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Introduction
Cataracts are still the most important cause of blindness in the
world and are a significant and increasing global problem. The mechanism
of cataract development is complicated and involves many factors.
For many years, we have been involved in the development of anti-cataract
treatments and investigating the mechanisms of cataract development.
Our previous studies on selenite-induced rat cataracts demonstrated
that the NO concentration in ocular tissue was significantly higher
than in normal rats. Inducible nitric oxide synthase (iNOS or NOS2)
is thought to play a role in the initiation of cataracts, and the
development of cataracts is prevented by NOS inhibitors[1].
We researched whether NO was produced by the ocular tissues or comes
from other parts of the body, so we selected human lens epithelial
cells (HLEC) for further study.
Inducible NOS, one of the three isoforms of NOS, catalyses the
oxidization of L-arginine and generates large amounts of
NO[2]; it does not require elevated intracellular Ca2+
levels for activation. In addition, its function lasts for a longer
period than that of constitutive NOSs (endothelial NOS, eNOS; neural
NOS, nNOS). Inducible NOS can be produced by treatment with agents,
such as cytokines, interferons and bacterial lipopolysaccharide
(LPS), in a wide variety of cell types such as macrophages, hepatocytes,
keratinocytes, endothelial cells, and epithelial cells[3-7].
In the eye, eNOS and nNOS are localized in the retina, ciliary body
and conjunctiva, and are thought to be involved in neurotransmission,
regulation of intraocular pressure and vasodilation, while iNOS
is involved in endotoxin-induced uveitis and inflammation of the
anterior segment of the eye, and inhibits the cellular proliferation
of retinal pigment epithelial cells[8,9]. Therefore,
the purpose of this study is to investigate the biological activity
of HLEC in iNOS expression and NO production.
Diethyldithiocarbamate (DDC), a potent free radical scavenger,
has been found to prevent selenite-induced opacity in cultured rat
lenses[10]. This was thought to be related to its anti-oxidant
effect. It has been reported that both NO and its derivative peroxynitrite
(ONOO-) inhibit mitochondrial respiration and may increase
oxygen radical production by mitochondria. Large or persistent levels
of NO promote mitochondrial oxidant formation, and inhibit the respiratory
chain complexes, probably by nitrosylating or oxidizing protein
thiols and removing iron from the iron-sulphur centres. Peroxynitrite
causes irreversible inhibition of mitochondrial respiration and
damage to a variety of mitochondrial components via oxidizing reactions,
most notably at complexes I and II of the electron transport chain,
ATPase, aconitases, and Mn-superoxide dismutase[11,12].
These damages lead to deficient energy metabolism. Usually, in the
initiation of cataractogenesis, deficient energy metabolism occurs
with mitochondria function disorder. Thus, the effect of DDC for
the prevention of NO overproduction was also investigated in this
study.
Materials and methods
Materials DDC and bovine serum albumin (BSA) were purchased
from Wako Pure Chemical Industries Ltd (Osaka, Japan). Dulbecco's
modified Eagle's medium (low glucose) (DMEM), fetal bovine serum
(FBS), 0.05% trypsin-EDTA solution and phosphate buffered saline
(PBS) were purchased from GIBCOTM Invitrogen Co (Tokyo,
Japan). Gentamicin reagent solution (10 g/L) was obtained from GIBCO
BRL Life Technologies (Japan). Interferon-污 (IFN-污) was purchased
from PeproTech EC Ltd (London, UK). LPS purified from Escherichia
coli was obtained from Sigma-Aldrich Co (St Louis, MO, USA).
Protease inhibitor cocktail tablets were obtained from Complete
Roche Applied Science (Japan). All the primers used were synthesized
by Sigma Genosys (Sigma Genosys, Japan, KK). Alkaline phosphatase
(AP)-conjugated anti-rabbit IgG and ProtoBlot II AP System with
Stabilized Substrate (BCIP/NBT) were obtained from Promega Co. All
other chemicals used, except where indicated, were of analytical
grade.
Cell culture and treatments The human lens epithelial cell
line SRA 01/04 was provided by Ibaraki Medical University, and maintained
in DMEM supplemented with 10% (v/v) heat-inactivated FBS and 10
mg/L gentamicin at 37 oC in a humidified CO2 incubator
(ESPEC BNA-111, TABAI ESPEC CORP, Japan) with 5% CO2.
Culture medium was changed every other day. The confluent cells
were separated and trypsinized with 0.05% trypsin-EDTA to produce
single cells, after which they were seeded at 4×104
cm-2 and allowed to form subcultures. Cell viability
was assessed by Trypan blue exclusion assay. Each treatment was
carried out on d 3 after seeding, when the cells were 80% confluent.
