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(Investigative Ophthalmology and Visual Science. 2001;42:1721-1727.)
© 2001 by The Association for Research in Vision and Ophthalmology, Inc.

Overexpression of H- and L-Ferritin Subunits in Lens Epithelial Cells: Fe Metabolism and Cellular Response to UVB Irradiation

Malgorzata Goralska, Benjamin L. Holley and M. Christine McGahan

From the Department of Anatomy, Physiology, and Radiology, College of Veterinary Medicine, North Carolina State University, Raleigh.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To determine the effect of changes in ferritin subunit makeup on Fe metabolism and the resistance of lens epithelial cells (LEC) to photo-oxidative stress.

METHODS. Cultured canine LEC were transiently transfected with pTargeT mammalian expression vector containing the whole coding sequence of H- or L-chain cDNA. The subunit composition of newly synthesized ferritin was analyzed by metabolic labeling and SDS-PAGE electrophoresis. Total ferritin concentration was measured by ELISA. Fe uptake and incorporation into ferritin was determined by incubating transfected cells with 59Fe-labeled transferrin followed by native PAGE electrophoresis. The effect of UV irradiation was assessed by cell count after exposure of transfected cells to UVB radiation.

RESULTS. Transfected cells differentially expressed H- and L-ferritin chains from cDNA under the control of CMV promoter; overexpression of L-chain was much greater than that of H-chain. The expressed chains assembled into ferritin molecules under in vitro and in vivo condition. The ferritin of H-transfectants incorporated significantly more Fe than those of L-transfectants. The UVB irradiation reduced cell number of L-transfectants by half, whereas H-chain transfectants were protected.

CONCLUSIONS. Post-transfectional expression of ferritin H- and L-chains in LEC appears to be regulated differentially. Overexpression of L-chain ferritin did not have a major effect on cellular Fe distribution and did not protect LEC against UV irradiation, whereas overexpression of H-chain resulted in increased storage of Fe in ferritin and protected cells from UV damage.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Ferritin, the ubiquitous Fe storage protein is a polymeric protein made up of 24 subunits of two types, heavy (H) and light (L), which can assemble in various proportions. The ratio of H- to L-chain is tissue specific. The two ferritin chains have approximately 50% amino acid sequence identity and exhibit functional specificity in incorporation and storage of Fe. The effect of altering the ratio of H- to L-chains on Fe storage has been extensively studied in vitro, but the functional relationship of H- and L-chains and Fe loading into ferritin in intact cells is not fully understood.

Recent findings of a mutation in ferritin synthesis in humans, which is associated with early bilateral cataract formation,1 2 emphasizes the importance of proper synthesis of this protein to normal lenticular homeostasis. This mutation results in unregulated synthesis of the L-chain, leading to significant overexpression of mainly L-chain ferritin in the absence of Fe overload.3 4 Thus, it is possible that dysregulation of ferritin synthesis results in the inability of the lens to properly store Fe. Increased availability of reactive Fe could result in oxidative damage and cataractogenesis.5 Even if the clinical symptoms of hereditary hyperferritinemia cataract syndrome (HHCS) are not caused by changes in Fe metabolism, it will be important to determine how the overexpression of L-chain causes this disease.

Because oxidative damage is a hallmark of cataractogenesis and virtually all oxidative damage is catalyzed by Fe, it is essential for a complete understanding of lenticular physiology to determine how changes in the H/L chain ferritin ratio alter Fe dynamics and the response of the cell to oxidative stress. The ability to exclusively increase the concentration of one of the two ferritin chains in LEC creates a model for studying these factors. To do this, we have cloned the coding regions of ferritin H- and L-chain cDNA into a mammalian expression vector. We have demonstrated the synthesis of each chain in an in vitro transcription translation system and the overexpression of each chain in primary lens epithelial cell cultures. In addition, we have found that altered expression of these chains significantly changes the ability of lens ferritin to incorporate Fe and substantially alters the cells’ susceptibility to UV damage.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
Eyes containing lenses without visible opacities were obtained from mixed breed dogs euthanatized at the Wake County, North Carolina Animal Shelter. Lenses were dissected, and the anterior capsules with adherent epithelial cells were removed and placed in tissue culture dishes. LEC were cultured in Dulbecco’s modified Eagle’s medium (Gibco BRL, Rockville, MD) containing 10% fetal bovine serum (Hyclone, Logan, UT) and Antibiotic-Antimycotic (Gibco BRL). Epithelial cells that grew out from the capsule onto the plate were dispersed by trypsinization, reseeded, and grown to confluence. After reaching confluence, cells from two to three lenses were combined and plated in six-well tissue culture plates for experiments. Ten years of experiments in this laboratory using LEC have not revealed any differences in any measured parameters that are breed or age related.

