Expression of divalent metal transporter 1 (DMT1) isoforms in first trimester human placenta and embryonic tissues

W.S. Chong1, P.C. Kwan1, L.Y. Chan1, P.Y. Chiu1, T.K. Cheung2 and T.K. Lau1,3

1 Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong SAR, People’s Republic of China and 2 Department of Gastroenterology and Hepatology, Princess Alexandra Hospital, Queensland, Australia

3 To whom the correspondence should be addressed. E-mail: tzekinlau{at}cuhk.edu.hk


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Divalent metal transporter 1 (DMT1) is a transmembrane glycoprotein which mediates the proton-coupled transport of a variety of divalent metal ions. Two isoforms, which differ by the presence (DMT1-IRE) or absence (DMT1-nonIRE) of an iron-responsive element (IRE) in their 3’ untranslated region, are implicated in apical iron transport and endosomal iron transport respectively. Although the expression pattern of DMT1 isoforms is tissue specific in adult, data regarding its expression in embryonic tissues are lacking. METHODS: Semiquantitative RT–PCR and immunohistochemistry were used to study the mRNA and protein expression of both DMT1 isoforms in embryonic tissues between 8 and 14 weeks gestational age. RESULTS: DMT1-IRE and DMT1-nonIRE expressions were ubiquitous in embryonic tissues examined. In the lung, statistically significant correlations were found between the levels of DMT1 isoform expression and gestational age. In the placenta, DMT1-IRE was the predominantly expressed isoform. Both isoform proteins were localized in embryonic epithelial cellular membrane. CONCLUSION: Both DMT1 isoforms are ubiquitously expressed in embryonic tissues in the first trimester. Predominant DMT1-IRE isoform expression in placenta suggests an iron-regulatory mechanism reminiscent of that in the adult duodenum. Epithelial distributions of both DMT1 isoforms are associated with the absorptive or excretory functions of the expressed tissues.

Key words: divalent metal transporter 1/early pregnancy/fetal tissues/human placenta/metal transport


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Divalent metal transporter (DMT1), also known as natural resistance-associated macrophage protein 2 (NRAMP2), divalent cation transporter 1 (DCT1) and solute carrier family 11, member 2 (SLC11A2), is a mammalian transmembrane proton-coupled metal-ion transporter which mediates the transport of multiple divalent metal ions but with its highest affinity for iron (Gunshin et al., 1997Go). Mutational studies in Belgrade rat and microcytic mouse (Fleming et al., 1997Go, 1998Go) together with the ectopic expression studies (Fleming et al., 1999Go; Levy et al., 1999Go; Canonne-Hergaux et al., 2001Go; Griffiths et al., 2001Go) demonstrated an important role of DMT1 in gastrointestinal iron uptake and endosomal iron transport. The discovery of DMT1 mRNA isoform with an iron-responsive element (IRE) in the 3’ untranslated region accounts for its ability to maintain iron homeostasis through regulation of its expression level in response to dietary iron status, whereas the DMT1-nonIRE isoform which lacks the IRE domain is devoid of this function (Lee et al., 1998Go). The proteins of DMT1-IRE and DMT1-nonIRE differ in their last C-terminal 18 or 25 amino acids and are found to have differential cell type specificity and distinct subcellular localization in the adult human (Canonne-Hergaux et al., 1999Go; Tabuchi et al., 2002Go; Tchernitchko et al., 2002Go). The predominance of DMT1-IRE expression in epithelial cell lines and DMT1-nonIRE expression in erythroid precursor cells suggests their role as apical iron transporter and endosomal iron transporter respectively. Despite the ubiquitous expression of both DMT1 isoforms in adult tissues (Gunshin et al., 2001Go), the relative expression levels of DMT1 isoform are tissue-specific which contributes to the different functions of DMT1-IRE and DMT1-nonIRE in gastrointestinal iron uptake and metal detoxification respectively.

Placenta is the sole organ that serves both absorptive and excretory functions of the growing fetus. DMT1 is perceived to participate in the traffic of divalent metal ions across the placental tissue. In human term placenta, DMT1 has been shown immunohistochemically to localize in cytoplasm and basal membrane of syncytiotrophoblast, which led to the speculation that the transport of ferrous iron from the endosome to the cytoplasm as well as across the basal membrane is mediated by DMT1 (Georgieff et al., 2000Go). However, information regarding placental DMT1 expression during early pregnancy is lacking.

