Changes in the transcriptional profile of transporters in the intestine along the anterior-posterior and crypt-villus axes

The absorption of biologically active compounds occurs via passive transcellular, paracellular and carrier-mediated transport mechanisms [1]. Analysis of the human genome sequence suggested the presence of 406 genes encoding ion channels and 883 genes encoding transporters [2]. Generally, these proteins establish the electrochemical gradient across membranes and provide the means for transporting electrolytes, amino acids, dipeptides, monosaccharides, monocarboxylic acids, organic cations, phosphates, nucleosides, and water-soluble vitamins [3, 4]. Frequently, transporters play a direct role in the absorption of bioactive compounds from the intestinal lumen. The bioavailability of some compounds can depend significantly on carrier-mediated systems and, thus, are sensitive to drug-drug and drug-food interactions; however, these interactions tend to be more relevant for bioactive molecules with low bioavailabilities [5].

The mRNA expression profiles of several functionally-defined transporter families have already been measured by real-time PCR [6, 7]; yet, for a majority of biologically active compounds it remains unknown which transporters play a role in their absorption. Although the human genome project has made the identification of most, if not all, genes encoding transporters and ion channels possible, only a few studies have focused on intestinal transporter expression using such genome-wide strategies [8–11]. Therefore, insight into the expression profiles of transporters along the intestinal tract enables a physiologically relevant assessment of their potential as drug- and nutrient-carriers.

Even less is known about the expression of transporters along the crypt-villus axis [12]. The extensive automated literature data mining by Olsen et al. [12] revealed that more transporters are known to be expressed in the villi than in the crypts. Many transporters that are villus-specific have been implicated in absorption processes, such as the oligopeptide transporter Slc15a1, facilitated glucose transporter Slc2a10, and the sodium/glucose cotransporter Slc5a1 [13]; whereas the crypt-specific, basolaterally expressed Na+/HCO3 co-transporter (Slc4a4) is essential for intestinal anion secretion. Similarly, the mulitrdrug resistance protein 1 (Abcc1), is crypt-specific, basolaterally expressed and acts as a secretion pump for various compounds [14, 15]. On the other hand, P-glycoprotein (Abcb1) is villus-specific, expressed on the apical site and acts as well as a secretion pump for a variety of drugs [5, 16]. Although exceptions may be found, one can assume that villus-specific transporters might be more efficient as mediators of absorption as their surface availability is more extensive.

Messenger RNA levels may not always correlate with the expression of encoded proteins [17]. Although use of proteomic techniques increasingly serves to resolve discrepancies between mRNA and protein levels, proteomics of integral membrane proteins still remains a challenge [18]. Also, protein levels do not necessarily correlate with protein activity. Various studies indicate that genomic profiling in combination with data mining of chemotoxicity databases can be an efficient strategy to identify new putative drug carriers [19, 20].

A first step in identifying genes relevant to drug absorption in the intestine is to obtain a molecular catalogue of all expressed mRNAs. In this study, we used the high-density oligomer microarray by Affymetrix to measure the mRNA expression levels of genes expressed in four intestinal regions (duodenum, jejunum, ileum, and colon) in the mouse. We identified all genes with transporter activity according to the Gene Ontology (GO) consortium system that are represented on the microarrays. Then, we examined specific transporter classes that are significantly changed along the intestine and compared our findings to publicly available human microarray data. Finally, the mRNA expression of some selected transporters was measured along the crypt-villus axis using laser capture microdissection.

A crucial step in classifying genes into different molecular functions is the use of a consistent and universal classification system and a precise annotation of Affymetrix probe sets. Therefore, we included the following features in our analysis: i) Mapping of single probes to all transcripts referred to in the UniGene database and attributing of annotation tags to each probe set, and ii) assigning of flags to differentially regulated probe sets. Similar to Chalifa-Caspi et al. [23], we have seen that the annotation provided by Affymetrix (NetAffx) is not entirely accurate. In the Affymetrix annotation a probe set is represented by at most one UniGene cluster, while, conducting our own mapping of Affymetrix probes onto UniGene, we observed that in a number of cases multiple UniGene identifiers can be associated to a given probe set.

Based on our quality criteria, 48 % of all probe sets in the murine genome were found to have a "high" quality annotation on the microarrays used in this study. Thus, selecting only "high" quality genes establishes a high degree of confidence regarding the correct annotation of probe sets.

We observed that the genes on the microarrays are not represented by an equal number of probe sets. Hence, by counting each probe set as a unit in the GO classification system the way it is done by a majority of GO classification programs such as MappFinder, OntoExpress and the one provided by NetAffx, a bias may be introduced [24, 25]. Moreover, to our knowledge, none of the programs publicly available provides a confidence assessment regarding the significantly regulated functional classes. Therefore, we developed a software, the so-called ISREC Ontologizer (Io), that takes these factors into account. The use of the flags (i.e., 1, F, G) provided with the Io software permits such an assessment to be made. As indicated in Figure 2, a significant portion of the results have a flag "F" and were, therefore, considered ambiguous. Some of these "flagged" results, based on UniGene identifiers, could be due to the fact that some of the probe sets are associated with the same UniGene cluster, but are specific for different splice variants. However, we observed a similar portion of ambiguous results (i.e., flag "F") when doing the classification based on RefSeq identifiers and concluded that the ambiguity associated with flagged genes may stem from probe design rather than the differential expression of splice variants [26].

