Osmotic Response Element Is Required for the Induction of Aldose
Reductase by Tumor Necrosis Factor-
*
Takeshi
Iwata
,
Sanai
Sato§,
Jose
Jimenez
,
Michelle
McGowan
,
Maria
Moroni
,
Anup
Dey¶,
Nobuhiro
Ibaraki
,
Venkat N.
Reddy**, and
Deborah
Carper

From the
Laboratory of Mechanisms of Ocular Diseases,
NEI, the § Laboratory of Ocular Therapeutics, NEI, and the
¶ Laboratory of Molecular Growth Regulation, NICHD, National
Institutes of Health, Bethesda, Maryland 20892, the
Department
of Ophthalmology, Nippon Medical School, 1-1-5, Sendagi, Bunkyo-ku,
Tokyo 113, Japan, and the ** University of Michigan, School of Medicine,
Kellogg Eye Center, Ann Arbor, Michigan 48105
 |
ABSTRACT |
Induction of aldose reductase (AR) was observed
in human cells treated with tumor necrosis factor-
(TNF-
). AR
protein expression increased severalfold in human liver cells after 1 day of exposure to 100 units/ml TNF-
. An increase in AR transcripts
was also observed in human liver cells after 3 h of TNF-
treatment, reaching a maximum level of 11-fold at 48 h. Among the
three inflammatory cytokines: TNF-
, interleukin-1, and
interferon-
, TNF-
(100 units/ml) gave the most induction of AR.
Differences in the pattern of AR induction were observed in human
liver, lens, and retinal pigment epithelial cells with increasing
concentrations of TNF-
. A similar pattern of AR promoter response
was observed between TNF-
and osmotically stressed human liver
cells. The deletion of the osmotic response element (ORE) abolished the
induction by TNF-
and osmotic stress. A point mutation that converts
ORE to a nuclear factor-
B (NF-
B) sequence abolished the osmotic response but maintained the TNF-
response. Electrophoretic gel mobility shift assays showed two NF-
B proteins, p50 and p52, capable
of binding ORE sequence, and gel shift Western assay detected NF-
B
proteins p50 and p65 in the ORE complex. Inhibitors of NF-
B signaling, lactacystin, and MG132 abolished the AR promoter response to
TNF-
.
 |
INTRODUCTION |
Aldose reductase (AR),1
the first enzyme in the sorbitol pathway, catalyzes the reduction of
sugars to alcohols. Human AR is expressed in many tissues, and is
especially high in heart, kidney, brain, and skeletal muscle (1).
Sorbitol accumulation by the action of AR has been suggested to cause
diabetic complications such as cataract, retinopathy, neuropathy, and
nephropathy (2-4). The inhibition of AR by AR inhibitors in diabetic
animal models, such as the galactose-fed rat (5) and the Otsuka
Long-Evans Tokushima Fatty rat (6, 7), has been successful in retarding or preventing such complications.
Accumulation of osmolytes such as sorbitol, myoinositol, betaine, and
glycerophosphorylcholine helps regulate osmotic pressure in renal
medullary cells during antidiuresis. It has been shown that sorbitol
accumulates in renal medulla cells and other cell types when cultured
in hypertonic medium (8, 9). Elevation of AR activity and gene
expression occur under these conditions. Recent reports describe the
involvement of two AR promoter cis-elements, the osmotic
response element (ORE) (10-12), similar to the tonicity-responsive enhancer element originally found in the dog betaine transporter gene
(13), and the aldose reductase enhancer element (14) in regulating
osmotic response and constitutive promoter activities. A signal
transduction study revealed that the p38 and the c-Jun N-terminal
kinase (SAPK/JNK) pathways are not necessary for the transcriptional
regulation of the AR promoter through ORE (15). ORE differs from the
NF-
B binding sequence by one base pair, but no detailed study has
been reported comparing the functional relationship between these two
elements. Recently, it was reported that the tonicity-responsive
enhancer element forms DNA-protein complexes of 200 kDa by
electrophoretic gel mobility shift assay (16).
