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Biological Research

Print version ISSN 0716-9760

Biol. Res. vol.39 no.1 Santiago  2006

http://dx.doi.org/10.4067/S0716-97602006000100004 

 

Biol Res 39: 25-37, 2006

ARTICLE

Gene chip analyses reveal differential genetic responses to iron deficiency in rat duodenum and jejunum

JAMES F COLLINS

Departments of Pediatrics and Nutritional Sciences, Steele Children's Research Center, University of Arizona, Tucson, Arizona, USA

Dirección para Correspondencia

 


ABSTRACT

Previous studies revealed novel genetic changes in the duodenal mucosa of iron-deprived rats during post-natal development. These observations are now extended to compare the genetic response to iron deficiency in the duodenum versus jejunum of 12-wk-old rats. cRNA samples were prepared from the duodenal and jejunal mucosa of three groups each of control and iron-deficient rats and hybridized with RAE 230A and 230B gene chips (Affymetrix). Stringent data reduction strategies were employed. Results showed that several genes were similarly induced in both gut segments, including DMT1, Dcytb, transferrin receptor 1, heme oxygenase 1, metallothionein, the Menkes copper ATPase (ATP7A), tripartitie motif protein 27, and the sodium-dependent vitamin C transporter. However, a subset of genes showed regulation in only one or the other gut segment. In duodenum only, gastrokine 1, trefoil factor 1 and claudin 2 were induced by iron-deficiency. Other genes previously identified were only regulated in the duodenum. Overall, these studies demonstrate similarities and distinct differences in the genetic response to iron deprivation in the duodenum versus jejunum and provide evidence that more distal gut segments also may play a role in increasing iron absorption in iron-deficiency anemia.

Key terms: ATP7A, copper transport, iron-deficiency anemia, microarray


INTRODUCTION

Iron is a critical element for many metabolic processes, including its participation as a cofactor for cytochromes and enzymes that transfer electrons (3). However, excess iron stores can lead to oxidative damage, as free iron readily participates in redox reactions within cells. Thus, body iron levels must be tightly controlled. Body iron stores normally are regulated at the level of absorption in the proximal small intestine (5), with the greatest overall absorption rates and adaptive responses to iron deficiency being observed in the duodenum (27). Several physiological effectors are known to modulate dietary iron absorption in mammals (13), including hepatic stores, erythroid, hypoxia, and inflammatory mediators, which act directly on the intestinal epithelium to control iron absorption and, thus, body iron levels. Hepcidin, a recently identified antimicrobial peptide, is currently recognized as the hormone responsible for regulating intestinal iron absorption. Hepcidin is synthesized in the liver and secreted into the circulation, and has been shown to decrease intestinal iron transport in mice (17). It has also been demonstrated recently that hepcidin expression is decreased in physiological situations where iron absorption is increased (e.g., hypoxia, iron deficiency, and anemia), whereas hepcidin production is upregulated under conditions in which intestinal iron transport is decreased (e.g., iron-overload such as occurs in hemochromatosis and inflammation) (21-23, 26). Additionally, hepcidin gene expression is related to body iron stores in normal humans (11) and iron feeding of rats results in a rapid increase in hepcidin levels, which decreases iron absorption (10).

Several proteins involved in duodenal iron transport recently have been identified. Duodenal cytochrome b (Dcytb) is a brush-border membrane ferric reductase that reduces dietary ferric iron to ferrous iron (19), which can then transported into epithelial cells by the divalent metal transporter 1 [DMT1; also called DCT1 (12) and nRAMP2 (24)]. Within enterocytes, iron is either complexed with ferritin or is trafficked to the basolateral membrane for export into the circulation by the coordinated action of the basolateral iron transport protein Iron Regulated Gene 1 [IREG1 (20); also called MTP1 (1) and ferroportin (7)]and hephaestin, which oxidizes iron for binding to transferrin and distribution throughout the body (25). However, a complete understanding of the molecular events associated with intestinal iron absorption has not yet been achieved. For example, microcytic anemia mice (9) and Belgrade rats (8) are able to absorb substantial amounts of dietary iron, despite the lack of normal DMT1 protein. Furthermore, sex-linked anemia (sla) mice, which have a deletion in the hephaestin gene, have substantial accumulation of iron within enterocytes and moderate-to-severe hypochromic, microcytic anemia (25). These mice, nevertheless, have the ability to absorb some dietary iron despite the possible mistargeting and subsequent degradation of the hephaestin protein (16).

