Identification of a Cytosolic
NADP+-dependent Isocitrate Dehydrogenase That
Is Preferentially Expressed in Bovine Corneal Epithelium
A CORNEAL EPITHELIAL CRYSTALLIN*
Lijie
Sun
,
Tung-Tien
Sun§, and
Robert M.
Lavker
¶
From the
Department of Dermatology, University of
Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 and
the § Epithelial Biology Unit, Ronald O. Perelman Department
of Dermatology, and the Departments of Pharmacology and Urology, Kaplan
Comprehensive Cancer Center, New York University School of Medicine,
New York, New York 10016
 |
ABSTRACT |
Recently, metabolic enzymes have been observed in
both the lens and corneal epithelium at levels greatly exceeding what
is necessary for normal metabolic functions. These proteins have been
termed taxon-specific crystallins and are thought to play a role in
maintaining tissue transparency. We report here that cytosolic
NADP+-dependent isocitrate dehydrogenase
(ICDH) represents a new corneal crystallin. Using suppression
subtractive hybridization, we identified a gene (with a deduced amino
acid sequence that showed 94% identity to rat cytosolic
NADP+-dependent ICDH) that is preferentially
expressed in bovine corneal epithelium. Northern blots established that
its mRNA level in the corneal epithelium was 31-, 39-, 133-, 230-, and 929-fold more than in the liver, bladder epithelium, stomach
epithelium, brain, and heart, respectively. This mRNA was detected
primarily in corneal epithelial basal cells by in situ
hybridization. SDS-polyacrylamide gel electrophoresis, two-dimensional
gel analysis, and Western blotting showed that this protein was
overexpressed in the corneal epithelium, constituting ~13% of the
total soluble bovine corneal epithelial proteins. Enzyme assays showed
a corresponding overabundance of this protein in bovine corneal
epithelium. Taken together, these data indicate that bovine cytosolic
ICDH fulfills the criteria for a corneal epithelial crystallin and may
be involved in maintaining corneal epithelial transparency.
 |
INTRODUCTION |
The corneal epithelium is a self-renewing stratified squamous
epithelial tissue that protects the underlying delicate structures of
the eye, supports a tear film, and maintains transparency so that light
can be transmitted to the interior of the eye. This latter quality is
essential since the cornea accounts for two-thirds of the refraction of
light in the eye. It is unclear how the corneal epithelium satisfies
the requirements of transparency. However, the current belief is that
high concentrations of metabolic enzymes within the corneal epithelium
may be involved in tissue transparency, light absorption, and
protection from UV-induced free radicals (1-4). These metabolic
enzymes were termed "corneal crystallins" (5) based on their
similarity to the situation in the lens (3, 6-8).
"Crystallins" are the major soluble proteins of the crystallin lens
(9-12). The
- and
/
-families of crystallins are ubiquitously found in all vertebrate lenses. The
-crystallins have sequence similarity to small heat-shock proteins (13) and are thought to be
molecular chaperones functioning to protect proteins from heat-induced
stresses (14). The
/
-crystallins are related to microbial
oxidative stress proteins (15), and alterations in this family of
crystallins have been implicated in lens opacity characterized by
cataracts (12). Taxon-specific crystallins are members of a diverse
group of metabolic enzymes that are expressed at much lower levels in
other tissues compared with the lens (9, 11, 16). These
species-specific crystallins represent the recruitment of enzymes to
serve the lens in a new structural role, and this phenomenon has been
termed "gene sharing" (7, 17).
In this paper, we have identified bovine cytosolic
NADP+-dependent isocitrate dehydrogenase
(ICDH)1 as an enzyme that
fits a gene-sharing profile. Using a combination of Northern and
Western blotting as well as enzyme assays, we show that this enzyme is
present in unusually high amounts in bovine corneal epithelium. Our
data suggest that this enzyme may be a new corneal crystallin (3, 6-8)
and thus may play a structural as well as a catalytic role in
contributing to corneal transparency.
 |
MATERIALS AND METHODS |
Identification of Corneal Epithelium-specific Molecules by
Suppression Subtractive Hybridization--
Fresh bovine bladder,
brain, eye, heart, liver, lung, stomach, and testis were obtained from
a local slaughterhouse and immediately placed on ice. The corneal
epithelium was removed by scraping the surface of the cornea with a
scalpel blade under a dissecting microscope and immediately frozen in
liquid nitrogen. The bladder and stomach epithelia were also removed by
scraping and frozen in liquid nitrogen. Portions of the other tissues
were frozen in liquid nitrogen. Total RNA from these tissues was
isolated using TRIzol reagent (Life Technologies, Inc.), and
poly(A)+ mRNA was isolated from total RNA preparations
using a QIAGEN OligotexTM mRNA kit.
