Interferon Consensus Sequence-binding Protein Is Constitutively
Expressed and Differentially Regulated in the Ocular Lens*
Wenmei
Li
,
Chandrasekharam N.
Nagineni
,
Hong
Ge§,
Bassey
Efiok¶,
Ana B.
Chepelinsky§, and
Charles E.
Egwuagu
From the Laboratories of
Immunology and
§ Molecular & Developmental Biology, NEI and the
¶ Laboratory of Molecular Hematology, NHLBI, National Institutes
of Health, Bethesda, Maryland 20892
 |
ABSTRACT |
Interferon signaling is mediated by STATs and
interferon regulatory factor (IRF) families of transcription factors.
Ten distinct IRFs have been described and most are expressed in a
variety of cells except for interferon consensus sequence-binding
protein (ICSBP) and lymphoid-specific IRF/Pip that are thought to be
exclusively expressed in lymphoid cells. We show here for the first
time that ICSBP is constitutively and inducibly expressed in the mouse
lens. In contrast to lymphoid cells with exclusive expression of ICSBP in the nucleus, ICSBP is present in both the cytoplasm and nucleus of
the lens cell. However, ICSBP in the nucleus is of lower apparent molecular weight. We further show that the ICSBP promoter is
constitutively bound by lens nuclear factors and that its activation
requires binding of additional factors including STAT1. Furthermore,
transcriptional activation of ICSBP gene by interferon
is
accompanied by selective nuclear localization of ICSBP in proliferating
epithelial cells but not in the nuclei of nondividing cells in the lens
fiber compartment. Constitutive and inducible expression of ICSBP in
the ocular lens and differential regulation of its subcellular
localization in the developing lens suggest that ICSBP may have
nonimmunity related functions and that the commonly held view that it
is lymphoid-specific be modified.
 |
INTRODUCTION |
Interferons (IFNs)1 are
a family of secreted proteins that are involved in the regulation of
diverse cellular processes (1). In addition to their well defined roles
in host defense against infectious agents, they have been associated
with the regulation of cellular immunity, cell growth (1-3), and
epithelial cell differentiation (4). The importance of IFNs is
underscored by the expression of their receptors in virtually all
mammalian cell types and by the fact that they regulate the expression
of more than 50 cellular genes (1, 5). Interaction of IFNs with their
cell surface receptor leads to activation of protein tyrosine kinases,
JAK1, JAK2, or Tyk2, which in turn phosphorylate and activate members
of a family of latent cytoplasmic transcription factors called STATs
(signal transducers and activators
of transcription) (6, 7). Phosphorylated STATs form homo-
or heterodimers that translocate to the nucleus where they bind to well
defined DNA sequences called GAS (gamma interferon
activation site) or ISREs
(IFN-stimulated response
elements) and activate the transcription of genes coding
for members of the interferon regulatory factor (IRF) family of
transcription factors (8, 9).
IRFs are important mediators of transcriptional activation or
repression of IFN-regulatable genes. They are characterized by a
115-amino acid N-terminal DNA-binding domain that interacts with ISRE
motifs of IFN-regulatable genes (9). Direct and indirect evidence
indicate that the C-terminal portion of IRFs contains a protein-protein
interaction domain able to function as transcriptional activators
and/or repressors (9, 10). Ten members of the IRF family have been
identified, and they include ICSBP, ISGF3
/p48, IRF-1, IRF-2, IRF-3,
IRF-4/lymphoid-specific IRF/Pip/ICSAT, IRF-5, IRF-6, IRF-7, and vIRF
(9). IRF-1 and IRF-2 are the best characterized members of this family
and were initially identified by studies of the transcriptional
regulation of the human IFN
gene (11, 12). They have subsequently
been shown to be key factors in the regulation of cell growth through
their effects on the cell cycle (2, 3). IRF-1 is a tumor suppressor
(13), whereas IRF-2 is oncogenic (14).
In contrast to IRF-1 and IRF-2, which are expressed in a variety of
cell types, two IRF members, interferon consensus sequence-binding protein (ICSBP) (15) and lymphoid-specific IRF/Pip (Pu.1
interaction partner) (16-18) are thought to be
expressed exclusively in cells of macrophage and lymphocyte lineages.
Constitutive expression of ICSBP is thought to be limited to B
lymphocytes, and mice with null mutation for the ICSBP gene develop
myelogenous leukemia-like syndrome, suggesting that ICSBP activities
may be restricted to lymphoid cells (15, 19).
We have previously reported the generation of transgenic mice with
targeted ectopic expression of IFN
in the lens under the direction
of the
A-crystallin promoter (20, 21). In these mice, the normal
pattern of endogenous lens gene expression is perturbed, and the
developmental fate of cells destined to become lens fiber cells is
altered. It was during the course of studies to establish a biological
link between expression of IFN
and the observed developmental
defects that we discovered that several IRFs are constitutively
expressed in the mouse lens. In this report, we present evidence that
ICSBP is constitutively and inducibly expressed in the mouse lens.
 |
EXPERIMENTAL PROCEDURES |
Animals--
BALB/c wild type (WT) mice were purchased from
Jackson Laboratories (Bar Harbor, ME). CD-1 WT mice were from Charles
River (Raleigh, NC). Generation of the BALB/c IFN
transgenic (TR)
mice has previously been described (20, 21). All animal procedures conformed to Institutional Guidelines and the Association for Research
in Vision and Ophthalmology Resolution on the Use of Animals in Research.