The culture medium was changed every other day for routine culture,
and then 1 h before each experiment. All cultures were carried out
in duplicate.
Semiquantitative reverse transcription-polymerase chain reaction
(RT-PCR) for iNOS mRNA evaluation Total cellular RNA was extracted
and purified from HLEC with the RNeasy Mini Kit and RNase-Free DNase
Set (QIAGEN KK, Tokyo, Japan) according to the manufacturer's instructions.
The RNA level was quantified by measuring the absorbance at 260
nm. The samples with a ratio of 260/280 nm greater than 1.8 were
used. RT-PCR was performed using the RNA PCR Kit (AWV Ver 2.1, TAKARA
BIO INC, Tokyo, Japan). As a control for calibrating an equivalent
amount of input cDNA, the mRNA level of constitutively expressed
G3PDH was determined in parallel aliquots of cDNA to control any
differences in cDNA synthesis efficiency. In general, total RNA
1 µg was added to a 20 µL volume of reaction medium for
reverse transcription, and all PCR procedures were performed using
a volume of 50 µL according to the manufacturer's instructions.
The primers used in this study were as follows: iNOS (sense, 5´-CCAGT
GACAC AGGAT GACCT TCAG-3´ and antisense, 5´-TGCCA TTGTT
GGTGG AGTAA CG-3´), and human G3PDH (sense, 5´-CATCA CCATC
TTCCA GGAGC GAGA-3´ and antisense, 5´-CCACC ACCCT GTTGC
TGTAG CCA-3´). The cycling conditions were: 35 cycles for iNOS
and 20 cycles for G3PDH at 94 oC for 30 s, 65 oC
for 30 s, and 72 oC for 1 min for amplifying 603 bp and
752 bp products, respectively. Finally, the extension of products
was performed at 72 oC for 10 min. PCR-amplified samples
were run on 1.5% agarose gel and visualized using ethidium bromide,
and then photographed (ImageMaster VDS-CL, Amersham Biosciences)
under UV light.
Western blotting for determination of iNOS protein At the
end of the treatment, cells were washed with ice-cold phosphate
buffer and removed from plates by a cell scraper and centrifuged
at 200×g for 5 min. The cell pellets were then suspended
in 200 µL lysis buffer (Tris·HCl 50 mmol/L, pH 7.6, protease
inhibitor cocktail tablet in PBS 150 mmol/L, Triton X-100 0.5%)
and lysed for 30 min on ice. The cell debris was removed by centrifugation
at 12 000×g at 4 oC for 20 min. The
protein concentration was determined using a protein assay kit (Bio-Rad
Laboratories, CA, USA) with BSA as the standard. A sample of total
protein (20 µg) was separated on an 8% polyacrylamide sodium
dodecyl sulfate (SDS) gel. The proteins were then transferred to
polyvinylidene difluoride (PVDF) membranes (BIO-RAD, CA, USA), using
a semi-dry transfer cell (TRans-Blot SD Semi-Dry Electrophoretic
Transfer Cell, BIO-RAD, CA, USA). The transfer buffer used in the
system contained Tris·HCl 25 mmol/L, glycine 192 mmol/L, methanol
20%, and SDS 0.0375%. After transfer, nonspecific sites on the membranes
were blocked with 5% non-fat dry milk in PBS. The blots were probed
with 1 mg/L rabbit anti-human iNOS polyclonal antibody (Santa Cruz
Biotechnology Inc, CA, USA) overnight at 4 oC followed
by washing several times with PBS-0.1% Tween 20 (PBST). A secondary
alkaline-phosphatase conjugated anti-rabbit IgG (1:7500 dilutions)
was added, and incubated for 1 h at room temperature. After washing
several times in PBST, iNOS proteins were visualized by BCIP/NBT
kit.
Results
Induction of iNOS mRNA The expression of iNOS has been
studied in most detail in murine macrophages where maximum expression
can be induced by a combination of IFN-污 and LPS[16].
Figure 1 showed that non-stimulated HLEC had undetectable levels
of iNOS mRNA (lane 1). Stimulation with LPS or IFN-污 alone for 3
h had a negligible effect on iNOS mRNA induction (lanes 2 and 3).
HLEC, pretreated with IFN-污 for 3 h, then washed twice with PBS,
followed by an incubation with LPS for 3 h, had a very thin band
(lane 4), whereas, cells treated with IFN-污 for 3 h followed by
co-incubation with LPS and IFN-污, produced a substantial amount
of iNOS mRNA (lane 5).
