 |
INTRODUCTION |
TUMOR NECROSIS FACTOR-
(TNF-
) exerts its effect
on cells by binding to two distinct receptors, TNF-R1 (55 kDa) and
TNF-R2 (75 kDa) (33). These two receptors bind TNF-
with high
affinity but differ in their intracellular domains and mediate distinct cellular responses (33). The type I receptor is thought to confer cytotoxic signals and mediate cell adhesion, whereas the type II
receptor has been linked to mitogenic responses (32, 34). Both receptor
types have been implicated in TNF-
-induced apoptosis (32). Signaling
pathways initiated by TNF-
include the activation of protein kinase
C (PKC), phospholipase A2
(PLA2), phosphatidylcholine phospholipase C, and plasma membrane-associated neutral and endosomal acidic sphingomyelinase (9, 27, 28, 35). TNF-
has also been shown to
induce serine-threonine and tyrosine phosphorylation of various
cellular proteins (11).
In cultured endothelial cells, the major effect of TNF-
is the
stimulation of the transcription of genes encoding the endothelial cell
adhesion molecules E-selectin (endothelial-leukocyte adhesion molecule
1, or ELAM-1), intercellular adhesion molecule 1 (ICAM-1), and vascular
cell adhesion molecule 1 (VCAM-1) (4). The promoter regions of these
genes contain nuclear factor (NF)-
B binding sites, which have been
implicated in the TNF-
-mediated induction of these genes (5).
Increased tissue levels of TNF-
and overexpression of these adhesion
molecules may play an important role in the pathogenesis of some
diseases, such as the proliferative phase of diabetic retinopathy (18).
TNF-
reduces proteoglycan synthesis, which may impair endothelial
barrier function (10), and induces production of reactive oxygen
species, which could increase oxidant-induced injury of the
endothelium, especially in diseases such as diabetes in which
antioxidant pathways are compromised (8). In these studies, we present
another effect of TNF-
on the endothelium. The accumulation of
myo-inositol and its subsequent
incorporation into phosphoinositides are significantly decreased by
TNF-
in large-vessel endothelial cells. The effect of TNF-
persists over an extended period of time and is preceded by a
downregulation of
Na+-myo-inositol
cotransporter (SMIT) mRNA levels. Because the normal metabolism of
myo-inositol is critical to
maintaining phosphoinositide synthesis and because several signal
transduction pathways utilize phosphoinositides, including the
phosphatidylinositol cycle and phosphatidylinositol 3-kinase, TNF-
may alter endothelial cell function by reducing
myo-inositol metabolism.
 |
MATERIALS AND METHODS |
Materials.
Chemicals, neutral red, interleukin (IL)-1
, IL-1
, and IL-2,
genistein, orthovanadate,
N-oleoylethanolamine, staurosporin, 4-bromophenacyl bromide, sphingosine, pyrrolidinedithiocarbamate, 7-amino-1-chloro-3-tosylamido-2-hepatanone (TLCK)
C2-ceramide, and
lipopolysaccharide (LPS) were from Sigma (St. Louis, MO) unless otherwise noted. Insulin-like growth factor I (IGF-I) was from Intergen
(Purchase, NY). IL-6 was from Bachem Bioscience (King of Prussia, PA).
Anti-p50, anti-p65, and anti-cAMP response element binding protein
(CREB) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Chloroform, methanol, isoamyl alcohol,
ethanol, Corning 75-cm2 flasks,
and Falcon six-well plates were from Fisher Scientific (Fair Lawn, NJ).
Sodium dodecyl sulfate was from British Drug House (Poole, UK).
Ethidium bromide and
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) were from Boehringher Mannheim (Indianapolis, IN). Pyridine, trimethylchlorosilane, and hexamethyldisilazane were from
Pierce (Rockford, IL). Transcription buffer, dithiothreitol, RNasin,
ATP, CTP, UTP, GTP, T7 RNA polymerase, and deoxyribonuclease were from
Promega (Madison, WI). The 18S antisense ribosomal RNA probe and
-actin mRNA probe were from Ambion (Austin, TX).
Myo-[2-3H]inositol,
[methyl-3H]choline
chloride,
L-[4,5-3H]leucine,
and [32P]UTP were from
Amersham (Arlington Heights, IL). Safety-Solve, cesium chloride, and
scintillation vials were from Research Products International (Mount Prospect, IL). Recombinant human
TNF-
and transforming growth factor-
(TGF-
) were from Research
& Development Systems (Minneapolis, MN). Wortmannin,
rapamycin, and calphostin C were from Calbiochem (La Jolla, CA).
PD-98059 was a kind gift from Dr. Alan Saltiel, Department of Signal
Transduction, Parke-Davis Pharmaceutical Division (Ann Arbor, MI).
SB-203580 was a kind gift from SmithKline Beecham Pharmaceuticals (King
of Prussia, PA). Cell culture medium was obtained from the
Diabetes-Endocrinology Research Center Cell Biology core, University of
Iowa (Iowa City, IA). Synthetic oligonucleotides used for gel mobility
shift assays were provided by Genosys (Woodlands, TX) through the
Diabetes-Endocrinology Research Center Molecular Biology Core,
University of Iowa.
Cell culture.
Murine MB114 cerebral microvessel endothelial (CME) cells were obtained
from Dr. Steven Moore, University of Iowa. The cells were grown in M199
medium supplemented with 10% heat-inactivated fetal bovine serum, 50 U/ml penicillin, 50 µg/ml streptomycin, and basal medium Eagle amino
acid and vitamin solutions. Bovine aortic endothelial (BAE) cells
originated from freshly slaughtered steers and were grown in
Dulbecco's minimal essential medium supplemented with 10%
heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 µg/ml
streptomycin, and 294 µg/ml glutamine. Bovine pulmonary artery (PA)
endothelial cells were kindly provided by Dr. Robert Bar, University of
Iowa, and were grown in the same medium as described above for the BAE
cells. Endothelial cells from bovine periaortic adipose tissue and
coronary artery were kindly provided by Dr. Robert Bar and were grown
in the same medium as described above for CME cells. Murine
neuroblastoma cells, NB41A3, were obtained from the American Type
Culture Collection and grown in Ham's F-10 medium supplemented with
2.5% heat-inactivated fetal bovine serum, 15% horse serum, 100 U/ml
penicillin, 100 µg/ml streptomycin, and 294 µg/ml glutamine. Human
skin fibroblasts were kindly provided by Dr. Robert Spanheimer,
University of Iowa, and were grown in minimal essential medium with
Earle's salts supplemented with 15% heat-inactivated fetal bovine
serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 294 µg/ml
glutamine, and basal medium Eagle amino acid and vitamin solutions.
Human Hep G2 hepatoma cells were kindly provided by Dr. Jeffrey Fields, University of Iowa, and grown in Eagle's medium supplemented with 10%
heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 µg/ml
streptomycin, and 294 µg/ml glutamine. Murine cortical collecting
duct and rat inner medullary collecting duct cells were kindly provided
by Dr. John Stokes, University of Iowa, and were grown in Dulbecco's
modified Eagle's medium-F-12 supplemented with 5%
heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 µg/ml
streptomycin, and 294 µg/ml glutamine. Rat C6 glioma cells and rat
smooth muscle cells were kindly provided by Dr. Arthur Spector,
University of Iowa. C6 glioma cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 294 µg/ml glutamine.
Rat smooth muscle cells were grown in Dulbecco's minimal essential
medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 294 µg/ml glutamine.
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 were fed
three times per week by replacing the medium. Endothelial cells between
passages 6 and 15 were used in these studies. 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 confluency. For other
studies, cells were seeded in either six-well plates or 25-, 75-, or
150-cm2 flasks. All studies were
conducted when cells reached confluency.
