Expression and function of DMT1 without IRE in C6 cells mediated by recombinant adenovirus

Abstract

Divalent metal transporter 1 (DMT1) is a ferrous iron import protein. The improper expression of DMT1 is involved in neurodegenerative diseases. In the present study, we constructed a recombinant adenovirus containing the gene of DMT1 without the iron response element (DMT1-IRE) and investigated its expression and function in the C6 glioma cell line. The DMT1-IRE gene, obtained by RT-PCR, was cloned into the shuttle plasmid pAdTrack-CMV containing green fluorescent protein (GFP) reporter gene. Linearized plasmid pAdTrack-CMV-DMT1-IRE was subsequently co-transformed into Escherichia coli (E. coli) BJ5183 cells along with an adenoviral backbone plasmid pAdEasy-1 after digestion with Pme I. Pac I-digested pAdEasy1-DMT1-IRE was then transfected into E1-transformed human embryonic kidney cells (HEK293 cells) , in which recombinant adenoviruses were generated within 7 to 10 days. The results demonstrated that we obtained the DMT1-IRE gene. pAdEasy1-DMT1-IRE yielded a large fragment, plus a smaller fragment of 4.5 kb after digestion with PacI. PCR confirmed pAdEasy1-DMT1-IRE contained gene DMT1-IRE, indicating the successful construction of recombinant adenovirus plasmid containing DMT1-IRE. GFP fluorescence further confirmed the generation of adenovirus. AdDMT1-IRE could efficiently infect C6 glioma cells. And cell viability decreased in Ad-DMT1-IRE infected cells after iron overload compared to the control. These results suggest that the over expressed DMT1-IRE can aggravate the iron induced cell death due to its iron influx function.

Keywords

divalent metal transporter 1 / recombinant adenovirus / homologous recombination / iron

Introduction

Recent researchers have proven that iron played a key role in the Parkinson disease (PD) and other neurodegenerative diseases. Increased iron content was found in the substantia nigra pars compacta (SNPc) of Parkinsonian patient brains [1-3]. However, the underlying mechanisms of iron accumulation in the SNPc remained unknown. The discovery of divalent metal transporter 1 (DMT1) is the most important breakthrough in the field of mammalian iron metabolism in recent years. DMT1, also named as nature resistance associated macrophage protein 2 (Nramp2) or divalent cation transporter 1 (DCT1), was first identified in 1995 in a screen for homologs of Nramp1, a protein involved in host defense [4]. DMT1 is considered as a major duodenal ferrous iron transporter [5-7]. High levels of DMT1 have been observed in the brain and expressed on neurons, astrocytes and microglia [8].

The evidence in our previous experiments proved that the expression of DMT1 increased in the substantial nigra (SN) where iron deposited in rodent PD models, suggesting its involvement in iron accumulation in the SN [9,10]. In the present study, a gene reconstruction technique was used to construct recombinant adenovirus expression vector encoding human gene of DMT1 without the iron response element (DMT1-IRE) and we investigated its function in C6 glioma cell line.

Materials and methods

Materials

The C6 glioma cell line was from the cell bank of the Chinese Academy of Science. Pme I and Pac I were from NEB. Hind III, Kpn I and PMD18-T simple vector were from Takara (Japan). E1-transformed human embryonic kidney cells (HEK293 cells), pAdtrack-CMV, pAdeasy1, DH10B, JM109 were reserved in our laboratory. Lipofectamine 2000 was from Promega (USA). FeSO₄·7H₂O and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were from Sigma (USA). Dulbecco’s modified Eagle’s medium (DMEM) was from Gibco (USA). Other chemicals and regents available were from local commercial sources.

Methods

Cell culture

HEK293 cells were maintained in DMEM growth medium supplemented with 10% fetal bovine serum (FBS), 100 unit/mL of penicillin, and 100 mg/mL of streptomycin at 37°C in 5% CO₂. C6 cells were cultured in DMEM growth medium supplemented with 10% FBS, 100 unit/mL penicillin and 100 μg/mL streptomycin at 37°C, in a humid 5% CO₂ and 95% air environment.

