Normalization of hyperosmotic-induced inositol uptake by renal
and endothelial cells is regulated by NF-
B
Mark A.
Yorek1,
Joyce A.
Dunlap1,
Wenli
Liu2, and
William L.
Lowe Jr.2
1 Department of Internal Medicine,
Diabetes-Endocrinology Research Center and Veterans Affairs Medical
Center, University of Iowa, Iowa City, Iowa 52246; and
2 Department of Medicine, Veterans Affairs
Lakeside Medical Center, and Northwestern University Medical
School, Chicago, Illinois 60611
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ABSTRACT |
Hyperosmolarity is a stress factor that
has been shown to cause an increase in the transcription of the
Na+-dependent myo-inositol cotransporter (SMIT).
However, regulation of the reversion of SMIT mRNA levels and
transporter activity following removal of hyperosmotic stress is less
understood. Previously we have shown that postinduction normalization
of SMIT mRNA levels and myo-inositol accumulation following
removal of hyperosmotic stress is inhibited by actinomycin D and
cycloheximide, suggesting that normalization requires RNA transcription
and protein synthesis. We now demonstrate that removal of hyperosmotic
stress causes an activation of the transcription factor NF-
B in
renal and endothelial cells. Inhibiting NF-
B activation with
pyrrolidine dithiocarbamate (PD) blocks the normalization of SMIT mRNA
levels and myo-inositol accumulation on removal of the cells
from hyperosmotic medium. These studies demonstrate that the
downregulation of the myo-inositol transporter following
reversal of hyperosmotic induction is regulated via the activation of
NF-
B.
myo-inositol; hyperosmolarity; nuclear factor
B; sodium-dependent myo-inositol cotransporter
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INTRODUCTION |
MYO-INOSITOL HAS AT LEAST two important
functions in mammalian cells. First, it plays an integral role in
signal transduction pathways by virtue of its incorporation into
phosphoinositides and subsequent release as second messengers on
activation of a phosphoinositide specific phospholipase C or
phosphatidylinositol 3-kinase (3, 12, 23). Second, myo-inositol
is an important osmolyte, serving to protect cells exposed to
hyperosmotic stress (4). This protective role is shared with other
osmolytes such as sorbitol, betaine, taurine, and
glycerophosphorylcholine; however, these osmolytes may differ in their
role as osmotic regulators because of their specific tissue
localization and mechanisms responsible for their
accumulation/metabolism (17, 26).
In most mammalian cells the intracellular concentration of
myo-inositol is maintained at levels many times higher than
circulating concentrations (7, 14). This gradient is regulated and
maintained by a Na+/myo-inositol cotransporter
(SMIT) that is widely expressed in mammalian cells and by an efflux
mechanism, which is poorly understood (8, 13). In mammalian cells,
hyperosmolarity is the most potent means to increase the activity of
the SMIT and, thus, myo-inositol accumulation (21, 25, 27). The
hyperosmolarity-induced increase in myo-inositol transport is
dependent on increased transcription of the SMIT gene (27). Although
osmotic regulation of the organic osmolytes is physiologically
important to renal cells, we and others have shown that hyperosmolarity
also regulates SMIT activity and mRNA levels in endothelial, neural,
and glial cells (15, 18, 21, 24, 25).
It has been shown that, following removal of the hyperosmotic stimulus,
the level of intracellular myo-inositol and
myo-inositol uptake by mammalian cells rapidly returns to
normal (15, 18, 24, 29). In previous studies, we demonstrated that the
reversion of myo-inositol accumulation and SMIT mRNA levels,
once the hyperosmotic stimulus had been removed, was inhibited by
actinomycin D and cycloheximide, suggesting that normalization of SMIT
activity and mRNA levels following hyperosmotic induction requires RNA transcription and protein synthesis (29). In the present study, we show
that removal of the hyperosmotic stimulus causes an activation of
nuclear factor
B (NF-
B) and that activation of this transcription factor is associated with the postinduction normalization of SMIT mRNA
levels and myo-inositol uptake.
