Potential conflict of interest: Nothing to report.
Hereditary hemochromatosis (HC) is commonly associated with homozygosity for the cysteine-to-tyrosine substitution at position 282 (C282Y) of the HFE protein. This mutation prevents HFE from binding beta2-microglobulin (beta2M) and reaching the cell surface. We have discovered that a widely used hepatoma cell line, Huh-7, carries a HFE mutation similar to that associated with human HC. By HFE gene sequencing of Huh-7 genomic DNA, we found a TAC nucleotide deletion (c. 691_693del) responsible for loss of a tyrosine at position 231 (p. Y231del) of the HFE protein. This mutation affects a conserved hydrophobic region in a loop connecting two beta strands that make up the alpha3 domain of HFE, not far from the 282 site. HFE was detected by western blot in HepG2 but not in Huh-7 cell membrane fractions. In WRL-68 cells expressing wild-type HFE, the HFE protein was largely found at the plasma membrane where it colocalizes with beta2M. On the contrary, the HFE-Y231del mutant, similarly to an exogenously expressed HFE-C282Y mutant, failed to reach the plasma membrane and did not colocalize with membrane-expressed beta2M. C282Y mutant HFE in HC is associated with inadequate hepcidin expression. We found that Huh-7 cells display lower hepcidin messenger RNA levels as compared to HepG2 cells, which carry a wild-type HFE. Interestingly, hepcidin messenger RNA levels increased significantly in Huh-7 cells stably expressing exogenous wild-type HFE at the plasma membrane. Conclusion: Huh-7 cells may represent a novel and valuable tool to investigate the role of altered HFE traffic in iron metabolism and pathogenesis of human HFE HC. (HEPATOLOGY 2010.)
Hereditary hemochromatosis (HC) is a hereditary autosomal recessive disease characterized by accumulation of iron in parenchymal tissues of various organs, with the risk of functional damage and disease.1
The classic form of HC is associated with a substitution of tyrosine for cysteine at position 282 (C282Y) of the HFE protein.2 HFE is a major histocompatibility class I–like protein whose ancestral peptide-binding groove is too narrow to allow antigen presentation.3 Newly synthesized HFE binds to beta2-microglobulin (beta2M),4 an event necessary for its expression on the cell surface and endosomal membranes, where it interacts with the receptor 1 for transferrin (TfR1).5, 6 The C282Y mutation, which is localized in the α3 domain of HFE, disrupts a disulfide bond in HFE that is critical for its binding to beta2M,4 and impairs proper HFE traffic and interaction with TfR1. This may cause inappropriate iron-sensing by the hepatocytes which fail to produce the required amount of hepcidin, a circulating peptide that normally limits iron transfer from the intestine and macrophages to the bloodstream.7 In fact, hepcidin is inappropriately low in human8, 9 and mouse HC.10 In spite of the recent progress in this area, the link between HFE and hepcidin expression in hepatocytes is still not clear.
While checking expression of different iron proteins in human cell lines, we discovered that a widely used hepatoma cell line, Huh-7, carries a mutation within the α3 domain of HFE close to the site where the C282Y mutation lies, and lacks HFE expression on the cell membrane, as occurs in classic C282Y-associated human HC.
Genomic DNA was extracted from the parent Huh-7 cell line, the Huh-7.5 and the BBVII clone derivatives,11, 12 and HepG2 cells. Polymerase chain reaction (PCR)-amplified fragments spanning the coding regions (exons plus intron-exon boundaries) of HFE were purified using the QIAquick PCR purification kit (Qiagen, Milan, Italy) and sequenced using the dye-terminator cycle-sequencing kit (Beckman Coulter, Milan, Italy). DNA fragments were then electrophoretically separated and analyzed with a CEQ 8000 XL Genetic Analysis System (Beckman Coulter).
The HFE complementary DNA (cDNA) was obtained by PCR as specified previously.13 The HFE coding region was excised by enzymatic digestion with PmeI in order to maintain the Myc epitope and was subcloned in the pcDNA3.1/Hygro plasmid (Invitrogen, San Giuliano Milanese, Italy). The C282Y and Y231del plasmids were obtained by mutagenesis using the QuickChange Site-Directed Mutagenesis kit (Stratagene, Milan, Italy).