Cloning Ferritin Light (L)- and Heavy (H)-Chain Genes (Coding Regions)
Whole coding sequences of dog H- and L-chain ferritin cDNAs were PCR amplified from dog lens epithelial cell mRNA. Primers were designed using known human ferritin gene sequences. The L-chain primers corresponded to nucleotides 1 to 18 (5'-ATGAGCTCCCAGATTCGT-3') of the coding strand and to the nucleotides 504 to 522 (5'-TTAGTCGTGCTTGAGAGTGAG-3') of the noncoding strand of ferritin L gene. The H-chain primers corresponded to nucleotides 1 to 20 (5'-ATGACGACCGCGTCCCCCTC-3') and 532 to 552 (5'-GTTTTGG TACAACTTATAGAAA-3') of the coding and noncoding strand of FH gene, respectively. After amplification with Taq polymerase, products were cloned into pTargeT mammalian expression vector (Promega, Madison, WI) by ligating to 3'-T overhangs of the plasmid. Dideoxy sequencing was used to determine the cDNA sequence. Amino acid sequence translations from the DNA sequence were obtained using Swiss protein database. Many PCR products were cloned and sequenced because of concerns about the presence of expressed pseudogenes6 7 and point mutations arising during the PCR process. Several expressed pseudogenes found in this process were eliminated by screening in the in vitro system. The H- and L-chain clones selected for this study expressed the correct size subunit in the in vitro transcription/translation system, assembled into holoferritin of the correct size, and, according to our sequence analysis, contained all the important functional groups as described in the Results section.

Transcription/Translation In Vitro
In vitro expression of ferritin genes was studied using TNT Coupled Reticulocyte Lysate System (Promega). One to 1.5 µg of the expression vector containing ferritin H- or L-chain cDNA was used in the assay, which was conducted according to the manufacturer’s protocol. The synthesized protein was analyzed by SDS-PAGE and by PAGE and autoradiography.

Transient Transfection of LEC with Recombinant Plasmid
Transfection conditions were optimized using a pEGFP (Clontech, Palo Alto, CA) expression vector containing the cDNA for Green Fluorescent Protein and Fugene 6 (Boehringer Mannheim, Indianapolis, IN). Under optimal conditions we were able to detect fluorescence in approximately 30% of the LEC, 48 hours after transfection. These conditions were than applied to the ferritin H- and L-chain transfections. LECs were plated in six-well tissue culture dish at 125,000 to 150,000 cells/well. The next day, cells were transfected with 2.0 to 2.5 µg plasmid DNA in 0.75 ml of DMEM containing 10% serum and 4 µl of Fugene 6. Twenty-four hours later the medium was changed to serum-free MEM, and cells were left to grow for additional 24 hours. MEM, which has no added Fe, was used to lower endogenous ferritin synthesis. Transfected cells were used to study de novo ferritin synthesis and Fe incorporation into ferritin. Treatment conditions are described in detail in the Results section and in the tables and figure legends.

Metabolic Labeling of Newly Synthesized Ferritin
Cells in each well of a six-well plate were labeled for 20 hours with 77 µCi of 35S-methionine in methionine-free DMEM under different experimental conditions. After incubation, the cells were rinsed with PBS and lysed on ice with 250 µl of 0.05 M Tris/HCl buffer (pH 8.0), which contained 0.15 M NaCl, protease inhibitor cocktail for mammalian cells (Sigma, St. Louis, MO), 0.02% sodium azide, and 1% Triton X-100. Ferritin was immunoprecipitated from 200 µl of the cell lysate with goat anti-horse ferritin antibody (ICN Biochemicals, Irvine, CA) and subsequent treatment with 10% Pansorbin (Calbiochem, La Jolla, CA). Ferritin was released from antibody–antigen complex by boiling for 2 minutes in denaturing/reducing SDS-PAGE loading buffer and electrophoresed on 10% SDS-PAGE gel using the tris/tricine buffer system. Radioactivity of dried gels was quantified in an Instant Imager (Packard-Canberra, Rockville, MD). The gels were autoradiographed, and the images were digitized using Deskscan II and annotated with Photofinish.

