Haemochromatosis is a genetic disorder of iron overload resulting from loss-of-function mutations in genes coding for the iron-regulatory proteins HFE (human leucocyte antigen-like protein involved in iron homoeostasis), transferrin receptor 2, ferroportin, hepcidin and HJV (haemojuvelin). Recent studies have established the expression of all of the five genes in the retina, indicating their importance in retinal iron homoeostasis. Previously, we demonstrated that HJV is expressed in RPE (retinal pigment epithelium), the outer and inner nuclear layers and the ganglion cell layer. In the present paper, we report on the consequences of Hjv deletion on the retina in mice. Hjv−/− mice at ≥18 months of age had increased iron accumulation in the retina with marked morphological damage compared with age-matched controls; these changes were not found in younger mice. The retinal phenotype in Hjv−/− mice included hyperplasia of RPE. We isolated RPE cells from wild-type and Hjv−/− mice and examined their growth patterns. Hjv−/− RPE cells were less senescent and exhibited a hyperproliferative phenotype. Hjv−/− RPE cells also showed up-regulation of Slc7a11 (solute carrier family 7 member 11 gene), which encodes the ‘transporter proper’ subunit xCT in the heterodimeric amino acid transporter xCT/4F2hc (cystine/glutamate exchanger). BMP6 (bone morphogenetic protein 6) could not induce hepcidin expression in Hjv−/− RPE cells, confirming that retinal cells require HJV for induction of hepcidin via BMP6 signalling. HJV is a glycosylphosphatidylinositol-anchored protein, and the membrane-associated HJV is necessary for BMP6-mediated activation of hepcidin promoter in RPE cells. Taken together, these results confirm the biological importance of HJV in the regulation of iron homoeostasis in the retina and in RPE.

INTRODUCTION

Iron is an essential nutrient obligatory for vital cellular functions. However, it can provoke oxidative stress and cellular dysfunction when it accumulates excessively in tissues. In recent years, we have been investigating the retinal expression of various genes involved in the maintenance of iron homoeostasis to understand the role of these genes in retinal health and disease [15]. The rationale for these investigations was the growing evidence that the retina is also susceptible to iron-induced oxidative damage similar to other organs. The presence of excess iron in retina has been demonstrated in patients with AMD (age-related macular degeneration) [6,7] and aceruloplasminaemia with associated macular degeneration [8]. Similarly, knockout mice with disruption of two iron-oxidizing enzymes, ceruloplasmin and hephaestin, have excessive iron accumulation in RPE (retinal pigment epithelium) [9,10]. Hereditary haemochromatosis, an autosomal recessive disorder of iron metabolism, is the most common genetic disease in Caucasians with homozygosity in the range of approximately 1 in 300. Haemochromatosis patients accumulate excess iron in various organs, including the liver, pancreas, kidney, heart, and brain [1114]. This results in hepatocarcinoma/cirrhosis, diabetes, nephropathy, cardiomyopathy and pituitary dysfunction. Almost 85% of the cases with haemochromatosis represent the classical form, which results in iron overload mainly in older patients due to mutations in HFE (human leucocyte antigen-like protein involved in iron homoeostasis). The remaining ~15% of mutations occur in hepcidin, HJV (haemojuvelin), ferroportin and TfR2 (transferrin receptor 2), all of which are also important determinants of iron homoeostasis. Mutations in HJV and hepcidin lead to iron overload at a much younger age, resulting in juvenile haemochromatosis [14].

Although there is unequivocal evidence for the accumulation of iron in various systemic organs in haemochromatosis in humans, with consequent dysfunction of these organs, little information is available on retinal involvement in this disease. Indeed, there have been only two reports of retinal iron accumulation in haemochromatosis patients [15,16]. The apparent lack of focus on the retina as a potential target organ for iron overload in haemochromatosis is probably due to the widespread notion that the blood–retinal barrier dissociates retinal iron status from systemic iron levels, thereby protecting the retina from the consequences of systemic iron overload. It is important to note that, for a number of years, a similar notion persisted with respect to brain. However, recent studies have provided evidence that although the brain is separated from the systemic circulation by the blood–brain barrier, iron accumulation in certain areas of the brain such as the basal ganglia occurs in patients with haemochromatosis [1720]. Similarly, in the last few years there has been overwhelming evidence from animal models for the involvement of the retina in haemochromatosis and other disorders of iron overload. We now know that the retina expresses all of the five genes involved in haemochromatosis [13,21]. TfR2 and ferroportin are expressed throughout the retina [1,21]. HFE, the most commonly mutated gene in haemochromatosis, is expressed predominantly in the basolateral membrane of RPE [1]. In our previous study, we demonstrated that Hfe−/− mice show retinal iron accumulation as they get older and have hyperproliferative RPE [4]. Hepcidin is expressed in RPE, Müller cells and photoreceptor cells [2]. In addition, hepcidin-knockout mice have age-dependent iron accumulation in the retina with consequent retinal degeneration [22]. The expression of HJV in the retina is widespread: the protein is found in RPE, Müller cells, photoreceptor cells and retinal ganglion cells [3]. In RPE, the expression of Hjv is polarized with exclusive localization in the apical membrane facing the subretinal space. Although mutations in HJV in humans result in a severe form of iron overload (juvenile haemochromatosis) and induce oxidative stress and cellular dysfunction in several systemic organs, there have been no reports of the consequences of these mutations on retinal health. In the present study, we used Hjv−/− mice to investigate the potential involvement of the retina as a target organ for iron overload in juvenile haemochromatosis and assess the consequences in terms of retinal phenotype.