In order to obtain vectors expressing the untagged forms of wild-type or mutant HFE proteins used in this study, we designed a specific oligonucleotide with an endogenous stop codon (5′-CAAGCTCGAGTCACTCACGTTCAGCTAAGA-3′) and used it to amplify and clone the relevant cDNAs in pcDNA3.1/Hygro.
Huh-7 cells were purchased from Japanese Collection of Research Bioresources (JCRB), Tokyo, Japan. The Huh-7.5 and BBVII derivative clones were kindly provided by Dr. C. Rice (New York, NY) and D. Moradpour (Lausanne, Switzerland). Huh-7 cells, HepG2 cells, and WRL-68 cells were cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum, L-glutamine, and antibiotics at 37°C, in a 5% CO2 environment.
WRL-68 cells stably expressing wild type and mutant HFE-Myc cDNAs were obtained by electroporation (Gene Pulser II; Bio-Rad, Milan, Italy) and antibiotic selection. Briefly, 30 μg of each plasmid were electroporated in WRL-68 cells and, after 72 hours, cells were cultured in DMEM with 400 μg/mL hygromicin for selection of stable transfectants.
Huh-7 cells stably expressing wild-type HFE or empty vector were obtained by transfecting cells with untagged wild-type HFE cDNA construct or empty vector by Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. At 72 hours after transfection, stable transfectants were selected in DMEM supplemented with 50 μg/mL hygromicin.
Immunofluorescence and Confocal Microscopy.
The WRL-68 cells stably transfected with HFE-Myc, HFE-C282Y-Myc, and HFE-Y231del-Myc plasmids were fixed in glass chamber slides with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 minutes at room temperature. After two washes with PBS, cells were permeabilized with 0,05% saponin in PBS for 6 minutes at 4°C; washed twice with PBS and blocked for 45 minutes in cow colostrum diluted 1:10 in PBS. After rinsing in PBS, the slides were subjected to immunofluorescence. In brief, after 1 hour of incubation with the primary antibody, slides were washed and incubated for 1 hour with the appropriate secondary antibody. Both antibodies were diluted in the blocking solution as specified below. The following antibodies were used in immunofluorescence studies: rabbit polyclonal anti-HFE CT16 (1:300 dilution, kindly provided by Dr. W. Sly, St. Louis, MO); anti-beta2M (1:220; Abcam, Cambridge, UK); mouse monoclonal anti-Myc (1:500; Invitrogen); AlexaFluor 488–conjugated goat anti-rabbit immunoglobulin G (IgG) (1:250; Molecular Probes, Invitrogen); AlexaFluor 594–conjugated goat anti-rabbit IgG (1:350;Molecular Probes, Invitrogen), and rabbit anti-mouse IgG tetramethyl rhodamine isothiocyanate–conjugate (1:40, Dako, Milan, Italy).
Confocal microscopy studies were carried out by using a laser-scanning microscope Leica DM IRE2. Images were collected at 512 pixel × 512 pixel resolution and a built-in software was used to reconstruct the images obtained from the confocal sections. The cell monolayers were optically sectioned in the z-axis, and the images were sequentially collected to minimize the crossover between channels.
Biotinylation of Membrane Proteins and Western Blot.
Biotinylation of the plasma membrane of various cell lines was performed using sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropinate (sulfo-NHS-SS-biotin; Pierce Chemical, Celbio, Pero MI, Italy) according to the manufacturer's instructions. Cells were detached on ice in lysis buffer: 150 mM NaCl, 10 mM ethylene diamine tetraacetic acid, 10 mM Tris (pH 7.4), protease inhibitor cocktail, and 1% Triton X-100 (Sigma-Aldrich, Milan, Italy). Biotinylated proteins (1 mg for HepG2 or Huh-7 cell or 250 μg for Huh-7 stable clones) were immunoprecipitated with Streptavidin Agarose Resins according to the manufacturer's instructions.
