From the Department of Biochemistry, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284
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ABSTRACT |
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Treatment of cultured rat Kupffer cells with
lipopolysaccharide (LPS) resulted in a time-dependent
increase in the expression of the inducible isoform of nitric-oxide
synthase (iNOS). Agents that elevated intracellular cAMP levels
(e.g. forskolin, dibutyryl cAMP, cholera toxin, and
isoproterenol) markedly decreased nitrite production and iNOS protein
formation by LPS-stimulated Kupffer cells. Furthermore, inhibition of
LPS-induced nitrite formation and iNOS protein levels by these agents
was enhanced in the presence of the phosphodiesterase inhibitor
3-isobutyl-1-methylxanthine. Forskolin, the most potent inhibitor
of LPS-induced nitrite formation by Kupffer cells, decreased iNOS
mRNA levels in a time-dependent manner. Time course
studies indicated that forskolin was most effective at inhibiting
LPS-induced nitrite formation and iNOS mRNA levels by Kupffer cells
when added before LPS. Message stability studies established that
forskolin did not enhance the rate of decay of LPS-induced iNOS
mRNA. Nuclear run-on assays revealed that forskolin decreased
LPS-induced transcription of the iNOS gene. Treatment of Kupffer cells
with LPS induced the translocation of the p65 subunit of nuclear factor
B (NF-
B) into the nucleus, and this process was abolished by
forskolin. In addition, the LPS-dependent degradation of
I
B
was not observed in forskolin-treated cells; the levels of the
p65 subunit of NF-
B were minimal in the nucleus at the same time.
Also, we observed that forskolin induced transcription of the I
B
gene in a time-dependent manner and in addition
up-regulated LPS-induced I
B
mRNA levels. Taken together, this
study indicates that the attenuation of LPS-induced iNOS formation in
Kupffer cells by elevated intracellular cAMP levels occurs by
preventing the degradation of I
B
which suppresses the activation
of NF-
B and inhibits the onset of transcription of the iNOS
gene.
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INTRODUCTION |
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Nitric oxide (NO)1 has
been identified as an important signaling molecule that is involved in
regulating a wide array of biological activities in neural, vascular,
and immune cellular systems (1). NO is generated from
L-arginine and molecular oxygen in the presence of the
enzyme NO synthase (NOS) (2). To date three distinct NOS isoforms have
been identified from molecular cloning and sequencing analyses (3). The
endothelial (eNOS) and neuronal (nNOS) isoforms are expressed
constitutively in endothelial and neuronal cells, respectively. The
amount of NO generated by these cell types is dependent upon the
cellular content of NOS (4). NO generated by endothelial cells plays an
important role in the control of vascular tone, whereas in neuronal
tissue NO acts to regulate cGMP-mediated neurotransmission (1, 2). The
third isoform of NOS, termed inducible NOS (iNOS), was first identified
in macrophages stimulated with interferon- and bacterial
lipopolysaccharide (LPS) (3). iNOS has been identified in a wide
variety of cell types including macrophages, mesangial cells, vascular
smooth muscle cells, keratinocytes, chondrocytes, osteoclasts, and
hepatocytes (1, 5). NO generated within these cells mediates macrophage cytotoxicity during host defense reactions, alterations in the contractile responses of mesangial cells, and in instances where NO
exceeds normal physiological levels, instigates the inhibition of
vascular smooth muscle tone, hepatocyte metabolism, and protein synthesis (6-8). Recent evidence indicates that elevated levels of NO
play a major role in the pathogenesis of several chronic disorders and
inflammatory processes. In particular, studies have indicated that an
overproduction of NO in response to LPS and cytokines contributes to
the development and prolongation of severe hypotension and peripheral
vasodilation observed during endotoxic shock (9).
The activities of nNOS and eNOS are regulated by rapid, transient elevations of intracellular free calcium which enhance the binding of calmodulin to the NOS enzyme resulting in NO release over a time frame of seconds and minutes (10). In contrast, the expression of iNOS is thought to be regulated primarily at the transcriptional level of the iNOS gene. Once induced, iNOS produces NO for periods of several hours or days. Thus, given the magnitude of the wide variety of inhibitory actions of NO, it is of considerable interest and even may be of some therapeutic utility to delineate the mechanism(s) by which the production and resultant activity of NOS can be controlled or attenuated.