During pregnancy, the developing embryo is highly vulnerable to inappropriate mineral status. Deleterious consequences may result from deficiency or overload of divalent metal ions, including iron (Gambling et al., 2003Go). Iron supplementation is usually recommended in the case of anaemia during pregnancy. However, a recent study showed that only supplementation given in early gestation is effective in rat (Gambling et al., 2004Go). Liver DMT1-IRE and DMT1-nonIRE were increased and decreased respectively in neonates born from an anaemic mother. This indicated that the expressions of DMT1 isoforms in fetal tissues are regulated by maternal iron status through gestation. As complex interactions of body iron status with the absorption of essential copper (Gambling et al. 2003Go), nickel (Tallkvist et al., 2003Go), manganese and cobalt (Forbes and Gros, 2003Go; Roth and Garrick, 2003Go) as well as nonessential toxic cadmium and lead (Bressler et al., 2004Go), are mediated by DMT1 in the human adult and the proved transport activities towards copper (Arredondo et al., 2003Go), cadmium and lead (Tallkvist et al., 2001Go; Bannon et al., 2002Go; Leazer et al., 2002Go; Park et al., 2002Go; Okubo et al., 2003Go) are mediated by DMT1, differential expression of DMT1 isoforms in embryonic tissues may have implications for the mechanism of mineral homeostasis during gestation. The aim of the present study was to investigate the expression pattern of DMT1 isoforms in a variety of embryonic organs in early gestation.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The Clinical Research Ethics Committee of The Chinese University of Hong Kong approved the study protocol and the study subjects gave informed consent for participation. Women who were admitted for surgical termination of pregnancy at <14 weeks of gestation for psychosocial reasons were invited to participate in this study. All women had been assessed previously in the outpatient clinic by a clinical gynaecologist to be eligible for a legal termination of pregnancy. None of the investigators were involved in the clinical decision as to whether the request for termination of pregnancy was accepted or not.

Tissue procurement and preservation
All women had suction termination of pregnancy according to the standard clinical protocol. If a subject consented to the study, an ultrasound scan was performed to measure the crown-rump length (CRL) of the embryo to confirm the gestational age, and a case would be excluded if major fetal abnormality was detected. All aborted tissues were collected and examined under a dissecting microscope. Individual organs of interest (placenta, intestine, kidney and lung) were identified, isolated, and rinsed in isotonic saline prior to further processing. Specimens procured were allocated to RT–PCR study (samples were snap-frozen in liquid nitrogen and stored at –70°C) or immunohistochemistry study (samples were fixed in 10% formalin for 24 h and subsequently for paraffin embedding).

Semiquantitative RT–PCR
Total RNA was isolated by RNeasy Protect Mini Kit (Qiagen GmbH, Hilden, Germany) and the first strand cDNA was obtained by reverse transcription from 1 g of extracted RNA by using GeneAmp® Gold RNA PCR Reagent Kit (PE Applied Biosystem) according to manufacturer’s instructions. The amplification of the cDNA was performed at 94°C for 1 minute, 65°C for 1 min, and 72°C for 2 min for 25 ({beta}-actin) or 30 (DMT1-IRE/DMT1-nonIRE) cycles depending on the set of primers used (Table I). The PCR products from each case were electrophoresed simultaneously on a 2% ethidium bromide-stained agarose gel. Resolved gel images were then captured and quantified by Gel Doc 1000 system (Bio-Rad Laboratories, CA, USA).


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Table I. Sequences of human oligonucleotide primers used in this study

 

Immunohistochemical staining
Rabbit anti-human DMT1-IRE antibodies and rabbit anti-rat DMT1-nonIRE antibodies were purchased from Alpha Diagnostic International Inc. (San Antonio, TX, USA). Immunohistochemcial reagents including Biotin-Blocking System, StreptABComplex/HRP Duet Mouse/Rabbit Kit and Liquid DAB Substrate-Chromogen System were purchased from Dako A/S (Glostrup, Denmark).