Some transporters are subject to post-translational modification and of course, one of the processing steps is translocation to the membrane. However, if one considers mRNA to be representative of function, then the majority of transporters can be expected to have similar activity along the length of the intestine. In this regard, it is interesting to note that a remarkable fraction of transporters annotated with "carrier activity" are differentially regulated along the A-P axis and could therefore significantly influence the absorption of carrier-mediated bioactive compounds. Our GO classification results indicate that, in the context of the whole transcriptome, transporters do not seem to be an especially dynamically regulated class, which differs slightly to the conclusions made by Bates and colleagues [9]. However, a direct comparison is difficult to make, as the classification system used by Bates et al. was not in accordance with the official GO classification system, and this may underlie the different biological conclusions made between the two studies. Furthermore, more importantly, while that study included only 4 % of all annotated transporters (6), this study covers 76 % and, provides the first extensive overview of the genomic profiles of transporters in the intestine. Similar to their study, the most pronounced difference between adjoining regions was observed for the ileum and the colon. Although, in general, the differentially expressed transporters were more highly expressed in the small intestine than in the colon, a surprisingly high number of transporters were identified as being more highly expressed in the colon than in the small intestine [13]. Many of these transporters have been described as highly expressed in the colon and involved in the bi-directional transport of electrolytes and fluids, which is the principal role of the colon [27]. However, the majority of known drug and nutrient transporters or carriers of bioactive compounds [28] are not differently expressed between the small intestine and the colon.

The exploitation of transporters, which are highly expressed in the colon or at similarly high levels as in the small intestine is especially interesting for compounds such as proteins and peptides, which are susceptible to the high enzymatic activity present in the small intestine [29]. For example, Slc6a14, which transports neutral and amino acids, might be an interesting candidate to be targeted for carrier-mediated absorption. Generally, we observed that most of the differentially regulated "carriers" were not specifically over-expressed in any of the three small intestinal segments, but overall more highly expressed in the small intestine versus the colon. Thus, the ileum, which contains smaller concentrations of pancreatic enzymes, could be a suitable tissue for compounds that are sensitive to the activity of pancreatic enzymes.

Genes classified as having "carrier", "channel", "ion transport" and "electron" and "neurotransmitter" activity are most likely to have different expression levels between the small intestine and colon and may, hence, play a more significant role in the difference of active drug and nutrient transport in these two anatomically distinct regions.

The expression profiles of the most strongly regulated genes (i.e. absolute fold change of 4 between the segments) agreed with previous findings in mice (cf. Table 2). Moreover, comparing transporter expression profiles in mice and humans suggests that gut transcriptional profiles obtained in mice are a valid estimator for the situation in humans (Table 2 and Table 3).

There are several methods that can be used to characterize the patterns of expression of a target gene. Traditionally, labeled antisense specific probes have been widely used for in situ hybridization to detect specific gene expression in the intestinal epithelium [12, 30]. This technique, however, is very labor-intensive when several candidates are being tested. There are several techniques used to isolate the epithelial layer from the lamina propria of the intestine [31]. These methods use mechanical procedures and detergents and/or EDTA to detach the epithelial layer. Separation of crypts from villi epithelium can be achieved following adequate separation procedures. However, alterations of gene expression patterns depending on the procedure as well as cross-contamination from different tissue compartments are possible. Similarly, laser dissection microscopy has been developed to isolate single cells [32]. A key step in this procedure is the selection of an adequate fixation procedure that prevents RNA degradation and preserves tissue histological integrity [33, 34]. The protocols used in the present study allows the assessment of a minimally altered gene expression pattern that is concordant with in situ hybridization experiments (M. Rumbo unpublished data).

Within this context, our data suggest that the changes in expression profiles measured in whole intestinal tissue extracts are a suitable predictor for changes in epithelial cell-only gene expression along the crypts and the villi; however, it should be mentioned that all genes in our comparison were already known to be expressed in the epithelium. This good correlation could reflect the fact that these genes are solely expressed in the epithelium or that the epithelium contributes much more to the isolated RNA than underlying layers of intestinal tissues and, therefore dominates the genomic profile of the intestine. Thus, for genes which are not solely expressed in the epithelium the prediction based on measurements in whole tissue might not be appropriate.

