1Departments of Pediatrics and 2Nutritional Sciences, Steele Memorial Children's Research Center, University of Arizona, Tucson, Arizona; and 3Department of Medicine and 4Division of Gastroenterology, University of Washington Medical Center, Seattle, Washington
Submitted 29 October 2004 ; accepted in final form 4 January 2005
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
iron deficiency anemia; ATP7a; copper transport; intestine; microarray; gene chip
Several proteins involved in duodenal iron absorption have recently been identified with the use of iron deprivation in murine models that leads to increased expression of genes related to iron transport. Duodenal cytochrome b (Dcytb) is a ferric reductase that reduces iron to Fe2+ (27), which is then transported into epithelial cells by the divalent metal transporter 1 [DMT1; also called DCT1 (16) and nRAMP2 (39)]. Within enterocytes, iron is either sequestered within protein complexes of ferritin or trafficked to the basolateral membrane for export into the circulation. Extrusion across the basolateral membrane is likely accomplished by the coordinated action of the basolateral iron transport protein iron-regulated gene 1 [IREG1 (28); also called MTP1 (1) and ferroportin (8)] and hephaestin, which oxidizes iron for binding to transferrin and distribution throughout the body in the circulation (40). Despite the identification of these described genes over the past several years, a complete understanding of the molecular events associated with intestinal iron absorption has not yet been achieved. This fact is exemplified by the microcytic anemia mice (11) and Belgrade rats (10), which are able to absorb substantial amounts of dietary iron despite the lack of normal DMT1. Additionally, sex-linked anemia (sla) mice, which have a deletion in the hephaestin gene that eliminates 194 amino acids in the putative protein, have substantial accumulation of iron within enterocytes and moderate to severe hypochromic, microcytic anemia (40). These mice nevertheless have the ability to absorb some dietary iron despite the possible mistargeting of the hephaestin protein to a supranuclear compartment (rather than the basolateral membrane) (23).
The goal of the current study was to use microarray techniques to examine changes in gene expression in the rat duodenum associated with iron deprivation. We accomplished this by depriving rats of dietary iron at different stages of development and then performing comparative gene chip analyses with cRNA derived from duodenal mucosa of groups of rats killed at 8 days, 21 days, 6 wk, 12 wk, and 36 wk of age. This novel approach allowed us to track the relative expression of iron-responsive genes longitudinally over the entire course of postnatal development. We also examined the effect of iron deficiency on known iron transport genes with gene chip and quantitative real-time PCR methods.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
For all studies, only male rats were used and groups of three to five animals were considered as one group (n = 1). Each experiment was repeated three times with samples derived from different groups of iron-deficient or iron-replete rats. Rats were anesthetized by CO2 narcosis, and blood was obtained by cardiac puncture. Blood was sent to the University of Arizona Animal Care Pathology Services laboratory for complete blood cell counts (CBC) with differential analysis of blood samples. Rats were then killed by cervical dislocation, and 25 in. of the small intestine (depending on age) just distal to the pyloric sphincter were removed. The intestinal segment was flushed with PBS and opened lengthwise, and light mucosal scrapes were taken. Approximately equal amounts of mucosal tissue were mixed in the same tube from all the rats in that group, with each individual sample being immediately frozen in liquid nitrogen. All samples were stored at 80°C until use. All animal procedures were approved by the University of Arizona Institutional Animal Care and Use Committee.
RNA purification. RNA was purified from mucosal tissue with TRIzol reagent (Invitrogen) as previously described (20). Total RNA (100 µg) was then further purified with the RNeasy Mini Kit (Qiagen) according to the manufacturer's suggested protocol. The RNA was eluted at the final step twice with the same 30 µl of RNAse-free water and quantified by ultraviolet (UV) spectrophotometry. RNA was then visualized by denaturing agarose gel electrophoresis, and RNA concentrations were adjusted by densitometry of the gel. Only high-quality RNA, as judged by intactness of the ribosomal bands, was used for gene chip analyses.
Preparation of samples for gene chip analyses. cRNA was produced from duodenal mucosa RNA samples essentially according to the manufacturer's instructions (Affymetrix; Expression Analysis Technical Manual). Experimental repetitions done in triplicate at each age were performed at the same time with cRNA samples derived from different groups of experimental rats that were either iron deficient or iron replete. RNA was purified from all six groups at each age simultaneously, followed by cRNA production, and then 1 µl of each cRNA sample was analyzed by gel electrophoresis. After gel electrophoresis, densitometry was performed and the most concentrated cRNA sample was quantified by UV spectrophotometry. Subsequently, the relative concentration of all other cRNA samples from that age group was calculated according to optical density of the most concentrated sample. Only cRNA samples that showed a smear of material from high to low molecular weight (e.g., significantly above and below the ribosomal RNA bands) were used for gene chip analyses. By these procedures, we ensured that equal amounts of high-quality cRNA at each age were hybridized with the gene chips.