Samples were heated to 95°C for 5 minutes and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions. All western signals were detected with ECL Advance (GE Healthcare, Milan, Italy) following the manufacturer's instructions. Western blot analysis was performed using either rabbit anti-CT16 antibody (1:30000) or mouse anti–pan-Cadherin (1:50000, Abcam) followed by either peroxidase-conjugated swine anti-rabbit IgG (1:10000; Sigma-Aldrich) or peroxidase-conjugated rabbit anti-mouse IgG (1:50000; Sigma-Aldrich).
Real-Time Quantitative Reverse Transcription PCR.
Total RNA from HepG2 and Huh-7 cells was extracted using TRIzol reagent according to the manufacturer's instructions (Invitrogen). Complementary DNA was generated by reverse transcription of 5 μg of total RNA with 100 ng random primers, 1 mM deoxyribonucleotide triphosphates and 200 U Moloney murine leukemia virus reverse transcriptase (Promega, Milan, Italy) in 1× reverse transcriptase buffer for 1 hour at 37°C. Expression of hepcidin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was analyzed using SYBR GreenER quantitative PCR SuperMix (Invitrogen). Primers were as follows: GAPDH_dir 5′-GGACCTGACCTGCCGTCTAG-3′, GAPDH_rev 5′-TAGCCCAGGATGCCCTTGAG-3′, HAMP_dir 5′-TGTTTTCCCACAACAGACGGG-3′, HAMP_rev 5′-CGCAGCAGAAAATGCAGATGG-3′.
Cycling conditions were 2 minutes at 50°C, 8 minutes and 30 seconds at 95°C, followed by 40 cycles of 15 seconds at 95°C, and 1 minute at 60°C. After 40 amplification cycles, threshold cycle values were automatically calculated using the default settings of the iCycler software (Bio-Rad Laboratories, version 3.0), and femtograms of starting cDNA were calculated from a standard curve covering a range of four orders of magnitude. Hepcidin and GAPDH standard curves ranged from 1 to 1000 femtograms per 25 μL reaction. At the end of the PCR run, melting curves of the amplified products were obtained and used to determine the specificity of the amplification reaction. The change of specific messenger RNA (mRNA) expression was reported as fold-increase as compared to that of control cells. All experiments were performed in triplicate, and repeated at least three times.
Data presented in the figures are mean ± standard deviation. Statistical differences of results were analyzed using the Student t test.
By direct HFE gene sequencing, we found that the Huh-7 parent cell line and its derivative clones Huh-7.5 and BBVII all carry a three–base pair (bp) TAC deletion at positions 691–693 of the HFE gene (Fig. 1A). This mutation results in the deletion of a tyrosine at position 231 (p.Y231del) of the HFE protein. Tyrosine 231 in human HFE is substituted for by phenylalanine in rodent Hfe. Both amino acids are aromatic and are conserved among different species (Fig. 1B). The 230 and 231 tyrosines are located in a loop connecting two beta strands that make up the alpha3 domain of HFE, close to the bottom of the alpha1-alpha2 platform.3 The 231 deletion is not far from the 282 site where the key hemochromatotic mutation lies (Fig. 1B). It is possible that if this tyrosine is deleted, the HFE chain is destabilized or not properly folded.
Because the presence of HFE at the plasma membrane is critical for its postulated role in iron metabolism,4 we specifically investigated whether this mutation would affect HFE traffic to the plasma membrane. Endogenous HFE is barely detectable in human hepatocytes by conventional immunohistochemical techniques, both in vivo14 and in Huh-7 and HepG2 cell lines in vitro (unpublished observations). Therefore, we used the biotin-streptavidin approach to detect HFE in cell membrane extracts by western blot in Huh-7 and HepG2 cells. By using a large amount of protein extracts and after long film exposure, we found that HFE is properly expressed at the plasma membrane in HepG2 cells but is absent in Huh-7 cells (Fig. 2A, compare the bound [B] fraction of HepG2 to Huh-7 cells). The HFE detected in the not-bound (NB in Fig. 2A) fraction in Huh-7 cells (and HepG2 cells) represents the non-immunoprecipitated cytoplasmic fraction of the protein.
Because mutant HFE in HC has been linked to the inadequate hepcidin expression by the liver, we compared hepcidin mRNA expression of Huh-7 cells to that of HepG2 cells, a hepatic cell line carrying a wild-type HFE. When cultured in the same experimental conditions, Huh-7 cells expressed significantly lower hepcidin mRNA than did the HepG2 cells (Fig. 2B).