Tumor necrosis factor-
(TNF-
) is an inflammatory cytokine that
has been classically studied as a molecule central to the pathogenesis
of infectious, inflammatory, and autoimmune diseases. TNF-
is
produced primarily by active macrophage and T-cells in response to
various stimuli. It is functional in both the transmembrane and the
secreted homotrimeric form (17, 18). In obese-induced insulin
resistance (19-25), TNF-
causes the reduction of insulin-stimulated tyrosine phosphorylation of the insulin receptor and its substrate insulin receptor substrate-1 (IRS-1) (26). Insulin-sensitizing agents
such as thiazolidinediones have been shown to reduce the inhibition of
tyrosine phosphorylation of IRS-1 by TNF-
(27). TNF-
also
activates nuclear factor-
B (NF-
B) by phosphorylation of the
NF-
B inhibitor I
B, which releases NF-
B to the nucleus in
homodimer or heterodimer forms (28, 29).
The striking similarity between ORE and the NF-
B binding sequence
has led us to investigate the effect of the activators of NF-
B on AR
transcription. In this paper we report a significant induction of AR by
TNF-
, which is mediated by NF-
B binding to the previously
described ORE. Deletion or mutation of this element has a significant
effect on the response of the AR promoter to both TNF-
and
hyperosmotic stress.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture and Cytokine Treatment--
The human liver cell
line (Chang liver; ATCC, Rockville, MD) was cultured in basal medium
with Earle's BSS (BME; Life Technologies, Inc.), 10% fetal bovine
serum (FBS) (Life Technologies, Inc.), and 50 µg/ml gentamycin (Life
Technologies, Inc.). Human lens cell line SRA01/04 (30) was cultured in
Dulbecco's modified Eagle's medium (Life Technologies, Inc.) with
10% FBS and 50 µg/ml gentamycin. Fourth passage human retinal
pigment epithelial (RPE) cells were cultured in Dulbecco's modified
Eagle's medium with 15% FBS, 50 µg/ml gentamycin, and 0.5 µg/ml
fungizone (Life Technologies, Inc.). TNF-
(Life Technologies, Inc.)
was added to the medium at various concentrations between 20 and 500 units/ml. The medium was replaced every 2 days.
Protein Extraction, SDS-Polyacrylamide Gel Electrophoresis
(PAGE), and Immunoblotting--
Liver cells were washed with
phosphate-buffered saline (Life Technologies, Inc.), and scraped,
pelleted, and resuspended in sodium dodecyl sulfate (SDS) gel loading
buffer (30 mM Tris-HCl, pH 6.8, 5% glycerol, 1% SDS,
2.5%
-mercaptoethanol, 0.05% bromphenol blue) for protein
analysis. Recombinant human muscle AR was obtained from Wako Pure
Chemical Industries Ltd. (Osaka, Japan). SDS-PAGE was performed on a
10% acrylamide gel according to the method of Laemmli (34) using the
Novex El9001-XCELL II Mini Cell system (Novex, San Diego, CA).
Phosphorylase b (94 kDa), bovine serum albumin (67 kDa),
ovalbumin (43 kDa), and carbonic anhydrase (30 kDa) were used as
molecular weight standards (Bio-Rad). Proteins were stained with
Coomassie Blue. For the immunoblotting, proteins were transferred from
the acrylamide gel onto a nitrocellulose membrane (Bio-Rad) using a
Panther semidry electroblotter (Owl Scientific, Cambridge, MA).
Nonspecific binding onto the nitrocellulose membrane was blocked with a
5% nonfat dry milk solution (Kirkegaard & Perry Laboratories,
Gaithersburg, MD) dissolved in PBS for 2 h at room temperature.
The nitrocellulose membrane was then incubated overnight at 4 °C in
PBS containing antisera (200-fold dilution) raised in goat against
purified human placental aldose reductase (35). After three washings
with PBS, the membrane was incubated with biotinylated rabbit IgG
against goat IgG and then with a mixture of avidin and biotinylated
peroxidase using a Vectastain ABC kit (Vector Laboratories, Inc.,
Burlingame, CA). The immunostaining was visualized by peroxidase
reaction with 4-chloro-1-naphthol.
RNA Extraction and Northern Blot Analysis--
Total RNA was
extracted from cells with the RNAzol B RNA isolation kit (Tel-Test,
Friendswood, TX) as described by the manufacturer. Total RNA was
separated on a formaldehyde gel containing 1% agarose and the RNA was
transferred to a Biotrans nylon membrane (ICN Biomedical, Irvine, CA)
for Northern analysis (36). Human AR cDNA (37) and human 18 S
cDNA (Ambion Inc., Austin, TX) were used as probes for
hybridization. Quantitation of relative AR expression was determined
using ImageQuant software (Molecular Dynamics, Sunnyvale, CA) after
normalization to 18 S ribosomal RNA.