Gene chip analyses. cRNA was fractionated, hybridization cocktails were prepared, and then rat genome RAE230A and RAE230B chips were hybridized with 10 µg of cRNA by standard Affymetrix protocols. Hyb cocktails were hybridized to only one chip and were then discarded. Chips were immediately washed and stained with the GeneChip Fluidics Station 400 (Affymetrix) utilizing the EukGE-WS2v4 fluidics protocol. After chips had been washed and stained, they were scanned twice with the Agilent Gene Array Scanner (Affymetrix).
Gene chip data analysis and reduction. After scanning the chips, absolute CHiP (.CHP) files were generated by Microarray Suite software (MAS, version 5.0.1.000 [EC] ; Affymetrix) with scaling set for all probe sets with a target value of 500 and normalization set for all probe sets. Other parameters were set at default values. Comparison CHiP files were then generated that compared all three high-Fe data sets individually with all three low-Fe data sets at each age, with all analysis settings remaining constant. All subsequent data analysis and reduction was performed with Data Mining Tool software (DMT, version 3.0; Affymetrix).
Within the DMT software, first Detection and Change was pivoted for all nine comparisons. To identify genes that showed increased expression with iron deprivation at each age, the Count and Percent function was then used with Present, Marginal, Increasing, and Marginally Increasing selected. Probe sets were then selected that showed increases in six to nine of the nine comparisons, and the probe set was saved. This new probe set was then selected, and signal log ratio (SLR) data were pivoted, followed by averaging the SLRs from the nine comparisons. Probes sets were then selected that had average SLRs of 0.6 (i.e., 1.5-fold increase) and higher. A similar strategy was used to identify genes that were downregulated with iron deficiency.
Probe sets from these analyses at each age were then uploaded into the Expression Queries folder on the Affymetrix web site through the NetAffx Analysis Center (http://www.affymetrix.com/analysis/index.affx), using the Batch Query function under the Query-Expression label. The Intersection tool was then used to identify probe sets that behaved similarly at the different ages. Subsequently, lists of probe set IDs of genes that increased or decreased across four or five ages were saved. These lists were then imported back into the DMT software, and SLR and signal intensity data from the high-Fe and low-Fe groups were pivoted and averaged. This overall data analysis and reduction strategy allowed us to come up with short lists of candidate duodenal genes that were regulated by body or enterocyte iron status.
Final gene chip data are presented in tables that show genes that increased or decreased expression across four or five ages studied (genes that were regulated in four of the ages are shown in Supplemental Tables 16, available online at http://ajpgi.physiology.org/cgi/content/full/00489.2004/DC1). Shown are gene name, gene symbol, GenBank accession number for the Affymetrix target sequence, and aliases/biological function. If the gene symbol is not known, "??" was placed in the table at that position. If the gene name is listed as "similar" to a gene, this is the name assigned by Affymetrix for that individual probe set. For some genes, the cDNA has not been cloned from rats, and if this is the case, percent homology to known mouse or human cDNA clones is shown. Furthermore, in some cases, homology was only found to mouse or human chromosomal regions, so 1020 kb of these regions were searched against DNA sequence databases to see what gene(s) was present in this region. If a known gene was present there, we listed these genes as "on the same chromosomal region." Other tables show gene symbol, GenBank accession number, the average fold change from the nine comparisons done with DMT software at each age, and the average expression levels from the three high-Fe groups and the three low-Fe groups at each age. All gene chip data can be found in the GEO repository under accession no. GSE1892 (http://www.ncbi.nlm.nih.gov/geo).
|
|
Statistical analysis. Data from blood analysis of rats, animal weights, gene chip quality control parameters, and real-time PCR data were analyzed by Student's t-test, and P values <0.05 were considered significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Rats were weighed before death, and data from each age and dietary group were averaged. Iron-deprived rats at 8 days (sucklings; 18.38 ± 1.25 g high Fe vs. 17.14 ± 1.05 g low Fe; P = 0.004), 21 days (weanlings; 59.98 ± 4.64 g high Fe vs. 43.81 ± 5.51 g low Fe; P < 0.0001), and 6 wk (168.33 ± 14.79 g high Fe vs. 134.33 ± 14.38 g low Fe; P < 0.0001) of age weighed less than their control groups that had been fed a high-Fe diet. However, 12-wk-old [375.29 ± 27.2 g high Fe vs. 361.63 ± 33.33 g low Fe; not significant (NS)] and 36-wk-old (512.90 ± 37.81 g high Fe vs. 544.63 ± 37.29 g low Fe; NS) rats showed no differences in weights between the iron-replete and iron-deficient groups.