MATERIALS AND METHODS

Materials

Reagents were obtained from the following sources: RNA extraction reagent (TRIzol®; Invitrogen), GeneAmp RT (reverse transcription)–PCR kit (Applied Biosystems), Taq polymerase kit (TaKaRa), Power Block™ (Biogenex), mouse monoclonal anti-RPE65 antibody (Abcam), rabbit polyclonal anti-HJV antibody (GenScript), goat anti-rabbit IgG coupled to Alexa Fluor® 568 and goat anti-mouse IgG coupled to Alexa Fluor® 488 (Molecular Probes), and JB-4 solutions (Polysciences). DMEM (Dulbecco's modified Eagle's medium)/F12 (Invitrogen) with 10% FBS (fetal bovine serum), 100 IU/ml penicillin and 100 μg/ml streptomycin was used for growing RPE cells. PI-PLC (phosphatidylinositol-specific phospholipase C) was obtained from Sigma–Aldrich. [3H]Glutamate (specific radioactivity, 43 Ci/mmol) was from American Radiolabeled Chemicals and [3H]thymidine (specific radioactivity, 74.8 Ci/mmol) was from PerkinElmer.

Animals

Breeding pairs of Hjv+/− mice on a 129/SvEvTac (129/S) background were kindly provided by Dr Nancy Andrews (Duke University School of Medicine, Durham, NC, U.S.A.). Genotyping was performed to identify wild-type (Hjv+/+), heterozygous (Hjv+/−) and homozygous recessive (Hjv−/−) mice in the litters. Age-matched wild-type (Hjv+/+) and knockout (Hjv−/−) male mice were selected from the same litters for comparison studies. All experimental procedures involving these animals adhered to the ‘Principles of Laboratory Animal Care’ (National Institutes of Health publication #85-23, revised in 1985) and were approved by the Institutional Committee for Animal Use in Research and Education.

Preparation of eye sections and morphometric analysis

Mice were killed by CO2 asphyxiation followed by cervical dislocation. For studies using frozen sections, eyes were enucleated and oriented in Tissue-Tek® O.C.T. compound so that the 10-μm-thick sections included the full length of the retina approximately along the horizontal meridian, passing through the ora serrata and the optic nerve in both the temporal and nasal hemispheres. After slow freezing, the cryosections were prepared and mounted on slides (Superfrost; Fisher Scientific). They were stained with H/E (haematoxylin and eosin) and used for morphometric studies. Additional cryosections were used for immunohistochemical studies.

H/E-stained sections of retina from different ages of Hjv−/− mice along with age-matched Hjv+/+ mice were used for systematic morphometric analysis with a fluorescence microscope (Axioplan2 equipped with an HRM camera and the Axiovision 4.6.3 program; Carl Zeiss Meditec). Measurements included the thicknesses of the total retina, RPE, IPL (inner plexiform layer), INL (inner nuclear layer), OPL (outer plexiform layer) and the photoreceptor inner/outer segments. The number of cell bodies in the GCL (ganglion cell layer) was quantified by counting cells from the temporal ora serrata to the nasal ora serrata and expressing the data as number of cells per 100 μm length of retina. Three measurements were made on each side (temporal and nasal) of the optic nerve at 200–300-μm intervals, resulting in six measurements obtained per eye. Two measurements on either side of the optic nerve were taken as central retina and the remaining four measurements from that eye were considered as peripheral retina. Both eyes were analysed in each mouse.

JB-4 plastic sections and histological analysis

Eyes were enucleated and fixed for 1 h at room temperature (22°C) in 2% (w/v) paraformaldehyde/0.2% glutaraldehyde in 0.1 M sodium cacodylate/HCl buffer (pH 7.4) in sucrose and post-fixed for 1 h with 1% tannic acid in 0.1 M sodium cacodylate buffer. Tissues were dehydrated using a graded series of ethanol solutions and infiltrated overnight in 1.25% benzoyl peroxide using the JB-4 embedding kit. The next day, the eyecups were oriented and embedded in plastic as recommended in the embedding kit. Sections, 3 μm thick, were cut and used for histological analysis after H/E staining.

Immunofluorescence analysis

Cryosections of mouse eyes were fixed in 4% (v/v) paraformaldehyde for 10 min, washed with 0.01 M PBS (pH 7.4) and blocked with 1× blocking agent for 60 min. Sections were then incubated overnight at 4°C with rabbit polyclonal anti-HJV antibody. Negative control sections were treated identically, but in the absence of the primary antibody. Sections were rinsed and incubated for 1 h with goat anti-rabbit IgG coupled to Alexa Fluor® 568 at a dilution of 1:1000. Coverslips were mounted after staining with Hoechst nuclear stain, and sections were examined with a wide-field epifluorescence microscope (Carl Zeiss Meditec). The RPE cell layer in retinal sections was identified using an antibody specific for RPE65, a marker for RPE cells.

Establishment of primary RPE cell cultures from mouse eyes

Primary cultures of RPE were prepared as described previously [24]. Age-matched Hjv+/+ and Hjv−/− mice were obtained from the same litter originating from the mating of heterozygous mice. Then 3-week-old mice were used to establish primary cultures of RPE. The purity of the cultures was verified as described previously by immunodetection of RPE65, a known marker for RPE cells [23].

ELISA for determination of ferritin levels

A mouse ferritin ELISA kit (Kamiya Biomedical Company) was used for quantification of ferritin levels in Hjv+/+ and Hjv−/− mouse retinas and RPE cells. The absorbance of the final reaction mixture was measured at 450 nm. A calibration curve was used as described in the manufacturer's instructions to determine the ferritin levels in these samples.

[3H]Thymidine-incorporation assay

Hjv+/+ and Hjv−/− primary RPE cells were plated at a density of 10000 cells per well in 24-well plates, and grown for 24 h in serum-containing medium. Cells were then serum-starved for 48 h, [3H]thymidine (1 μCi per well) was added to the wells and the cells were incubated for 12 h. The cells were then washed twice with 5% trichloroacetic acid, the resultant precipitate was solubilized in 0.1 M NaOH and the radioactivity was measured using a scintillation counter.