In the presence of LPS, Kupffer cells, the resident macrophage found in the sinusoids of the liver, produce large amounts of nitrite and nitrate, the stable end products of the NO pathway (11). It has become apparent recently that overproduction of NO by hepatic cells plays a major role in hepatic injury/necrosis associated with endotoxic shock. Kupffer cells also synthesize and release several cytokines in response to LPS which in turn stimulate neighboring hepatocytes to generate NO (8). The consequent overproduction of NO in the liver results in profound degenerative changes observed in hepatocytes (12). These changes include a decrease in total protein synthesis, cellular proliferation, and an increase in cGMP formation (13). The induction of iNOS by LPS in Kupffer cells requires the initiation of gene expression and de novo protein synthesis over a period of several hours. It is unclear whether classical second messengers such as cAMP are involved in iNOS gene expression and NO formation. Recent studies have indicated that agents that elevate levels of cAMP improve circulatory function in animal models of endotoxic shock; in particular isoproterenol was found to inhibit the development of vascular hyporeactivity in the endotoxic rat (14, 15). In contrast, certain in vitro studies have shown that elevation of cAMP caused an induction of iNOS, whereas in other studies increased levels of cAMP caused a reduction in iNOS (6, 7, 16). The present study was designed to investigate whether iNOS gene expression and/or enzymatic activity is regulated by elevated levels of cAMP in cultured rat Kupffer cells.
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EXPERIMENTAL PROCEDURES |
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Isolation and Primary Culture of Rat Kupffer Cells-- Following enzymatic digestion of the rat liver, Kupffer cells were isolated by centrifugal elutriation as described previously (17). The viability of the Kupffer cell preparation was greater than 95% as determined by trypan blue exclusion. Freshly isolated Kupffer cells were maintained at 37 °C in RPMI 1640 culture medium (Life Technologies, Inc.) supplemented with 25 mM HEPES, L-glutamine, and 10% fetal bovine serum (Hyclone Laboratories, Inc., Logan, UT), 112 units/ml penicillin, and 112 units/ml streptomycin in 24-well plates or 60-mm tissue culture dishes. All cells were incubated in an atmosphere of 90% air and 10% CO2. On the 2nd day of culture the RPMI medium was changed. For experimental purposes, Kupffer cells were used within 3-4 days of their establishment in culture.
Measurement of Nitrite Formation-- Production of NO by iNOS was quantified by measuring the accumulation of nitrite in the culture medium using the Griess reaction (18). Kupffer cells were cultured in 24-well plastic tissue culture plates at a density of 1 × 106 cells/ml (1 ml/well) at 37 °C. After 4 days in culture the cells were washed, and complete medium without phenol red was added to each well. The cells were then exposed to several cAMP-elevating agents. After a specified incubation interval the medium from each well was removed. The nitrate in each sample was reduced to nitrite using the method described by Grisham et al. (19). Samples were then mixed with an equal volume of the Griess reagent (1% sulfanilamide, 5% H3PO4, 0.1% naphthylethylenediamine dihydrochloride) and incubated at room temperature for 10 min. The absorbance of each sample was measured spectrophotometrically at 543 nm using sodium nitrite as a standard. Nitrite formation was expressed as nmol/106 cells. Values denote the mean ± S.D. of quadruplicate determinations from at least two separate experiments, unless otherwise stated.
Preparation of Whole Cell, Nuclear, and Cytoplasmic
Extracts--
Kupffer cells were plated at a density of 1 × 107 cells/60-mm dish. After 3 days in culture, the cells
were rinsed with fresh medium and stimulated with LPS alone or with
cAMP-elevating compounds. Following treatment, the cells were rinsed
with phosphate-buffered saline three times; then 500 µl of lysis
buffer (50 mM Tris-HCl, pH 7.4) containing 5 mM
EDTA, 5 mM EGTA, 1 µM leupeptin, 1 µM pepstatin A, 1 µM aprotinin, and 1 µM phenylmethylsulfonyl fluoride was added to each dish,
and the cells were scraped quickly. The resulting cell suspensions were
subjected to three rapid freeze-thaw-vortex cycles to disrupt the
Kupffer cells completely. A 100-µl sample of the broken cell
preparation was used for protein quantitation, and the remainder of the
sample was stored at 80 °C until analyzed.