Luciferase Promoter Constructs and Luciferase Assay--
The
human AR promoter was isolated from a phage human genomic library
(CLONTECH) using the human cDNA as a probe
(37). The 5' flanking sequence of the gene was sequenced up to
3.7
kilobase pairs (LARK Technologies, Houston, TX). The promoter fragments were amplified by polymerase chain reaction and subcloned into the pGL3
basic luciferase reporter vector (Promega, Madison, WI) and sequenced.
Point mutation, 3-bp mutation, and deletion of ORE in construct 8 were
introduced by primers 5'-GAA AGC ACC AAA TGG GAA ATC ACC GGC ATG G-3',
5'-TTA AAG AAA GCA CCA AAT AAA AAA TCA ACG GCA TGG AGT TTA GAG-3', and
5'-TTT AAA GAA AGC ACC AAA CGG CAT GGA GTT TAG AGA-3', respectively,
using the transformer site-directed mutagenesis kit
(CLONTECH). A normal human liver cell (Chang liver)
was cultured in 35-mm diameter culture wells (Falcon 3046) with 2 ml of
medium. When cells were 60% confluent, 2 µg of luciferase construct
plasmid and 0.2 µg of pSV-
-galactosidase vector were transfected
by the CaCl2 precipitation method (Profection mammalian
transfection system, Promega). After 48 h, six wells per construct
were replaced with fresh medium with three of the wells being
supplemented with TNF-
to a final concentration of 500 units/ml. For
osmotic stress experiments, three wells out of six were replaced by
hypertonic medium (medium supplemented with 150 mM NaCl).
Cells were harvested after 12 h, followed by luciferase assay and
-galactosidase assay according to the manufacturer's instructions
(Dual-Light, Tropix, Bedford, MA). The NF-
B inhibitors, MG132
(BIOMOL Research Laboratories, Plymouth Meeting, PA) (31, 32) and
Lactacystin (BIOMOL Research Laboratories) (33) were added 2 h
prior to the TNF-
treatment at a final concentration of 6 and 20 µM, respectively.
Whole Cell Extract Preparation for Electrophoretic Gel Mobility
Shift Assay (EGMSA)--
Whole cell extracts were prepared as
described previously (38). Human liver cells were cultured in 10-cm
diameter dishes to 90% confluence. The cells were then grown in normal
medium, medium with TNF-
(100 units/ml), or hypertonic medium for 30 min to 6 h. After treatment, cells were pelleted, washed with PBS,
and resuspended in two packed cell volumes of Buffer C (20 mM Hepes, pH 7.9, containing 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 25% v/v glycerol, 2 mM
proteinase inhibitor AEBSF). The cells were snap-frozen in ethanol/dry
ice. Frozen cells were quickly thawed for homogenization and
centrifuged for 1 h at 34,000 × g. The
supernatant was frozen at -70 °C until use for EGMSA or
electrophoretic gel mobility shift Western analysis (EGMSWA).