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MATERIALS AND METHODS |
Materials. Chemicals were from Sigma (St. Louis, MO) unless
otherwise noted. Ethanol, chloroform, isoamyl alcohol, Corning 75-cm2 flasks, and Falcon six-well plates were from Fisher
Scientific (Fair Lawn, NJ). Phenol was from Bethesda Research
Laboratories (Gaithersburg, MD). Ethidium bromide and proteinase K were
from Boehringer Mannheim (Indianapolis, IN). SDS was from BDH (Poole, England). Pyridine, trimethylchlorosilane, and hexamethyldisilazane were from Pierce (Rockford, IL). Acrylamide, bis-acrylamide, dextran sulfate, and N,N,N',N'-tetramethylethylenediamine
were from Bio-Rad (Hercules, CA). Transcription buffer, dithiothreitol,
RNasin, ATP, CTP, UTP, GTP, T7 and SP6 RNA polymerase, and
deoxyribonuclease were from Promega (Madison, WI).
Myo-[2-3H]inositol,
[
-32P]dATP, and
[32P]UTP were from Amersham (Arlington Heights,
IL). Safety-Solve, cesium chloride, and scintillation vials were from
RPI (Mount Prospect, IL). cDNA probes for the
-actin gene were
obtained from Ambion (Austin, TX). Media were obtained from the
Diabetes Endocrinology Research Center, University of Iowa (Iowa City, IA).
Cell culture. Bovine aortic endothelial (BAE) cells originated
from freshly slaughtered steers and were grown in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 294 µg/ml glutamine as previously described
(8, 13). Rat inner medullary collecting duct (IMCD) cells were kindly
provided by Dr. John Stokes, University of Iowa, and were grown in
DMEM/F-12 medium supplemented with 5% heat-inactivated fetal bovine
serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 294 µg/ml
glutamine as previously described (25, 29). All cells were propagated
in Corning 75-cm2 flasks in an incubator maintained at
37°C with 5% CO2 in humidified air. Cells were passed
weekly at a dilution ranging from 1:10 to 1:20 and fed three times per
week by replacing the medium. For myo-inositol accumulation
studies, the cells were seeded onto Falcon six-well cluster plates, and
assays were conducted in triplicate when the cells reached confluence.
For the SMIT mRNA studies and electrophoretic mobility shift assays,
the cells were seeded in Corning 25 or 75 cm2 flasks.
Myo-inositol accumulation. For these studies IMCD and BAE cells
were exposed to medium containing 150 mM raffinose (490 mosM) for 24 h
and then washed with isotonic medium (~300 mosM) and resuspended in
isotonic medium or this medium containing 100 µM PD for 16 h. Cells
were preincubated with PD for 1 h before resuspension in isotonic
medium. Myo-inositol accumulation was also determined in cells
maintained in isotonic medium or exposed to hyperosmotic medium for 24 h. Myo-inositol accumulation was determined as previously described (25, 29).
Quantification of SMIT mRNA levels. To quantify SMIT mRNA
levels, IMCD and BAE cells were incubated in medium containing 150 mM
raffinose for 24 h and then washed with isotonic medium and resuspended
in isotonic medium or this medium containing 100 µM PD for 6 h. Cells
were preincubated with PD for 1 h before resuspension in isotonic
medium. SMIT mRNA levels were also determined in cells that were
maintained in isotonic medium or hyperosmotic medium for the 24-h
period. SMIT mRNA levels were quantified using a solution
hybridization-RNase protection assay as previously described (25, 29).
Briefly, for the IMCD cells, 32P-labeled antisense SMIT
mRNAs were transcribed using SP6 RNA polymerase and a rat SMIT cDNA
construct in pGEM-3Zf(+) that had been linearized with EcoR I
(26). Due to the high degree of homology of the SMIT gene, the
32P-labeled antisense murine SMIT mRNA probe was found to
hybridize with bovine RNA to give a protected band similar in size to
the protected RNA band derived using RNA prepared from murine cells. Thus, to determine SMIT mRNA levels in BAE cells,
32P-labeled antisense SMIT mRNAs were transcribed using T7
RNA polymerase and a murine SMIT cDNA construct in pGEM-3Zf(+) that had
been linearized with Hind III. Details of the cloning
of the murine and rat SMIT cDNAs have been described previously (25,
26). Antisense SMIT mRNA was incubated at 45°C in 75%
formamide-0.4 M NaCl with 20 µg of total RNA. After 16 h incubation,
the samples were digested with RNases A and T1. The
protected double-stranded hybrids were collected by ethanol
precipitation and electrophoresed through an 8% polyacrylamide-8 M
urea denaturing gel. To confirm equal loading of the gel,
-actin
mRNA levels were determined simultaneously with the use of commercially
available
-actin antisense control templates. The antisense
-actin RNA probes were generated per the manufacturer's
instructions using T7 polymerase. A sufficient quantity of each of the
antisense SMIT mRNA and
-actin probes was added to each sample to
insure the presence of an excess of labeled antisense RNA (25). SMIT
mRNA was represented as a single band on the autoradiogram of the gel,
with the intensity of the band being proportional to the SMIT mRNA
level in the sample. SMIT mRNA levels were quantified by scanning
densitometry of the autoradiogram using a GS 300 transmittance/reflectance scanning densitometer (Hoefer, San Francisco,
CA) interfaced with a model HP 3396A integrator and standardized to the
intensity of the
-actin mRNA band.