To study traffic of wild-type and mutant p.Y231del HFE and confirm the defective localization of this mutant HFE, we also used well-differentiated human embryonic liver cells, WRL-68 cells, which stably expressed wild-type and mutant HFE-Myc. By using confocal microscopy, we found that wild-type HFE-Myc, as recognized by both the anti-CT16 (which detects both exogenous and endogenous HFE)4 and anti-Myc (which detects only exogenous HFE) antibodies, is localized at the plasma membrane, where endogenous beta2M is also detected (Fig. 3A). The HFE-C282Y-Myc mutant, as expected and originally described by Waheed et al.,4 was not expressed at the plasma membrane (Fig. 3B). The pattern of the HFE-Y231del-Myc mutant was identical to that of the HFE-C282Y-Myc mutant, with exclusive intracellular localization, and lack of colocalization with membrane-expressed beta2M (Fig. 3C), indicating defective HFE traffic to the cell surface.
To investigate whether lower hepcidin expression in Huh-7 cells is related to the lack of HFE protein at the plasma membrane, we stably expressed an untagged wild-type HFE in Huh-7 cells. We found that after exogenous expression of wild-type HFE in Huh-7 cells, HFE was properly routed to the cell membrane, as shown in the bound (B) fraction of Fig. 4A, and hepcidin mRNA increased significantly as compared to cells stably transfected with an empty plasmid or with HFE C282Y or Y231del mutant plasmids (Fig. 4B).
We have found that the original Huh-7 cell line provided by the JCRB where the cell line was first established, and two derivative clones developed in viral research laboratories,11, 12 carry a HFE mutation that impairs protein traffic to the plasma membrane, as occurs in classic HFE-HC, and is associated with lower hepcidin expression. The Huh-7 cell line was established in 1985 from a hepatoma arisen in a 57-year-old Japanese male.15, 16 The patient was negative for HCV. Unfortunately, no data are available on the iron status of this subject. C282Y-HFE HC affects populations of north European descent.17 Yet, rare pathogenic HFE mutations have been also reported in other ethnic groups, including South Asian populations.18, 19 Interestingly, Mendes and colleagues have reported non-C282Y iron-overloaded Portuguese subjects carrying a Y230F HFE mutation.20 This suggests that, in humans, the HFE region where the 230–231 aromatic amino acids lie may also be important for HFE function in iron metabolism.
It is noteworthy that Huh-7 cells have represented the long-sought cellular system that has finally allowed detailed molecular studies of hepatitis C virus (HCV). In fact, among a variety of tested cell systems, HCV replication appears to be restricted to the Huh-7 cell line, indicating that a favorable cellular environment exists within these cells.21 The Huh-7.5 clone has been derived from replicon-containing Huh-7 cells cured of HCV RNA with interferon alpha.11, 12 Interestingly, recent studies have suggested that reduced expression of classical class I major histocompatibility complex and HFE may provide viruses with an efficient tool for altering cellular metabolism and escaping certain immune responses. Ben-Arieh and colleagues22 have reported that both the stability and assembly of HFE complexes could be modified by the human cytomegalovirus viral protein US2, which targeted HFE molecules for degradation by the proteasome. Seemingly, the human immunodeficiency virus-1 protein Nef (negative regulatory factor) reroutes HFE to a perinuclear structure that overlaps the trans-Golgi network, causing a 90% reduction of surface HFE in human macrophages.23 Whether the defective HFE pathway in Huh-7 cells described here may favor HCV replication is presently unclear.
In conclusion, the Huh-7 cell line carries a HFE mutation that, as occurs in human C282Y-HFE HC, involves a critical protein domain and impairs HFE traffic to the plasma membrane. Huh-7 cells have been used in a variety of studies, including studies on iron metabolism. We have shown here that the Huh-7 cell line represents a novel tool to investigate the effect of altered HFE traffic on iron metabolism and, possibly, help clarifying the pathogenesis of HC.
We wish to thank Pamela Bjorkman, California Institute of Technology, Pasadena, CA, for insightful comments and discussion.