EGMSA and EGMSWA--
Whole cell extracts (2-6 µg of protein)
were incubated with 0.1 pmol of 32P-labeled oligonucleotide
(105 cpm/reaction mixture) in EGMSA binding buffer (20 mM Tris-HCl (pH 7.5), 1 mM MgCl2,
0.2 mM EDTA, 0.5 mM dithiothreitol, 5% (v/v) glycerol, and 2 µg of poly(dI-dC)) for 30 min at room temperature. The oligomers used were: probe P1, 5'-AAA TGG AAA ATC ACC GGC-3'; probe
P2, 5'-AAA TGG GAA ATC ACC GGC-3'; and probe P3, 5'-AAA TTT AAA AAA ACC
GGC-3'. For the competition experiments, extracts were preincubated at
4 °C for 30 min with a 100-fold molar excess of unlabeled oligomers
prior to the addition of labeled probe. Recombinant NF-
B proteins
p50 and p52 used in EGMSA were purchased from Promega. EGMSWA was
performed by transfer of DNA-protein complexes from a polyacrylamide
gel to a nitrocellulose membrane (Schleicher & Schuell) using a
Trans-Blot SD semi-dry cell (Bio-Rad). The DNA-protein complexes on the
membrane were visualized by staining with a solution of 1% Ponceau S
(Sigma) and 1% acetic acid. Nonspecific binding onto the
nitrocellulose membrane was blocked with a 5% nonfat dry milk solution
(Kirkegaard & Perry Laboratories, Gaithersburg, MD) for 2 h at
room temperature. The nitrocellulose membrane was then separated into
five separate and identical lanes and incubated for 2 h with
antisera (1,000-fold dilution) raised in rabbits against the human
NF-
B proteins p50, p52, p65, Rel-B, and c-Rel (Santa Cruz
Biotechnology, Santa Cruz, CA). After three washings with PBS, the
membrane was incubated with horseradish peroxidase-conjugated anti-rabbit IgG (Santa Cruz). The immunostaining was visualized by the
chemiluminescent detection kit (Phospho-HRP Western detection kit;
Santa Cruz) as described by the manufacturer.
 |
RESULTS |
Induction of AR by TNF-
--
Human liver cells were exposed to
100 units/ml TNF-
from 1 to 5 days. AR protein expression increased
in liver cells as early as 1 day (Fig.
1). This increase was observed throughout
the 5 days of the experiment. Northern blot analysis of human AR in liver cells treated for various times with TNF-
indicates that AR
mRNA increases by 3 h with a maximum 11-fold increase at
48 h (Fig. 2). The effect of
interferon-
, interleukin-1
, and tumor necrosis factor-
(TNF-
) were compared (Fig. 3). TNF-
treatment gave the highest increase in liver cells compared with other
cytokines. No induction was observed with interferon-
treatment.

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Fig. 1.
Western blot analysis of AR expression in
human liver cells exposed to TNF- (100 units/ml) for 1-5 days. A, SDS-PAGE and Coomassie Blue
staining of 7 µg of liver cell proteins and 75 ng of pure human AR
protein. B, Western blot analysis using human AR antibody.
Lane 1, control day 1; lane
2, TNF- day 1; lane 3, TNF- day
3; lane 4, TNF- day 5; lane
5, control day 5; lane 6, purified
recombinant human muscle AR (7.5 ng). Arrow indicates the
position of AR protein. 43 K and 30 K indicate
mobility of molecular size markers (ovalbumin and carbonic anhydrase,
respectively) in kilodaltons.
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Fig. 2.
Northern analysis of AR expression in human
liver cells exposed to TNF- (100 units/ml) for
0-48 h. A, Northern blot of human liver AR. Ten µg of
total RNA was separated on an agarose gel and transferred to a nylon
membrane. Human AR cDNA was used as a probe. B,
quantitation of relative human AR expression by ImageQuant software
(Molecular Dynamics) after normalization to 18 S ribosomal RNA. The
relative expression was calculated by setting control (0 h) at 1. C, Northern blot of human 18 S ribosomal RNA. D,
ethidium bromide staining of total RNA separated on agarose gel. Time
points in hours (h) are indicated. 48C indicates
48-h control.
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Fig. 3.
Northern analysis of AR expression in human
liver cells exposed to different inflammatory cytokines (100 units/ml)
for 12 h. A, Northern blot of human AR. B,
comparison of AR expression relative to control, which was set at 1. C, Northern blot of human 18 S ribosomal RNA. D,
ethidium bromide staining of total RNA separated on agarose gel.
|
|
Northern analyses were performed on liver, lens, and RPE cells treated
for 12 h with various concentrations of TNF-
from 0 to 500 units/ml (Fig. 4). In liver cells, a
linear increase was observed with increasing TNF-
concentrations. In
contrast, lens cells showed no induction until the TNF-
concentration reached 100 units/ml. This elevated level did not change
with 500 units/ml TNF-
. In RPE cells, a sigmoidal pattern of
induction was observed.

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Fig. 4.