Electrophoretic mobility shift assay. IMCD or BAE cells were
incubated in isotonic medium or hyperosmotic medium for 24 h followed
by incubation for 1 h in isotonic medium in the absence or presence of
100 µM PD. Before this experiment, cells induced by hyperosmolarity
were resuspended in isotonic medium for 15-180 min to determine
the time course for activation of NF-
B by reversal of hyperosmotic
induction. Following these incubations, cells were washed and harvested
using PBS at 4°C and low-speed centrifugation. The cells were then
resuspended in 1.5 ml of buffer A (10.0 mM HEPES, pH 8.0, 1.5 mM MgCl2, 10.0 mM KCl, 0.5 mM dithiothreitol, 300 mM
sucrose, 0.1% Nonidet P-40, 1 µg/ml of pepstatin, antipain, chymostatin, and aprotinin, 0.1 µg/ml leupeptin, and 0.5 mM
phenylmethylsulfonyl fluoride) and incubated on ice for 5 min. The
crude nuclear pellet was then collected by microcentrifugation for 2 min at 4°C. Afterward, the pellet was quickly washed with
buffer A and resuspended in buffer B (20 mM HEPES, pH
8.0, 20% glycerol, 100 mM KCl, 100 mM NaCl, 0.2 mM EDTA, 0.5 mM
phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, 1 µg/ml of
pepstatin, antipain, chymostatin, and aprotinin, and 0.1 µg/ml
leupeptin). The isolated nuclei were sonicated for 10 s at 4°C and
clarified by microcentrifugation. Protein concentration of the extract
was determined, and the extract was stored at
70°C until
assayed. For gel mobility shift assays, annealed oligonucleotides
containing the consensus sequence for NF-
B
(5'-TTTCGCGGGGACTTTCCCGCGC-3';
5'-TTTGCGCGGGAAAGTCCCCGCG-3') and the E-box of the
adenovirus major late transcription factor promoter
(5'-ATAGGTGTAGGCCACGTGACCGGGTGT-3';
5'-ACACCCGGTCACGTG-3') were radiolabeled with
[
-32P]dATP and unlabeled dGTP, dCTP, and
dTTP using Klenow DNA polymerase and gel purified as previously
described (30). Fifteen micrograms of nuclear extract protein were
preincubated for 10 min at 25°C with 1-µg
poly(dIdC) · poly(dIdC) under ionic conditions.
Radiolabeled probe (5 × 104 cpm, ~2 ng) was added
to each 20-µl reaction and incubated for 15 min at 37°C. For a
nonspecific control, a 50-fold excess of unlabeled oligonucleotide was
included in some incubations. For supershift analysis, nuclear extracts
from IMCD and BAE cells incubated in control medium, hyperosmolarity
medium, or hyperosmolarity medium followed by incubation in isotonic
medium (reversal) were preincubated for 15 min at room temperature with
1 µg of NF-
B anti-p50 or anti-p65 rabbit polyclonal antibody
(Santa Cruz, CA). Afterward, radiolabeled oligonucleotide was added and
examined as described above. Samples were analyzed on a 5%
nondenaturing polyacrylamide gel in 0.5×
tris(hydroxymethyl)aminomethane-borate-EDTA [45 mM
tris(hydroxymethyl)aminomethane-borate, 1 mM EDTA, pH 8.0] and
electrophoresed at 115 V for 3 h at 25°C. Gels were then dried, and
autoradiographs were exposed for the appropriate period at
70°C with intensifying screens.