We previously performed extensive gene chip analyses to characterize the genetic response to iron-deprivation in the duodenum of rats throughout post-natal development (6). These observations are now extended to compare the genetic response to iron-deficiency in the duodenum versus jejunum of 12-wk-old rats. Current results demonstrate that several genes were similarly induced in both gut segments, including known iron-responsive genes and novel genes previously identified. However, a subset of genes showed regulation in only one or the other gut segment. Overall, these studies demonstrate similarities and distinct differences in the genetic response to iron deprivation in the duodenum versus jejunum, and provide evidence that more distal gut segments may also play a role in increasing iron absorption in conditions of iron deficiency.

MATERIALS AND METHODS

Experimental animals

Sprague Dawley rats were obtained from Harlan (Madison, WI) and were housed in the University of Arizona Animal Care facility. Modified AIN-93G rodent diets were obtained from Dyets Inc. (Bethlehem, PA), which contained either 198 ppm Fe (DYET# 115135; control diet with the same Fe content as standard rat chow) or 3 ppm Fe (DYET# 115102; called Low Fe diet). The diets were identical except for the addition of pure ferric citrate to the control diet. Tap water was tested initially for iron content and did not show any detectable iron. Rats were supplied with food and tap water ad libitum. To produce 12-week-old, iron-deficient rats, three-week-old weanling rats were placed on control or low Fe diets for 9 weeks and then sacrificed. For all studies, only male rats were used, and groups of 4-5 animals were considered as one group (n=1). The experiments were repeated three times with samples derived from different groups of control or iron-deficient 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 CBC with differential analysis of blood samples. Rats were then sacrificed by cervical dislocation and the first ~15 cm of the gut was removed just distal to the pyloric sphincter and designated duodenum. The remaining proximal one half of the small bowel was removed and designated jejunum. The jejunal segments were ~3 times longer than the duodenal segments. The intestinal segments were flushed with PBS, 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. Snap-frozen mucosal scrapings were stored at -80o C until use. The University of Arizona Institutional Animal Care and Use Committee approved all animal procedures.

RNA purification

RNA was purified from mucosal tissue with Trizol reagent (Invitrogen) as previously described (6, 14). Total RNA (100 μg) was further purified utilizing the RNeasy Mini Kit (Qiagen) according to the manufacturer's suggested protocol. The RNA was eluted at the final step twice with the same 30μl of RNAse-free water, quantified by UV spectrophotometry, and visualized by denaturing agarose gel electrophoresis. RNA concentrations were then adjusted by densitometry of the gel. Only high-quality RNA, as judged by intactness of the ribosomal bands, was utilized 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 were performed at the same time with cRNA samples derived from different groups of control or iron-deficient, experimental rats. RNA was purified from all six groups from each gut segment (3 control and 3 iron-deficient) simultaneously followed by cRNA production, and then 1 ml of each cRNA sample was analyzed by denaturing, agarose gel electrophoresis. Subsequently, densitometry of the gel was performed, and the most concentrated cRNA sample was quantified by UV spectrophotometry. Then, 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 utilized for gene chip analyses. By these procedures, we ensured that equal amounts of high-quality cRNA were hybridized with the gene chips.

Gene chip data analysis

cRNA was fractionated and hybridization cocktails were prepared, and then rat genome RAE230A and RAE230B chips were hybridized with 10 μg of cRNA as previously described (6). 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). Resulting data were analyzed with high stringency parameters as previously described (6), with 9 total comparisons being performed between each control and iron-deficient group.