Suppression subtractive hybridization was performed using a
CLONTECH PCR-SelectTM cDNA
Subtraction kit according to the manufacturer's protocol. Briefly,
bovine corneal epithelium was chosen as the tester, and the seven other
tissues (bladder and stomach epithelia, brain, heart, liver, lung, and
testis) served as the driver. The double-stranded cDNA synthesized
from both tester and driver mRNAs was digested with a four-cutter
restriction enzyme (RsaI). The RsaI-digested tester cDNA was ligated to two types of adapters, and hybridization was performed twice among the different adapter-ligated tester cDNAs and driver cDNA. Only the cDNAs with different
adapters at both ends were amplified by the polymerase chain reaction
(PCR). The PCR products were subsequently cloned into the pCRII vector using a TA Cloning kit (Invitrogen) and transformed into
Escherichia coli. For differential screening, the
transformed colonies were selected randomly. Colony or dot-blot
hybridization was performed with forward- and reverse-subtracted
cDNAs as probes using a CLONTECH PCR-Select
Differential Screening kit. The reverse-subtracted probe was made by
subtractive hybridization performed with the original tester cDNA
as a driver and the driver cDNA as a tester. Clones that hybridized
with only the forward-subtracted probe were selected for further analysis.
DNA Sequence Analysis--
Differentially expressed clones
obtained from the corneal epithelial subtractive cDNA library were
subjected to DNA sequencing and GenBankTM analysis. To
amplify the missing RsaI fragment of bovine ICDH, PCR was
conducted using 5'-GCTACGATTTAGGCATAG-3' (nucleotides 209-226 on
fragment a in Fig. 1) as the 5'-primer and
5'-TGGGCCACCATGTCATCA-3' (nucleotides 857 to 840 on fragment
c in Fig. 1) as the 3'-primer in a thermal cycler (MJ Research,
Inc.). The PCR product was subcloned into the pCRII vector and
sequenced on both strands. The resulting sequences were compared with
the GenBankTM data base using a BLAST or FASTA program.
Northern Blot Hybridization--
Poly(A)+ RNAs (0.5 µg) from various bovine tissues were used as templates to synthesize
double-stranded cDNA using a SMARTTM PCR cDNA
Synthesis kit (CLONTECH). The synthesized cDNA
was electrophoresed on a 1% agarose gel, transferred onto a positively
charged nylon membrane (Roche Molecular Biochemicals), and
UV-cross-linked (18). The membrane was prehybridized at 72 °C in
ExpressHybTM hybridization solution
(CLONTECH) for 1 h and hybridized in fresh buffer with denatured random primer-labeled probes at 72 °C
overnight. After hybridization, the blot was sequentially washed in 2×
SSC and 0.5% SDS for 20 min twice at 68 °C and in 0.2× SSC and
0.5% SDS for 20 min twice at 68 °C and exposed to a PhosphorImager screen overnight. Intensities of signals were quantified by
densitometry using the ImageQuant program (Molecular Dynamics,
Inc.).
Western Blotting--
Fresh corneal epithelium and the other
tissues were cut into pieces and homogenized in 4 volumes of cold 0.25 M sucrose solution with a glass homogenizer. The homogenate
was first centrifuged at 1000 rpm for 10 min to remove cell debris and
then at 14,000 rpm for 10 min. The supernatant was saved as the
cytosolic fraction, and the pellet represented the mitochondrial
fraction (19). Total soluble lysates were made by sonicating the
tissues in 0.25 M sucrose solution for three periods of
20 s on ice and centrifuged at 14,000 rpm for 10 min. Protein
concentration was determined in triplicate using a Bio-Rad protein
assay (20).