Cell Culture and IFN
Treatment--
The murine lens
epithelial cell line,
TN4-1 (22), kindly provided by Dr. Paul
Russell (NEI, NIH, Bethesda, MD), was grown in Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml
streptomycin. The CRLE2 and 1AMLE 6 mouse epithelial cell lines (23),
kind gifts from Dr. Christina M. Sax (NEI, NIH), were propagated in
minimum essential medium supplemented with 5% rabbit serum, 5% fetal
bovine serum, 2 mM glutamine, 100 units/ml penicillin, and
100 µg/ml streptomycin. The cells were treated with murine
recombinant IFN
(Life Technologies, Inc.) at a concentration of 100 units/ml for 2 h at 37 °C, 5% CO2. Some cells were
propagated in medium containing the protein synthesis inhibitor,
cycloheximide (CHX) (Sigma) at 35 µg/ml for 30 min followed by
addition of IFN
and incubation for 2 h.
Reverse Transcribed PCR--
Lenses from 6-week-old WT or TR
mouse littermates were carefully dissected and washed before RNA
isolation to avoid any possible contamination by other tissues. Total
RNA was isolated from the lenses or cultured lens cells as recommended
for the TRIzol Reagent (Life Technologies, Inc.). All RNA samples were
digested with RNase-free DNase 1 (Life Technologies, Inc.) for 30 min
and purified by phenol/chloroform extraction and precipitation in 0.4 M LiCl. cDNA synthesis was performed at 42 °C for
1 h with 10 µg of total RNA, 0.3 µg of
oligo(dT)(12-16) and 1000 units Superscript Reverse
Transcriptase II (Life Technologies, Inc.) in a final volume of 50 µl. For each RNA preparation, a negative control reaction was
performed without reverse transcriptase. After purification of the
cDNA, hot start PCR assays were performed with AmpliTaq Gold DNA
polymerase (Perkin-Elmer). Samples were incubated at 95 °C for 10 min to activate the AmpliTaq Gold, and amplification was carried out
for 25 cycles at 94 °C for 45 s, 63 °C for 45 s, and
72 °C for 45 s, and this was followed by a final 10-min extension at 72 °C. All the primer pairs used for PCR amplifications spanned at least one intron, making it possible to distinguish between
amplification products derived from cDNA and those resulting from
any contaminating genomic DNA templates. The sequence of the PCR
primers used are: for mouse G3PDH,
5'-TGAAGGTCGGTGTGAACGGATTTGGC-3' and
5'-CATGTAGGCCATGAGGTCCACCAC-3' (24), and for mouse ICSBP, 5'-GCTGCGG
CAGTGGCTGATCGAACAGATCG-3' and 5'-AGTGGCAGGCCTGCACTGGGCTGCTG-3' (25). For Southern blot analysis, the amplified fragments were electrophoresed in agarose gels, transferred onto Hybond N+ nylon membrane (Amersham Pharmacia Biotech), and probed with fluorescein-dUTP 3'-end-labeled oligonucleotide probe, internal to the corresponding PCR
primers. Probe labeling and signal detection were performed with the
ECL 3'-oligolabeling and detection system (Amersham Pharmacia Biotech).
Northern Blot Analysis--
Total RNA (30 µg) was fractionated
on a 0.8% agarose-formaldehyde gel, transferred to Hybond
N+ membrane (Amersham Pharmacia Biotech), and hybridized
for 12 h at 65 °C in hybridization solution containing 5 × 106 cpm/ml of probe as described (26). ICSBP or
-actin-specific cDNA fragments were labeled to high specific
activity (>108 cpm/µg) with [
-32P]dCTP
by random priming (oligolabeling kit; Amersham Pharmacia Biotech) and
used as hybridization probes. After two high stringency washes in
0.1 × SSC, 0.1% SDS at 65 °C, signals were detected by
autoradiography at
70 °C. with Kodak X-Omat AR film and Cronex intensifying screens.
Western Blot Analysis--
Lenses derived from 6-week-old WT or
TR mouse littermates were disrupted in 50 mM Tris-HCl, pH
7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium
deoxycholate, 2 µM leupeptin, 2 µM
pepstatin, 0.1 µM aprotinin, 1 mM
4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, 0.5 mM phenylmethylsulfonyl fluoride, and 1 µM E-64 on ice. Extracts were clarified by centrifugation, and protein levels were determined by Coomassie Blue dye binding method as recommended for Coomassie Plus Protein Assay Reagent (Pierce). For
analysis of the lens epithelial cell lines, cells were cultured for
2 h in medium alone or medium containing 100 units/ml IFN
. The
cells were lysed and fractionated to cytosolic and nuclear fractions as
described previously (27). Nuclear and cytosolic fractions were also
obtained from the EL4 lymphoma cell line (ATCC TIB-39) (American Type
Culture Collection, Manassas, VA) and BALB/c mouse spleen cells. All
samples were heated for 10 min at 95 °C in 1× sample buffer and
electrophoresed in 10% SDS/polyacrylamide gel. The gel was
electroblotted onto polyvinylidene fluoride membrane, blocked with 5%
nonfat milk, and probed with either goat anti-mouse ICSBP polyclonal
antibodies (1:2000) from Santa Cruz Biotech (Santa Cruz, CA) or a
rabbit anti-mouse ICSBP polyclonal antibody (1:2000) from
Zymed Laboratories Inc. (San Francisco, CA). Mouse
A-crystallin-specific antibody was kindly provided by Sam Zigler
(NEI, NIH). Preimmune serum was also used in parallel as control.
Signals were detected with horseradish peroxidase-conjugated secondary
F(ab')2 antibodies using the ECL system (Amersham Pharmacia Biotech).