Senescence assay

A senescence assay was performed in Hjv+/+ and Hjv−/− primary RPE cells using a commercially available senescence β-galactosidase staining kit (Cell Signaling Technology). The development of a blue colour after incubation with the reagents provided in the kit identifies the senescent cells.

RT–PCR

RNA was isolated from wild-type and Hjv−/− primary RPE cells and used for RT–PCR using the GeneAmp RT–PCR kit. PCR primers were designed on the basis of the sequence information available in GenBank® for mouse cDNAs. The following primers were used: mouse HFE forward, 5′- GGCTTCTGGAGATATGGTTAT-3′, and reverse, 5′-GACTCCACTGATGATTCCGATA-3′; mouse hepcidin forward, 5′-GCACCACCTATCTCCATCAACAGA-3′, and reverse, 5′-GGTCAGGATGTGGCTCTAGGCTAT-3′; mouse HJV forward, 5′- GGCTGAGGTGGACAATCTTC-3′, and reverse, 5′- GAACAAAGAGGGCCGAAAG-3′; mouse TfR1 forward, 5′-GCCCAAGTATTCTCAGATATGAT-3′, and reverse, 5′-TAGAAGTAGCACGGAAGTAGTCTC -3′; mouse TfR2 forward, 5′-GAGGATCCGGAAGTCTACTGTC-3′, and reverse, 5′- TCGATGCACGCAAAGATGTTACTG-3′; mouse DMT1 (divalent metal transporter 1) forward, 5′- CACTATCATGGCCCTCACGTTT-3′, and reverse, 5′-GCTGCAGGCCCGAAGTAACA-3′; mouse β2M (β2-microglobulin) forward, 5′-CCGAACATACTGAACTGCTAC-3′, and reverse, 5′- CATACTGGCATGCTTAACTCT-3′; mouse xCT forward, 5′- AAGTGGTTCAGACGATTATCAG-3′, and reverse 5′-AAGAAACGTGGTAGAGGAATG-3′; mouse 4F2hc forward, 5′-CTCCCAGGAAGATTTTAAAGACCTTCT-3′, and reverse, 5′-TTCATTTTGGTGGCTACAATGTCAG-3′. Mouse 18S primers were used as an internal control. PCR was performed using a commercially available Taq polymerase kit.

Assay of system xc (cystine/glutamate exchanger) transport activity

The heterodimeric amino acid transporter xCT/4F2hc is responsible for the activity of the amino acid transport system known as xc. It is an Na+-independent system that mediates the cellular entry of cystine in exchange for intracellular glutamate under physiological conditions. However, we routinely measure the activity of this transporter by cellular uptake of [3H]glutamate under Na+-free conditions [24,25]. Under these conditions, xc mediates the cellular entry of [3H]glutamate in exchange for intracellular unlabelled glutamate. Transport activity was measured using a Na+-free uptake buffer (25 mM Hepes/Tris, pH 7.5, 140 mM N-methyl-D-glucamine chloride, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4 and 5 mM glucose). L-[3H]Glutamate was used as the substrate for uptake experiments. Uptake was initiated by the addition of 250 μl of uptake buffer containing 2.5 μM glutamate spiked with 2 μCi/ml [3H]glutamate. Cells were incubated for 15 min at 37°C, after which time the buffer was removed and the cells were washed twice with ice-cold uptake buffer. The cells were then solubilized with 0.5 ml of 1% SDS/0.2 M NaOH, and radioactivity was determined by liquid scintillation spectrometry. Protein was measured using the Bio-Rad protein assay reagent. Non-carrier-mediated uptake (i.e the diffusional component) was determined by measuring the uptake of [3H]glutamate under identical conditions, but in the presence of excess unlabelled glutamate (5 mM). The diffusional component was less than 10% of total uptake. The transport activity of xc was calculated by subtracting the diffusional component from total uptake.

Western blotting

Plasma-membrane proteins from Hjv+/+ and Hjv−/− RPE cells were prepared using a Pierce cell-surface protein isolation kit (Thermo Fisher Scientific). Protein lysates were subjected to SDS/PAGE (10% gel). The proteins were then transferred on to a PVDF membrane and probed with specific antibodies. The positive bands were detected with appropriate secondary antibodies coupled to horseradish peroxidase. The signals were developed using an enhanced-chemiluminescence detection kit (Thermo Fisher Scientific).

Cleavage of cell-surface HJV by PI-PLC

ARPE19 cells (A.T.C.C.), a human RPE cell line, were cultured in DMEM/F12 medium, supplemented with 10% (v/v) FBS, 100 IU/ml penicillin and 100 μg/ml streptomycin. For PI-PLC treatment, the cells were seeded in 24-well plates with coverslips in DMEM/F12 and incubated in the presence or absence of PI-PLC (Sigma) at a concentration of 1 unit/ml for 2 h at 37°C in a 5% CO2 incubator. Non-permeabilized cells were then stained for immunodetection of HJV using a rabbit anti-HJV antibody (GenScript) at 1:100 dilution.

Generation of hepcidin-specific promoter–reporter constructs

The human hepcidin-specific promoter–EGFP (enhanced green fluorescent protein) construct was generated by first subcloning the 2-kb hepcidin promoter (obtained by PCR using human genomic DNA as the template) into the pGEM-T Easy vector. The promoter insert was then released by digestion with HindIII/XhoI, and the insert was subcloned into pUIIR3EGFP vector. The primers used for PCR were 5′-ATACTCGAGACTCTCACCCAGGCTGGG-3′ (sense) and 5′-AAGCTTCATCGTGCCGTCTGTCTGGCT-3′ (antisense).