Proteins (10 µg) from cytosolic or total fractions of the various
bovine tissues were separated on 12.5% SDS-polyacrylamide gels. Gels
were stained with either Coomassie G-250 (Pierce GelCode blue stain
reagent) or silver nitrate using a Silver Stain Plus kit (Bio-Rad) to
detect proteins, or proteins were transferred to a nitrocellulose
membrane using a Mini Trans-Blot electrophoretic transfer cell
(Bio-Rad). The membrane was stained with Ponceau S solution. The blot
was incubated with a rabbit polyclonal antibody against rat cytosolic
NADP+-dependent isocitrate dehydrogenase
(kindly provided by Drs. Gary T. Jennings and L. McAlister-Henn) and
detected with peroxidase-linked second antibody (Amersham Pharmacia Biotech).
Two-dimensional Gel Electrophoresis--
Thirty micrograms of
total soluble protein isolated from the corneal epithelium was loaded
on isoelectric focusing tube gels (4% acrylamide, 9.2 M
urea, 2% ampholytes (pH 3-10), and 2% Nonidet P-40) and run at 200 V
for 20 min, followed by 450 V for 3 h. After focusing, the rod
gels were equilibrated for 10 min in a buffer containing 62 mM Tris phosphate (pH 6.8), 10% glycerol, 0.7 M
-mercaptoethanol, 2% SDS, and 0.005% bromphenol
blue. The protein was then resolved in the second dimension by 12.5%
SDS-PAGE until the dye reached the end of the gel. The proteins were
either silver-stained or transferred to Immobilon-P membrane (Millipore Corp.) by electroblotting (21, 22).
Enzymatic Activity Assays--
The activity of
NADP+-dependent ICDH from the corneal
epithelium and other tissues was assayed at 25 °C in a 1-ml reaction mixture composed of 0.1 M Tris-Cl (pH 8.0), 3 mM MgCl2, 0.5 mM NADP+,
1.5 mM DL-isocitrate, and 10 µg/ml cytosolic
proteins (19). Glutamate dehydrogenase activity was measured at
25 °C upon addition of 2 mM
-ketoglutarate, 50 mM NH4Cl, and 50 µM NADH.
-Ketoglutarate dehydrogenase activity was monitored by adding 2.5 mM NAD+, 200 µM thiamine
pyrophosphate, 130 µM CoASH, and 2.0 mM
-ketoglutarate (23). The rate of NADPH production was measured
spectrophotometrically at 340 nm. The enzyme activity is expressed in
A/min/mg of protein.
In Situ Hybridization--
A 649-base pair DNA fragment
(fragment h in Fig. 1) of bovine cytosolic
NADP+-dependent isocitrate dehydrogenase was
amplified by PCR and subcloned into pCRII. Digestion with
XhoI and transcription with Sp6 RNA polymerase were utilized
for antisense probes, and digestion with SpeI and
transcription with T7 RNA polymerase were used for sense probes. RNA
probes were prepared using 35S-labeled UTP. In
situ hybridization was conducted as described (24).
 |
RESULTS |
To identify genes preferentially expressed in bovine corneal
epithelium, we prepared a corneal epithelial subtractive library using
the suppression subtractive hybridization technique (25). Common
messages of the corneal epithelium were subtracted using driver
cDNAs of the bladder epithelium, brain, heart, liver, lung, stomach
epithelium, and testis. From the corneal epithelial subtractive cDNA library, 275 clones were picked and probed with the
forward-subtracted cDNAs (corneal epithelium-specific) or the
reverse-subtracted cDNA ("specific" for the seven tissues)
(26). About 40% of the clones gave much stronger signals with
forward-subtracted probes, indicating that these clones representing
mRNA are cornea-enriched. Among these cornea-specific clones were
those encoding the K3 and K12 cornea-specific keratins (27-37) as well
as aldehyde dehydrogenase class 3 (ALDH3), the major water-soluble
protein in mammalian corneas and one of the putative corneal epithelial
crystallins (1-6). It should be noted that the number of
cornea-enriched clones obtained using suppression subtractive
hybridization is not strictly correlated with the frequency of the
cDNAs. For example, we found three, two, and two clones for K3
keratin, K12 keratin, and ALDH3, respectively, all major components of
the corneal epithelium. Remarkably, 55% of the clones were homologous
to various regions of rat cytosolic
NADP+-dependent ICDH (Figs.