Final microarray data are presented in tables that show the following: 1) previously described differentially expressed genes (6) that increased or decreased in both gut segments (Tables I-IV); 2) unique genes that were induced or repressed in only one gut segment (Table V and VII); and 3) unique genes that were increased or decreased in both gut segments (Table VI). Shown are gene name, gene symbol, GenBank accession # for the Affymetrix target sequence, and biological function and any known aliases. 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. Further, in some cases, homology was only found to mouse or human chromosomal regions, so 10-20 kb of these regions were searched against DNA sequence databases to see what gene(s) was present in this region. If a single known gene was present there, we listed these genes as "on the same chromosomal region." Other tables show gene name, gene symbol, GenBank accession #, average fold increase from the 9 comparisons, and the average expression levels from the 3 control and the 3 low Fe groups for each gut segment. Gene chip data have been deposited in the GEO repository under accession #GSE1892 (duodenum) and #GSE2269 (jejunum) (http://www.ncbi.nlm.nih.gov/geo).

RESULTS

Rat health status

Blood analysis of the rats revealed that iron-deprived rats at 12 weeks of age showed signs of hypochromic, microcytic anemia (6). These signs included decreased red blood cell counts, hemoglobin levels, hematocrit, mean corpuscular volume, and mean corpuscular hemoglobin. Further, red blood cell distribution width was increased significantly in iron-deficient rats at 12 weeks of age. Rats were weighed before sacrifice, and data were averaged from each dietary group. Iron-deprived, 12-week-old rats were not different in weight from controls, despite being highly iron deficient (6).

Gene chip control parameters

The key quality control parameters for the gene chip experiments presented in this manuscript are background, raw Q, scale factor, β-actin 3'/5' ratio, GAPDH 3'/5' ratio, and percent present calls (Affymetrix). Data from the RAE230A and RAE230B gene chips, which were hybridized with the 12-week-old duodenal samples, were reported previously (6), and all were within acceptable ranges. Data from three 230A and three 230B chips from the jejunum for either control or low Fe diets were averaged, and the means ± standard deviation 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 two low-iron groups that were hybridized with RAE 230A gene chips; however, all other control parameters were within acceptable ranges for these samples). Scale factor was less than two-fold different between data sets that were compared to one another, and this parameter also was within the manufacturer's suggested guidelines. And finally, β-actin and GAPDH 3'/5' ratios, were significantly below the 3.0 threshold (with the exception of one low-iron sample that was hybridized with a RAE 230B gene chip; however, all other quality-control parameters were within acceptable ranges for this sample). Furthermore, present/absent calls were very similar between experimental repetitions. Thus, all data obtained from these experiments were considered valid.

Gene chip data

Genes expressed differentially in control versus iron-deficient rats were examined and were classified as either increased or decreased. We found that some genes increased or decreased in both gut segments and also that some other genes increased or decreased in one or the other gut segment. The following cutoffs were used to prepare tables for this report: 1) for genes previously shown to be regulated at 4 or 5 different post-natal ages from sucklings to adults (6), a 1.5-fold cutoff was implemented; 2) for novel genes that were regulated in both duodenum and jejunum, a 2.0-fold cutoff was used; and 3) for novel genes that were regulated in only one gut segment, a 3.0-fold cutoff was used. Table I shows genes that were reported previously to be induced at several post-natal ages in the duodenum that are now shown also to be responsive in the jejunum. These included DMT1, Dcytb, transferrin receptor 1, metallothionein, tripartite motif protein 27, the Menkes copper ATPase (ATP7A), glycerol-3-phosphate acyltransferase, factor-responsive smooth muscle protein, phosphoglucomutase-related protein, acidic calponin 3, glutathione peroxidase 2, integrin alpha 6, early growth response 1, sodium-dependent vitamin C transporter, selective LIM binding factor, small nuclear RNA activating complex, polypeptide 1, amyotrophic lateral sclerosis 2, laminin gamma 2 chain precursor, peripheral myelin protein 22, heme oxygenase 1, neural precursor cell expressed developmentally downregulated 9 (NEDD9), prolyl 4-hydroxylase alpha subunit, sepiapterin reductase, cytotoxic T lymphocyte-associated protein beta 2 precursor, and farnesyl transferase beta subunit.

