated and, thus, the physiological meaning of the differential regulation of the expression of these claudin genes by the trans-10, cis-12 isomer of CLA is as yet unclear. Furthermore, tight junctions are regulated in part by their affiliation with the F-actin cytoskeleton (53). Interestingly, genes involved in the regulation of actin cytoskeleton (myosin, heavy chain 14) and paracellular permeability by Rho (ras homlog gene family, member B) were upregulated. A number of cytoplasmic scaffolding molecules associated with the tight junctions such as RAB3B and related RAS viral oncogene homolog were upregulated, whereas erythrocyte membrane protein band 4.1-like 2 and CDK4 were downregulated. These proteins are thought to have a regulatory role in the formation of a diffusion barrier (54) and may underpin the effects of CLA on paracellular permeability.

In conclusion, this is the first study to our knowledge to use whole genome-wide expression analysis to examine the isomer-specific effects of CLA in an intestinal cell model. Whereas cis-9, trans-10 CLA had no effect, trans-10, cis-12 CLA had a major impact on global gene expression. This microarray-based study has highlighted a number of gene expression patterns of relevance to the 2 important intestinal processes (carcinogenesis and Ca transport), which were modulated by trans-10, cis-12 CLA. These may help our mechanistic understanding of the role of this bioactive food component in promoting gut function and health. The trans-10, cis-12 CLA-induced alterations in expression of these genes (and indeed of other novel genes), which may be of potential importance in chemopreventative and Ca transport mechanisms, warrant further investigation and should be followed by functional studies.


   FOOTNOTES
 
1 Supported by Safefood, The Food Safety Promotion Board, Ireland and by the EU-funded European Nutrigenomics Organisation (NuGO) Network of Excellence. Back

2 Author disclosures: E. F. Murphy, G. J. Hooiveld, M. Muller, R. A. Calogero, and K. D. Cashman, no conflicts of interest. Back

3 Supplemental Figures 1–3, Supplemental Tables 1–3, and supplemental text are available with the online posting of this paper at jn.nutrition.org. Back

7 Abbreviations used: CLA, conjugated linoleic acid; CDK, cyclin-dependent kinase; p21, cyclin-dependent kinase inhibitor 1A; ES, enrichment score; FDR, false discovery rate; GO, gene ontology; GSEA, gene set enrichment analysis; IQR, interquartile; PCA, principal component analysis; qRT-PCR, quantitative RT-PCR; TRPV6, transient receptor potential channel, subfamily V, member 6; VDR, vitamin D receptor. Back

Manuscript received 30 January 2007. Initial review completed 8 March 2007. Revision accepted 10 September 2007.


   LITERATURE CITED
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 

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