Data analysis. Data for myo-inositol accumulation are
reported as nanomoles per milligram cell protein. Significance of
differences was determined by ANOVA and Student's t-test. For
analysis of SMIT mRNA levels, statistical comparisons for significance
were performed using the one-tailed multiple-comparison procedure of Dunnett.
 |
RESULTS |
Activation of NF-
B following postinduction
normalization of hyperosmolarity. Previously we had shown that
normalization of SMIT mRNA levels and increased accumulation of
myo-inositol by mammalian cells exposed to hyperosmotic
conditions were prevented by actinomycin D and cycloheximide (29). This
suggests that turnover of SMIT mRNA levels and activity following
hyperosmotic induction requires RNA transcription and protein
synthesis. Moreover, we have also shown that tumor necrosis factor-
(TNF-
) activates NF-
B and causes a decrease in SMIT mRNA levels
and myo-inositol accumulation by cultured endothelial cells
(30). The effect of TNF-
on SMIT mRNA levels and transport activity
was prevented by various inhibitors of NF-
B activation such as
pyrrolidine dithiocarbamate, genistein, and
N
-p-tosyl-L-lysine chloromethyl ketone
(TLCK) (30). To further examine the regulation of the normalization of SMIT mRNA levels and myo-inositol transporter activity following removal of hyperosmotic induction, we investigated whether the transcription factor NF-
B regulates the postinduction normalization of SMIT mRNA levels and activity. For the present studies, we chose to examine the effect of reversal of hyperosmotic induction on NF-
B activation and SMIT mRNA levels and transporter activity using a cultured renal cell line (rat IMCD cells) and BAE
cells. Less extensive studies were also conducted with murine cerebral
microvessel endothelial cells and murine cortical collecting duct
cells, and results similar to those observed with IMCD and BAE cells
were obtained.
Data in Fig. 1 demonstrate
that exposing IMCD and BAE cells to hyperosmolarity for 24 h causes a
small decrease in NF-
B activity compared with the basal activity of
NF-
B observed in cells maintained in isotonic medium. Transferring
cells from the hyperosmotic medium to isotonic medium causes a
time-dependent activation of NF-
B compared with basal activity.
Within 30-60 min after reversal of the hyperosmotic conditions,
NF-
B is maximally activated. After 60 min, NF-
B activity begins
to decline and approaches basal levels of activity after 180 min (data
not shown). Data in Fig. 2 demonstrate that
DNA protein binding for an unrelated probe containing the consensus
sequence for the E-box of the adenovirus major late transcription
factor promoter was not affected by hyperosmolarity or reversal of the
hyperosmotic condition (30). Data in Fig. 3
show that a 50-fold excess of unlabeled NF-
B oligonucleotide competed for binding of the NF-
B-labeled probe in nuclear extracts prepared from IMCD and BAE cells incubated in control medium, hyperosmotic medium, or hyperosmotic medium followed by isotonic medium
for 1 h. Data in Fig. 4 show that, in
nuclear extracts prepared from IMCD and BAE cells incubated in control
medium, hyperosmotic medium, or hyperosmotic medium followed by
isotonic medium for 1 h, the NF-
B p65 antibody caused a gel
retardation (supershift) in the binding complex (see arrow). In
contrast, the NF-
B p50 antibody had no effect. This suggests that
p65 is the major NF-
B isoform activated by hyperosmotic reversion.
This was the same NF-
B isoform activated by TNF-
in cultured BAE cells (30). In a separate study we found that the activity of the
transcription factor AP-1 was not affected by removing the cells from
hyperosmolarity (data not shown).

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Fig. 1.
Effect of reversal of hyperosmolarity on the activation of NF- B in
inner medullary collecting duct (IMCD) and bovine aortic endothelial
(BAE) cells. To induce hyperosmolarity, cells were incubated for 24 h
in medium containing 150 mM raffinose. Afterward, the cells were
resuspended in isotonic medium for 15-60 min. Control and
hyperosmolarity-conditioned cells were incubated in isotonic medium or
medium containing 150 mM raffinose, respectively, for the entire 24-h
period. After these incubations, nuclear extracts were prepared, and
gel mobility shift assays performed as described in MATERIALS AND
METHODS. Representative autoradiograph from a single experiment
that was repeated at least 4 times with similar results.