TABLE I

Genes that increased in duodenum and jejunum of 12-wk-old, iron-deficient rats


Gene Name

Symbol Acc. # Biological Function/ Aliases

Transferrin Receptor

TFRC M58040

Iron Homeostasis; Transferrin-Iron Endocytosis

Farnesyl Transferase Beta Subunit

FNTB M69056 Catalytic Activity; Post-Translational

Farnesylation of Proteins

     
Cytotoxic T Lymphocyte-Associated      

Protein 2 Beta Precursor (85% to mouse)

CTLA2B

AI230591 Immunity
Sepiapterin Reductase SPR M36410 Metabolism
Prolyl 4-Hydroxylase Alpha Subunit P4HA1 BI274401

Procollagen-Proline 4-Dioxygenase
Activity: HIF 1-α regulation

Neural Precursor Cell Expressed

     

Developmentally Downregulated 9

NEDD9

BF555968 Cell Adhesion Molecule Activity;
      Regulation of Cell Growth
Heme Oxygenase 1 HMOX1 NM_012580 Heme Oxidation
Peripheral Myelin Protein 22 PMP22 AA943163 Cell Cycle Arrest
Similar to Laminin Gamma 2       

Chain Precursor

LAMC2 BM385282 Inflammatory Response Pathway;
Cell Adhesion

Tripartite Motif Protein 27

     
(95% similar to mouse) TRIM27 NM_009054 Oncogenesis; Formation of Cellular
Compartments
Amyotrophic Lateral Sclerosis 2      

(Juvenile) Chr. Reg., Cand. 13

ALS2CR13 AA945854 Unknown
Small Nuclear RNA Activating Complex      

Polypeptide 1 (84% similar to mouse)

SNAPC1 BI294596

Regulation of Transcription,
DNA-dependent

Sodium-Dependent Vitamin C

     
Transporter (97% similar to mouse) SLC23A2 BI275077 Vitamin C Transport

Selective LIM Binding Factor,

     
Rat Homolog SLB NM_053792 Modulation of Activity of Specific LIM
Transcription Factors
Divalent Metal Ion Transporter 1,      
all +/-IRE transcripts DMT1 AF029757 Divalent Metal Ion Transport
Duodenal Cytochrome B (91% similar) CYBRD1 BF419070 Rat Sproutin; Ferric-Reductase
Duodenal Cytochrome B (on same      

chromosomal region)

CYBRD1 AI010267 Ferric-Reductase
Transferrin Receptor TFRC BF417032 Iron Ion Homeostasis; Transferrin-Iron
Endocytosis
Metallothionein 1 and 2

MT1/2

BM383531 Metal Ion Binding; Copper Binding
Tripartite Motif Protein 27 (86% similar) TRIM27 BI294862 Oncogenesis; Formation of Cellular
Compartments
ATPase, Cu++ Transporting,      

Alpha Polypeptide

ATP7A AI136839 Copper Ion Efflux from Enterocytes
Divalent Metal Ion Transporter, +IRE      

transcripts only

DMT1 NM_013173 Divalent Metal Ion Transport
Metallothionein  MT1/2 AF411318 Metal Ion Binding
Glycerol-3-Phosphate Acyltransferase      

(87% similar)

GPAM BG666882 Glycerolipid Synthesis
Factor-Responsive Smooth Muscle Protein SM20 NM_019371 Apoptosis; Protein Metabolism;
Oxidoreductase

Phosphoglucomutase-Related Protein
(on same chr. region)

PGM-RP AI412174 Cell Adhesion; Carbohydrate
Metabolism/ Aciculin
Glutathione Peroxidase 2 (Gastrointestinal) GPX2 AA800587 Defense Against Oxidative Stress
Integrin, Alpha 6 ITGA6 BE110753

Cell Adhesion; Cell Surface Mediated
Signaling

Early Growth Response 1 EGR1 NM_012551 Regulation of Transcription,
DNA-Dependent
Calponin 3, Acidic CNN3 BI274457 Actin/Calmodulin Binding

Table II presents genes that were shown previously to be decreased at 4 or 5 different post-natal ages, which are now shown also to be decreased in the jejunum. Tables III and IV show average expression levels and fold changes of genes listed in Tables I and II. Table V shows fold changes of unique genes that are induced in the duodenum only. Some of these genes are as previously described (6) and also may be presented in Table I and III; however, they represent novel probe sets for these specific genes. Finally, Tables VI and VII show fold changes of unique genes that were increased in both gut segments (Table VI) or decreased (Table VII) in only one or the other gut segment.