Paraffin-embedded specimens were cut into 5 m thick sections and mounted on 3-aminopropyltriethoxysilane-coated glass slides. Sections were de-waxed in xlyene, rehydrated through serial graded ethanols, and rinsed in running tap water. Sections were incubated in 3% H2O2 in methanol for 10 min to quench endogenous peroxidase activity prior to trypsin (0.25 g/ml; Gibco BRL, Gaitherburg, MD, USA) digestion at 37°C for 10 min. To block non-specific antibody binding, sections were incubated with 20% goat serum in 0.5% bovine serum albumin in phosphate-buffered saline (PBS) for 20 min, and subsequently incubated with rabbit anti-rat DMT1-nonIRE antibody (1:100) or rabbit anti-human DMT1-IRE antibody (1:200) overnight at 4°C in a humidity chamber. Negative control sections were replaced with rabbit IgG at the same dilution as corresponding primary antibodies used. Specific staining of the primary antibodies for both isoforms were tested by preincubating the antibodies with blocking peptide (1:20; Alpha Diagnostic International Inc., San Antonio, TX, USA) for 24 h at 4°C. Adjacent sections were incubated with biotinylated goat IgG and subsequently horseradish peroxide-conjugated streptavidin according to the instructions stated in StreptABComplex/HRP Duet Mouse/Rabbit Kit. Antibody-bound sites were revealed by incubation with Liquid DAB Substrate-Chromogen System for 6 min and counterstained with Methyl Green. Slides were dehydrated with butan-1-ol, cleared in xylene and mounted with cover glass using DPX mountant (Fluka, Milwaukee, WI, USA) before microscopic examination.

Statistical analysis
All statistical calculations were performed using Statistical Package for Social Sciences for Windows version 11.01 (SPSS Inc., USA). Spearman rank coefficient of correlation was calculated to determine any association between mRNA expression levels and gestational age in different tissue types. While non-parametric Kruskal-Wallis H-test was used for comparisons of DMT1 expression levels among tissues, Mann-Whitney U-test was used for comparisons of mRNA expression levels between DMT1 isoforms in the same tissue. P < 0.05 was considered statistically significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Sample statistics
There were a total of 99 cases of singleton pregnancies ranging from 8.0 to 13.5 weeks of gestation (mean ± SD 10.75 ± 1.58) recruited for this study. Among them, 34 cases were allocated to RT–PCR while the other 65 cases were used for immunohistochemistry. As a complete tissue set was not always obtainable in each case, the sample number for each tissue involved in each group varied. For RT–PCR study, there were 17 samples of intestine and 13 samples each of placenta, kidney and lung. Among samples for immunohistochemistry, there were 27 intestinal samples, 33 samples of placenta, 27 samples of kidney, and 32 samples of lung.

Embryonic tissue distribution of DMT1 isoform mRNA
To determine if both DMT1 isoforms were expressed in various embryonic tissues, PCR amplification was performed with specific primers for individual DMT1 isoform in intestine, kidney, lung and placenta. For each tissue, independent amplifications were carried out in triplicate in parallel to both DMT1 isoforms and {beta}-actin.

Our results showed that both DMT1 mRNA isoforms are ubiquitously expressed at varying levels in all tissues examined (Figure 1). After normalization using individual {beta}-actin intensity as internal reference, the expression of DMT1-IRE was found to be significantly higher than that of DMT1-nonIRE isoform in the placenta (P < 0.05) (Figure 2). No significant difference was detected between the DMT1-IRE and DMT1-nonIRE isoforms in the lung, kidney and intestine.



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Figure 1. Representative photograph of separated RT-PCR products encoding for two isoforms of divalent metal transporter-1 (DMT-1) and ß-actin in various embryonic tissues. Total RNA was extracted from the embryonic intestine (I), placenta (P), kidney (K) and lung (L) and was subjected to RT–PCR. The PCR products of a typical week 13 case were visualized on the same gel using the Gel Doc 1000 System.

 


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Figure 2. Relative gene expression levels of divalent metal transporter-1 (DMT-1) isoforms in various embryonic tissues. Densitometric mRNA quantities were normalized by endogenous {beta}-actin levels. The results are represented as box plots. *P< 0.05.

 

Effect of gestational age on the level of DMT1 expression
In fetal lung tissue samples, there was significant positive correlation between gestational age and expression of DMT1-IRE (rs = 0.634, P < 0.05), and between gestational age and expression of DMT1-nonIRE (rs = 0.626, P < 0.05) (Figure 3A, B). The levels of DMT1-IRE and DMT1-nonIRE expression increased with increasing gestational age. In the placenta, kidney and intestine, the levels of DMT1-IRE and DMT1-nonIRE mRNA expression were independent of gestational ages. In the kidney (Figure 3C, D), the associations between the isoform expression levels and gestational age were not statistically significant.