Total RNA extraction and nested reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was isolated by using Trizol reagent (Invitrogen, USA) from frozen tissues of human duodenum according to the manufacturer’s instructions. Then 2 μg of total RNA was reversed transcribed in a 20 μL reaction using reverse-transcription system (Promega, USA). In order to obtain a full-length gene, nested PCR was used. Primers were designed by computer software (Primer Premier 5.0). In the first round of amplification, the primers were 5'-AGGGTACCTCTAAGAACTCAGCCACTCAG-3', 3'-GGATACCCGAACACAGAACTTCGAACCA-5'. The diluted (1∶100) resulting product was used as template for secondary PCR reactions. The secondary forward primer was changed to 5'-AAGGTACCACCATGGTGCTGGGTCCTG-3', which included a Kpn I site. The reverse primer for DMT1-IRE was 3'-GTCTGCAAAACTTGTTTTCCGTTTTCGAACG-5', which introduced a Hind III restriction site. High-fidelity PCR reactions were ensured by using a mixture of Taq DNA polymerase (TaKaRa, Japan) and high-fidelity DNA polymerase pyrobest (TaKaRa, Japan) in a ratio of 9∶1. Thermocycling was carried out as follows: 94°C for 5 min, then 30 cycles of 94°C for 1 min, 63°C for 1 min and 72°C for 2 min, followed by 72°C for 10 min. The PCR products were cloned into a PMD18-T simple vector (Takara, Japan) for sequencing.

Construction of recombinant expression vector encoding human DMT1-IRE

Human DMT1-IRE gene was digested from PMD18-T simple vector with Kpn I and Hind III, and then cloned into the shuttle plasmid pAdTrack-CMV containing GFP reporter gene. Linearized resultant vector pAdCMV-DMT1-IRE cleaved by Pme I was subsequently co-transformed into Escherichia coli (E. coli) strain BJ5183 cells along with a supercoiled adenoviral backbone plasmid pAdEasy-1. Recombinants were selected for kanamycin resistance and confirmed by PCR and multiple restriction endonuclease analyses. Candidate clones usually yielded a large fragment, plus a smaller fragment of 3.0 kb or 4.5 kb according to different recombination.

Viral package and amplification

HEK 293cells (E1-transformed human embryonic kidney cells) were seeded at 2×10⁶ cells/mL per flask 24 hours prior to transfection. On the day of transfection, recombinant adenovirus vector pAdDMT1-IRE was cleaved by Pac I to expose inverted terminal repeats, and then was transfected into HEK293 cells with lipofectamine 2000 according to manufacture’s manual and in which recombinant adenovirus was generated within 7 to 10 days. GFP expression was visualized by fluorescence microscopy at the indicated time thereafter. Then, four cycles of freeze/thaw/vortex were performed on harvested cells, and the lysate was centrifuged at 1000 r/min for 10 min. The recombinant adenovirus was named as AdDMT1-IRE. The recombinant adenovirus was amplified in HEK293 cells and stored at -80°C.

Infection of C6 cells

C6 cells were resuspended in DMEM with 5% FBS supplement and seeded at a density of 2×10⁵ cells/mL 24 hours prior to infection. When the confluence was about 50% to 70%, the cells were infected with recombinant adenovirus AdDMT1-IRE, and GFP fluorescence was observed at the indicated time to verify the infective efficiency.

The expression of DMT1 detected by RT-PCR in AdDMT1-IRE infected cells

After infection with recombinant adenovirus AdDMT1-IRE for 36 hours, RNA was isolated from infected C6 cells using Trizol reagent. We amplified 380 bp with the primers (sense: 5'-TAGGTACCATGCAGTATCCCAAGGTCCCAC-3', antisense: 5'-AGCAAGCTTTTAGCCCAC AGCCTGTTCAATC-3'). The samples were heated at 95°C for 5 min and the subsequent cycles were performed at three temperature steps: 94°C for 30 s, 54°C for 30 s, 72°C for 45 s. After 32 cycles an additional 10 min for extension was carried out. Ethidium bromide stained gels were scanned and qualified using Tanon Image Software; DMT1 levels were normalized with respect to GAPDH levels.

MTT assay

C6 cells were detached with 0.25% trypsin plus 0.02% ethylenediamine tetraacetic acid (EDTA), resuspended in DMEM with 2% FBS supplement and seeded at a density of 2×10⁴ cells/mL onto 96-well plates. After attachment, ferrous iron (200 μmol/L) was added to DMEM without serum supplement for the subsequent 24 hours. After the cells were incubated in MTT (5 mg/mL) for 4 hours, cell injury was measured by colorimetric assay (TECAN, Austria).

Statistical analysis

Each experiment was performed at least three times and the results analyzed by SPSS were presented as \overline{x}\pm s. T test was used to compare differences between means. A P value less than 0.05 was considered to be statistically significant.