Western Blot Analysis for iNOS-- Before SDS-PAGE the disrupted cell suspensions were dried using a Savant vacuum centrifuge. Sample pellets were solubilized in buffer containing 0.1 M dithiothreitol, 50% glycerol, 0.5 M Tris, pH 6.8, 2.5 mM pyronine Y, and 20% SDS and subjected to SDS-PAGE (7.5% gel) using the buffer system of Laemmli (21). The separated proteins were transferred electrophoretically to polyvinylidene difluoride membranes, using a semidry transfer blot system, and the membranes were soaked for 1 h in Tris-buffered saline, pH 7.4, containing 5% non-fat dried milk powder and incubated for 24 h with anti-iNOS antibody in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.01% Tween 20, and 1% bovine serum albumin. The blots were then incubated with horseradish peroxidase-labeled goat anti-mouse IgG in the same buffer for 2 h. Finally, the blots were rinsed in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.01% Tween 20. Peroxidase-labeled proteins were visualized by incubation with the peroxidase color development reagent 3,3'-diaminobenzidine and hydrogen peroxide.
Western Blot Analysis for p65 and IB
--
Cytoplasmic and
nuclear samples were resolved using SDS-PAGE (11% gel). The separated
proteins were electrotransferred to polyvinylidene difluoride
membranes. The membranes were then incubated in blocking buffer
(Tris-buffered saline, pH 7.4) containing 10% non-fat dried milk
powder for 1 h and then exposed to diluted primary antibodies
against the p65 subunit of NF-
B or I
B
overnight at 4 °C.
The membranes were incubated for 1 h at room temperature with
5,000-fold diluted horseradish peroxidase-conjugated anti-rabbit IgG
antibody. Protein bands were visualized using an enhanced chemiluminescence (ECL) assay kit.
Northern Blot Analysis--
Kupffer cells were plated at a
density of 1 × 107 cells/60-mm dish. After 3 days in
culture and the appropriate treatment, total RNA from cultured rat
Kupffer cells was isolated using TRIzol reagent (Life Technologies,
Inc.). RNA (3-4 µg) was separated by electrophoresis on a 0.8%
agarose, 2.2 M formaldehyde gel and transferred using a
Possiblot (Stratagene, La Jolla, CA) onto a Magna nylon membrane
(Microns Separations Inc. Westborough, MA). A full-length murine iNOS
cDNA probe kindly provided by Dr. S. H. Snyder (The Johns Hopkins
University School of Medicine, Baltimore) or I-B cDNA kindly
provided by Dr. A. Baldwin Jr. (University of North Carolina, Chapel
Hill) was labeled with a multiprime DNA labeling system using
[
-32P]dCTP (specific activity, 3,000 Ci/mmol).
Northern blot hybridizations were performed in 50% formamide, 1 M NaCl, 10% dextran sulfate, 50 mM Tris-HCl,
pH 7.5, 0.1% sodium pyrophosphate, and 0.2% Denhardt's solution at
42 °C for 16 h. The membranes were washed twice in 2 × SSC, 1% SDS at 65 °C for 20 min, twice in 0.1 × SSC, 0.1% SDS at 55 °C for 15 min, and finally at room temperature in 0.1 × SSC for 15 min. Radioactivity was visualized using a
PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Control
hybridizations were performed using a 32P end-labeled
oligonucleotide complementary to rat 18 S rRNA.
Nuclear Run-on Analysis--
Nuclei were isolated from treated
Kupffer cells (5-6 × 107) according to standard
protocols (22). Briefly, cells were rinsed once in ice-cold
phosphate-buffered saline, scraped gently in 6 ml of phosphate-buffered
saline, and centrifuged at 800 × g at 4 °C for 5 min. Lysis buffer (10 mM Tris-Cl, 10 mM NaCl, 3 mM MgCl2, 0.5% (v/v) Nonidet P-40, 2 ml) was
added followed by vortexing for 10 s to disrupt the cell pellets.
The cells were incubated on ice for 5 min and nuclei pelleted by
centrifugation at 800 × g at 4 °C for 5 min. The
pelleted nuclei were resuspended in storage buffer (50 mM
Tris-HCl, 5 mM MgCl2, 0.1 mM EDTA,
40% (v/v) glycerol), frozen, and then stored in liquid nitrogen until needed. For in vitro transcription, freshly thawed nuclei
were incubated in reaction buffer (10 mM Tris-HCl; 5 mM MgCl2; 0.3 M KCl; 100 mM ATP, CTP, GTP; 1 M dithiothreitol; 10 mCi/ml
[-32P]UTP (3,000 mCi/mmol, Amersham Corp.), shaking
for 30 min at 30 °C. The reaction was quenched by the addition of
RNase-free DNase I (10 mg/ml, Worthington) and proteinase K (20 mg/ml,
Ambion, Austin, TX) and incubated for 30 min at 42 °C. Nascent
labeled RNAs were purified by extraction with phenol/chloroform and two sequential precipitations with ammonium acetate. Equal amounts of
32P-labeled RNA were resuspended in 1 ml of TES/NaCl
solution (10 mM TES, pH 7.4, 10 mM EDTA, 0.2%
SDS, 0.3 M NaCl) and hybridized for 60 h at 65 °C
to denatured DNA probes immobilized on nitrocellulose membranes.
