Overexpression of H- and L-ferritin subunits in lens epithelial cells: Fe metabolism and cellular response to UVB irradiation.
ABSTRACT 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.
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 (59)Fe-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.
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.
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.
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ABSTRACT: Iron has emerged as a significant cause of neurotoxicity in several neurodegenerative conditions including Alzheimer's disease (AD), Parkinson's disease (PD), sporadic Creutzfeldt-Jakob disease (sCJD), and others. In some cases the underlying cause of iron mis-metabolism is known, while in others our understanding is at best incomplete. Recent evidence implicating key proteins involved in the pathogenesis of AD, PD, and sCJD in cellular iron metabolism suggest that imbalance of brain iron homeostasis associated with these disorders is a direct consequence of disease pathogenesis. A complete understanding of the molecular events leading to this phenotype is lacking partly because of the complex regulation of iron homeostasis within the brain. Since systemic organs and the brain share several iron regulatory mechanisms and iron modulating proteins, dysfunction of a specific pathway or selective absence of iron modulating protein(s) in systemic organs has provided important insight into the maintenance of iron homeostasis within the brain. Here we review recent information on the regulation of iron uptake and utilization in systemic organs and within the complex environment of the brain, with particular emphasis on the underlying mechanisms leading to brain iron mis-metabolism in specific neurodegenerative conditions. Mouse models that have been instrumental in understanding systemic and brain disorders associated with iron mis-metabolism are also described, followed by current therapeutic strategies aimed at restoring brain iron homeostasis in different neurodegenerative conditions. We conclude by highlighting important gaps in our understanding of brain iron metabolism and mis-metabolism, particularly in the context of neurodegenerative disorders.Antioxidants & Redox Signaling 07/2013; · 8.20 Impact Factor
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ABSTRACT: Hypoxia inducible factor (HIF) regulates expression of over 60 genes by binding to hypoxia response elements (HRE) located upstream of the transcriptional start sites. Many genes encoding proteins involved in iron transport and homeostasis are regulated by HIF. Expression of iron handling proteins can also be translationally regulated by binding of iron regulatory protein (IRP) to iron responsive elements (IREs) on the mRNA of ferritin chains and transferrin receptor (TfR). Lens epithelial cells (LEC) function in a low oxygen environment. This increases the risk of iron catalyzed formation of reactive oxygen species (ROS) and oxidative cell damage. We examined changes in expression of ferritin (iron storage protein) and Tf/TfR1 (iron uptake proteins) in LEC cultured under hypoxic conditions. Ferritin consists of 24 subunits of two types, heavy (H-chain) and light (L-chain) assembled in a cell specific ratio. Real-time PCR showed that 24 h exposure to hypoxia lowered transcription of both ferritin chains by over 50% when compared with normoxic LEC. However it increased the level of ferritin chain proteins (20% average). We previously found that 6 h exposure of LEC to hypoxia increased the concentration of cytosolic iron which would stimulate translation of ferritin chains. This elevated ferritin concentration increased the iron storage capacity of LEC. Hypoxic LEC labeled with 59FeTf incorporated 70% more iron into ferritin after 6 h as compared to normoxic LEC. Exposure of LEC to hypoxia for 24 h reduced the concentration of TfR1 in cell lysates. As a result, hypoxic LEC internalized less Tf at this later time point. Incorporation of 59Fe into ferritin of hypoxic LEC after 24 h did not differ from that of normoxic LEC due to lower 59FeTf uptake. This study showed that hypoxia acutely increased iron storage capacity and lowered iron uptake due to changes in expression of iron handling proteins. These changes may better protect LEC against oxidative stress by limiting iron-catalyzed ROS formation in the low oxygen environment in which the lens resides.Experimental Eye Research 01/2014; · 3.03 Impact Factor
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ABSTRACT: Iron is an abundant transition metal that is essential for life, being associated with many enzyme and oxygen carrier proteins involved in a variety of fundamental cellular processes. At the same time, the metal is potentially toxic due to its capacity to engage in the catalytic production of noxious reactive oxygen species. The control of iron availability in the cells is largely dependent on ferritins, ubiquitous proteins with storage and detoxification capacity. In mammals, cytosolic ferritins are composed of two types of subunits, the H and the L chain, assembled to form a 24-mer spherical cage. Ferritin is present also in mitochondria, in the form of a complex with 24 identical chains. Even though the proteins have been known for a long time, their study is a very active and interesting field yet. In this review, we will focus our attention to mammalian cytosolic and mitochondrial ferritins, describing the most recent advancement regarding their storage and antioxidant function, the effects of their genetic mutations in human pathology, and also the possible involvement in non-iron-related activities. We will also discuss recent evidence connecting ferritins and the toxicity of iron in a set of neurodegenerative disorder characterized by focal cerebral siderosis.Archives of toxicology. 08/2014;
Overexpression of H- and L-Ferritin Subunits in Lens
Epithelial Cells: Fe Metabolism and Cellular
Response to UVB Irradiation
Małgorzata Goralska, Benjamin L. Holley, and M. Christine McGahan
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 incorpo-
ration into ferritin was determined by incubating transfected
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-transfec-
tants incorporated significantly more Fe than those of L-trans-
fectants. The UVB irradiation reduced cell number of L-trans-
fectants 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. Overex-
pression 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 in-
creased storage of Fe in ferritin and protected cells from UV
damage. (Invest Ophthalmol Vis Sci. 2001;42:1721–1727)
59Fe-labeled transferrin followed by native PAGE
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 relation-
ship of H- and L-chains and Fe loading into ferritin in intact cells
is not fully understood.