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Figure 3. Relative gene expression levels of divalent metal transporter-1 (DMT-1)-iron-responsive element (IRE) (A, C) and DMT1-nonIRE (B, D) in lung and kidney among different gestational ages. Messenger RNA levels of DMT1-IRE and DMT1-nonIRE in developing lung (A, B) increased with gestational age. In contrast, mRNA levels of both isoforms in developing kidney (C, D) do not correlate with gestational age. NS = not significant.

 

Embryonic tissue distribution of DMT1 isoforms
Placenta
In the placenta, positive staining of DMT1-IRE (Figure 4C) and DMT1-nonIRE (Figure 4D) was prominent in plasma membrane and cytoplasm of syncytiotrophoblast. Diffuse staining was also found in cytotrophoblast and other stromal cells of villous core regions. Rabbit IgG eliminated staining in negative control sections (Figure 7A).



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Figure 4. Immunohistochemical staining of divalent metal transporter-1 (DMT-1) isoforms of placental tissues at term (A, B) and gestational week 13 (C–D). Syncytiotroplast obtained from term placenta stained positively for DMT1-iron-responsive element (IRE) (A) and DMT1-nonIRE (B). Intense IRE (C) and nonIRE (D) staining are localized both in the cytoplasm and plasma membrane of syncytiotrophoplast. Degenerative syncytiotrophoplast were not stained (arrow). Magnification: x400. Bar = 25 m.

 


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Figure 7. Negative control for the placenta (A), the intestine (B), the kidney (C), and the lung (D) showed no background staining. Magnification: x200. Bar = 50 m.

 

Kidney
Developing embryonic kidney tissues demonstrated the presence of both DMT1 isoform in cellular membrane of early glomeruli and the epithelium of newly formed collecting tubules (Figure 5). The pattern of isoform staining was similar. Negative control showed no background staining (Figure 7C).



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Figure 5. Photomicrographs of iron-responsive element (IRE) and nonIRE isoform staining of developing kidney at 13th gestational week. Intense staining of both isoforms is seen in developing tubules (T) but only sparsely in glomeruli (G) (A, B; magnification: x400; Bar = 25 m). Diffuse staining of both isoforms was also observed in mesenchyme. Higher magnification (x1000; bar = 25 m) revealed staining for both isoforms localized in tubular membrane (C, D) and also cytoplasm of developing glomeruli (E, F).

 

Intestine
Positive staining of both isoforms was found in intestinal epithelium. Intense staining of DMT1-IRE was localized in plasma membrane of developing enterocytes (Figure 6A). And the signal intensity of DMT1-nonIRE was observed evenly on both sides of enterocytes (Figure 6B). Negative control showed no background staining (Figure 7B).



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Figure 6. Representative photomicrographs demonstrate the immunohistochemical staining of divalent metal transporter-1 (DMT-1)-iron-responsive element (IRE) (A, C) and DMT1-nonIRE (B, D) of developing intestine and lung at 13th gestational week. (A) Staining of IRE is prominent in the luminal membrane of developing villous enterocytes (filled triangle). (B) Intense membranous staining of nonIRE isoform is also seen over the enterocyte surfaces along the villus–crypt axis. In embryonic lung, the epithelial membranes of developing bronchioli (Br) are stained positively for IRE (C) and non-IRE (D) isoforms. Mesenchymal tissues are negatively stained. Magnification: x400. Bar = 25 m.

 

Lung
The positive staining of pulmonary tissues in glandular phase showed the membranous localization of both DMT1 isoforms in developing brochioli epithelium (Figure 6C, D). Mesenchymal tissues were not stained. Spatial distributions of both isoforms were similar. Negative control sections showed no background staining (Figure 7D).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study, we have demonstrated for the first time the expression and distribution of DMT1-IRE and DMT1-nonIRE in human embryonic intestine, kidney, lung and placental tissues during early pregnancy. The expression of both DMT1 isoforms studied at transcriptional and translational levels is ubiquitous in the embryonic tissues, as early as the 8th gestational week. The mRNA expression levels of DMT1-IRE and DMT1-nonIRE varied in different tissues. However, the predominant expression of both isoforms in kidney as reported previously (Hubert and Hentze, 2002Go; Tchernitchko et al., 2002Go) in rodent adult was not shown. This difference in the expression pattern of DMT1 isoforms observed among embryonic and adult tissues may suggest differential regulatory and functional roles of DMT1 at different stages of development.

