Early studies suggested that only the proximal region of the mammalian small intestine is involved in iron transport and in the adaptive response to iron-deficiency (27
). In fact, known iron-responsive genes such as DMT1 have been described to be induced in only the proximal portion of the gut (4
). However, the current data clearly show that DMT1 and duodenal cytochrome b, genes known to be involved in transepithelial iron transport, also are strongly induced at the gene level in more distal intestinal segments of iron-deficient rats. This is a significant finding, as induction of mRNA expression for both of these genes is known to translate into increased protein levels. Many other novel genes induced by iron-deficiency also were regulated similarly in both gut segments, and some of these genes may play unidentified roles in intestinal iron and metal ion homeostasis. Overall, the current data demonstrate that induction of iron transport-related genes was equivalent or greater in jejunum as compared to duodenum.
Our previous studies indicated that the gene chips are highly reliable and accurate, as real-time PCR confirmed changes in expression of many of the genes that are now shown to also be regulated in more distal portions of the small intestine (6
). Furthermore, in the current investigation, gene chip data were analyzed utilizing very strict reduction strategies to minimize the possibility of reporting false positives. Additionally, these presented data include many genes that have been reported to be regulated by dietary iron intake levels by other investigators using different techniques, such as Northern and Western blots and immuno-histochemical methods. Thus, the current presented data have a very high probability of being accurate.
We previously found that the brush-border membrane iron transport-related genes (DMT1 and Dcytb) were more strongly and consistently induced with iron deprivation than the genes encoding the basolateral membrane proteins, hephaestin and IREG1 (6
). This observation is now extended to show the same trend in the jejunum of 12-week-old, iron-deficient rats. We further demonstrated that a host of other genes also was induced in the duodenum of iron-deprived rats, and we now show that many of these genes are also induced in the jejunum of 12-week-old, iron-deficient rats. These genes include the Menkes copper ATPase (APT7A) and metallothionein. This may suggest that under iron-deficient conditions, DMT1 functions to transport copper into enterocytes, which leads to induction of ATP7A and metallothionein. We have demonstrated that DMTl and ATP7A are also strongly induced at the protein level, an that ATP7A is present in brush-border and basolateral membrane domains in the duodenum of iron-deficient rats (Ravia et al., 2005). Aditionally, a brush-border membrane ferric reductase has been reported to reduce dietary copper (15
), and DMT1 has been shown to transport reduced (e.g., cuprous) copper (2
). These observations thus may explain why iron-deficient rats have significantly increased liver copper levels. Moreover, the fact that these genetic responses are conserved throughout the duodenum and
jejunum strongly suggests a functional coupling between iron and copper transport-related genes in the iron-deficient state.
Other novel genes also were induced by iron, deprivation in both the duodenum and jejunum of 12-week-old rats. Some of these genes encode proteins with known roles in intestinal iron homeostasis, including transferrin receptor 1, heme oxygenase 1, and prolyl 4-hydroxylase. Additionally, another strongly induced gene in both gut segments was the sodium-dependent vitamin C transporter, which may increase vitamin C absorption, from the interstitial fluids and which, in turn, could enhance the reducing capacity of a brush-border membrane ferric reductase (15
). Other genes with unknown roles in iron homoeostasis also were induced in duodenum and jejunum, including tripartite motif protein 27 and integrin alpha 6. Tripartite motif protein 27 is one of the most consistently induced genes in our previous and the current studies, along with DMT1, Dcytb and APT7A, suggesting potential physiological relevance. Integrin alpha 6 is very strongly expressed and also very consistently induced by iron, deprivation, and we have noticed that for highly expressed genes, the gene chips tend to underestimate changes in expression. Thus, tripartite motif protein 27 and integrin alpha 6 are of particular interest for further study.
A host of other genes showed induction of at least three-fold in only the duodenum, while no genes were found to be induced at least three-fold only in the jejunum. These data suggest that the effects of iron-deficiency are more profound in the proximal small intestine, as judged by the number of genes being differentially expressed. Some genes induced solely in the duodenum were reported previously (6
); however, different probe sets representing these genes were identified in the current studies. These genes include aquaporin 4 and two probes sets recognizing SRY-BOX containing gene 9 (SOX9). Altogether, three distinct probe sets showed strong induction of SOX9 in only the duodenum of 12-week-old, iron-deficient rats. Other novel genes uniquely induced in the duodenum have not been associated previously with intestinal iron transport or metal homeostasis; however, some of these genes, such as gastrokine 1, claudin 2, and trefoil factor 1, are known to play important roles in gut physiology. Their relationship to iron deficiency is currently unknown. Another group of novel genes was found to be induced in both gut segments or uniquely decreased in one or the other gut segment, but involvement of these genes in iron transport and gut homeostasis is unknown.
Oligonucleotide microarray techniques have been utilized in many areas of mammalian physiology to identify novel genes involved in various metabolic processes. Surprisingly, this experimental approach had not been utilized to explore the effect of iron deprivation on the global expression of genes in the small intestinal epithelium, until our previous studies were reported in January of 2005 (6
). However, Marzullo et al. (18
) recently utilized differential display reverse-transcription PCR to identify differentially expressed transcripts in the intestine of iron-deficient rats. Their studies did not identify any of the genes reported in the current communication, with the exception of DMT1, whereas our previous and current studies did not identify the genes they reported (cytochrome C oxidase [COX} subunit II mitochondrial gene, and serum and glucorticoid-regulated kinase). These discrepancies may be due to the different experimental methods used or to differences in experimental design. It also should be noted that many genes involved in intestinal iron transport have been identified by techniques designed to identify differentially expressed transcripts [e.g., Dcytb identification by cDNA subtraction (19
)] or by methods designed to detect changes in protein function [i.e., expression cloning of DMT1 in Xenopus
In summary, the current analysis of over 28,000 rat transcripts demonstrates that many genes are similarly regulated in the duodenum and jejunum of iron-deficient rats. The fact that some known iron-responsive genes were strongly induced in both gut segments was unexpected, as previous studies have suggested that only the proximal intestine is responsible for iron transport. Also of particular interest is the fact that the Menkes copper ATPase and metallothionein were coordinately regulated along with DMT1 and Dcytb, suggesting a “functional coupling” of these genes. These observations further demonstrate the complex nature of intestinal iron transport and metal ion homeostasis.