Northern analyses of AR expression in liver,
lens, and RPE cells treated with various TNF-
concentrations. Human liver, lens, and RPE cells were
exposed to different concentrations of TNF- (0-500 units/ml) for
12 h. A, Northern blot of human AR. B, AR
induction with increasing TNF- concentrations is indicated as a
ratio based on control (0 units/ml) as 1. C, Northern blot
of human 18 S ribosomal RNA. D, ethidium bromide staining of
total RNA separated on agarose gel.
|
|
Identification of Region Important for Both TNF-
and Osmotic
Response of the Human AR Promoter--
Thirteen reporter constructs
containing sequential deletions of the human AR promoter were
transfected into human liver cells that were treated with TNF-
or
stressed with hypertonic medium for 12 h (Fig.
5). TNF-
treatment increased the AR
promoter activity 3.3-5.5-fold in constructs 1-8
compared with the constitutive activity of the control cells, while
deletion of ORE in construct 9 abolished the TNF-
response. Osmotic
stress increased the promoter activity 4.0-10.8-fold in constructs
1-8 compared with the constitutive activity of the control cells,
while deletion of ORE in construct 9 abolished the osmotic response.
This experiment indicates that the important promoter region for
TNF-
response and osmotic stress response is between constructs 8 and 9.


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Fig. 5.
Comparison of human AR promoter activity in
transfected human liver cells by TNF-
treatment and hyperosmotic stress. A, scheme of 13 luciferase (LUC) reporter constructs of the human AR
promoter between -3.7 kilobase pairs and +31 bp used for transfection
in human liver cells. B, graph of relative luciferase
activity of TNF- -treated cells (black bars)
and control cells (white bars) based on
calculation in C. C, relative luciferase activity
of TNF- -treated and control cells. T/C indicates the
ratio of the promoter activity of TNF- -treated cells over control
cells. D, graph of relative luciferase activity of
osmotically stressed hypertonic (black bars) and
control cells (white bars). E,
relative luciferase activity of osmotically stressed hypertonic cells
and control cells. H/C indicates the ratio of the promoter
activity of osmotically stressed cells over control cells (+).
Bar graphs represent the mean ± standard
deviation (error bar); n = 3. n indicates the number of wells used for this particular
experiment.
|
|
ORE Is Required for the TNF-
Response of the AR
Promoter--
To further characterize the AR promoter region between
constructs 8 and 9, a small deletion was made between sequences -1,160 bp to -1,150 bp of construct 8, which eliminated the entire ORE (Fig.
6). This deletion abolished the TNF-
response and osmotic response of the AR promoter. It also had an
inhibitory effect on the constitutive promoter activity. A point
mutation from adenine to guanine at position -1,157 bp in construct 8 was made to convert ORE to the NF-
B sequence (41). This change
abolished the osmotic response but not the TNF-
response of the AR
promoter. The construct with mutations of two 5' guanines (-1,159 bp,
1,158 bp) and one 3' cytosine (
1,150 bp) had the same effect as the
deletion construct.

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Fig. 6.
The effect of ORE deletion or point mutation
on human AR promoter activity. AR promoter activity was evaluated
using constructs with a deletion of the entire ORE, 3-bp mutation
( 1,159, 1,158, and 1,150 bp), and a point mutation (-1,157 bp)
in reporter construct 8. A, schematic of four luciferase
reporter constructs used for the transfection. Filled
gray box indicates the ORE sequence
(A, constructs 1, 2, and 3). Solid
arrow indicates the location of the mutations to ORE
sequence (A, constructs 2 and 3). B, relative
luciferase activity of the AR promoter in cells exposed to hypertonic
medium or TNF- treatment. White box indicates
control, gray box indicates hypertonic, and
black box indicates TNF- -treated cells.
Bar graphs represent the mean ± standard
deviation (error bar); n = 3. n indicates the number of wells used for this particular
experiment.
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ORE Complex in EGMSA Contains NF-
B
Proteins--
Electrophoretic gel mobility shift Western assays were
performed to determine whether the ORE complexes contain NF-
B
proteins (Fig. 7). EGMSA of ORE was
performed using extracts of human liver cells that had been treated
with TNF-
. The ORE complex was transferred to a nitrocellulose
membrane and Western analysis was performed with NF-
B antibodies
specific for p50, p52, p65, Rel-B, and c-Rel. The p50 antibody reacted
with the fastest migrating ORE complex (Fig. 7, arrow
5), while the p65 antibody reacted with two other ORE
complexes (Fig. 7, arrows 1 and
2).

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Fig. 7.