Following hybridization, the filters were washed twice in 2 × SSC
at 65 °C for 1 h, once at 37 °C in 2 × SSC containing RNase A (10 mg/ml, Ambion) for 30 min, and once in 2 × SSC at 37 °C for 1 h. Radioactivity was visualized using a
PhosphorImager.
Materials--
Anti-mac iNOS was obtained from
Transduction Laboratories (Lexington, KY). Rabbit polyclonal
antibodies raised against p65 and IB
were purchased from Santa
Cruz Biotech Inc. (Santa Cruz, CA). Goat anti-mouse and goat
anti-rabbit IgG horseradish peroxidase conjugate and prestained
SDS-PAGE standards were obtained from Bio-Rad. Bacterial LPS (from
Escherichia coli, serotype 011:B4), dibutyryl cAMP, cholera
toxin, forskolin, isoproterenol, IBMX, and actinomycin D were purchased
from Sigma.
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RESULTS |
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cAMP-elevating Agents Inhibit LPS-induced Nitrite and iNOS Protein
Formation by Kupffer Cells--
Cultured unstimulated Kupffer cells
exhibited a low basal level of nitrite production, whereas LPS caused
an 8-fold increase in nitrite formation during the 24-h observation
period (Table I). The addition of various
cAMP-elevating agents attenuated LPS-induced nitrite formation by
Kupffer cells, each in a dose-dependent manner (data not
shown). The effect of the highest concentration of each agent used in
this study is noted in Table I. The diterpene forskolin activates
adenylate cyclase directly, resulting in an increase of intracellular
cAMP levels; forskolin inhibited LPS-induced nitrite formation
strongly. The membrane-permeable cAMP analog dibutyryl cAMP reduced
LPS-induced nitrite formation by 34%. Both isoproterenol and cholera
toxin stimulate adenylate cyclase via the stimulatory G-protein
Gs, isoproterenol by binding to cell surface -adrenergic
receptors and cholera toxin by ADP-ribosylation of Gs. We
have used these agents previously to demonstrate
cAMP-dependent changes in platelet-activating factor
binding in Kupffer cells, and we have confirmed that isoproterenol
causes an increase in intracellular cAMP in these cells (23). It is
important to note that none of the cAMP-elevating agents (at the
indicated concentrations) caused 1) any significant change in the rate
of formation of nitrite above the control value when added to Kupffer
cells in the absence of LPS (Table I) and 2) any morphological
alterations or detachment of Kupffer cells after an incubation period
of 24 h. In addition, LPS-induced nitrite formation by Kupffer
cells in the combined presence of the aforementioned cAMP-elevating
agents and the phosphodiesterase inhibitor IBMX was inhibited to a
greater degree than in the absence of IBMX (Table I). Thus
cAMP-elevating agents clearly are capable of decreasing LPS-induced
iNOS activity in Kupffer cells.
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Forskolin Inhibited LPS-induced Nitrite Formation and iNOS mRNA Levels in a Time-dependent Manner-- Kupffer cells were treated with either LPS or LPS and forskolin; nitrite formation was measured at the time intervals indicated in Fig. 2A. In agreement with other research groups, LPS stimulated nitrite formation by Kupffer cells in a time-dependent fashion. The inhibitory effects of forskolin on LPS-induced nitrite formation by Kupffer cells became apparent after 6-8 h of treatment and continued up to 24 h. The time dependence of LPS-induced iNOS mRNA accumulation in Kupffer cells is shown in Fig. 2, B and D; the iNOS mRNA level increased rapidly between 3 and 6 h and then declined by 24 h. The addition of forskolin caused a considerable decrease in the accumulation of iNOS mRNA, and the levels of message were barely detectable at 24 h (Fig. 2, C and D).