Immunohistochemistry--
Seventeen day mouse embryos were fixed
in 4% paraformaldehyde and embedded in Ameraffin tissue embedding
medium (Baxter). Tissue sections (5 µm) were deparaffinized in
xylene, rehydrated through a graded alcohol series, and used for
immunostaining by the avidin-biotin-peroxidase complex method (Vector
Laboratories, Burlingame, CA.). After preincubation for 30 min with 2%
blocking serum, sections were incubated for 2 h at room
temperature with antibodies (2 µg/ml) specific to mouse ICSBP (Santa
Cruz). Control sections received the appropriate normal serum. In
addition, antibody specificity control experiments were carried out by
incubating the primary antibody with a 10-fold excess of a blocking
peptide specific for the immunogenic epitope (ICSBP amino acid
407-425) for 2 h. The neutralized antibody was then used for
immunostaining reactions with control tissue sections. All sections
were subsequently incubated with biotinylated secondary antibody for 30 min at room temperature, and signal was visualized with
diaminobenzidine-H2O2 as recommended (Vector).
In some experiments, sections were counterstained with hematoxylin.
Electrophoretic Mobility Shift Assay--
Lens nuclear extracts
were prepared either from 1-3-day-old WT CD-1 mouse lenses or cultured
lens epithelial cells as described previously (28). Buffer used for
nuclear protein extraction contained the following protease inhibitors:
2 µM leupeptin, 2 µM pepstatin, 0.1 µM aprotinin, 1 mM
4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, 0.5 mM phenylmethylsulfonyl fluoride, and 1 µM
E-64. Protein concentration was determined by the Coomassie Blue dye
binding as recommended for Coomassie Plus Protein Assay Reagen kit
(Pierce), and extracts were stored at
70 °C until use. Nuclear
extracts (10 µg) in binding buffer (20 mM HEPES, pH 7.9, 50 mM KCl, 10% glycerol, 0.5 mM
dithiothreitol, 0.1 mM EDTA) containing 0.14 µg/µl
poly(dI-dC) were incubated on ice for 10 min. 32P-Labeled
double-stranded DNA probe (50,000 cpm) was added and incubated for an
additional 20 min on ice. Samples were electrophoresed in 4%
polyacrylamide gel in 0.5 × Tris borate-EDTA buffer. For competition experiments, the nuclear extract was preincubated with
unlabeled probe and poly(dI-dC) for 20 min on ice prior to the addition
of labeled probe. The sequences used for the double-stranded probes or
competitors are: ICSBP pIRE/GAS, 5'-AGTGATTTCTCGGAAAGAGAGCGCTTC-3' (
175 to
149), and ICSBP-IRE, 5'-GTAAAGAGA GAAAAGGACTC-3' (-217 to
198) (25). For supershift assays, STAT1, STAT2, STAT3, or STAT4
antibody (Upstate Biotechnology Inc., Lake Placid, NY) was added to the
binding buffer containing the nuclear extract and preincubated on ice
for 10 min. The 32P-labeled probe was then added, and the
entire mixture was incubated for an additional 20 min on ice before electrophoresis.
 |
RESULTS |
Constitutive and Inducible Expression of ICSBP in the Mouse
Lens--
We had previously generated TR mice with ectopic expression
of IFN
in the lens to study the paracrine effects of IFN
in the
eye (20, 21). In this study, we examined mRNA and protein levels of
IFN
-inducible transcription factors in the lenses of WT and TR mouse
littermates to determine whether there is any correlation between
enhanced expression of members of the IRF family of transcription
factors and the abnormal lens phenotype observed in our TR mice. We
found that both IRF-1 and IRF-2 are constitutively expressed in the
lens and the levels of IRF-1 is markedly enhanced in the TR mouse lens
(data not shown). Most surprising, we found ICSBP to be constitutively
and inducibly expressed in the mouse lens as indicated by reverse
transcribed PCR and Western blot analyses (Fig.
1). These results have been confirmed by
six independent experiments, and the authenticity of the ICSBP
transcripts has been verified by cDNA sequencing; the nucleotide
sequence of the ICSBP transcripts isolated from the lens is identical
to published sequences reported for ICSBP derived from mouse
hematopoietic cells (15). Detection of ICSBP transcripts in the WT lens
was unexpected because constitutive transcription of the ICSBP gene is
thought to be restricted to B-lymphocytes (15, 19, 27). Analysis of rat
and bovine lenses reveal that the ICSBP protein and mRNA are also
expressed in these species (data not shown). This is the first time
that constitutive expression or transcriptional activation of the ICSBP
gene has been demonstrated in mammalian cells that are not directly
involved in immunological responses.

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Fig. 1.
ICSBP is constitutively expressed in mouse
lens. Six-week-old BALB/c WT or IFN TR mouse lenses were used
to prepare whole lens extracts or poly(A) RNAs. A, reverse
transcribed PCR and Southern blot analyses for expression of ICSBP and
G3PDH mRNAs as indicated under "Experimental Procedures." Sizes
of the amplified DNA fragments are: ICSBP, 627 base pairs; G3PDH, 983 base pairs. B, whole lens extracts from WT (75 µg
protein/lane) and IFN TR mouse (30 µg protein/lane) littermates
were immunoblotted with antibodies specific for mouse ICSBP or
A-crystallin.
|
|
Spatial Localization of ICSBP Is Differentially Regulated in the
Lens by IFN
--
As the vertebrate lens is comprised of
undifferentiated, proliferating lens epithelial cells and terminally
differentiated fiber cells, we sought to determine the spatial
localization of cells expressing ICSBP in the lens. Fig.