Identification of
NF- B proteins complexed with ORE. EGMSA
was performed on cell extracts from cells stressed with hypertonic
medium or TNF- exposure, followed by Western blot analysis with
NF- B antibodies to p50, p52, p65, Rel-B, or c-Rel. A,
EGMSA of cell extracts from human liver cells treated with hypertonic
medium or TNF- were mixed with ORE probe. C, control;
O, hypertonic; T, TNF- . Numbered
arrows indicate the complexes formed. B, ORE
complexes formed in cell extracts from TNF- -treated cells were
separated on native PAGE followed by Western blot analysis with NF- B
antibodies. Numbered arrows indicate bands
detected by NF- B antibodies, which match the location of the
complexes detected by EGMSA.
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|
Common ORE complexes were observed between control, osmotically
stressed, and TNF-
-treated cell extracts (Fig. 7A). ORE
complex 1 was observed specifically with TNF-
-treated cell extracts, while ORE complex 2 was observed in the TNF-
-treated and osmotically stressed cell extracts. ORE complex 3 was observed in control and
TNF-
-treated cell extracts. ORE complex 4 was observed only in the
osmotically stressed cell extracts. ORE complex 5 was observed in all
cell conditions.
An additional EGMSA for ORE was performed with commercially available
recombinant pure p50 and p52 proteins (Fig.
8). Probe P1, which contains the wild
type sequence of ORE, bound both NF-
B p50 and p52. Probe P2, which
contains a point mutation converting ORE to the NF-
B sequence at
position -1,157 bp, also bound NF-
B p50 and p52 at the same binding
strength. No binding for probe P3, which contains a random 4-bp
mutation, was observed.

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Fig. 8.
EGMSA of recombinant
NF- B proteins p50 and p52. EGMSA was
performed with two recombinant NF- B proteins (p50, p52) and three
probes. Probe P1, wild type sequence of ORE;
probe P2, point mutation of probe P1 which
converts ORE sequence to NF- B binding sequence; probe
P3, 4-bp mutation of probe P1.
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Inhibition of NF-
B Signaling Abolished TNF-
Response of
AR--
NF-
B signaling inhibitors lactacystin and MG132 were added
to transfected liver cells exposed to TNF-
in order to determine the
response of the human AR promoter. Both inhibitors had no effect on the
constitutive AR promoter activity but abolished the TNF-
response
(Fig. 9).

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Fig. 9.
Effect of NF- B
inhibitors on AR promoter response induced by
TNF- . Lactacystin and MG132 were added to
liver cells 2 h prior to the addition of TNF- at a final
concentration of 20 and 6 µM, respectively.
Open boxes indicate luciferase activity of AR
promoter without TNF- treatment ( ), and filled
boxes indicate with TNF- treatment (+). Bar
graphs represent the mean ± standard deviation
(error bar); n = 3. n
indicates the number of wells used for this particular
experiment.
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 |
DISCUSSION |
In this paper we have reported that the inflammatory cytokine
TNF-
is an inducer of human AR. Recent studies indicate that TNF-
may be involved in obese related diabetic complications (19-25).
TNF-
is higher in obese patients, and it has been shown to block
phosphorylation of IRS-1 (26). AR induction by TNF-
may contribute
to the severity of diabetic complications in obese patients. Further
studies on the relationship between obesity, TNF-
, and severity of
diabetic complications are being investigated.
Induction of AR by TNF-
was observed in several cell types including
human liver, lens, and retinal pigment epithelial cells. The only
difference in AR induction between the cell types was observed with
increasing concentrations of TNF-
, where a linear, threshold, or
sigmoidal response was observed for liver, lens, or RPE cells,
respectively. As observed with liver cells, AR in lens and retinal
pigment epithelial cells was induced the most by TNF-
compared with
treatment with INF-
or interleukin-1
(data not shown).
We (14) and others (10-12) have identified a cis-element
(ORE) required for the osmotic response of AR in several species. This
element is similar to a previously reported tonicity-responsive enhancer element (13) and to an element in the osmoregulating myoinositol transporter gene recently reported by Zhou et
al. (39). The ORE sequence differs by only one nucleotide from the NF-
B binding sequence (40). In our study, we evaluated the relationship between ORE and NF-
B during induction of AR protein and
gene expression. The time required for the up-regulation of AR
transcription by the NF-
B activator TNF-
was similar to the previously described osmotic response (9); however, AR protein expression was much faster for the TNF-
response compared with the
osmotic response. This result suggests the possibility of a common
transcriptional regulation for both TNF-
and osmotic response but a
different regulation for translation of AR.