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Kinetics of Inhibition of LPS-induced Nitrite and iNOS mRNA Formation by Forskolin-- Forskolin was added to Kupffer cells before, at the same time as, or at different times after the addition of LPS to determine the optimum time for the inhibition of LPS-induced nitrite production and iNOS mRNA levels. Nitrite accumulation in the culture medium was measured 24 h after the addition of LPS. Fig. 3A shows that maximal suppression of LPS-induced nitrite formation by Kupffer cells occurred when forskolin was present 1 h before the addition of LPS. When forskolin was added after LPS, its inhibitory effect decreased gradually with time. Fig. 3B depicts a similar experiment except iNOS mRNA was isolated and analyzed by Northern blotting. When Kupffer cells were pretreated with forskolin for 1 h before the addition of LPS, the level of iNOS mRNA formed after 6 h was barely detectable. However, the intensities of the iNOS mRNA bands increased when forskolin was added at the same time as LPS and subsequently 2 and 4 h after the addition of LPS.
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Effects of LPS and Forskolin on the Half-life of iNOS
mRNA--
To examine whether forskolin attenuated LPS-induced
steady-state levels of iNOS mRNA by decreasing its stability, we
assessed the effects of forskolin on the half-life of LPS-induced iNOS mRNA by coincubation of Kupffer cells with the transcriptional inhibitor actinomycin D. Kupffer cells were treated with LPS in the
presence and absence of forskolin for 6 h to induce maximal iNOS
mRNA accumulation. Actinomycin D (10 ng/ml) was added to the cells
at this point to inhibit further transcription. At different times
after the addition of actinomycin D, total RNA was isolated and
examined by Northern analysis. To allow for differences in loading, the
signal density of each RNA sample hybridized to the iNOS probe was
corrected by that hybridized to the 18 S probe. Fig.
4 shows the decay of iNOS mRNA as ln
(relative intensity) against time. Under these conditions, the
half-life of iNOS mRNA can be calculated as (ln (2)/gradient of
regression line). The calculated half-lives of iNOS mRNA in
LPS-stimulated Kupffer cells in the absence and presence of forskolin
were 2.4 and 2.3 h, respectively; therefore, the reduction of
LPS-induced iNOS mRNA levels by forskolin in Kupffer cells was not
caused by a decrease in message stability.
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Effect of Forskolin on Transcription of the iNOS Gene--
iNOS
gene transcription in Kupffer cells was measured directly using a
nuclear run-on assay to confirm that forskolin caused inhibition of
this process. Kupffer cells were incubated either alone, or with LPS or
LPS and forskolin for 3 h and 5 h; cells were lysed and
nuclei isolated. The transcription of iNOS and -actin by isolated
nuclei was determined by hybridizing the elongated, labeled RNA
transcripts to iNOS- and
-actin-specific cDNA fragments that had
been slot blotted onto a nitrocellulose membrane. Fig. 5A shows that iNOS gene
transcription was barely detectable in control cells (lane
1), was increased greatly by 3-h exposure to LPS (lane
2), and that pretreatment with forskolin for 1 h attenuated
the LPS effect (lane 3). In a similar experiment using a 5-h
stimulation with LPS, a 1-h pretreatment with forskolin caused no
apparent attenuation of iNOS gene transcription (compare lanes
2 and 3 in Fig. 5B). These results are in
agreement with time-dependent changes in iNOS mRNA.
Fig. 2D shows that after a 3-h stimulation with LPS a pulse
of iNOS gene transcription has just commenced, and the effect of added
forskolin will be maximal, i.e. the gradients of the curves
with and without forskolin differ considerably. After 5 h of LPS
stimulation the pulse of iNOS gene transcription is ending; mRNA
levels fall after 6 h. At this time the effect of added forskolin
will be minimal; the gradients of the curves with and without forskolin
will differ very little as both approach their highest values. Taken
together, the above findings indicated that forskolin attenuates
LPS-induced iNOS mRNA formation, mainly at the transcriptional
level.
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Forskolin Decreased the Translocation of the p65 Subunit of NF-B
from the Cytoplasm into the Nucleus--
Recent studies by Xie
et al. (24) have shown that activation of the transcription
factor NF-
B and its binding to the promoter region of the iNOS gene
are critical steps in the induction of iNOS synthesis by LPS in
macrophages. In addition, Tran-Thi et al. (25) reported
NF-
B-binding activity in Kupffer cells treated previously with LPS.
In view of these findings, we decided to investigate the effect of
forskolin on NF-
B activation in the presence and absence of LPS.
Kupffer cells were either untreated, treated with LPS for 30 min,
pretreated with forskolin for 1 h and then stimulated with LPS for
30 min, or pretreated with forskolin for 1 h and then stimulated
with vehicle for 30 min; cytoplasmic and nuclear proteins were then
isolated. A representative experiment is depicted in Fig.