Gene chip control parameters.
The key quality control parameters for the gene chip experiments presented in this manuscript are background, raw Q, scale factor, -actin 3'-to-5' ratio, GAPDH 3'-to-5' ratio, and percent present calls (Affymetrix). Data from three 230A and 230B chips at each age and for either high- or low-Fe diet were averaged, and the means ± SD were determined. Average backgrounds were all below the threshold of 100, and average raw Q values were all below the 3.5 threshold (with the exception of the 12-wk low-Fe group, which was slightly above 3.5; however, all other control parameters were within acceptable ranges for this group). Scale factor was less than threefold different between data sets that were compared with one another, and this parameter was also within the manufacturer's suggested guidelines. Finally,
-actin and GAPDH 3'-to-5' ratios were significantly below the 3.0 threshold, and percent present calls were similar between experiments.
Gene chip data.
Genes expressed differentially in iron-replete vs. iron-deficient states were examined and were classified as either increased or decreased. We found that some genes increased or decreased at all five ages studied and also that some other genes increased or decreased in four of the five ages studied. Table 2 shows genes that increased in all five ages studied. These include DMT1, Dcytb, transferrin receptor 1, metallothionein, tripartite motif protein 27, the Menkes copper ATPase (ATP7a), glycerol-3-phosphate acyltransferase, factor-responsive smooth muscle protein, pyruvate carboxylase, phosphoglucomutase-related protein, acidic calponin 3, glutathione peroxidase 3, lipocalin interacting membrane receptor, gap junction protein 2, retinoblastoma binding protein 7, progressive ankylosis, and integrin
6. Genes that showed increased expression in all ages except sucklings (e.g., 8 days of age) and genes that were upregulated at all ages except in 36-wk-old rats are shown in Supplemental Tables 1 and 3. The corresponding average fold increases from the nine comparisons at each age and average expression levels in the high- and low-Fe groups for genes that increased expression at all five ages are presented in Table 3, and the same data for genes that increased in four out of five ages is presented in Supplemental Tables 2 and 4. Genes that showed decreased expression at all five ages are shown in Table 4, and average fold decreases and expression levels for these genes are presented in Table 5. These genes were aminopeptidase A, death-associated protein (similar to), monoamine oxidase A, and lysosomal apyrase-like 2. Genes that decreased at all ages except in sucklings are shown in Supplemental Table 5, and the average fold decrease and expression levels for these genes are presented in Supplemental Table 6.
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The key finding of our studies was that the brush-border iron transport machinery (e.g., DMT1 and Dcytb) was significantly induced with iron deprivation throughout postnatal development, whereas the genes encoding the basolateral proteins thought to be necessary for iron extrusion from intestinal epithelial cells (e.g., IREG and hephaestin) were not as significantly or consistently upregulated. However, these lesser changes in expression of IREG1 and hephaestin could translate into significant changes in protein activity and may thus have true physiological importance. Other genes known to be involved in iron metabolism were also induced with iron deprivation [e.g., heme oxygenase 1 (HO1) and transferrin receptor 1 (TFR1)]. Furthermore, we identified iron-dependent regulation of many genes that have not previously been associated with iron metabolism. Some of these genes, such as the Menkes copper ATPase (ATP7a), the sodium-dependent vitamin C transporter, and others, may be directly involved in intestinal iron absorption, as they were induced not only in iron-deficient, anemic rats but also in iron-deprived rats that did not display a noticeable iron-deficient phenotype (e.g., the 36-wk-old group).
To ensure that our studies accurately duplicated iron deficiency in humans, we developed a natural feeding strategy with diets that contained high (198 ppm; actually levels identical to commercial rodent chows) and low (3 ppm) Fe levels. This approach has been used extensively by many investigators to decipher various aspects of intestinal iron absorption, as many of the key genes involved in this process are regulated by cellular or body iron status (12, 29, 37).
Several genes were induced at all five ages studied (Table 2). Some of these genes have been shown previously to be iron responsive; however, many are novel. DMT1 and Dcytb showed strong induction at all ages studied, which was confirmed by real-time PCR (Table 6). Our data thus strongly suggest that at least some functional aspects of intestinal iron transport and its upregulation in the iron-deficient state are conserved among sucklings, weanlings, and older rats. Furthermore, our findings confirm previous studies showing increased DMT1 and Dcytb mRNA expression levels in adult, iron-deficient rodents (5, 16, 27, 46).