Myo-inositol accumulation and cellular
myo-inositol determination.
For myo-inositol accumulation
determination, cells were incubated in M199 serum-free medium
containing 0.5% bovine serum albumin for 1-24 h in the absence or
presence of 0.01-10 ng/ml TNF-
. Afterwards, the cells were
washed with serum-free medium and then incubated for 10-60 min in
2 ml of serum-free medium containing myo-[2-3H]inositol.
The myo-inositol concentration of the
serum-free medium was 11.4 µM. After the incubation, cells were
quickly washed two times with ice-cold 10 mM HEPES buffer, pH 7.4, containing 128 mM NaCl, 5.2 mM KCl, 2.1 mM
CaCl2, 2.9 mM
MgSO4, and 5 mM glucose and were
collected by scraping the cells in 1.5 ml water. The cell suspension
was sonicated for 5 s, and samples were taken to determine protein
content and myo-inositol accumulation.
Myo-inositol accumulation was
determined by taking duplicate aliquots of the cell suspension and
measuring the radioactivity present using a Beckman LS8100 (Fullerton,
CA) liquid scintillation counter. Protein content was determined in
duplicate aliquots of the cell suspension using a modification of the
Lowry method (17). For many of these studies,
myo-inositol accumulation was
determined after a 1-h incubation. In these studies, the term
"myo-inositol accumulation"
represents the cellular accumulation of radioactivity derived from
myo-[2-3H]inositol
during the incubation and does not represent the initial rate of
myo-inositol transport by the cells,
nor does it account for any
myo-[2-3H]inositol
taken up by the cells and then secreted during the 1-h incubation
period. TNF-
did not cause an increase in the turnover of
radioactivity after metabolic labeling of the cells with
myo-[2-3H]inositol.
Therefore, secretion of
myo-[2-3H]inositol
during the 1-h incubation period likely has little effect on these
results. Besides turnover, the uptake of
myo-inositol over a 1-h period could
be influenced by other factors such as changes in intracellular
metabolism; therefore, myo-inositol
uptake studies using shorter incubation periods as well as kinetic
analysis of high-affinity myo-inositol
transport were conducted to validate the results obtained from studies
using a 1-h incubation period.
Besides TNF-
, the effect of other cytokines and growth factors on
myo-inositol accumulation by cultured
endothelial cells was examined. For these studies, the cells were
incubated in serum-free medium for 16 h in the absence or presence of
IL-1
, IL-1
, IGF-I, IL-2, IL-6, or TGF-
at the concentrations
indicated in the legend of Fig. 4. Afterwards, the
accumulation of myo-inositol was
determined as described above (see Myo-inositol
accumulation and cellular myo-inositol
determination). To examine the effect of
C2-ceramide, sphingosine, or LPS
on myo-inositol accumulation, cells
were incubated in serum-free medium in the absence or presence of
10-100 µM C2-ceramide, sphingosine (50 µM), or 1 µg/ml LPS for 16 h, and, afterwards, the
accumulation of myo-inositol was
examined as described above. In studies examining the effect of
inhibitors on the TNF-
-mediated decrease of
myo-inositol accumulation, the cells
were preincubated for 1 h in serum-free medium in the absence or
presence of each inhibitor at the concentration indicated in Table 2.
Afterwards, 5 ng/ml TNF-
was added, and the incubation was continued
for an additional 15 h. The accumulation of
myo-inositol was then determined as
described above. To examine the combined effect of TNF-
and
hyperosmolarity on myo-inositol
accumulation, cells were preincubated in serum-free medium containing
TNF-
(5 ng/ml) for 1 h before the addition of 150 mM raffinose
[to induce hyperosmolarity (36)]. Other cells were
incubated in serum-free medium alone or this medium containing TNF-
(5 ng/ml) or 150 mM raffinose. After 24 h, the cells were washed, and
myo-inositol accumulation was
determined by incubating the cells for 1 h in osmotically matched HEPES
buffer containing 10 µM
myo-[2-3H]inositol.
To examine myo-inositol accumulation
and incorporation into phosphoinositides, cells were incubated in
serum-free medium containing 5 ng/ml TNF-
for 16 h followed by an
incubation in serum-free medium containing
myo-[2-3H]inositol
for 6 h. Afterwards, samples were taken to determine total
myo-inositol accumulation and protein
content as described above, as well as the amount of
myo2-3H]inositol
incorporated into phosphoinositides. For the latter determination, an
aliquot of the cell suspension (0.5 ml) was extracted with 10 ml of
chloroform-methanol-HCl (2:1:0.015), and the lipid (organic) phase
containing the phospholipid fraction was separated from the aqueous
fraction by the addition of 2 ml of acidic saline and by mixing. The
lower phase was collected in a scintillation vial, and the chloroform
was evaporated in a fume hood followed by the addition of scintillation
solution and determination of the amount of radioactivity present in
the lipid fraction. Myo-inositol
accumulation and incorporation into phosphoinositides were calculated
as nanomoles per milligrams of cell protein. To determine the effect of
TNF-
on Na+-dependent and
Na+-independent
myo-inositol uptake, cells were
incubated in HEPES buffer. For
Na+-independent
myo-inositol uptake determination, the
cells were washed and incubated in this buffer containing choline
chloride in place of NaCl. Kinetic parameters for high-affinity
myo-inositol transport were determined
by incubating cells for 5 min in HEPES buffer with or without NaCl and
containing 5-100 µM
myo-[2-3H]inositol,
as previously described (37). Uptake of
myo-inositol occurring in the absence
of NaCl was subtracted from uptake in the presence of NaCl before
determining the apparent (indicated by the prime)
K'm and maximal
velocity (V'max) for
high-affinity myo-inositol transport
(37). For the latter two studies, cells were treated with or without
TNF-
before the examination of
myo-inositol uptake.
To examine the effect of TNF-
on choline accumulation and
incorporation into phospholipid and leucine accumulation and
incorporation into cell proteins, BAE cells were incubated in
serum-free medium in the absence or presence of TNF-
(5 ng/ml) for
16 h. Afterwards, the cells were incubated for an additional 6 h in
serum-free medium containing either
[methyl-3H]choline
chloride or
L-[4,5-3H]leucine.
The total accumulation of choline or leucine was determined as
described for myo-inositol. Choline
incorporation into phospholipid was determined as described, and
leucine incorporation into cell protein was determined by perchloric
acid (PCA) precipitating the protein from an aliquot of
the cell suspension followed by centrifugation and washing of the
pellet with an additional 10% PCA. Afterwards, the amount of
radioactivity in the cell pellet was determined. For some
studies, the cells were incubated with either
[methyl-3H]choline
chloride or
L-[4,5-3H]leucine
for 15-60 min to examine the effect of TNF-
on choline or
leucine accumulation after a shorter incubation period. As indicated
above, these studies did not examine the effect of TNF-
on the
initial rate of choline or leucine uptake.
To determine the effect of TNF-
on intracellular
myo-inositol content, cells were grown
in 25-cm2 flasks to confluency and
then incubated in serum-free medium in the absence or presence of
TNF-
(5 ng/ml) for 24 h. Afterwards, the cells were washed with
glucose-free HEPES buffer, collected in water, and sonicated. Aliquots
of the cell suspension were taken for protein determination and
derivatization as described previously (36). The derivatized samples
were then chromatographed on a temperature-programmed Hewlett-Packard
5890 gas chromatograph (Palo Alto, CA) interfaced with a model HP3390A
integrator. The initial temperature of 180°C was maintained for 2 min and was then increased at 4°C/min to a final temperature of
225°C, which was maintained for 5 min. The column consisted of 3%
SE-30 on Supelcoport (Supelco, Bellefonte, PA). An authentic
myo-inositol standard was run to
verify its elution time, and methyl
-D-mannopyranoside was added
as an internal standard (36). The
myo-inositol content was calculated as
nanomoles per milligram of cell protein.