2 shows ICSBP localization in embryonic
day 17 TR and WT mouse eye sections using polyclonal antibodies
specific to mouse ICSBP. In these experiments, three serial sections
were fixed onto the same glass slide; one section served as a negative
control and was incubated with normal preimmune serum, another section
received the primary antibody, and the third section received the
primary antibody and 10-fold excess of a neutralizing peptide specific
to an immunogenic epitope of mouse ICSBP. In all six independent
experiments performed, the experimental sections showed identical
antibody-staining patterns, whereas the negative control section
consistently showed no immunological reactivity. The section containing
10-fold molar excess of the peptide consistently showed no significant
immunoreactivity. In some experiments the amount of the peptide was
varied, and the neutralizing effect of the blocking peptide was found
to be dose-dependent. As shown in Fig. 2, ICSBP protein is
present in both the cytoplasm and nuclei of lens cells. In the TR mouse
lens, intense nuclear localization of ICSBP is observed in cells at the
lens equator (Fig. 2, C and D) and anterior
epithelia (Fig. 2F) but not in nuclei of the cells at the
lens fiber compartment (white arrows in Fig. 2, D
and F). In the WT lens, the amount of ICSBP in the nucleus
is very low and not easily detectable. However, cytoplasmic ICSBP is
easily detectable, and the level in the lens epithelia appears to be
higher compared with that of the fiber compartment (arrowhead in Fig. 2E). We obtained similar
results using eye sections of mice at days 16-20 of embryonic
development (data not shown).

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Fig. 2.
Immunohistochemical localization of ICSBP in
the developing mouse lens. Embryonic day 17 eye sections from WT
BALB/c mouse (E) and IFN TR BALB/c mouse (A D
and F) were incubated with affinity purified anti-mouse
ICSBP antibody. Control section (A) was incubated with the
primary antibody and 10-fold molar excess of an ICSBP peptide
corresponding to the immunogenic epitope (mouse ICSBP amino acids
407-425). The anterior of the lens is oriented to the top.
ICSBP immunostaining of nuclei of cells in the lens epithelia
(F) and cells at the lens equator (C and
D) is indicated by black arrows; nuclei in the
fiber mass do not show significant DAB staining (white
arrows in D and F). The sections in
D-F are counter-stained with hematoxylin and eosin.
f, lens fiber; r, retina; arrowhead,
anterior epithelia; A and B, original
magnification, 200×; C-F, original magnification,
1000×.
|
|
Cultured Lens Epithelial Cells Constitutively and Inducibly Express
ICSBP--
As indicated by our immunolocalization studies, a
significant amount of ICSBP expression occurs in the WT lens epithelia, and there is selective accumulation of the ICSBP protein in the nuclei
of epithelial cells of the TR mouse lens. To confirm these results we
examined well characterized lens epithelial cell lines for ICSBP
expression. Three lens cell lines,
TN4-1, CRLE2, and 1AMLE6 were
therefore treated with mouse IFN
in either the presence or absence
of CHX and analyzed for constitutive or inducible expression of ICSBP.
RNA was isolated from the various treatment groups and used for
Northern analyses. In each of the cell lines, two ICSBP mRNAs of
3.0 and 1.7 kilobases are detected (Fig.
3A), and their sizes are
similar to those of mouse ICSBP transcripts in hematopoietic cells (15,
25). In cells treated with IFN
, a significant increase in ICSBP is
observed, indicating activation of the gene by IFN
. Treatment with
CHX prior to addition of IFN
had no effects on the level of ICSBP
transcripts, suggesting that inducible transcription of lens ICSBP
mRNAs does not require de novo protein synthesis. In
addition, cells that were treated for 2 h with IFN
and
untreated cells were fractionated into cytoplasmic and nuclear
fractions and analyzed for the presence of the ICSBP protein by Western blotting. As indicated in Fig. 3B, the ICSBP protein is
detected in both the cytoplasm and nucleus. However, the ICSBP present in the nucleus migrates faster on SDS/polyacrylamide gel
electrophoresis and appears to be of a lower apparent molecular weight.
Cells that were not treated with IFN
were found to contain more
ICSBP in the cytoplasm than in the nucleus. After treatment with
IFN
, both higher and lower molecular weight ICSBP species are
detected in the cytoplasm. However, the lower molecular weight species present in the nucleus is significantly increased. As shown in Fig.
3B, the ICSBP species detected in the lens cell nucleus
co-migrates on SDS/polyacrylamide gel electrophoresis with the ICSBP of
mouse lymphoid cells. Consistent with previous reports (27), neither mouse spleen cells nor the lymphoma cell line EL4 contain ICSBP in the
cytoplasm.

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Fig. 3.
ICSBP is constitutively and inducibly
expressed in cultured lens epithelial cells. A,
TN4-1, CRLE2, and 1AMLE6 lens epithelial cell lines were cultured
with (lanes 2, 5, and 8) or without
IFN (lanes 1, 4, and 7) or with
IFN and CHX (lanes 3, 6, and 9).
Northern blot analysis (30 µg RNA/lane) was performed and
hybridization was with 32P-labeled ICSBP or -actin
cDNA probe as described under "Experimental Procedures." Sizes
of the respective transcripts are indicated by the arrows to
the right. B, detection of ICSBP by Western blot
analysis. Lens cells cultured in medium with (+) or without ( )
IFN , EL4 mouse lymphoma cells, and purified BALB/c mouse spleen
cells were fractionated into cytosolic (Cyt) or nuclear
(Nuc) fractions and analyzed for ICSBP expression as
described under "Experimental Procedures." kb,
kilobases.
|
|
Constitutive and IFN
-inducible Lens Nuclear Factors Bind to
ICSBP-GAS--
Transcriptional activation of the ICSBP gene by IFN
is mediated by the binding of activated STAT1 homodimers in the nucleus to the conserved cis regulatory palindromic
IFN
-responsive GAS element, pIRE/GAS, present in the ICSBP gene at
positions
147 to
175 (25). We therefore tested by electrophoretic
mobility shift assay whether endogenous lens nuclear factors are able
to bind pIRE/GAS. Analysis performed using nuclear extracts derived from WT mouse lens is shown in Fig.