DMT1 is known to have two 3' splice variants, one containing an IRE (+IRE) and the other not containing this element (IRE) (15). Our data showed stronger induction of DMT1 mRNA expression at all ages studied, as detected with an Affymetrix probe set that recognizes both 3' splice variants (+/ IRE) as opposed to a probe set that only recognizes the +IRE transcript (Table 1). Both of these probe sets recognize both 5' splice variants that have been recently described (21). These data suggest that iron deficiency in rats leads to transcriptional induction of DMT1, which induces expression of both 3' splice variants. This induction would be independent of the possible regulation of DMT1 mediated through the iron regulatory protein (IRP) pathway, which may involve +IRE transcript stabilization via the IRE (15). Additionally, transcriptional induction during iron deficiency may be related to the 5' slice variants (21).
Although expression of DMT1 and Dcytb was strongly induced, other genes encoding basolaterally expressed proteins involved in iron transport (e.g., IREG1 and hephaestin) did not show induction by the microarray studies. Previous studies have yielded conflicting results in iron-deficient rodents (6, 28, 33, 40). Therefore, we sought to confirm this observation by real-time PCR (Table 6). IREG1 and hephaestin mRNA expression by real-time PCR was increased at some of the ages studied, although induction was inconsistent and significantly lower than the induction of the genes encoding the brush-border iron transport proteins. Overall, these data lend support to prior observations suggesting that the mucosal transfer step is more highly regulated in intestinal iron absorption and that efflux across the basolateral membrane of enterocytes may be less sensitive to iron deficiency (29, 34).
Other known iron-responsive genes, namely HO1 and TFR1, were also induced in iron-deficient rats at all ages studied (for TFR1) or at all ages except in sucklings (for HO1). TFR1 mRNA levels are known to be increased in iron deficiency in rat duodenum (25). Increased TfR1 mRNA expression in the iron-deficient state is thought to be mediated by the IRP/IRE system (17). HO1 encodes an intracellular protein that is involved in releasing iron from the heme molecule, once heme has been endocytosed by enterocytes (30). The heme absorption pathway represents a distinct mechanism for intestinal iron absorption. It is thus noteworthy that HO1 is induced in iron-deficient rats at several ages, suggesting that this pathway is also of physiological importance in rodents and that heme iron absorption may also be induced by iron deficiency.
Another gene of potential interest that was induced at all five ages was tripartite motif protein 27 (TRIM27; also called Ret finger protein). TRIM27 is of particular interest, because it was one of only a few genes that were shown to be induced across all five ages when data were analyzed with the most stringent parameters. Furthermore, a second probe set that recognizes this gene showed induction at four of the five ages (see Supplemental Tables 2 and 4). TRIM27 has not been previously shown to be expressed in the duodenal mucosa; therefore, its physiological role in the intestine is unknown.
We also noted strong induction of basolateral membrane copper ATPase (ATP7a) gene expression in iron-deprived rats of all ages examined, which is a novel and potentially important finding. ATP7a induction was seen at all ages, and results were similar to those seen for other iron-responsive genes such as DMT1 and Dcytb, which showed not only strong induction but also very high expression levels (Tables 1 and 2). Furthermore, we detected an approximately four to fivefold increase in liver copper levels in 7- to 12-wk-old iron-deficient rats (data not shown), and previous observations have also shown increased body copper levels in iron-deficient rats (32, 36) and humans (9). The current study thus provides intriguing evidence that may explain copper loading in the iron-deficient state.
We speculate that under iron-deficient conditions, more copper is transported into enterocytes by DMT1, leading to induction of ATP7a, which responds in turn to increased intracellular copper levels. DMT1 has been shown in vitro to transport copper in colonic carcinoma (Caco-2) cells (2), and it is also known to transport a number of heavy metal ions in various model transport systems (16). Additional evidence suggesting that DMT1 is responsible for copper absorption during iron deficiency comes from the fact that a known, high-affinity brush-border copper transporter [CTR1 (35)] did not show induction by the gene chip analyses at any age studied. Furthermore, the strong induction of metallothionein, which can bind copper intracellularly and has been shown to be important for intestinal copper homeostasis (22), also supports the concept that enterocytes absorb more copper during iron deficiency.
Another gene that was induced across all five ages by iron-deprivation is the lipocalin interacting membrane receptor (LIMR). This observation is potentially significant, as a recent study suggested that lipocalin [24p3/Ngal (neutrophil gelatinase-associated lipocalin)] may be able to mediate iron absorption by some cell types (45). Another recent report has suggested that LIMR may be a prototype of a new family of endocytic receptors (43). Additionally, Goetz et al. (14) proposed that NGAL (e.g., lipocalin) participates in the antibacterial iron-depletion strategy of the innate immune system. Thus it is possible that lipocalin plays a role in iron homeostasis in the gut.