Neutral red assay.
A neutral red assay was used to measure cytopathogenicity (7). BAE and
murine CME cells were incubated in serum-free medium in the absence or
presence of TNF-
(5 ng/ml) for 16 h. Afterwards, 3 µl of 1%
neutral red were added, and the cells were incubated for an additional
2 h at 37°C. The medium was then removed by aspiration, and
the cell monolayer was washed three times with HEPES buffer. The
neutral red was extracted with 1 ml of 50% ethanol and 50 mM sodium
citrate, pH 4.2, and absorbance was measured at 540 nm. The data
were calculated as percent of control.
Ceramide assay.
After treatment of BAE or murine CME cells with TNF-
for 5-45
min, the cells were extracted with chloroform-methanol-HCl as
described. To determine ceramide levels, the lipid fraction was
incubated with diacylglycerol kinase as described previously (19). The
major lipid products of the phosphorylation reaction were phosphatidic
acid (from diacylglycerol) and ceramide 1-phosphate (from ceramide).
These products were completely resolved by thin-layer chromatography,
using chloroform-acetone-methanol-acetic acid-water (10:4:2:2:1) as
solvent, and were visualized by autoradiography. The data were
calculated as picomoles of ceramide per nanomoles of lipid phosphorus,
based on a ceramide standard curve generated with
C6-ceramide.
Electrophoretic mobility shift assay.
Confluent cells were treated with 5 ng/ml TNF-
or hyperosmolarity in
serum-free medium for 15 or 30 min. For some studies, cells were
pretreated with genistein (50 µM), TLCK (50 µM), calphostin C (1 µM), or pyrrolidinedithiocarbamate (100 µM) for 1 h before the
addition of TNF-
, and the incubations were then continued for an
additional 30 min. Afterwards, the cells were washed and harvested with
phosphate-buffered saline (PBS) at 4°C and with slow-speed
centrifugation. The cells were resuspended in 4 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 each
pepstatin, antipain, chymostatin, and aprotinin, 0.1 µg/ml leupeptin,
and 0.5 mM phenylmethylsulfonyl fluoride) and left on ice for 5 min.
The crude nuclear pellet was then collected by microcentrifugation for
2 min at 4°C. Afterwards, 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 each
pepstatin, antipain, chymostatin, and aprotinin, and 0.1 µg/ml
leupeptin). The nuclei were sonicated for 10 s at 4°C and clarified
by microcentrifugation. Protein concentration of the extract was
determined using the method of Lowry (17), and the extract was stored
at
70°C. For gel mobility shift assays, annealed
oligonucleotides containing the consensus sequence for NF-
B
(5'-TTTCGCGGGGACTTTCCCGCGC-3';
5'-TTTGCGCGGGAAAGTCCCCGCG-3'), mutant NF-
B
(5'-TTTCG
GG
TTCCCGCGC-3';
5'-TTTGCGCGGG
GT
CGCG-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 or
Taq polymerase and gel purified.
Twenty micrograms of nuclear extract were preincubated for 10 min at
25°C with 1 µg poly(dIdC) · poly(dIdC) under
ionic conditions. For a nonspecific control, a 10- or 50-fold excess of
unlabeled oligonucleotide was included in some incubations.
Radiolabeled probe (5 × 104
counts/min, ~2 ng) was added to each 20-µl reaction and incubated for 15 min at 37°C. 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
80°C with intensifying screens. For supershift analysis,
nuclear extracts from TNF-
-treated BAE or PA endothelial cells were
preincubated for 15 min at room temperature with 1 µg of anti-p50,
anti-p65, or anti-CREB rabbit polyclonal antibodies. Afterwards, the
radiolabeled oligonucleotides were added and examined as described
above.
RNA isolation and gel electrophoresis.
RNA was prepared using the guanidine isothiocyanate-cesium chloride
method. RNA was quantitated by measuring the absorbance at 260 nm, and
the integrity of the RNA and accuracy of quantification were confirmed
by size separating the RNA by denaturing gel electrophoresis and
comparing the intensity of the 18S and 28S ribosomal RNA bands after
ethidium bromide staining of the gel.
Quantification of SMIT mRNA levels.
SMIT mRNA levels in CME cells were quantified using a solution
hybridization-ribonuclease (RNase) protection assay as previously described (36). Briefly,
32P-labeled antisense SMIT mRNAs
were transcribed, using T7 RNA polymerase and a SMIT cDNA construct in
pGEM-3Zf(+) that had been linearized with Hind
III. Antisense SMIT mRNAs were then incubated at 45°C in 75%
formamide-0.4 M NaCl with 20 µg of total RNA. After a 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, 18S ribosomal RNA was determined simultaneously
with the use of a commercially available 18S antisense control template
that binds to an 80-nucleotide fragment from a conserved region of the
18S ribosomal RNA. The antisense 18S RNAs were generated per the
manufacturer's instructions using T7 polymerase. A sufficient quantity
of each of the antisense SMIT mRNA and 18S rRNA probes was added to
each sample to ensure the presence of an excess of labeled antisense RNA. These data were obtained empirically by conducting an RNase protection assay using 20-80 µg of total RNA and a constant
amount of antisense SMIT mRNA or 18S rRNA probe. When the assay was
conducted, with use of an amount of antisense RNA that was similar to
that used in the RNase protection assays, a linear response was seen when up to 80 µg of total RNA was used in the assay. This represents four times the amount of total RNA that was used in the RNase protection assays reported in this study. SMIT mRNAs were 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 (Hewlett-Packard) integrator and standardized to the intensity of
the 18S rRNA band.
Northern blot analysis.
To determine BAE cell SMIT mRNA levels, Northern blot analyses were
conducted as described previously (38). Briefly, RNA was separated by
electrophoresis using a 1% agarose-formaldehyde denaturing gel as
described and blotted to a nylon membrane for 16 h in 20× saline
sodium citrate. After blotting, the nylon membrane was
baked at 80°C for 1.5 h under vacuum. The blot was then subjected to hybridization with a
32P-labeled 626-base pair SMIT
cDNA that was generated using a random primed DNA labeling kit
(Boehringer Mannheim) or a
32P-labeled
-actin genomic DNA
probe, according to previously described procedures (38). After
hybridization, the blot was subjected to a stringent wash procedure
(38). Afterwards, the blot was subjected to autoradiography, and the
level of the SMIT mRNA was quantified by scanning densitometry and
standardized to the intensity of the
-actin mRNA.
Data analysis.
Data for myo-inositol accumulation are
reported as nanomoles per milligrams cell protein or as percent of
control. Statistical comparisons for significance were performed using
an unpaired Student's t-test or
Dunnett's analysis at a P value of
0.05.
 |
RESULTS |
Effect of TNF-
on
myo-inositol accumulation.
Previously we had shown that hyperosmolarity increased
myo-inositol accumulation in cultured
endothelial cells and that the increase in accumulation was preceded by
an increase in SMIT mRNA levels (36). From these studies, we concluded
that exposing cultured endothelial cells to a hyperosmotic medium
increased SMIT gene and protein expression, resulting in an increase in myo-inositol accumulation. The signal
transduction pathway thought to be responsible for mediating some of
the effects of the hyperosmotic response is the mitogen-activated
protein (MAP) kinase family of protein kinases. Exposing mammalian
cells to hyperosmolarity has been shown to activate multiple members of
the MAP kinase family, including the extracellular signal-regulated
kinases (ERK), c-Jun NH2-terminal
kinase (JNK) and p38 kinase (14). Because TNF-
also activates ERK,
JNK, and p38 kinase, we examined the effect of TNF-
on SMIT mRNA
levels and myo-inositol accumulation (14, 24). Shown in Fig. 1 is the effect of
TNF-
(10 ng/ml) on myo-inositol
accumulation by a variety of cultured mammalian cells. Cells were
incubated in serum-free medium in the absence or presence of TNF-
for 16 h. Afterwards, myo-inositol
accumulation was determined by incubating the cells for 1 h in fresh
serum-free medium containing
myo-[2-3H]inositol.