4A. Two prominent DNA-protein complexes are formed with the pIRE/GAS probe, and formation of the
complexes is competed by the unlabeled probe (Fig. 4A,
lane 3), indicating that the interaction is specific. A DNA
element located in the ICSBP gene at positions
191 to
217 and 22 base pairs upstream from the mouse pIRE/GAS site (25) contains a minimum ISRE motif (GAAANN) resembling the IRE or Pu box. This sequence, referred to as ICSBP-IRE (25), was used as a competitor to
further characterize the ICSBP/GAS binding activities in the lens. The
ICSBP-IRE probe allowed us to detect lens factors that bind to other
DNA elements in the ICSBP promoter besides its GAS site. As shown in
Fig. 4A, the IRE probe competed for the complex labeled
b but not with the a complex (lane 4),
indicating that there are factors in the lens that constitutively
interact with GAS, as well as, non-GAS elements of the ICSBP
promoter.

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Fig. 4.
A, lens nuclear factors interact with
ICSBP GAS element. Lens nuclear extracts where prepared from newborn
CD-1 mouse lenses and electrophoretic mobility shift assay analysis was
performed using the pIRE/GAS (lane 2) as described under
"Experimental Procedures." For the competition assays 100-fold
excess of the unlabeled probe pIRE/GAS (lane 3) or ICSBP-IRE
(lane 4) was incubated with the nuclear extract and labeled
pIRE/GAS probe. DNA-protein complexes (a and b)
are indicated by arrows to the left. Lane
1, labeled probe incubated without nuclear extract. B,
ICSBP-GAS-binding nuclear factors are induced by IFN in lens cells.
Nuclear extracts (10 µg of protein) from TN4-1 lens cells treated
with IFN (lanes 3, 7, 10, and
13) or with IFN and CHX (lanes 4,
8, 11, and 14) or without IFN
(lanes 2, 6, 9, and 12)
were incubated with 32P-labeled ICSBP-GAS (lanes
5-14) or ICSBP-IRE (lanes 1-4) probe. Incubation of
extracts with unlabeled competitor probe, ICSBP-GAS (lanes
12-14) or ICSBP-IRE (lanes 9-11). Lanes 1 and 5, labeled probe incubated without nuclear extract.
IFN -induced protein-DNA complex is indicated as c;
complex formed either in the presence or absence of IFN treatment is
indicated as d.
|
|
To further characterize the interaction between the ICSBP/pIRE and
ICSBP-IRE elements with lens nuclear factors, we analyzed nuclear
extracts derived from the various lens cell lines before and after
treatment with IFN
. Typical results obtained from these studies are
shown in Fig. 4B. Either probe formed a common complex (d) that is competed away by a 100-fold excess of either
probe. Similar to results obtained using lens nuclear extracts,
formation of this complex is also observed in extracts from cells that
were not treated with IFN
(lanes 2 and 6),
confirming that lens nuclear factors constitutively bind to these
elements. An additional retarded band (c) was detected with
the pIRE/GAS probe after treatment of the cells with IFN
(Fig.
4B, compare lanes 6 and 7), suggesting that these DNA binding activities are IFN
-inducible factors. pIRE/GAS-binding factors are also detected in cells treated with IFN
and CHX, indicating that expression of the binding activities does not
require de novo protein synthesis (Fig. 4B,
lane 8). Competition experiments with 100-fold excess of
unlabeled probes revealed that the complex formed with the ICSBP pIRE
motif after induction by IFN
is specific (lanes 13 and
14); the ICSBP-IRE probe is neither able to form the
c complex nor compete in the formation of the c
complex (lanes 10 and 11).
In hematopoietic cells, activation of ICSBP by IFN
is mediated by
STAT1 binding to the pIRE/GAS element. It was therefore of interest to
determine whether the pIRE/GAS binding activity in the lens extracts is
STAT1 and/or any of the other members of the STAT family. Anti-STAT1
antibody supershifted the pIRE/GAS-protein complex (c) (data
not shown). Antibodies specific to STAT2, STAT3, or STAT4 did not
affect the c complex, suggesting that activation of ICSBP
gene transcription in the lens is also mediated by STAT1.
 |
DISCUSSION |
Members of the IRF family of transcription factors differ in the
range of cell types they are normally expressed in, their physiological
inducers, and the distinct biological processes they affect (9). The
IRF proteins identified to date include transcriptional activators
(IRF-1 and ISGF3
), transcriptional repressors (IRF-2 and ICSBP), and
other members (lymphoid-specific IRF, IRF-3, IRF-5, IRF-6, IRF-7, and
vIRF) whose functions are less well understood (1, 9). With the recent
demonstration of a virally encoded homologue of cellular IRFs (29), it
is likely that more IRFs will be identified and that previously
described members would be found to possess new functions. The results
of this study provide the first demonstration that the IRF member, ICSBP, is constitutively and inducibly expressed in the embryonic and
adult mouse lens. Expression of ICSBP in the lens is unequivocally demonstrated at the RNA and protein levels, and its authenticity has
been confirmed by cDNA sequencing. The ICSBP protein and mRNA are also expressed in rat and bovine lenses, suggesting that other mammals constitutively express ICSBP in their lenses.