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Fig. 2.
Effect of reversal of hyperosmolarity on the electrophoretic mobility
shift assay for the adenovirus major late transcription factor promoter
(E-box) in IMCD and BAE cells. Cells were incubated as described in
Fig. 1. Afterward, nuclear extracts were prepared and gel mobility
shift assays performed as described in MATERIALS AND
METHODS. Representative autoradiograph from a single experiment
that was repeated at least 4 times with similar results.
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Fig. 3.
Competitive electrophoretic mobility shift assay of NF- B. Cells were
incubated as described in Fig. 1. The time point for the reversal
period (isotonic) was selected as 1 h. Nuclear extracts prepared from
these cells were incubated with or without a 50-fold excess of
unlabeled NF- B oligonucleotide. Afterward, the gel mobility shift
assay was performed as described in the MATERIALS AND
METHODS.
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Fig. 4.
Electrophoretic mobility supershift assay of NF- B. Cells were
incubated as described in Fig. 1. The time point for the reversal
period (isotonic) was selected as 1 h. Nuclear extracts prepared from
these cells were preincubated with or without 1 µg of the NF- B
antibody p50 or p65 for 15 min at room temperature. Afterward,
radiolabeled NF- B oligonucleotide probe was added to the incubation
mixture and the gel mobility shift assay performed as described in
MATERIALS AND METHODS. Arrow, the NF- B p65 antibody
caused a gel retardation (supershift) in the binding
complex.
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The activation of NF-
B following removal of IMCD and BAE cells from
hyperosmotic medium to isotonic medium was completely prevented by PD
(100 µM) and to a lesser extent by TLCK (50 µM; Fig.
5). In contrast, genistein (50 µM) and
sulfasalazine (100 µM) were not effective in preventing the
activation of NF-
B when hyperosmotic-conditioned cells were
transferred to isotonic medium.

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Fig. 5.
Effect of reversal of hyperosmolarity in the absence or presence of
genistein (Gen), pyrrolidine dithiocarbamate (PD),
N -p-tosyl-L-lysine chloromethyl ketone
(TLCK), or sulfasalazine (Sul) on the activation of NF- B in IMCD and
BAE cells. Cells were incubated in medium as described in Fig. 1,
except that, for 1 h before the transfer of cells treated with
hyperosmotic medium to isotonic medium, some cells were pretreated with
50 µM genistein, 100 µM PD, 50 µM TLCK, or 100 µM
sulfasalazine. After this 1-h preincubation, cells pretreated with
inhibitors were resuspended in isotonic medium containing the same
concentration of each inhibitor and the incubation continued for 1 h.
Other hyperosmolarity-conditioned cells were resuspended in isotonic
medium alone for 1 h. Control and hyperosmolarity-conditioned cells
were incubated in isotonic medium or medium containing 150 mM
raffinose, respectively, for the entire 24-h period. After these
incubations, nuclear extracts were prepared and gel mobility shift
assays performed as described in MATERIALS AND METHODS.
Representative autoradiograph from a single experiment that was
repeated at least 4 times with similar results.
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Effect of NF-
B activation on SMIT mRNA levels and
myo-inositol transport activity following hyperosmotic postinduction
normalization. Incubating IMCD and BAE cells for 24 h in medium
containing 150 mM raffinose (hyperosmolarity) caused a significant
increase in SMIT mRNA levels (Figs. 6 and
7). Returning IMCD and BAE cells that were
exposed for 24 h to hyperosmotic medium to normal medium for 6 h
(isotonic) resulted in normalization of SMIT mRNA levels. The
normalization of SMIT mRNA levels following hyperosmotic induction and
resuspension in isotonic medium was prevented in part by the addition
of 100 µM PD to the isotonic medium. Exposing control IMCD or BAE
cells for 6 h to isotonic medium containing 100 µM PD had no effect
on SMIT mRNA levels. After 6 h incubation in isotonic medium containing
100 µM PD, SMIT mRNA levels in IMCD and BAE cells were 88 ± 17 and
117 ± 11% of control, respectively. For these studies we focused on
the effects of PD, because studies presented in Fig. 5 demonstrated
that it was most effective in preventing the activation of NF-
B when
hyperosmotic-conditioned cells were transferred to isotonic medium.