59Fe Uptake and Incorporation into Ferritin
LEC were rinsed and preincubated for 1 hour in serum free DMEM to remove Tf that was bound to the membrane. Human apoTf (Boehringer Mannheim) was labeled with 59Fe-nitrilotriacetic acid as previously described.8 Cells were incubated with 59Fe-labeled transferrin (5.2 µM) and treated as described in the figure legends. After treatment, the cells were rinsed with ice-cold PBS and lysed with 500 µl of a hypotonic 10 mM Tris/HCl buffer (pH 7.4) containing protease inhibitor cocktail (Sigma). Total 59Fe uptake was determined by counting the lysates in a gamma counter (1480 Wallac Wizard; Wallac OY, Turku, Finland). The lysates were then centrifuged at 30,000g for 30 minutes. Proteins in the supernatant were precipitated with -20°C acetone and recovered by centrifugation at 15,000g. The dried pellets were resuspended in PBS. The resusupended pellets were analyzed by separation on an 8% PAGE gel. The gels were dried, and the radioactivity in the bands was quantified, autoradiographed, and imaged as described above in Transient Transfection of LEC with Recombinant Plasmid. Ferritin content of the remaining resuspended pellets was determined by ELISA.

Quantification of Ferritin in Cell Lysates by ELISA
Total ferritin concentration in the cells was determined by a simple sandwich ELISA as described previously.9 Assay samples were obtained from the Fe incorporation and metabolic labeling experiments. Dog liver ferritin (New England Immunology Associates, Cambridge, MA) was used as a standard. Goat anti-horse ferritin (ICN Biomedical) and HRP-labeled goat anti-horse ferritin antibodies were used to perform the assay with ABTS (KPL) as a substrate. The optical density was read at 405 nm in a 7520 Microplate Reader (Cambridge Technology, Cambridge, MA).

UVB Irradiation
Transfected LECs were exposed to UVB at a dose of 30 mJ/cm2 (wavelength maximum, 312 nm). Cells growing in six-well tissue culture dishes in serum-free DMEM were irradiated with a UVB lamp (model EB 280C; Fisher Scientific, Pittsburgh, PA) for 45 seconds. Twenty hours later cells in each well were trypsinized and counted on a hemacytometer. Control cells were from a parallel, nonirradiated plate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Nucleotide Sequences of Dog Ferritin H- and L-Chains
Ferritin H- and L-chain cDNA was cloned into the mammalian expression vector, pTarget. Sequences of the entire coding regions of dog lens apoferritin H- and L-subunits were analyzed and compared with corresponding sequences of human ferritin using PCGene. The dog H cDNA consists of 552 nucleotides and shows 64% homology to the dog L cDNA, which contains 522 nucleotides in the coding region (Fig. 1) . The dog and human H cDNA shows a striking degree (93.3%) of identity. The L cDNA sequences demonstrated slightly less (88.1%) homology. This is the first report of nucleotide sequences for the whole coding regions of dog H- and L-ferritin chains.



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  		Figure 1. Comparison of the coding region cDNA sequences of canine H- and L-chain ferritin.

 
The dog H-ferritin cDNA sequence encodes 183 amino acids and is very similar to that of human H-ferritin (95.6%; Fig. 2 ). Seven amino acids that make up the human ferritin iron binding motif10 are also conserved in the dog sequence. Primary amino acid sequences of dog and human L-ferritin are less similar (85.6% homology). Dog L-ferritin chain has 173 residues and is two amino acids shorter than human L-chain. Despite these differences, dog L-ferritin contains conserved residues such as the leucines involved in intersubunit interactions found in human and horse L-ferritin.11



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  		Figure 2. Comparison of ferritin H- and L-chain amino acid sequences from dog and human. The amino acid sequences of dog ferritin were deduced from dog cDNA sequences and compared with those of humans using PC gene.

 
In Vitro Expression of Dog Ferritin Subunits
To test the ability of these cloned coding regions to express and assemble into homopolymers, both genes were expressed in vitro in the TNT Coupled Reticulocyte Lysate System. Autoradiographs of the synthesized proteins demonstrated the presence of a single band in each of reaction mixtures corresponding either to ferritin L- or H-chain when analyzed by SDS-PAGE under reducing conditions (Fig. 3A) . Electrophoretic analysis in a nondenaturing PAGE gel showed that the synthesized chains assemble into ferritin homopolymers of different molecular weights (Fig. 3B) . The clones of both genes were then used for transfection of LEC.