1 and 3).

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Fig. 1.
Schematic diagram of bovine
NADP+-dependent ICDH cDNA.
Fragments a-g represent clones obtained from the bovine
subtractive cDNA library; the number of clone obtained is shown in
parentheses. Fragment f, labeled with an
asterisk, was used as a probe for Northern blot
hybridization. Fragment h was amplified by PCR and used as a
probe for in situ hybridization. The black box
represents the open reading frame (ORF), and R represents
the RsaI restriction site. nt, nucleotides.
|
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The bovine cytosolic ICDH cDNA is 1682 nucleotides long and has an
open reading frame encoding 414 amino acids (Fig.
2). This protein contains the isocitrate
and isopropylmalate dehydrogenase signature: a glycine-rich stretch of
residues located in the C-terminal section. The last three amino acids
at the C-terminal end, AKL, were found in proteins that are targeted to
peroxisomes (38). Comparison of the amino acid sequence of bovine
cytosolic ICDH with that of rat ICDH showed 94% identity, and this
enzyme is highly homologous to mitochondrial ICDH from other species
(Fig. 3). Northern blot analysis showed
that the corneal epithelium contains a much higher level of ICDH than
other tissues (Fig. 4). Quantification of
the Northern blot revealed that the expression level of the mRNA in
corneal epithelium is ~31-, 39-, 133-, 230-, and 929-fold more than
in the liver, bladder and stomach epithelia, brain, and heart,
respectively.

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Fig. 2.
Nucleotide and deduced amino acid sequences
of the cDNA encoding bovine ICDH. Putative protein kinase C
phosphorylation sites are underlined, and the putative
peroxisomal targeting signal is double-underlined. The
isocitrate and isopropylmalate dehydrogenase signature is indicated by
a dotted line. The stop codon is marked with an
asterisk.
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Fig. 3.
Alignment of ICDH sequences.
Numbers indicate the amino acid positions.
Asterisks designate conserved residues for at least five
sequences. Dashes represent gaps for optimal alignment.
bICDH, bovine cytosolic
NADP+-dependent ICDH;
IDHC_RAT, rat cytosolic
NADP+-dependent ICDH;
IDHP_HUMAN, human mitochondrial
NADP+-specific ICDH; IDHP_BOVIN,
bovine mitochondrial NADP+-dependent ICDH;
IDHP_PIG, pig mitochondrial
NADP+-specific ICDH; IDHP_SOYBN,
soybean oxalosuccinate NADP+-dependent ICDH;
IDHC_YEAST, yeast
NADP+-dependent ICDH.
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Fig. 4.
Northern blot analysis of ICDH mRNAs from
various bovine tissues. Upper panel, mRNAs from
various bovine tissues were hybridized with a cDNA fragment of ICDH
(fragment f in Fig. 1). Note the intense signal in the
corneal epithelium. Lower panel, the probe on the same blot
was stripped off, and the blot was rehybridized with a
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA
probe as a control.
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Western blot analysis with a polyclonal antibody against rat cytosolic
ICDH revealed an extremely strong 43-kDa bovine corneal epithelial
protein, which was minimal in other tissues (Fig.
5a). Analysis of proteins
using (extra-long) high resolution SDS-PAGE revealed the presence of an
intense 43-kDa band in both the total and cytosolic corneal epithelial
proteins, with little or none in the bladder, heart, kidney, or liver
(Fig. 5b). Further analysis of total corneal epithelial
proteins employing two-dimensional gel electrophoresis (Fig.
6a) in combination with
Western blotting (Fig. 6b) revealed only a single protein in
the 43-kDa band. An even stronger band was detected in the corneal
epithelium at 54 kDa and most likely represented ALDH3, a previously
identified major corneal epithelial crystallin that constitutes a
maximum of 40% of the total soluble protein (2, 3, 6, 39, 40). Quantitation of the densities of the bands indicated an ALDH3/ICDH ratio of 3.2. This suggests that ICDH constitutes ~13% of the total
bovine corneal epithelial protein. This concentration of ICDH may be
considered within the levels that have been reported for corneal
crystallins (7).