Treatment of retinal cells with exogenous BMP6 (bone morphogenetic protein 6)

Cells were serum-starved for 6 h by culturing in the presence of 1% FBS and then treated with 100 ng/ml of recombinant human BMP6 (R&D Systems) in normal medium for 12 h.

RESULTS

Age-dependent morphological changes in the retina in Hjv−/− mice

To analyse the function of Hjv in the retina, Hjv−/− mouse retinas of different ages were used to compare with age-matched Hjv+/+ retinas stained with H/E. We did not find any morphological differences in the retina between the two genotypes in younger mice. In JB-4 plastic sections, we found drastic morphological changes in the retinas of Hjv−/− mice at ≥18 months of age compared with the age-matched Hjv+/+ controls, both in the central and peripheral retina (Figure 1A). We performed a systematic morphometric analysis of the retinas from frozen sections. The morphological changes evident in Hjv−/− mice include significant loss of ganglion cells and increase in RPE cell thickness, specifically in the central retina (Figure 1B). The loss of ganglion cells in 18-month-old Hjv−/− mouse retinas was also evident in JB-4 plastic sections (Figure 1A).

Deletion of Hjv in mice causes significant morphological changes in retina

Figure 1
Deletion of Hjv in mice causes significant morphological changes in retina

(A) Representative H/E-stained JB-4 plastic sections of Hjv+/+ and Hjv−/− mouse retinas of 18-month-old mice. (B) H/E-stained retinal frozen sections from 18-month-old Hjv+/+ and Hjv−/− mice were used for morphometric analysis. Histograms showing RPE thickness and number of cell bodies in the GCL per 100 μm length of retina calculated using measurements from central retina and peripheral retina of Hjv+/+ and Hjv−/− mice. Results are means±S.E.M.of measurements from retinas of six Hjv+/+ and six Hjv−/− mice (12 eyes each). *P<0.05.

Figure 1
Deletion of Hjv in mice causes significant morphological changes in retina

(A) Representative H/E-stained JB-4 plastic sections of Hjv+/+ and Hjv−/− mouse retinas of 18-month-old mice. (B) H/E-stained retinal frozen sections from 18-month-old Hjv+/+ and Hjv−/− mice were used for morphometric analysis. Histograms showing RPE thickness and number of cell bodies in the GCL per 100 μm length of retina calculated using measurements from central retina and peripheral retina of Hjv+/+ and Hjv−/− mice. Results are means±S.E.M.of measurements from retinas of six Hjv+/+ and six Hjv−/− mice (12 eyes each). *P<0.05.

Our previous studies and reports from other groups have shown that iron accumulation in the retina is associated with RPE hypertrophy and hyperplasia [4,9,22]. These studies used the ceruloplasmin/hephaestin double-knockout mice as well as Hfe- and hepcidin-knockout mice as models of retinal iron overload. Since loss-of-function mutations in HJV also lead to excessive iron accumulation in various tissues in humans, we examined the RPE cell layer in the retinas of Hjv−/− mice (18-month-old). Age-matched retinas from Hjv+/+ mice were used for comparison. Hjv knockout was confirmed by the presence and absence of HJV immunostaining in wild-type and Hjv−/− retinas respectively (results not shown). There were focal areas of RPE hyperplasia in Hjv−/− mouse retina as evidenced by the presence of multinucleated regions in the RPE cell layer when examined after immunostaining with RPE65 (a marker for RPE) and the nuclear stain Hoechst (Figure 2A). There were many regions with hyperplasia in the RPE of 18-month-old Hjv−/− mouse retinas, as shown in Figure 2(B).

Hjv−/− mice display focal areas of RPE hyperplasia

Figure 2
Hjv−/− mice display focal areas of RPE hyperplasia

(A) Immunostaining of 18-month-old Hjv+/+ and Hjv−/− mouse retinas with anti-RPE65 antibody showing focal areas of RPE hyperplasia in the knockout mice. Left-hand panels show staining for RPE65 (green), middle panels represent Hoechst nuclear staining, and right-hand panels are merged images. Retinal sections treated similarly, but in the absence of the primary antibody, were used as a negative control (results not shown). The insets in the right-hand panels represent a higher magnification of the RPE cell layer. (B) Additional representative regions of RPE hyperplasia in Hjv−/− mice retinas.

Figure 2
Hjv−/− mice display focal areas of RPE hyperplasia

(A) Immunostaining of 18-month-old Hjv+/+ and Hjv−/− mouse retinas with anti-RPE65 antibody showing focal areas of RPE hyperplasia in the knockout mice. Left-hand panels show staining for RPE65 (green), middle panels represent Hoechst nuclear staining, and right-hand panels are merged images. Retinal sections treated similarly, but in the absence of the primary antibody, were used as a negative control (results not shown). The insets in the right-hand panels represent a higher magnification of the RPE cell layer. (B) Additional representative regions of RPE hyperplasia in Hjv−/− mice retinas.

Iron accumulation in Hjv−/− mouse retina

HJV is an important iron-regulatory protein; loss-of-function mutations in this protein result in iron overload at a much younger age and consequently lead to juvenile haemochromatosis. The levels of cytosolic iron-storage protein ferritin are regulated by intracellular iron through IRP1 (iron-regulatory protein 1) and IRP2. An increase in intracellular iron leads to increased ferritin mRNA translation, resulting in increased ferritin protein levels [26,27]. Ferritin is a 24-subunit protein complex composed of H (heavy) and L (light) chains. To determine whether the retinal morphological changes observed in Hjv−/− mice are accompanied by increased iron accumulation, ferritin levels in retinas and primary RPE cells from Hjv−/− mice and age-matched Hjv+/+ mice were measured using ELISA (Figure 3A). The ferritin levels were significantly higher in Hjv−/− mice compared with the controls. We also examined the changes in other proteins involved in iron homoeostasis in 3-month- and 8-month-old Hjv−/− mouse retinas. Hfe and hepcidin expression were lower in Hjv−/− mouse retinas (Figure 3B), indicating additional mechanisms of iron accumulation in Hjv−/− mouse retinas. In addition, the expression of the transferrin receptors TfR1 and TfR2, as well as that of the iron-importing transporter DMT1, decreased in Hjv−/− mouse retina (Figure 3B). An increase in intracellular iron is known to decrease the expression of TfRs and DMT1. Thus changes in the expression levels of TfR1, TfR2 and DMT1 also provide evidence for increased iron accumulation in Hjv−/− mouse retinas.