6. Panel A represents
cytoplasmic extracts, and panel B represents nuclear
extracts. In untreated cells (lane 1) the p65 subunit of
NF-
B was located mainly in the cytoplasm. A residual amount of p65
was detected in the nuclear extract. In contrast, LPS (lane
2) increased greatly the amount of p65 detected in the nucleus of
Kupffer cells. Interestingly, in forskolin-treated Kupffer cells, both
in the presence (lane 3) and absence (lane 4) of
LPS, the amount of p65 detected in the nuclear extract was greatly
diminished compared with LPS-treated cells (lane 2).
Practically all of the p65 was retained in the cytoplasm (lanes
3 and 4), suggesting that forskolin interfered with the
translocation of p65 from the cytoplasm into the nucleus.
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Effects of Forskolin on the LPS-induced Degradation of
IB
--
NF-
B proteins reside in the cytoplasm of resting
cells complexed to a family of inhibitory proteins designated I
B,
which includes I
B
and I
B
subunits. Activation of NF-
B,
which results in the translocation of the free protein into the
nucleus, is preceded by a rapid phosphorylation and proteolytic
degradation of I
B subunits (26). Using Western blot analysis we
examined the levels of I
B
protein, a major form of I
B in the
cytoplasmic extracts of untreated and treated Kupffer cells. A
representative experiment is depicted in Fig.
7. In cytoplasmic extracts from untreated
Kupffer cells (lane 1), an I
B
-specific antibody
detected an intense single band with a molecular mass of about 45 kDa. In contrast, after 30 min of treatment of Kupffer cells with LPS (lane 2), the amount of I
B
protein detected was
reduced greatly compared with that in untreated cells. However, in
forskolin-treated Kupffer cells both in the presence (lane
3) and absence (lane 4) of LPS, the levels of I
B
detected in the cytoplasm were comparable to the levels of the protein
formed in untreated cells (compare lanes 1, 3,
and 4). This finding strongly indicated that in the presence
of forskolin the I
B
protein in the cytoplasm of Kupffer cells
remains intact and remains complexed to the NF-
B proteins.
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Effect of Forskolin and LPS on IB
mRNA Levels in Kupffer
Cells--
The above results showed that in the presence of forskolin,
the integrity of I
B
protein in the cytoplasm of Kupffer cells was
maintained. We then investigated whether forskolin actually initiated
I
B
gene expression in Kupffer cells. Kupffer cells were
stimulated with either forskolin or LPS or LPS and forskolin and at the
times indicated in Fig. 8. RNA was
isolated and employed in Northern blot analyses. The mRNA for
I
B
in Kupffer cells appeared as a single band at approximately
1.6 kilobases. It is important to note here that in untreated Kupffer
cells the level of I
B
mRNA was undetectable (data not shown).
In the presence of forskolin (Fig. 8, panels A and
D), I
B
mRNA levels peaked after 2 h of
stimulation and then declined to negligible levels by 6 h. In
contrast, in the presence of LPS (panels B and
D), I
B
mRNA levels increased just after 30 min of
stimulation, reached a peak by 1 h, and then remained elevated for
up to 6 h. When Kupffer cells were pretreated for 1 h with
forskolin and then stimulated with LPS over a period of 6 h
(panels C and D), it was observed that forskolin
up-regulated LPS-induced I
B
mRNA levels for up to 4 h
after the addition of LPS (Fig. 8D). These findings suggest
that forskolin has the capacity to induce I
B
mRNA synthesis
and in turn increase the levels of protein in the cytoplasm of Kupffer
cells. In the event of pretreating Kupffer cells with forskolin and
subsequent stimulation by LPS the cytoplasmic levels of I
B
are
maintained and thereby attenuate the translocation of NF-
B into the
nucleus.
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DISCUSSION |
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The release of NO by Kupffer cells under conditions of endotoxic shock represents an important contribution to the pathophysiology of the liver (27). Thus it is of considerable interest to investigate the intracellular signaling pathways that regulate the induction/suppression of iNOS gene expression. In Kupffer cells, agents that elevate intracellular cAMP levels attenuated both LPS-induced nitrite formation (Table I) and LPS-induced iNOS protein (Fig. 1). Furthermore, in the presence of forskolin LPS-induced iNOS mRNA formation was suppressed greatly (Fig. 2, C and D). This suggested that elevated levels of cAMP may have 1) directly suppressed the onset of iNOS gene transcription, 2) decreased transcription of the iNOS gene, or 3) destabilized iNOS mRNA following transcription. The half-life of iNOS mRNA was essentially the same in the presence or absence of forskolin, eliminating the possibility of altered mRNA stability (Fig. 4). Experiments in which forskolin was added to cultured Kupffer cells before, with, or at different times after LPS indicated that maximal inhibition of iNOS mRNA required the presence of forskolin before the addition of LPS (Fig. 3B). This favors alternative 1) above, i.e. suppression of the initiation of transcription. Direct measurement of iNOS gene transcription showed that at 3 h after LPS stimulation forskolin decreased (Fig. 5A) the transcriptional process substantially, whereas by 5 h after LPS stimulation forskolin had little effect.