 

DISCUSSION

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.

TABLE II

Genes that decreased in duodenum and jejunum of 12-wk-old, iron-deficient rats

Gene Name

Symbol Acc. # Biological Function/ Aliases

Monoamine Oxidase A

MAOA D00688 Oxidation of Monoamine
Neurotransmitters and Hormones
Solute Carrier Family 34, Member 2

SLC34A2

NM_053380 Sodium-Dependent Phosphate
Cotransporter; Pi Absorption/NaPi-IIb

3-Hydroxy-3-Methylglutaryl-Coenzyme

     

A Synthase 2

HMGCS2 M33648 Acetyl-CoA Metabolism; Cholesterol
Biosynthesis

Glucose-6-Phosphatase, Catalytic

G6PC U07993 Glycogen Biosynthesis; Glycolysis and
Gluconeogenesis

Phosphoenolpyruvate Carboxykinase,

     

Cytosolic (GTP)

PCK1 BI277460 Gluconeogenesis; Lipid Metabolism

Glucose-6-Phosphatase, Catalytic

G6PC NM_013098 Glycogen Biosynthesis; Glycolysis and
Gluconeogenesis

UDP-Glucuronosyltransferase

UGT2B12 U27518 Glucuronidation of Endobiotics and
Xenobiotics

Cyclin-Dependent Kinase

     

Inhibitor 1C, p57

CDKN1C AI013919 Tumor Repressor Activity; Regulation
of Cell Proliferation, Differentiation
UDP-Glucuronosyltransferase

UGT2B12

NM_031980 Glucuronidation of Endobiotics and
Xenobiotics

Solute Carrier Family 2, Member 5

SLC2A5 NM_031741

Facilitated Glucose, Fructose
Transporter/ GLUT 5

UDP-Glucuronosyltransferase 2B3
Precursor, Microsomal

UDPGT M31109 Glucuronidation of Endobiotics and
Xenobiotics
Non-Oncogenic Rho GTPase-Specific
GTP Exchange Factor
AKAP13 AI407536 Cell Growth and Maintenance/A kinase
(PRKA) Anchor Protein 13
Hypoxia Inducible Factor 1,      

Alpha Subunit

HIF1A NM_024359

Regulation of Transcription,

      DNA-Dependent; Response to Hypoxia

 

TABLE III

Changes in expression levels of genes that increased in duodenum and jejunum


   

Duodenum

Jejunum

Gene Name

Symbol
Acc. #
Fold
Inc.
Control
Low
Fe
Fold
Inc.
Control
Low
Fe

Transferrin Receptor
TFRC
M58040
6.1
144
670
5.1
153
484

Transferrin Receptor

TFRC
BF417032
6.1
642
4241
7.3
602
4114

Metallothionein 1 and 2

MT1/2
BM383531
4.6
1093
3432
2.9
648
1667

Tripartite Motif Protein 27

(86% similar)

TRIM27
BI294862
4.3
122
447
2.8
87
355
Metallothionein
MT1/2
AF411318
4.3
2114
8166
3.1
1854
6169
Divalent Metal Ion Transporter 1,
all +/-IRE transcripts
DMT1
AF029757
4.1
1892
7236
3.8
1088
3641
Cytotoxic T Lymphocyte-Assoc.