Iron levels are increased in Hjv−/− mouse retina

Figure 3
Iron levels are increased in Hjv−/− mouse retina

(A) Ferritin levels were determined by ELISA in tissue lysates from 18-month-old Hjv+/+ and age-matched Hjv−/− mouse retinal tissues (n=5) and primary RPE cell preparations (n=4). Results are means±S.E.M. *P<0.001. (B) RT–PCR analysis of iron-regulatory genes in 3-month-old and 8-month-old Hjv+/+ (WT) and Hjv−/− (KO) mouse retinas.

Figure 3
Iron levels are increased in Hjv−/− mouse retina

(A) Ferritin levels were determined by ELISA in tissue lysates from 18-month-old Hjv+/+ and age-matched Hjv−/− mouse retinal tissues (n=5) and primary RPE cell preparations (n=4). Results are means±S.E.M. *P<0.001. (B) RT–PCR analysis of iron-regulatory genes in 3-month-old and 8-month-old Hjv+/+ (WT) and Hjv−/− (KO) mouse retinas.

Hyperproliferation of Hjv−/− primary RPE cells

Hjv−/− retinas in situ showed evidence of RPE hyperplasia. Therefore we isolated primary RPE cells from Hjv+/+ and Hjv−/− mouse retinas and examined their growth and proliferation pattern. Using a β-galactosidase senescence assay, we found that Hjv−/− RPE cells senesce at a much lower rate than Hjv+/+ RPE cells grown under identical conditions and passage number (Figure 4A). We then used a thymidine-incorporation assay to compare the proliferation of Hjv+/+ and Hjv−/− RPE cells. Thymidine incorporation is a measure of DNA synthesis and hence cell proliferation. The proliferation rate of Hjv−/− RPE cells was significantly greater (~3-fold) than that of wild-type RPE cells (Figure 4B).

RPE cells from Hjv−/− mice exhibit a decreased senescence and hyperproliferative phenotype

Figure 4
RPE cells from Hjv−/− mice exhibit a decreased senescence and hyperproliferative phenotype

(A) Senescence assay in Hjv−/− primary RPE cells compared with Hjv+/+ primary RPE cells. Blue colour indicates senescent cells. (B) [3H]Thymidine-incorporation assay with Hjv+/+ and Hjv−/− RPE cells. Results are means±S.E.M. (n=12). *P<0.001. KO, knockout; WT, wild-type.

Figure 4
RPE cells from Hjv−/− mice exhibit a decreased senescence and hyperproliferative phenotype

(A) Senescence assay in Hjv−/− primary RPE cells compared with Hjv+/+ primary RPE cells. Blue colour indicates senescent cells. (B) [3H]Thymidine-incorporation assay with Hjv+/+ and Hjv−/− RPE cells. Results are means±S.E.M. (n=12). *P<0.001. KO, knockout; WT, wild-type.

Up-regulation of xCT mRNA and protein in Hjv−/− primary RPE cells

Several previous studies, including our previous study on Hfe−/− mouse retina, have implicated the cystine/glutamate exchanger in cell-cycle progression [4,2831]. Slc7a11 (solute carrier family 7 member 11) encodes the ‘transporter proper’ subunit xCT in the heterodimeric amino acid transport system xc that mediates the cellular uptake of cystine coupled with the efflux of glutamate. The other subunit is 4F2hc, which functions as a chaperone for xCT for optimal recruitment into the plasma membrane. Since cellular cysteine is the rate-limiting factor for glutathione synthesis, xc is one of the key determinants of cellular glutathione status. RT–PCR was performed for xCT and 4F2hc using RNA from Hjv+/+ and Hjv−/− RPE cells. RT–PCR showed that xCT mRNA expression was higher in Hjv−/− RPE cells, whereas 4F2hc mRNA levels remained unchanged (Figure 5A). We also monitored the levels of xCT protein in the plasma membrane of Hjv+/+ and Hjv−/− RPE cells by Western blotting. The protein levels in these cells changed in parallel with their mRNA levels (Figure 5B).

The cystine/glutamate exchanger xCT is up-regulated in Hjv−/− RPE cells

Figure 5
The cystine/glutamate exchanger xCT is up-regulated in Hjv−/− RPE cells

(A) RT–PCR analysis of mRNA transcripts specific for xCT and 4F2hc in Hjv+/+ and Hjv−/− RPE cells. 18S was used as an internal control. (B) Western blot for xCT protein in Hjv+/+ and Hjv−/− RPE cells. β-actin was used as a loading control.

Figure 5
The cystine/glutamate exchanger xCT is up-regulated in Hjv−/− RPE cells

(A) RT–PCR analysis of mRNA transcripts specific for xCT and 4F2hc in Hjv+/+ and Hjv−/− RPE cells. 18S was used as an internal control. (B) Western blot for xCT protein in Hjv+/+ and Hjv−/− RPE cells. β-actin was used as a loading control.