The promoter region of the recently cloned rat gene encoding iNOS has
been found to contain consensus sequences for the binding of numerous
transcription factors (28). Activation of these factors is critical in
the induction of iNOS by LPS or cytokines. One transcription factor of
paramount importance required during the induction of iNOS by LPS in
macrophages is NF-B. The promoter region of the rat gene encoding
iNOS contains two copies of the NF-
B binding site consensus sequence
(24). NF-
B is an inducible, ubiquitous transcription factor present
in the cytoplasm of cells. It is composed of a dimer of p50 and p65
(Rel A) subunits. In resting cells the NF-
B complexes are
sequestered in the cytoplasm by association with a family of inhibitory
proteins which includes mainly I
B
and I
B
(26). Activation
of NF-
B can be initiated by a variety of agents including mitogens
such as phorbol 12-myristate 13-acetate and inflammatory cytokines such
as LPS and tumor necrosis factor
. After cellular activation the
I
B
proteins undergo phosphorylation and subsequent proteolytic
degradation via the ubiquitin pathway (29). Free NF-
B protein
rapidly translocates into the nucleus and binds to its consensus DNA
sequence(s) (26) regulating a variety of genes responsible for
immunological and inflammatory reactions (30).
As noted above, pretreatment of Kupffer cells by forskolin resulted in
maximal attenuation of LPS-induced iNOS mRNA in Kupffer cells. We
surmised that forskolin inhibited the LPS-stimulated nuclear
translocation of NF-B in Kupffer cells, resulting in decreased
transcription of the iNOS gene and consequently decreased steady-state
levels of iNOS mRNA. Western blot analysis of cytoplasmic and
nuclear extracts (Fig. 6) confirmed that in Kupffer cells forskolin
pretreatment greatly reduced the amount of NF-
B (p65 subunit) which
was translocated to the nucleus by LPS stimulation. A similar result
was obtained using cytoplasmic and nuclear extracts from Kupffer cells
that had been treated only with forskolin. These findings confirmed our
conjecture that forskolin inhibits LPS-induced iNOS mRNA formation
in Kupffer cells by functionally inactivating NF-
B.
Attenuation of NF-B activity by forskolin could occur by two
alternative pathways: either forskolin could directly prevent the
degradation of I
B
proteins and thus the translocation of NF-
B
into the nucleus, or forskolin could cause cytoplasmic retention of
NF-
B by some other mechanism without preventing degradation of the
I
B
protein. LPS treatment of Kupffer cells decreased levels of
cytoplasmic I
B
, and this decrease coincided with the appearance
of the p65 subunit of NF-
B in the nucleus. In cytoplasmic extracts
of Kupffer cells that had been pretreated with forskolin there appeared
to be no decrease in I
B
upon stimulation with LPS (compare
lanes 2 and 3, Fig. 7). These results indicate
that forskolin protects I
B
presumably by preventing its
phosphorylation and degradation.
Nuclear NF-B itself can induce the expression of the I
B
gene,
thus rapidly replenishing the depleted I
B
protein pool in the
cytoplasm of cells (31). The newly synthesized I
B
proteins then
complex with NF-
B in the cytoplasm, limiting its translocation into
the nucleus. This I
B homeostasis operates in LPS-stimulated Kupffer
cells, where I
B
mRNA levels remained elevated for longer than
6 h (Fig. 8, panel B); it provides the most likely
explanation for why LPS-induced iNOS mRNA levels in Kupffer cells
are not expressed indefinitely but were observed to decrease
substantially by 24 h (Fig. 2, B and D).
Forskolin alone induced transient I
B
mRNA formation in
Kupffer cells, with the mRNA levels peaking after 2 h of
stimulation and decreasing to control values by 6 h (Fig. 8,
panels A and D). Interestingly, pretreatment of
Kupffer cells for 1 h with forskolin up-regulated LPS-induced
I
B
mRNA levels (Fig. 8, panels C and
D). It is important to note that maximal up-regulation of
I
B
mRNA levels in Kupffer cells pretreated with forskolin for
1 h and then stimulated with LPS occurred after 1 h (Fig. 8,
panel D), but in fact the Kupffer cells had been in contact
with forskolin for 2 h, at which time forskolin induces maximum
amounts of I
B
mRNA in Kupffer cells (Fig. 8D). It
may be of importance to consider that the time at which maximal
inhibition of LPS-induced iNOS mRNA levels in Kupffer cells occurs
is when the cells have been pretreated with forskolin for 1 h
compared with the situation where forskolin was added after LPS (Fig.