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  		Figure 3. Electrophoretic analysis of ferritin synthesized in in vitro transcription/translation reticulocyte lysate system. The synthesized protein was electrophoresed in 15% SDS-PAGE (A) under reducing conditions and 8% native PAGE (B).

 
Overexpression of L- and H-Ferritin Chains in LEC
Analysis of the subunit composition of newly synthesized ferritin in LEC, 68 hours after transfection, showed a sevenfold increased expression of L-chain in L-transfected cells in comparison to those transfected with plasmid and H-ferritin (Fig. 4) . H-transfectants demonstrated significantly higher ferritin H-chain levels (20% above of that of the control). The level of L-ferritin in H-transfected cells as well as the level of H-ferritin in L-transfectants did not change in comparison to the control. The H/L ratio of newly synthesized ferritin in control, plasmid-transfected LEC, revealed a 2.70 times higher level of H-chain than L-chain of ferritin. Transfection with H-chain increased the ratio to 3.22 and transfection with L-chain drastically reduced the ratio to 0.34, which indicated vast overexpression of L-subunit type in L-transfectants.



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  		Figure 4. Subunit makeup of newly synthesized ferritin of differentially transfected LEC. Forty-eight hours after transfection, LEC were incubated for additional 20 hours in serum-free, methionine-free DMEM to which 35S-methionine was added. Cells were subsequently lysed, and ferritin was immunoprecipitated and run on a denaturing/reducing 10% SDS-PAGE using tris/tricine buffer system. The results presented in the histogram are the means ± SEM of five experiments (PL, pTarget; FH, ferritin heavy chain cDNA; FL, ferritin light chain cDNA). *P < 0.05, significantly different from plasmid H-chain; ^P < 0.05, significantly different from plasmid L-chain.

 
Total ferritin from nontransfected LEC and liver was heat purified, electrophoresed on 15% SDS-PAGE under reducing conditions, and stained with Coomassie blue. In LEC there was a higher concentration of H- than L-chain, corroborating the data of higher de novo synthesis of H-chain in control, plasmid-transfected LEC (Fig. 5) . L-chain ferritin predominates in the liver extracts as has been known for many years.



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  		Figure 5. Electrophoretic analysis of heat purified ferritin from nontransfected LEC. Cells were lysed and heated in 75°C for 10 minutes in 40% methanol. The lysates were centrifuged, and ferritin-containing supernatants were precipitated with 50% acetone. Protein pellets were dissolved and electrophoresed on 15% SDS-PAGE gel under reducing condition. The gels were stained with Coomassie blue stain.

 
To determine whether newly overexpressed ferritin subunits remain in the form of free chains or are assembled into the ferritin molecule, the samples were analyzed using nondenaturing PAGE. Newly synthesized, 35S-methionine–labeled ferritin was purified from the cell lysates by precipitation of nonferritin lysate proteins with methanol at 75°C and subsequently electrophoresed on 8% PAGE gel. Autoradiograms show the presence of a single band with mobility similar to that of horse spleen ferritin standard, but only in L-transfectants (Fig. 6) . Cells transfected with plasmid and H-chain had no detectable assembled ferritin, probably because of the combination of both lower concentration of the protein and some loss of it during methanol purification.



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  		Figure 6. Electrophoretic analysis of newly synthesized ferritin in nondenaturing condition. Forty-eight hours after transfection, differentially transfected LEC were incubated for additional 20 hours in serum-free, methionine-free DMEM containing 35S-methionine. Cells were subsequently lysed and heated at 75°C for 10 minutes in 40% methanol. The lysates were centrifuged, and ferritin-containing supernatants were precipitated with 50% acetone. Protein pellets were dissolved and electrophoresed on a 8% native PAGE. The experiment was repeated twice and the gel shown is a representative experiment (PL, pTarget; FH, ferritin heavy chain cDNA; FL, ferritin light chain cDNA).