In contrast to the effect of hyperosmolarity, TNF-
significantly
reduced myo-inositol accumulation in
BAE cells (column D), bovine PA
endothelial cells (column E), and
murine CME cells (column H). In
contrast, TNF-
did not affect
myo-inositol accumulation by human
skin fibroblasts (column A), rat
smooth muscle cells (column B),
human Hep G2 cells (column C), rat
C6 glioma cells (column F), murine
neuroblastoma cells (column G),
murine cortical collecting duct cells (column
J), or rat inner medullary collecting duct cells
(column K).
Myo-inositol accumulation was
significantly increased by TNF-
in bovine microvessel endothelial
cells derived from periaortic adipose tissue and bovine coronary artery
endothelial cells (columns I and
L, respectively). The studies reported
here were conducted using serum-free medium; however, when cells were incubated with or without TNF-
in their normal culture medium followed by a determination of
myo-inositol accumulation, similar results were obtained (data not shown).

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Fig. 1.
Effect of tumor necrosis factor- (TNF- ) on
myo-inositol accumulation in cultured
mammalian cells. Cells were grown in 6-well plates until confluency and
then incubated for 16 h in serum-free medium containing 0.5% bovine
serum albumin in the absence or presence of 10 ng/ml TNF- .
Afterwards, cells were washed and incubated for 1 h in serum-free
medium containing 11.4 µM
myo-[2-3H]inositol,
and myo-inositol accumulation was
determined as described (see MATERIALS AND
METHODS). Accumulation of
myo-inositol is expressed as a
percentage of myo-inositol
accumulation in respective control cells, which were not exposed to
TNF- , and is defined here as 100%. Cells used in these studies and
basal accumulation of myo-inositol in
1 h (expressed as nmol/mg protein) by each were as follows:
A, human skin fibroblasts, 1.07 ± 0.09; B,
rat smooth muscle cells, 0.95 ± 0.04; C, human Hep G2
cells, 0.91 ± 0.04; D, bovine aorta endothelial (BAE)
cells, 0.56 ± 0.06; E, bovine pulmonary artery (PA)
endothelial cells, 0.48 ± 0.03; F, rat C6 glioma cells,
1.03 ± 0.09; G, murine neuroblastoma cells, 1.00 ± 0.09; H, murine cerebral microvessel endothelial (CME)
cells, 0.62 ± 0.06; I, bovine periaortic adipose
microvessel endothelial cells, 1.83 ± 0.27; J, murine
cortical collecting duct cells, 0.89 ± 0.18; K, rat
inner medullary collecting duct cells, 0.87 ± 0.09; and
L, bovine coronary artery endothelial cells, 0.21 ± 0.07. Each value is mean ± SE of 9 separate experiments.
* Significant decrease (P < 0.05) compared with control;
# significant increase
(P < 0.05) compared with control.
|
|
The concentration curve and time course dependence for the
TNF-
-mediated decrease in
myo-inositol accumulation were
examined in murine CME, BAE, and bovine PA endothelial cells (Fig.
2). These studies demonstrated that the
effect of TNF-
on myo-inositol accumulation was dose dependent and that a maximum inhibition of
myo-inositol accumulation was achieved
with 5 ng/ml TNF-
. The response to TNF-
was also time dependent.
A 6-h incubation with 5 ng/ml TNF-
was sufficient to cause a maximal
inhibition of myo-inositol
accumulation in murine CME and bovine PA endothelial cells, whereas a
24-h incubation was necessary for BAE cells. This variability in time
dependence is likely due to the different turnover rates of the SMIT
protein in each cell type. To validate the results obtained using a 1-h
incubation period with
myo2-3H]inositol,
we examined the effect of TNF-
on
myo-inositol uptake by murine CME,
bovine PA endothelial, and BAE cells after a 10-60 min incubation
(Fig. 3). Even though
myo-inositol accumulation by the cells
over this time period was not precisely linear, the results clearly
demonstrate that myo-inositol
accumulation is significantly decreased by TNF-
.

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Fig. 2.
Concentration- and time-dependent decrease in
myo-inositol accumulation by TNF- .
Murine CME, BAE, and bovine PA endothelial cells were grown in 6-well
plates and then exposed to various concentrations of TNF- (0-10
ng/ml) (A) or to 5 ng/ml TNF- for
1-24 h (B).
Myo-inositol accumulation was then
determined as described in MATERIALS AND
METHODS. Myo-inositol
accumulation is expressed as percentage of control, which is defined as
100%. Basal myo-inositol accumulation
by murine CME, BAE, and bovine PA endothelial cells after a 1-h
incubation was 0.48 ± 0.02, 0.31 ± 0.03, and 0.32 ± 0.03 nmol/mg protein, respectively. Each value is the mean ± SE of 9 separate experiments.
* Myo-inositol accumulation
significantly decreased (P < 0.05)
compared with control.
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Fig. 3.
Effect of TNF- on myo-inositol
accumulation. Murine CME (A,
n = 6), bovine PA endothelial
(B, n = 6), and BAE (C,
n = 9) cells were grown in 6-well
plates to near confluency and then incubated for 16 h in serum-free
medium in absence ( ) or presence (+) of TNF- (5 ng/ml).
Myo-inositol accumulation, expressed
as nmol/mg protein, was then determined in triplicate after a 10- to
60-min incubation as described.
* Myo-inositol accumulation
significantly decreased (P < 0.05)
compared with control.
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To determine the specificity of the effect of TNF-
on
myo-inositol accumulation, we examined
the effect of growth factors and other cytokines on
myo-inositol accumulation by bovine PA endothelial, BAE, and murine CME cells (Fig.
4). After a 16-h incubation in serum-free
medium, TNF-
(5 ng/ml, condition
1), IL-1
(10 ng/ml, condition
3) and TGF-
(5 ng/ml, condition
7) significantly decreased
myo-inositol accumulation by bovine PA endothelial and BAE cells.
Myo-inositol accumulation by murine CME cells was reduced by only TNF-
. In contrast, IGF-1 (50 ng/ml, condition 2), IL-1
(10 ng/ml,
condition 4), IL-2 (10 ng/ml,
condition 5), and IL-6 (10 ng/ml,
condition 6) did not alter
myo-inositol accumulation.

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Fig. 4.
Effect of cytokines and growth factors on
myo-inositol accumulation. Bovine PA
endothelial, BAE, or murine CME cells were grown in 6-well plates to
near confluency and then incubated for 16 h in serum-free medium in
absence or presence of TNF- (5 ng/ml, condition
1), insulin-like growth factor I (50 ng/ml,
condition 2), interleukin (IL)-1
(10 ng/ml, condition 3), IL-1 (10 ng/ml, condition 4), IL-2 (10 ng/ml,
condition 5), IL-6 (10 ng/ml,
condition 6), or transforming growth
factor- (5 ng/ml, condition 7).
Myo-inositol accumulation was then
determined as described in MATERIALS AND
METHODS. Myo-inositol
accumulation is expressed as percentage of control, which is defined as
100%. Each value is mean ± SE of 6 separate experiments.
* Myo-inositol accumulation
significantly decreased (P < 0.05)
compared with control.