Levels of
-actin mRNA were unchanged by these incubation conditions
(Fig. 6).

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Fig. 6.
Effect of reversal of hyperosmolarity in the absence or presence of PD
on Na+-dependent myo-inositol cotransporter (SMIT)
mRNA levels in IMCD and BAE cells. Cells were incubated in medium as
described in Fig. 1, except that, for 1 h before the transfer of
hyperosmolarity-conditioned cells to isotonic medium, one set of cells
were pretreated with 100 µM PD. After this 1-h preincubation, cells
pretreated with PD were resuspended in isotonic medium containing 100 µM PD, and the incubation continued for another 6 h. Other
hyperosmolarity-conditioned cells were resuspended in isotonic medium
alone for 6 h. Control and hyperosmolarity-conditioned cells were
incubated in isotonic medium or medium containing 150 mM raffinose,
respectively, for the entire 30-h period. After these incubations, RNA
was isolated and SMIT and -actin mRNA levels determined as described
in MATERIALS AND METHODS. Representative autoradiograph
from a single experiment that was repeated 6 times.
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Fig. 7.
Effect of reversal of hyperosmolarity in the absence or presence of PD
on SMIT mRNA levels in IMCD and BAE cells. The experiment described in
Fig. 6 was repeated 6 times and the data presented as a percentage of
control with the level of SMIT mRNA in control cells assigned a value
of 100%. SMIT mRNA levels were standardized using -actin mRNA. Each
value is mean ± SE from 6 separate experiments. * P < 0.05, compared with control. + P < 0.05, compared with isotonic treated cells. Hyper, hypertonic; Iso,
isotonic.
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Consistent with the effect of hyperosmotic medium on SMIT mRNA levels,
treatment of IMCD and BAE cells for 24 h with medium containing 150 mM
raffinose (hyperosmolarity) caused a significant increase in
myo-inositol accumulation (Table
1). Returning cells incubated
in hyperosmotic medium for 24 h to isotonic medium for 24 h resulted in
a normalization of myo-inositol accumulation that was prevented
by the addition of 100 µM PD to the isotonic medium. Exposing control
IMCD and BAE cells for 24 h to isotonic medium containing 100 µM PD
had no effect on myo-inositol accumulation.
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Table 1.
Effect of pyrrolidine dithiocarbamate on reversibility of
hyperosmotic-induced myo-inositol accumulation by IMCD and BAE cells
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DISCUSSION |
Cells react to increased osmolarity with numerous changes in gene
expression. The specific genes affected differ between species, but the
known osmoprotective effects of the gene products are remarkably
similar, particularly with regard to cellular accumulation of
compatible organic osmolytes (5). The transcription of the gene
encoding the Na+-dependent myo-inositol
cotransporter (SMIT) is increased by hyperosmolarity, and, following
exposure to hyperosmotic conditions, many cells have been shown to
increase myo-inositol accumulation, suggesting that
myo-inositol is an important organic osmolyte (6, 10, 15, 18,
21, 24, 25, 27). After removal of the hyperosmotic stress, SMIT mRNA
levels and, subsequently, cellular myo-inositol content and
transport rapidly return to normal (15, 18, 24, 25, 29). However, the
process regulating the normalization of SMIT mRNA levels and
myo-inositol transport following removal of hyperosmotic stress
in not well understood. Returning cells from a hyperosmotic medium to
isotonic medium has been shown to activate a myo-inositol
efflux pathway that is Na+ independent and inhibited by
quinidine, quinine, anion transport blockers, and
cis-unsaturated fatty acids (2, 11, 22). In our studies, we
have examined the effect of postinduction normalization of
hyperosmolarity on SMIT mRNA levels and myo-inositol
transporter activity. Previously we have shown that postinduction
normalization of SMIT mRNA levels and myo-inositol accumulation
requires RNA and protein synthesis (29).