Protein 2 Beta Precursor
(85% similar to mouse)

CTLA2B
AI230591
4
797
2334
1.8
533
941
Farnesyltransferase Beta Subunit

FNTB

M69056
3.7
715
2848
1.7
360
679
Glycerol-3-Phosphate
Acyltransferase (87% similar
to mouse)
GPAM
BG666882
3.4
2114
8166
1.6
316
505
Heme Oxygenase 1
HMOX1
NM_012580
3.2
730
2602
2.4
582
1449
Amyotrophic Lateral Sclerosis 2

(Juvenile) Chr. Reg., Cand. 13

ALS2CR13
AA945854
3.2
975
2975
2
905
1545
ATPase, Cu++ Transporting,

Alpha Polypeptide

ATP7A
AI136839
3.1

4891

15697
2.5
2467
6615
Sepiapterin Reductase
SPR
M36410
3

452

1575
1.9
410
859
Proliferating Cell Nuclear Antigen

PCNA

NM_022381
3
1978
6746
1.9
1038
2113
Neural Precursor Cell

Expressed Developmentally

Downregulated 9

NEDD9

BF555968
2.9
2032
6373
1.5
2371
3527
Early Growth Response 1
EGR1
NM_012551
2.9
559
1759
1.6
821

1268

Prolyl 4-Hydroxylase Alpha Subunit
P4HA1
BI274401
2.8
147
384
2.7
172
415

Tripartite Motif Protein 27
(95% similar to mouse)

TRIM27
NM_009054
2.7
591
1292
2.1
551
938
Small Nuclear RNA Activating

Complex, Polypeptide 1

(84% to mouse)

SNAPC1
BI294596
2.5
394
1130
2.3
213

512

Duodenal Cytochrome B (on same
chromosomal region)
CYBRD1
AI010267
2.5
2817
5602
19
90
1968
Similar to Laminin Gamma 2

Chain Precursor

LAMC2

BM385282
2.4
1254
3642
1.7
1753
3203
Phosphoglucomutase-Related

Protein (on same chr. region)

PGM-RP
AI412174
2.3
868
1675
1.6
1116
1717
Peripheral Myelin Protein 22
PMP22
AA943163
2.2
1585
3549
2.4
2065
4764

Factor-Responsive Smooth

Muscle Protein

SM20
NM_019371
2.2
1035
2241
2
1075
2138
Glutathione Peroxidase 2

(Gastrointestinal)

GPX2
AA800587
2
4566
9172

1.9

2167
4015
Sodium-Dependent Vitamin C

Transporter (88% similar to
mouse)

SLC23A1/2
BI275077
1.8
1265
1926
2.1
667

1244

Duodenal Cytochrome B

(91% similar)

CYBRD1
BF419070
1.7
6470
10246
21.7
303
4981
Divalent Metal Ion Transporter,

+IRE transcripts only

DMT1
NM_013173
1.6
7452
13341
4.2
2815
12758
Integrin, Alpha 6
ITGA6
BE110753
1.6
12335
20379
1.6
7234
14678
Selective LIM Binding Factor,

Rat Homolog

SLB
NM_053792
1.5
1542
2502
1.6
1267

2227

Calponin 3, Acidic
CNN3
BI274457
3.2
561
2043
2
207
379

 

TABLE IV

Changes in expression levels of genes that decreased in duodenum and jejunum


   

Duodenum

Jejunum
 
Gene Name Symbol Acc. # Fold
Dec.
Control Low
Fe
Fold
Dec.
Control Low
Fe

Solute Carrier Family 34,                

Member 2

SLC34A2 NM_053380 18.9 838 59 1.7 3005

1573

3-Hydroxy-3-Methylglutaryl-                

Coenzyme A Synthase 2

HMGCS2 M33648 9.7 292 32 2.6 839

259

Glucose-6-Phosphatase, Catalytic

G6PC U07993 5.5 823 227 2.7 1224

475

Phosphoenolpyruvate                

Carboxykinase, Cytosolic (GTP)

PCK1 BI277460 4.6 2650 475 1.6 5659

3094

Cyclin-Dependent Kinase                

Inhibitor 1C, p57

CDKN1C AI013919 3.9 1149 363 2.2 1453 743
UDP-Glucuronosyltransferase UGT2B12 U27518 3.7 3263 858 2 466 183
Glucose-6-Phosphatase,                

Catalytic

G6PC NM_013098 3.4 2894 632 2.1 3413

1669

UDP-Glucuronosyltransferase UGT2B12 NM_031980 3.4 4538 1125 2.3 1586

656

Solute Carrier Family 2,                

Member 5

SLC2A5 NM_031741 2.9 3974 1331 1.7 2820 1744
UDP-Glucuronosyltransferase 2                

B3 Precursor, Microsomal

UDPGT M31109 2.4 916 382 1.9 772 396
Non-Oncogenic Rho                

GTPase-Specific GTP Exchange
Factor (93% similar to human)


AKAP13


AI407536

2.4

362

132

1.6

848


462

Monoamine Oxidase A MAOA D00688 2 1756 888 1.9 544 308
Hypoxia Inducible Factor 1,                

Alpha Subunit

HIF1A NM_024359 1.9 1263 643 2 1913 1050

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.