Functional activity of system xc in wild-type and Hjv−/− RPE cells

To obtain functional evidence for the increase in the expression of Slc7a11 in Hjv−/− RPE cells, we compared the activity of system xc between wild-type and Hjv−/− RPE cells. Under the experimental conditions employed during the measurement, xc mediates the cellular uptake of [3H]glutamate in exchange for intracellular unlabelled glutamate. The Na+-independent glutamate uptake, which is a measure of xc functional activity, was ~3-fold higher in Hjv−/− RPE cells than in Hjv+/+ RPE cells (Figure 6A). To confirm that the observed glutamate uptake occurred via system xc, we performed substrate selectivity studies (Figure 6B). Uptake of [3H]glutamate in Hjv+/+ and Hjv−/− RPE cells was inhibited by unlabelled glutamate and cystine, but not by valine and aspartate. This mirrors the substrate specificity of system xc, strongly suggesting that the observed uptake of glutamate in RPE cells occurred principally via system xc.

The activity of the cystine/glutamate exchanger xc is increased in Hjv−/− RPE cells

Figure 6
The activity of the cystine/glutamate exchanger xc is increased in Hjv−/− RPE cells

(A) Uptake of glutamate in Hjv+/+ and Hjv−/− RPE cells. Uptake of [3H]glutamate (2.5 μM) was measured for 15 min at 37°C in the absence of Na+ as a readout of xc transport activity. Results are means±S.E.M. (n=8) and represent transport activity specific for system xc. *P<0.001. (B) Substrate selectivity of glutamate uptake in Hjv+/+ and Hjv−/− RPE cells. Uptake of [3H]glutamate (2.5 μM) was measured in Hjv+/+ and Hjv−/− RPE cells in the absence of Na+ for 15 min at 37°C in the absence or presence of the unlabelled amino acids glutamate, cystine, valine and aspartate, each at a concentration of 1 mM. *P<0.001 (n=6). Again, the results (means±S.E.M.) represent only the transport activity specific for xc.

Figure 6
The activity of the cystine/glutamate exchanger xc is increased in Hjv−/− RPE cells

(A) Uptake of glutamate in Hjv+/+ and Hjv−/− RPE cells. Uptake of [3H]glutamate (2.5 μM) was measured for 15 min at 37°C in the absence of Na+ as a readout of xc transport activity. Results are means±S.E.M. (n=8) and represent transport activity specific for system xc. *P<0.001. (B) Substrate selectivity of glutamate uptake in Hjv+/+ and Hjv−/− RPE cells. Uptake of [3H]glutamate (2.5 μM) was measured in Hjv+/+ and Hjv−/− RPE cells in the absence of Na+ for 15 min at 37°C in the absence or presence of the unlabelled amino acids glutamate, cystine, valine and aspartate, each at a concentration of 1 mM. *P<0.001 (n=6). Again, the results (means±S.E.M.) represent only the transport activity specific for xc.

BMP signalling in Hjv−/− retinal cells

BMP6 has been shown to be a co-ligand along with HJV for BMP receptor-mediated signalling involved in the induction of hepcidin expression and consequently in the modulation of iron homoeostasis [3235]. We treated Hjv+/+ and Hjv−/− primary RPE cells with BMP6 and monitored the expression of hepcidin by RT–PCR. We found that Hjv+/+ RPE cells had increased hepcidin mRNA levels after BMP6 treatment; in contrast, Hjv−/− primary RPE cells did not have any change in hepcidin expression (Figure 7A), confirming the need for HJV in BMP signalling. HJV is a GPI (glycosylphosphatidylinositol)-anchored protein, and hence treatment with PI-PLC, an enzyme that specifically hydrolyses the anchoring lipid, releases the GPI-anchored HJV from the membrane [36]. We confirmed the anchoring of HJV to GPI in ARPE19 cells. Treatment of non-permeabilized ARPE19 cells with PI-PLC led to a significant reduction in membrane-associated HJV (Figure 7B). We then transfected ARPE19 cells with a hepcidin-specific promoter–EGFP construct to assess the role of BMP6 on the promoter activity. Addition of BMP6 to these transfected cells induced the expression of GFP (Figure 7C). However, when these cells were treated with PI-PLC to remove the membrane-anchored HJV, BMP6 failed to activate the hepcidin promoter, as evidenced from the lack of expression of the reporter GFP (Figure 7C). The experiments were repeated with three independent transfections, and four different regions were imaged for each experiment. The number of GFP-positive cells per field of view was counted and the data as a quantitative representation of the reporter activity in control ARPE19 cells (i.e. in the absence of PI-PLC) with and without treatment with BMP6 are given in Figure 7(D). The results from hepcidin-specific promoter activity were corroborated by RT–PCR for hepcidin mRNA levels (results not shown).

Regulation of hepcidin expression by BMP6 in RPE cells requires HJV

Figure 7
Regulation of hepcidin expression by BMP6 in RPE cells requires HJV

(A) RT–PCR analysis of hepcidin expression in the absence or presence of BMP6 in Hjv+/+ and Hjv−/− RPE cells. The histogram is a quantitative representation of hepcidin expression after normalizing with 18S internal control. Results are means±S.E.M. (n=3). *P<0.001. (B) ARPE19 cells, a human RPE cell line, were treated with (right-hand panel) or without (left-hand panel) PI-PLC and then immunostained using an anti-HJV antibody without permeabilization. Experiments were repeated three times with consistent results. No staining was detected when anti-HJV antibody was not added in the negative control (results not shown). (C) Effects of BMP6 in the presence or absence of membrane-associated HJV on hepcidin promoter activity using an EGFP reporter assay in ARPE19 cells. HJV was removed from the cell surface of ARPE19 cells by treatment with PI-PLC. (D) Quantitative representation of the number of GFP-positive cells per field of view in control cells and in BMP6-treated cells, both in the absence of PI-PLC (three independent transfections with the reporter construct and examination of four different sections per experiment). There were no GFP-positive cells, with or without BMP6 treatment, when PI-PLC was used. Results are means±S.E.M..