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Fig. 5.
a, Western blotting of bovine cytosolic
proteins with an antibody against ICDH. Ten micrograms of bovine total
and cytosolic proteins was resolved by SDS-PAGE (12.5% acrylamide) and
blotted with an antibody against rat cytosolic
NADP+-dependent ICDH. B,
C, H, K, and L represent
bladder epithelium, corneal epithelium, heart, kidney, and liver,
respectively. b, SDS-PAGE of bovine cytosolic proteins from
various tissues. Ten micrograms of total and cytosolic proteins from
various bovine tissues was resolved by SDS-PAGE and silver-stained. The
43-kDa ICDH (marked with asterisks) accumulated in the total
and cytosolic fractions of the corneal epithelium (lanes 2 and 7). The prominent band at ~54 kDa seen in the corneal
extract (marked with plus signs; lanes 2 and
7) most likely represents ALDH3. Lane 5 represents molecular mass markers (from the top: 220, 97, 66, 46, 30, and 21 kDa).
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Fig. 6.
Analysis of total corneal epithelial proteins
by two-dimensional gel electrophoresis (a) in
combination with Western blotting (b). Thirty
micrograms of total bovine corneal epithelial proteins was resolved in
the first dimension by isoelectric focusing (IEF) in
ampholytes (pH 3-10) and in the second dimension on a 12.5%
SDS-polyacrylamide gel. The protein spot that is circled in
the silver-stained gel (a) represents ICDH as determined by
Western blotting using an antibody against the rat enzyme
(b).
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Similar to our mRNA and protein findings, a much higher ICDH
enzymatic activity was detected in bovine corneal epithelium compared
with all other tissues (Fig.
7a). However, the activities of the downstream enzymes of ICDH, glutamate dehydrogenase, and
-ketoglutarate dehydrogenase were not significantly higher in the
corneal epithelium when compared with the bladder, heart, and liver
(Fig. 7, b and c).

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Fig. 7.
Enzymatic activities of ICDH
(a), glutamate dehydrogenase (b),
and -ketoglutarate dehydrogenase
(c) in various bovine tissues. Activities were
calculated as A/min/mg of protein, and values were
normalized to that of the corneal epithelium. The results are the mean
of three separate experiments. Note the marked increase in ICDH
activity in the corneal epithelium (a) compared with the
other tissues; no differences were noted, however, in glutamate
dehydrogenase (b) and -ketoglutarate dehydrogenase (c)
activities among the corneal epithelium, bladder, heart, and
liver.
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To determine whether high levels of ICDH are found in the corneal
epithelia from other species, we prepared cytosolic extracts from
human, rabbit, rat, and mouse corneal epithelia. In these species, ICDH
activity in the cornea was not significantly higher than that in the
heart, kidney, liver, pancreas, or skin, indicating that high levels of
ICDH may be bovine-specific (Fig.
8).

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Fig. 8.
ICDH activities in different species.
The ICDH activities of human, mouse, rat, and rabbit tissues were
assayed and calculated as A/min/mg of protein. The
results represent the mean of three separate experiments for human and
duplicate experiments for rat and rabbit.
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In situ hybridization showed that ICDH mRNA was present
mainly in the basal cells of the corneal epithelium (Fig.
9, c and d) and
that ICDH expression stopped abruptly at the corneal/limbal junction
(Fig. 9, e and f). No detectable signal was seen
over corneal fibroblasts (keratocytes) or endothelial cells (data not shown).

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Fig. 9.
ICDH mRNA is detected in basal and wing
cells of central corneal epithelium. Bovine corneal
(a-d) and limbal (e and f) epithelia
were processed for in situ hybridization with
35S-labeled antisense (c-f) and sense
(a and b) probes to ICDH. A strong signal for
ICDH mRNA was detected in basal (B) and wing
(W) cells, but not in the superficial (S) cells,
of the corneal epithelium (c and d). Little if
any signal for ICDH mRNA was seen in the limbal epithelium
(e and f). a, c, and
e are micrographs taken under bright-field, and
b, d, and f were taken under
dark-field.