Constitutive expression of ICSBP is thought to be limited to B
lymphocytes and is not observed in virgin or resting T cells, macrophages, bone marrow, or thymus (15, 27, 30). However, transcription of the ICSBP gene has been shown to be inducible by
either IFN
or antigenic stimulation in T cells and macrophages but
not nonhematopoietic cells (27, 30). As part of the immunologically privileged environment of the anterior chamber of the eye, the avascular adult lens has no interactions with the immune system and
thus would not be expected to come in direct contact with immunological
effector molecules. What then is the functional relevance in the lens
of ICSBP, a transcription factor whose functions are thought to be
restricted to the ontogenesis and regulation of immunological responses
by macrophages and lymphocytes? It is of note that ICSBP expression has
been reported in the chicken (31, 32). The avian protein is expressed
not only in cells of the lymphoid system but also in fibroblasts,
suggesting that the transcriptional regulatory activities of ICSBP may
not be restricted to lymphocytes and hematopoietic cells.
The data presented here suggest that the expression of the ICSBP gene
in the lens may be under regulation by endogenous lens nuclear factors
that constitutively bind cis regulatory DNA elements present
in the mouse ICSBP promoter. The data further reveal that transcriptional activation of the ICSBP gene in lens cells is mediated
by the additional binding of IFN
-activable factors to the GAS
element in the proximal ICSBP promoter. Supershift assays using
antibodies specific to various members of the STAT family of
transcription factors revealed that STAT1 is one of the
IFN
-inducible binding activities. Although our results indicate that
the ICSBP protein in the lens possesses essential characteristics
previously described for ICSBP in hematopoietic cells, electrophoretic
mobility shift assay analyses using ICSBP-pIRE/GAS or ICSBP-IRE probe
reveal subtle differences in the nuclear factors that interact with the ICSBP gene in lymphoid and lens cells. Whereas in EL4 lymphoid cells
specific binding to either the pIRE/GAS or IRE probe was observed only
after treatment with IFN
(25), lens nuclear factors bind to either
element constitutively, as well as, after IFN
treatment.
Furthermore, the IFN
-inducible binding activity in lens cells appear
to be distinct from those in lymphoid cells because the former is
resistant to CHX and consisted of multiple supershifted bands (data not
shown), whereas the latter is sensitive to CHX, consists of a single
band, and requires de novo protein synthesis (25).
Differential sensitivity to CHX of IFN
-inducible factors that
interact with the ICSBP-GAS element suggests that distinct factors
regulate ICSBP gene in different cell types.
In hematopoietic cells, expression of ICSBP is primarily localized in
the nucleus (27). Here we show that ICSBP is present in both the
cytoplasm and nucleus of lens epithelial cells. In fact, in the
unstimulated lens cell, the level of the ICSBP protein in the cytoplasm
is higher than in the nucleus. Our immunolocalization studies on
embryonic WT mouse eye sections further show that the level of ICSBP
expression is significantly higher in the epithelial compartment of the
lens, which exclusively contains undifferentiated, proliferating cells.
Similar to lens epithelial cells in culture, the ICSBP protein is
predominantly localized in the cytoplasm. However, significant nuclear
localization of the ICSBP protein is observed in response to IFN
signaling. This occurs exclusively in the proliferating lens cells but
not in nondividing cells at the lens fiber compartment, suggesting that
response to STAT1 signaling in the lens may be restricted to the lens
epithelia. Interestingly, the ICSBP proteins in the cytoplasmic and
nuclear compartments differ in size. The ICSBP protein in the cytoplasm is of a higher apparent molecular weight, suggesting that nuclear localization of ICSBP is accompanied by post-translational
modification. We are currently examining whether the lower molecular
weight ICSBP species detected in the lens cell nucleus derives from
dephosphorylation of ICSBP or if it is the product of an alternatively
spliced ICSBP transcript.
Previous studies have shown that ICSBP is constitutively phosphorylated
and that phosphorylation events can modulate the ability of ICSBP to
bind DNA; ICSBP can bind DNA either through its association with other
IRFs or directly when it is not tyrosine-phosphorylated (33, 34).
Cytoplasmic expression of ICSBP in the normal lens and differential
regulation of its spatial and subcellular localization in response to
extracellular stimuli suggest that ICSBP may have distinct functions at
different subcellular and/or spatial locations. Thus, the nuclear ICSBP
may function as a transcription factor in the proliferating epithelia,
whereas the ICSBP in the cytoplasm may have regulatory roles through
protein-protein interactions with other IRFs, such as IRF-1 and IRF-2,
which are also present in the cytoplasm of lens
cells.2 It is important to
note that selective localization to either the cytoplasm or nucleus has
also been observed for other lens proteins. For example, the
cyclin-dependent kinase 5, a protein associated with the
elongation and denucleation of differentiating fiber cells, is present
in the cytoplasm of both epithelial and fiber cells of the rat lens.
However, in the final stages of lens differentiation, immediately prior
to denucleation, the cyclin-dependent kinase 5 protein
localizes to the nuclei of fiber cells (35). Furthermore, cyclin B2
localizes to the cytoplasm of proliferating cells in the epithelia and
to the nuclei of primary fibers of the chicken lens (36). Differential
localization of proteins to distinct subcellular compartments may
therefore be an important regulatory mechanism by which specific
biochemical pathways are temporally and spatially segregated in the
vertebrate lens.