Thirteen reporter constructs of the human AR promoter were transfected
into human liver cells to determine the critical promoter region for
TNF-
response. Deletion of constructs 8 and 9 totally abolished the
TNF-
response, which shares the same critical region as the osmotic
response. The AR response to TNF-
treatment in all reporter
constructs was similar to those of the osmotic stress, which suggests a
possible common transcriptional mechanism for both responses.
Inhibition of both TNF-
and osmotic response by the deletion of ORE
in construct 8 confirmed the involvement of this element as well as the
importance of this element in both responses. A point mutation in
construct 8, which converts the ORE sequence to the NF-
B sequence,
abolished the osmotic response as previously reported (10) but did not
effect the TNF-
response as shown in this study. This result
indicates that ORE requires adenine instead of guanine at position
-1,157 bp in the human AR promoter for osmotic response, but does not
require this nucleotide for the TNF-
response. Two 5' guanines and
one 3' cytosine in ORE, which match the critical nucleotides of NF-
B
binding sequence, were mutated to confirm the binding of NF-
B
protein. This 3-bp mutation construct had the same effect as the
deletion construct, which not only confirms the binding of NF-
B
protein but also confirms the importance of this binding site. A
summary of the NF-
B/ORE site is shown in Fig.
10. Binding of two commercially available NF-
B proteins, p50 and p52, was not affected by the point
mutation converting ORE to an NF-
B sequence. The evidence for common
and different ORE complexes found between control, osmotic stress, and
TNF-
in EGMSA suggests that a rearrangement of transcription factors
on the ORE are required for the initiation of transcription by
different stimuli. Inhibitors MG-132 and lactacystin used for
suppression of NF-
B activity inhibit proteasome degradation of I
B
when phosphorylated by I
B kinase. The inhibition of TNF-
response
by both inhibitors support the involvement of NF-
B as a signaling
molecule for the TNF-
response of the AR promoter through ORE.

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Fig. 10.
Schematic summary of promoter region
required for TNF- response of human AR.
Bent arrows indicate the position of 5' end of
two luciferase reporter constructs. Underlined sequence
indicates the ORE; vertical arrow shows the
position of the nucleotide that differs between ORE and the NF- B
sequence; and the sequences in the open box show
the possible NF- B sequences (40). Asterisk indicates the
nucleotides mutated for the 3-bp mutation construct (Fig. 6).
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This is the first study to identify specific transcription factor
binding to ORE. Our results clearly indicate that NF-
B proteins p50
and p65 bind to ORE and that ORE responds to multiple signaling for AR
transcription. The similarity of ORE complexes found between osmotic
and TNF-
-treated cells may suggest involvement of NF-
B in the
osmotic response of AR. A recent report showed that osmotic stress of
HeLa cells causes the tumor necrosis factor receptor to cluster on the
surface of the cell membrane (41). Together, these observations suggest
a possible relationship between cytokine and osmotic response in the
cell. However, abolishment of osmotic response by conversion of ORE
sequence to NF-
B sequence rules out the possibility that the NF-
B
transcription factor is directly responsible for ORE activation in
human cells under osmotic stress.
 |
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: NEI, National
Institutes of Health, 9000 Rockville Pike, Bldg. 6, Rm. 232, Bethesda, MD 20892. Tel.: 301-496-2144; Fax: 301-496-1759; E-mail:
debbie{at}helix.nih.gov.
 |
ABBREVIATIONS |
The abbreviations used are:
AR, aldose
reductase;
TNF-
, tumor necrosis factor-
;
IRS-1, insulin receptor
substrate-1;
ORE, osmotic response element;
NF-
B, nuclear
factor-
B;
FBS, fetal bovine serum;
PAGE, polyacrylamide gel
electrophoresis;
PBS, phosphate-buffered saline;
bp, base pair(s);
RPE, retinal pigment epithelial;
EGMSA, electrophoretic gel mobility shift
assay;
EGMSWA, electrophoretic gel mobility shift Western
analysis.
 |
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