erritin, the ubiquitous Fe storage protein is a polymeric
protein made up of 24 subunits of two types, heavy (H) and
Recent findings of a mutation in ferritin synthesis in hu-
mans, which is associated with early bilateral cataract forma-
tion,1,2emphasizes 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.3,4Thus, 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.5Even 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 dis-
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 overexpres-
sion 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 dam-
MATERIALS AND METHODS
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 (Hy-
clone, 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 experi-
ments 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?-ATGAGCTCCCAGAT-
TCGT-3?) of the coding strand and to the nucleotides 504 to 522
From the Department of Anatomy, Physiology, and Radiology,
College of Veterinary Medicine, North Carolina State University, Ra-
Supported by National Institutes of Health Grant EY04900 and
Funds from State of North Carolina.
Submitted for publication December 21, 2000; accepted March
Commercial relationships policy: N.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked “advertise-
ment” 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
Investigative Ophthalmology & Visual Science, July 2001, Vol. 42, No. 8
Copyright © Association for Research in Vision and Ophthalmology
(5?-TTAGTCGTGCTTGAGAGTGAG-3?) of the noncoding strand of fer-
ritin 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 se-
quenced because of concerns about the presence of expressed pseu-
dogenes6,7and 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 transcrip-
tion/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 ?g 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
Transient Transfection of LEC with
Transfection conditions were optimized using a pEGFP (Clontech, Palo
Alto, CA) expression vector containing the cDNA for Green Fluores-
cent 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 transfec-
tions. 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
?g plasmid DNA in 0.75 ml of DMEM containing 10% serum and 4 ?l
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 syn-
thesis and Fe incorporation into ferritin. Treatment conditions are
described in detail in the Results section and in the tables and figure
Metabolic Labeling of Newly Synthesized Ferritin
Cells in each well of a six-well plate were labeled for 20 hours with 77
?Ci of35S-methionine in methionine-free DMEM under different ex-
perimental conditions. After incubation, the cells were rinsed with PBS
and lysed on ice with 250 ?l 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 ?l 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 electro-
phoresed 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 Photofin-
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 (Boehr-
inger Mannheim) was labeled with59Fe-nitrilotriacetic acid as previ-
ously described.8Cells were incubated with59Fe-labeled transferrin
(5.2 ?M) and treated as described in the figure legends. After treat-
ment, the cells were rinsed with ice-cold PBS and lysed with 500 ?l of
a hypotonic 10 mM Tris/HCl buffer (pH 7.4) containing protease
inhibitor cocktail (Sigma). Total59Fe uptake was determined by count-
ing 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 radioac-
tivity 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.9Assay 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,
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
Nucleotide Sequences of Dog Ferritin H-
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 fer-
ritin using PCGene. The dog H cDNA consists of 552 nucleo-
tides 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 iden-
tity. 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.
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 bind-
ing motif10are 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
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
1722Goralska et al.
IOVS, July 2001, Vol. 42, No. 8
in vitro in the TNT Coupled Reticulocyte Lysate System. Auto-
radiographs of the synthesized proteins demonstrated the pres-
ence of a single band in each of reaction mixtures correspond-
ing 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 mo-
lecular weights (Fig. 3B). The clones of both genes were then
used for transfection of LEC.
Overexpression of L- and H-Ferritin Chains
Analysis of the subunit composition of newly synthesized
ferritin in LEC, 68 hours after transfection, showed a seven-
fold 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
nine H- and L-chain ferritin.
Comparison of the coding region cDNA sequences of ca-
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.
Comparison of ferritin H- and L-chain amino acid sequences
transcription/translation reticulocyte lysate system. The synthesized
protein was electrophoresed in 15% SDS-PAGE (A) under reducing
conditions and 8% native PAGE (B).
Electrophoretic analysis of ferritin synthesized in in vitro
tially transfected LEC. Forty-eight hours after transfection, LEC were
incubated for additional 20 hours in serum-free, methionine-free
DMEM to which35S-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; ˆP ?
0.05, significantly different from plasmid L-chain.