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 |
DISCUSSION |
High Levels of Corneal Epithelial ICDH Indicate a Dual Role for
This Enzyme--
The idea that a gene encoding a single protein may
acquire and maintain a second function without duplication and without loss of the primary function is known as gene sharing (16, 17). This
concept, originally formulated from studies on the lens crystallins (for reviews, see Refs. 7 and 8), appears to apply to many systems. For
example, in Tetrahymena, a cytoskeletal 14-nm
filament-forming protein involved in oral morphogenesis and in
pronuclear behavior during conjugation was also shown to be a
mitochondrial enzyme (41). Gene sharing has also been shown to occur in
the corneal epithelium, where enzymes such as ALDH3, transketolase, and
cyclophilin have been recruited as major corneal proteins that may
serve structural as well as catalytic roles (6, 42). In this study, we
demonstrate that ICDH is present in bovine corneal epithelium in an
amount that far exceeds what would be necessary for its conventional enzymatic requirements. The ICDH family of enzymes is found in a
majority of species and has a wide variety of functions. For example,
NADP+-dependent ICDHs are present in
mitochondria, peroxisomes, and the cytoplasm. The mitochondrial enzyme
catalyzes the reduction of isocitrate to
-ketoglutarate, which is
central to energy production (ATP) in the citric acid cycle. In the
ovary, the cytosolic form is thought to supply NADPH for fatty acid
synthesis and for fatty acid chain elongation and desaturation
reactions (43). In yeast, the peroxisomal form is thought to function
in a reduction/oxidation shuttle during the degradation of
polyunsaturated fatty acids (44, 45). This enzyme is also involved in
the synthesis of certain amino acids such as glutamate. We reasoned
that if the high ICDH levels in the corneal epithelium observed here
were solely for metabolic events in glutamate biosynthesis, increases in corneal glutamate dehydrogenase and
-ketoglutarate dehydrogenase activities would be expected, as these are the downstream enzymes in
this biosynthetic pathway. This was not the case; and thus, we conclude
that corneal epithelial ICDH must perform some other functional role.
Bovine Cytosolic ICDH May be Regarded as a Corneal Epithelial
Crystallin--
Metabolic enzymes have been observed in corneal
epithelia from a wide variety of species in amounts that are in excess
of what would be required for a catalytic role and have been termed corneal crystallins (5, 7). The most prevalent corneal crystallin is
ALDH3, found in the corneal epithelia of all mammalian species tested
to date (1-4, 6). In addition to ALDH3, human corneal epithelial cells
express aldehyde dehydrogenase class 1 (46), whereas bovine corneal
epithelial cells express ALDHx (47). Recently, transketolase has been
found at high levels in mouse, human, and bovine corneal epithelia (6,
42). All of the proteins that have thus far been classified as corneal
crystallins are metabolic enzymes that are abundantly expressed
(5-40% of the total soluble protein) and often are taxon-specific
(for reviews, see Refs. 7 and 8). Our data suggest that in cattle, ICDH fulfills the enzyme crystallin profile of these aforementioned putative
corneal crystallins for several reasons. 1) The mRNA levels of ICDH
in the corneal epithelium ranged from 31- to 929-fold higher than those
observed in the liver and heart, respectively (Fig. 4). 2) In agreement
with the mRNA data, corneal epithelial ICDH protein levels far
exceeded those of other tissues as determined by SDS-PAGE and Western
blotting (Figs. 5 and 6). 3) Within bovine corneal epithelium, ICDH
constituted up to 13% of the total soluble protein (Figs. 5 and 6). 4)
The abundance of ICDH greatly exceeded what was required for its
enzymatic function (Fig. 7). 5) The abundant levels of ICDH detected in
bovine corneal epithelium were not observed in human, mouse, rat, and
rabbit corneal epithelia (Fig. 8).
ICDH May Be a Bovine Corneal Epithelial Differentiation
Product--
It is well established that corneal and limbal basal
cells are biochemically distinct. Schermer et al. (32)
demonstrated that K3, a major keratin expressed during advanced stages
of corneal epithelial differentiation, is expressed only in the
suprabasal cells of the limbal epithelium, but uniformly in the central
corneal epithelium. This provided the first evidence that limbal basal cells are biochemically more primitive than corneal epithelial basal
cells. Our present observation of a strong signal for ICDH mRNA in
basal (and wing) cells of bovine corneal epithelium, but not in limbal
epithelial basal cells, further distinguishes these two basal cell
populations. This ICDH basal cell distribution pattern is analogous to
that of the K3/K12 keratin pair and suggests that ICDH may be
considered as a bovine corneal epithelial differentiation product. In
contrast,
-enolase has been demonstrated to be expressed predominantly in the more primitive limbal basal cells (48). The
biochemical differences observed between limbal and corneal epithelial
basal cells formed the basis of a model in which corneal epithelial
stem cells were postulated to be located in the basal layer of the
limbal epithelium (32). This model has received strong support from a
series of kinetic (49-51), cell culture (52-55), and
wounding/perturbation (49, 51, 56-59) experiments.