3B), which coincides with maximal I
B
mRNA levels
in the cells. In contrast, when forskolin was added to Kupffer cells
several hours after LPS, the transcriptional and translational pathways
of iNOS formation are already well established, and thus there is no
opportunity for forskolin to exert its inhibitory mode of action on
iNOS synthesis.
Recent reports have shown that LPS stimulation of Kupffer cells
activates NF-B within 1 h, both in vitro (32, 33)
and in the intact rat (34). Moreover, CD18/ICAM-1-dependent
NF-
B activation leads to nitric oxide production in Kupffer cells
(35). To our knowledge, the present report is the first detailed
analysis of the NF-
B control system during iNOS response to
endotoxin in the Kupffer cell. Also, this is the first report to
characterize the effects of cAMP on this regulatory system; a previous
study using Kupffer cells reported no effect of dibutyryl cAMP on
LPS-stimulated iNOS induction (36).
The effects of cAMP on iNOS production are of increasing interest since
the first report (37) that cAMP-elevating agents induced iNOS in
cultured vascular smooth muscle cells and that this induction was
synergistic with that elicited by inflammatory cytokines. cAMP
elevation has been shown to have similar effects in renal mesangial
cells (6) and in rat brown adipoctyes (38). Although cAMP alone does
not induce iNOS in unstimulated cardiac myocytes, it augments iNOS
induction in interleukin 1-stimulated cells (39). NF-
B/Rel is
regulated positively by the cAMP cascade to help initiate iNOS gene
expression in response to LPS stimulation of the macrophage line RAW
264.7, and inhibition of adenylate cyclase attenuates the activation of
iNOS in these cells (40). In 3T3 fibroblasts different signaling
pathways including elevation of cAMP lead to the induction of iNOS by
NF-
B mediation (41). In contrast, elevation of cellular cAMP has
been shown to down-regulate iNOS in endotoxin-activated cultures of rat
microglia (quiescent brain macrophages (42)), rat primary astrocytes
(43), and J774 cells (murine macrophage line (44)).
Our present findings suggest that pretreatment of Kupffer cells with
forskolin both prevents the degradation of IB
and induces I
B
mRNA formation, thereby increasing the pool of I
B
proteins in the cytoplasm. When these same cells are stimulated with
LPS, degradation of I
B
proteins that are complexed with NF-
B
subunits occurs, but the newly activated NF-
B proteins reassociate
with the newly synthesized I
B
proteins rapidly, thus limiting
their translocation into the nucleus. A reduced translocation of
NF-
B proteins into the nucleus results in decreased iNOS gene
transcription and consequently low levels of iNOS formation.
The ability of cAMP to attenuate LPS-induced NO formation by Kupffer
cells provides a model in which to characterize the intracellular signaling pathways that regulate iNOS gene expression in the liver under conditions of endotoxic shock. Oxidant stress up-regulates and
antioxidants down-regulate NF-B, and hypoxia alters cellular signal
transduction in shock and sepsis (45). Treatment of Kupffer cells
in vitro which models hypoxia/ischemia may permit systemic shock to be evaluated separately from simple endotoxin exposure; it
will be of interest to determine whether these secondary shock-related effects on NF-
B are abrogated by cAMP elevation.
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ACKNOWLEDGEMENTS |
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We thank Lynnette Walters for isolating the Kupffer cells and for typing this manuscript. We are grateful to Dr. Katherine M. Howard for helpful comments and to Dr. Stephen A. K. Harvey for thoughtful criticism of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant DK-33538 and by Robert A. Welch Foundation Grant AQ-0728.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry,
The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78284.
1
The abbreviations used are: NO, nitric oxide;
NOS, nitric-oxide synthase; eNOS, nNOS, and iNOS, endothelial,
neuronal, and inducible NOS, respectively; LPS, lipopolysaccharide;
PAGE, polyacrylamide gel electrophoresis; NF-B, nuclear factor
B;
TES,
2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]]amino}ethanesulfonic acid; IBMX, 3- isobutyl-1-methylxanthine.
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REFERENCES |
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