Increasing the amount of LPS from 1 µg/L to 1 mg/L resulted
in a corresponding increase of iNOS mRNA levels (Figure 2A). With
the co-incubation time increase, the intensity of the iNOS bands
tended to increase (Figure 2B). On the contrary, extending the pretreatment
time of IFN-污 from 1 d to 2 d did not produce a stronger iNOS band
(Figure 2C, lane 5 vs 3).
Inhibitory effect of DDC on iNOS expression Figure 3 shows
the effect of DDC on iNOS mRNA expression. Incubation with LPS,
IFN-污, or DDC alone failed to produce iNOS mRNA or protein, while
the combination of LPS and IFN-污 resulted in the expression of iNOS
(lane 4), and the expression was inhibited by adding DDC to the
culture medium (lane 5). However, the induction of iNOS by LPS and
IFN-污 was attenuated in HLEC that had been loaded with DDC. In addition,
the inhibitory effect was enhanced at increased DDC concentrations
from 10 µmol/L to 1 mmol/L (Figure 4).
Discussion
The present research reports the expression of iNOS mRNA in HLEC
stimulated with LPS and IFN-污. The co-incubation of both factors
is needed for a quick cellular response and large amount of iNOS
mRNA production, compared with stimulation with LPS or IFN-污 alone
or successively. The synergistic effect between LPS and IFN-污, as
far as the transcription of iNOS mRNA is concerned, has been extensively
investigated in mouse macrophages[15-17]. LPS is a common
initiator of inflammation, triggering tyrosine phosphorylation and
activation of mitogen-activated protein kinases (MAPKs)[18].
MAPKs phosphorylate and regulate a variety of transcription factors,
leading to inflammatory gene expression[19]. In several
cell types, LPS also activates transcription nuclear factor kappa
B (NF-百B) and regulates iNOS gene expression[20]. The
synthesis of iNOS is mainly regulated at the transcriptional level.
The promoter region of the iNOS gene from different species has
been reported to contain binding sites for several transcription
factors. Those known to be active include kB sites located both
in the enhancer and basal promoter[20,21] , two juxtaposed
enhancer-linked IFN-stimulated response elements (ISREs), the distal
one of which is a strong activator[20,21], while the
proximal one is a weak activator of transcription[22]
, and an octamer element in the basal promoter[21]. An
IFN-污 activated site (GAS) is necessary for full expression of the
mouse iNOS gene in response to IFN-污 and LPS. The binding of Stat1a
to the GAS of the iNOS promoter is required for optimal induction
of the iNOS gene by IFN-污 and LPS[23]. Also, iNOS gene
expression appears to require the simultaneous presence of all transcription
factors binding to their enhancers. When all are present, transcription
is enhanced; when any one of them is absent, the transcription level
is reduced or completely stopped. In the case of humans, transcriptional
regulation is a crucial factor in the initiation of cytokine-stimulated
NO production by human iNOS[24]. The two activator protein-1
sites, as well as the upstream NF-百B site, are important for LPS
and IFN-污 stimulation of human iNOS induction.
Activation of NF-百B seems to be an essential step for iNOS induction
in most cell types, and the inhibitory effect of DDC on the transcriptional
activity of the iNOS gene may be due to an effect on the activation
of transcription factor NF-百B, which regulates the expression of
a variety of genes essential for cellular immune response, inflammation,
growth, and development. Because dithiocarbamates are well-known
antioxidants and NF-百B inhibitors[25,26], and DDC, a
dithiocarbamate analogue, exhibits concentration-dependent biphasic
effects in inhibiting NF-百B activation in cerebral endothelial cells[27],
that is, it inhibits NF-百B activation at low but not high concentrations
(>500 µmol/L). The present studies investigated the inhibitory
effect of DDC at concentrations ranging from 10 µmol/L to 1
mmol/L, and no biphasic effect was observed. If a biphasic effect
does exist in HLEC, the "high concentration" should be
more than 1 mmol/L. Our previous studies on the ocular bioavailability
of disulfiram (DSF), which is a dimer of DDC and quickly converted
into DDC by catalysis of the protein mercapto group in the cornea
and aqueous humor, demonstrated that the concentration of DDC in
the aqueous humor was not more than 100 µmol/L after topical
administration. Thus, if DSF is used as an anticataract agent for
the prevention and treatment of cataracts, the main problem we need
to consider is how to enhance the corneal permeability and bioavailability
of DSF preparations. Whether the anticataract effect of DDC is due
to its inhibition of NF-百B or not, the signal transduction pathway
linking stimulation of iNOS mRNA in HLEC needs to be clarified by
further investigations.
In conclusion, the present study reports the expression of iNOS
in HLEC, which shows that co-stimulation of IFN-污 and LPS is required,
while the expression is inhibited by DDC.
Acknowledgment
We are most grateful to Dr David JACK for comments and suggestions
on our manuscript.
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