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Determination of the effect of TNF-
on
Na+-dependent
myo-inositol uptake by BAE cells
showed that TNF-
had no effect on
Na+-independent
myo-inositol uptake.
Na+-independent and
Na+-dependent
myo-inositol uptakes by BAE cells
after a 10-min incubation period in buffer containing 10 µM
myo-inositol were 0.017 ± 0.006 and 0.143 ± 0.007 nmol/mg protein, respectively (mean ± SE,
n = 6). After treatment for 16 h with
5 ng/ml TNF-
, Na+-independent
and Na+-dependent
myo-inositol uptakes were 0.014 ± 0.002 and 0.071 ± 0.007 nmol/mg protein
(P < 0.05), respectively. Similar
results were obtained with bovine PA endothelial and CME cells (data
not shown). Because the above studies (Figs. 1-4) did not address
the effect of TNF-
on the initial rate of
myo-inositol uptake, kinetic analysis
of the effect of TNF-
on high-affinity
myo-inositol transport was conducted.
These studies demonstrated that TNF-
caused a significant decrease
in V'max with no change in K'm.
K'm for
high-affinity myo-inositol transport
by BAE cells treated with and without TNF-
for 16 h was 60.3 ± 4.8 and 65.2 ± 12.0 µM, respectively, and V'max was 210.7 ± 12.5 and 143.8 ± 15.2 pmol · mg
protein
1 · min
1,
respectively (mean ± SE, n = 3) (P < 0.05). Studies
conducted with bovine PA endothelial cells gave similar results (data
not shown).
Because phosphoinositide production is linked to
myo-inositol uptake, we examined the
effect of TNF-
on myo-inositol
accumulation and incorporation into phosphoinositides in BAE cells.
After a 6-h incubation, myo-inositol
accumulation and incorporation into phosphoinositides were decreased by
60 and 40%, respectively, in cells treated with 5 ng/ml TNF-
for 16 h (Table 1). The free myo-inositol content was also
significantly decreased after a 24-h incubation in serum-free medium
containing 5 ng/ml TNF-
.
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Table 1.
Effect of TNF- on myo-inositol accumulation and
incorporation into phospholipids and intracellular myo-inositol content
in bovine aorta endothelial cells
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To determine whether the TNF-
-induced decrease in
myo-inositol accumulation is due to an
increase in myo-inositol efflux, BAE
cells were prelabeled with
myo-[2-3H]inositol
for 16 h and were then washed and incubated in the absence or presence
of TNF-
for up to 24 h. At 1, 3, 6, and 24 h, samples were collected
to determine the amount of
myo-[2-3H]inositol
remaining in the cells and appearing in the medium. Results from this
study demonstrated that myo-inositol
efflux was not increased by TNF-
and that >60% of the
myo2-3H]inositol
taken up by the cells during the pulse period remained cell associated
after 24 h (data not shown).
Recovery of myo-inositol accumulation.
Data in Fig. 5 show the recovery of
myo-inositol accumulation by cells
preincubated in serum-free medium containing TNF-
(5 ng/ml) for 16 h
followed by a 24-h incubation in serum-free medium alone for murine
CME, BAE, and bovine PA endothelial cells. Myo-inositol accumulation was
significantly improved in cells exposed to TNF-
after a 24-h washout
or recovery period compared with TNF-
-treated cells; however,
myo-inositol accumulation by the
reverted cells remained significantly decreased compared with control
cells. The recovery ranged from 20 to 75% after the 24-h incubation.

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Fig. 5.
Reversion of effect of TNF- on
myo-inositol accumulation by murine
CME, BAE, and bovine PA endothelial cells. Cells were grown in 6-well
plates and then incubated for 16 h in serum-free medium (control) or
same medium containing 5 ng/ml TNF- . Afterwards, some cells
incubated in medium containing TNF- were washed and then incubated
for 24 h in normal serum-free medium.
Myo-inositol accumulation was then
determined by incubating cells for 1 h in serum-free medium containing
myo-[2-3H]inositol.
Myo-inositol accumulation is expressed
as nmol/mg protein. Each value is mean ± SE of 6 separate
experiments. * Myo-inositol
accumulation significantly decreased
(P < 0.05) compared with control;
+ myo-inositol
accumulation significantly increased
(P < 0.05) compared with
TNF- -treated cells.
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Effect of TNF-
on choline and leucine accumulation.
To determine whether the effect of TNF-
on
myo-inositol accumulation was specific
or whether TNF-
affected the accumulation of other molecules
required for membrane or protein synthesis, we examined the effect of
TNF-
on choline accumulation and incorporation into phospholipid and
L-leucine accumulation and
incorporation into cellular proteins. For these studies, BAE or murine
CME cells were incubated in serum-free medium in the absence or
presence of TNF-
(5 ng/ml) for 16 h followed by a 6-h
incubation in serum-free medium containing
[methyl-3H]choline
chloride or
L-[4,5-3H]leucine.
Results from these studies showed that choline accumulation and
incorporation into phospholipid and
L-leucine accumulation and
incorporation into cellular proteins were not altered by TNF-
(data
not shown). In other studies, we examined the accumulation of choline
and leucine over a 15- to 60-min incubation period after
treatment of BAE, bovine PA endothelial, or CME cells with or without
TNF-
for 16 h. These studies also indicated that TNF-
did not
alter choline or leucine accumulation (data not shown). Therefore, the TNF-
-induced decrease in
myo-inositol accumulation is
apparently not due to a general defect in the cellular uptake of
phospholipid or protein synthesis substrates. However, it must be noted
that these studies did not examine the effect of TNF-
on the initial
rate of choline or leucine uptake by the cells.
It is unlikely that the TNF-
-induced decrease in
myo-inositol accumulation is a
secondary event due to TNF-
-induced apoptosis (12, 23). In a study
using neutral red, no significant difference between the uptake of the
dye by normal or TNF-
-treated cells was observed. BAE cells treated
with 0.1, 0.5, or 5.0 ng/ml TNF-
for 16 h absorbed 113 ± 9, 84 ± 9, and 79 ± 11% (P > 0.30) of the dye compared with normal cells. In addition, there was no change in the amount of neutral red absorbed by murine CME cells treated with 5 ng/ml TNF-
compared with control cells (data not shown).
Effect of TNF-
and/or hyperosmolarity on
myo-inositol accumulation.
Previously, we had shown that exposing cultured endothelial cells to
hyperosmotic conditions by the addition of 150 mM raffinose (~490
mosM) to serum-free medium for 6-24 h caused an increase in
myo-inositol accumulation that was
preceded by an increase in SMIT mRNA levels (36). Because TNF-
and
hyperosmolarity apparently have contrasting effects on
myo-inositol accumulation in cultured
endothelial cells, we examined the effect of TNF-
on the
hyperosmolarity-induced increase in
myo-inositol accumulation. For these
studies, murine CME, BAE, or bovine PA endothelial cells were exposed
to serum-free medium containing 5 ng/ml TNF-
(T), 150 mM raffinose
(R) or a combination of the two (T + R) for 16 h. The cells
receiving TNF-
and raffinose (T + R) were preincubated with TNF-
for 1 h before the addition of raffinose. Afterwards, myo-inositol accumulation
was determined by incubating the cells for 1 h in osmotically matched
buffer containing 10 µM
myo-[2-3H]inositol.
Figure 6 shows that hyperosmolarity (R)
increased myo-inositol accumulation
compared with control in all three types of endothelial cells, whereas
TNF-
caused a significant decrease in
myo-inositol accumulation. The
addition of TNF-
to the hyperosmotic medium reduced the
hyperosmolarity-induced increase in
myo-inositol accumulation by
30-50%.

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Fig. 6.