Both DMT1 isoforms were expressed in placental tissue of early pregnancy. Our immunohistochemical study demonstrated that they localized primarily in plasma membrane of syncytiotrophoblast which is consistent with previous findings in term human placenta (Georgieff et al., 2000Go) even though the investigator had not studied the expression of different isoforms. Moreover, the predominant mRNA expression of DMT1-IRE in placenta suggests that DMT1 expression is iron-regulatory and that its expression might increase in case of iron deficiency. The placental DMT1 isoform expression pattern shown in this study is analogous to that found in mature duodenal enterocytes which are specialized for absorptive function and where the DMT1-IRE species predominates (Canonne-Hergaux et al., 1999Go; Griffiths et al., 2001Go). Intestinal tract of the first trimester is known to take up macromolecules from the lumen. However, the observed DMT1-IRE predominant expression pattern of adult intestine was not observed in developing intestine during first trimester. Instead, both mRNA isoform expression levels were found in comparable quantities with similar membranous localization in intestinal epithelium. The difference in isoform expression patterns between the fetal and adult intestinal tissue is not unexpected because the fetal intestine is not the primary site of iron absorption. The distribution pattern of DMT1 isoforms in the fetal placenta and intestine strongly suggests that the DMT1-IRE has a regulatory function in iron absorption.

In this study, mRNA expression levels of both isoforms in the lung were found to increase with advancing gestational age. Our immunohistochemical data show that both DMT1 isoforms localized specifically to the plasma membrane of bronchioli epithelium but not in the mesenchyme. Specific epithelial DMT1 staining provides clues concerning the correlation between mRNA expression level of both DMT1 isoform and gestational age. In the first trimester, fetal lung is in the pseudoglandular stage of development and undergoes aggressive branching morphogenesis (Harding, 1994Go). Therefore, the proportion of epithelium to mesenchyme is expected to increase with gestational age. Since DMT1 expression was limited to epithelial tissues, this might explain the positive correlation observed between the DMT1 isoform expression levels and gestational ages in our study. As pulmonary fluid is actively secreted in the first trimester lung, the expression of DMT1 isoforms in epithelial lining suggest that it might participate in the excretion or re-absorption of a variety of metal ions which are important to metal ion homeostasis of the developing fetus. The DMT1 isoform expression profile in the lung is in parallel to that found in adult glandular tissues which are involved in excretion or re-absorption processes (Canonne-Hergaux et al., 2000Go; Tchernitchko et al., 2000; Koch et al., 2003Go).

In the developing kidney, expression of both DMT1 isoforms in the tubular epithelium of the present study is in agreement with that in adult kidney, indicating that the regulation of metal ion excretion and reabsorption occurs as early as in the first trimester fetus (Brace et al., 1998Go; Ferguson et al., 2001Go). Although the transition of mesonephric to metanephric nephrons during the first trimester is characterized by differentiation of mesenchymal to epithelial metanephron (Brophy and Robillard, 2004Go) which suggested the same patterns of both DMT1 isoforms levels as that observed in the lung during gestation, discrete correlations between the DMT1 expression levels and gestational ages were not evident in kidney as shown in this study. It may be because the developing nephric mesenchyme also expressed both DMT1 isoforms at low levels as revealed by the diffuse, faintly stained areas in immunohistochemistry. However, the expression of DMT1 in metanephron persists in mature kidney. It has previously been shown to be expressed in collecting duct, thick ascending loop of Henle, and distal convoluted tubules (Ferguson et al., 2001Go).

In conclusion, we have shown that both DMT1 isoforms are ubiquitously expressed in the embryonic placenta, lung, kidney and intestine as early as the 8th gestational week. Specific epithelial distribution of both DMT1 isoforms in the embryonic tissues participating in the excretion or re-absorption of metal ions suggests the importance of DMT1 in maintaining metal ion homeostasis in the developing fetus. Predominant DMT1-IRE isoform expression in fetal placenta as shown in this study and the adult intestine as described previously (Hubert and Hentze, 2002Go) suggest a critical role of this protein isoform in iron-regulatory absorption pathway of divalent metal ion in these tissues. Further studies on the relationship between the maternal plasma iron concentration and DMT1 isoform expression levels in syncytiotrophoblast may be valuable to determine the importance of DMT1 in iron-regulatory divalent metal ion absorption pathway. Given that there are complex interactions between iron and other metal micronutrients through the common DMT1 transport pathway, further work is required to examine the possible effect of their interactions on fetal growth and development, especially during iron supplementation in early pregnancy.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on May 13, 2005; resubmitted on June 23, 2005; accepted on July 6, 2005.





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