Also of potential physiological relevance was the induction of the sodium-dependent vitamin C transporter (SVCT) in the four older age groups. Induction of this gene was modest (between 1.6- and 2.7-fold), as detected by two distinct probe sets on the chips. Although both probe sets showed homology to both SVCT1 and SVCT2, we surmise that we were likely detecting SVCT1, because only SVCT1 (and not SVCT2) has been shown to be expressed in the intestinal epithelium (38). Furthermore, a recent study showed increased intracellular ascorbic acid levels in intestinal epithelial cells of iron-deficient rats (3). This is of significance because the Dcytb protein is thought to bind ascorbic acid intracellularly, which likely serves as an electron donor to facilitate iron reduction for transport across the apical membranes of enterocytes via DMT1. Our data are thus consistent with the hypothesis that increased absorption of vitamin C may be important for enhanced iron absorption during iron deficiency.
In addition to induction of a large number of genes, iron deficiency led to decreased expression of some genes, although the number of genes that were downregulated was smaller than the number of genes that were upregulated. Two such genes decreased by iron deficiency were monoamine oxidases A and B, which have recently been localized to multiple cell types within the human duodenal mucosa (31). The monoamine oxidases (MAO) are known to be involved in oxidation of neurotransmitters and hormones, and interestingly, both of these proteins have been previously associated with dopamine metabolism and oxidative stress. In fact, a recent paper has described the MAO-mediated metabolism of dopamine as a potential cause of oxidative stress and has discussed the role of ferrous and ferric ions in relation to Parkinson disease (19). Our data have demonstrated a strong reduction in mRNA expression of these genes during dietary iron deprivation, and thus the role of these proteins in the duodenum during iron deficiency warrants further study.
Several genes of potential interest were downregulated at four of the five ages studied, including Na-Pi-IIb (SLC34A2; up to 19-fold at 6- and 12 wk of age). This enterocyte-specific, apically expressed protein is thought to be responsible for intestinal Pi absorption (44), and the mammalian duodenum is thought to have the greatest ability to absorb dietary Pi (41). This finding may be significant, as decreased Pi levels could have detrimental effects on many metabolic processes. Several other transporters showed downregulation with iron deprivation including GLUT 5 (SLC2A5; a facilitated glucose/fructose transporter), a zinc transporter (SLC39A8), a sulfate transporter (SLC26A2), a sodium-dependent vitamin transporter (SLC5A6), and a monocarboxylic acid transporter (SLC16A1). These data suggest that iron deficiency may have an effect on membrane transport processes in the small intestinal epithelium.
Another recent study by Marzullo et al. (26) has utilized a differential display, reverse-transcription approach to identify iron-regulated genes in the rat small intestine. The authors studied 9- to 10-wk-old rats that had been fed either a diet containing 38.4 ppm Fe (control) or an iron-deficient diet containing 14.7 ppm Fe for 32 days. Interestingly, these authors found that cytochrome-c oxidase subunit II mitochondrial and serum- and glucocorticoid-regulated kinase genes were regulated by iron status. These genes were not regulated by iron status in the current studies. However, results between the two studies are hard to compare because the following experimental parameters were different: age of the animals, Fe content of the diets, length of feeding the diets, the specific gut segment used (small intestine vs. duodenum), and preparation of the tissue (mucosal scrape vs. whole intestine).
In conclusion, we used microarray techniques to discover novel genes associated with intestinal iron absorption. Comparative gene chip analysis is a plausible approach to this end, as many genes known to be involved in iron transport are induced at the mRNA level by iron deprivation. In some cases this increase in mRNA levels is due to well-described alterations in transcript stability through the IRP/IRE regulatory system (i.e., ferritin, transferrin receptor, possibly DMT1 and IREG1), but transcriptional events must also be important. We identified novel genes that are very likely to be critical for iron absorption. These include the copper ATPase (ATP7a), lipocalin interacting membrane receptor, the sodium-dependent vitamin C transporter (SVCT1), tripartite motif protein 27, and the ABC transporter ABCG2 (breast cancer-resistance protein), all of which were strongly induced in the setting of iron deficiency. A range of other genes, whose roles in gut physiology and iron transport are unknown, also appear to be regulated by body or enterocyte iron status. Our findings thus demonstrate that iron deprivation results in a large spectrum of differentially expressed genes in the duodenal epithelium, and identification of these genetic changes is likely to increase our understanding of the complex physiology of intestinal iron homeostasis.
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|