Effect of TNF- , hyperosmolarity, or combination of TNF- and
hyperosmolarity on myo-inositol
accumulation. Murine CME, BAE, and bovine PA endothelial cells were
grown in 6-well plates and then incubated for 16 h in serum-free medium
(C) or same medium containing TNF- (T, 5 ng/ml), 150 mM raffinose
(R, ~490 mosM) or combination of TNF- and 150 mM raffinose (T + R). Osmolarity of isotonic medium was ~310 mosM. Cells that received
combination of TNF- and 150 mM raffinose were preincubated for 1 h
with TNF- before addition of raffinose.
Myo-inositol accumulation was then
determined by incubating cells for 1 h in osmotically matched HEPES
buffer containing 10 µM
myo-inositol.
Myo-inositol accumulation is expressed
as nmol/mg protein (prot). Each value is mean ± SE of 9 separate
experiments. * Myo-inositol
accumulation significantly decreased
(P < 0.05) compared with C;
+ myo-inositol
accumulation significantly increased
(P < 0.05) compared with C;
# myo-inositol
accumulation significantly decreased
(P < 0.05) compared with
hyperosmotic treated cells (R).
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Effect of inhibitors, LPS, C2-ceramide,
and sphingosine on myo-inositol
accumulation.
Incubation of BAE cells with TNF-
(5 ng/ml) for 10 min caused a
fourfold increase in ceramide levels [75.8 ± 6.7 (control cells) to 318.9 ± 28.7 pmol/nmol lipid phosphorus, (TNF-
-treated cells) P < 0.05, n = 6]. Ceramide levels were
also significantly increased by about twofold in CME cells (data not
shown). To determine whether the generation of ceramide was responsible
for the TNF-
-induced decrease in
myo-inositol accumulation, we examined
the effect of C2-ceramide on
myo-inositol accumulation by murine
CME and BAE cells and compared it to the effect of TNF-
. For these
studies, the cells were incubated for 16 h in serum-free medium
containing 10-100 µM
C2-ceramide or 5 ng/ml TNF-
.
Afterwards, myo-inositol accumulation
was determined by incubating the cells for 1 h in serum-free medium
containing
myo-[2-3H]inositol.
Data in Fig. 7 demonstrate that
C2-ceramide, in a concentration-dependent fashion, caused a significant decrease in
myo-inositol accumulation by murine
CME and BAE cells. These results suggest that ceramide production may
be mediating the effect of TNF-
on
myo-inositol accumulation. In contrast
to the effect of C2-ceramide on
myo-inositol accumulation, incubating BAE, bovine PA endothelial, or CME cells for 16 h in serum-free medium
containing sphingosine (50 µM) inhibited
myo-inositol accumulation by <10%
in bovine PA endothelial and CME cells and by <15% in BAE cells
(data not shown). None of these changes achieved statistical significance.

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Fig. 7.
Effect of C2-ceramide on
myo-inositol accumulation. Murine CME
or BAE cells were grown in 6-well plates and then incubated for 16 h in
serum-free medium (control) or same medium containing 10-100 µM
C2-ceramide or 5 ng/ml TNF- .
Myo-inositol accumulation was then
determined by incubating cells for 1 h in serum-free medium containing
myo-[2-3H]inositol.
Myo-inositol accumulation is expressed
as nmol/mg protein. Each value is mean ± SE of 9 separate
experiments. * Myo-inositol
accumulation significantly decreased
(P < 0.05) compared with
control.
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As mentioned above, TNF-
activates a variety of signal transduction
pathways, including PKC, PLA2, and
protein phosphorylation of serine-threonine and tyrosine residues (9,
11, 27, 28, 35). To determine the signal transduction pathway(s)
responsible for the TNF-
-induced inhibition of
myo-inositol accumulation, we examined
the effect of various inhibitors of these signal transduction pathways
and inhibitors of phosphorylation, protease degradation, and
phosphatases on the TNF-
-induced inhibition of
myo-inositol accumulation in BAE cells
(Table 2). In many cells, the effect of
TNF-
is mediated through the activation of the transcription factor
NF-
B, which is activated by the degradation of I-
B protein (4).
Therefore, we also examined the effect of inhibitors of NF-
B
activation on the TNF-
-induced inhibition of
myo-inositol accumulation in BAE
cells. Inhibitors of ERK1 (PD-98059), p38 kinase (SB-203580), p70 S6
kinase (rapamycin), or phosphatidylinositol 3-kinase (wortmannin) did
not affect the TNF-
-induced decrease in
myo-inositol accumulation.
Pyrrolidinedithiocarbamate, an inhibitor of NF-
B activation, blocked
the TNF-
-induced decrease in
myo-inositol accumulation. TLCK, a
protease inhibitor used to block I-
B-
degradation, prevented the
TNF-
-induced decrease in
myo-inositol accumulation. Genistein,
a protein tyrosine kinase inhibitor used to block the phosphorylation
of I-
B-
, partially blocked the TNF-
-induced decrease in
myo-inositol accumulation.
Orthovanadate, a protein tyrosine phosphatase inhibitor, did not affect
the TNF-
-induced decrease in
myo-inositol accumulation. Activation
of PKC-
has been reported to regulate the binding of NF-
B to DNA
in nuclear extracts, possibly through the phosphorylation and
subsequent degradation of I-
B-
(26). Calphostin C has been shown
to inhibit PKC-
, whereas staurosporine, a nonspecific inhibitor of
PKC, does not inhibit PKC-
(26). Consistent with these observations, calphostin C, but not staurosporine, partially inhibited the effect of
TNF-
on myo-inositol accumulation.
N-oleoylethanolamine and 4-bromophenacyl bromide, inhibitors of ceramidase and
PLA2 activity, respectively, had
no effect on the TNF-
-induced decrease in
myo-inositol accumulation. LPS
mimicked the effect of TNF-
on
myo-inositol accumulation, and their
combined effects were not additive.
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Table 2.
Myo-inositol accumulation by bovine aortic endothelial cells: effect of
lipopolysaccharide and inhibitors of kinases, phosphatases,
NF- B activation, phospholipase A2, and
ceramidase on TNF- activity
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SMIT mRNA levels.
Given our previous results, which stated that the effect of
hyperosmolarity on myo-inositol
accumulation occurred at the level of SMIT gene expression (36), we
next examined the effect of TNF-
on SMIT mRNA levels. Data in Fig.
8 demonstrate the effect of
hyperosmolarity, TNF-
, and
C2-ceramide on SMIT mRNA levels in
murine CME cells. As previously reported, hyperosmolarity stimulated a
large increase in SMIT mRNA levels compared with the level in control
cells incubated in isotonic medium (36). In contrast, treatment with
TNF-
and C2-ceramide
significantly decreased SMIT mRNA levels by 50-70%. The insert
shows a representative autoradiograph of SMIT mRNA and 18S rRNA.
Examination of the time course of the effect of TNF-
and
C2-ceramide on SMIT mRNA levels in
murine CME cells demonstrated that a maximum decrease in SMIT mRNA
levels occurred after a 6-h incubation with TNF-
or
C2-ceramide (data not shown).
Analysis of SMIT mRNA levels in BAE cells by Northern blot analysis
demonstrated that SMIT mRNA levels in cells exposed to TNF-
(5 ng/ml) for 16 h were reduced to 20% of control levels, whereas SMIT
mRNA levels in cells exposed to hyperosmotic medium for 16 h were
increased over 10-fold, after correction for
-actin mRNA levels
(Fig. 9).

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Fig. 8.