Implications in Maintaining Corneal Epithelial Transparency--
A
high concentration of enzyme crystallins in the lens is believed to
contribute to the lens' high refractive index and focusing power
(reviewed extensively in Refs. 7, 8, 11, 12, 15, 16, and 60), and a
similar role has been proposed for the corneal crystallins (7, 8). In
support of this idea, a recent study demonstrated that following
wounding or excimer photorefractive keratectomy, rabbit keratocytes
assumed a reflective spindle-shaped morphology that contributed to
corneal haze (61). Rabbit keratocytes normally have a high expression
of proteins homologous to transketolase and ALDH1, which together
represent 30% of the total water-soluble cellular proteins and thus
may be considered as keratocyte crystallins. A 67% specific decrease
in these keratocyte proteins accompanied the change in keratocyte shape
following wounding, suggesting that these enzymes might function in
regulating cellular refractive index and transparency (61). It is
tempting to speculate that in bovine corneal epithelium, ICDH functions
in a similar manner.
Presently, it is not clear what regulates the expression of ICDH. Both
ALDH3 and transketolase are up-regulated during postnatal development
of murine corneal epithelium, coinciding with eye opening (42). This
has led to the suggestion that UV light-generated oxidative stress may
be one of the inductive factors in corneal crystallin expression.
Support for this idea comes from a recent study that demonstrated that
higher ALDH3 mRNA levels were detected in corneas of mice exposed
to a 12-h light/dark cycle compared with those of age-matched animals
raised in the dark (62).
As the first line of defense against environmental insults, the corneal
epithelium is continuously exposed to UV radiation. One defense
mechanism against UV damage in corneal epithelial cells is nuclear
ferritin, which has been shown to protect avian corneal epithelial
cells from UV-induced DNA strand breaks (63, 64). Corneal crystallins,
acting as UV absorbers, have been proposed as another defense mechanism
against UV-induced photodamage (2, 65, 66). In this respect, it is
interesting to note that bovine corneal epithelium is thus far unique
in that it contains three metabolic enzymes that may serve as corneal
crystallins: ALDH3 (2, 3), transketolase (42, 66), and ICDH (this work). The abundant amounts of these three cytosolic enzymes in bovine
corneal epithelium are indicative that this tissue has extremely high
concentrations of NADPH-producing enzymes. NADPH is an effective
scavenger of free radicals and H2O2 resulting from excessive UV radiation. In support of this idea, high levels of
ICDH, malic dehydrogenase, and glucose-6-phosphate dehydrogenase were
postulated to be involved in defending the lens against oxidative damage (60). Whether UV stimulation regulates ICDH is not known. Since
there is a clear association between development of eye cancer in
cattle and increasing levels of radiation (67), ICDH, in addition to
ALDH3 and transketolase, may be a protective factor designed to
counteract the deleterious effects of UV radiation.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Gary T. Jennings and Lee
McAlister-Henn for generously providing the antibody against rat
cytosolic NADP+-dependent ICDH, Dr. Norman
Schechter for advice and helpful discussions, Dr. Pamela J. Jensen for
critical review of the manuscript, and Dorothy Campbell for preparation
of the histological samples.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant EY6769 (to R. M. L.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) IT259176 and AF136009.
¶
To whom correspondence and reprint requests should be
addressed: Dept. of Dermatology, University of Pennsylvania School of Medicine, Clinical Research Bldg./235A, 415 Curie Blvd., Philadelphia, PA 19104. Tel.: 215-898-3232; Fax: 215-573-2143; E-mail:
lavker{at}mail.med.upenn.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
ICDH, isocitrate
dehydrogenase;
PCR, polymerase chain reaction;
PAGE, polyacrylamide gel
electrophoresis;
ALDH, aldehyde dehydrogenase.
 |
REFERENCES |
-
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