 
To determine accumulation of ferritin during the entire 68 hours after transfection, ferritin concentration was measured by ELISA. Both H- and L-transfected cells had higher levels of ferritin in cell lysates than plasmid-transfected LEC (Fig. 7) . For H-transfectants, despite the relatively lower level of overexpression as determined in the metabolic labeling experiments, there was a 100% increase in the amount of ferritin within these cells at the 68-hour time point. The concentration of ferritin in L-transfected cells was 20-fold greater than the control and reached 180 ng/ml of lysate. These data indicate that although H-chain maybe expressed less efficiently in LEC than L-chain, H-chain overexpression significantly increases LEC ferritin concentration.



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  		Figure 7. Ferritin quantification using ELISA. Samples were obtained from Fe incorporation experiments. Cell lysates of differentially transfected cells were acetone precipitated, and protein pellets were dissolved in PBS and analyzed by both ELISA and separation on 8% PAGE gel. The results of ELISA are the means ± SEM of 14 experiments (PL, pTarget; FH, ferritin heavy chain cDNA; FL, ferritin light chain cDNA). *P < 0.05, significantly different from PL.

 
In Vivo Iron Incorporation into Ferritin of Transfected LEC
Forty-eight hours after transfection, the cells were incubated with 59Fe-saturated transferrin for an additional 20 hours. There were no differences in total Fe uptake by all transfected cells (data not shown). Nondenaturing PAGE analysis of the cell lysates showed the presence of 59Fe in bands that comigrated with ferritin and transferrin standards and in a diffuse band of low molecular weight (<3000 Da; Fig. 8A ). Ferritin from H-transfectants incorporated significantly more, 1.7 times, 59Fe than ferritin of plasmid transfectants. Ferritin of L-transfectants contained an amount of 59Fe similar to that of control and a significantly lower amount than the H-transfectants, despite the much higher level of ferritin in L-transfectants compared with H-transfectants. When the incorporation of 59Fe was expressed as a ratio of Fe to ferritin protein, L-transfectants had profoundly (20-fold) lower incorporation than plasmid and H-transfectants (Fig. 8B) .



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  		Figure 8. Electrophoretic analysis of 59Fe incorporation into ferritin, transferrin, and low-molecular-weight pool of differentially transfected LEC. The incorporation of 59Fe into ferritin was expressed as relative counts (A) and 59Fe cpm/ng ferritin (B). Forty-eight hours after transfection, LEC were labeled in serum-free DMEM with 59Fe-saturated transferrin for an additional 20 hours. Cells were lysed, lysates were centrifuged, and protein supernatants were precipitated with 50% acetone. Dissolved proteins were electrophoresed on 8% native PAGE. The same samples of dissolved proteins were used for ferritin determination by ELISA. The results are the means ± SEM of 12 experiments (PL, pTarget; FH, ferritin heavy chain cDNA; FL, ferritin light chain cDNA). (A) *P < 0.05, significantly different from PL; ^P < 0.05, significantly different from FL. (B) *P < 0.05, significantly different from PL and FH.

 
UV Irradiation of Differently Transfected LEC
To test the ability of modified ferritin to protect lens cells from UV damage, cell cultures of differentially transfected cells were irradiated with UVB light, and cell counts were compared with those of the parallel nontreated wells (Fig. 9) . There were no differences among cell counts of all transfected, nonirradiated control groups. Irradiation significantly decreased the number of plasmid and L-transfected cells. The difference was bigger for L-transfectants (53% fewer cells) than for plasmid transfectants (43%) in comparison to nonirradiated L- and plasmid transfectants, respectively. However, H-transfectants appeared to be protected against UV damage because the number of UV-irradiated cells was not different from control, nonirradiated cells.



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  		Figure 9. Changes in cell count of differentially transfected cells after UVB irradiation. Forty-eight hours after transfection, cells growing in six-well tissue culture dishes were exposed to UVB for 45 seconds (30 mJ/cm2). Twenty hours later cells were trypsinized and counted on a hemacytometer. The results are the means ± SEM of 10 experiments (PL, pTarget; FH, ferritin heavy chain cDNA; FL, ferritin light chain cDNA). *P < 0.05, significantly different from corresponding nonirradiated group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study we used expression vectors containing canine H- or L-chain ferritin cDNA to investigate the ability of overexpressed ferritin chains to assemble into ferritin and the effect of altered subunit ratios on important physiological functions, including Fe storage in ferritin and the ability of cells to withstand UV irradiation. This is the first study to show overexpression of H- and L-chain ferritin subunits in transiently transfected primary cell cultures.