Although several studies indicate that ICSBP is primarily a
transcriptional repressor in lymphoid and hematopoietic cells, the
exact mechanisms by which it mediates its regulatory activities on gene
transcription is largely unknown. Much is still to be learned about its
interaction with other IRFs and the cross-talk between members of the
IRF family. The noninnervated and avascular vertebrate lens has
traditionally been found to be a useful in vivo experimental
system for studying the function of several proteins because of its
anatomic position within the eye, an organ that is segregated from the
rest of body, and the fact that it consists of only two cell types of a
common embryonic origin. Our demonstration that ICSBP is constitutively
expressed in the lens therefore provides a unique opportunity to study
its nonimmunity-related functions without many confounding effects
associated with analysis of more complex tissues that are also exposed
to lymphoid and hematopoietic cells. Further studies are needed to
identify IRF-regulated proteins in the lens and how they are regulated
by ICSBP and the other IRFs present in this tissue. It is equally
important to elucidate the functional relevance of nuclear and
cytoplasmic ICSBP and the regulatory mechanisms that may control
its sequestration in the cytoplasm and subsequent translocation
to the nucleus in response to extracellular stimuli.
 |
ACKNOWLEDGEMENTS |
We are grateful to Drs. Peggy Zelenka, Graeme
Wistow, and Jen-Yue Tsai for critical reading of the manuscript; Drs.
Paul Russell and Christina M. Sax for providing the lens epithelial
cell lines; Dr. Samuel Zigler for providing the mouse
A-crystallin-specific antibody; Nicole Newman for preparation of the
histological sections; Ricardo Dreyfus, Shauna Everett, Wayne Randolph,
John Ward, Gary Best, and Hassan Ennaciri for photographic assistance;
and Rashid Mahdi for assistance in DNA and reverse transcribed PCR analysis.
 |
FOOTNOTES |
*
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.
To whom correspondence should be addressed: Lab. of
Immunology, National Eye Institute, 10/10N116, 10 Center Dr., 1858, NIH, Bethesda, MD 20892.
2
W. Li, C. N. Nagineni, H. Ge, B. Efiok,
A. B. Chepelinsky, and C. E. Egwuagu, unpublished data.
 |
ABBREVIATIONS |
The abbreviations used are:
IFN, interferon;
IRF, interferon regulatory factor;
ICSBP, interferon consensus
sequence-binding protein;
WT, wild type;
TR, transgenic;
CHX, cycloheximide;
PCR, polymerase chain reaction.
 |
REFERENCES |
-
Boehm, U.,
Klamp, T.,
and Howard, J. C.
(1997)
Annu. Rev. Immunol.
15,
749-795[CrossRef][Medline]
[Order article via Infotrieve]
-
Taniguchi, T.,
Harada, H.,
and Lamphier, M.
(1995)
L. Cancer Res. Clin. Oncol.
121,
516-520
-
Vaughan, P. S.,
van Wijnen, A. J.,
Stein, J. L.,
and Stein, G. S.
(1997)
J. Mol. Med.
75,
348-359[CrossRef][Medline]
[Order article via Infotrieve]
-
Saunders, N. A.,
and Jetten, A. M.
(1994)
J. Biol. Chem.
269,
2016-2022[Abstract/Free Full Text]
-
Valente, G.,
Ozmen, L.,
Novelli, F.,
Geuna, M.,
Palestro, G.,
Forni, G.,
and Garotta, G.
(1992)
Eur. J. Immunol.
22,
2403-2412[Medline]
[Order article via Infotrieve]
-
Darnell, J. E., Jr.
(1997)
Science
277,
1630-1635[Abstract/Free Full Text]
-
Schindler, C.,
and Darnell, J. E., Jr.
(1995)
Annu. Rev. Biochem.
64,
621-651[CrossRef][Medline]
[Order article via Infotrieve]
-
Decker, T.,
Kovarik, P.,
and Meinke, A.
(1997)
J. Interferon Cytokine Res.
17,
121-134[Medline]
[Order article via Infotrieve]
-
Nguyen, H.,
Hiscott, J.,
and Pitha, P. M.
(1997)
Cytokine Growth Factor Rev.
8,
293-312[CrossRef][Medline]
[Order article via Infotrieve]
-
Sharf, R.,
Azriel, A.,
Lejbkowicz, F.,
Winograd, S. S.,
Ehrlich, R.,
and Levi, B.-Z.
(1995)
J. Biol. Chem.
270,
13063-13069[Abstract/Free Full Text]
-
Miyamoto, M.,
Fujita, T.,
Kimura, Y.,
Maruyama, M.,
Harada, H.,
Sudo, Y.,
Miyata, T.,
and Taniguchi, T.
(1988)
Cell
54,
903-913[Medline]
[Order article via Infotrieve]
-
Harada, H.,
Fujita, T.,
Miyamoto, M.,
Kimura, Y.,
Maruyama, M.,
Furia, A.,
Miyata, T.,
and Taniguchi, T.
(1989)
Cell
58,
729-739[Medline]
[Order article via Infotrieve]
-
Taniguchi, T.
(1997)
J. Cell. Physiol.
173,
128-130[CrossRef][Medline]
[Order article via Infotrieve]
-
Vaughan, P. S.,
Aziz, F.,
van Wijnen, A. J.,
Wu, S.,
Harada, H.,
Taniguchi, T.,
Soprano, K. J.,
Stein, J. L.,
and Stein, G. S.
(1995)
Nature
377,
362-365[CrossRef][Medline]
[Order article via Infotrieve]
-
Driggers, P. H.,
Ennist, D. L.,
Gleason, S. L.,
Mak, W.-H.,
Marks, M. S.,
Levi, B.-Z.,
Flanagan, J. R.,
Appella, E.,
and Ozato, K.
(1990)
Proc. Natl. Acad. Sci. U. S. A.
87,
3743-3747[Abstract]
-
Eisenbeis, C. F.,
Singh, H.,
and Storb, U.
(1995)
Genes Dev.