For some compounds, depending on the enzymatic stability and solubilization characteristics, targeting of carriers in distinct intestinal regions may selectively improve their absorption and bioavailability. Thus, we analyzed the expression profiles of known drug carriers along the intestine. The major part of inorganic phosphorus (P_i) absorption from the small intestine occurs via a Na⁺-dependent phosphate co-transporter, NaP_i-II_b encoded by the gene SLC34A2. In vivo pharmacokinetic studies in rats have indicated that foscarnet is transported across the enterocytes by the NaP_i-II_b system and thus acts as a competitive inhibitor of P_i uptake [28]. Slc34a2 mRNA has been shown to be expressed in the small intestine and the colon [35], which is in accordance with our findings. Our studies further demonstrate that the expression significantly increases along the A-P axis of the small intestine. As a consequence, the most efficient transport rate is expected to arise in the ileum. In addition to the findings by Hilfiker et al. [35] that Slc34a2 protein is expressed on the apical membrane of mature enterocytes in the duodenum, we observed that Slc34a2 mRNA is villus-specific along the whole intestine except for the duodenum, reinforcing the concept that transporters expressed in the villi epithelium tend to be implicated more in absorptive processes.

Slc10a2 (ASBT), the primary carrier for Na⁺-dependent bile salt uptake from the intestinal lumen by the ileum, has been shown to be present in the brush-border membrane of the terminal part of the ileum, but little is known concerning its regulation along the intestine [36, 37]. Various studies indicate that based on substrate examples ASBT could be an important target for increasing the bioavailability of various bile acid conjugates [28]. Our findings confirm that Slc10a2 is especially highly expressed in the ileum compared to proximal small intestinal regions; however a comparable level was measured in the colon. Interestingly, only in the ileum was Slc10a2 identified as being villus-specific which could be an indication for its specific role in the ileum.

The four members of the ABC families, Abcg8, Abca1, Abcc1 and Abcc3, that we studied are known to act as secretion pumps in the intestine [14, 38, 39]. In accordance with earlier findings, Abcc3 expression increases from the anterior to posterior part of the intestine [10, 40]. On the other hand, our findings for Abcc3 did not indicate an increase in mRNA expression from the crypts to the villi as shown by Rost et al. [40] in rats. The mRNA levels of Abcc1 along the crypt-villus axis were in accordance with protein expression [14]. Generally, all four members may play a significant role along the whole small intestine as secretion pumps, whereas in the large intestine, Abcg8 is not expected to be involved in the secretion of compounds.

The transporter Slc28a2 is known to be involved in the transport of nucleoside analogues such as some antiviral compounds [41]. Nucleosides are relatively hydrophilic molecules and their ability to be transported across cell membranes is a critical determinant of their metabolism [42]. Our data indicate that similar uptake rates can be expected along the whole intestine when targeting this carrier, as it is similarly expressed in the gut. On the other hand, Slc2a1, which was significantly (p ≤ 0.05) more highly expressed in the colon, might be an appropriate drug-carrier for compounds sensitive to pancreatic enzymatic degradation. It has to be noted though that the protein product of SLC2A1, GLUT1, is not detectable in healthy human colon tissue, but only in colon cancer serving as a marker for poor prognosis [43, 44]. Thus, it may be an interesting drug-target/drug carrier in individuals with colon cancers.

In contrast to GLUT1, SLC5A8 has been shown to be highly expressed in the colon and to act as a tumor suppressor gene. SLC5A8 is silenced by methylation in most human colon tumors and reintroduction of this gene leads to growth suppression [21]. Recent studies have shown that SLC5A8 acts as a Na+-coupled transporter for short chain fatty acids and monocarboxylates. Its transport rate is strongly inhibited by various drugs such as ibuprofen. We have shown for the first time that this gene is expressed at similar levels in the distal regions of the gut and seems to be preferentially expressed in the villi of the small intestinal enterocytes. Summarizing, this Na+-coupled transporter may be involved in the active transport of known monocarboxylate drugs and serve as a valuable drug-carrier for new drugs along the whole intestine.

In conclusion, we have characterized the mRNA expression profiles of 76 % of all currently known transporters along the A-P axis of the gut. We have identified various transporters that could serve as carriers for biologically active compounds. The significant regional specificities are likely to correspond to functional differences along the length of the intestinal tract.

Finally, we have presented a comprehensive analysis of regional variations in gene expression using whole gut tissue that is sufficiently sensitive to provide a good assessment of relative changes in regional gene expression in epithelial cells; however, for the functionality of a given transporter to be unequivocally determined, it will be critical to examine these epithelial cells in a cell-type specific manner.

In the following, the term "transporter" will refer to all genes that are classified as genes with "transporter activity" according to the GO system. At the date of analysis the ATP-binding cassette (ABC) transporters were not classified as genes with "transporter activity". Thus, this class has not been integrated the GO analysis. However, in the analysis of expression levels on a gene by gene basis the ABC transporters have been integrated.

Please note that according to the official human HUGO and mouse MGI system we will refer to human gene symbols in upper case, and to mouse genes with a first letter in capital and the subsequent ones in lower case.