TABLE V

Unique genes that increased only in duodenum in response to iron deficiency


Fold
Increase

Gene Title
Symbol
Acc. #
Biological Function/ Aliases

9.1
Aquaporin 4
AQP4
NM_012825
Water Transport
7.7
Gastrokine 1
GKN1
AI639014

Role in Mucosal Protection
Postulated/ Foveolin, CA11

6.7
Membrane-Spanning 4-Domains,
 

Subfamily A, Member 7
(89% to mouse)

MS4A7
AI408286
Unknown
6.7
Unknown EST Clone
???
AI044560

Unknown

5.4
Cyclin D1
CCND1
X75207

Regulation of Cell Cycle;
Wnt Signaling

5.2
Highly Sim. to Mouse Nucleolar
Protein Family A, Member 1

AI579023

Unknown
5.0
SRY-Box Containing Gene 9
 

(97% to mouse)

SOX9
AI072788

Chondrogenesis; Testis
Determining Pathway

4.6
Similar to Cryptdin-7
 

Precursor (95% to rat)

LOC290857
AI639089
Paneth Cell Corticostatin/ Defensin
4.4
Unknown EST Clone
???
BI298306

Unknown

4.3
RT1 Class II, Locus Db1
RT1-DB1
BI279526

MHC Class II Protein Complex;
Immunity

4.3
Similar to Lrp2bp-Pending
 

Protein

LOC361149

BI291430

Low Density Lipoprotein Receptor-
Related Protein Binding Protein
4.1
Solute Carrier Family 12,
 

Member 2

SLC12A2
NM_031798
Na+/K+/2Cl- Cotransporter
4.0
Mink-Related Peptide 2
KCNE3
NM_022235

Potassium Channel Regulator
Activity

3.9

Myristoylated Alanine Rich

 

Protein Kinase C Substrate

MARCKS
BE111706
Actin Binding; Calmodulin Binding;
Kinase Activity
3.9
Trans-Acting Transcription
 

Factor 6 (94% to mouse)

SP6
BI278449
Transcriptional Regulation of Gene
Expression
3.6
Myristoylated Alanine Rich
 

Protein Kinase C Substrate

MARCKS
BE111706
Actin Binding; Calmodulin Binding;
Kinase Activity
3.5
Immunoglobulin Joining Chain
 

(89% to mouse)

IGJ
AA817898

Immune Response

3.4
Stearoyl-Coenzyme A
 

Desaturase 2

SCD2
BE107760
Desaturation of Saturated Fatty
Acyl-CoAs
3.4
Brain Specific Protein Homolog
 
(95% to mouse)
???
AI171466
Unknown
3.4
Similar to Phosphoinositide-3
 

-Kinase, Class 2, Beta
Polypeptide

PI3K-C2B
BE105801
Signal Transduction
3.3
Claudin 2 (93% to mouse)
CLDN2
BM392116
Tight Junction; Integral Membrane
Protein
3.2
SRY-Box Containing Gene 9
 

(93% to mouse)

SOX9
AI548994
Chondrogenesis; Testis Determining
Pathway
3.0
Putative Small Membrane
 

Protein NID67

NID67
AF313411

Neurogenesis; Ion Channel Activity

3.0
Protein phosphatase 1, regulatory
(inhibitor) subunit 1B
PPP1R1B
AA942959


Intracellular Signaling; Protein
Phosphatase Inhibitor Activity

3.0
Deleted in Liver Cancer 1
DLC1
AI176713

Hepatocarcinogenesis;
Oncosuppressive Activity
3.0
Similar to TG interacting factor
TFIG
BM392224
TGFB-induced factor (TALE family
homeobox)
3.0
Trefoil Factor 1
TFF1
NM_057129