We have previously shown that the activation of NF-
B by the cytokine
TNF-
may regulate the expression and transport activity of the SMIT
(28, 30). In studies with cultured BAE cells and 3T3-L1 adipocytes, we
demonstrated that TNF-
activates NF-
B and causes a decrease in
SMIT mRNA levels and myo-inositol accumulation. Blocking the
activation of NF-
B prevented the decrease in SMIT mRNA levels and
myo-inositol accumulation mediated by TNF-
. Therefore, there
is a precedent for mediation by NF-
B of SMIT mRNA levels and
myo-inositol accumulation in cultured cells. This is further supported by the present studies that showed that removal of
hyperosmotic stress was followed by a transient increase in NF-
B
activity and a decrease in SMIT mRNA levels and myo-inositol
accumulation by cultured renal and endothelial cells. However, one
difference between the effect of TNF-
and reversal of hyperosmotic
induction on NF-
B activity is that TNF-
causes a prolonged
activation of NF-
B, whereas the activation of NF-
B by reversal of
hyperosmolarity lasts for only hours. In these studies, we found that
PD was most effective in preventing the activation of NF-
B following
removal of hyperosmotic stress. In these studies, TLCK, a protease
inhibitor, was also effective in blocking the activation of NF-
B
following removal of hyperosmotic stress. The addition of PD also
significantly inhibited the decrease in SMIT mRNA levels and
myo-inositol accumulation following postinduction
normalization. Other compounds that have been described to inhibit
NF-
B activation, including genistein, a tyrosine kinase inhibitor,
and sulfasalazine, were not effective in preventing the activation of
NF-
B and subsequent decrease in SMIT mRNA levels and
myo-inositol accumulation following postinduction normalization
(data not shown). PD has been generally classified as an antioxidant
(20). However, the mechanism for the inhibitory effect of PD on NF-
B
activation has been reported to involve inhibition of binding of the
transcription factor to DNA rather than an effect on the activation
process (16). This mode of inhibition may explain why PD was the more
potent inhibitor of NF-
B activation compared with the other
compounds we tested. This result would also suggest that the activation
of NF-
B by TNF-
and subsequent downregulation of SMIT mRNA levels
and activity and the activation of NF-
B following removal of
hyperosmolarity might be mediated by independent mechanisms.
NF-
B is primarily an activator of gene transcription. Therefore, the
most likely explanation for the downregulation by NF-
B of SMIT mRNA
levels and transporter activity is the regulation by NF-
B of the
expression of proteins that regulate SMIT mRNA stability and/or protein
turnover. Further studies are necessary to determine whether the
3'-untranslated region of the SMIT gene contains AU-enriched
sequences that have been described to be recognition signals for
trans-acting factors (regulatory proteins) that may affect the
half-life of SMIT mRNA (1, 20). Another possible explanation for the
decrease in myo-inositol uptake due to the reversal of
hyperosmotic stress is the activation of either protein kinase A or
protein kinase C. Preston et al. (19) in studies with MDCK cells
demonstrated that activators of protein kinase A or protein kinase C
reduce myo-inositol uptake in cells exposed to either isotonic
or hypertonic conditions. However, there are conflicting reports
regarding the possible role of protein kinase C in the
posttranslational regulation of SMIT activity. Guzman and Crews (9)
have reported that hyperglycemia increases myo-inositol
transport in cultured mesangial cells by a mechanism mediated by the
activation of protein kinase C. Furthermore, we have shown that phorbol
myristate acetate, an activator of protein kinase C, did not affect
myo-inositol uptake by 3T3-L1 adipocytes (28). It has not been
reported whether hyperosmolarity or hypotonicity alters protein kinase
A or protein kinase C activity. Thus it seems unlikely that increased
protein kinase A or protein kinase C activity is responsible for
mediating the effects of reversal of hyperosmolarity on SMIT mRNA
levels and transporter activity. Nonetheless, at this time we can only
state that the downregulation of SMIT mRNA levels and transporter
activity by reversal of hyperosmotic induction parallels the activation
of NF-
B and that inhibition of NF-
B activity prevents the decrease.
In summary, these studies demonstrate that postinduction normalization
of hyperosmotic-induced SMIT mRNA levels and transporter activity is
associated with the activation of NF-
B in cultured renal and
endothelial cells.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-45453, a Diabetes Center grant
from the Medical Research Service of the Department of Veterans Affairs
and Juvenile Diabetes Foundation, and a Merit Review grant from the
Department of Veterans Affairs.
 |
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. A. Yorek, 3 E
17 Veterans Affairs Medical Center, Iowa City, IA 52246 (E-mail:
myorek{at}icva.gov).
Received 5 August 1999; accepted in final form 6 December 1999.
 |
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