Figure 7
Regulation of hepcidin expression by BMP6 in RPE cells requires HJV

(A) RT–PCR analysis of hepcidin expression in the absence or presence of BMP6 in Hjv+/+ and Hjv−/− RPE cells. The histogram is a quantitative representation of hepcidin expression after normalizing with 18S internal control. Results are means±S.E.M. (n=3). *P<0.001. (B) ARPE19 cells, a human RPE cell line, were treated with (right-hand panel) or without (left-hand panel) PI-PLC and then immunostained using an anti-HJV antibody without permeabilization. Experiments were repeated three times with consistent results. No staining was detected when anti-HJV antibody was not added in the negative control (results not shown). (C) Effects of BMP6 in the presence or absence of membrane-associated HJV on hepcidin promoter activity using an EGFP reporter assay in ARPE19 cells. HJV was removed from the cell surface of ARPE19 cells by treatment with PI-PLC. (D) Quantitative representation of the number of GFP-positive cells per field of view in control cells and in BMP6-treated cells, both in the absence of PI-PLC (three independent transfections with the reporter construct and examination of four different sections per experiment). There were no GFP-positive cells, with or without BMP6 treatment, when PI-PLC was used. Results are means±S.E.M..

DISCUSSION

Retinas in patients with aceruloplasminaemia [8], as well as in ceruloplasmin/hephaestin double-knockout mice [9,10], show marked morphological changes demonstrating that excessive iron accumulation is detrimental to the retina. Recent studies with mouse models have also brought to light the retinal involvement in haemochromatosis, a highly prevalent autosomal-recessive genetic disorder in humans, which is associated with iron overload in the circulation as well as in a variety of organs [4,22]. To date, there have been only two studies, reported almost four decades ago, showing evidence of excessive iron accumulation in the retina in patients with haemochromatosis [15,16]. We have reported previously that Hfe−/− mice, a widely used animal model of haemochromatosis, have increased iron accumulation with characteristic hypertrophic and hyperproliferative RPE [4]. Another group of investigators have also reported on the age-dependent iron accumulation and retinal degeneration in hepcidin−/− mice, another model of haemochromatosis [22]. Since all five genes associated with haemochromatosis are expressed in retina, and Hfe- and hepcidin−/− mice have retinal damage, it seems reasonable that the disease involves the retina as a target organ for excessive iron accumulation, as was demonstrated in patients with haemochromatosis many years ago [15,16]. In the present study, we used a different mouse model of haemochromatosis, namely the Hjv−/− mouse, to investigate the relevance of the disease to retinal iron homoeostasis. To date, this represents the third mouse model to be used to study retinal involvement in haemochromatosis.

In the present study, we show for the first time that there are marked morphological changes in the retina of 18-month and older Hjv−/− mice. It is important to note that the retinal changes seen in 18-month-old Hjv−/− mice are not evident in younger mice. Interestingly, Hfe−/− mice also had iron accumulation and retinal disruption at ≥18 months of age. On the basis of these results, we speculated that human patients with haemochromatosis resulting from loss-of-function mutations in HFE would develop iron overload in the retina at later stages of their life. Patients with mutations in HJV or hepcidin develop severe iron overload at much younger ages (juvenile haemochromatosis). Although the systemic iron overload in Hjv−/− mice occurs by 10 weeks of age, we find retinal iron accumulation and morphological changes only in older mice. Interestingly, hepcidin-knockout mice have also been recently reported to develop iron accumulation and retinal degeneration when they are 18 months or older [22]. It is known that HJV regulates iron homoeostasis through hepcidin. Hence it is not surprising that, just like hepcidin−/− mice, Hjv−/− mice also have retinal degeneration only when they get older. Although juvenile haemochromatosis patients with mutations in HJV or hepcidin develop other systemic disorders at a much younger age, they might be susceptible to retinal problems only at later stages.

The morphological changes in Hjv−/− mice include an increase in RPE thickness and a decreased number of cells in the ganglion cell layer, both of which are predominant, especially in the central retina. There were marked disruptions of the INL and ONL (outer nuclear layer). This morphological damage in the old Hjv−/− mouse retina was due to increased iron accumulation, as was evident from ferritin levels. In addition, we found that Hjv−/− mouse retina had decreased expression of the iron-regulatory proteins TfR1 and DMT1, which are known to be down-regulated under conditions of increased intracellular iron levels. Whether these morphological changes accompanied by increased iron in the retina are associated with detectable changes in retinal function needs to be investigated. We also provide evidence of hyperplasia in the RPE cell layer. Iron accumulation in the retina observed in the ceruloplasmin/hephaestin double-knockout mouse and hepcidin-knockout mice are associated with hypertrophy and hyperplasia of RPE [9,22]. We reported a similar phenomenon in the Hfe−/− haemochromatosis mouse [4]. In the present study, we find the same phenotype in Hjv−/− mice. These findings demonstrate that excessive iron accumulation underlies the cellular and growth-pattern changes seen in RPE. Similar to Hfe−/− mice, the hyperplasia phenotype of RPE observed in vivo in the intact retina of the Hjv−/− mouse is also seen with primary RPE cell cultures established from Hjv−/− mouse retinas. Many studies including our previous study have shown that Slc7a11 plays a critical role in cell proliferation [4,37,38]. The expression of this transporter is up-regulated in Hjv−/− RPE cells, suggesting that cystine uptake might play an important role in the hyperproliferative phenotype of Hjv−/− cells. The increased expression of Slc7a11 that codes for the ‘transporter proper’ subunit xCT in the cystine/glutamate exchange system xc is demonstrable at the mRNA level, protein level and functional level in Hjv−/− RPE cells.