Effect of hyperosmolarity, TNF- , and
C2-ceramide on
Na+-myo-inositol
cotransporter (SMIT) mRNA levels in murine CME cells. Cells were grown
in 75-cm2 flasks to confluency and
then incubated for 6 h in either serum-free medium or medium containing
TNF- (5 ng/ml), C2-ceramide (50 µM), or 150 mM raffinose (~490 mosM). Afterwards, RNA was isolated
and SMIT mRNA levels were determined as described in
MATERIALS AND METHODS. SMIT mRNA
levels were standardized using 18S rRNA levels. Data are presented as a
percentage of control, with level of SMIT mRNA in control cells
assigned a value of 1. Each value is mean ± SE of a minimum of 5 separate determinations. Inset shows a representative
autoradiograph of levels of SMIT mRNA and 18S rRNA. * SMIT mRNA
levels significantly decreased (P < 0.05) compared with control,
# SMIT mRNA levels
significantly increased (P < 0.05)
compared with control.
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Fig. 9.
Northern blot analysis of BAE cell SMIT mRNA levels: effect of TNF-
and hyperosmolarity. Cells were grown in
75-cm2 flasks to confluency and
then incubated for 16 h in serum-free medium or same medium containing
TNF- (5 ng/ml) or 150 mM raffinose (~490 mosM). RNA was isolated,
and SMIT and -actin mRNA levels were determined as described in
MATERIALS AND METHODS.
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Electrophoretic mobility shift assay.
The above data suggest that the effect of TNF-
on
myo-inositol accumulation is
associated with the activation of NF-
B. To determine whether TNF-
activated NF-
B in BAE and bovine PA endothelial cells, the ability
of TNF-
to stimulate NF-
B binding to its consensus DNA binding
site was examined. Data in Fig. 10
demonstrate that a 15- or 30-min incubation with TNF-
(5 ng/ml)
increased NF-
B in nuclear extracts from BAE or bovine PA endothelial
cells. In contrast, an oligonucleotide probe containing a mutated
NF-
B consensus sequence did not produce a gel-shifted band when
nuclear extracts from TNF-
-treated cells were used (data not shown). 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 TNF-
treatment (Fig. 10). In
contrast, treating BAE or bovine PA endothelial cells for 15 or 30 min
with hyperosmotic medium had no effect on NF-
B activation (data not
shown). Data in Fig. 11 show that a 10- or 50-fold excess of unlabeled NF-
B oligonucleotide competed for
binding of the NF-
B-labeled probe in nuclear extracts prepared from
BAE and bovine PA endothelial cells treated with TNF-
. The mutant
NF-
B oligonucleotide exhibited weak competition for the NF-
B gel
shift, and a nonspecific unlabeled oligonucleotide containing the E-box of the adenovirus major late transcription factor promoter had no
effect on NF-
B oligonucleotide binding. As shown in Table 2,
we found that calphostin C, genistein, pyrrolidinedithiocarbamate, and
TLCK, each to varying degrees, were able to prevent the TNF-
-induced inhibition of myo-inositol
accumulation by BAE cells. To determine whether the effects of these
compounds may be related to blocking the TNF-
-induced
activation of NF-
B, we conducted an electrophoretic mobility shift
assay using nuclear extracts prepared from untreated and
TNF-
-treated (5 ng/ml, 30 min) cells as well as from cells pretreated for 1 h with each of these inhibitors followed by a 30-min
incubation with TNF-
. Data in Fig. 12
(representative of 3 separate experiments) show that TNF-
activation
of NF-
B in BAE cells is partially reduced by calphostin C and
pyrrolidinedithiocarbamate and totally blocked by TLCK. Genistein
appeared not to decrease TNF-
activation of NF-
B. NF-
B is a
member of the Rel family of transcriptional regulatory proteins which
includes p50 (NF-
B1), p52 (NF-
B2), Rel A (p65), cRel, and Rel B. The existence of more than one of these members in a cell may result in
the appearance of more than one NF-
B binding complex. Data in Fig.
10 show that nuclear extracts from TNF-
-treated BAE cells form two
complexes, a faster-migrating lower complex and a predominant upper
complex. This may be due to the presence of more than one isoform of
NF-
B in BAE cells. However, this has not been a consistent finding, as shown in Fig. 13. Data in Fig. 13 show
that in nuclear extracts prepared from BAE and bovine PA endothelial
cells treated with TNF-
for 30 min, the p65 antibody caused a gel
retardation (supershift) in the binding complex. In contrast, p50
antibody caused only a minimal gel retardation. As a control, data in
Fig. 13 show that an antibody to CREB had no effect. These results
suggest that p65 is the major NF-
B form activated by TNF-
in
these cells.

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Fig. 10.
Electrophoretic mobility shift assay of nuclear factor (NF)- B, using
nuclear extracts from BAE and bovine PA endothelial cells. Cells were
grown in 150-cm2 flasks to
confluency and then incubated for 15 (15") or 30 (30") min in
medium with or without 5 ng/ml TNF- . Cells were then harvested,
nuclear extracts were prepared, and gel mobility shift assays were
performed as described in MATERIALS AND
METHODS. For these studies, radiolabeled
oligonucleotide probes containing the consensus sequence for NF- B
(top) or E-box of the adenovirus
major late transcription factor promoter
(bottom) were used.
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Fig. 11.
Competition electrophoretic mobility shift assay of NF- B. Nuclear
extracts prepared from BAE and bovine PA endothelial cells treated for
30 min with 5 ng/ml TNF- were used to determine binding competition
by a 10- (10×) or 50-fold (50×) excess of unlabeled
oligonucleotide for NF- B, mutated (m) NF- B, or E-box of the
adenovirus major late transcription factor promoter, using radiolabeled
oligonucleotide probe containing the consensus sequence for NF- B.
Gel mobility shift assay was performed as described in
MATERIALS AND METHODS.
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Fig. 12.
Effect of calphostin C, genistein, pyrrolidinedithiocarbamate (PD), and
7-amino-1-chloro-3-tosylamido-2-hepatanone (TLCK) on TNF- -induced
activation of NF- B in BAE cells. Cells were grown to confluency in
150-cm2 flasks and then incubated
for 30 min with TNF- (5 ng/ml) in absence or presence of inhibitors.
Cells receiving inhibitors were preincubated for 1 h with each
inhibitor before addition of TNF- . Afterwards, cells were harvested,
nuclear extracts were prepared, and gel mobility shift assays were
performed as described in MATERIALS AND
METHODS. For these studies, radiolabeled
oligonucleotide probes containing the consensus sequence for NF- B
(top) or E-box of the adenovirus
major late transcription factor promoter
(bottom) were used.
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Fig. 13.
Electrophoretic mobility supershift assay of NF- B. BAE and bovine PA
endothelial cells were grown to confluency in
150-cm2 flasks and then incubated
for 30 min with TNF- (5 ng/ml). Afterwards, cells were harvested,
nuclear extracts were prepared, and gel mobility supershift assays were
performed as described in MATERIALS AND
METHODS. For these studies, nuclear extracts were
preincubated with or without 1 µg of antibodies anti-p50, anti-p65,
or anti-CREB before addition of radiolabeled oligonucleotide probe
containing the consensus sequence for NF- B.
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 |
DISCUSSION |
Most mammalian cells acquire
myo-inositol through an active,
Na+-dependent cotransporter
(SMIT), and the internal concentration of
myo-inositol is generally 5- to
500-fold higher than the level of
myo-inositol in plasma or
extracellular fluid (2). Myo-inositol is an important component of membranes and, in the form of
phosphoinositides, is an integral part of several signal transduction
pathways (1, 20, 21). In addition, in renal and other cells,
myo-inositol is one of several organic
osmolytes that are important for maintaining an osmotic balance during
periods of osmotic stress (3, 36).