Subunit Composition of Lens Ferritin
The tissue-specific ratio of H- and L-chains in ferritin, well known for tissues such as spleen, liver, or heart, has not been determined for normal, healthy LEC. Electrophoretic analysis of the newly synthesized subunits from control cells showed that dog LEC synthesized more H- than L-chain (H/L ratio, 2.7). The Coomassie-stained SDS-PAGE gel of heat-purified ferritin corroborates this. However, an exact quantification of H/L-chain ratio in assembled LEC ferritin could not be definitively measured because the species-specific antibodies against each of the subunits were not available (other species antibodies kindly supplied by Paolo Santambrogio [Milan, Italy] were not cross-reactive).

Differential Overexpression of Ferritin Chains
Transfected LEC did efficiently express H- and L-chain cDNA under the control of the CMV promoter, although the cells did not overexpress both chains to the same degree. Overexpression of L-chain was much greater than that of H-chain under the same conditions. It has been shown that L- and H-chain genes can express differentially during development or cellular differentiation12 13 14 15 through posttranscriptionally regulated molecular mechanisms not fully understood. One of the better known mechanisms of posttranscriptional regulation is iron-dependent regulation of ferritin synthesis through IRP proteins, which act as a translational repressor by binding to a 28-base sequence in the 5' untranslated region of ferritin mRNA (iron response element [IRE]).16 Both H- and L-ferritin chain mRNAs carry an iron-responsive element.

Many different mutations in L-chain IRE in humans inhibit IRP binding, resulting in great accumulation of overexpressed L-chain–rich ferritin in cells (including the lens) and serum and are associated with early onset bilateral cataracts known as HHCS.2 17 18 19 There are no reports of clinical symptoms caused by unregulated H-chain overexpression. Perhaps these changes would be lethal, although it is possible that there are specific, posttranscriptional regulatory mechanisms preventing cells from the H-chain overexpression. Accumulation of H-chain leading to profound overproduction of H-chain–rich ferritin could deplete cells of available Fe because H-chain–rich ferritin is a very efficient Fe chelator. Indeed, an Fe-deficient phenotype was created by overexpression of H-chain ferritin in HeLa cells.20 It has been demonstrated recently that the 3' untranslated region of mRNA of H-chain contains sequences interacting with cytosolic RNA binding factors, which change the stability of H-chain mRNA.21 22 The plasmid construct used in the present study contained exclusively the coding regions of both H- and L-chains; therefore, the regulatory elements of mRNAs could not be responsible for differential overexpression of ferritin chains in transfected LEC. It is possible though that lack of the sequences involved in regulation of H-chain message stability may result in much higher degradation of H-chain message in comparison to L-chain. Preferential expression of L-chain has been reported by others. For example, the disproportionately high level of L-chain mRNA was found in cataractous lenses of humans and guinea pigs,23 although there was no overexpression of L-chain protein. The greater expression of ferritin L- over H-chain was also reported for transiently transfected fibroblasts.24

The lower overexpression of H- compared with L-chain in transfected LEC could also result from more rapid turnover of this subunit because of the lower stability of ferritin H-chain protein.25 26 However, the relatively higher total accumulation of ferritin during the entire 68 hours after transfection in H- versus L-transfected LEC as measured by ELISA does not bolster this hypothesis. Although there was only a 20% increase in de novo synthesis of the H-subunit during the 20-hour labeling period, there was a twofold higher concentration of ferritin in LEC 68 hours after transfection was initiated. This is similar to the increased amount of ferritin accumulated after 2 to 3 days in stably transfected HeLa cells.20 There was much greater de novo synthesis (700% compared with control) of L-chain in LEC, which led to a 20-fold increase in ferritin concentration at the 68-hour time point. Contrary to earlier findings by Picard et al.27 and Corsi et al.,24 overexpression of H-chain in transfected dog LEC had no effect on synthesis of endogenous L-chain. This may be due to lower overall H-chain expression in our system.