The present study also shows that loss-of-function of HJV affects not only the RPE cell layer, but also other cellular layers of the retina. The changes observed in the retina in the mouse model of juvenile haemochromatosis may have relevance to AMD. AMD is a major cause of gradual bilateral loss of central vision in elderly people [29]. There is overwhelming evidence in support of a genetic component in the aetiology of AMD [30,31]. Several genes have been identified either as the cause of, or contributors to, AMD-associated retinal pathology, but still the aetiology of the disease in most cases remains unknown. Irrespective of the genetic factors, patients with AMD show evidence of excessive iron accumulation in retina [6,7]. Iron is a pro-oxidant and increased concentration of free iron in cells can cause oxidative stress. The Fenton reaction is the primary mechanism underlying the iron-dependent oxidative stress in which Fe2+ mediates the conversion of H2O2 into the highly reactive hydroxyl radical. Since AMD occurs mostly in older patients and also since oxidation-induced cellular damage increases with age, the possible contribution of oxidative stress to the aetiology of AMD has been speculated previously [31]. Published reports on patients with haemochromatosis [15,16] and recent findings from studies with haemochromatosis mouse models (Hfe- and hepcidin-knockout mice) [4,22], along with our present study on a Hjv−/− mouse model, clearly show that iron does accumulate in the retina in this disease. This raises the possibility that the genetic disease haemochromatosis may promote the progression and pathological features of AMD in humans. The increasing evidence for the presence of excessive iron in AMD, emerging reports on the retinal involvement in haemochromatosis, and the RPE hyperplasia as a common feature found in haemochromatosis mouse models and AMD patients strongly suggest that further studies to investigate the potential association between haemochromatosis and AMD are highly warranted.

Hepcidin is an important regulator of systemic iron homo-eostasis, and hepcidin deficiency induces severe iron overload [22]. HJV is a member of the RGM (repulsive guidance molecule) family, which also includes the BMP co-receptors RGMa and DRAGON (also called RGMb). Mutations in HJV (also called HFE2 or RGMc) cause severe iron overload and correlate with low hepcidin levels, suggesting that HJV positively regulates hepcidin expression [32]. Membrane-anchored HJV has been shown to up-regulate the expression of hepcidin through the BMP signalling pathway by acting as a BMP co-receptor [32]. HJV is a GPI-anchored protein that can be cleaved by the furin family of pro-protein convertases, which releases a soluble form of HJV that suppresses BMP signalling and hepcidin expression by acting as a bait that competes with membrane-bound HJV for BMP ligands [33]. In the present study, we found that HJV expressed in RPE cells is indeed a GPI-anchored protein, as is evident from the findings that treatment of the cells with PI-PLC resulted in the loss of membrane-bound HJV. Among the different BMP ligands, BMP6 has been shown to play a key role as a ligand for HJV and as an endogenous regulator of hepcidin expression and iron homoeostasis in vivo [34,35]. In the present study, we have shown for the first time that, in RPE cells, addition of BMP6 activates hepcidin expression and hepcidin-promoter activity only in the presence of HJV, and more specifically, only in the presence of membrane-bound HJV. This confirms that retina requires membrane-anchored HJV to act as a co-receptor of BMP6 for the regulation of hepcidin expression within the retina and consequently for the regulation of retinal iron homoeostasis.

In summary, deletion of the gene coding for the iron-regulatory protein HJV in mice leads to excessive iron accumulation with consequent age-related morphological disruption in the retina. Deletion of Hjv in RPE cells leads to a hyperproliferative phenotype both in vivo and in vitro. Up-regulation of the cystine/glutamate exchanger is a probable contributor to the enhanced proliferation of Hjv−/− RPE cells. Similar to the published reports on Hfe−/− and hepcidin−/− mouse models of haemochromatosis, the present study suggests that mutations, dysregulation or dysfunction of HJV in humans may also have a role in the aetiology and/or progression of AMD. In addition, the present study also shows that HJV is expressed as a GPI-anchored protein in retinal cells and that the membrane-bound form of this protein is obligatory for BMP6-mediated hepcidin expression in the retina.

Abbreviations

     
  • AMD

    age-related macular degeneration

  •  
  • BMP

    bone morphogenic protein

  •  
  • DMEM

    (Dulbecco's modified Eagle's medium

  •  
  • DMT

    divalent metal transporter

  •  
  • EGFP

    enhanced green fluorescent protein

  •  
  • FBS

    fetal bovine serum

  •  
  • GCL

    ganglion cell layer

  •  
  • GPI

    glycosylphosphatidylinositol

  •  
  • H/E

    haematoxylin and eosin

  •  
  • HFE

    human leucocyte antigen-like protein involved in iron homoeostasis

  •  
  • HJV

    haemojuvelin

  •  
  • INL

    inner nuclear layer

  •  
  • IRP

    iron-regulatory protein

  •  
  • ONL

    outer nuclear layer

  •  
  • PI-PLC

    phosphatidylinositol-specific phospholipase C

  •  
  • RGM

    repulsive guidance molecule

  •  
  • RPE

    retinal pigment epithelium

  •  
  • RT

    reverse transcription

  •  
  • TfR

    transferrin receptor

  •  
  • Slc

    solute carrier

  •  
  • xc

    cystine/glutamate exchanger

AUTHOR CONTRIBUTION

Jaya Gnana-Prakasam designed and performed experiments and wrote the paper. Amany Tawfik performed the morphometric analysis. Michelle Romej carried out the experiments with BMP6. Sudha Ananth analysed xCT transport activity. Pamela Martin performed the immunofluorescence analysis. Sylvia Smith and Vadivel Ganapathy designed experiments and wrote the paper.

FUNDING

This work was supported by the National Institutes of Health [grant number EY019672].

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