Subunit makeup of newly synthesized ferritin of differen-
IOVS, July 2001, Vol. 42, No. 8
Ferritin and Fe in Lens Epithelial Cells1723
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 com-
parison 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. Trans-
fection with H-chain increased the ratio to 3.22 and trans-
fection with L-chain drastically reduced the ratio to 0.34,
which indicated vast overexpression of L-subunit type in
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 predomi-
nates in the liver extracts as has been known for many
To determine whether newly overexpressed ferritin sub-
units remain in the form of free chains or are assembled into
the ferritin molecule, the samples were analyzed using nonde-
naturing PAGE. Newly synthesized,35S-methionine–labeled fer-
ritin was purified from the cell lysates by precipitation of
nonferritin lysate proteins with methanol at 75°C and subse-
quently 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 combi-
nation of both lower concentration of the protein and some
loss of it during methanol purification.
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 overex-
pression 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 con-
trol 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 fer-
In Vivo Iron Incorporation into Ferritin of
Forty-eight hours after transfection, the cells were incubated
There were no differences in total Fe uptake by all trans-
fected cells (data not shown). Nondenaturing PAGE analysis
of the cell lysates showed the presence of59Fe in bands that
comigrated with ferritin and transferrin standards and in a
diffuse band of low molecular weight (?3000 Da; Fig. 8A).
more, 1.7 times,59Fe than ferritin of plasmid transfectants.
Ferritin of L-transfectants contained an amount of59Fe sim-
ilar 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
ferritin protein, L-transfectants had profoundly (20-fold)
lower incorporation than plasmid and H-transfectants
59Fe-saturated transferrin for an additional 20 hours.
59Fe was expressed as a ratio of Fe to
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 transfec-
transfected 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.
Electrophoretic analysis of heat purified ferritin from non-
nondenaturing condition. Forty-eight hours after transfection, differen-
tially transfected LEC were incubated for additional 20 hours in serum-
free, methionine-free DMEM containing
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 re-
peated twice and the gel shown is a representative experiment (PL,
pTarget; FH, ferritin heavy chain cDNA; FL, ferritin light chain cDNA).
Electrophoretic analysis of newly synthesized ferritin in
35S-methionine. Cells were
from Fe incorporation experiments. Cell lysates of differentially trans-
fected cells were acetone precipitated, and protein pellets were dis-
solved 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.
Ferritin quantification using ELISA. Samples were obtained
1724 Goralska et al.
IOVS, July 2001, Vol. 42, No. 8
tants (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, nonirradi-
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 func-
tions, including Fe storage in ferritin and the ability of cells to
withstand UV irradiation. This is the first study to show over-
expression 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
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. Overexpres-
sion 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
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 pro-
teins, 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]).16Both H- and L-ferritin
chain mRNAs carry an iron-responsive element.
transferrin, and low-molecular-weight pool of differentially transfected
LEC. The incorporation of59Fe into ferritin was expressed as relative
counts (A) and59Fe cpm/ng ferritin (B). Forty-eight hours after trans-
fection, LEC were labeled in serum-free DMEM with
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 deter-
mination 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; ˆP ? 0.05,
significantly different from FL. (B) *P ? 0.05, significantly different
from PL and FH.
Electrophoretic analysis of59Fe incorporation into ferritin,
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 nonirra-
Changes in cell count of differentially transfected cells after
IOVS, July 2001, Vol. 42, No. 8
Ferritin and Fe in Lens Epithelial Cells1725
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.2,17–19There 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 pheno-
type was created by overexpression of H-chain ferritin in HeLa
cells.20It has been demonstrated recently that the 3? untrans-
lated region of mRNA of H-chain contains sequences interact-
ing with cytosolic RNA binding factors, which change the
stability of H-chain mRNA.21,22The 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 overexpres-
sion 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 expres-
sion 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,23although there
was no overexpression of L-chain protein. The greater expres-
sion of ferritin L- over H-chain was also reported for transiently
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.25,26However, 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.20There 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.27and Corsi et al.,24overexpression 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 as-
semble in vivo into ferritin molecules, we transfected LEC with
either H- or L-chain cDNA,35S-methionine–labeled and subse-
quently purified ferritin from cell lysates using the heat resis-
tance 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 overex-
pressed ferritin chain can either be incorporated into endoge-
nous ferritin, changing its subunit composition or can assem-
ble into homopolymeric ferritin as has been demonstrated in
primate fibroblastoid cells (COS-7) transfected with human
ferritin chains.24Which of these mechanisms takes place in
transfected LEC needs to be further examined.
Fe Metabolism in LEC with Ferritin of Different
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 differ-
ent ferritin subunit makeup.
The overexpression of H-chain in LEC increased incorpora-
tion 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.27,28Much 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 incor-
poration into L-transfectant’s ferritin. It has been shown in
vitro that decreasing H-chain content in ferritin recombinants
lowers Fe incorporation.29,30Therefore, 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 fer-
ritin, 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.32–34In a more
recent study,20overexpression of H-chain ferritin altered intra-
cellular 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.35L-transfectants were not pro-
tected 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 overex-
pression 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 ex-
tracted lens.36The 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 mea-
surements 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
1726 Goralska et al.
IOVS, July 2001, Vol. 42, No. 8
Fe-catalyzed free radical reactions contribute to cataractogen-
esis 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 opportu-
nity for further investigation.
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Ferritin and Fe in Lens Epithelial Cells1727