Increased osmotic stress activates different members of the MAP kinase
family of signal transduction proteins, including ERK, JNK, and p38
kinase (14, 15). Cytokines, such as TNF-
, have been shown to
regulate the activity of these same signal transduction proteins (14,
15). Therefore, we designed studies to determine the effect of TNF-
on SMIT mRNA levels and myo-inositol
accumulation, anticipating that the effect of TNF-
on
myo-inositol accumulation would be
similar to the effect of hyperosmolarity. However, in contrast to the
effect of hyperosmolarity, TNF-
significantly decreased
myo-inositol accumulation and SMIT
mRNA levels in cultured large-vessel endothelial cells and CME cells.
The effect of TNF-
on myo-inositol
accumulation was mimicked by LPS, IL-1
, and TGF-
in large-vessel
endothelial cells. In addition, the effect of TNF-
on
myo-inositol accumulation was highly
selective, in that TNF-
did not decrease the accumulation of
myo-inositol by a wide variety of
other mammalian cultured cells, including microvessel endothelial cells
derived from periaortic adipose tissue and coronary artery endothelial
cells. The reason for these differences is not known. However, it seems
unlikely that the lack of an effect by TNF-
on
myo-inositol accumulation by the
unaffected cells was due to an absence of TNF-
receptors, because
TNF-
interacts with a wide variety of cell types and most cells
express specific TNF-
receptors on their cell surface (12).
Stimulation of sphingomyelinase by TNF-
causes the release of two
second messengers, ceramide and sphingosine (19, 27, 28, 35). Our
studies showed that the inhibition of sphingosine production by
N-oleoylethanolamine did not influence
the effect of TNF-
on myo-inositol
accumulation (26). In addition, incubating endothelial cells with
sphingosine had no significant effect on myo-inositol accumulation. In
contrast, the addition of
C2-ceramide to the medium mimicked
the effect of TNF-
on SMIT mRNA levels and
myo-inositol accumulation. These
results suggest that the production of ceramide is responsible for the
TNF-
-induced decrease in SMIT mRNA levels and
myo-inositol accumulation.
The activation of ERK1, p38 kinase, S6 kinase, phosphatidylinositol
3-kinase, or PLA2 by TNF-
are
not necessary for the inhibition of
myo-inositol accumulation by TNF-
.
Studies by Kwon et al. (13) have indicated that activation of the MAP
kinase cascade does not contribute to the hyperosmotic induction of
myo-inositol or betaine uptake by
Madin-Darby canine kidney cells. Taken together, these studies suggest
that the activation of the MAP kinase cascade may not contribute to the
regulation of myo-inositol
accumulation by osmotic stress or cytokines. However, it remains to be
determined whether the activation of JNK may have a role in mediating
myo-inositol accumulation by TNF-
.
Lee et al. (16) have shown that in HeLa cells TNF-
activates MAP
kinase/ERK kinase kinase 1, a kinase in the pathway of
JNK activation. JNK, in turn, have also been shown to phosphorylate
I-
B-
, resulting in activation of NF-
B.
Our data show that TNF-
activates NF-
B in BAE and bovine PA
endothelial cells and that the activation of NF-
B is associated with
the effect of TNF-
on SMIT mRNA levels and
myo-inositol accumulation.
Staurosporine, a nonspecific inhibitor of PKC, did not affect the
decrease in myo-inositol accumulation
by TNF-
(26). In contrast, the TNF-
-induced decrease in
myo-inositol accumulation was
partially prevented by calphostin C, which inhibits PKC-
, an isoform
of PKC that is insensitive to staurosporine and has been shown to
stimulate binding of NF-
B to DNA (26). Moreover,
pyrrolidinedithiocarbamate, an inhibitor of NF-
B activation, and
genistein and TLCK, a protein tyrosine kinase and protease inhibitor,
respectively, also significantly reduced the TNF-
-induced decrease
in myo-inositol accumulation. These
latter two agents could inhibit the proteolytic degradation of I-
B
and thereby inhibit the activation of NF-
B. Consistent with their
effects on myo-inositol accumulation,
TLCK, calphostin C, and pyrrolidinedithiocarbamate prevented the
TNF-
-induced activation of NF-
B to different degrees in BAE
cells. In contrast, genistein, which partially prevented the
TNF-
-induced decrease in
myo-inositol accumulation, did not prevent the TNF-
-mediated activation of NF-
B. The reason for this
inconsistency is unknown and requires further investigation. Nonetheless, these data suggest that TNF-
activates NF-
B by a
mechanism that could be dependent on the activation of PKC-
and that
NF-
B, in turn, may directly or indirectly decrease the expression of
the SMIT gene and reduce SMIT protein levels. TNF-
has been shown to
repress transcription of several genes, including the vascular
endothelial growth factor receptor gene in endothelial cells (22) and
the CCAAT-enhancer binding protein and glucose transporter isoform 4 genes in 3T3-L1 adipocytes (30). However, it has
not been shown that the activation of NF-
B by TNF-
is directly
responsible for downregulating the expression of these genes or the
SMIT gene. NF-
B is primarily an activator of gene transcription. It
is possible that TNF-
-induced activation of NF-
B is an
independent event and not related to the TNF-
-mediated regulation of
SMIT mRNA levels or myo-inositol
accumulation. Second, it is possible that NF-
B activation is
increasing the transcription of a protein(s) that regulates SMIT mRNA
stability and/or protein turnover and thus is a secondary event
contributing to the posttranscriptional regulation of the SMIT. To
unequivocally answer this question, nuclear run-on assays are currently
being conducted as well as studies of the 5'-flanking region of
the SMIT gene, which has not yet been completely sequenced (25).
Therefore, at this time we can only state that the downregulation of
SMIT mRNA levels and myo-inositol
accumulation by TNF-
parallels the activation of NF-
B in
endothelial cells.
In summary, our studies show that TNF-
regulates SMIT mRNA levels
and myo-inositol accumulation in
cultured large-vessel endothelial cells and CME cells. TNF-
-induced
ceramide production appears to be a proximal event in this effect,
whereas the studies demonstrating that inhibitors of NF-
B activation
partially prevent the TNF-
-induced decrease in
myo-inositol accumulation suggest that
a distal event in the effect of TNF-
may include NF-
B activation. Elucidation of the signaling pathways linking NF-
B activation with
ceramide production and regulation of SMIT mRNA levels and myo-inositol accumulation will require
further investigation. The contrasting effects of TNF-
and
hyperosmolarity on the regulation of SMIT mRNA levels and
myo-inositol accumulation suggest
separate regulatory pathways. One difference is that hyperosmolarity,
unlike TNF-
, does not activate NF-
B. To date, the
5'-flanking region of the SMIT gene has not been cloned, and it
is unknown whether the putative "osmotic response element" (ORE)
of the SMIT gene is similar to the ORE of the aldose reductase gene or
the tonicity-sensitive element of the betaine transporter gene (6, 25,
31). Additional studies, such as cloning and characterization of the
5'-flanking region of the SMIT gene, will be necessary to advance
our understanding of the regulation of
myo-inositol metabolism.
Interestingly, TNF-
has a variety of effects in endothelial cells,
including the regulation of the expression of cell adhesion molecules,
proteoglycan synthesis, cell permeability, and proliferation (4, 10).
Whether changes in myo-inositol
metabolism are associated with these TNF-
-mediated events in
endothelial cells is currently under investigation. As mentioned
previously, myo-inositol metabolism is
associated with membrane synthesis and is required for signal
transduction pathways, so changes in
myo-inositol metabolism could have a
wide range of effects on the endothelium.
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-45453 and DK-25295 and by a
Merit Review Grant from the Department of Veterans Affairs.
Address for reprint requests: M. A. Yorek, 3E17 Veterans Affairs
Medical Center, Iowa City, IA 52246.