Maintenance of Gastrointestinal
Epithelium; Growth Factor Activity
3.0
Insulin-like Growth Factor
 

Binding Protein 3

IGFBP3
NM_012588

Regulation of Cell Growth;
Insulin-like Growth Factor Binding


 

TABLE VI

Unique genes that increased in duodenum and jejunum


Fold Increase
Duodenum Jejunum

Gene Name
Symbol
Acc. #
Biological Function/ Aliases

2.7

2.0

Eukaryotic Elongation
Factor-2 Kinase


EEF2K

U93849

Protein Amino Acid Phosphorylation
3.1
2.3
Zinc Finger Protein 503
ZFP503
BF560938

DNA Binding: Transcriptional
Regulation

2.0

2.5
Unknown EST Clone
??
AA818159
Unknown

3.2

2.0
Serologically Defined
Colon Cancer
Antigen 33


SDCCAG33


BE117444


Transcription Factor Activity

2.0

2.5
Unknown EST Clone
??
BF387865

Unknown

2.3

2.1
Unknown EST Clone
??
AA850472

Unknown

2.4

2.0

Similar to Retinoic
Acid Inducible
Protein 3



LOC312790


BI276110


Unknown

3.5

4.2
RT1 Class II,
Locus Bb

RT1-BB

AI715202

MHC Class II Protein complex

2.4

2.1
RhoB gene
RHOB
NM_022542


Rho Protein Signal Transduction;
Cell Growth and Maintenance

2.9
2.0
Unknown EST Clone
??
BF558827
Unknown

2.2

2.2
Pyruvate Dehydrogenase
Kinase, Isoenzyme 4
(86% to mouse)

 

PDK4

 

AI175045

 

Muscle Metabolism

3.1

2.0
Retinol Binding
Protein 7, Cellular
(89% to mouse)

 

RBP7

 

BI283223

 


Novel Member of the Cellular
Retinoid Binding Protein Family


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 oocytes (12)].

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.

Table VII

Unique genes that decreased in duodenum or jejunum

 

Duodenum
Fold Decrease

Gene Name Symbol Acc. # Biological Function/Aliases
 

4.5

Unknown EST Clone

??? BI281129 Unknown
4.4
Similar to Death-Associated
Protein (83% to human)

LOC362136

AI716676

Unknown
4.3
Cytochrome P450, 2B19 CYP2B15 AI454613 Monooxygenase Activity
3.6
FAT mRNA FAT NM_031561
Transport of Long-Chain Fatty
Acids/cd 36 antigen
3.4
Unknown EST Clone ??? BI279036 Unknown
3.3

Phosphatase, Orphan 1
(96% to human)

PHOSPHO1 AI112954

Putative Phosphatase Activity

3.2
Similar to RAB30 RAB30 BM392291

Oncogenesis; Member of RAS
Oncogene Family

3.1
Unknown EST Clone ??? BI277131 Unknown

3.1

Unknown EST Clone LOC308556 BI290815 Unknown
3.1
Solute Carrier Family
43, Member 2
SLC43A2 BF394311
Na+-Dependent System-L-like
Amino Acid Transporter
3.0
Pancreatic
Lipase-Related Protein 2
PNLIPRP2 NM_057206
Lipid Metabolism;
Triacylglycerol Lipase Activity
3.0
S100 Calcium Binding
Protein A9
S100A9 NM_053587
Calcium Ion Binding/
Calgranulin B

Jejunum
Fold Decrease
Gene Name Symbol Acc. # Biological Function/Aliases

4.9
Carbonic Anhydrase 3 CA3 NM_019292
One-Carbon Compound
Metabolism; Zinc Ion Binding

 

ACKNOWLEDGEMENTS

These studies were supported by NIH Grant 1 R21 DK 068349 (to JFC) and HRSA Grant (1 C76 HR 00432-01).

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Received: February 14, 2005. Accepted: March 27, 2005.