Assembly of Overexpressed H- and L-Ferritin Chain into a Ferritin Polymer
To analyze the ability of overexpressed ferritin chains to assemble in vivo into ferritin molecules, we transfected LEC with either H- or L-chain cDNA, 35S-methionine–labeled and subsequently purified ferritin from cell lysates using the heat resistance properties of this protein. Electrophoretic analysis in nondenaturing PAGE showed that L-transfectants contained assembled ferritin, which most likely consisted predominantly of the overexpressed L-chain, although this conclusion was not evaluated by immunoblotting. The ferritin in plasmid- and H-transfectants was not detectable under these conditions, which is likely due to both the lower concentration of ferritin in these cells and some loss during purification. The overexpressed ferritin chain can either be incorporated into endogenous ferritin, changing its subunit composition or can assemble into homopolymeric ferritin as has been demonstrated in primate fibroblastoid cells (COS-7) transfected with human ferritin chains.24 Which of these mechanisms takes place in transfected LEC needs to be further examined.

Fe Metabolism in LEC with Ferritin of Different Subunit Composition
Although all tissues from patients with HHCS were shown to have large excess of L-chain ferritin, the lens is the only known tissue where this overexpression is associated with clinical symptoms of disease. There are many studies on how changes in ferritin subunit composition alter the ability of the protein to safely store Fe, although most of them were conducted either in vitro or in vivo but not in lenticular tissue. In the present study we developed the model that allowed us to study the Fe metabolism and physiology of lens epithelial cells with different ferritin subunit makeup.

The overexpression of H-chain in LEC increased incorporation of Fe into ferritin above control levels. However, we were not able to demonstrate any changes in Fe uptake into the cells, Fe content of transferrin, or the low-molecular-weight pool as was reported for cultured erythroid cells.27 28 Much greater overexpression of L-chain did not have an effect on the Fe content of the cells’ Fe pool and did not change the Fe incorporation into L-transfectant’s ferritin. It has been shown in vitro that decreasing H-chain content in ferritin recombinants lowers Fe incorporation.29 30 Therefore, we speculate that overexpression of L-chain may not significantly decrease the H/L-chain ratio of endogenous ferritin but that an excess of overexpressed L-chain may assemble into homopolymeric ferritin, which has a low capacity for storage Fe. Thus, the great overexpression of L-chain that we obtained in LEC did not have a major effect on the parameters of cellular Fe metabolism that were measured. These results are in concordance with earlier observations that overexpressed L-chain ferritin in HHCS was not associated with alteration in Fe metabolism and that ferritin in the lens from an HHCS patient was Fe poor.31

Protective Effect of Ferritin H-Chain against Damage from UV Irradiation
It has been shown that cells overexpressing ferritin develop resistance to oxidative stress and that the subunit makeup of ferritin plays an important role in the process.32 33 34 In a more recent study,20 overexpression of H-chain ferritin altered intracellular Fe dynamics and provided substantial protection against hydrogen peroxide–induced damage. In our present study, overexpression of H-chain also altered Fe dynamics and protected LEC against UVB. This protective effect of H-chain overexpression is likely due to the measured increase in ferritin Fe sequestration, because Fe is known to be involved in UV-induced photo-oxidative stress.35 L-transfectants were not protected despite a large increase in ferritin synthesis. However, this increased ferritin synthesis was not accompanied by an increase in Fe incorporation. Because the cell number in the non-UV treated L-transfectant was similar to that of control during a 68-hour period, we concluded that L-chain overexpression had no overtly toxic effect on LEC. The mechanism of lowered resistance of L-transfected cells to photo-oxidative stress and the protective effect of H-chain overexpression needs to be further examined.

A recent investigation of a single lens from a human patient with HHCS revealed aggregates of L-chain ferritin in the extracted lens.36 The authors conclude that such aggregates could contribute to the opacities seen in these patients and suggest that Fe storage and oxidative damage may not be contributing factors. However, this study did not include measurements of these parameters. In our present investigation, altered ferritin subunit ratios, resulting in changes in the ability of the lens to safely store Fe could lead to the conclusion that Fe-catalyzed free radical reactions contribute to cataractogenesis in HHCS. The data presented here do not substantiate the hypothesis that changes in Fe metabolism are responsible for this pathologic condition. The species- and tissue-specific model developed in the present study, which mimics the condition found in pathologic lenses, creates a good opportunity for further investigation.


   Footnotes
 
Supported by National Institutes of Health Grant EY04900 and Funds from State of North Carolina.

Submitted for publication December 21, 2000; accepted March 23, 2001.

Commercial relationships policy: N.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: M. Christine McGahan, Department of Anatomy, Physiology, and Radiology, College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough Street, Raleigh, NC 27606. chris_mcgahan{at}ncsu.edu


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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