Results

Construction of recombinant adenovirus plasmid containing human DMT1 gene

Human DMT1-IRE gene was amplified by RT-PCR (Fig. 1a). Then the product was confirmed by sequencing. DMT1 gene in the plasmid pAdCMV-DMT1-IRE was confirmed by PCR (Fig. 1b) and endonuclease analyses (Fig. 1c). Recombinant adenovirus plasmid encoding DMT1-IRE was confirmed by Pac I endonuclease analysis (Fig. 1d), pAdDMT 1-IRE could yield a large fragment plus a smaller fragment of 4.5 kb, indicating the successful construction of recombinant adenovirus expression plasmid.

Recombinant adenovirus package and virus amplification

Seven to ten days after transfection of plasmid pAd-DMT1-IRE into HEK293 cells, GFP fluorescence expression was observed and increased time-dependently (Fig. 2). Recombinant adenovirus AdDMT1-IRE was obtained after package and amplification in HEK293 cells.

DMT1 expression increased in Ad-DMT 1-IRE infected C6 cells.

The mRNA levels of DMT1 were observed by RT-PCR after infection with recombinant adenovirus AdDMT1-IRE for 36 hours. Data showed that DMT1 mRNA levels increased significantly after infection with AdDMT1-IRE compared with the control (Fig. 3).

Recombinant adenovirus mediated increased DMT1-IRE expression decreased cell viability after iron overload

The cell viability assay was performed to detect iron induced cell death. A significant reduction of cell viability was observed when AdDMT1-IRE infected C6 cells were treated with 200 μmol/L ferrous iron compared to that of control (Fig. 4).

Discussion

Elevated iron has been reported in the brain of Parkinsonian patients. Iron levels increase significantly in SNPc, however no obvious changes have been observed in substantia nigra pars reticulata (SNr) [11,12]. Due to the cytotoxicity of ferrous iron by Fenton reaction, iron accumulation has been a key factor in the etiology of PD. Evidence has proved that iron accumulation played a key role in the etiology and pathology in neurodegenerative disease, such as PD.

DMT1, previously known as Nramp2, was first identified in 1995, in a screen for a homolog of Nramp1, a protein involved in host defense [4]. The biological function of DMT1 has been determined through phenotypic analysis of Microcytic anemia (mk) mice and Belgrade (b) rats which has spontaneous mutations in DMT1 [6,13]. These mutations converted amino acid 185 from glycine to arginine (G185R), and resulted in severe impairment of iron transport function [4]. Both animals have deficits in the iron transport from the gut lumen into the absorptive enterocyte, and from plasma transferrin into erythroid precursors. This indicates that DMT1 is important for both of the iron transport ways. DMT1 transports divalent (ferrous) iron, most likely produced by the reduction of ferric iron at the cell surface, or within an endosomal compartment [5]. DMT1 has been shown to be expressed in the brain, suggesting a role for this transporter in brain iron uptake [15]. In this study we have constructed recombinant adenovirus encoding human DMT1-IRE successfully as an experimental model of high DMT1 expression with AdEasy system.

Adenovirus-mediated genome transfer by using AdEasy system has distinct advantages. For example, it can carry long exogenous DNA fragments, permit the expression of recombinant proteins in most mammalian cell lines and tissues, accurately express and modify the recombinant protein. More importantly, because the infection does not interfere with the host genome, it shows a low risk to induce alteration of the host DNA. So it has been widely used to develop research on gene expression [16-18]. Additionally, high titers of viruses and high levels of transgene expression could generally be obtained [19].

In our study, C6 glioma cell line was used as a model to investigate the expression and function of DMT1-IRE in the glial cells. After infection with AdDMT1-IRE, RT-PCR was used to confirm the expression of DMT1-IRE in C6 cells. As mentioned above, DMT1 is an iron importer protein, responsible for ferrous iron influx. Excessive ferrous iron can damage cells by Fenton reaction. To determine whether increased DMT1-IRE could induce more ferrous iron influx and aggravate iron induced cell death, we detected the cell viability in C6 cells after ferrous iron overload by MTT assay. We demonstrated that the cell viability in AdDMT1-IRE infected cells decreased significantly compared to the control after iron overload. This confirmed our hypothesis that increased expression of DMT1-IRE in C6 glioma cells could increase the ferrous iron influx and aggravate the iron induced cell damage.

In summary, we concluded that DMT1-IRE mediated by recombinant adenovirus could be expressed in C6 cells and could increase iron induced cell death.

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