9,
1377-1387[Abstract]
-
Matsuyama, T.,
Grossman, A.,
Mittrucker, H.-W.,
Siderovski, D. P.,
Kiefer, F.,
Kawakami, T.,
Richardson, C. D.,
Taniguchi, T.,
Yoshinaga, S. K.,
and Mak, T. W.
(1995)
Nucleic Acids Res.
23,
2127-2136[Abstract]
-
Yamagata, T.,
Nishida, J.,
Tanaka, T.,
Sakai, R.,
Mitani, K.,
Yoshida, M.,
Taniguchi, T.,
Yazaki, Y.,
and Hirai, H.
(1996)
Mol. Cell. Biol.
16,
1283-1294[Abstract]
-
Holtschke, E.,
Lohler, J.,
Kanno, Y.,
Fehr, T.,
Giese, N.,
Rosenbauer, F.,
Lou, J.,
Knobeloch, K.,
Gabriele, L.,
Waring, J. F.,
Bachmann, M. F.,
Zinkernagel, R. M.,
Morse, H. C., III,
Ozato, K.,
and Horak, I.
(1996)
Cell
87,
307-317[Medline]
[Order article via Infotrieve]
-
Egwuagu, C. E.,
Sztein, J.,
Chan, C. C.,
Reid, W.,
Mahdi, R.,
Nussenblatt, R. B.,
and Chepelinsky, A. B.
(1994)
Invest. Ophthalmol. Visual Sci.
35,
332-341[Abstract]
-
Egwuagu, C. E.,
Sztein, J.,
Chan, C. C.,
Mahdi, R.,
Nussenblatt, R. B.,
and Chepelinsky, A. B.
(1994)
Dev. Biol.
166,
557-568[CrossRef][Medline]
[Order article via Infotrieve]
-
Yamada, T.,
Nakamura, T.,
Westphal, H.,
and Russell, P.
(1990)
Curr. Eye Res.
9,
31-37[Medline]
[Order article via Infotrieve]
-
Sax, C. M.,
Dziedzic, D. C.,
Piatigorsky, J.,
and Reddan, J. R.
(1995)
Exp. Eye Res.
61,
125-127[Medline]
[Order article via Infotrieve]
-
Sabath, D. E.,
Broome, H. E.,
and Prystowsky, M. B.
(1990)
Gene (Amst.)
91,
185-191[CrossRef][Medline]
[Order article via Infotrieve]
-
Kanno, Y.,
Kozak, C. A.,
Schindler, C.,
Driggers, P. H.,
Ennist, D. L.,
Gleason, S. L.,
Darnell, J. E., Jr.,
and Ozato, K.
(1993)
Mol. Cell. Biol.
3,
3951-3963
-
Ausubel, F. M.,
Brent, R.,
Kingston, R. E.,
Moore, D. D.,
Seidman, J. G.,
Smith, J. A.,
and Stuhl, K.
(1995)
Current Protocols in Molecular Biology, pp. 4.9-4.10, John Wiley & Sons, Inc., New York
-
Politis, A. D.,
Ozato, K.,
Coligan, J. E.,
and Vogel, S. N.
(1994)
J. Immunol.
152,
2270-2278[Abstract/Free Full Text]
-
Schreiber, E.,
Matthias, P.,
Muller, M. M.,
and Schaffner, W.
(1989)
Nucleic Acids Res.
17,
6419[Medline]
[Order article via Infotrieve]
-
Moore, P. S.,
Boshoff, C.,
Weiss, R. A.,
and Chang, Y.
(1996)
Science
274,
1739-1744[Abstract/Free Full Text]
-
Nelson, N.,
Kanno, Y.,
Hong, C.,
Cantursi, C.,
Fujita, T.,
Fowlkes, B. J.,
O'Connell, E.,
Hu-li, J.,
Paul, W. E.,
Jankovic, D.,
Sher, A. F.,
Coligan, J. E.,
Thorton, A.,
Appella, E.,
Yang, Y.,
and Ozato, K.
(1996)
J. Immunol.
156,
3711-3720[Abstract]
-
Junwirth, C.,
Rebbert, M.,
Ozato, K.,
Degen, H. J.,
Schultz, U.,
and Dawid, I. B.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
3105-3109[Abstract]
-
Dosch, E.,
Zoller, B.,
Redmann-Muller, I.,
Nanda, I.,
Schmid, M.,
Viciano-Gofferge, A.,
and Jungwirth, C.
(1998)
Gene (Amst.)
210,
265-275[CrossRef][Medline]
[Order article via Infotrieve]
-
Sharf, R.,
Meraro, D.,
Azriel, A.,
Thornton, A. M.,
Ozato, K.,
Petricoin, E. F.,
Larner, A. C.,
Schaper, F.,
Hauser, H.,
and Levi, B.-Z.
(1997)
J. Biol. Chem.
272,
9785-9792[Abstract/Free Full Text]
-
Bovolenta, C.,
Driggers, P. H.,
Marks, M. S.,
Medin, J. A.,
Politis, A. D.,
Vogel, S. N.,
Levy, D. E.,
Sakaguchi, K.,
Appella, E.,
Coligan, J. E.,
and Ozato, K.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
5046-5050[Abstract]
-
Gao, C. Y.,
Zakeri, Z.,
Zhu, Y.,
He, H.,
and Zelenka, P. S.
(1997)
Dev. Genet.
20,
267-275[CrossRef][Medline]
[Order article via Infotrieve]
-
Gao, C. Y.,
Bassnett, S.,
and Zelenka, P. S.
(1995)
Dev. Biol.
169,
185-194[CrossRef][Medline]
[Order article via Infotrieve]
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